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Sample records for adenosine deaminase inhibitor

  1. 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 ...

  2. [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.

  3. Non-linear quantitative structure-activity relationship for adenine derivatives as competitive inhibitors of adenosine deaminase

    SciTech Connect

    Sadat Hayatshahi, Sayyed Hamed; Khajeh, Khosro

    2005-12-16

    Logistic regression and artificial neural networks have been developed as two non-linear models to establish quantitative structure-activity relationships between structural descriptors and biochemical activity of adenosine based competitive inhibitors, toward adenosine deaminase. The training set included 24 compounds with known k {sub i} values. The models were trained to solve two-class problems. Unlike the previous work in which multiple linear regression was used, the highest of positive charge on the molecules was recognized to be in close relation with their inhibition activity, while the electric charge on atom N1 of adenosine was found to be a poor descriptor. Consequently, the previously developed equation was improved and the newly formed one could predict the class of 91.66% of compounds correctly. Also optimized 2-3-1 and 3-4-1 neural networks could increase this rate to 95.83%.

  4. 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 ...

  5. A new strategy based on pharmacophore-based virtual screening in adenosine deaminase inhibitors detection and in-vitro study

    PubMed Central

    2012-01-01

    Background and the purpose of the study Adenosine deaminase (ADA) inhibition not only may be applied for the treatment of ischemic injury, hypertension, lymphomas and leukaemia, but also they have been considered as anti- inflammatory drugs. On the other hand according to literatures, ADA inhibitors without a nucleoside framework would improve pharmacokinetics and decrease toxicity. Hence we have carried out a rational pharmacophore design for non-nucleoside inhibitors filtration. Methods A merged pharmacophore model based on the most potent non-nucleoside inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and natural products were generated and applied for compounds filtration. The effects of filtrated compounds based on pharmacophore and docking studies investigated on ADA by UV and Fluorescence spectroscopy techniques. Results Extracted compounds were find efficiently inhibit ADA, and the most potent (2) shows an inhibition constant equal to 20 μM. Besides, Fluorescence spectroscopy studies revealed that enzyme 3D structure bear further change in lower concentrations of compound 2. Conclusion 3 non-nucleoside inhibitors for ADA are presented. According to obtained results from UV and fluorescence spectroscopy, such interesting pharmacophore template with multiple approaches will help us to extract or design compound with desired properties. PMID:23351306

  6. 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

  7. Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity

    PubMed Central

    Larson, Eric T.; Deng, Wei; Krumm, Brian E.; Napuli, Alberto; Mueller, Natascha; Van Voorhis, Wesley C.; Buckner, Frederick S.; Fan, Erkang; Lauricella, Angela; DeTitta, George; Luft, Joseph; Zucker, Frank; Hol, Wim G. J.; Verlinde, Christophe L. M. J.; Merritt, Ethan A.

    2008-01-01

    Summary Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling. For this reason, plasmodial adenosine deaminase accepts a wider range of substrates, as it is responsible for deamination of both adenosine and 5′-methylthioadenosine. The latter substrate is not accepted by mammalian ADA homologs. The structural basis for this natural difference in specificity between plasmodial and mammalian ADA has not been well understood. We now report crystal structures of Plasmodium vivax adenosine deaminase in complex with adenosine, guanosine, and the picomolar inhibitor 2′-deoxycoformycin. These structures highlight a drastic conformational change in plasmodial ADA upon substrate-binding that has not been observed for mammalian ADA enzymes. Further, these complexes illuminate the structural basis for the differential substrate specificity and potential drug selectivity between mammalian and parasite enzymes. PMID:18602399

  8. Development of a capillary electrophoresis method for analyzing adenosine deaminase and purine nucleoside phosphorylase and its application in inhibitor screening.

    PubMed

    Qi, Yanfei; Li, Youxin; Bao, James J

    2016-08-01

    A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity. PMID:27173606

  9. 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

  10. Structures of Substrate-And Inhibitor-Bound Adenosine Deaminase From a Human Malaria Parasite Show a Dramatic Conformational Change And Shed Light on Drug Selectivity

    SciTech Connect

    Larson, E.T.; Deng, W.; Krumm, B.E.; Napuli, A.; Mueller, N.; Voorhis, W.C.Van; Buckner, F.S.; Fan, E.; Lauricella, A.; DeTitta, G.; Luft, J.; Zucker, F.; Hol, W.G.J.; Verlinde, C.L.M.J.; Merritt, E.A.

    2009-05-20

    Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore, interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling. For this reason, plasmodial ADA accepts a wider range of substrates, as it is responsible for deamination of both adenosine and 5{prime}-methylthioadenosine. The latter substrate is not accepted by mammalian ADA homologs. The structural basis for this natural difference in specificity between plasmodial and mammalian ADA has not been well understood. We now report crystal structures of Plasmodium vivax ADA in complex with adenosine, guanosine, and the picomolar inhibitor 2{prime}-deoxycoformycin. These structures highlight a drastic conformational change in plasmodial ADA upon substrate binding that has not been observed for mammalian ADA enzymes. Further, these complexes illuminate the structural basis for the differential substrate specificity and potential drug selectivity between mammalian and parasite enzymes.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. Adenosine-5'-phosphate deaminase. A novel herbicide target.

    PubMed Central

    Dancer, J E; Hughes, R G; Lindell, S D

    1997-01-01

    The isolation of carbocyclic coformycin as the herbicidally active component from a fermentation of Saccharothrix species was described previously (B.D. Bush, G.V. Fitchett, D.A. Gates, D. Langley [1993] Phytochemistry 32: 737-739). Here we report that the primary mode of action of carbocyclic coformycin has been identified as inhibition of the enzyme AMP deaminase (EC 3.5.4.6) following phosphorylation at the 5' hydroxyl on the carbocyclic ring in vivo. When pea (Pisum sativum L. var Onward) seedlings are treated with carbocyclic coformycin, there is a very rapid and dramatic increase in ATP levels, indicating a perturbation in purine metabolism. Investigation of the enzymes of purine metabolism showed a decrease in the extractable activity of AMP deaminase that correlates with a strong, noncovalent association of the phosphorylated natural product with the protein. The 5'-phosphate analog of the carbocyclic coformycin was synthesized and shown to be a potent, tight binding inhibitor of AMP deaminase isolated from pea seedlings. Through the use of a synthetic radiolabeled marker, rapid conversion of carbocyclic coformycin to the 5'-phosphate analog could be demonstrated in vivo. It is proposed that inhibition of AMP deaminase leads to the death of the plant through perturbation of the intracellular ATP pool. PMID:9159944

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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.

  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. 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

  6. 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.

  7. Investigation into effects of antipsychotics on ectonucleotidase and adenosine deaminase in zebrafish brain.

    PubMed

    Seibt, Kelly Juliana; Oliveira, Renata da Luz; Bogo, Mauricio Reis; Senger, Mario Roberto; Bonan, Carla Denise

    2015-12-01

    Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35%), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38%) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq, entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug.

  8. DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase.

    PubMed

    Zhang, Kai; Wang, Ke; Xie, Minhao; Zhu, Xue; Xu, Lan; Yang, Runlin; Huang, Biao; Zhu, Xiaoli

    2014-02-15

    A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies.

  9. Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

    PubMed Central

    Baggott, J E; Vaughn, W H; Hudson, B B

    1986-01-01

    With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements

  10. 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.

  11. 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

  12. Long-Term Expression of Human Adenosine Deaminase in Vascular Smooth Muscle Cells of Rats: A Model for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Lynch, Carmel M.; Clowes, Monika M.; Osborne, William R. A.; Clowes, Alexander W.; Dusty Miller, A.

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli β-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  13. 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

  14. Disruption of the adenosine deaminase gene causes hepatocellular impairment and perinatal lethality in mice.

    PubMed Central

    Wakamiya, M; Blackburn, M R; Jurecic, R; McArthur, M J; Geske, R S; Cartwright, J; Mitani, K; Vaishnav, S; Belmont, J W; Kellems, R E

    1995-01-01

    We have generated mice with a null mutation at the Ada locus, which encodes the purine catabolic enzyme adenosine deaminase (ADA, EC 3.5.4.4). ADA-deficient fetuses exhibited hepatocellular impairment and died perinatally. Their lymphoid tissues were not largely affected. Accumulation of ADA substrates was detectable in ADA-deficient conceptuses as early as 12.5 days postcoitum, dramatically increasing during late in utero development, and is the likely cause of liver damage and fetal death. The results presented here demonstrate that ADA is important for the homeostatic maintenance of purines in mice. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7731963

  15. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    SciTech Connect

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  16. 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

  17. A 24-Year Enzyme Replacement Therapy in an Adenosine-deaminase-Deficient Patient.

    PubMed

    Tartibi, Hana M; Hershfield, Michael S; Bahna, Sami L

    2016-01-01

    Severe combined immunodeficiency (SCID) is a fatal childhood disease unless immune reconstitution is performed early in life, with either hematopoietic stem cell transplantation or gene therapy. One of its subtypes is caused by adenosine deaminase (ADA) enzyme deficiency, which leads to the accumulation of toxic metabolites that impair lymphocyte development and function. With the development of polyethylene glycol-conjugated adenosine deaminase (PEG-ADA) enzyme replacement therapy, many ADA-deficient children with SCID who could not receive a hematopoietic stem cell transplantation or gene therapy survived and had longer and healthier lives. We report a 24-year course of treatment in a patient who was diagnosed with ADA deficiency at 4 months of age. The patient was treated with PEG-ADA, which was the only therapy available for him. The patient's plasma ADA level was regularly monitored and the PEG-ADA dose adjusted accordingly. This treatment has resulted in near-normalization of lymphocyte counts, and his clinical course has been associated with only minor to moderate infections. Thus far, he has had no manifestations of autoimmune or lymphoproliferative disorders. This patient is among the longest to be maintained on PEG-ADA enzyme replacement therapy. PMID:26684479

  18. Transition state structure of E. coli tRNA-specific adenosine deaminase.

    PubMed

    Luo, Minkui; Schramm, Vern L

    2008-02-27

    Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single tRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNA(Arg2) was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1'-(3)H], [5'-(3)H2], [1'-(14)C], [6-(13)C], [6-(15)N], [6-(13)C, 6-(15)N] and [1-(15)N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-(13)C]-, [6-(15)N]-, [1-(15)N]-, [1'-(3)H]-, [5'-(3)H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late SNAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-(15)N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 A in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 A, respectively. The large inverse [5'-(3)H] KIE of -3.7% and modest normal [1'-(3)H] KIE of 1.4% indicate that significant ribosyl 5'-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition

  19. Crystal Structure of Staphylococcus aureus tRNA Adenosine Deaminase TadA in Complex with RNA

    SciTech Connect

    Losey,H.; Ruthenburg, A.; Verdine, G.

    2006-01-01

    Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

  20. Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

    PubMed

    Hortle, Elinor; Nijagal, Brunda; Bauer, Denis C; Jensen, Lora M; Ahn, Seong Beom; Cockburn, Ian A; Lampkin, Shelley; Tull, Dedreia; McConville, Malcolm J; McMorran, Brendan J; Foote, Simon J; Burgio, Gaetan

    2016-09-01

    The factors that determine red blood cell (RBC) lifespan and the rate of RBC aging have not been fully elucidated. In several genetic conditions, including sickle cell disease, thalassemia, and G6PD deficiency, erythrocyte lifespan is significantly shortened. Many of these diseases are also associated with protection from severe malaria, suggesting a role for accelerated RBC senescence and clearance in malaria resistance. Here, we report a novel, N-ethyl-N-nitrosourea-induced mutation that causes a gain of function in adenosine 5'-monophosphate deaminase (AMPD3). Mice carrying the mutation exhibit rapid RBC turnover, with increased erythropoiesis, dramatically shortened RBC lifespan, and signs of increased RBC senescence/eryptosis, suggesting a key role for AMPD3 in determining RBC half-life. Mice were also found to be resistant to infection with the rodent malaria Plasmodium chabaudi. We propose that resistance to P. chabaudi is mediated by increased RBC turnover and higher rates of erythropoiesis during infection. PMID:27465915

  1. Intracellular localization of differentially regulated RNA-specific adenosine deaminase isoforms in inflammation.

    PubMed

    Yang, Jing-Hua; Nie, Yongzhan; Zhao, Qingchuan; Su, Yingjun; Pypaert, Marc; Su, Haili; Rabinovici, Reuven

    2003-11-14

    Adenosine-to-inosine (A-to-I) RNA editing is a post-transcriptional process that amplifies the repertoire of protein production. Recently, the induction of this process through up-regulation of the editing enzyme RNA-specific adenosine deaminase 1 (ADAR1) was documented during acute inflammation. Here we report that the inflammation-induced up-regulation of ADAR1 involves differential production and intracellular localization of several isoforms with distinct RNA-binding domains and localization signals. These include the full-length ADAR1 (p150) and two functionally active short isoforms (p80 and p110). ADAR1 p80 starts at a methionine 519 (M519) due to alternative splicing in exon 2, which deletes the putative nuclear localization signal, the Z-DNA binding domain, and the entire RNA binding domain I. ADAR1 p110 is the mouse homologue of the human ADAR1 110-kDa variant (M246), which retains the second half of the Z-DNA binding domain, all RNA binding domains, and the deaminase domain. Additional variations are found in the third RNA binding domain of ADAR1; they are differentially regulated during inflammation, generating isoforms with different levels of activities. Studies in several cell types transfected with ADAR1-EGFP chimeras demonstrated that the p150 and p80 variants are localized in the cytoplasm and nucleolus, respectively. In agreement with this observation, endogenous ADAR1 was identified in the cytoplasm and nucleolus of mouse splenocytes and HeLa cells. Since the ADAR1 variants are differentially regulated during acute inflammation, it suggests that the localization of these variants and of A-to-I RNA editing in the cytoplasm, nucleus, and nucleolus is intracellularly reorganized in response to inflammatory stimulation. PMID:12954622

  2. Expression of a functional human adenosine deaminase in transgenic tobacco plants.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2013-06-01

    An inherited disorder, adenosine deaminase deficiency is a form of severe combined immunodeficiency, which is ultimately caused by an absence of adenosine deaminase (ADA), a key enzyme of the purine salvage pathway. The absence of ADA-activity in sufferers eventually results in a dysfunctional immune system due to the build-up of toxic metabolites. To date, this has been treated with mixed success, using PEG-ADA, made from purified bovine ADA coupled to polyethylene glycol. It is likely, however, that an enzyme replacement therapy protocol based on recombinant human ADA would be a more effective treatment for this disease. Therefore, as a preliminary step to produce biologically active human ADA in transgenic tobacco plants a human ADA cDNA has been inserted into a plant expression vector under the control of the CaMV 35S promoter and both human and TMV 5' UTR control regions. Plant vector expression constructs have been used to transform tobacco plants via Agrobacterium-mediated transformation. Genomic DNA, RNA and protein blot analyses have demonstrated the integration of the cDNA construct into the plant nuclear genome and the expression of recombinant ADA mRNA and protein in transgenic tobacco leaves. Western blot analysis has also revealed that human and recombinant ADA have a similar size of approximately 41 kDa. ADA-specific activities of between 0.001 and 0.003 units per mg total soluble protein were measured in crude extracts isolated from transformed tobacco plant leaves. PMID:23264022

  3. Evaluation of Serum Adenosine Deaminase in Cystic Fibrosis Patients in an Iranian Referral Hospital

    PubMed Central

    Farahmand, Fatemeh; Tajdini, Parisa; Falahi, Gholamhossein; Shams, Sedigheh; Mahmoudi, Shima

    2016-01-01

    Background: Adenosine, a signaling nucleoside, is controlled in part by the enzyme adenosine deaminase (ADA). There are rare reports on the role of adenosine levels and ADA in cystic fibrosis (CF) patients. Objectives: The aim of this study was to assess serum ADA in CF patients in order to find whether the severity of lung disease in CF is related to significant changes of ADA or not. Patients and Methods: Venous blood serum ADA was measured in CF patients (3-15 years) and 49 healthy children (3-15 years) referred to Children’s Medical Center. Classification of respiratory and gastrointestinal disease severity in CF patients as well as Body Mass Index (BMI) was performed. The results were compared with values obtained from healthy children matched for age and gender. Results: This study included 49 children of both genders (20 females and 29 males) with CF (mean age: 6.36 ± 2.22 years). Mean serum ADA in CF patients group and control group was 9.38 ± 2.72 and 16.04 ± 1.27, respectively (P value = 0.001). Mean serum ADA in CF patients with normal BMI was higher than in patients with low BMI (P value = 0.002). Conclusions: In this study the lower serum level of ADA was seen in CF patients compared to control group. The clinical symptoms, especially respiratory symptoms, in CF patients might be associated with reduction of serum ADA and rising serum adenosine; therefore, further studies on the use of ADA enzyme therapy in CF patients are highly recommended. PMID:27617063

  4. The ADA*2 allele of the adenosine deaminase gene (20q13.11) and recurrent spontaneous abortions: an age-dependent association

    PubMed Central

    Nunes, Daniela Prudente Teixeira; Spegiorin, Lígia Cosentino Junqueira Franco; de Mattos, Cinara Cássia Brandão; Oliani, Antonio Helio; Vaz-Oliani, Denise Cristina Mós; de Mattos, Luiz Carlos

    2011-01-01

    OBJECTIVE: Adenosine deaminase acts on adenosine and deoxyadenosine metabolism and modulates the immune response. The adenosine deaminase G22A polymorphism (20q.11.33) influences the level of adenosine deaminase enzyme expression, which seems to play a key role in maintaining pregnancy. The adenosine deaminase 2 phenotype has been associated with a protective effect against recurrent spontaneous abortions in European Caucasian women. The aim of this study was to investigate whether the G22A polymorphism of the adenosine deaminase gene is associated with recurrent spontaneous abortions in Brazilian women. METHODS: A total of 311 women were recruited to form two groups: G1, with a history of recurrent spontaneous abortions (N = 129), and G2, without a history of abortions (N = 182). Genomic DNA was extracted from peripheral blood with a commercial kit and PCR-RFLP analysis was used to identify the G22A genetic polymorphism. Fisher's exact test and odds ratio values were used to compare the proportions of adenosine deaminase genotypes and alleles between women with and without a history of recurrent spontaneous abortion (p<0.05). The differences between mean values for categorical data were calculated using unpaired t tests. The Hardy-Weinberg equilibrium was assessed with a chi-square test. RESULTS: Statistically significant differences were identified for the frequencies of adenosine deaminase genotypes and alleles between the G1 and G2 groups when adjusted for maternal age. CONCLUSIONS: The results suggest that the adenosine deaminase *2 allele is associated with a low risk for recurrent spontaneous abortions, but this association is dependent on older age. PMID:22086524

  5. The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

    PubMed Central

    Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

    2000-01-01

    Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

  6. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis.

    PubMed

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; Santos, Odelta dos; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana

    2015-11-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival.

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

    PubMed

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

    2016-07-01

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

  8. Modulatory effect of iron chelators on adenosine deaminase activity and gene expression in Trichomonas vaginalis

    PubMed Central

    Primon-Barros, Muriel; Rigo, Graziela Vargas; Frasson, Amanda Piccoli; dos Santos, Odelta; Smiderle, Lisiane; Almeida, Silvana; Macedo, Alexandre José; Tasca, Tiana

    2015-01-01

    Trichomonas vaginalis is a flagellate protozoan that parasitises the urogenital human tract and causes trichomoniasis. During the infection, the acquisition of nutrients, such as iron and purine and pyrimidine nucleosides, is essential for the survival of the parasite. The enzymes for purinergic signalling, including adenosine deaminase (ADA), which degrades adenosine to inosine, have been characterised in T. vaginalis. In the evaluation of the ADA profile in different T. vaginalis isolates treated with different iron sources or with limited iron availability, a decrease in activity and an increase in ADA gene expression after iron limitation by 2,2-bipyridyl and ferrozine chelators were observed. This supported the hypothesis that iron can modulate the activity of the enzymes involved in purinergic signalling. Under bovine serum limitation conditions, no significant differences were observed. The results obtained in this study allow for the assessment of important aspects of ADA and contribute to a better understanding of the purinergic system in T. vaginalis and the role of iron in establishing infection and parasite survival. PMID:26517498

  9. The effect of adenosine deaminase inhibition on the A1 adenosinergic and M2 muscarinergic control of contractility in eu- and hyperthyroid guinea pig atria.

    PubMed

    Pak, Krisztian; Zsuga, Judit; Kepes, Zita; Erdei, Tamas; Varga, Balazs; Juhasz, Bela; Szentmiklosi, Andras Jozsef; Gesztelyi, Rudolf

    2015-08-01

    The A1 adenosine and M2 muscarinic receptors exert protective (including energy consumption limiting) effects in the heart. We investigated the influence of adenosine deaminase (ADA) inhibition on a representative energy consumption limiting function, the direct negative inotropic effect elicited by the A1 adenosinergic and M2 muscarinergic systems, in eu- and hyperthyroid atria. Furthermore, we compared the change in the interstitial adenosine level caused by ADA inhibition and nucleoside transport blockade, two well-established processes to stimulate the cell surface A1 adenosine receptors, in both thyroid states. A classical isolated organ technique was applied supplemented with the receptorial responsiveness method (RRM), a concentration estimating procedure. Via measuring the contractile force, the direct negative inotropic capacity of N(6)-cyclopentyladenosine, a selective A1 receptor agonist, and methacholine, a muscarinic receptor agonist, was determined on the left atria isolated from 8-day solvent- and thyroxine-treated guinea pigs in the presence and absence of 2'-deoxycoformycin, a selective ADA inhibitor, and NBTI, a selective nucleoside transporter inhibitor. We found that ADA inhibition (but not nucleoside transport blockade) increased the signal amplification of the A1 adenosinergic (but not M2 muscarinergic) system. This action of ADA inhibition developed in both thyroid states, but it was greater in hyperthyroidism. Nevertheless, ADA inhibition produced a smaller rise in the interstitial adenosine concentration than nucleoside transport blockade did in both thyroid states. Our results indicate that ADA inhibition, besides increasing the interstitial adenosine level, intensifies the atrial A1 adenosinergic function in another (thyroid hormone-sensitive) way, suggesting a new mechanism of action of ADA inhibition. PMID:25877465

  10. Adenosine deaminase activity in serum and lymphocytes of rats infected with Sporothrix schenckii.

    PubMed

    Castro, Verônica S P; Pimentel, Victor C; Da Silva, Aleksandro S; Thomé, Gustavo R; Wolkmer, Patrícia; Castro, Jorge L C; Costa, Márcio M; da Silva, Cássia B; Oliveira, Daniele C; Alves, Sydney H; Schetinger, Maria R C; Lopes, Sonia T A; Mazzanti, Cinthia M

    2012-07-01

    Sporotrichosis is a fungal infection of subcutaneous or chronic evolution, inflammatory lesions characterized by their pyogranulomatous aspect, caused by the dimorphic fungus Sporothrix schenckii. Adenosine deaminase (ADA) is a "key" enzyme in the purine metabolism, promoting the deamination of adenosine, an important anti-inflammatory molecule. The increase in ADA activity has been demonstrated in several inflammatory conditions; however, there are no data in the literature associated with this fungal infection. The objective of this study was to evaluate the activity of serum ADA (S-ADA) and lymphocytes (L-ADA) of rats infected with S. schenckii. We used seventy-eight rats divided into two groups. In the first experiment, rats were infected subcutaneously and in the second experiment, infected intraperitoneally. Blood samples for hematologic evaluation and activities of S-ADA and L-ADA were performed at days 15, 30, and 40 post-infection (PI) to assess disease progression. In the second experiment, it was observed an acute decrease in activity of S-ADA and L-ADA (P < 0.05), suggesting a compensatory mechanism in an attempt to protect the host from excessive tissue damage. With chronicity of disease the rats in the first and second experiment at 30 days PI showed an increased activity of L-ADA (P < 0.05), promoting an inflammatory response in an attempt to combat the spread of the agent. Thus, it is suggested that infection with S. schenckii alters the activities of S-ADA in experimentally infected rats, demonstrating the involvement of this enzyme in the pathogenesis of sporotrichosis.

  11. ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY.

    PubMed

    Bravo-Tobar, Iván Darío; Nello-Pérez, Carlota; Fernández, Alí; Mogollón, Nora; Pérez, Mary Carmen; Verde, Juan; Concepción, Juan Luis; Rodriguez-Bonfante, Claudina; Bonfante-Cabarcas, Rafael

    2015-01-01

    Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease.

  12. ADENOSINE DEAMINASE ACTIVITY AND SERUM C-REACTIVE PROTEIN AS PROGNOSTIC MARKERS OF CHAGAS DISEASE SEVERITY

    PubMed Central

    BRAVO-TOBAR, Iván Darío; NELLO-PÉREZ, Carlota; FERNÁNDEZ, Alí; MOGOLLÓN, Nora; PÉREZ, Mary Carmen; VERDE, Juan; CONCEPCIÓN, Juan Luis; RODRIGUEZ-BONFANTE, Claudina; BONFANTE-CABARCAS, Rafael

    2015-01-01

    SUMMARY Chagas disease is a public health problem worldwide. The availability of diagnostic tools to predict the development of chronic Chagas cardiomyopathy is crucial to reduce morbidity and mortality. Here we analyze the prognostic value of adenosine deaminase serum activity (ADA) and C-reactive protein serum levels (CRP) in chagasic individuals. One hundred and ten individuals, 28 healthy and 82 chagasic patients were divided according to disease severity in phase I (n = 35), II (n = 29), and III (n = 18). A complete medical history, 12-lead electrocardiogram, chest X-ray, and M-mode echocardiogram were performed on each individual. Diagnosis of Chagas disease was confirmed by ELISA and MABA using recombinant antigens; ADA was determined spectrophotometrically and CRP by ELISA. The results have shown that CRP and ADA increased linearly in relation to disease phase, CRP being significantly higher in phase III and ADA at all phases. Also, CRP and ADA were positively correlated with echocardiographic parameters of cardiac remodeling and with electrocardiographic abnormalities, and negatively with ejection fraction. CRP and ADA were higher in patients with cardiothoracic index ≥ 50%, while ADA was higher in patients with ventricular repolarization disturbances. Finally, CRP was positively correlated with ADA. In conclusion, ADA and CRP are prognostic markers of cardiac dysfunction and remodeling in Chagas disease. PMID:26603224

  13. Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

    PubMed Central

    Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

    2014-01-01

    While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

  14. The PurR mutation of Drosophila melanogaster confers resistance to purine and 2,6-diaminopurine by elevating adenosine deaminase activity.

    PubMed

    Dutton, F L; Chovnick, A

    1990-01-01

    Media supplemented with purine (7H-imidazo[4,5-d]pyrimidine) or the purine analogue 2,6-diaminopurine (DAP) can be employed to select several classes of purine-resistant variants from mutagenized cultures of Drosophila. One class results in elevated resistance to purine and diaminopurine which is correlated with elevated activity of the enzyme adenosine deaminase (adenosine aminohydrolase = EC 3.5.4.4). The first member of this class, Pur R, maps to position 82 +/- in the right arm of the second chromosome. The Pur R mutation causes an elevation of adenosine deaminase (ADA) enzyme activity, apparently by altering a thermolabile, ADA-specific repressor. Pur R may thus encode a negative regulator of adenosine deaminase activity similar to the ADA-binding protein found in mammalian systems.

  15. Oxidative Stress Biomarkers and Adenosine Deaminase over the Alopecic Area of the Patients with Alopecia Areata

    PubMed Central

    Öztürk, Perihan; Arıcan, Özer; Kurutaş, Ergül Belge; Mülayim, Kamil

    2016-01-01

    Background: Alopecia areata (AA) is an autoimmune, T-cell mediated, and chronic inflammatory disorder. The pathological mechanisms of disease are unclear, but oxidative stress may be involved. To our knowledge, no studies have examined the oxidative stress levels or biomarkers within the lesional area and skin surface in patients with AA. Similarly, adenosine deaminase (ADA) has not been characterized in AA. Aims: Therefore, we aimed to define ADA levels and the factors involved in oxidative stress from scalp-scrapes of patients with AA. Study Design: Case-control study. Method: A total of 60 patients (30 diagnosed AA patients and 30 healthy controls) were included in the study. ADA as well as oxidative stress factors, including malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) were analyzed from scalp-scrapes in both groups and quantified by spectrophotometry. Results: Activities of SOD (p=0.000), CAT (p=0.033), and ADA (p=0.004) as well as levels of GSH (p=0.000) and MDA (p=0.032) in patients with AA were higher than the controls statistically significant. Conclusion: Based on these results, factors associated with oxidative stress were elevated in AA patient scalp-scrapes compared to controls and may have a defined role the disease pathogenesis. Alterations in the activities of antioxidant enzymes from AA patient scraping samples may be a local effect of elevated oxidative stress levels. In this disease, oxidative stress may affect not only hair follicle but also any layers of the skin. PMID:27403388

  16. Adenosine deaminase in CSF and pleural fluid for diagnosis of tubercular meningitis and pulmonary tuberculosis.

    PubMed

    Nepal, A K; Gyawali, N; Poudel, B; Mahato, R V; Lamsal, M; Gurung, R; Baral, N; Majhi, S

    2012-12-01

    Tuberculosis (TB) is one of the most common infectious diseases in developing countries including Nepal. Delay in diagnosis and treatment of tuberculosis results in poor prognosis of the disease. This study was conducted to estimate diagnostic cut off values of Adenosine Deaminase (ADA) in cerebrospinal fluid (CSF) and pleural fluid and to evaluate the sensitivity, specificity, positive and negative predictive values ofADA in pleural fluid and CSF from patients with tuberculous and non-tuberculous disease. A total of 98 body fluid (CSF: 24, Pleural fluid: 74) specimens were received for the estimation of ADA. ADA activity was measured at 37 degrees C by spectrophotometric method of Guisti and Galanti, 1984 at 625nm wavelength. Among the patients enrolled for the study subjects for which CSF were received (n = 24) included 8 tuberculous meningitis (TBM), and 16 non-tubercular meningitis (NTM). Pleural fluid samples (n = 74) were received from 19 pulmonary TB with pleural effusion, 17 PTB without pleural effusion and 37 of non-tuberculous disease patients. CSF ADA activity were (11. 1 +/- 2.03 IU/L) and (5.3 +/- +1.89 IU/L) (p <00001) in TM and non-NTM groups and Pleural fluid ADA activity were (10 +/- 22.18 IU/L) and (23.79 +/- 11.62 IU/L) (p < 0.001) in PTB and non-TB groups respectively. ADA test in body fluids, which is simple, cost-effective and sensitive, specific for the tubercular disease is recommended to perform before forwarding the cumbersome and expensive procedures like culture and PCR for TB diagnosis. PMID:24579533

  17. Diagnostic value of sputum adenosine deaminase (ADA) level in pulmonary tuberculosis

    PubMed Central

    Binesh, Fariba; Jalali, Hadi; Zare, Mohammad Reza; Behravan, Farhad; Tafti, Arefeh Dehghani; Behnaz, Fatemah; Tabatabaee, Mohammad; Shahcheraghi, Seyed Hossein

    2016-01-01

    Introduction Tuberculosis is still a considerable health problem in many countries. Rapid diagnosis of this disease is important, and adenosine deaminase (ADA) has been used as a diagnostic test. The aim of this study was to assess the diagnostic value of ADA in the sputum of patients with pulmonary tuberculosis. Methods The current study included 40 patients with pulmonary tuberculosis (culture positive, smear ±) and 42 patients with non tuberculosis pulmonary diseases (culture negative). ADA was measured on all of the samples. Results The median value of ADA in non-tuberculosis patients was 2.94 (4.2) U/L and 4.01 (6.54) U/L in tuberculosis patients, but this difference was not statistically significant (p=0.100). The cut-off point of 3.1 U/L had a sensitivity of 61% and a specificity of 53%, the cut-off point of 2.81 U/L had a sensitivity of 64% and a specificity of 50% and the cut-off point of 2.78 U/L had a sensitivity of 65% and a specificity of 48%. The positive predictive values for cut-off points of 3.1, 2.81 and 2.78 U/L were 55.7%, 57.44% and 69.23%, respectively. The negative predictive values for the abovementioned cut-off points were 56.75%, 57.14% and 55.88%, respectively. Conclusion Our results showed that sputum ADA test is neither specific nor sensitive. Because of its low sensitivity and specificity, determination of sputum ADA for the diagnosis of pulmonary tuberculosis is not recommended. PMID:27482515

  18. Mutations in the human adenosine deaminase gene that affect protein structure and RNA splicing

    SciTech Connect

    Akeson, A.L.; Wiginton, D.A.; States, C.J.; Perme, C.M.; Dusing, M.R.; Hutton, J.J.

    1987-08-01

    Adenosine deaminase deficiency is one cause of the genetic disease severe combined immunodeficiency. To identify mutations responsible for ADA deficiency, the authors synthesized cDNAs to ADA mRNAs from two cell lines, GM2756 and GM2825A, derived from ADA-deficient immunodeficient patients. Sequence analysis of GM2756 cDNA clones revealed a different point mutation in each allele that causes amino acid changes of alanine to valine and arginine to histidine. One allele of GM2825A also has a point mutation that causes an alanine to valine substitution. The other allele of GM2825A was found to produce an mRNA in which exon 4 had been spliced out but had no other detrimental mutations. S1 nuclease mapping of GM2825A mRNA showed equal abundance of the full-length ADA mRNA and the ADA mRNA that was missing exon 4. Several of the ADA cDNA clones extended 5' of the major initiation start site, indicating multiple start sites for ADA transcription. The point mutations in GM2756 and GM2825A and the absence of exon 4 in GM2825A appear to be directly responsible for the ADA deficiency. Comparison of a number of normal and mutant ADA cDNA sequences showed a number of changes in the third base of codons. These change do not affect the amino acid sequence. Analyses of ADA cDNAs from different cell lines detected aberrant RNA species that either included intron 7 or excluded exon 7. Their presence is a result of aberrant splicing of pre-mRNAs and is not related to mutations that cause ADA deficiency.

  19. Ascitic fluid gamma interferon concentrations and adenosine deaminase activity in tuberculous peritonitis.

    PubMed Central

    Sathar, M A; Simjee, A E; Coovadia, Y M; Soni, P N; Moola, S A; Insam, B; Makumbi, F

    1995-01-01

    The gamma interferon (gamma-IFN) concentration and the adenosine deaminase (ADA) activity were evaluated in 30 patients with tuberculous peritonitis, 21 patients with ascites due to a malignant disorder, and 41 patients with cirrhosis. The gamma-IFN concentrations were significantly higher (p < 0.0001) in tuberculous peritonitis patients (mean: 6.70 U/ml) than in the malignant (mean: 3.10 U/ml) and cirrhotic (mean: 3.08 U/ml) groups. Use of a cut off value of > or = 3.2 U/ml gave the assay a sensitivity of 93% (25 of 27), a specificity of 98% (54 of 55), positive (P+) and negative (P-) predictive values of 96% and a test accuracy of 96%. The ADA activity was significantly (p < 0.0001) higher in the tuberculous peritonitis group (mean: 101.84 U/l) than in the control groups (cirrhosis (mean: 13.49 U/l) and malignancy (mean: 19.35 U/l)). A cut off value of > 30 U/l gave the ADA test a sensitivity of 93% (26 of 28) a specificity of 96% (51 of 53), a (P+) value of 93%, a (P-) value of 96%, and a test accuracy of 95%. There was a significant (p < 0.0001) correlation (r = 0.72) between ADA activity and gamma-IFN values in patients with tuberculous peritonitis. These results show that a high concentration of gamma-IFN in ascitic fluid is as valuable as the ADA activity in the diagnosis of tuberculous peritonitis. Both are rapid non-invasive diagnostic tests for tuberculous peritonitis. PMID:7698702

  20. Hyperbilirubinemia and rapid fatal hepatic failure in severe combined immunodeficiency caused by adenosine deaminase deficiency (ADA-SCID).

    PubMed

    Kühl, J S; Schwarz, K; Münch, A; Schmugge, M; Pekrun, A; Meisel, C; Wahn, V; Ebell, W; von Bernuth, H

    2011-03-01

    Adenosin deaminase (ADA) deficiency is the cause for Severe Combined Immunodeficiency (SCID) in about 15% of patients with SCID, often presenting as T (-)B (-)NK (-)SCID. Treatment options for ADA-SCID are enzyme replacement, bone marrow transplantation or gene therapy. We here describe the first patient with ADA-SCID and fatal hepatic failure despite bone marrow transplantation from a 10/10 HLA identical related donor. As patients with ADA-SCID may be at yet underestimated increased risk for rapid hepatic failure we speculate whether hepatitis in ADA-SCID should lead to the immediate treatment with enzyme replacement by pegylated ADA.

  1. Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4-Mediated Entry of the Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Raj, V. Stalin; Smits, Saskia L.; Provacia, Lisette B.; van den Brand, Judith M. A.; Wiersma, Lidewij; Ouwendijk, Werner J. D.; Bestebroer, Theo M.; Spronken, Monique I.; van Amerongen, Geert; Rottier, Peter J. M.; Fouchier, Ron A. M.; Bosch, Berend Jan; Osterhaus, Albert D.M.E.

    2014-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. PMID:24257613

  2. Adenosine deaminase acts as a natural antagonist for dipeptidyl peptidase 4-mediated entry of the Middle East respiratory syndrome coronavirus.

    PubMed

    Raj, V Stalin; Smits, Saskia L; Provacia, Lisette B; van den Brand, Judith M A; Wiersma, Lidewij; Ouwendijk, Werner J D; Bestebroer, Theo M; Spronken, Monique I; van Amerongen, Geert; Rottier, Peter J M; Fouchier, Ron A M; Bosch, Berend Jan; Osterhaus, Albert D M E; Haagmans, Bart L

    2014-02-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection. PMID:24257613

  3. Pleural effusion adenosine deaminase: a candidate biomarker to discriminate between Gram-negative and Gram-positive bacterial infections of the pleural space

    PubMed Central

    Li, Ruolin; Wang, Junli; Wang, Xinfeng; Wang, Maoshui

    2016-01-01

    OBJECTIVES: Delay in the treatment of pleural infection may contribute to its high mortality. In this retrospective study, we aimed to evaluate the diagnostic accuracy of pleural adenosine deaminase in discrimination between Gram-negative and Gram-positive bacterial infections of the pleural space prior to selecting antibiotics. METHODS: A total of 76 patients were enrolled and grouped into subgroups according to Gram staining: 1) patients with Gram-negative bacterial infections, aged 53.2±18.6 years old, of whom 44.7% had empyemas and 2) patients with Gram-positive bacterial infections, aged 53.5±21.5 years old, of whom 63.1% had empyemas. The pleural effusion was sampled by thoracocentesis and then sent for adenosine deaminase testing, biochemical testing and microbiological culture. The Mann-Whitney U test was used to examine the differences in adenosine deaminase levels between the groups. Correlations between adenosine deaminase and specified variables were also quantified using Spearman’s correlation coefficient. Moreover, receiver operator characteristic analysis was performed to evaluate the diagnostic accuracy of pleural effusion adenosine deaminase. RESULTS: Mean pleural adenosine deaminase levels differed significantly between Gram-negative and Gram-positive bacterial infections of the pleural space (191.8±32.1 U/L vs 81.0±16.9 U/L, p<0.01). The area under the receiver operator characteristic curve was 0.689 (95% confidence interval: 0.570, 0.792, p<0.01) at the cutoff value of 86 U/L. Additionally, pleural adenosine deaminase had a sensitivity of 63.2% (46.0-78.2%); a specificity of 73.7% (56.9-86.6%); positive and negative likelihood ratios of 2.18 and 0.50, respectively; and positive and negative predictive values of 70.6% and 66.7%, respectively. CONCLUSIONS: Pleural effusion adenosine deaminase is a helpful alternative biomarker for early and quick discrimination of Gram-negative from Gram-positive bacterial infections of the pleural space

  4. Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes

    SciTech Connect

    Ku, Min-Je; Lee, Won-Ho; Nam, Ki-hyun; Rhee, Kyeong-hee; Lee, Ki-Seog; Kim, Eunice EunKyung; Yu, Myung-Hee; Hwang, Kwang Yeon

    2005-04-01

    The tRNA-specific adenosine deaminase from the pathogenic bacteria S. pyogenes has been overexpressed and crystallized. The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn{sup 2+} ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 Å using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 Å using synchrotron radiation. The crystal belongs to the tetragonal space group P4{sub 2}2{sub 1}2, with unit-cell parameters a = b = 81.042, c = 81.270 Å. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (V{sub M}) of 3.3 Å{sup 3} Da{sup −1} and a solvent content of 62.7%.

  5. The Effect of Acute Exercise upon Adenosin Deaminase Oxidant and Antioxidant Activity

    ERIC Educational Resources Information Center

    Kafkas, M. Emin; Karabulut, Aysun Bay; Sahin, Armagan; Otlu, Onder; Savas, Seyfi; Aytac, Aylin

    2012-01-01

    The purpose of this study was to determine the changes of MDA, glutation (GSH), Adenozine deaminase (ADA) and superoxidase dismutaze (SOD) levels with exercise training in obese middle-aged women (body mass index, MMI [greater than or equal to] 30.0). Twelve obese middle-aged women participated in this study. The descriptive statistics of some of…

  6. A double origin electrophoretic method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II.

    PubMed

    Murch, R S; Gambel, A M; Kearney, J J

    1986-10-01

    A rapid, reliable method for the simultaneous separation of adenosine deaminase, adenylate kinase, and carbonic anhydrase II by agarose gel electrophoresis is presented. This method uses a double origin sample application system. Unreduced sample extracts for adenylate kinase analysis are applied 13.0 cm from the anode. Reduced sample extracts for the remaining proteins of interest are applied 7.0 cm from the anode. The use of applicator foils and an increased voltage gradient result in superior resolution, linearity, and band sharpness of the allozyme patterns. Further, there is no masking of the adenylate kinase 2 band as a result of the use of a reducing agent, and carbonic anhydrase II is resolved without interference from hemoglobin as has been observed with other multisystem methods.

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-06-01

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

  9. Syzygium cumini extract decrease adenosine deaminase, 5'nucleotidase activities and oxidative damage in platelets of diabetic patients.

    PubMed

    De Bona, Karine S; Bellé, Luziane P; Sari, Marcel H; Thomé, Gustavo; Schetinger, Maria R C; Morsch, Vera M; Boligon, Aline; Athayde, Margareth L; Pigatto, Aline S; Moretto, Maria B

    2010-01-01

    Diabetes mellitus, a chronic metabolic disorder, has assumed epidemic proportions and its long-term complications can have devastating consequences. The oxidative stress in diabetes was greatly increased due to prolonged exposure to hyperglycemia and impairment of oxidant/antioxidant equilibrium. Syzygium cumini is being widely used to treat diabetes by the traditional practitioners over many centuries. Adenosine deaminase (ADA) and 5'-Nucleotidase (5'NT) are enzymes of purine nucleoside metabolism that play an important role in the regulation of adenosine (Ado) levels. In this study, we investigated the effect of Syzygium cumini aqueous leaves extract (ASc) on ADA and 5'NT activities and on parameters of oxidative stress under in vitro conditions, using platelets of patients with Type 2 diabetes mellitus. Platelet-Rich Plasma (PRP) was assayed by ADA, 5'NT, Catalase (CAT), Superoxide Dismutase (SOD) activities and Thiobarbituric acid reactive substances (TBARS) levels. We observed that ADA, 5'NT activities and TBARS levels were significantly higher when compared to the control group, and ASc (100 and 200 μg/mL) prevented these effects. Our study demonstrates that ASc was able to remove oxidant species generated in diabetic conditions and modulates in the Ado levels. Then, ASc may promote a compensatory response in platelet function, improving the susceptibility-induced by the diabetes mellitus. PMID:21063110

  10. Alterations in the cholinesterase and adenosine deaminase activities and inflammation biomarker levels in patients with multiple sclerosis.

    PubMed

    Polachini, C R N; Spanevello, R M; Casali, E A; Zanini, D; Pereira, L B; Martins, C C; Baldissareli, J; Cardoso, A M; Duarte, M F; da Costa, P; Prado, A L C; Schetinger, M R C; Morsch, V M

    2014-04-25

    Multiple sclerosis (MS) is one of the main chronic inflammatory diseases of the CNS that cause functional disability in young adults. It has unknown etiology characterized by the infiltration of lymphocytes and macrophages into the brain. The aim of this study was to evaluate the acetylcholinesterase (AChE) activity in lymphocytes and whole blood, as well as butyrylcholinesterase (BChE) and adenosine deaminase (ADA) activities in serum. We also checked the levels of nucleotides, nucleosides, biomarkers of inflammation such as cytokines (interleukin (IL)-1, IL-6, interferon (IFN)-γ, tumor necrosis factor-alpha (TNF-α) and IL-10) and C-reactive protein (CRP) in serum from 29 patients with the relapsing-remitting form of MS (RRMS) and 29 healthy subjects as the control group. Results showed that AChE in lymphocytes and whole blood as well as BChE, and ADA activities in serum were significantly increased in RRMS patients when compared to the control group (P<0.05). In addition, we observed a decrease in ATP levels and a significant increase in the levels of ADP, AMP, adenosine and inosine in serum from RRMS patients in relation to the healthy subjects (P<0.05). Results also demonstrated an increase in the IFN-γ, TNF-α, IL-1, IL-6 and CRP (P<0.05) and a significant decrease in the IL-10 (P<0.0001) in RRMS patients when compared to control. Our results suggest that alterations in the biomarkers of inflammation and hydrolysis of nucleotides and nucleosides may contribute to the understanding of the neurological dysfunction of RRMS patients.

  11. High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2015-01-01

    Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L(-1) , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct.

  12. High-yield production of apoplast-directed human adenosine deaminase in transgenic tobacco BY-2 cell suspensions.

    PubMed

    Singhabahu, Sanjeewa; George, John; Bringloe, David

    2015-01-01

    Adenosine deaminase (ADA) deficiency, where a deleterious mutation in the ADA gene of patients results in a dysfunctional immune system, is ultimately caused by an absence of ADA. Over the last 25 years the disease has been treated with PEG-ADA, made from purified bovine ADA coupled with polyethylene glycol (PEG). However, it is thought that an enzyme replacement therapy protocol based on recombinant human ADA would probably be a more effective treatment. With this end in mind, a human ADA cDNA was inserted into plant expression vectors used to transform tobacco plant cell suspensions. Transgenic calli expressing constructs containing apoplast-directing signals showed significantly higher levels of recombinant ADA expression than calli transformed with cytosolic constructs. The most significant ADA activities, however, were measured in the media of transgenic cell suspensions prepared from high expressing transformed calli: where incorporation of a signal for arabinogalactan addition to ADA led to a recombinant protein yield of approximately 16 mg L(-1) , a 336-fold increase over ADA produced by cell suspensions transformed with a cytosolic construct. PMID:24825606

  13. 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.

  14. Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

    PubMed

    Akinyemi, Ayodele Jacob; Thomé, Gustavo Roberto; Morsch, Vera Maria; Bottari, Nathieli B; Baldissarelli, Jucimara; de Oliveira, Lizielle Souza; Goularte, Jeferson Ferraz; Belló-Klein, Adriane; Oboh, Ganiyu; Schetinger, Maria Rosa Chitolina

    2016-07-01

    Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of l-NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l-NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension-derived complications associated to platelet hyperactivity. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27151061

  15. Plasma Adenosine Deaminase Enzyme Reduces with Treatment of Pulmonary Tuberculosis in Nigerian Patients: Indication for Diagnosis and Treatment Monitoring.

    PubMed

    Ige, O; Edem, V F; Arinola, O G

    2016-01-01

    Tuberculosis(TB)-specific host biomarkers for diagnosis and monitoring of treatment response have been identified as priorities for TB research. Macrophage and T cell lymphocytes play vital roles in Mycobacterium tuberculosis immune response and their associated biomarkers could form good candidates for diagnosis and treatment monitoring. The enzyme adenosine deaminase (ADA) is produced mainly by monocytes and macrophages and increase in biological fluids in the course of infection with microorganisms infecting macrophages. This study comprised sixty-eight (68) participants; twenty-four (24) multi-drug-resistant TB(MDR-TB) patients, twenty-four (24) drug-sensitive TB patients(DS-TB) and twenty (20) non-TB apparently healthy individuals. Five (5) milliliters of blood was drawn before commencement of chemotherapy and 6 anti-TB therapy. In DSTB and MDR-TB patients before commencement of chemotherapy and 6 months of anti-TB treatment, the mean plasma levels of ADA were significantly increased compared with control. At 6 months of anti-TB chemotherapy of DSTB or MDR TB patients, ADA level was significantly decreased compared with before chemotherapy. Plasma ADA in DSTB patients before and 6 months of chemotherapy were not significantly different compared MDR TB patients. Plasma ADA level is a promising biomarker for the screening and treatment monitoring of pulmonary tuberculosis but not to differentiate MDR TB from DSTB patients. PMID:27574764

  16. A Study on the Serum Adenosine Deaminase Activity in Patients with Typhoid Fever and Other Febrile Illnesses

    PubMed Central

    Ketavarapu, Sameera; Ramani G., Uma; Modi, Prabhavathi

    2013-01-01

    Background: Adenosine Deaminase (ADA) has been suggested to be an important enzyme which is associated with the cell mediated immunity, but its clinical significance in typhoid fever has not yet been characterized. The present study was taken up to evaluate the serum ADA activity in patients of typhoid fever. The levels of ADA were also measured in the patients who were suffering from other febrile illnesses. Material and Method: This was a case control study. The subjects who were included in this study were divided into 3 groups. Group A consisted of 50 normal healthy individuals who served as the controls. Group B consisted of 50 patients, both males and females of all age groups, who were suffering from culture positive typhoid fever. Group C consisted of 50 patients who were suffering from febrile illnesses other than typhoid fever like viral fever, gastro enteritis, malaria, tonsillitis, upper respiratory tract infections, etc. The serum levels of ADA were estimated in all the subjects who were under study. Results: The serum ADA level was found to be increased in the patients of typhoid fever as compared to that in those with other febrile illnesses and in the controls. Conclusion: From the present study, it can be concluded that there was a statistically significant increase in the serum ADA levels in the patients with typhoid. PMID:23730630

  17. Novel deletion and a new missense mutation (Glu 217 Lys) at the catalytic site in two adenosine deaminase alleles of a patient with neonatal onset adenosine deaminase severe combined immunodeficiency

    SciTech Connect

    Hirschhorn, R.; Nicknam, M.N.; Eng, F.; Yang, D.R.; Borkowsky, W. )

    1992-11-01

    Mutations at the adenosine deaminase (ADA) locus result in a spectrum of disorders, encompassing a fulminant neonatal onset severe combined immunodeficiency (SCID) and childhood onset immunodeficiency, as well as apparently normal immune function. The extent of accumulation of the toxic metabolite, deoxyATP, correlates directly with severity of disease. The authors have now determined the mutations on both alleles of a child with fulminant, neonatal onset ADA SCID and accumulation of extremely high concentrations of deoxyATP. The genotype was consistent with the severely affected phenotype. One allele carried a large deletion that arose by non-homologous recombination and included the first five exons and promoter region. The second allele carried a missense mutation (G[sup 649]A) resulting in replacement of Glu[sup 217], an amino acid involved in the catalytic site, by Lys and predicting a major alteration in charge. Expression of the mutant cDNA on Cos cells confirmed that the mutation abolished enzyme activity. The authors have previously reported that a missense mutation at the preceding codon is similarly associated with neonatal onset ADA SCID and accumulation of extremely high deoxyATP. These findings suggest that genotype-phenotype correlations may be apparent for ADA SCID, despite the role that random variation in exposure to environmental pathogens may play in the initial phenotype. Such genotype-phenotype correlations may be important to consider in evaluating results of ongoing trials of [open quotes]gene[close quotes] and enzyme replacement therapy. 50 refs., 5 figs., 2 tabs.

  18. Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

    PubMed

    Čulić, Ognjen; Cordero, Mario D; Žanić-Grubišić, Tihana; Somborac-Bačura, Anita; Pučar, Lara Batičić; Detel, Dijana; Varljen, Jadranka; Barišić, Karmela

    2016-10-01

    Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656-0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

  19. Global regulation of alternative splicing by adenosine deaminase acting on RNA (ADAR)

    PubMed Central

    Solomon, Oz; Oren, Shirley; Safran, Michal; Deshet-Unger, Naamit; Akiva, Pinchas; Jacob-Hirsch, Jasmine; Cesarkas, Karen; Kabesa, Reut; Amariglio, Ninette; Unger, Ron; Rechavi, Gideon; Eyal, Eran

    2013-01-01

    Alternative mRNA splicing is a major mechanism for gene regulation and transcriptome diversity. Despite the extent of the phenomenon, the regulation and specificity of the splicing machinery are only partially understood. Adenosine-to-inosine (A-to-I) RNA editing of pre-mRNA by ADAR enzymes has been linked to splicing regulation in several cases. Here we used bioinformatics approaches, RNA-seq and exon-specific microarray of ADAR knockdown cells to globally examine how ADAR and its A-to-I RNA editing activity influence alternative mRNA splicing. Although A-to-I RNA editing only rarely targets canonical splicing acceptor, donor, and branch sites, it was found to affect splicing regulatory elements (SREs) within exons. Cassette exons were found to be significantly enriched with A-to-I RNA editing sites compared with constitutive exons. RNA-seq and exon-specific microarray revealed that ADAR knockdown in hepatocarcinoma and myelogenous leukemia cell lines leads to global changes in gene expression, with hundreds of genes changing their splicing patterns in both cell lines. This global change in splicing pattern cannot be explained by putative editing sites alone. Genes showing significant changes in their splicing pattern are frequently involved in RNA processing and splicing activity. Analysis of recently published RNA-seq data from glioblastoma cell lines showed similar results. Our global analysis reveals that ADAR plays a major role in splicing regulation. Although direct editing of the splicing motifs does occur, we suggest it is not likely to be the primary mechanism for ADAR-mediated regulation of alternative splicing. Rather, this regulation is achieved by modulating trans-acting factors involved in the splicing machinery. PMID:23474544

  20. 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

  1. 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.

  2. 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

  3. 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

  4. A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains.

    PubMed Central

    Chen, C X; Cho, D S; Wang, Q; Lai, F; Carter, K C; Nishikura, K

    2000-01-01

    Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing. PMID:10836796

  5. Role of caffeic acid phenethyl ester on mitomycin C induced clastogenesis: analysis of chromosome aberrations, micronucleus, mitotic index and adenosine deaminase activity in vivo.

    PubMed

    Sulaiman, Ghassan Mohammad

    2012-05-01

    The aim of the present investigation is to determine whether the caffeic acid phenethyl ester (CAPE) in combination with mitomycine-C (MMC) can ameliorate MMC-induced clastogenesis in the bone marrow cells of mice. The scoring of chromosomal aberrations, mitotic activity and micronuclei were undertaken in the current study as markers of clastogenicity. The action of CAPE in adenosine deaminase enzyme (ADA) activities of serum, thymus and spleen were also investigated. The animals were orally administered CAPE alone at the doses 5 or 10 mg kg b.wt.(-1) for 5 days then sacrificed 24 hours after the CAPE administration. MMC was administered to mice either alone at a single dose (2 mg kg b.wt.(-1)) by intraperitoneal injection, before or after CAPE treatment. Pre or post - treatment with two doses of CAPE significantly decreased the number of chromosomal aberrations, micronuclei and adapted the mitotic activity reduction in the bone marrow cells of mice induced by MMC when compared with only MMC given group. In addition, combination treatment with MMC caused a significant decrease in the activities of ADA in serum, thymus and spleen. The results of this study showed that ADA activity probably related to high levels of reactive oxygen species. This study concluded that the protective effect of CAPE against MMC clastogenesis resides at least in part, in its antioxidant effects.

  6. Safety and Efficacy of Suicide Gene Therapy with Adenosine Deaminase 5-Fluorocytosine Silmutaneously in in Vitro Cultures of Melanoma and Retinal Cell Lines

    PubMed Central

    Sakkas, Antonios; Zarogoulidis, Paul; Domvri, Kalliopi; Hohenforst-Schmidt, Wolfgang; Bougiouklis, Dimitris; Kakolyris, Stylianos; Zarampoukas, Thomas; Kioumis, Ioannis; Pitsiou, Georgia; Huang, Haidong; Li, Qiang; Meditskou, Soultana; Tsiouda, Theodora; Pezirkianidis, Nikolaos; Zarogoulidis, Konstantinos

    2014-01-01

    Local treatment as a treatment modality is gaining increased general acceptance over time. Novel drugs and methodologies of local administration are being investigated in an effort to achieve disease local control. Suicide gene therapy is a method that has been investigated as a local treatment with simultaneously distant disease control. In our current experiment we purchased HTB-70 (melanoma cell line, derived from metastatic axillary node) and CRL-2302 (human retinal epithelium) were from ATCC LGC Standards and Ancotil®, 2.5 g/250 ml (1 g/00ml) (5-Flucytosine) MEDA; Pharmaceuticals Ltd. UK. Adenosine Cytosine Deaminase (Ad.CD) was also used in order to convert the pro-drug 5-Flucytosine to the active 5-Fluoracil. Three different concentrations of 5-Flucytosine (5-FC) were administered (0.2ml, 0.8ml and 1.2ml). At indicated time-points (4h, 8h and 24h) cell viability and apoptosis were measured. Our concept was to investigate whether suicide gene therapy with Ad. CD-5-FC could be used with safety and efficiency as a future local treatment for melanoma located in the eye cavity. Indeed, our results indicated that in every 5-FC administration had mild cytotoxicity for the retinal cells, while increased apoptosis was observed for the melanoma cell line. PMID:24799955

  7. Polyethylene Glycol-Conjugated Adenosine Deaminase (ADA) Therapy Provides Temporary Immune Reconstitution to a Child with Delayed-Onset ADA Deficiency

    PubMed Central

    Lainka, Elke; Hershfield, Michael S.; Santisteban, Ines; Bali, Pawan; Seibt, Annette; Neubert, Jennifer; Friedrich, Wilhelm; Niehues, Tim

    2005-01-01

    We describe the effects of polyethylene glycol-conjugated adenosine deaminase (ADA) replacement therapy on lymphocyte counts, activation, apoptosis, proliferation, and cytokine secretion in a 14-month-old girl with “delayed-onset” ADA deficiency and marked immunodysregulation. Pretreatment lymphopenia affected T cells (CD4, 150/μl; CD8, 459/μl), B cells (16/μl), and NK cells (55/μl). T cells were uniformly activated and largely apoptotic (CD4, 59%; CD8, 82%); and T-cell-dependent cytokine levels in plasma were elevated, including the levels of interleukin 2 (IL-2; 26 pg/ml), IL-4 (81 pg/ml), IL-5 (46 pg/ml), gamma interferon (1,430 pg/ml), tumor necrosis factor alpha (210 pg/ml), and IL-10 (168 pg/ml). Mitogen-stimulated peripheral blood mononuclear cells show reduced IL-2 secretion and proliferation. During the first 5 months of therapy there was clinical improvement and partial immune reconstitution, with nearly normal lymphocyte subset numbers, reduced T-cell activation and CD4-cell apoptosis, and decreased plasma cytokine levels. In parallel, IL-2 secretion and the lymphocyte mitogenic response improved. Between 4 and 7 months, immunoglobulin G antibodies to bovine ADA developed and resulted in the complete reversal of immune recovery. PMID:16002636

  8. Activity of cholinesterases, pyruvate kinase and adenosine deaminase in rats experimentally infected by Fasciola hepatica: Influences of these enzymes on inflammatory response and pathological findings.

    PubMed

    Baldissera, Matheus D; Bottari, Nathieli B; Mendes, Ricardo E; Schwertz, Claiton I; Lucca, Neuber J; Dalenogare, Diessica; Bochi, Guilherme V; Moresco, Rafael N; Morsch, Vera M; Schetinger, Maria R C; Rech, Virginia C; Jaques, Jeandre A; Da Silva, Aleksandro S

    2015-11-01

    The aim of this study was to investigate acetylcholinesterase (AChE) in total blood and liver tissue; butyrylcholinesterase (BChE) in serum and liver tissue; adenosine deaminase (ADA) in serum and liver tissue; and pyruvate kinase (PK) in liver tissue of rats experimentally infected by Fasciola hepatica. Animals were divided into two groups with 12 animals each, as follows: group A (uninfected) and group B (infected). Samples were collected at 20 (A1 and B1;n=6 each) and 150 (A2 and B2; n=6 each) days post-infection (PI). Infected animals showed an increase in AChE activity in whole blood and a decrease in AChE activity in liver homogenates (P<0.05) at 20 and 150 days PI. BChE and PK activities were decreased (P<0.05) in serum and liver homogenates of infected animals at 150 days PI. ADA activity was decreased in serum at 20 and 150 days PI, while in liver homogenates it was only decreased at 150 days PI (P<0.05). Aspartate aminotransferase and alanine aminotransferase activities in serum were increased (P<0.05), while concentrations of total protein and albumin were decreased (P<0.05) when compared to control. The histological analysis revealed fibrous perihepatitis and necrosis. Therefore, we conclude that the liver fluke is associated with cholinergic and purinergic dysfunctions, which in turn may influence the pathogenesis of the disease.

  9. Chicken embryo fibroblasts exposed to weak, time-varying magnetic fields share cell proliferation, adenosine deaminase activity, and membrane characteristics of transformed cells

    SciTech Connect

    Parola, A.H.; Porat, N.; Kiesow, L.A. )

    1993-01-01

    Chicken embryo fibroblasts (CEF) exposed to a sinusoidally varying magnetic field (SVMF) (100 Hz, 700 microT, for 24 h) showed a remarkable rise of segmental rotational relaxation rate of adenosine deaminase (ADA, EC 3.5.4.4) as determined by multifrequency phase fluorometry. Pyrene-labeled, small subunit ADA was applied to cultured (normal) CEF, which have available and abundant ADA complexing protein (ADCP) on their plasma membranes. Sine-wave-modulated fluorometry of the pyrene yielded a profile of phase angle vs. modulation frequency. In SVMF-treated cells and in Rous-sarcoma-virus (RSV) transformed cells the differential phase values at low modulation frequencies of the excitation are remarkably reduced. This effect is magnetic rather than thermal, because the temperature was carefully controlled and monitored; nevertheless to further check this matter we studied CEF, infected by the RSV-Ts68 temperature-sensitive mutant (36 degrees C transformed, 41 degrees C revertant). When grown at 36 degrees C in the SVMF, cells did not show the slightest trend towards reversion, as would be expected had there been local heating. Concomitant with the increased segmental rotational relaxation rate of ADA, there was a decrease in fluorescence lifetime and a slight, yet significant, increase in membrane lipid microfluidity. These biophysical observations prompted us to examine the effect of SVMF on cell proliferation and ADA activity (a malignancy marker): higher rates of cell proliferation and reduced specific activity of ADA were observed.

  10. Aqueous seed extract of Syzygium cumini inhibits the dipeptidyl peptidase IV and adenosine deaminase activities, but it does not change the CD26 expression in lymphocytes in vitro.

    PubMed

    Bellé, Luziane Potrich; Bitencourt, Paula Eliete Rodrigues; Abdalla, Faida Husein; Bona, Karine Santos de; Peres, Alessandra; Maders, Liési Diones Konzen; Moretto, Maria Beatriz

    2013-03-01

    Syzygium cumini (Sc) have been intensively studied in the last years due its beneficial effects including anti-diabetic and anti-inflammatory potential. Thus, the aim of this study was to evaluate the effect of aqueous seed extract of Sc (ASc) in the activity of enzymes involved in lymphocyte functions. To perform this study, we isolated lymphocytes from healthy donors. Lymphocytes were exposed to 10, 30, and 100 mg/mL of ASc during 4 and 6 h and adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), and acetylcholinesterase (AChE) activities as well as CD26 expression and cellular viability were evaluated. ASc inhibited the ADA and DPP-IV activities without alteration in the CD26 expression (DPP-IV protein). No alterations were observed in the AChE activity or in the cell viability. These results indicate that the inhibition of the DPP-IV and ADA activities was dependent on the time of exposition to ASc. We suggest that ASc exhibits immunomodulatory properties probably via the pathway of DPP-IV-ADA complex, contributing to the understanding of these proceedings in the purinergic signaling. PMID:22798209

  11. 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

  12. Adenosine deaminase isoenzyme levels in patients with human T-cell lymphotropic virus type 1 and human immunodeficiency virus type 1 infections.

    PubMed Central

    Tsuboi, I; Sagawa, K; Shichijo, S; Yokoyama, M M; Ou, D W; Wiederhold, M D

    1995-01-01

    In serum, the enzyme adenosine deaminase (ADA) is known to be divided into two isoenzymes, ADA1 and ADA2, which have different molecular weights and kinetic properties. The present study investigated ADA isoenzyme levels in the sera of patients infected with retroviruses associated with adult T-cell leukemia (ATL), human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy (HAM), and AIDS, ADA isoenzyme activities were found to be significantly (P < 0.001) higher in the sera of patients with ATL, HAM, and AIDS than in the sera of healthy controls. In the case of the ADA subtypes in the sera of patients with ATL, ADA1 activity was significantly (P < 0.001) elevated in patients with the acute and lymphoma types of ATL compared with that in patients with the chronic and smoldering types of ATL. ADA2 activity was significantly elevated in the sera of patients with the acute, lymphoma, and chronic types of ATL (P < 0.001) compared with that in patients with smoldering ATL and HTLV-1 carriers. In the case of patients with human immunodeficiency virus type 1 (HIV-1) infection, ADA1 and ADA2 activities in the sera of patients with AIDS and HIV-1 antibody-positive individuals were significantly (P < 0.001) higher than those in the sera of HIV-1 antibody-negative individuals. A significant elevation in ADA2 activity was also seen in the sera of AIDS patients (P < 0.01) compared with that in the sera of HIV-1 antibody-positive individuals. These results suggest that the magnitude of elevation of ADA isoenzyme levels in serum correlates well with the clinical conditions of the patients with these diseases. Measurement of the activities of ADA isoenzymes may therefore provide an additional parameter for distinguishing the subtypes of ATL and may prove to be useful as prognostic and therapeutic monitors in diseases associated with HTLV-1 and HIV-1 infections. PMID:8548545

  13. Double-stranded-RNA-specific adenosine deaminase 1 (ADAR1) is proposed to contribute to the adaptation of equine infectious anemia virus from horses to donkeys.

    PubMed

    Tang, Yan-Dong; Zhang, Xiang; Na, Lei; Wang, Xue-Feng; Fu, Li-Hua; Zhu, Chun-Hui; Wang, Xiaojun; Zhou, Jian-Hua

    2016-10-01

    Equine infectious anemia virus (EIAV) is a member of the genus Lentivirus of the family Retroviridae. Horses are the most susceptible equids to EIAV infection and are therefore the primary hosts of this virus. In contrast, infected donkeys do not develop clinically active equine infectious anemia (EIA). This phenomenon is similar to what has been observed with HIV-1, which fails to induce AIDS in non-human primates. Interestingly, Shen et al. developed a donkey-tropic pathogenic virus strain (EIAVDV117, DV117) by serially passaging a horse-tropic pathogenic strain, EIAVLN40 (LN40), in donkeys. LN40, which was generated by passaging a field isolate in horses, displayed enhanced virulence in horses but caused no clinical symptoms in donkeys. Infection with DV117 induced acute EIA in nearly 100 % of donkeys. Genomic analysis of DV117 revealed a significantly higher frequency of A-to-G substitutions when compared to LN40. Furthermore, detailed analysis of dinucleotide editing showed that A-to-G mutations had a preference for 5'TpA and 5'ApA. These results strongly implicated the activity of the adenosine deaminase, ADAR1, in this type of mutation. Further investigation demonstrated that overexpression of donkey ADAR1 increased A-to-G mutations within the genome of EIAV. Together with our previous finding that multiple mutations in multiple genes are generated in DV117 during its adaptation from horses to donkeys, the present study suggests that ADAR1-induced A-to-G mutations occur during virus adaption to related new hosts contributing to the alteration of EIAV host tropism. PMID:27383210

  14. Evaluation of adenosine deaminase activity and antibody to Mycobacterium tuberculosis antigen 5 in cerebrospinal fluid and the radioactive bromide partition test for the early diagnosis of tuberculosis meningitis.

    PubMed Central

    Coovadia, Y M; Dawood, A; Ellis, M E; Coovadia, H M; Daniel, T M

    1986-01-01

    A number of different biochemical and serological tests have been described recently for the early and accurate diagnosis of tuberculous meningitis. None of these tests has yet gained widespread acceptance in clinical medicine or in microbiology laboratories. To investigate this problem we evaluated adenosine deaminase activity (ADA), an enzyme linked immunosorbent assay (ELISA) that detects antibody to antigen 5 of Mycobacterium tuberculosis, and the radioactive bromide partition test (BPT) in the cerebrospinal fluid (CSF). Cerebrospinal fluid specimens from children with tuberculous, pyogenic, and viral meningitis as well as from patients with pulmonary tuberculosis without meningitis and from controls with normal CSFs were included inn the study. In addition, we estimated ADAs in serum samples from selected children in these groups. The sensitivity and specificity of the three tests evaluated in the CSF were: ADA assay 73% and 71%; BPT 92% and 92%; and ELISA for antibody to antigen 5, 53% and 90%, 40% and 94%, and 27% and 100%, respectively, at tires of more than or equal to 1:20, 1:40, and 1:80. The serum ADA was lower (11.0 +/- 6.15 IU/l) in children with tuberculous meningitis when compared with those with pulmonary tuberculosis alone (25.8 +/- 20.9 IU/l). The BPT was found to be the most reliable test in the early differentiation of tuberculous from other causes of meningitis and remained abnormal for a period of up to five months after the beginning of treatment. Accordingly, we believe that the BPT should be used in conjunction with bacterial and fungal antigen detection systems for the initial differentiation of clinically suspicious tuberculous meningitis from Gram or culture negative cases, or both, of bacterial and fungal meningitis. PMID:3087296

  15. Double-stranded-RNA-specific adenosine deaminase 1 (ADAR1) is proposed to contribute to the adaptation of equine infectious anemia virus from horses to donkeys.

    PubMed

    Tang, Yan-Dong; Zhang, Xiang; Na, Lei; Wang, Xue-Feng; Fu, Li-Hua; Zhu, Chun-Hui; Wang, Xiaojun; Zhou, Jian-Hua

    2016-10-01

    Equine infectious anemia virus (EIAV) is a member of the genus Lentivirus of the family Retroviridae. Horses are the most susceptible equids to EIAV infection and are therefore the primary hosts of this virus. In contrast, infected donkeys do not develop clinically active equine infectious anemia (EIA). This phenomenon is similar to what has been observed with HIV-1, which fails to induce AIDS in non-human primates. Interestingly, Shen et al. developed a donkey-tropic pathogenic virus strain (EIAVDV117, DV117) by serially passaging a horse-tropic pathogenic strain, EIAVLN40 (LN40), in donkeys. LN40, which was generated by passaging a field isolate in horses, displayed enhanced virulence in horses but caused no clinical symptoms in donkeys. Infection with DV117 induced acute EIA in nearly 100 % of donkeys. Genomic analysis of DV117 revealed a significantly higher frequency of A-to-G substitutions when compared to LN40. Furthermore, detailed analysis of dinucleotide editing showed that A-to-G mutations had a preference for 5'TpA and 5'ApA. These results strongly implicated the activity of the adenosine deaminase, ADAR1, in this type of mutation. Further investigation demonstrated that overexpression of donkey ADAR1 increased A-to-G mutations within the genome of EIAV. Together with our previous finding that multiple mutations in multiple genes are generated in DV117 during its adaptation from horses to donkeys, the present study suggests that ADAR1-induced A-to-G mutations occur during virus adaption to related new hosts contributing to the alteration of EIAV host tropism.

  16. A combination of the QuantiFERON-TB Gold In-Tube assay and the detection of adenosine deaminase improves the diagnosis of tuberculous pleural effusion

    PubMed Central

    Liu, Yuanyuan; Ou, Qinfang; Zheng, Jian; Shen, Lei; Zhang, Bingyan; Weng, Xinhua; Shao, Lingyun; Gao, Yan; Zhang, Wenhong

    2016-01-01

    The differential diagnosis of tuberculous pleural effusion (TPE) and malignant pleural effusion (MPE) remains difficult despite the availability of numerous diagnostic tools. The current study aimed to evaluate the performance of the whole blood QuantiFERON-TB Gold In-Tube (QFT-GIT) assay and conventional laboratory biomarkers in differential diagnosis of TPE and MPE in high tuberculosis prevalence areas. A total of 117 patients with pleural effusions were recruited, including 91 with TPE and 26 with MPE. All of the patients were tested with QFT-GIT, and the conventional biomarkers in both blood and pleural effusion were detected. The level of antigen-stimulated QFT-GIT in the whole blood of TPE patients was significantly higher than that of MPE (2.89 vs 0.33 IU/mL, P<0.0001). The sensitivity and specificity of QFT-GIT for the diagnosis of TPE were 93.0% and 60.0%, respectively. Among the biomarkers in blood and pleural effusion, pleural adenosine deaminase (ADA) was the most prominent biomarker, with a cutoff value of 15.35 IU/L. The sensitivity and specificity for the diagnosis of TPE were 93.4% and 96.2%, respectively. The diagnostic classification tree from the combination of these two biomarkers was 97.8% sensitive and 92.3% specific. Ultimately, the combination of whole blood QFT-GIT with pleural ADA improved both the specificity and positive predictive value to 100%. Thus, QFT-GIT is not superior to pleural ADA in the differential diagnosis of TPE and MPE. Combined whole blood QFT-GIT and pleural ADA detection can improve the diagnosis of TPE. PMID:27485497

  17. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    PubMed Central

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-01-01

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. 20/34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access substrate DNA cytosines. PMID:22181350

  18. First-In-Class Small Molecule Inhibitors of the Single-Strand DNA Cytosine Deaminase APOBEC3G

    SciTech Connect

    Li, Ming; Shandilya, Shivender M.D.; Carpenter, Michael A.; Rathore, Anurag; Brown, William L.; Perkins, Angela L.; Harki, Daniel A.; Solberg, Jonathan; Hook, Derek J.; Pandey, Krishan K.; Parniak, Michael A.; Johnson, Jeffrey R.; Krogan, Nevan J.; Somasundaran, Mohan; Ali, Akbar; Schiffer, Celia A.; Harris, Reuben S.

    2012-04-04

    APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.

  19. 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.

  20. 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

  1. 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

  2. Carbocyclic adenosine analogues as S-adenosylhomocysteine hydrolase inhibitors and antiviral agents: recent advances.

    PubMed

    De Clercq, E

    1998-01-01

    Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5'-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6'-R-alkyl (i.e., 6'-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process. PMID:9708366

  3. Synthesis and biological evaluation of pteridine and pyrazolopyrimidine based adenosine kinase inhibitors.

    PubMed

    Gomtsyan, Arthur; Didomenico, Stanley; Lee, Chih-Hung; Stewart, Andrew O; Bhagwat, Shripad S; Kowaluk, Elizabeth A; Jarvis, Michael F

    2004-08-16

    Three new approaches have been tested to modify existing pyridopyrimidine and alkynylpyrimidine classes of nonnucleoside adenosine kinase inhibitors 2 and 3. 4-Amino-substituted pteridines 8a-e were generally less active than corresponding 5- and 6-substituted pyridopyrimidines 2. Pyrazolopyrimidine 13c with IC(50)=7.5 nM was superior to its open chain alkynylpyrimidine analog 13g (IC(50)=22 nM) while pyrrolopyrimidines such as 17a were inactive.

  4. 5,6,7-trisubstituted 4-aminopyrido[2,3-d]pyrimidines as novel inhibitors of adenosine kinase.

    PubMed

    Perner, Richard J; Gu, Yu-Gui; Lee, Chih-Hung; Bayburt, Erol K; McKie, Jeffery; Alexander, Karen M; Kohlhaas, Kathy L; Wismer, Carol T; Mikusa, Joe; Jarvis, Michael F; Kowaluk, Elizabeth A; Bhagwat, Shripad S

    2003-11-20

    The synthesis and structure-activity relationship of a series of 5,6,7-trisubstituted 4-aminopyrido[2,3-d]pyrimidines as novel nonnucleoside adenosine kinase inhibitors is described. A variety of alkyl, aryl, and heteroaryl substituents were found to be tolerated at the C5, C6, and C7 positions of the pyridopyrimidine core. These studies have led to the identification of analogues that are potent inhibitors of adenosine kinase with in vivo analgesic activity.

  5. Adenosine Analog NITD008 Is a Potent Inhibitor of Zika Virus

    PubMed Central

    Deng, Yong-Qiang; Zhang, Na-Na; Li, Chun-Feng; Tian, Min; Hao, Jia-Nan; Xie, Xu-Ping; Shi, Pei-Yong; Qin, Cheng-Feng

    2016-01-01

    The ongoing Zika virus (ZIKV) outbreaks have raised global concerns due to its unexpected clinical manifestations. Antiviral development is of high priority in response to the ZIKV emergency. In this study, we report that an adenosine analog NITD008 has potent in vitro and in vivo antiviral activity against ZIKV. The compound can effectively inhibit the historical and contemporary ZIKV strains in cultures as well as significantly reduce viremia and prevent mortality in A129 mice. Our results have demonstrated that NITD008 is potent inhibitor of ZIKV and can be used as reference inhibitor for future ZIKV antiviral drug screen and discovery. PMID:27747251

  6. Overexpression of adenosine deaminase acting on RNA 1 in chordoma tissues is associated with chordoma pathogenesis by reducing miR-125a and miR-10a expression

    PubMed Central

    KUANG, LEI; LV, GUOHUA; WANG, BING; LI, LEI; DAI, YULIANG; LI, YAWEI

    2015-01-01

    Chordoma is a rare, slow-growing primary malignant neoplasm of the axial skeleton, which arises from the remnants of the notochord. Emerging evidence suggests that microRNAs (miRs) are dysregulated in chordoma tissues and crucially involved in chordoma pathogenesis. In the present study, the expression of 11 candidate miRs were analyzed in chordoma tissues and miR-10a and miR-125a were found to be significantly downregulated compared with controls. Notably, the expression of the primary transcripts, pri-miR-125a and pri-miR-10a was unaltered, suggesting that disturbed microRNA expression may be induced by altered pri-miRNA processing. Previous studies have indicated that disturbed adenosine deaminase acting on RNA (ADAR) expression is able to alter mRNA and miRNA adenosine to inosine (A-to-I) levels associated with cancer pathogenesis. Therefore, the expression of ADAR1 and ADAR2 was analyzed in chordoma tissues. It was found that ADAR1 was significantly overexpressed, which was accompanied by enhanced pre-miR-10a and pri-miR-125a A-to-I editing. These findings suggest that ADAR2 overexpression causes enhanced pre-miR-10a and pri-miR-125a A-to-I editing, which alters mature miR-10a and miR-125a expression and may contribute to chordoma pathogenesis. PMID:25673044

  7. 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

  8. Molecular Shape Analysis-Guided Virtual Screening Platform for Adenosine Kinase Inhibitors.

    PubMed

    Bhutoria, Savita; Das, Ballari; Ghoshal, Nanda

    2016-01-01

    We propose a new application of molecular shape descriptors in hierarchical selection during virtual screening (VS). Here, a structure-based pharmacophore and docking-guided VS protocol have been evolved to identify inhibitors against adenosine kinase (AK). The knowledge gained on the shape requirements has been extrapolated in classifying active and inactive molecules against this target. This classification enabled us to pick the appropriate ligand conformation in the binding site. We have suggested a set of hierarchical filters for VS, from a simple molecular shape analysis (MSA) descriptor-based recursive models to docking scores. This approach permits a systematic study to understand the importance of spatial requirements and limitations for inhibitors against AK. Finally, the guidelines on how to select compounds for AK to achieve success have been highlighted. The utility of this approach has been suggested by giving an example of database screening for plausible active compounds. PMID:27478367

  9. Molecular Shape Analysis-Guided Virtual Screening Platform for Adenosine Kinase Inhibitors

    PubMed Central

    Bhutoria, Savita; Das, Ballari; Ghoshal, Nanda

    2016-01-01

    We propose a new application of molecular shape descriptors in hierarchical selection during virtual screening (VS). Here, a structure-based pharmacophore and docking-guided VS protocol have been evolved to identify inhibitors against adenosine kinase (AK). The knowledge gained on the shape requirements has been extrapolated in classifying active and inactive molecules against this target. This classification enabled us to pick the appropriate ligand conformation in the binding site. We have suggested a set of hierarchical filters for VS, from a simple molecular shape analysis (MSA) descriptor-based recursive models to docking scores. This approach permits a systematic study to understand the importance of spatial requirements and limitations for inhibitors against AK. Finally, the guidelines on how to select compounds for AK to achieve success have been highlighted. The utility of this approach has been suggested by giving an example of database screening for plausible active compounds. PMID:27478367

  10. Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

    NASA Astrophysics Data System (ADS)

    Jiang, Keyong; Sun, Shujuan; Liu, Mei; Wang, Baojie; Meng, Xiaolin; Wang, Lei

    2013-01-01

    AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) ( P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) ( P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

  11. Determination of Plaque Inhibitory Activity of Adenine Arabinoside (9-β-d-Arabinofuranosyladenine) for Herpesviruses Using an Adenosine Deaminase Inhibitor

    PubMed Central

    Bryson, Yvonne; Connor, James D.; Sweetman, Lawrence; Carey, Sharen; Stuckey, Margaret A.; Buchanan, Robert

    1974-01-01

    The in vitro susceptibility of type 1 and type 2 strains of Herpesvirus hominis to 9-β-d-arabinofuranosyladenine (adenine arabinoside, ara-A) was measured in a system where deamination was inhibited. Under these conditions, it was possible to measure the activity of low concentrations of ara-A. It was determined that plaque inhibitory concentration for type 1 viruses was less than 3 μg/ml for all strains tested. The plaque inhibitory concentration for 7 of 10 type 2 strains was also less than 3 μg/ml. The method used identified and controlled the interaction between antiviral agent (ara-A) and the indicator system, human skin fibroblastic cells. Otherwise, metabolism of ara-A resulted in rapid enzymatic degradation and loss of antiviral activity. PMID:15828177

  12. Organization of the Mouse RNA-specific Adenosine Deaminase Adar1 Gene 5’-Region and Demonstration of STAT1-independent, STAT2-dependent Transcriptional Activation by Interferon

    PubMed Central

    George, Cyril X.; Das, Sonali; Samuel, Charles E.

    2008-01-01

    The p150 form of the RNA-specific adenosine deaminase ADAR1 is interferon-inducible and catalyzes A-to-I editing of viral and cellular RNAs. We have characterized mouse genomic clones containing the promoter regions required for Adar1 gene transcription and analyzed interferon induction of the p150 protein using mutant mouse cell lines. Transient transfection analyses using reporter constructs led to the identification of three promoters, one interferon-inducible (PA) and two constitutively active (PB and PC). The TATA-less PA promoter, characterized by the presence of a consensus ISRE element and a PKR kinase KCS-like element, directed interferon-inducible reporter expression in rodent and human cells. Interferon induction of p150 was impaired in mouse cells deficient in IFNAR receptor, JAK1 kinase or STAT2 but not STAT1. Whereas Adar1 gene organization involving multiple promoters and alternative exon 1 structures was highly preserved, sequences of the promoters and exon 1 structures were not well conserved between human and mouse. PMID:18774582

  13. 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

  14. Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

    PubMed Central

    Pfaller, Christian K.; Mastorakos, George M.; Matchett, William E.; Ma, Xiao; Samuel, Charles E.

    2015-01-01

    ABSTRACT Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and

  15. 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.

  16. Trifunctional Agents as a Design Strategy for Tailoring Ligand Properties: Irreversible Inhibitors of A1 Adenosine Receptors†

    PubMed Central

    Boring, Daniel L.; Ji, Xiao-Duo; Zimmet, Jeff; Taylor, Kirk E.; Stiles, Gary L.

    2012-01-01

    The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]-oxy]phenyl]-l,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H]CGS21680 (2-[[2-[4-(2-carboxyethyl)phenyl]ethyl]amino]adenosine-5′-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversibly binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein. PMID:1868116

  17. Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deaminase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene. Amendment to clinical research project, Project 90-C-195, January 10, 1992.

    PubMed

    Blaese, R M; Culver, K W; Chang, L; Anderson, W F; Mullen, C; Nienhuis, A; Carter, C; Dunbar, C; Leitman, S; Berger, M

    1993-08-01

    Significant increases in lymphocyte adenosine deaminase activity, T cell numbers and immune function have been achieved in the two children with SCID thus far treated with autologous T cells genetically-corrected by retroviral-mediated insertion of a normal ADA gene. Although the data obtained to date demonstrate that the use of ADA gene corrected peripheral T cells appears to be an effective treatment for ADA(-)SCID, it is theoretically preferable to try to develop a treatment for these children that will result in stem cell gene correction. The genetic correction of T cell progenitors with long-term immune reconstituting ability would be more desirable because repeated infusions of genetically altered cells should not be necessary and the generation of a more complete repertoire of T cell specificities might also be possible. Furthermore, the present treatment protocol involves indefinite continuation of enzyme replacement treatment with PEG-ADA. The demonstration of ADA gene expression in the progeny of transduced stem cells may simplify the decision concerning cessation of this very costly enzyme treatment (approximately $250,000/yr./patient). Recent evidence suggests that a small fraction of bone marrow or peripheral blood mononuclear cells bearing the CD34 antigen contains hematopoietic stem cells with both lymphoid and myeloid reconstituting ability. We propose in this amendment to supplement the infusion of human ADA gene-transduced autologous T cells in children with ADA(-)SCID with autologous peripheral blood CD34+ cells transduced with a second, readily distinguishable ADA vector.(ABSTRACT TRUNCATED AT 250 WORDS)

  18. Carrier frequency of a nonsense mutation in the adenosine deaminase (ADA) gene implies a high incidence of ADA-deficient severe combined immunodeficiency (SCID) in Somalia and a single, common haplotype indicates common ancestry.

    PubMed

    Sanchez, Juan J; Monaghan, Gemma; Børsting, Claus; Norbury, Gail; Morling, Niels; Gaspar, H Bobby

    2007-05-01

    Inherited adenosine deaminase (ADA) deficiency is a rare metabolic disorder that causes immunodeficiency, varying from severe combined immunodeficiency (SCID) in the majority of cases to a less severe form in a small minority of patients. Five patients of Somali origin from four unrelated families, with severe ADA-SCID, were registered in the Greater London area. Patients and their parents were investigated for the nonsense mutation Q3X (ADA c7C>T), two missense mutations K80R (ADA c239A>G) and R142Q (ADA c425G>A), and a TAAA repeat located at the 3' end of an Alu element (AluVpA) positioned 1.1 kb upstream of the ADA transcription start site. All patients were homozygous for the haplotype ADA-7T/ADA-239G/ADA-425G/AluVpA7. Among 207 Somali immigrants to Denmark, the frequency of ADA c7C>T and the maximum likelihood estimate of the frequency of the haplotype ADA-7T/ADA-239G/ADA-425G/AluVpA7 were both 0.012 (carrier frequency 2.4%). Based on the analysis of AluVpA alleles, the ADA c7C/T mutation was estimated to be approximately 7,100 years old. Approximately 1 out of 5 - 10000 Somali children will be born with ADA deficiency due to an ADA c7C/T mutation, although within certain clans the frequency may be significantly higher. ADA-SCID may be a frequent immunodeficiency disorder in Somalia, but will be underdiagnosed due to the prevailing socioeconomic and nutritional deprivation.

  19. Diagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for tuberculous pericarditis compared to adenosine deaminase and unstimulated interferon-γ in a high burden setting: a prospective study

    PubMed Central

    2014-01-01

    Background Tuberculous pericarditis (TBP) is associated with high morbidity and mortality, and is an important treatable cause of heart failure in developing countries. Tuberculous aetiology of pericarditis is difficult to diagnose promptly. The utility of the new quantitative PCR test (Xpert MTB/RIF) for the diagnosis of TBP is unknown. This study sought to evaluate the diagnostic accuracy of the Xpert MTB/RIF test compared to pericardial adenosine deaminase (ADA) and unstimulated interferon-gamma (uIFNγ) in suspected TBP. Methods From October 2009 through September 2012, 151 consecutive patients with suspected TBP were enrolled at a single centre in Cape Town, South Africa. Mycobacterium tuberculosis culture and/or pericardial histology served as the reference standard for definite TBP. Receiver-operating-characteristic curve analysis was used for selection of ADA and uIFNγ cut-points. Results Of the participants, 49% (74/151) were classified as definite TBP, 33% (50/151) as probable TBP and 18% (27/151) as non TBP. A total of 105 (74%) participants were human immunodeficiency virus (HIV) positive. Xpert-MTB/RIF had a sensitivity and specificity (95% confidence interval (CI)) of 63.8% (52.4% to 75.1%) and 100% (85.6% to 100%), respectively. Concentration of pericardial fluid by centrifugation and using standard sample processing did not improve Xpert MTB/RIF accuracy. ADA (≥35 IU/L) and uIFNγ (≥44 pg/ml) both had a sensitivity of 95.7% (88.1% to 98.5%) and a negative likelihood ratio of 0.05 (0.02 to 0.10). However, the specificity and positive likelihood ratio of uIFNγ was higher than ADA (96.3% (81.7% to 99.3%) and 25.8 (3.6 to 183.4) versus 84% (65.4% to 93.6%) and 6.0 (3.7 to 9.8); P = 0.03) at an estimated background prevalence of TB of 30%. The sensitivity and negative predictive value of both uIFNγ and ADA were higher than Xpert-MT/RIF (P < 0.001). Conclusions uIFNγ offers superior accuracy for the diagnosis of microbiologically

  20. Genetics Home Reference: adenosine deaminase deficiency

    MedlinePlus

    ... SCID lack virtually all immune protection from bacteria, viruses, and fungi. They are prone to repeated and persistent infections that can be very serious or life-threatening. These infections are often caused by "opportunistic" ...

  1. 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

  2. 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.

  3. Trypanocidal activity of 8-methyl-5'-{[(Z)-4-aminobut-2-enyl]-(methylamino)}adenosine (Genz-644131), an adenosylmethionine decarboxylase inhibitor.

    PubMed

    Bacchi, Cyrus J; Barker, Robert H; Rodriguez, Aixa; Hirth, Bradford; Rattendi, Donna; Yarlett, Nigel; Hendrick, Clifford L; Sybertz, Edmund

    2009-08-01

    Genzyme 644131, 8-methyl-5'-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

  4. Synthesis and biological evaluation of 6,7-disubstituted 4-aminopyrido[2,3-d]pyrimidines as adenosine kinase inhibitors.

    PubMed

    Perner, Richard J; Lee, Chih-Hung; Jiang, Meiqun; Gu, Yu-Gui; Didomenico, Stanley; Bayburt, Erol K; Alexander, Karen M; Kohlhaas, Kathy L; Jarvis, Michael F; Kowaluk, Elizabeth L; Bhagwat, Shripad S

    2005-06-01

    The synthesis and structure-activity relationship of a series of 6,7-disubstituted 4-aminopyrido[2,3-d]pyrimidines as novel non-nucleoside adenosine kinase inhibitors is described. A variety of substituents, primarily aryl, at the C6 and C7 positions of the pyridopyrimidine core were found to yield analogues that are potent inhibitors of adenosine kinase. In contrast to the 5,7-disubstituted and 5,6,7-trisubstituted pyridopyrimidine series, these analogues exhibited only modest potency to inhibit AK in intact cells.

  5. 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

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

    PubMed

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

    2014-09-01

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

  7. 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

  8. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    PubMed

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP. PMID:27596420

  9. Enzymatic production of 5'-inosinic acid by AMP deaminase from a newly isolated Aspergillus oryzae.

    PubMed

    Li, Shubo; Chen, Leitao; Hu, Yangjun; Fang, Guohui; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2017-02-01

    5'-adenylic acid deaminase (AMP deaminase), an important enzyme for the food industry, can catalyze the irreversible hydrolysis of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia. In this study, a new strain was screened that efficiently produces 3191.6U/g of AMP deaminase at 32°C. After purification, the optimal temperature and pH of the AMP deaminase were found to be 40°C and 6.0, respectively, but it was partially inhibited by Fe(3+), Cu(2+), Al(3+), and Zn(2+). With amplification of the AMP deaminase production system, 6mL of crude enzyme could produce 2.00mg/g of IMP from 2.04mg/g of dried yeast with an 84.8% molar yield after 40min. These results provide a new insight into AMP deaminase production and offer a potential platform for producing 5'-IMP.

  10. 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.

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. Effects of adenosine derivatives on cytotoxic T lymphocyte (CTL) and primary antibody responses in mice

    SciTech Connect

    Kandil, O.; Nakic, M.; Nelson, J.A.

    1986-03-05

    Accumulation of adenosine (Ado) and/or deoxyadenosine (dAdo) in adenosine deaminase deficiency is thought to mediate the immunodeficiency associated with the genetic disease in humans. To study the mechanism by which immune function is impaired, we are using analogs which are not substrates for the deaminase. The analogs and their doses (mg/kg/day, IP) are: an Ado analog, Tubercidin (2); and two dAdo analogs, 2-chlorodeoxy-adenosine (50) and 2-fluoro, arabinosyl AMP (250). CTL were generated by injecting C57B1/6 mice with 2.5 x 10/sup 7/ P815 mastocytoma cells, IP. Groups of mice were then treated once daily on days 9-11 with the analogs. On day 12, the animals were sacrificed and spleen cell suspensions were enriched for CTL by passage through nylon wool columns. CTL were measured against /sup 51/Cr-labeled P815 targets using a standard /sup 51/Cr-release assay. Primary antibody response was assessed in AKR mice following immunization with a T-dependent (sheep erythrocytes) or T-independent (TNP-Ficoll) antigen. Animals were treated with the analogs on days 1-3. On day 4, spleen cells were removed and assayed for antibody response in a plaque-forming assay. The dAdo analogs, but not tubercidin, inhibited the immune functions at these near-maximally tolerated doses. Treatment of the mice with the deaminase inhibitor, deoxycoformycin (1 mg/kg/day), was not inhibitory to these responses.

  17. 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

  18. Combined treatment of adenosine nucleoside inhibitor NITD008 and histone deacetylase inhibitor vorinostat represents an immunotherapy strategy to ameliorate West Nile virus infection

    PubMed Central

    Nelson, Jacob; Roe, Kelsey; Orillo, Beverly; Shi, Pei-Yong; Verma, Saguna

    2015-01-01

    West Nile virus (WNV), a member of the Flaviviridae family, is the leading cause of viral encephalitis in the United States. Despite efforts to control the spread of WNV, there has been an increase in the number of outbreaks and clinical cases with neurological problems. There are no antiviral compounds currently in trials for WNV. NITD008 is an adenosine analogue inhibitor that interrupts the RNA-dependent RNA polymerase of flaviviruses. While NITD008 has shown promise as an antiviral for dengue virus, the ability of this drug to block WNV replication is only limited to Vero cells. Neuroinflammation is also a major cause of the WNV-associated pathology, therefore we evaluated the effect of NITD008 and a newly characterized anti-inflammatory drug vorinostat (SAHA), a histone deacetylase inhibitor, on WNV replication and disease progression in a mouse model. When administered at 10 and 25 mg/kg at days 1 to 6 after WNV infection in C57BL/6 mice, NITD008 conferred complete protection from clinical symptoms and death, which correlated with reduced viral load in the serum and restriction of virus-CNS entry. Delay of NITD008 treatment to days 3 to 6 and days 5 to 9 after infection, when WNV replication was high in the periphery and brain, resulted in the gradual loss of protection against WNV infection. However, co-treatment with SAHA and NITD008 during the CNS phase of disease improved disease outcome significantly by reducing inflammation and neuronal death. Our results support potential synergistic effect of combination therapy of NITD008 with SAHA for the treatment of WNV encephalitis. PMID:26225754

  19. Combined treatment of adenosine nucleoside inhibitor NITD008 and histone deacetylase inhibitor vorinostat represents an immunotherapy strategy to ameliorate West Nile virus infection.

    PubMed

    Nelson, Jacob; Roe, Kelsey; Orillo, Beverly; Shi, Pei-Yong; Verma, Saguna

    2015-10-01

    West Nile virus (WNV), a member of the Flaviviridae family, is the leading cause of viral encephalitis in the United States. Despite efforts to control the spread of WNV, there has been an increase in the number of outbreaks and clinical cases with neurological problems. There are no antiviral compounds currently in trials for WNV. NITD008 is an adenosine analogue inhibitor that interrupts the RNA-dependent RNA polymerase of flaviviruses. Previous studies demonstrated NITD008 as a potent antiviral for dengue virus, however this drug was associated with preclinical toxicity. The ability of NITD008 to block WNV replication is only shown in Vero cells. Neuroinflammation is also a major cause of the WNV-associated pathology, therefore we evaluated the effect of NITD008 and a newly characterized anti-inflammatory drug vorinostat (SAHA), a histone deacetylase inhibitor, on WNV replication and disease progression in a mouse model. When administered at 10 and 25mg/kg at days 1-6 after WNV infection in C57BL/6 mice, NITD008 conferred complete protection from clinical symptoms and death, which correlated with reduced viral load in the serum and restriction of virus-CNS entry. Delay of NITD008 treatment to days 3-6 and days 5-9 after infection, when WNV replication was high in the periphery and brain, resulted in the gradual loss of protection against WNV infection. However, co-treatment with SAHA and NITD008 during the CNS phase of disease improved disease outcome significantly by reducing inflammation and neuronal death. Our results support potential synergistic effect of combination therapy of NITD008 with SAHA for the treatment of WNV encephalitis.

  20. 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

  1. Isozyme-specific enzyme inhibitors. 14. 5'(R)-C-[(L-homocystein-S-yl)methyl]adenosine 5'-(beta,gamma-imidotriphosphate), a potent inhibitor of rat methionine adenosyltransferases.

    PubMed

    Kappler, F; Vrudhula, V M; Hampton, A

    1987-09-01

    The title compound is a covalent adduct of L-methionine (Met) and beta,gamma-imido-ATP. In its synthesis the N-Boc derivative of 5'(R)-C-(aminomethyl)-N6-benzoyl-5'-O-tosyl-2',3'-O- isopropylidenadenosine was converted by the successive actions of CF3CO2H and HNO2 into the corresponding 5'(R)-C-hydroxymethyl derivative. Treatment of this with disodium L-homocysteinate led to attack of sulfur at C6', apparently via a 5',6'-epoxide, and to total stereoselective inversion at C5' to furnish, after debenzoylation, 5'(R)-C-(L-homocystein-S-ylmethyl)-2',3'-O-isopropylidene ade nosine. The 5' configuration was established by conversion of this into the known 5'(S)-C-methyl-2',3'-O-isopropylidene adenosine with Raney nickel. The alpha-amino acid residue was protected as an N-Boc methyl ester, after which the 5'-hydroxyl was phosphorylated with benzyl phosphate and dicyclohexylcarbodiimide. The phosphoanhydride bond with inorganic imidodiphosphate was then created by established methods. Finally, blocking groups were removed under conditions that gave the desired adduct with no racemization of its L-methionine residue. It was a potent inhibitor [KM(ATP)/Ki = 1080; KM(Met)/Ki = 7.7] of the M-2 (normal tissue) form of rat methionine adenosyltransferase and of the M-T (hepatoma tissue) form [KM(ATP)/Ki = 670; KM(Met)/Ki = 22]. Inhibitions were competitive with respect to ATP or to L-methionine, indicating a dual substrate site mode of binding to the enzyme forms.

  2. Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and inhibitor-sensitivity in response to catabolite repression.

    PubMed Central

    Lloyd, D; Edwards, S W

    1976-01-01

    1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN'-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN'-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and

  3. Induction of oral tremor in mice by the acetylcholinesterase inhibitor galantamine: Reversal with adenosine A2A antagonism.

    PubMed

    Podurgiel, Samantha J; Spencer, Tiahna; Kovner, Rotem; Baqi, Younis; Müller, Christa E; Correa, Merce; Salamone, John D

    2016-01-01

    Tremulous jaw movements (TJMs) have become a commonly used rat model of Parkinsonian tremor. TJMs can be induced by a number of neurochemical conditions that parallel those seen in human Parkinsonism, including DA depletion, DA antagonism, and cholinomimetic administration, and can be reduced by various antiparkinsonian agents. TJMs typically occur in bursts with the peak frequency in the range of 3-7.5 Hz, which is similar to the Parkinsonian tremor frequency range. While the vast majority of this work has been done using rats, current efforts have focused on extending the TJM model to mice. The aim of the present studies was to establish a mouse model of Parkinsonian resting tremor using the anticholinesterase galantamine, and to investigate the effects of adenosine A2A antagonism on galantamine-induced TJMs. Galantamine significantly induced TJMs in a dose-dependent manner (0.5, 1.0, 1.5, 2.0, 2.5 mg/kg IP). The TJMs tended to occur in bursts in the 3-7.5 Hz frequency range, with a peak frequency of approximately 6 Hz. Systemic administration of the adenosine A2A antagonist MSX-3 (2.5, 5.0, 10.0 mg/kg) significantly attenuated galantamine-induced TJMs. Co-administration of MSX-3 also altered the local frequency of galantamine-induced TJMs, decreasing the peak frequency from approximately 6 Hz to 5 Hz, though the vast majority of TJMs remained in the frequency range characteristic of Parkinsonian resting tremor. These results indicate that adenosine A2A antagonism is capable of reducing anticholinesterase-induced TJMs in mice. Extending the TJM model to mice gives researchers an additional avenue for investigating drug-induced Parkinsonism and tremorogenesis, and could be a useful addition to the study of motor abnormalities observed in mouse genetic models of Parkinsonism. PMID:26459156

  4. Plant Purine Nucleoside Catabolism Employs a Guanosine Deaminase Required for the Generation of Xanthosine in Arabidopsis[W

    PubMed Central

    Dahncke, Kathleen; Witte, Claus-Peter

    2013-01-01

    Purine nucleotide catabolism is common to most organisms and involves a guanine deaminase to convert guanine to xanthine in animals, invertebrates, and microorganisms. Using metabolomic analysis of mutants, we demonstrate that Arabidopsis thaliana uses an alternative catabolic route employing a highly specific guanosine deaminase (GSDA) not reported from any organism so far. The enzyme is ubiquitously expressed and deaminates exclusively guanosine and 2’-deoxyguanosine but no other aminated purines, pyrimidines, or pterines. GSDA belongs to the cytidine/deoxycytidylate deaminase family of proteins together with a deaminase involved in riboflavin biosynthesis, the chloroplastic tRNA adenosine deaminase Arg and a predicted tRNA-specific adenosine deaminase 2 in A. thaliana. GSDA is conserved in plants, including the moss Physcomitrella patens, but is absent in the algae and outside the plant kingdom. Our data show that xanthosine is exclusively generated through the deamination of guanosine by GSDA in A. thaliana, excluding other possible sources like the dephosphorylation of xanthosine monophosphate. Like the nucleoside hydrolases NUCLEOSIDE HYDROLASE1 (NSH1) and NSH2, GSDA is located in the cytosol, indicating that GMP catabolism to xanthine proceeds in a mostly cytosolic pathway via guanosine and xanthosine. Possible implications for the biosynthetic route of purine alkaloids (caffeine and theobromine) and ureides in other plants are discussed. PMID:24130159

  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. Resistance of an adenosine kinase-deficient human lymphoblastoid cell line to effects of deoxyadenosine on growth, S-adenosylhomocysteine hydrolase inactivation, and dATP accumulation.

    PubMed

    Hershfield, M S; Kredich, N M

    1980-07-01

    Accumulation of dATP derived from 2'-deoxyadenosine (dAdo), causing inhibition of ribonucleotide reductase and depletion of the other deoxynucleotide substrates required for DNA synthesis, has been suggested as the cause of the lymphopenia and immune defect in inheritable deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). dAdo also inactivates the enzyme S-adenosylhomocysteine hydrolase (AdoHcyase; S-adenosyl-L-homocystein hydrolase EC 3.3.1.1) which is involved in the catabolism of S-adenosyl-L-homocysteine (AdoHcy), both a product and a potent inhibitor of S-adenosylmethionine-dependent transmethylation. We have tried to determine whether inactivation of AdoHcyase might also contribute to dAdo toxicity to adenosine deaminase-inhibited cells. dAdo rapidly inactivates intracellular AdoHcyase and causes the accumulation of AdoHcy in WI-L2 human B lymphoblastoid cells. Low concentrations of adenosine (Ado), which block binding of dAdo to purified AdoHcyase, prevented inactivation of intracellular AdoHcyase and also lessened the growth-inhibitory effect of dAdo. A mutant of this cell line which lacks Ado kinase and accumulated endogenously synthesized Ado was resistant to the effects of dAdo on both growth and AdoHcyase activity. The mutant also accumulated far less dATP from dAdo than did its parent and was resistant to the inhibitory effect of dAdo on DNA synthesis, indicating the Ado kinase is involved in dAdo phosphorylation in these cells. Combinations of deoxycytidine, thymidine, and deoxyguanosine that could prevent dATP-mediated depletion of deoxynucleotide pools but not AdoHcyase inactivation were less effective than Ado in preventing dAdo toxicity to normal lymphoblasts. Our results suggest that inactivation of AdoHcyase, as well as dATP accumulation, contributes to dAdo toxicity.

  7. Resistance of an adenosine kinase-deficient human lymphoblastoid cell line to effects of deoxyadenosine on growth, S-adenosylhomocysteine hydrolase inactivation, and dATP accumulation.

    PubMed Central

    Hershfield, M S; Kredich, N M

    1980-01-01

    Accumulation of dATP derived from 2'-deoxyadenosine (dAdo), causing inhibition of ribonucleotide reductase and depletion of the other deoxynucleotide substrates required for DNA synthesis, has been suggested as the cause of the lymphopenia and immune defect in inheritable deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4). dAdo also inactivates the enzyme S-adenosylhomocysteine hydrolase (AdoHcyase; S-adenosyl-L-homocystein hydrolase EC 3.3.1.1) which is involved in the catabolism of S-adenosyl-L-homocysteine (AdoHcy), both a product and a potent inhibitor of S-adenosylmethionine-dependent transmethylation. We have tried to determine whether inactivation of AdoHcyase might also contribute to dAdo toxicity to adenosine deaminase-inhibited cells. dAdo rapidly inactivates intracellular AdoHcyase and causes the accumulation of AdoHcy in WI-L2 human B lymphoblastoid cells. Low concentrations of adenosine (Ado), which block binding of dAdo to purified AdoHcyase, prevented inactivation of intracellular AdoHcyase and also lessened the growth-inhibitory effect of dAdo. A mutant of this cell line which lacks Ado kinase and accumulated endogenously synthesized Ado was resistant to the effects of dAdo on both growth and AdoHcyase activity. The mutant also accumulated far less dATP from dAdo than did its parent and was resistant to the inhibitory effect of dAdo on DNA synthesis, indicating the Ado kinase is involved in dAdo phosphorylation in these cells. Combinations of deoxycytidine, thymidine, and deoxyguanosine that could prevent dATP-mediated depletion of deoxynucleotide pools but not AdoHcyase inactivation were less effective than Ado in preventing dAdo toxicity to normal lymphoblasts. Our results suggest that inactivation of AdoHcyase, as well as dATP accumulation, contributes to dAdo toxicity. PMID:6254019

  8. Membrane bound pyrophosphatase and P-type adenosine triphosphatase of Leishmania donovani as possible chemotherapeutic targets: similarities and differences in inhibitor sensitivities.

    PubMed

    Sen, S S; Bhuyan, N R; Lakshman, K; Roy, A K; Chakraborty, B; Bera, T

    2009-12-01

    The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H+-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N'-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F(1)F(o)-ATPase inhibitor quercetin. ATPase activity was stimulated by K+ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design. PMID:19961421

  9. 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.

  10. Adenosine in the tuberomammillary nucleus inhibits the histaminergic system via A1 receptors and promotes non-rapid eye movement sleep.

    PubMed

    Oishi, Yo; Huang, Zhi-Li; Fredholm, Bertil B; Urade, Yoshihiro; Hayaishi, Osamu

    2008-12-16

    Adenosine has been proposed to promote sleep through A(1) receptors (A(1)R's) and/or A(2A) receptors in the brain. We previously reported that A(2A) receptors mediate the sleep-promoting effect of prostaglandin D(2), an endogenous sleep-inducing substance, and that activation of these receptors induces sleep and blockade of them by caffeine results in wakefulness. On the other hand, A(1)R has been suggested to increase sleep by inhibition of the cholinergic region of the basal forebrain. However, the role and target sites of A(1)R in sleep-wake regulation remained controversial. In this study, immunohistochemistry revealed that A(1)R was expressed in histaminergic neurons of the rat tuberomammillary nucleus (TMN). In vivo microdialysis showed that the histamine release in the frontal cortex was decreased by microinjection into the TMN of N(6)-cyclopentyladenosine (CPA), an A(1)R agonist, adenosine or coformycin, an inhibitor of adenosine deaminase, which catabolizes adenosine to inosine. Bilateral injection of CPA into the rat TMN significantly increased the amount and the delta power density of non-rapid eye movement (non-REM; NREM) sleep but did not affect REM sleep. CPA-promoted sleep was observed in WT mice but not in KO mice for A(1)R or histamine H(1) receptor, indicating that the NREM sleep promoted by A(1)R-specific agonist depended on the histaminergic system. Furthermore, the bilateral injection of adenosine or coformycin into the rat TMN increased NREM sleep, which was completely abolished by coadministration of 1,3-dimethyl-8-cyclopenthylxanthine, a selective A(1)R antagonist. These results indicate that endogenous adenosine in the TMN suppresses the histaminergic system via A(1)R to promote NREM sleep.

  11. Regulation of glucosamine-6-phosphate deaminase synthesis in yeast.

    PubMed

    Singh, B; Datta, A

    1979-02-19

    A basal level of glucosamine-6-phosphate deaminase is detected in yeast cells grown on glucose. However, a burst of enzyme production occurs in the presence of N-acetylglucosamine in pathogenic Candida albicans and non-pathogenic Saccharomyces cervisiae. The enzyme synthesis stops and its concentration in the cells declines rapidly as soon as N-acetylglucosamine is removed from the medium. Experiments with RNA- and protein-synthesis inhibitors indicate that the appearance of new enzyme activity is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibition of DNA synthesis either by hydroxyurea or by mitomycin-C does not impair the synthesis of glucosamine-6-phosphate deaminase. PMID:369615

  12. Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

    PubMed Central

    Jones, R M; Jordan, P M

    1994-01-01

    Porphobilinogen deaminase (EC 4.3.1.8) has been purified to homogeneity (16,000-fold) from the plant Arabidopsis thaliana in yields of 8%. The deaminase is a monomer of M(r) 35,000, as shown by SDS/PAGE, and 31,000, using gel-filtration chromatography. The pure enzyme has a Vmax. of 4.5 mumol/h per mg and a Km of 17 +/- 4 microM. Determination of the pI and pH optimum revealed values of 5.2 and 8.0 respectively. The sequence of the N-terminus was found to be NH2-XVAVEQKTRTAI. The deaminase is heat-stable up to 70 degrees C and is inhibited by NH3 and hydroxylamine. The enzyme is inactivated by arginine-, histidine- and lysine-specific reagents. Incubation with the substrate analogue and suicide inhibitor, 2-bromoporphobilinogen, results in chain termination and in inactivation. Images Figure 1 PMID:8192681

  13. 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

  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. 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.

  16. 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

  17. 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

  18. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    PubMed

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants. PMID:25674802

  19. 1-Aminocyclopropane-1-Carboxylate Deaminase from Pseudomonas stutzeri A1501 Facilitates the Growth of Rice in the Presence of Salt or Heavy Metals.

    PubMed

    Han, Yunlei; Wang, Rui; Yang, Zhirong; Zhan, Yuhua; Ma, Yao; Ping, Shuzhen; Zhang, Liwen; Lin, Min; Yan, Yongliang

    2015-07-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase, which is encoded by some bacteria, can reduce the amount of ethylene, a root elongation inhibitor, and stimulate the growth of plants under various environmental stresses. The presence of ACC deaminase activity and the regulation of ACC in several rhizospheric bacteria have been reported. The nitrogen-fixing Pseudomonas stutzeri A1501 is capable of endophytic association with rice plants and promotes the growth of rice. However, the functional identification of ACC deaminase has not been performed. In this study, the proposed effect of ACC deaminase in P. stutzeri A1501 was investigated. Genome mining showed that P. stutzeri A1501 carries a single gene encoding ACC deaminase, designated acdS. The acdS mutant was devoid of ACC deaminase activity and was less resistant to NaCl and NiCl2 compared with the wild-type. Furthermore, inactivation of acdS greatly impaired its nitrogenase activity under salt stress conditions. It was also observed that mutation of the acdS gene led to loss of the ability to promote the growth of rice under salt or heavy metal stress. Taken together, this study illustrates the essential role of ACC deaminase, not only in enhancing the salt or heavy metal tolerance of bacteria but also in improving the growth of plants, and provides a theoretical basis for studying the interaction between plant growth-promoting rhizobacteria and plants.

  20. 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.

  1. Myoadenylate deaminase deficiency. Functional and metabolic abnormalities associated with disruption of the purine nucleotide cycle.

    PubMed Central

    Sabina, R L; Swain, J L; Olanow, C W; Bradley, W G; Fishbein, W N; DiMauro, S; Holmes, E W

    1984-01-01

    To assess the role of the purine nucleotide cycle in human skeletal muscle function, we evaluated 10 patients with AMP deaminase deficiency (myoadenylate deaminase deficiency; MDD). 4 MDD and 19 non-MDD controls participated in an exercise protocol. The latter group was composed of a patient cohort (n = 8) exhibiting a constellation of symptoms similar to those of the MDD patients, i.e., postexertional aches, cramps, and pains; as well as a cohort of normal, unconditioned volunteers (n = 11). The individuals with MDD fatigued after performing only 28% as much work as their non-MDD counterparts. Muscle biopsies were obtained from the four MDD patients and the eight non-MDD patients at rest and following exercise to the point of fatigue. Creatine phosphate content fell to a comparable extent in the MDD (69%) and non-MDD (52%) patients at the onset of fatigue. Following exercise the 34% decrease in ATP content of muscle from the non-MDD subjects was significantly greater than the 6% decrease in ATP noted in muscle from the MDD patients (P = 0.048). Only one of four MDD patients had a measurable drop in ATP compared with seven of eight non-MDD patients. At end-exercise the muscle content of inosine 5'-monophosphate (IMP), a product of AMP deaminase, was 13-fold greater in the non-MDD patients than that observed in the MDD group (P = 0.008). Adenosine content of muscle from the MDD patients increased 16-fold following exercise, while there was only a twofold increase in adenosine content of muscle from the non-MDD patients (P = 0.028). Those non-MDD patients in whom the decrease in ATP content following exercise was measurable exhibited a stoichiometric increase in IMP, and total purine content of the muscle did not change significantly. The one MDD patient in whom the decrease in ATP was measurable, did not exhibit a stoichiometric increase in IMP. Although the adenosine content increased 13-fold in this patient, only 48% of the ATP catabolized could be accounted for

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

    PubMed

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

    2016-11-15

    The impact of different concentrations of Na(+), K(+), Ca(2+), Mg(2+), Fe(2+), and Zn(2+) on the degradation of adenosine triphosphate (ATP) and the influence of these ions on the activity of adenosine monophosphate deaminase (AMP-deaminase) and acid phosphatase (ACP) in common carp fillets (in vivo) during 4°C storage was examined. The content of ATP, inosine monophosphate (IMP), and hypoxanthine (Hx), and the activity of AMP-deaminase and ACP were determined. Results indicated that the effects of different concentrations of six kinds of metal ions on AMP-deaminase and ACP were not the same. Na(+), K(+), Fe(2+), and Zn(2+) enhanced AMP-deaminase activity, which led to the rapid degradation of ATP and to the generation of a large quantity of IMP within a short time. Ca(2+) and Mg(2+) delayed the change in AMP-deaminase and ACP activity in carp and caused a further delay in the degradation of ATP. Fe(2+) and Zn(2+) inhibited ACP activity, which reduced the decomposition of IMP and the formation of Hx. PMID:27283700

  3. Rescue of the Orphan Enzyme Isoguanine Deaminase

    SciTech Connect

    D Hitchcock; A Fedorov; E Fedorov; L Dangott; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from Escherichia coli was shown to catalyze the deamination of isoguanine (2-oxoadenine) to xanthine. Isoguanine is an oxidation product of adenine in DNA that is mutagenic to the cell. The isoguanine deaminase activity in E. coli was partially purified by ammonium sulfate fractionation, gel filtration, and anion exchange chromatography. The active protein was identified by peptide mass fingerprint analysis as cytosine deaminase. The kinetic constants for the deamination of isoguanine at pH 7.7 are as follows: k{sub cat} = 49 s{sup -1}, K{sub m} = 72 {micro}M, and k{sub cat}/K{sub m} = 6.7 x 10{sup 5} M{sup -1} s{sup -1}. The kinetic constants for the deamination of cytosine are as follows: k{sub cat} = 45 s{sup -1}, K{sub m} = 302 {micro}M, and k{sub cat}/K{sub m} = 1.5 x 10{sup 5} M{sup -1} s{sup -1}. Under these reaction conditions, isoguanine is the better substrate for cytosine deaminase. The three-dimensional structure of CDA was determined with isoguanine in the active site.

  4. 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

  5. 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.

  6. 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.

  7. Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

    SciTech Connect

    Scott, A.I.; Roessner, C.A.; Stolowich, N.J.; Karuso, P.; Williams, H.J.; Grant, S.K.; Gonzalez, M.D.; Hoshino, T. )

    1988-10-18

    The active site of porphobilinogen (PBG){sup 1} deaminase from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-242, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA{sup {minus}} strain of E. coli the enzyme was enriched from (5-{sup 13}C)ALA and examined by {sup 1}H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked heat to tail and terminating in a CH{sub 2}-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-(2,11-{sup 13}C{sub 2})PBG reveals that the aminomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the {alpha}-free pyrrole. NMR spectroscopy of the ES{sub 2} complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the {alpha}-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

  8. Theophylline and adenosine modulate the inflammatory functions of the human neutrophil by exerting an opposing influence on the stimulus-induced increase in intracellular calcium

    SciTech Connect

    Schmeichel Morley, C.J.

    1988-01-01

    Based on evidence that endogenously-produced adenosine inhibited neutrophil responses, the influence of methylxanthine bronchodilators on neutrophil responses stimulated in vitro by n-formyl-methionyl-leucyl-phenylalanine (fMLP) was examined. At concentrations between 10/sup /minus/5/ M and 10/sup /minus/4/ M, theophylline potentiated lysosomal enzyme release by 30 to 50%, superoxide anion formation by 30 to 60%, and neutrophil aggregation. Theophylline at concentrations >10/sup /minus/4/ M inhibited the same responses by >90%. Adenosine deaminase mimicked, whereas adenosine reversed the theophylline potentiation. A potential role for calcium in the modulation of the neutrophil responses by theophylline and adenosine was explored. Theophylline enhanced by >150% the fMLP-stimulated increase in cytoplasmic calcium concentration ((Ca/sup 2 +/)/sub i/) at time points between 5 and 90 sec as measured by Fura-2. Adenosine deaminase induced a comparable enhancement, whereas 3 /times/ 10/sup /minus/7/ M adenosine and 10/sup /minus/7/ M N-ethylcarboxamideadenosine decreased the (Ca/sup 2 +/)/sub i/ in fMLP-stimulated neutrophils. Extracellular calcium was not required for the opposing influences of theophylline and adenosine and neither compound altered fMLP-stimulated /sup 45/Ca uptake at the early time points.

  9. Inhibitors

    MedlinePlus

    ... Community Counts Blood Safety Inhibitors Articles & Key Findings Free Materials Videos Starting the Conversation Playing it Safe A Look at Hemophilia Joint Range of Motion My Story Links to Other Websites ...

  10. Determination of 1-aminocycopropane-1-carboxylic acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings.

    PubMed

    Penrose, D M; Moffatt, B A; Glick, B R

    2001-01-01

    Previously, it was proposed that plant growth-promoting bacteria that possess the enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, can reduce the amount of ethylene produced by a plant and thereby promote root elongation. To test this model, canola seeds were imbibed in the presence of the chemical ethylene inhibitor, 2-aminoethoxyvinyl glycine (AVG), various strains of plant growth-promoting bacteria, and a psychrophilic bacterium containing an ACC deaminase gene on a broad host range plasmid. The extent of root elongation and levels of ACC, the immediate precursor of ethylene, were measured in the canola seedling roots. A modification of the Waters AccQ.Tag Amino Acid Analysis Method was used to quantify ACC in the root extracts. It was found that, in the presence of the ethylene inhibitor, AVG, or any one of several ACC deaminase-containing strains of bacteria, the growth of canola seedling roots was enhanced and the ACC levels in these roots were lowered.

  11. Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors

    SciTech Connect

    Lund, Kaleb C. Wallace, Kendall B.

    2008-01-01

    Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 {mu}M), AZT monophosphate (150 {mu}M), and 2',3'-dideoxycytidine (ddC; 1 {mu}M) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 {mu}M) and ddC (1 {mu}M). In the presence of succinate + cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-{gamma} activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug.

  12. [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.

  13. 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

  14. Beyond SHM and CSR: AID and related cytidine deaminases in the host response to viral infection.

    PubMed

    Rosenberg, Brad R; Papavasiliou, F Nina

    2007-01-01

    As the primary effector of immunoglobulin somatic hypermutation (SHM) and class switch recombination (CSR), activation-induced cytidine deaminase (AID) serves an important function in the adaptive immune response. Recent advances have demonstrated that AID and a group of closely related cytidine deaminases, the APOBEC3 proteins, also act in the innate host response to viral infection. Antiviral activity was first attributed to APOBEC3G as a potent inhibitor of HIV. It is now apparent that the targets of the APOBEC3 proteins extend beyond HIV, with family members acting against a wide variety of viruses as well as host-encoded retrotransposable genetic elements. Although it appears to function through a different mechanism, AID also possesses antiviral properties. Independent of its antibody diversification functions, AID protects against transformation by Abelson murine leukemia virus (Ab-MLV), an oncogenic retrovirus. Additionally, AID has been implicated in the host response to other pathogenic viruses. These emerging roles for the AID/APOBEC cytidine deaminases in viral infection suggest an intriguing evolutionary connection of innate and adaptive immune mechanisms.

  15. Yeast Cytosine Deaminase Mutants with Increased Thermostability Impart Sensitivity to 5-Fluorocytosine

    PubMed Central

    Stolworthy, Tiffany S.; Korkegian, Aaron M.; Willmon, Candice L.; Ardiani, Andressa; Cundiff, Jennifer; Stoddard, Barry L.; Black, Margaret E.

    2008-01-01

    SUMMARY Prodrug gene therapy (PGT) is a treatment strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme capable of converting a nontoxic prodrug into a potent cytotoxin. One of the most promising PGT enzymes is cytosine deaminase (CD), a microbial salvage enzyme that converts cytosine to uracil. CD also converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU), an inhibitor of DNA synthesis and RNA function. Over 150 studies of cytosine deaminase-mediated PGT applications have been reported since 2000, all using wild-type enzymes. However, various forms of cytosine deaminase are limited by inefficient turnover of 5FC and/or limited thermostability. In a previous study we stabilized and extended the half-life of yeast cytosine deaminase (yCD) by repacking of its hydrophobic core at several positions distant from the active site. Here we report that random mutagenesis of residues selected based on alignment with similar enzymes, followed by selection for enhanced sensitization to 5FC, also produces an enzyme variant (yCD-D92E) with elevated Tm values and increased activity half-life. The new mutation is located at the enzyme's dimer interface, indicating that independent mutational pathways can lead to an increase in the temperature that induces protein unfolding and aggregation in thermal denaturation experiments measured by circular dichroism spectroscopy, and an increase in the half-life of enzyme activity at physiological temperature, as well as more subtle effect on enzyme kinetics. Each independently derived set of mutations significantly improves the enzyme's performance in PGT assays both in cell culture and in animal models. PMID:18291415

  16. 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.

  17. 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.

  18. 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

  19. AID/APOBEC deaminases and cancer

    PubMed Central

    Rebhandl, Stefan; Huemer, Michael; Greil, Richard; Geisberger, Roland

    2015-01-01

    Mutations are the basis for evolution and the development of genetic diseases. Especially in cancer, somatic mutations in oncogenes and tumor suppressor genes alongside the occurrence of passenger mutations have been observed by recent deep-sequencing approaches. While mutations have long been considered random events induced by DNA-replication errors or by DNA damaging agents, genome sequencing led to the discovery of non-random mutation signatures in many human cancer. Common non-random mutations comprise DNA strand-biased mutation showers and mutations restricted to certain DNA motifs, which recently have become attributed to the activity of the AID/APOBEC family of DNA deaminases. Hence, APOBEC enzymes, which have evolved as key players in natural and adaptive immunity, have been proposed to contribute to cancer development and clonal evolution of cancer by inducing collateral genomic damage due to their DNA deaminating activity. This review focuses on how mutagenic events through AID/APOBEC deaminases may contribute to cancer development. PMID:26097867

  20. Deoxycytidine monophosphate deaminase in Acetabularia: properties and regulation in the early generative phase.

    PubMed

    Bannwarth, H; Ikehara, N; Schweiger, H G

    1982-06-01

    The occurrence of a dCMP deaminase in Acetabularia mediterranea has been demonstrated. The enzyme which is found in a particulate fraction is substantially stimulated by the addition of dCTP. The activity of the enzyme is increased at the beginning of the generative phase in nucleate as well as in anucleate cells. This regulation is due to de novo synthesis of the enzyme. By means of inhibitor studies, it has been shown that the enzyme is translated on 70S ribosomes of and coced for in cell organelles.

  1. Combining molecular dynamics and docking simulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv.

    PubMed

    Timmers, Luís Fernando Saraiva Macedo; Ducati, Rodrigo Gay; Sánchez-Quitian, Zilpa Adriana; Basso, Luiz Augusto; Santos, Diógenes Santiago; de Azevedo, Walter Filgueira

    2012-02-01

    Cytidine Deaminase (CD) is an evolutionarily conserved enzyme that participates in the pyrimidine salvage pathway recycling cytidine and deoxycytidine into uridine and deoxyuridine, respectively. Here, our goal is to apply computational techniques in the pursuit of potential inhibitors of Mycobacterium tuberculosis CD (MtCDA) enzyme activity. Molecular docking simulation was applied to find the possible hit compounds. Molecular dynamics simulations were also carried out to investigate the physically relevant motions involved in the protein-ligand recognition process, aiming at providing estimates for free energy of binding. The proposed approach was capable of identifying a potential inhibitor, which was experimentally confirmed by IC(50) evaluation. Our findings open up the possibility to extend this protocol to different databases in order to find new potential inhibitors for promising targets based on a rational drug design process.

  2. Bis(morpholino-1,3,5-triazine) derivatives: potent adenosine 5'-triphosphate competitive phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibitors: discovery of compound 26 (PKI-587), a highly efficacious dual inhibitor.

    PubMed

    Venkatesan, Aranapakam M; Dehnhardt, Christoph M; Delos Santos, Efren; Chen, Zecheng; Dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Khafizova, Gulnaz; Brooijmans, Natasja; Mallon, Robert; Hollander, Irwin; Feldberg, Larry; Lucas, Judy; Yu, Ker; Gibbons, James; Abraham, Robert T; Chaudhary, Inder; Mansour, Tarek S

    2010-03-25

    The PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism. It has been recognized recently that inhibiting this pathway might provide a viable therapy for cancer. A series of bis(morpholino-1,3,5-triazine) derivatives were prepared and optimized to provide the highly efficacious PI3K/mTOR inhibitor 1-(4-{[4-(dimethylamino)piperidin-1-yl]carbonyl}phenyl)-3-[4-(4,6-dimorpholin-4-yl-1,3,5-triazin-2-yl)phenyl]urea 26 (PKI-587). Compound 26 has shown excellent activity in vitro and in vivo, with antitumor efficacy in both subcutaneous and orthotopic xenograft tumor models when administered intravenously. The structure-activity relationships and the in vitro and in vivo activity of analogues in this series are described.

  3. 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.

  4. Pleural effusion: Role of pleural fluid cytology, adenosine deaminase level, and pleural biopsy in diagnosis

    PubMed Central

    Biswas, Biswajit; Sharma, Sudershan Kumar; Negi, Rameshwar Singh; Gupta, Neelam; Jaswal, Virender Mohan Singh; Niranjan, Narsimhalu

    2016-01-01

    Objective: The present study is designed to evaluate the role of pleural fluid analysis in diagnosing pleural diseases and to study the advantages and disadvantages of thoracocentasis and pleural biopsy. Materials and Methods: We prospectively included 66 consecutive indoor patients over a duration of 1 year. Pleural fluid was collected and cytological smears were made from the fluid. Plural biopsy was done in the same patient by Cope needle. Adequate pleural biopsy tissue yielding specific diagnosis was obtained in 47 (71.2%) cases. Results: Tuberculosis was the commonest nonneoplastic lesion followed by chronic nonspecific pleuritis comprising 60% and 33.3% of the nonneoplastic cases respectively and tuberculosis was predominantly diagnosed in the younger age group. Majority (70.8%) of malignancy cases were in the age group of >50-70. Adenocarcinoma was found to be the commonest (66.7%) malignant neoplasm in the pleurae followed by small-cell carcinoma (20.8%). Conclusion: Pleural biopsy is a useful and minimally invasive procedure. It is more sensitive and specific than pleural fluid smears.

  5. Evolution of the deaminase fold and multiple origins of eukaryotic editing and mutagenic nucleic acid deaminases from bacterial toxin systems

    PubMed Central

    Iyer, Lakshminarayan M.; Zhang, Dapeng; Rogozin, Igor B.; Aravind, L.

    2011-01-01

    The deaminase-like fold includes, in addition to nucleic acid/nucleotide deaminases, several catalytic domains such as the JAB domain, and others involved in nucleotide and ADP-ribose metabolism. Using sensitive sequence and structural comparison methods, we develop a comprehensive natural classification of the deaminase-like fold and show that its ancestral version was likely to operate on nucleotides or nucleic acids. Consequently, we present evidence that a specific group of JAB domains are likely to possess a DNA repair function, distinct from the previously known deubiquitinating peptidase activity. We also identified numerous previously unknown clades of nucleic acid deaminases. Using inference based on contextual information, we suggest that most of these clades are toxin domains of two distinct classes of bacterial toxin systems, namely polymorphic toxins implicated in bacterial interstrain competition and those that target distantly related cells. Genome context information suggests that these toxins might be delivered via diverse secretory systems, such as Type V, Type VI, PVC and a novel PrsW-like intramembrane peptidase-dependent mechanism. We propose that certain deaminase toxins might be deployed by diverse extracellular and intracellular pathogens as also endosymbionts as effectors targeting nucleic acids of host cells. Our analysis suggests that these toxin deaminases have been acquired by eukaryotes on several independent occasions and recruited as organellar or nucleo-cytoplasmic RNA modifiers, operating on tRNAs, mRNAs and short non-coding RNAs, and also as mutators of hyper-variable genes, viruses and selfish elements. This scenario potentially explains the origin of mutagenic AID/APOBEC-like deaminases, including novel versions from Caenorhabditis, Nematostella and diverse algae and a large class of fast-evolving fungal deaminases. These observations greatly expand the distribution of possible unidentified mutagenic processes catalyzed by

  6. 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

  7. 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.

  8. The catalase activity of diiron adenine deaminase.

    PubMed

    Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima; Kumaran, Desigan; Tichy, Shane E; Gheyi, Tarun; Zheng, Xiaojing; Bain, Kevin; Groshong, Chris; Emtage, Spencer; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Lindahl, Paul A; Raushel, Frank M

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn(2+) before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO(4). Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe(II) /Fe(II) ]-ADE catalyzed the conversion of H(2)O(2) to O(2) and H(2)O. The values of k(cat) and k(cat)/K(m) for the catalase activity are 200 s(-1) and 2.4 × 10(4) M(-1) s(-1), respectively. [Fe(II)/Fe(II)]-ADE underwent more than 100 turnovers with H(2)O(2) before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g(ave) = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H(2)O(2) by [Fe(II)/Fe(II)]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS. PMID:21998098

  9. The effects of N-acylhomoserine lactones, β-lactam antibiotics and adenosine on biofilm formation in the multi-β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7.

    PubMed

    Kusada, Hiroyuki; Hanada, Satoshi; Kamagata, Yoichi; Kimura, Nobutada

    2014-07-01

    Bacteria in the natural ecosystem frequently live as adherent communities called biofilms. Some chemical compounds are known to affect biofilm formation. We investigated the effect of exogenous small molecules, N-acylhomoserine lactones (AHLs), β-lactam antibiotics, and adenosine, on biofilm formation in the β-lactam antibiotic-resistant bacterium Acidovorax sp. strain MR-S7. Biofilm formation was induced by the addition of various types of AHL isomers and β-lactam antibiotics, whereas the addition of adenosine strongly interfered with the biofilm formation. A gene (macP) encoding adenosine deaminase (that converts adenosine to inosine controlling intracellular adenosine concentration) was successfully cloned from MR-S7 genome and heterologously expressed in Escherichia coli. The purified MacP protein clearly catalyzed the deamination of adenosine to produce inosine. A transcriptional analysis revealed that biofilm-inducing molecules, an AHL and a β-lactam antibiotic, strongly induced not only biofilm formation but also adenosine deaminase gene expression, suggesting that an elaborate gene regulation network for biofilm formation is present in the β-lactam antibiotic-resistant bacterium studied here.

  10. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    PubMed Central

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  11. 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.

  12. 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

  13. 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

  14. Purification and characterization of (-)(/sup 125/I)hydroxyphenylisopropyladenosine, an adenosine R-site agonist radioligand and theoretical analysis of mixed stereoisomer radioligand binding

    SciTech Connect

    Linden, J.

    1984-11-01

    (-)-N6-(R-4-Hydroxyphenylisopropyl)adenosine (HPIA) was iodinated with NaI and trace /sup 125/I. Mono- and diiodinated reaction products and the starting material were separated by high pressure liquid chromatography and the structures of the reaction products were verified by NMR. (-)-N6-(R-Phenylisopropyl)adenosine (PIA), IHPIA, and I2HPIA decreased rat atrial contractility with ED50 values of 24, 28, and 33 nM, respectively. The contractile effects of these compounds were competitively blocked by theophylline (KI . 7.9 microM), but were not affected by adenosine deaminase. IHPIA also inhibited (-)isoproterenol-stimulated cyclic AMP accumulation in adipocytes with an ED50 (10 nM) and to an extent (83%) nearly identical to PIA. (/sup 125/I)HPIA prepared using carrier-free /sup 125/I bound to adenosine receptors on membranes from rat cerebral cortex, adipocyte ghosts, and heart ventricles. Binding was inhibited stereospecifically by PIA and by other adenosine analogues and alkylxanthines. The KD of (/sup 125/I)HPIA determined kinetically using brain membranes at 21 degrees was 0.94 nM in good agreement with the equilibrium determination of 1.94 nM. The density of adenosine receptors in brain membranes was found to be 871 fmol/mg of protein. When normalized to protein, the density of receptors in heart membranes and adipocyte ghosts, respectively, was found to be 39- and 2.3-fold less than in brain membranes. It was concluded that (/sup 125/I)HPIA can be rapidly synthesized and purified, binds to adenosine R-sites and is an agonist radioligand resistant to adenosine deaminase. Computer modeling of the equilibrium binding resulting from the use of mixed stereoisomers of a radioligand indicates that the combined use of (-)(/sup 125/I)HPIA and (+)(/sup 125/I)HPIA would result in the generation of nonlinear Scatchard plots.

  15. 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

  16. Mycoplasma hyorhinis-encoded cytidine deaminase efficiently inactivates cytosine-based anticancer drugs

    PubMed Central

    Vande Voorde, Johan; Vervaeke, Peter; Liekens, Sandra; Balzarini, Jan

    2015-01-01

    Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency. PMID:26322268

  17. The catalase activity of diiron adenine deaminase

    SciTech Connect

    Kamat S. S.; Swaminathan S.; Holmes-Hampton, G. P.; Bagaria, A.; Kumaran, D.; Tichy, S. E.; Gheyi, T.; Zheng, X.; Bain, K.; Groshong, C.; Emtage, S.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-12-01

    Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn{sup 2+} before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO{sub 4}. Inductively coupled plasma mass spectrometry and Moessbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe{sup II}/Fe{sup II}]-ADE catalyzed the conversion of H{sub 2}O{sub 2} to O{sub 2} and H{sub 2}O. The values of k{sub cat} and k{sub cat}/K{sub m} for the catalase activity are 200 s{sup -1} and 2.4 x 10{sup 4} M{sup -1} s{sup -1}, respectively. [Fe{sup II}/Fe{sup II}]-ADE underwent more than 100 turnovers with H{sub 2}O{sub 2} before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g{sub ave} = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H{sub 2}O{sub 2} by [Fe{sup II}/Fe{sup II}]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. AMP deaminase histochemical activity and immunofluorescent isozyme localization in rat skeletal muscle

    NASA Technical Reports Server (NTRS)

    Thompson, J. L.; Sabina, R. L.; Ogasawara, N.; Riley, D. A.

    1992-01-01

    The cellular distribution of AMP deaminase (AMPda) isozymes was documented for rat soleus and plantaris muscles, utilizing immunofluorescence microscopy and immunoprecipitation methods. AMPda is a ubiquitous enzyme existing as three distinct isozymes, A, B and C, which were initially purified from skeletal muscle, liver (and kidney), and heart, respectively. AMPda-A is primarily concentrated subsarcolemmally and intermyofibrillarly within muscle cells, while isozymes B and C are concentrated within non-myofiber elements of muscle tissue. AMPda-B is principally associated with connective tissues surrounding neural elements and the muscle spindle capsule, and AMPda-C is predominantly associated with circulatory elements, such as arterial and venous walls, capillary endothelium, and red blood cells. These specific localizations, combined with documented differences in kinetic properties, suggest multiple functional roles for the AMPda isozymes or temporal segregation of similar AMPda functions. Linkage of the AMPda substrate with adenosine production pathways at the AMP level and the localization of isozyme-C in vascular tissue suggest a regulatory role in the microcirculation.

  4. Guanine deaminase functions as dihydropterin deaminase in the biosynthesis of aurodrosopterin, a minor red eye pigment of Drosophila.

    PubMed

    Kim, Jaekwang; Park, Sang Ick; Ahn, Chiyoung; Kim, Heuijong; Yim, Jeongbin

    2009-08-28

    Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 degrees C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (k(cat)/K(m)) for guanine (8.6 x 10(6) m(-1).s(-1)) was 860-fold higher than that for 7,8-dihydropterin (1.0 x 10(4) m(-1).s(-1)). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. PMID:19567870

  5. ACC deaminase activity in avirulent Agrobacterium tumefaciens D3.

    PubMed

    Hao, Youai; Charles, Trevor C; Glick, Bernard R

    2011-04-01

    Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58. PMID:21491979

  6. Preparation of a dual-enzyme co-immobilized capillary microreactor and simultaneous screening of multiple enzyme inhibitors by capillary electrophoresis.

    PubMed

    Lin, Pingtan; Zhao, Shulin; Lu, Xin; Ye, Fanggui; Wang, Hengshan

    2013-08-01

    A CE method based on a dual-enzyme co-immobilized capillary microreactor was developed for the simultaneous screening of multiple enzyme inhibitors. The capillary microreactor was prepared by co-immobilizing adenosine deaminase and xanthine oxidase on the inner wall at the inlet end of the separation capillary. The enzymes were first immobilized on gold nanoparticles, and the functionalized gold nanoparticles were then assembled on the inner wall at the inlet end of the separation capillary treated with polyethyleneimine. With the developed CE method, the substrates and products were baseline separated within 3 min. The activity of the immobilized enzyme can be directly detected by measuring the peak height of the products. A statistical parameter Z' factor was recommended for evaluation of the accuracy of a drug screening system. In the present study, it was calculated to be larger than 0.5, implying a good accuracy. Finally, screening a small compound library containing two known enzyme inhibitors and 20 natural extracts by the proposed method was demonstrated. The known inhibitors were identified, and some natural extracts were found to be positive for two-enzyme inhibition by the present method.

  7. 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.

  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. 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

  10. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression

    PubMed Central

    Ning, Chen; Wen, Jiaming; Zhang, Yujin; Dai, Yingbo; Wang, Wei; Zhang, Weiru; Qi, Lin; Grenz, Almut; Eltzschig, Holger K.; Blackburn, Michael R.; Kellems, Rodney E.; Xia, Yang

    2014-01-01

    Priapism is featured with prolonged and painful penile erection and is prevalent among males with sickle cell disease (SCD). The disorder is a dangerous urological and hematological emergency since it is associated with ischemic tissue damage and erectile disability. Here we report that phosphodiesterase-5 (PDE5) gene expression and PDE activity is significantly reduced in penile tissues of two independent priapic models: SCD mice and adenosine deaminase (ADA)-deficient mice. Moreover, using ADA enzyme therapy to reduce adenosine or a specific antagonist to block A2B adenosine receptor (ADORA2B) signaling, we successfully attenuated priapism in both ADA−/− and SCD mice by restoring penile PDE5 gene expression to normal levels. This finding led us to further discover that excess adenosine signaling via ADORA2B activation directly reduces PDE5 gene expression in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. Overall, we reveal that excess adenosine-mediated ADORA2B signaling underlies reduced penile PDE activity by decreasing PDE5 gene expression in a HIF-1α-dependent manner and provide new insight for the pathogenesis of priapism and novel therapies for the disease.—Ning, C., Wen, J., Zhang, Y., Dai, Y., Wang, W., Zhang, W., Qi, L., Grenz, A., Eltzschig, H. K., Blackburn, M. R., Kellems, R. E., Xia, Y. Excess adenosine A2B receptor signaling contributes to priapism through HIF-1α mediated reduction of PDE5 gene expression. PMID:24614760

  11. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    Kamat, S.S.; Swaminathan, S.; Bagaria, A.; Kumaran, D.; Holmes-Hampton, G. P.; Fan, H.; Sali, A.; Sauder, J. M.; Burley, S. K.; Lindahl, P. A.; Raushel, F. M.

    2011-03-22

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with kcat and kcat/Km values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction mechanism and the

  12. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

    S Kamat; A Bagaria; D Kumaran; G Holmes-Hampton; H Fan; A Sali; J Sauder; S Burley; P Lindahl; et. al.

    2011-12-31

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (k{sub cat} = 2.0 s{sup -1}; k{sub cat}/K{sub m} = 2.5 x 10{sup 3} M{sup -1} s{sup -1}). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn{sup 2+} prior to induction, the purified enzyme was substantially more active for the deamination of adenine with k{sub cat} and k{sub cat}/K{sub m} values of 200 s{sup -1} and 5 x 10{sup 5} M{sup -1} s{sup -1}, respectively. The apoenzyme was prepared and reconstituted with Fe{sup 2+}, Zn{sup 2+}, or Mn{sup 2+}. In each case, two enzyme equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member of the deaminase subfamily of the amidohydrolase superfamily to utilize a binuclear metal center for the catalysis of a deamination reaction. [Fe{sup II}/Fe{sup II}]-ADE was oxidized to [Fe{sup III}/Fe{sup III}]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [Fe{sup III}/Fe{sup III}]-ADE with dithionite restored the deaminase activity, and thus, the diferrous form of the enzyme is essential for catalytic activity. No evidence of spin coupling between metal ions was evident by electron paramagnetic resonance or Moessbauer spectroscopy. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 {angstrom} resolution, and adenine was modeled into the active site on the basis of homology to other members of the amidohydrolase superfamily. On the basis of the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH-rate profiles, and solvent viscosity were utilized to propose a chemical reaction

  13. 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

  14. Highly Potent and Selective Ectonucleotide Pyrophosphatase/Phosphodiesterase I Inhibitors Based on an Adenosine 5′-(α or γ)- Thio-(α,β- or β,γ)-methylenetriphosphate Scaffold

    PubMed Central

    Nadel, Yael; Lecka, Joanna; Gilad, Yocheved; Ben-David, Gal; Förster, Daniel; Reiser, Georg; Kenigsberg, Sarah; Camden, Jean; Weisman, Gary A.; Senderowitz, Hanoch; Sévigny, Jean; Fischer, Bilha

    2015-01-01

    Aberrant nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is associated with chondrocalcinosis, osteoarthritis, and type 2 diabetes. The potential of NPP1 inhibitors as therapeutic agents, and the scarceness of their structure–activity relationship, encouraged us to develop new NPP1 inhibitors. Specifically, we synthesized ATP-α-thio-β,γ- CH2 (1), ATP-α-thio-β,γ-CCl2 (2), ATP-α-CH2-γ-thio (3), and 8-SH-ATP (4) and established their resistance to hydrolysis by NPP1,3 and NTPDase1,2,3,8 (<5% hydrolysis) (NTPDase = ectonucleoside triphosphate diphosphohydrolase). Analogues 1–3 at 100 μM inhibited thymidine 5′-monophosphate p-nitrophenyl ester hydrolysis by NPP1 and NPP3 by >90% and 23–43%, respectively, and only slightly affected (0–40%) hydrolysis of ATP by NTPDase1,2,3,8. Analogue 3 is the most potent NPP1 inhibitor currently known, Ki = 20 nM and IC50 = 0.39 μM. Analogue 2a is a selective NPP1 inhibitor with Ki = 685 nM and IC50 = 0.57 μM. Analogues 1–3 were found mostly to be nonagonists of P2Y1/P2Y2/P2Y11 receptors. Docking analogues 1–3 into the NPP1 model suggested that activity correlates with the number of H-bonds with binding site residues. In conclusion, we propose analogues 2a and 3 as highly promising NPP1 inhibitors. PMID:24846781

  15. 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.

  16. 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.

  17. 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

  18. The current structural and functional understanding of APOBEC deaminases.

    PubMed

    Bransteitter, Ronda; Prochnow, Courtney; Chen, Xiaojiang S

    2009-10-01

    The apolipoprotein B mRNA-editing enzyme catalytic polypeptide (APOBEC) family of cytidine deaminases has emerged as an intensively studied field as a result of their important biological functions. These enzymes are involved in lipid metabolism, antibody diversification, and the inhibition of retrotransposons, retroviruses, and some DNA viruses. The APOBEC proteins function in these roles by deaminating single-stranded (ss) DNA or RNA. There are two high-resolution crystal structures available for the APOBEC family, Apo2 and the C-terminal catalytic domain (CD2) of Apo3G or Apo3G-CD2 [Holden et al. (Nature 456:121-124, 2008); Prochnow et al. (Nature 445:447-451, 2007)]. Additionally, the structure of Apo3G-CD2 has also been determined using NMR [Chen et al. (Nature 452:116-119, 2008); Furukawa et al. (EMBO J 28:440-451, 2009); Harjes et al. (J Mol Biol, 2009)]. A detailed structural analysis of the APOBEC proteins and a comparison to other zinc-coordinating deaminases can facilitate our understanding of how APOBEC proteins bind nucleic acids, recognize substrates, and form oligomers. Here, we review the recent development of structural and functional studies that apply to Apo3G as well as the APOBEC deaminase family. PMID:19547914

  19. 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.

  20. The effect of calcium removal on the suppression by adenosine of epileptiform activity in the hippocampus: demonstration of desensitization.

    PubMed Central

    Hosseinzadeh, H.; Stone, T. W.

    1994-01-01

    1. Previous work has suggested that presynaptic effects of adenosine may be dependent on divalent cations. The present study was undertaken to determine whether a similar requirement existed at postsynaptic sites. 2. Extracellular recordings were made in the CA1 pyramidal cell layer of rat hippocampal slices following orthodromic stimulation of Schaffer collateral fibres in stratum radiatum or antidromic stimulation of the alveus. In antidromic stimulation experiments, CaCl2 was omitted (calcium-free medium) or reduced to 0.24 mM (low calcium medium) and in some experiments MgSO4 was increased to 2 mM. Kynurenic acid at concentrations of 1 and 5 mM in calcium-free medium and 1 mM in low calcium medium had no effect on secondary spike size. 3. Adenosine and baclofen induced a concentration-dependent reduction in the amplitude of orthodromic potentials with maximum effects at 20 and 5 microM respectively. 4. In nominally calcium-free medium, bursts of multiple population spikes were obtained in response to antidromic stimulation. Adenosine had little effect in reducing the secondary spike amplitude. At high concentration (2 mM) an initial depression was seen which declined within 3-5 min. 5. Sensitivity to adenosine was restored in low calcium medium or by raising magnesium. Although raising the divalent cation concentration increased the inhibitory effect of adenosine, desensitization was still seen. 6. 2-Chloroadenosine (100-500 microM) and R-PIA (50 microM), which are not substrates for either the nucleoside transporters or adenosine deaminase, were inactive in the absence of calcium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8032657

  1. Vitamins and monothiols efficacy in the restoration of adenosine nucleotide degradation enzymes altered during methylmercury intoxication

    SciTech Connect

    Sood, P.P.; Bapu, C.; Vijayalakshmi, K.

    1995-12-31

    Male albino mice were intoxicated with a daily dose of 1 mg/kg of methylmercury chloride (MMC) for 7 days, and were treated thereafter with glutathione, N-acetyl-DL-homocysteine thiolactone, vitamin B complex, and vitamin E, either alone or in combinations for the next 7 days. The animals were sacrificed on the eighth day, with the exception of one group that was kept without toxic exposure for an additional 7 days and sacrificed on the fifteenth day. Brain, spinal cord, kidney, and liver of the animals were examined for changes in adenosine deaminase and 5{prime} nucleotidase. We found a severe inhibition of these enzymes during MMC intoxication and significant recovery during monothiols and vitamins administration, indicating the effectiveness of these agents in methylmercury detoxication. 26 refs., 2 figs.

  2. 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

  3. The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

    PubMed Central

    Cohen, F. R.; Lazareno, S.; Birdsall, N. J.

    1996-01-01

    1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H

  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. 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.

  6. Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes

    PubMed Central

    Liu, Chengqian; Skaldin, Maksym; Wu, Chengxiang; Lu, Yuanan; Zavialov, Andrey V.

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens. PMID:27510152

  7. Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.

    PubMed

    Liu, Chengqian; Skaldin, Maksym; Wu, Chengxiang; Lu, Yuanan; Zavialov, Andrey V

    2016-01-01

    Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens. PMID:27510152

  8. 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).

  9. 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.

  10. 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

  11. Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements.

    PubMed

    Cisneros, David A; Montero-Morán, Gabriela M; Lara-González, Samuel; Calcagno, Mario L

    2004-01-01

    Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.

  12. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    EPA Science Inventory

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

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

    PubMed Central

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

    1999-01-01

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

  14. Characterization of the Porphobilinogen Deaminase Deficiency in Acute Intermittent Porphyria

    PubMed Central

    Anderson, Peter M.; Reddy, Raman M.; Anderson, Karl E.; Desnick, Robert J.

    1981-01-01

    The molecular pathology of the porphobilinogen (PBG)-deaminase deficiency in heterozygotes for acute intermittent porphyria (AIP) was investigated by means of biochemical and immunologic techniques. The stable enzyme-substrate intermediates (A, B, C, D, and E) of PBG-deaminase were separated by anion-exchange chromatography of erythrocyte lysates from heterozygotes for AIP and normal individuals. In normal lysates, the intermediates eluted in a characteristic pattern with decreasing amounts of activity (A > B > C > D > E), the combined A and B intermediates representing >75% of total recovered activity. In contrast, two different profiles were observed in lysates from heterozygotes for AIP. In most heterozygotes, the elution profile was similar to that of normal individuals, but each intermediate was reduced ∼50%. A second profile in which the C intermediate had disproportionately higher activity than the A or B intermediates was observed in asymptomatic heterozygotes with high urinary levels of PBG (>5 μg/ml) as well as in heterozygotes during acute attacks. These findings suggested that the C intermediate (the dipyrrole-enzyme intermediate) may be rate limiting in the stepwise conversion of the monopyrrole, PBG, to the linear tetrapyrrole, hydroxymethylbilane. To investigate further the nature of the enzymatic defect in AIP, sensitive immunotitration and immunoelectrophoretic assays were developed with the aid of a rabbit anti-human PBG-deaminase IgG preparation produced against the homogeneous enzyme. Equal amounts of erythrocyte lysate activity from 32 heterozygotes for AIP from 22 unrelated families and 35 normal individuals were immunoelectrophoresed. There were no detectable differences in the amounts of cross-reactive immunologic material (CRIM) in lysates from the normal individuals and 25 heterozygotes from 21 of the 22 unrelated families with AIP. In contrast, when equal enzymatic activities were coimmunoelectrophoresed, all seven heterozygotes from

  15. Regulation of Activation Induced Deaminase (AID) by Estrogen.

    PubMed

    Pauklin, Siim

    2016-01-01

    Regulation of Activation Induced Deaminase (AID) by the hormone estrogen has important implications for understanding adaptive immune responses as well as the involvement of AID in autoimmune diseases and tumorigenesis. This chapter describes the general laboratory techniques for analyzing AID expression and activity induced by estrogen, focusing on the isolation and preparation of cells for hormone treatment and the subsequent analysis of AID responsiveness to estrogen at the RNA level and for determining the regulation of AID activity via estrogen by analyzing Ig switch circle transcripts and mutations in switch region loci.

  16. Biosynthesis of riboflavin. Characterization of the product of the deaminase.

    PubMed

    Nielsen, P; Bacher, A

    1981-12-15

    The 2'5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate deaminase was partially purified from cell extracts of Candida guilliermondii ATCC 9058. The enzyme requires Mg2+ for activity. Maximal activity was observed at pH 7,3. The enzyme converts its substrate, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate, to 2,5-diamino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate. This labile compound was treated with diacetyl and the resulting 6,7-dimethyl-8-ribityllumazine 5'-phosphate was identified by comparison with a synthetic sample. PMID:7317443

  17. Homozygosity for a novel adenosine deaminase (ADA) nonsense mutation (Q3>X) in a child with severe combined immunodeficiency (SCID)

    SciTech Connect

    Santisteban, I.; Arrendondo-Vega, F.X.; Kelly, S. |

    1994-09-01

    A Somali girl was diagnosed with ADA-deficient SCID at 7 mo; she responded well to PEG-ADA replacement and is now 3.3 yr old. ADA mRNA was undetectable (Northern) in her cultured T cells, but was present in T cells of her parents and two sibs. All PCR-amplified exon 1 genomic clones from the patient had a C>T transition at bp 7 relative to the start of translation, replacing Gln at codon 3 (AGA) with a termination codon (TGA, Q3>X). Patient cDNA (prepared by RT-PCR with a 5{prime} primer that covered codons 1-7) had a previously described polymorphism, K80>R, but was otherwise normal, indicating that no other coding mutations were present. A predicted new genomic BfaI restriction site was used to establish her homozygosity for Q3>X and to analyze genotypes of family members. We also analyzed the segregation of a variable Alu polyA-associated TAAA repeat (AluVpA) situated 5{prime} of the ADA gene. Three different AluVpA alleles were found, one of which was only present in the father and was not associated with his Q3>X allele. Because the father`s RBCs had only {approximately}15% of normal ADA activity, we analyzed his ADA cDNA. We found a G>A transition at bp 425 that substitutes Gln for Arg142, a solvent-accessible residue, and eliminates a BsmAI site in exon 5. ADA activity of the R142>Q in vitro translation product was 20-25% of wild type ADA translation product, suggesting that R142>Q is a new {open_quote}partial{close_quote} ADA deficiency mutation. As expected, Q3>X mRNA did not yield a detectable in vitro translation product. We conclude that the patient`s father is a compound heterozygote carrying the ADA Q3>X/R142>Q genotype. {open_quote}Partial{close_quote} ADA deficiency unassociated with immunodeficiency is relatively common in individuals of African descent. The present findings and previous observations suggest that {open_quote}partial{close_quote} ADA deficiency may have had an evolutionary advantage.

  18. Assignment of the DPP4 gene encoding adenosine deaminase binding protein (CD26/dipeptidylpeptidase IV) to 2q23

    SciTech Connect

    Mathew, S.; Morrison, M.E.; Murty, V.V.V.S.

    1994-07-01

    FISH was performed on chromosome preparations obtained from phytohemagglutinin-stimulated human blood lymphocytes. cDNA encoding ADAbp was isolated from the SK-RC-28 human renal cell carcinomas cell line using PCR technique and was cloned in pSVK3 plasmid for use as a probe. The PCR primers were constructed from the known nucleotide sequence of CD26, and the cDNA product was extracted from nucleotides 1 to 2344. The vector containing the probe was labeled by nick-translation with biotin-11-dUTP. Hybridization to chromosome spreads, washings, detection with FITC-conjugated avidin, selection and photography of metaphases, analysis of signals, and banding were performed according to the described method.

  19. New Insights into 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Phylogeny, Evolution and Ecological Significance

    PubMed Central

    Nascimento, Francisco X.; Rossi, Márcio J.; Soares, Cláudio R. F. S.; McConkey, Brendan J.; Glick, Bernard R.

    2014-01-01

    The main objective of this work is the study of the phylogeny, evolution and ecological importance of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, the activity of which represents one of the most important and studied mechanisms used by plant growth–promoting microorganisms. The ACC deaminase gene and its regulatory elements presence in completely sequenced organisms was verified by multiple searches in diverse databases, and based on the data obtained a comprehensive analysis was conducted. Strain habitat, origin and ACC deaminase activity were taken into account when analyzing the results. In order to unveil ACC deaminase origin, evolution and relationships with other closely related pyridoxal phosphate (PLP) dependent enzymes a phylogenetic analysis was also performed. The data obtained show that ACC deaminase is mostly prevalent in some Bacteria, Fungi and members of Stramenopiles. Contrary to previous reports, we show that ACC deaminase genes are predominantly vertically inherited in various bacterial and fungal classes. Still, results suggest a considerable degree of horizontal gene transfer events, including interkingdom transfer events. A model for ACC deaminase origin and evolution is also proposed. This study also confirms the previous reports suggesting that the Lrp-like regulatory protein AcdR is a common mechanism regulating ACC deaminase expression in Proteobacteria, however, we also show that other regulatory mechanisms may be present in some Proteobacteria and other bacterial phyla. In this study we provide a more complete view of the role for ACC deaminase than was previously available. The results show that ACC deaminase may not only be related to plant growth promotion abilities, but may also play multiple roles in microorganism's developmental processes. Hence, exploring the origin and functioning of this enzyme may be the key in a variety of important agricultural and biotechnological applications. PMID:24905353

  20. 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

  1. 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.

  2. Role of A2B Adenosine Receptors in Regulation of Paracrine Functions of Stem Cell Antigen 1-Positive Cardiac Stromal Cells

    PubMed Central

    Ryzhov, Sergey; Goldstein, Anna E.; Novitskiy, Sergey V.; Blackburn, Michael R.; Biaggioni, Italo

    2012-01-01

    The existence of multipotent cardiac stromal cells expressing stem cell antigen (Sca)-1 has been reported, and their proangiogenic properties have been demonstrated in myocardial infarction models. In this study, we tested the hypothesis that stimulation of adenosine receptors on cardiac Sca-1+ cells up-regulates their secretion of proangiogenic factors. We found that Sca-1 is expressed in subsets of mouse cardiac stromal CD31− and endothelial CD31+ cells. The population of Sca-1+CD31+ endothelial cells was significantly reduced, whereas the population of Sca-1+CD31− stromal cells was increased 1 week after myocardial infarction, indicating their relative functional importance in this pathophysiological process. An increase in adenosine levels in adenosine deaminase-deficient mice in vivo significantly augmented vascular endothelial growth factor (VEGF) production in cardiac Sca-1+CD31− stromal cells but not in Sca-1+CD31+ endothelial cells. We found that mouse cardiac Sca-1+CD31− stromal cells predominantly express mRNA encoding A2B adenosine receptors. Stimulation of adenosine receptors significantly increased interleukin (IL)-6, CXCL1 (a mouse ortholog of human IL-8), and VEGF release from these cells. Using conditionally immortalized Sca-1+CD31− stromal cells obtained from wild-type and A2B receptor knockout mouse hearts, we demonstrated that A2B receptors are essential for adenosine-dependent up-regulation of their paracrine functions. We found that the human heart also harbors a population of stromal cells similar to the mouse cardiac Sca-1+CD31− stromal cells that increase release of IL-6, IL-8, and VEGF in response to A2B receptor stimulation. Thus, our study identified A2B adenosine receptors on cardiac stromal cells as potential targets for up-regulation of proangiogenic factors in the ischemic heart. PMID:22431204

  3. Homodimerization of adenosine A₁ receptors in brain cortex explains the biphasic effects of caffeine.

    PubMed

    Gracia, Eduard; Moreno, Estefania; Cortés, Antoni; Lluís, Carme; Mallol, Josefa; McCormick, Peter J; Canela, Enric I; Casadó, Vicent

    2013-08-01

    Using bioluminescence resonance energy transfer and proximity ligation assays, we obtained the first direct evidence that adenosine A₁ receptors (A₁Rs) form homomers not only in cell cultures but also in brain cortex. By radioligand binding experiments in the absence or in the presence of the A₁Rs allosteric modulator, adenosine deaminase, and by using the two-state dimer receptor model to fit binding data, we demonstrated that the protomer-protomer interactions in the A₁R homomers account for some of the pharmacological characteristics of agonist and antagonist binding to A₁Rs. These pharmacological properties include the appearance of cooperativity in agonist binding, the change from a biphasic saturation curve to a monophasic curve in self-competition experiments and the molecular cross-talk detected when two different specific molecules bind to the receptor. In this last case, we discovered that caffeine binding to one protomer increases the agonist affinity for the other protomer in the A₁R homomer, a pharmacological characteristic that correlates with the low caffeine concentrations-induced activation of agonist-promoted A₁R signaling. This pharmacological property can explain the biphasic effects reported at low and high concentration of caffeine on locomotor activity.

  4. 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

  5. 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.

  6. Cytidine deaminases from B. subtilis and E. coli: compensating effects of changing zinc coordination and quaternary structure.

    PubMed

    Carlow, D C; Carter, C W; Mejlhede, N; Neuhard, J; Wolfenden, R

    1999-09-21

    Cytidine deaminase from E. coli is a dimer of identical subunits (M(r) = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtilis is a tetramer of identical subunits (M(r) = 14 800). After purification from an overexpressing strain, the enzyme from B. subtilis is found to contain a single atom of zinc per enzyme subunit by flame atomic absorption spectroscopy. Fluorescence titration indicates that each of the four subunits contains a binding site for the transition state analogue inhibitor 5-fluoro-3,4-dihydrouridine. A region of amino acid sequence homology, containing residues that are involved in zinc coordination in the enzyme from E. coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B. H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468]. This pattern of zinc coordination appears to be novel for a hydrolytic enzyme, and might be expected to reduce the reactivity of the active site substantially compared with that of the enzyme from E. coli (His-102, Cys-129, and Cys-132). Instead, the B. subtilis and E. coli enzymes are found to be similar in their activities, and also in their relative binding affinities for a series of structurally related inhibitors with binding affinities that span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence alignments, together with model building, suggest one possible mechanism of compensation.

  7. Inhibitors of phosphodiesterases PDE2, PDE3, and PDE4 do not increase the sinoatrial tachycardia of noradrenaline and prostaglandin PGE₁ in mice.

    PubMed

    Galindo-Tovar, Alejandro; Vargas, María Luisa; Kaumann, Alberto J

    2016-02-01

    Phosphodiesterases PDE2, PDE3, and PDE4 are expressed in murine sinoatrial cells. PDE3 and/or PDE4 reduce heart rate but apparently do not influence the tachycardia mediated through sinoatrial β1- and β2-adrenoceptors despite the high content of sinoatrial cAMP. The function of PDE2 is, however, uncertain. Prostaglandin PGE1 elicits sinoatrial tachycardia through EP receptors, but the control by phosphodiesterases is unknown. We investigated on spontaneously beating right atria of mice the effects of the PDE2 inhibitors Bay 60-7550 and EHNA on basal beating and the tachycardia produced by noradrenaline (3 nM) and PGE1 (1 μM). Bay 60-7550 (1 μM), but not EHNA (10 μM), increased basal sinoatrial beating. EHNA also failed to produce tachycardia in the presence of the adenosine deaminase inhibitor 2'-deoxycoformycin (10 μM), remaining inconclusive whether PDE2 reduces basal sinoatrial beating. Rolipram (10 μM) and cilostamide (300 nM) caused moderate tachycardia. The tachycardia evoked by Bay 60-7550 was similar in the absence and presence of rolipram. Noradrenaline elicited stable tachycardia that was not increased by Bay 60-7550. A stable tachycardia caused by PGE1 was not increased by the inhibitors of PDE2, PDE3, and PDE4. Unlike PDE3 and PDE4 which reduce murine basal sinoatrial beating, a possible effect of PDE2 needs further research. The stable tachycardia produced by noradrenaline and PGE1, together with the lack potentiation by the inhibitors of PDE2, PDE3, and PDE4, suggests that cAMP generated at the receptor compartments is hardly hydrolyzed by these phophodiesterases. Evidence from human volunteers is consistent with this proposal.

  8. 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

  9. 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.

  10. Processive DNA Demethylation via DNA Deaminase-Induced Lesion Resolution

    PubMed Central

    Morgan, Hugh; Incorvaia, Elisabetta; Rangam, Gopinath; Dean, Wendy; Santos, Fatima; Reik, Wolf; Petersen-Mahrt, Svend K.

    2014-01-01

    Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated. PMID:25025377

  11. AMP-deaminase from thymus of patients with myasthenia gravis.

    PubMed

    Rybakowska, I; Szydłowska, M; Szrok, S; Bakuła, S; Kaletha, K

    2015-01-01

    Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes.

  12. Photodynamic therapy-driven induction of suicide cytosine deaminase gene.

    PubMed

    Bil, Jacek; Wlodarski, Pawel; Winiarska, Magdalena; Kurzaj, Zuzanna; Issat, Tadeusz; Jozkowicz, Alicja; Wegiel, Barbara; Dulak, Jozef; Golab, Jakub

    2010-04-28

    Photodynamic therapy (PDT) of tumors is associated with induction of hypoxia that results in activation of hypoxia-inducible factors (HIFs). Several observations indicate that increased HIFs transcriptional activity in tumor cells is associated with cytoprotective responses that limit cytotoxic effectiveness of PDT. Therefore, we decided to examine whether this cytoprotective mechanism could be intentionally used for designing more efficient tumor cell cytotoxicity. To this end we transfected tumor cells with a plasmid vector carrying a suicide cytosine deaminase gene driven by a promoter containing hypoxia response elements (HRE). The presence of such a genetic molecular beacon rendered tumor cells sensitive to cytotoxic effects of a non-toxic prodrug 5-fluorocytosine (5-FC). The results of this study provides a proof of concept that inducible cytoprotective mechanisms can be exploited to render tumor cells more susceptible to cytotoxic effects of prodrugs activated by products of suicide genes.

  13. 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

  14. 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

  15. 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

  16. 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

  17. The pentose moiety of adenosine and inosine is an important energy source for the fermented-meat starter culture Lactobacillus sakei CTC 494.

    PubMed

    Rimaux, T; Vrancken, G; Vuylsteke, B; De Vuyst, L; Leroy, F

    2011-09-01

    The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.

  18. The Pentose Moiety of Adenosine and Inosine Is an Important Energy Source for the Fermented-Meat Starter Culture Lactobacillus sakei CTC 494▿

    PubMed Central

    Rimaux, T.; Vrancken, G.; Vuylsteke, B.; De Vuyst, L.; Leroy, F.

    2011-01-01

    The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment

  19. 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

  20. 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

  1. Cytidine deaminase activity in synovial fluid of patients with rheumatoid arthritis: relation to lactoferrin, acidosis, and cartilage proteoglycan release.

    PubMed Central

    Månsson, B; Geborek, P; Saxne, T; Björnsson, S

    1990-01-01

    It is claimed that cytidine deaminase activity reflects local granulocyte turnover or activity in the synovial fluid of patients with rheumatoid arthritis, but cytidine deaminase is not a granulocyte specific enzyme. Lactoferrin is a granulocyte specific protein that is released from the secondary granulae during activation. We measured cytidine deaminase activity and lactoferrin concentrations in 33 rheumatic synovial fluid samples. Cytidine deaminase activity and lactoferrin concentrations correlated closely, indicating that both analyses reflect similar events in the joint-that is, result in their release from granulocytes. Cytidine deaminase activity and granulocyte concentrations correlated less closely, suggesting that there are additional factors besides the cell number which contribute to this release. Joint acidosis may be one such factor, as pH and cytidine deaminase activity correlated inversely. There was no association with synovial fluid proteoglycan concentrations, a marker of cartilage degradation. PMID:2396864

  2. 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.

  3. Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed

    Belimov, A A; Safronova, V I; Sergeyeva, T A; Egorova, T N; Matveyeva, V A; Tsyganov, V E; Borisov, A Y; Tikhonovich, I A; Kluge, C; Preisfeld, A; Dietz, K J; Stepanok, V V

    2001-07-01

    Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals. The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. by determination of 16S rRNA gene sequences. The root elongation of Indian mustard and rape (Brassica napus var. oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively. The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 microM CdCl2 in the nutrient solution. The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis. A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil. The biomass of pea cv. Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture. The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions. The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions.

  4. Characterization of plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed

    Belimov, A A; Safronova, V I; Sergeyeva, T A; Egorova, T N; Matveyeva, V A; Tsyganov, V E; Borisov, A Y; Tikhonovich, I A; Kluge, C; Preisfeld, A; Dietz, K J; Stepanok, V V

    2001-07-01

    Fifteen bacterial strains containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase were isolated from the rhizoplane of pea (Pisum sativum L.) and Indian mustard (Brassica juncea L.) grown in different soils and a long-standing sewage sludge contaminated with heavy metals. The isolated strains were characterized and assigned to various genera and species, such as Pseudomonas brassicacearum, Pseudomonas marginalis, Pseudomonas oryzihabitans, Pseudomonas putida, Pseudomonas sp., Alcaligenes xylosoxidans, Alcaligenes sp., Variovorax paradoxus, Bacillus pumilus, and Rhodococcus sp. by determination of 16S rRNA gene sequences. The root elongation of Indian mustard and rape (Brassica napus var. oleifera L.) germinating seedlings was stimulated by inoculation with 8 and 13 isolated strains, respectively. The bacteria were tolerant to cadmium toxicity and stimulated root elongation of rape seedlings in the presence of 300 microM CdCl2 in the nutrient solution. The effect of ACC-utilising bacteria on root elongation correlated with the impact of aminoethoxyvinylglycine and silver ions, chemical inhibitors of ethylene biosynthesis. A significant improvement in the growth of rape caused by inoculation with certain selected strains was also observed in pot experiments, when the plants were cultivated in cadmium-supplemented soil. The biomass of pea cv. Sparkle and its ethylene sensitive mutant E2 (sym5), in particular, was increased through inoculation with certain strains of ACC-utilising bacteria in pot experiments in quartz sand culture. The beneficial effect of the bacteria on plant growth varied significantly depending on individual bacterial strains, plant genotype, and growth conditions. The results suggest that plant growth promoting rhizobacteria containing ACC deaminase are present in various soils and offer promise as a bacterial inoculum for improvement of plant growth, particularly under unfavourable environmental conditions. PMID:11547884

  5. Discovery and Structure Determination of the Orphan Enzyme Isoxanthopterin Deaminase

    SciTech Connect

    Hall, R.S.; Swaminathan, S.; Agarwal, R.; Hitchcock, D.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a (gi|44585104) and NYSGXRC-9236b (gi|44611670), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 {angstrom} resolution (Protein Data Bank entry 2PAJ). This protein folds as a distorted ({beta}/{alpha}){sub 8} barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s{sup -1}, 8.0 {micro}M, and 1.3 x 10{sup 5} M{sup -1} s{sup -1} (k{sub cat}, K{sub m}, and k{sub cat}/K{sub m}, respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site

  6. Discovery and structure determination of the orphan enzyme isoxanthopterin deaminase .

    PubMed

    Hall, Richard S; Agarwal, Rakhi; Hitchcock, Daniel; Sauder, J Michael; Burley, Stephen K; Swaminathan, Subramanyam; Raushel, Frank M

    2010-05-25

    Two previously uncharacterized proteins have been identified that efficiently catalyze the deamination of isoxanthopterin and pterin 6-carboxylate. The genes encoding these two enzymes, NYSGXRC-9339a ( gi|44585104 ) and NYSGXRC-9236b ( gi|44611670 ), were first identified from DNA isolated from the Sargasso Sea as part of the Global Ocean Sampling Project. The genes were synthesized, and the proteins were subsequently expressed and purified. The X-ray structure of Sgx9339a was determined at 2.7 A resolution (Protein Data Bank entry 2PAJ ). This protein folds as a distorted (beta/alpha)(8) barrel and contains a single zinc ion in the active site. These enzymes are members of the amidohydrolase superfamily and belong to cog0402 within the clusters of orthologous groups (COG). Enzymes in cog0402 have previously been shown to catalyze the deamination of guanine, cytosine, S-adenosylhomocysteine, and 8-oxoguanine. A small compound library of pteridines, purines, and pyrimidines was used to probe catalytic activity. The only substrates identified in this search were isoxanthopterin and pterin 6-carboxylate. The kinetic constants for the deamination of isoxanthopterin with Sgx9339a were determined to be 1.0 s(-1), 8.0 muM, and 1.3 x 10(5) M(-1) s(-1) (k(cat), K(m), and k(cat)/K(m), respectively). The active site of Sgx9339a most closely resembles the active site for 8-oxoguanine deaminase (Protein Data Bank entry 2UZ9 ). A model for substrate recognition of isoxanthopterin by Sgx9339a was proposed on the basis of the binding of guanine and xanthine in the active site of guanine deaminase. Residues critical for substrate binding appear to be conserved glutamine and tyrosine residues that form hydrogen bonds with the carbonyl oxygen at C4, a conserved threonine residue that forms hydrogen bonds with N5, and another conserved threonine residue that forms hydrogen bonds with the carbonyl group at C7. These conserved active site residues were used to identify 24 other genes

  7. Restriction of Porcine Endogenous Retrovirus by Porcine APOBEC3 Cytidine Deaminases

    PubMed Central

    Dörrschuck, Eva; Fischer, Nicole; Bravo, Ignacio G.; Hanschmann, Kay-Martin; Kuiper, Heidi; Spötter, Andreas; Möller, Ronny; Cichutek, Klaus; Münk, Carsten; Tönjes, Ralf R.

    2011-01-01

    Xenotransplantation of porcine cells, tissues, and organs shows promise to surmount the shortage of human donor materials. Among the barriers to pig-to-human xenotransplantation are porcine endogenous retroviruses (PERV) since functional representatives of the two polytropic classes, PERV-A and PERV-B, are able to infect human embryonic kidney cells in vitro, suggesting that a xenozoonosis in vivo could occur. To assess the capacity of human and porcine cells to counteract PERV infections, we analyzed human and porcine APOBEC3 (A3) proteins. This multigene family of cytidine deaminases contributes to the cellular intrinsic immunity and act as potent inhibitors of retroviruses and retrotransposons. Our data show that the porcine A3 gene locus on chromosome 5 consists of the two single-domain genes A3Z2 and A3Z3. The evolutionary relationships of the A3Z3 genes reflect the evolutionary history of mammals. The two A3 genes encode at least four different mRNAs: A3Z2, A3Z3, A3Z2-Z3, and A3Z2-Z3 splice variant A (SVA). Porcine and human A3s have been tested toward their antiretroviral activity against PERV and murine leukemia virus (MuLV) using novel single-round reporter viruses. The porcine A3Z2, A3Z3 and A3Z2-Z3 were packaged into PERV particles and inhibited PERV replication in a dose-dependent manner. The antiretroviral effect correlated with editing by the porcine A3s with a trinucleotide preference for 5′ TGC for A3Z2 and A3Z2-Z3 and 5′ CAC for A3Z3. These results strongly imply that human and porcine A3s could inhibit PERV replication in vivo, thereby reducing the risk of infection of human cells by PERV in the context of pig-to-human xenotransplantation. PMID:21307203

  8. Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

    PubMed

    Alvarez-Añorve, Laura I; Alonzo, Diego A; Mora-Lugo, Rodrigo; Lara-González, Samuel; Bustos-Jaimes, Ismael; Plumbridge, Jacqueline; Calcagno, Mario L

    2011-12-01

    The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

  9. 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.

  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. 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

  12. The ONIOM molecular dynamics method for biochemical applications: cytidine deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-03-22

    Abstract We derived and implemented the ONIOM-molecular dynamics (MD) method for biochemical applications. The implementation allows the characterization of the functions of the real enzymes taking account of their thermal motion. In this method, the direct MD is performed by calculating the ONIOM energy and gradients of the system on the fly. We describe the first application of this ONOM-MD method to cytidine deaminase. The environmental effects on the substrate in the active site are examined. The ONIOM-MD simulations show that the product uridine is strongly perturbed by the thermal motion of the environment and dissociates easily from the active site. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  13. Three-Dimensional Structure and Catalytic Mechanism of Cytosine Deaminase

    SciTech Connect

    R Hall; A Fedorov; C Xu; E Fedorov; S Almo; F Raushel

    2011-12-31

    Cytosine deaminase (CDA) from E. coli is a member of the amidohydrolase superfamily. The structure of the zinc-activated enzyme was determined in the presence of phosphonocytosine, a mimic of the tetrahedral reaction intermediate. This compound inhibits the deamination of cytosine with a K{sub i} of 52 nM. The zinc- and iron-containing enzymes were characterized to determine the effect of the divalent cations on activation of the hydrolytic water. Fe-CDA loses activity at low pH with a kinetic pKa of 6.0, and Zn-CDA has a kinetic pKa of 7.3. Mutation of Gln-156 decreased the catalytic activity by more than 5 orders of magnitude, supporting its role in substrate binding. Mutation of Glu-217, Asp-313, and His-246 significantly decreased catalytic activity supporting the role of these three residues in activation of the hydrolytic water molecule and facilitation of proton transfer reactions. A library of potential substrates was used to probe the structural determinants responsible for catalytic activity. CDA was able to catalyze the deamination of isocytosine and the hydrolysis of 3-oxauracil. Large inverse solvent isotope effects were obtained on k{sub cat} and k{sub cat}/K{sub m}, consistent with the formation of a low-barrier hydrogen bond during the conversion of cytosine to uracil. A chemical mechanism for substrate deamination by CDA was proposed.

  14. Creative deaminases, self-inflicted damage, and genome evolution.

    PubMed

    Conticello, Silvestro G

    2012-09-01

    Organisms minimize genetic damage through complex pathways of DNA repair. Yet a gene family--the AID/APOBECs--has evolved in vertebrates with the sole purpose of producing targeted damage in DNA/RNA molecules through cytosine deamination. They likely originated from deaminases involved in A>I editing in tRNAs. AID, the archetypal AID/APOBEC, is the trigger of the somatic diversification processes of the antibody genes. Its homologs may have been associated with the immune system even before the evolution of the antibody genes. The APOBEC3s, arising from duplication of AID, are involved in the restriction of exogenous/endogenous threats such as retroviruses and mobile elements. Another family member, APOBEC1, has (re)acquired the ability to target RNA while maintaining its ability to act on DNA. The AID/APOBECs have shaped the evolution of vertebrate genomes, but their ability to mutate nucleic acids is a double-edged sword: AID is a key player in lymphoproliferative diseases by triggering mutations and chromosomal translocations in B cells, and there is increasing evidence suggesting that other AID/APOBECs could be involved in cancer development as well.

  15. 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.

  16. 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

  17. Structural and biological function of NYD-SP15 as a new member of cytidine deaminases.

    PubMed

    Xu, Yidan; Li, Lei; Li, Jianmin; Liu, Qinghuai

    2016-05-25

    Recent studies were mainly focus on the cytidine deaminase family genes, which contained a lot of members that varied on the function of catalytic deamination in RNA or DNA and were involved in the process of growth maintenance, host immunity, retroviral infection, tumorigenesis, and drug resistance with a feature of C-U deamination. In this study, we identified a new member of cytidine deaminase family, NYD-SP15. Previous work showed that the deduced structure of the protein contained two dCMP_cyt_deam domains, which were involved in zinc ion binding. NYD-SP15 was expressed variably in a wide range of tissues, indicating its worthy biological function and creative significances. Sequence analysis, RT-PCR, western blot, flow cytometry, direct-site mutation and GST pull-down assay were performed to analyze the construction and function of NYD-SP15. The results in our studies showed that NYD-SP15 was closely related to deoxycytidylate deaminase and cytidine deaminase, with authentic cytidine deaminase activity in vivo and vitro as well as homo dimerization effects. NYD-SP15 contained nuclear localization sequence (NLS) and nuclear export-signal (NES) and could dynamically shuttle between the nucleus and cytoplasm. Furthermore, NYD-SP15 gene over-expression reduced the cells growth and blocked G1 to S phase, which implied a potential inhibition effect on cell growth. PMID:26945630

  18. ACC deaminase genes are conserved among Mesorhizobium species able to nodulate the same host plant.

    PubMed

    Nascimento, Francisco X; Brígido, Clarisse; Glick, Bernard R; Oliveira, Solange

    2012-11-01

    Rhizobia strains expressing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase have been reported to display an augmented symbiotic performance as a consequence of lowering the plant ethylene levels that inhibit the nodulation process. Genes encoding ACC deaminase (acdS) have been studied in Rhizobium spp.; however, not much is known about the presence of acdS genes in Mesorhizobium spp. The aim of this study was to assess the prevalence and phylogeny of acdS genes in Mesorhizobium strains including a collection of chickpea-nodulating mesorhizobia from Portugal. ACC deaminase genes were detected in 10 of 12 mesorhizobia type strains as well as in 18 of 18 chickpea Mesorhizobium isolates studied in this work. No ACC deaminase activity was detected in any Mesorhizobium strain tested under free-living conditions. Despite the lack of ACC deaminase activity, it was possible to demonstrate that in Mesorhizobium ciceri UPM-Ca7(T) , the acdS gene is transcribed under symbiotic conditions. Phylogenetic analysis indicates that strains belonging to different species of Mesorhizobium, but nodulating the same host plant, have similar acdS genes, suggesting that acdS genes are horizontally acquired by transfer of the symbiosis island. This data, together with analysis of the symbiosis islands from completely sequenced Mesorhizobium genomes, suggest the presence of the acdS gene in a Mesorhizobium common ancestor that possessed this gene in a unique symbiosis island.

  19. Molecular epidemiology and diagnosis of PBG deaminase gene defects in acute intermittent porphyria.

    PubMed Central

    Puy, H; Deybach, J C; Lamoril, J; Robreau, A M; Da Silva, V; Gouya, L; Grandchamp, B; Nordmann, Y

    1997-01-01

    Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis. Images Figure 1 Figure 2 PMID:9199558

  20. Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture.

    PubMed

    Saleem, Muhammad; Arshad, Muhammad; Hussain, Sarfraz; Bhatti, Ahmad Saeed

    2007-10-01

    Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into alpha-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.

  1. Nucleoside deaminase: an enzymatic marker for stress erythropoiesis in the mouse

    PubMed Central

    Rothman, Ivan K.; Zanjani, Esmail D.; Gordon, Albert S.; Silber, Robert

    1970-01-01

    The level of nucleoside deaminase was determined in extracts of mouse tissues obtained during a period of accelerated erythropoiesis induced by hypoxia, hemorrhage, or the injection of phenylhydrazine. Under these conditions a striking (10- to 100-fold) elevation of the enzyme activity occurred in the spleen. Similar results were obtained with the injection of purified erythropoietin. In control animals, only a trace of nucleoside deaminase activity was detected in the blood. During the reticulocyte response which followed erythropoietic stimulation, there was a sharp increase in the blood level of nucleoside deaminase, which rose up to 120 times that of control animals. By differential centrifugation, the enzyme was localized to the reticulocyte-rich fraction. Erythrocyte nucleoside deaminase remained elevated even after the reticulocyte count had fallen to normal in the phenylhydrazine-treated mice or to zero after the cessation of hypoxia. There was a very gradual decline in the enzyme activity in the blood which fell to the barely detectable control levels about 45 days after the initial reticulocyte response, a time period which corresponds to the survival of the mouse red blood cell. The persistence of high levels of nucleoside deaminase for the full life span of a generation of erythrocytes formed during stress, viewed in contrast to the virtual absence of the enzyme from normal erythrocytes of all ages, represents an enzymatic difference between the normal red blood cell and the cell produced under conditions of accelerated erythropoiesis. PMID:5475986

  2. Adenosine (ADO) released during orthodromic stimulation of the frog sympathetic ganglion inhibits phosphatidylinositol turnover (PI) associated with synaptic transmission

    SciTech Connect

    Curnish, R.; Bencherif, M.; Rubio, R.; Berne, R.M.

    1986-03-05

    The authors have previously demonstrated that /sup 3/H-purine release was enhanced during synaptic activation of the prelabelled frog sympathetic ganglion. In addition, during orthodromic stimulation, there is an increased /sup 3/H-inositol release (an index of PI) that occurs during the poststimulation period and not during the period of stimulation. They hypothesized that endogenous ADO inhibits PI turnover during orthodromic stimulation. To test this hypothesis (1) they performed experiments to directly measure ADO release in the extracellular fluid by placing the ganglion in a 5 ..mu..l drop of Ringer's and let it come to equilibrium with the interstitial fluid, (2) they destroyed endogenous ADO by suffusing adenosine deaminase (ADA) during the stimulation period. Their results show (1) orthodromic stimulation increases release of ADO into the bathing medium, (2) ADA induced an increase of PI during the stimulation period in contrast to an increase seen only during the poststimulation period when ADA was omitted. They conclude that there is dual control of PI during synaptic activity, a stimulatory effect (cause unknown) and a short lived inhibitory effect that is probably caused by adenosine.

  3. ADAR-related activation of adenosine-to-inosine RNA editing during regeneration.

    PubMed

    Witman, Nevin M; Behm, Mikaela; Ohman, Marie; Morrison, Jamie I

    2013-08-15

    Urodele amphibians possess an amazing regenerative capacity that requires the activation of cellular plasticity in differentiated cells and progenitor/stem cells. Many aspects of regeneration in Urodele amphibians recapitulate development, making it unlikely that gene regulatory pathways which are essential for development are mutually exclusive from those necessary for regeneration. One such post-transcriptional gene regulatory pathway, which has been previously shown to be essential for functional metazoan development, is RNA editing. RNA editing catalyses discrete nucleotide changes in RNA transcripts, creating a molecular diversity that could create an enticing connection to the activated cellular plasticity found in newts during regeneration. To assess whether RNA editing occurs during regeneration, we demonstrated that GABRA3 and ADAR2 mRNA transcripts are edited in uninjured and regenerating tissues. Full open-reading frame sequences for ADAR1 and ADAR2, two enzymes responsible for adenosine-to-inosine RNA editing, were cloned from newt brain cDNA and exhibited a strong resemblance to ADAR (adenosine deaminase, RNA-specific) enzymes discovered in mammals. We demonstrated that ADAR1 and ADAR2 mRNA expression levels are differentially expressed during different phases of regeneration in multiple tissues, whereas protein expression levels remain unaltered. In addition, we have characterized a fascinating nucleocytoplasmic shuttling of ADAR1 in a variety of different cell types during regeneration, which could provide a mechanism for controlling RNA editing, without altering translational output of the editing enzyme. The link between RNA editing and regeneration provides further insights into how lower organisms, such as the newt, can activate essential molecular pathways via the discrete alteration of RNA sequences. PMID:23534823

  4. Adenosine A1 receptors mediate inhibition of cAMP formation in vitro in the pontine, REM sleep induction zone.

    PubMed

    Marks, Gerald A; Birabil, Christian G; Speciale, Samuel G

    2005-11-01

    Microinjection of adenosine A1 receptor agonist or an inhibitor of adenylyl cyclase into the caudal, oral pontine reticular formation (PnOc) of the rat induces a long-lasting increase in REM sleep. Here, we report significant inhibition of forskolin-stimulated cAMP in dissected pontine tissue slices containing the PnOc incubated with the A1 receptor agonist, cyclohexaladenosine (10(-8) M). These data are consistent with adenosine A1 receptor agonist actions on REM sleep mediated through inhibition of cAMP.

  5. 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

  6. 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.

  7. 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

  8. Biological function of activation-induced cytidine deaminase (AID).

    PubMed

    Kumar, Ritu; DiMenna, Lauren J; Chaudhuri, Jayanta; Evans, Todd

    2014-01-01

    Activation-induced Cytidine Deaminase (AID) is an essential regulator of B cell diversification, but its full range of action has until recently been an enigma. Based on homology, it was originally proposed to be an RNA-editing enzyme, but so far, no RNA substrates are known. Rather, it functions by deaminating cytidine, and in this manner, coupled with base-excision repair or mismatch repair machinery, it is a natural mutator. This allows it to play a central role in adaptive immunity, whereby it initiates the processes of class switch recombination and somatic hypermutation to help generate a diverse and high-affinity repertoire of immunoglobulin isotypes. More recently, it has been appreciated that methylated cytidine, already known as a key epigenetic mark on DNA controlling gene expression, can also be a target for AID modification. Coupled with repair machinery, this can facilitate the active removal of methylated DNA. This activity can impact the process of cellular reprogramming, including transition of a somatic cell to pluripotency, which requires major reshuffling of epigenetic memory. Thus, seemingly disparate roles for AID in controlling immune diversity and epigenetic memory have a common mechanistic basis. However, the very activity that is so useful for B cell diversity and cellular reprogramming is dangerous for the integrity of the genome. Thus, AID expression and activity is tightly regulated, and deregulation is associated with diseases including cancer. Here, we review the range of AID functions with a focus on its mechanisms of action and regulation. Major questions remain to be answered concerning how and when AID is targeted to specific loci and how this impacts development and disease.

  9. Reprogramming of microRNAs by adenosine-to-inosine editing and the selective elimination of edited microRNA precursors in mouse oocytes and preimplantation embryos.

    PubMed

    García-López, Jesús; Hourcade, Juan de Dios; Del Mazo, Jesús

    2013-05-01

    Adenosine deaminases-acting-on-RNA (ADAR) proteins induce adenosine-to-inosine editing in double-stranded RNA molecules. This editing generates RNA diversity at the post-transcriptional level, and it has been implicated in the control of cell differentiation and development. The editing of microRNA (miRNA) precursors, along with Tudor-SN (Snd1) activity, could lead to the elimination of selected miRNAs and reprogram miRNA activity. Here, we report the dynamics of adenosine-to-inosine editing in miRNA precursors and their selected elimination during mouse preimplantation development. Adar1p110 and Snd1 were found to be strongly but differentially expressed in oocytes and zygotes with respect to later pre-implantation stages. When the biogenesis of miR-151 was assessed, the majority of miR-151 precursors was edited and subsequently eliminated during early development. Deep sequencing of this and other miRNAs confirmed that, in general, edited precursors were selectively eliminated at early post-zygotic stages. Moreover, in oocytes and throughout the zygote-to-blastocyst stages, Tudor-SN accumulated in newly discovered aggregates termed 'T bodies'. These results provide new insight into how editing and Tudor-SN-mediated elimination of miRNA precursors is regulated during early development.

  10. Adenosine (ADO) receptors in the PC12 phenochromocytoma cell line: characterization by radioligand binding

    SciTech Connect

    Williams, M.; Abreu, M.; Noronha-Blob, L.

    1986-03-01

    PC12 cells contain an ado-sensitive adenylate cyclase which is exclusively A-2 in nature. Binding of A-1 selective radioligands (Cyclohexylado; CHA and Cyclopentylado; CPA) to PC12 membranes is minimal and irreproducible. In contrast, the non-selective A-1/A-2 agonist NECA (5'-N-ethylcarboxamidoado) binds with high affinity to two sites in adenosine-deaminase pretreated PC12 membranes. Using computerized curve fitting, the first site had a Kd value of 5.2 +/- 1.5 nM (mean +/- s.d.; n = 8) and an apparent Bmax of 205 fmoles/mg prot. Due to large increases in non-specific binding, the second site could not be resolved but had a Kd in the range of 1 ..mu..M. Pharmacological analysis of specific NECA binding (5 nM) gave the rank order activity profile: NECA < MECA = 2-CADO > CHA > R-PIA > CPA S-PIA > AMP = ADP = ATP. Binding was also xanthine sensitive with PACPX > 8-PT > 8-PST. The ratio of activity for the R and S diastereomers of PIA was 10, consistent with the labeling of A-2-type ado receptors in the PC12 cell line.

  11. 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

  12. Structure-activity relationship of heterobase-modified 2'-C-methyl ribonucleosides as inhibitors of hepatitis C virus RNA replication.

    PubMed

    Eldrup, Anne B; Prhavc, Marija; Brooks, Jennifer; Bhat, Balkrishen; Prakash, Thazha P; Song, Quanlai; Bera, Sanjib; Bhat, Neelima; Dande, Prasad; Cook, P Dan; Bennett, C Frank; Carroll, Steven S; Ball, Richard G; Bosserman, Michele; Burlein, Christine; Colwell, Lawrence F; Fay, John F; Flores, Osvaldo A; Getty, Krista; LaFemina, Robert L; Leone, Joseph; MacCoss, Malcolm; McMasters, Daniel R; Tomassini, Joanne E; Von Langen, Derek; Wolanski, Bohdan; Olsen, David B

    2004-10-01

    Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility. PMID:15456273

  13. The Effect of Eplerenone on Adenosine Formation in Humans In Vivo: A Double-Blinded Randomised Controlled Study

    PubMed Central

    van den Berg, T. N. A.; Deinum, Jaap; Bilos, Albert; Donders, A. Rogier T.; Rongen, Gerard A.; Riksen, Niels P.

    2014-01-01

    Background It has been suggested that mineralocorticoid receptor antagonists have direct cardioprotective properties, because these drugs reduce mortality in patients with heart failure. In murine models of myocardial infarction, mineralocorticoid receptor antagonists reduce infarct size. Using gene deletion and pharmacological approaches, it has been shown that extracellular formation of the endogenous nucleoside adenosine is crucial for this protective effect. We now aim to translate this finding to humans, by investigating the effects of the selective mineralocorticoid receptor antagonist eplerenone on the vasodilator effect of the adenosine uptake inhibitor dipyridamole, which is a well-validated surrogate marker for extracellular adenosine formation. Methods and Results In a randomised, double-blinded, placebo-controlled, cross-over study we measured the forearm blood flow response to the intrabrachial administration of dipyridamole in 14 healthy male subjects before and after treatment with placebo or eplerenone (50 mg bid for 8 days). The forearm blood flow during administration of dipyridamole (10, 30 and 100 µg·min−1·dl−1) was 1.63 (0.60), 2.13 (1.51) and 2.71 (1.32) ml·dl−1·min−1 during placebo use, versus 2.00 (1.45), 2.68 (1.87) and 3.22 (1.94) ml·dl−1·min−1 during eplerenone treatment (median (interquartile range); P = 0.51). Concomitant administration of the adenosine receptor antagonist caffeine attenuated dipyridamole-induced vasodilation to a similar extent in both groups. The forearm blood flow response to forearm ischemia, as a stimulus for increased formation of adenosine, was similar during both conditions. Conclusion In a dosage of 50 mg bid, eplerenone does not augment extracellular adenosine formation in healthy human subjects. Therefore, it is unlikely that an increased extracellular adenosine formation contributes to the cardioprotective effect of mineralocorticoid receptor antagonists. Trial Registration Clinical

  14. Interleukin-1 and tumor necrosis factor-α trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID).

    PubMed

    Watashi, Koichi; Liang, Guoxin; Iwamoto, Masashi; Marusawa, Hiroyuki; Uchida, Nanako; Daito, Takuji; Kitamura, Kouichi; Muramatsu, Masamichi; Ohashi, Hirofumi; Kiyohara, Tomoko; Suzuki, Ryosuke; Li, Jisu; Tong, Shuping; Tanaka, Yasuhito; Murata, Kazumoto; Aizaki, Hideki; Wakita, Takaji

    2013-11-01

    Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection.

  15. Interleukin-1 and Tumor Necrosis Factor-α Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)*

    PubMed Central

    Watashi, Koichi; Liang, Guoxin; Iwamoto, Masashi; Marusawa, Hiroyuki; Uchida, Nanako; Daito, Takuji; Kitamura, Kouichi; Muramatsu, Masamichi; Ohashi, Hirofumi; Kiyohara, Tomoko; Suzuki, Ryosuke; Li, Jisu; Tong, Shuping; Tanaka, Yasuhito; Murata, Kazumoto; Aizaki, Hideki; Wakita, Takaji

    2013-01-01

    Virus infection is restricted by intracellular immune responses in host cells, and this is typically modulated by stimulation of cytokines. The cytokines and host factors that determine the host cell restriction against hepatitis B virus (HBV) infection are not well understood. We screened 36 cytokines and chemokines to determine which were able to reduce the susceptibility of HepaRG cells to HBV infection. Here, we found that pretreatment with IL-1β and TNFα remarkably reduced the host cell susceptibility to HBV infection. This effect was mediated by activation of the NF-κB signaling pathway. A cytidine deaminase, activation-induced cytidine deaminase (AID), was up-regulated by both IL-1β and TNFα in a variety of hepatocyte cell lines and primary human hepatocytes. Another deaminase APOBEC3G was not induced by these proinflammatory cytokines. Knockdown of AID expression impaired the anti-HBV effect of IL-1β, and overexpression of AID antagonized HBV infection, suggesting that AID was one of the responsible factors for the anti-HBV activity of IL-1/TNFα. Although AID induced hypermutation of HBV DNA, this activity was dispensable for the anti-HBV activity. The antiviral effect of IL-1/TNFα was also observed on different HBV genotypes but not on hepatitis C virus. These results demonstrate that proinflammatory cytokines IL-1/TNFα trigger a novel antiviral mechanism involving AID to regulate host cell permissiveness to HBV infection. PMID:24025329

  16. The cardiac effects of a novel A1-adenosine receptor agonist in guinea pig isolated heart.

    PubMed

    Belardinelli, L; Lu, J; Dennis, D; Martens, J; Shryock, J C

    1994-12-01

    Adenosine increases atrioventricular (AV) nodal conduction time and is used for termination of AV nodal re-entrant tachycardias, but it is rapidly metabolized. The purposes of the present study were to characterize the cardiac actions and effects of an orally active and stable adenosine analog, N6-cyclohexyl-2-O-methyladenosine (SDZ WAG-994) and to evaluate its potential as an antiarrhythmic agent. Guinea pig hearts were isolated and perfused with oxygenated Krebs-Henseleit solution. SDZ WAG-994 slowed the atrial rate and prolonged the AV nodal conduction time of spontaneously beating hearts in a concentration-dependent manner. The EC50 values for the negative chronotropic and dromotropic effects of SDZ WAG-994 were 0.69 +/- 0.04 and 1.49 +/- 0.54 microM, respectively. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.2 microM) significantly antagonized SDZ WAG-994-induced stimulus-to-His bundle (S-H) interval prolongation. The negative dromotropic effect of SDZ WAG-994 showed very strong frequency dependence. In hearts paced at an atrial cycle length of 300 msec (200 beats/min), the EC50 value of SDZ WAG-994 to prolong the S-H interval was 3.7-fold lower (0.40 +/- 0.02 microM) than in unpaced hearts, and at atrial pacing cycle lengths of 500 and 250 msec, 0.3 microM SDZ WAG-994 prolonged the S-H interval by 8 and 26 msec, respectively. SDZ WAG-994 also decreased coronary perfusion pressure (EC50 = 1.50 +/- 0.80 microM); this effect of SDZ WAG-994 was attenuated by adenosine deaminase and by 8-cyclopentyltheophylline (2 microM). Radioligand binding assays revealed that SDZ WAG-994 had a 280-fold greater affinity for A1- than for A2a receptors of the guinea pig brain. The marked frequency dependence of the negative dromotropic effect of SDZ WAG-994 suggests that this A1 agonist may be highly effective in the termination of AV nodal re-entrant tachycardias. PMID:7996449

  17. Pharmacological prevention of reperfusion injury in acute myocardial infarction. A potential role for adenosine as a therapeutic agent.

    PubMed

    Quintana, Miguel; Kahan, Thomas; Hjemdahl, Paul

    2004-01-01

    The concept of reperfusion injury, although first recognized from animal studies, is now recognized as a clinical phenomenon that may result in microvascular damage, no-reflow phenomenon, myocardial stunning, myocardial hibernation and ischemic preconditioning. The final consequence of this event is left ventricular (LV) systolic dysfunction leading to increased morbidity and mortality. The typical clinical case of reperfusion injury occurs in acute myocardial infarction (MI) with ST segment elevation in which an occlusion of a major epicardial coronary artery is followed by recanalization of the artery. This may occur either spontaneously or by means of thrombolysis and/or by primary percutaneous coronary intervention (PCI) with efficient platelet inhibition by aspirin (acetylsalicylic acid), clopidogrel and glycoprotein IIb/IIIa inhibitors. Although the pathophysiology of reperfusion injury is complex, the major role that neutrophils play in this process is well known. Neutrophils generate free radicals, degranulation products, arachidonic acid metabolites and platelet-activating factors that interact with endothelial cells, inducing endothelial injury and neutralization of nitrous oxide vasodilator capacity. Adenosine, through its multi-targeted pharmacological actions, is able to inhibit some of the above-mentioned detrimental effects. The net protective of adenosine in in vivo models of reperfusion injury is the reduction of the infarct size, the improvement of the regional myocardial blood flow and of the regional function of the ischemic area. Additionally, adenosine preserves the post-ischemic coronary flow reserve, coronary blood flow and the post-ischemic regional contractility. In small-scale studies in patients with acute MI, treatment with adenosine has been associated with smaller infarcts, less no-reflow phenomenon and improved LV function. During elective PCI adenosine reduced ST segment shifts, lactate production and ischemic symptoms. During the

  18. 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.

  19. 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.

  20. (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine binding to A1 adenosine receptors of intact rat ventricular myocytes

    SciTech Connect

    Martens, D.; Lohse, M.J.; Schwabe, U.

    1988-09-01

    The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine, and S-N(6)-phenylisopropyladenosine gave monophasic displacement curves with Ki values of 50 nM, 440 nM, and 4,300 nM, which agreed well with the GTP-inducible low affinity state in cardiac membranes. The low affinity for agonists was not due to agonist-induced desensitization, and correlated well with the corresponding IC50 values for the inhibition of cyclic AMP accumulation by isoprenaline. It is suggested that only a low affinity state of A1 receptors can be detected in intact rat myocytes due to the presence of high concentrations of guanine nucleotides in intact cells.

  1. 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.

  2. Aberrant activation-induced cytidine deaminase expression in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia.

    PubMed

    Shi, Yang; Zhao, Xiaoxian; Durkin, Lisa; Rogers, Heesun Joyce; Hsi, Eric D

    2016-06-01

    Activation-induced cytidine deaminase (AID) is expressed in germinal center B cells and plays a critical role in somatic hypermutation and class-switch recombination of immunoglobulin genes. Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) carries a poor prognosis and is specifically treated with tyrosine kinase inhibitors. Interestingly, AID has been shown to be aberrantly expressed and functional in Ph+ ALL and is thought to contribute to genetic instability. We hypothesized that AID might be detectable in routinely processed bone marrow biopsies by immunohistochemistry (IHC) and assist in identifying Ph+ ALL. We found that AID was expressed in 26 (70%) of 37 cases of Ph+ ALL but only 1 (2.9%) of 38 cases of Ph- ALL cases. There was a significant difference in AID expression between these 2 ALL groups (P < .001, Fisher exact test). The expression of AID was confirmed by RT-PCR (reverse-transcriptase polymerase chain reaction) and correlated with IHC scoring. AID protein is expressed in a large proportion of Ph+ ALL cases at levels detectable by IHC in clinical samples and might be useful to rapidly identify cases likely to have a BCR/ABL1 fusion. PMID:26980048

  3. High-throughput mutagenesis reveals functional determinants for DNA targeting by activation-induced deaminase

    PubMed Central

    Gajula, Kiran S.; Huwe, Peter J.; Mo, Charlie Y.; Crawford, Daniel J.; Stivers, James T.; Radhakrishnan, Ravi; Kohli, Rahul M.

    2014-01-01

    Antibody maturation is a critical immune process governed by the enzyme activation-induced deaminase (AID), a member of the AID/APOBEC DNA deaminase family. AID/APOBEC deaminases preferentially target cytosine within distinct preferred sequence motifs in DNA, with specificity largely conferred by a small 9–11 residue protein loop that differs among family members. Here, we aimed to determine the key functional characteristics of this protein loop in AID and to thereby inform our understanding of the mode of DNA engagement. To this end, we developed a methodology (Sat-Sel-Seq) that couples saturation mutagenesis at each position across the targeting loop, with iterative functional selection and next-generation sequencing. This high-throughput mutational analysis revealed dominant characteristics for residues within the loop and additionally yielded enzymatic variants that enhance deaminase activity. To rationalize these functional requirements, we performed molecular dynamics simulations that suggest that AID and its hyperactive variants can engage DNA in multiple specific modes. These findings align with AID's competing requirements for specificity and flexibility to efficiently drive antibody maturation. Beyond insights into the AID-DNA interface, our Sat-Sel-Seq approach also serves to further expand the repertoire of techniques for deep positional scanning and may find general utility for high-throughput analysis of protein function. PMID:25064858

  4. Improved cytotoxic effects of Salmonella-producing cytosine deaminase in tumour cells

    PubMed Central

    Mesa-Pereira, Beatriz; Medina, Carlos; Camacho, Eva María; Flores, Amando; Santero, Eduardo

    2015-01-01

    In order to increase the cytotoxic activity of a Salmonella strain carrying a salicylate-inducible expression system that controls cytosine deaminase production, we have modified both, the vector and the producer bacterium. First, the translation rates of the expression module containing the Escherichia coli codA gene cloned under the control of the Pm promoter have been improved by using the T7 phage gene 10 ribosome binding site sequence and replacing the original GUG start codon by AUG. Second, to increase the time span in which cytosine deaminase may be produced by the bacteria in the presence of 5-fluorocytosine, a 5-fluorouracyl resistant Salmonella strain has been constructed by deleting its upp gene sequence. This new Salmonella strain shows increased cytosine deaminase activity and, after infecting tumour cell cultures, increased cytotoxic and bystander effects under standard induction conditions. In addition, we have generated a purD mutation in the producer strain to control its intracellular proliferation by the presence of adenine and avoid the intrinsic Salmonella cell death induction. This strategy allows the analysis and comparison of the cytotoxic effects of cytosine deaminase produced by different Salmonella strains in tumour cell cultures. PMID:25227763

  5. Role for Free Isoleucine or Glycyl-Leucine in the Repression of Threonine Deaminase in Escherichia coli

    PubMed Central

    Wasmuth, John J.; Umbarger, H. E.

    1974-01-01

    In Escherichia coli, the three branched-chain amino acid activating enzymes appear to be essential for multivalent repression of the isoleucine- and valine-forming enzymes. The results of experiments with a mutant, strain CU18, having an altered threonine deaminase, indicate that free isoleucine and some form of threonine deaminase (the product of the ilvA gene) are also involved in multivalent repression. This strain exhibits abnormally high derepressibility but normal repressibility of its ilv gene products, and its threonine deaminase is inhibited only by high concentrations of isoleucine. In strain CU18, the isoleucine analogue, thiaisoleucine, is incapable of replacing isoleucine in the multivalent repression of the ilv genes, whereas the analogue can fully replace the natural amino acid in repression in other strains examined. The dipeptide, glycyl-leucine, which, like isoleucine, is a heterotropic negative effector of threonine deaminase but is not a substrate for isoleucyl-transfer ribonucleic acid synthetase, can completely prevent the accumulation of threonine deaminase-forming potential during isoleucine starvation in strains with normal threonine deaminases. It can not, however, prevent such accumulation in strain CU18 or in other strains in which threonine deaminase is insensitive to any concentration of isoleucine. PMID:4587610

  6. Extracellular purine metabolism and signaling of CD73-derived adenosine in murine Treg and Teff cells.

    PubMed

    Romio, Michael; Reinbeck, Benjamin; Bongardt, Sabine; Hüls, Sandra; Burghoff, Sandra; Schrader, Jürgen

    2011-08-01

    CD73-derived adenosine acts as potent inhibitor of inflammation, and regulatory T cells (Treg) have been shown to express CD73 as a novel marker. This study explored the role of endogenously formed adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and effector T cells (Teff) including key enzymes/purinergic receptors of extracellular ATP catabolism. Stimulating murine splenocytes and CD4(+) T cells with anti-CD3/anti-CD28 significantly upregulated activated NF-κB in CD73(-/-) T cells (wild type: 4.36 ± 0.21; CD73(-/-): 6.58 ± 0.75; n = 4; P = 0.029). This was associated with an augmented release of proinflammatory cytokines IL-2, TNF-α, and IFN-γ. Similar changes were observed with the CD73 inhibitor APCP (50 μM) on NF-κB and IFN-γ in wild-type CD4(+) T-cells. Treatment of stimulated CD4(+) T-cells with adenosine (25 μM) potently reduced IFN-γ release which is mediated by adenosine A2a receptors (A2aR). AMP (50 μM) also reduced cytokine release which was not inhibited by APCP. In Teff, A2aR activation (CGS21680) potently inhibited the release of IL-1, IL-2, IL-3, IL-4, IL-12, IL-13, IFN-γ, TNF-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), CCL3, and CCL4. However, in Treg, CGS21680 did not alter cytokine/chemokine release. In summary, CD73-derived adenosine tonically inhibits active NF-κB in CD4(+) T-cells, thereby modulating the release of a broad spectrum of proinflammatory cytokines and chemokines. Downregulation of P2X7 and upregulation of CD73 in Treg after antigenic stimulation may be an important mechanism to maintain the ability of Treg to generate immunosuppressive adenosine.

  7. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

  8. 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

  9. 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

  10. 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.

  11. cAMP-independent dilation of coronary arterioles to adenosine : role of nitric oxide, G proteins, and K(ATP) channels.

    PubMed

    Hein, T W; Kuo, L

    1999-10-01

    Adenosine is known to play an important role in the regulation of coronary blood flow during metabolic stress. However, there is sparse information on the mechanism of adenosine-induced dilation at the microcirculatory levels. In the present study, we examined the role of endothelial nitric oxide (NO), G proteins, cyclic nucleotides, and potassium channels in coronary arteriolar dilation to adenosine. Pig subepicardial coronary arterioles (50 to 100 microm in diameter) were isolated, cannulated, and pressurized to 60 cm H(2)O without flow for in vitro study. The arterioles developed basal tone and dilated dose dependently to adenosine. Disruption of endothelium, blocking of endothelial ATP-sensitive potassium (K(ATP)) channels by glibenclamide, and inhibition of NO synthase by N(G)-nitro-L-arginine methyl ester and of soluble guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one produced identical attenuation of vasodilation to adenosine. Combined administration of these inhibitors did not further attenuate the vasodilatory response. Production of NO from coronary arterioles was significantly increased by adenosine. Pertussis toxin, but not cholera toxin, significantly inhibited vasodilation to adenosine, and this inhibitory effect was only evident in vessels with an intact endothelium. Tetraethylammonium, glibenclamide, and a high concentration of extraluminal KCl abolished vasodilation of denuded vessels to adenosine; however, inhibition of calcium-activated potassium channels by iberiotoxin had no effect on this dilation. Rp-8-Br-cAMPS, a cAMP antagonist, inhibited vasodilation to cAMP analog 8-Br-cAMP but failed to block adenosine-induced dilation. Furthermore, vasodilations to 8-Br-cAMP and sodium nitroprusside were not inhibited by glibenclamide, indicating that cAMP- and cGMP-induced dilations are not mediated by the activation of K(ATP) channels. These results suggest that adenosine activates both endothelial and smooth muscle pathways to exert

  12. N6-(2-Hydroxyethyl)-Adenosine Exhibits Insecticidal Activity against Plutella xylostella via Adenosine Receptors

    PubMed Central

    Fang, Ming; Chai, Yiqiu; Chen, Guanjv; Wang, Huidong; Huang, Bo

    2016-01-01

    The diamondback moth, Plutella xylostella, is one of the most important pests of cruciferous crops. We have earlier shown that N6-(2-hydroxyethyl)-adenosine (HEA) exhibits insecticidal activity against P. xylostella. In the present study we investigated the possible mechanism of insecticidal action of HEA on P. xylostella. HEA is a derivative of adenosine, therefore, we speculated whether it acts via P. xylostella adenosine receptor (PxAdoR). We used RNAi approach to silence PxAdoR gene and used antagonist of denosine receptor (AdoR) to study the insecticidal effect of HEA. We cloned the whole sequence of PxAdoR gene. A BLAST search using NCBI protein database showed a 61% identity with the Drosophila adenosine receptor (DmAdoR) and a 32–35% identity with human AdoR. Though the amino acids sequence of PxAdoR was different compared to other adenosine receptors, most of the amino acids that are known to be important for adenosine receptor ligand binding and signaling were present. However, only 30% binding sites key residues was similar between PxAdoR and A1R. HEA, at a dose of 1 mg/mL, was found to be lethal to the second-instar larvae of P. xylostella, and a significant reduction of mortality and growth inhibition ratio were obtained when HEA was administered to the larvae along with PxAdoR-dsRNA or antagonist of AdoR (SCH58261) for 36, 48, or 60 h. Especially at 48 h, the rate of growth inhibition of the PxAdoR knockdown group was 3.5-fold less than that of the HEA group, and the corrected mortality of SCH58261 group was reduced almost 2-fold compared with the HEA group. Our findings show that HEA may exert its insecticidal activity against P. xylostella larvae via acting on PxAdoR. PMID:27668428

  13. 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.

  14. Amelioration of high salinity stress damage by plant growth-promoting bacterial endophytes that contain ACC deaminase.

    PubMed

    Ali, Shimaila; Charles, Trevor C; Glick, Bernard R

    2014-07-01

    Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC) deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudomonas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity). Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase containing bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a greater number of flowers and buds than the other treatments. Since the only difference between wild-type and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not normally suitable for the majority of crops due to their high salt contents.

  15. 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.

  16. 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.

  17. 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.

  18. Histone deacetylases 6 increases the cyclic adenosine monophosphate level and promotes renal cyst growth.

    PubMed

    Wu, Ming; Mei, Changlin

    2016-07-01

    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by abnormal enhanced cell proliferation and fluid secretion, which are triggered by increased levels of cyclic adenosine monophosphate (cAMP). Cebotaru et al. showed that a HDAC6 inhibitor reduced the cAMP level and inhibited cyst formation in Pkd1 knockout mice, which may become a new potential therapeutic agent for ADPKD. This study also raised several intriguing questions that might advance our understanding of the molecular pathogenesis of ADPKD. PMID:27312442

  19. GIRK channel activation via adenosine or muscarinic receptors has similar effects on rat atrial electrophysiology.

    PubMed

    Wang, Xiaodong; Liang, Bo; Skibsbye, Lasse; Olesen, Søren-Peter; Grunnet, Morten; Jespersen, Thomas

    2013-08-01

    G protein-coupled inwardly rectifying K⁺ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M₂ receptors and adenosine A₁ receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD₉₀), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD₉₀ and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD₉₀ and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A₁ and M₂ receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms. PMID:23609329

  20. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    PubMed

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems.

  1. Assessing the effects of heavy metals in ACC deaminase and IAA production on plant growth-promoting bacteria.

    PubMed

    Carlos, Mendoza-Hernández José; Stefani, Perea-Vélez Yazmin; Janette, Arriola-Morales; Melani, Martínez-Simón Sara; Gabriela, Pérez-Osorio

    2016-01-01

    This study poses a methodology in order to simultaneously quantify ACC deaminase and IAA levels in the same culture medium. Ten bacterial strains isolated from plant rhizosphere naturally settled in mining residues were chosen. These bacterial strains were characterized as PGPB, and all of them showed at least three characteristics (indole-3 acetic acid and siderophore production, ACC deaminase enzyme activity, and inorganic phosphate solubilization). Taxonomic identification showed that the strains belong to Enterobacter, Serratia, Klebsiella, and Escherichia genera. Similarly, both the ACC deaminase enzyme activity and the IAA synthesis in the presence of Cu, As, Pb, Ni, Cd, and Mn were measured. The results showed that both the ACC deaminase enzyme activity and the IAA synthesis were higher with the Pb, As, and Cu treatments than with the Escherichia N16, Enterobacter K131, Enterobacter N9, and Serratia K120 control treatments. On the other hand, Ni, Cd, and Mn negatively affected both the ACC deaminase enzyme activity and the IAA production on every bacterium except on the Klebsiella Mc173 strain. Serratia K120 bacterium got a positive correlation between ACC deaminase and IAA in the presence of every heavy metal, and it also promoted Helianthus annuus plant growth, showing a potential use in phytoremediation systems. PMID:27296962

  2. 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.

  3. 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...

  4. Amplified fluorescence detection of adenosine via catalyzed hairpin assembly and host-guest interactions between β-cyclodextrin polymer and pyrene.

    PubMed

    Huang, Haihua; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Guo, Qiuping; Huang, Jin; Liu, Jianbo; Song, Chunxia

    2016-04-21

    Nowadays, enzyme-free nucleic acid-based signal amplification strategies are frequently utilized in the design of biosensors due to their excellent sensitivity. Developing more extended analytical methods is fundamental for basic studies in the biological and biomedical fields. Herein, we introduce an enzyme-free amplified detection strategy for the small molecule adenosine. The approach is based on adenosine-aptamer binding triggered catalyzed hairpin assembly and host-guest interactions between β-cyclodextrin polymer (β-CDP) and pyrene. Two hairpin probes (probe H1 and probe H2) and an aptamer-trigger/inhibitor duplex probe were employed in the system and the pyrene-labeled probe H1 was chosen as the signal unit. In the absence of adenosine, the aptamer-trigger was inhibited by the inhibitor strand. The hairpin probes were in the closed hairpin formation without activation of the trigger strand. Pyrene labeled at the 5'-termini of the single-stranded stem of probe H1 could be easily trapped in the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to a significant fluorescence enhancement. Once the hairpin assembly was catalyzed by the adenosine-aptamer binding event, a hybridized DNA duplex H1/H2 was created continuously. Pyrene labeled at the 5'-termini of the DNA duplex H1/H2 finds it difficult to enter the cavity of β-CDP due to steric hindrance, leading to a weaker fluorescence signal. Thus, the target could be detected by this simple mix-and-detect amplification method without a need for expensive and perishable protein enzymes. As low as 42 nM of adenosine was detected by this assay, which is comparable to that of some reported colorimetric methods. Meanwhile, the proposed method was further successfully applied to detect adenosine in human serum samples, showing great potential for adenosine detection from complex fluids.

  5. A Cytidine Deaminase Edits C to U in Transfer RNAs in Archaea

    PubMed Central

    Randau, Lennart; Stanley, Bradford J.; Kohlway, Andrew; Mechta, Sarah; Xiong, Yong; Söll, Dieter

    2010-01-01

    All canonical transfer RNAs (tRNAs) have a uridine at position 8, involved in maintaining tRNA tertiary structure. However, the hyperthermophilic archaeon Methanopyrus kandleri harbors 30 (out of 34) tRNA genes with cytidine at position 8. Here, we demonstrate C-to-U editing at this location in the tRNA’s tertiary core, and present the crystal structure of a tRNA-specific cytidine deaminase, CDAT8, which has the cytidine deaminase domain linked to a tRNA-binding THUMP domain. CDAT8 is specific for C deamination at position 8, requires only the acceptor stem hairpin for activity, and belongs to a unique family within the “cytidine deaminase–like” superfamily. The presence of this C-to-U editing enzyme guarantees the proper folding and functionality of all M. kandleri tRNAs. PMID:19407206

  6. AID and APOBEC deaminases: balancing DNA damage in epigenetics and immunity.

    PubMed

    Franchini, Don-Marc; Petersen-Mahrt, Svend K

    2014-01-01

    DNA mutations and genomic recombinations are the origin of oncogenesis, yet parts of developmental programs as well as immunity are intimately linked to, or even depend on, such DNA damages. Therefore, the balance between deleterious DNA damages and organismal survival utilizing DNA editing (modification and repair) is in continuous flux. The cytosine deaminases AID/APOBEC are a DNA editing family and actively participate in various biological processes. In conjunction with altered DNA repair, the mutagenic potential of the family allows for APOBEC3 proteins to restrict viral infection and transposons propagation, while AID can induce somatic hypermutation and class switch recombination in antibody genes. On the other hand, the synergy between effective DNA repair and the nonmutagenic potential of the DNA deaminases can induce local DNA demethylation to support epigenetic cellular identity. Here, we review the current state of knowledge on the mechanisms of action of the AID/APOBEC family in immunity and epigenetics.

  7. Adenosine Kinase Inhibition Protects against Cranial Radiation-Induced Cognitive Dysfunction

    PubMed Central

    Acharya, Munjal M.; Baulch, Janet E.; Lusardi, Theresa A.; Allen, Barrett. D.; Chmielewski, Nicole N.; Baddour, Al Anoud D.; Limoli, Charles L.; Boison, Detlev

    2016-01-01

    Clinical radiation therapy for the treatment of CNS cancers leads to unintended and debilitating impairments in cognition. Radiation-induced cognitive dysfunction is long lasting; however, the underlying molecular and cellular mechanisms are still not well established. Since ionizing radiation causes microglial and astroglial activation, we hypothesized that maladaptive changes in astrocyte function might be implicated in radiation-induced cognitive dysfunction. Among other gliotransmitters, astrocytes control the availability of adenosine, an endogenous neuroprotectant and modulator of cognition, via metabolic clearance through adenosine kinase (ADK). Adult rats exposed to cranial irradiation (10 Gy) showed significant declines in performance of hippocampal-dependent cognitive function tasks [novel place recognition, novel object recognition (NOR), and contextual fear conditioning (FC)] 1 month after exposure to ionizing radiation using a clinically relevant regimen. Irradiated rats spent less time exploring a novel place or object. Cranial irradiation also led to reduction in freezing behavior compared to controls in the FC task. Importantly, immunohistochemical analyses of irradiated brains showed significant elevation of ADK immunoreactivity in the hippocampus that was related to astrogliosis and increased expression of glial fibrillary acidic protein (GFAP). Conversely, rats treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 3.1 mg/kg, i.p., for 6 days) prior to cranial irradiation showed significantly improved behavioral performance in all cognitive tasks 1 month post exposure. Treatment with 5-ITU attenuated radiation-induced astrogliosis and elevated ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism where preservation of extracellular adenosine can exert neuroprotection against radiation-induced pathology. These innovative findings link radiation-induced changes in cognition and CNS functionality to altered

  8. 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

  9. Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

    PubMed

    Stearns, Jennifer C; Shah, Saleh; Greenberg, Bruce M; Dixon, D George; Glick, Bernard R

    2005-07-01

    Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.

  10. Bacteria with ACC deaminase can promote plant growth and help to feed the world.

    PubMed

    Glick, Bernard R

    2014-01-20

    To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture. PMID:24095256

  11. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    PubMed

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique. PMID:25983132

  12. Structure of the DNA deaminase domain of the HIV-1 restriction factor APOBEC3G.

    PubMed

    Chen, Kuan-Ming; Harjes, Elena; Gross, Phillip J; Fahmy, Amr; Lu, Yongjian; Shindo, Keisuke; Harris, Reuben S; Matsuo, Hiroshi

    2008-03-01

    The human APOBEC3G (apolipoprotein B messenger-RNA-editing enzyme, catalytic polypeptide-like 3G) protein is a single-strand DNA deaminase that inhibits the replication of human immunodeficiency virus-1 (HIV-1), other retroviruses and retrotransposons. APOBEC3G anti-viral activity is circumvented by most retroelements, such as through degradation by HIV-1 Vif. APOBEC3G is a member of a family of polynucleotide cytosine deaminases, several of which also target distinct physiological substrates. For instance, APOBEC1 edits APOB mRNA and AID deaminates antibody gene DNA. Although structures of other family members exist, none of these proteins has elicited polynucleotide cytosine deaminase or anti-viral activity. Here we report a solution structure of the human APOBEC3G catalytic domain. Five alpha-helices, including two that form the zinc-coordinating active site, are arranged over a hydrophobic platform consisting of five beta-strands. NMR DNA titration experiments, computational modelling, phylogenetic conservation and Escherichia coli-based activity assays combine to suggest a DNA-binding model in which a brim of positively charged residues positions the target cytosine for catalysis. The structure of the APOBEC3G catalytic domain will help us to understand functions of other family members and interactions that occur with pathogenic proteins such as HIV-1 Vif.

  13. Alternative Induction of Meiotic Recombination From Single-Base Lesions of DNA Deaminases

    PubMed Central

    Pauklin, Siim; Burkert, Julia S.; Martin, Julie; Osman, Fekret; Weller, Sandra; Boulton, Simon J.; Whitby, Matthew C.; Petersen-Mahrt, Svend K.

    2009-01-01

    Meiotic recombination enhances genetic diversity as well as ensures proper segregation of homologous chromosomes, requiring Spo11-initiated double-strand breaks (DSBs). DNA deaminases act on regions of single-stranded DNA and deaminate cytosine to uracil (dU). In the immunoglobulin locus, this lesion will initiate point mutations, gene conversion, and DNA recombination. To begin to delineate the effect of induced base lesions on meiosis, we analyzed the effect of expressing DNA deaminases (activation-induced deaminase, AID, and APOBEC3C) in germ cells. We show that meiotic dU:dG lesions can partially rescue a spo11Δ phenotype in yeast and worm. In rec12 Schizosaccharomyces pombe, AID expression increased proper chromosome segregation, thereby enhancing spore viability, and induced low-frequency meiotic crossovers. Expression of AID in the germ cells of Caenorhabditis elegans spo-11 induced meiotic RAD-51 foci formation and chromosomal bivalency and segregation, as well as an increase in viability. RNAi experiments showed that this rescue was dependent on uracil DNA-glycosylase (Ung). Furthermore, unlike ionizing radiation-induced spo-11 rescue, AID expression did not induce large numbers of DSBs during the rescue. This suggests that the products of DNA deamination and base excision repair, such as uracil, an abasic site, or a single-stranded nick, are sufficient to initiate and alter meiotic recombination in uni- and multicellular organisms. PMID:19237686

  14. Bacteria with ACC deaminase can promote plant growth and help to feed the world.

    PubMed

    Glick, Bernard R

    2014-01-20

    To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture.

  15. Various effects of fluorescent bacteria of the genus Pseudomonas containing ACC deaminase on wheat seedling growth.

    PubMed

    Magnucka, Elżbieta G; Pietr, Stanisław J

    2015-12-01

    The study evaluates the effect of rhizobacteria having 1-aminocyclopropane-1-carboxylate deaminase (ACCd) on the development of wheat seedlings. This enzyme has been proposed to play a key role in microbe-plant association. Three fluorescent pseudomonads containing this deaminase were selected from 70 strains of pseudomonads isolated from rhizosphere of wheat (Triticum aestivum L.) and rape (Brassica napus L.). These bacteria, varied significantly in the ability to both biosynthesize auxins and hydrolyze ACC. Among them, Pseudomonas brassicacearum subsp. brassicacearum strain RZ310 presented the highest activities of ACC deaminase during 96h of growth in liquid Dworkin and Foster (DF) salt medium. Additionally, this rape rhizosphere strain did not produce indoles. Two other isolates, Pseudomonas sp. PO283 and Pseudomonas sp. PO366, secreted auxins only in the presence of their precursor. Phylogenetic analysis of the 16S rRNA gene and four other protein-encoding genes indicated that these wheat rhizosphere isolates belonged to the fluorescent Pseudomonas group. Moreover, the effects of these strains on wheat seedling growth under in vitro conditions were markedly dependent on both their cell suspensions used to grain inoculation and nutrient conditions. Strains tested had beneficial influence on wheat seedlings mainly at low cell densities. In addition, access to nutrients markedly changed bacteria action on cereal growth. Their presence generally favored the positive effects of pseudomonads on length and the estimated biomasses of wheat coleoptiles. Despite these general rules, impacts of each isolate on the growth parameters of cereal seedlings were unique.

  16. 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

  17. 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.

  18. Lipolysis in diabetic adipocytes: differences in response to growth hormone and adenosine.

    PubMed

    Solomon, S S; Schwartz, Y; Rawlinson, T

    1987-09-01

    The sensitivity to lipolytic agents is altered in diabetic vs. control animals. Because of its role as a diabetogenic hormone and its ability to elicit lipolysis, GH was studied in isolated fat cells (IFC) from control and streptozotocin-diabetic (STZ-DM) rats. IFCs from the epididymal fat of 150 to 200-g normal and STZ-DM Holtzman rats were prepared by collagenase digestion. Lipolysis was measured by glycerol release after either incubation or perifusion with the following concentrations: epinephrine (EPI), 0.01-0.1 microM; theophylline, 0.01-1.0 mg/ml; adenosine deaminase (ADA), and bovine GH (bGH), 0.01-1.0 microgram/ml. Rats, rendered diabetic by STZ (65 mg/kg), were used on day 3. In a dose-response study comparing glycerol release from control and STZ-DM IFC, IFC were preincubated with 1.0 microgram/ml bGH and then incubated with varying concentrations of EPI or bGH. In STZ-DM, we noted increased lipolytic sensitivity to low concentrations of EPI or bGH. Furthermore, in perifusion, STZ-DM IFC did not require obligatory preincubation with bGH for optimal glycerol release. The addition of ADA increased glycerol release from incubated IFC (STZ-DM and controls). In both systems an enhanced lipolytic response to theophylline was seen in the presence of bGH in control and STZ-DM. It was thus concluded that IFC from normal animals do not respond to GH without preincubation. IFC from STZ-DM rats show a lipolytic response to GH without preincubation. Preincubation with GH increases the lipolytic response of IFC from STZ-DM to all lipolytic agents compared to control responses. In addition, ADA greatly enhanced lipolysis in IFC from STZ-DM compared to that in controls. Together these data demonstrate enhanced sensitivity to both lipolytic stimuli and adenosine suppression of lipolysis in IFC from STZ-DM. PMID:3622374

  19. [60]Fullerene derivative modulates adenosine and metabotropic glutamate receptors gene expression: a possible protective effect against hypoxia

    PubMed Central

    2014-01-01

    Background Glutamate, the main excitatory neurotransmitter, is involved in learning and memory processes but at higher concentration results excitotoxic causing degeneration and neuronal death. Adenosine is a nucleoside that exhibit neuroprotective effects by modulating of glutamate release. Hypoxic and related oxidative conditions, in which adenosine and metabotropic glutamate receptors are involved, have been demonstrated to contribute to neurodegenerative processes occurring in certain human pathologies. Results Human neuroblastoma cells (SH-SY5Y) were used to evaluate the long time (24, 48 and 72 hours) effects of a [60]fullerene hydrosoluble derivative (t3ss) as potential inhibitor of hypoxic insult. Low oxygen concentration (5% O2) caused cell death, which was avoided by t3ss exposure in a concentration dependent manner. In addition, gene expression analysis by real time PCR of adenosine A1, A2A and A2B and metabotropic glutamate 1 and 5 receptors revealed that t3ss significantly increased A1 and mGlu1 expression in hypoxic conditions. Moreover, t3ss prevented the hypoxia-induced increase in A2A mRNA expression. Conclusions As t3ss causes overexpression of adenosine A1 and metabotropic glutamate receptors which have been shown to be neuroprotective, our results point to a radical scavenger protective effect of t3ss through the enhancement of these neuroprotective receptors expression. Therefore, the utility of these nanoparticles as therapeutic target to avoid degeneration and cell death of neurodegenerative diseases is suggested. PMID:25123848

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth.

    PubMed

    Jalili, Farzad; Khavazi, Kazem; Pazira, Ebrahim; Nejati, Alireza; Rahmani, Hadi Asadi; Sadaghiani, Hasan Rasuli; Miransari, Mohammad

    2009-04-01

    Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.

  5. Lipopolysaccharide rapidly modifies adenosine receptor transcripts in murine and human macrophages: role of NF-kappaB in A(2A) adenosine receptor induction.

    PubMed

    Murphree, Lauren J; Sullivan, Gail W; Marshall, Melissa A; Linden, Joel

    2005-11-01

    The A(2A) adenosine receptor (A(2A)AR) mediates anti-inflammatory actions of adenosine in a variety of cell types. LPS (lipopolysaccharide) was reported to induce a small (<2-fold) increase in the expression of A(2A)AR mRNA in human monocytes and monocytic cell lines. We investigated the effects of LPS on the expression of adenosine receptor mRNAs in primary mouse IPMPhi (intraperitoneal macrophages), human macrophages and Wehi-3 cells. Treatment with 10 ng/ml LPS for 4 h produced a >100-fold increase in A(2A)AR mRNA. LPS-induced increases in mRNA for A(2A)AR and TNFalpha (tumour necrosis factor alpha) are reduced by 90% in IPMPhi pretreated with the NF-kappaB (nuclear factor kappaB) inhibitor, BAY 11-7082 {(E)3-[(4-methylphenyl)sulphonyl]-2-propenenitrile; 10 microM}. In Wehi-3 cells exposed to LPS, A(2A)AR and A(2B)AR transcripts are elevated by 290- and 10-fold respectively, the A(1)AR transcript is unchanged and the A(3)AR transcript is decreased by 67%. The induction of A(2A)AR mRNA by LPS is detectable after 1 h, reaches a peak at 6 h at 600 times control and remains elevated beyond 24 h. The ED50 (effective dose) of LPS is 2.3 ng/ml. A(2A)AR receptor number, measured by 125I-ZM241385 binding to whole cells, is undetectable in naïve cells and increases linearly at a rate of 23 receptors x cell(-1) x min(-1) to a B(max) of 348 fmol/mg (28000 receptors/cell) in 20 h. The increase in receptor number is correlated with an increase in the potency of an A(2A) agonist (4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester; referred to as ATL146e) to stimulate cAMP in these cells. After LPS pretreatment, the potency of the A(2A) agonist, ATL146e, to reduce TNFalpha release from IPMPhi was increased by 200-fold. The results support the hypothesis that regulation of adenosine receptor expression, especially up-regulation of the A(2A)AR, is part of a delayed feedback mechanism

  6. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    PubMed

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

    2016-01-01

    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

  7. Studies on the roles of ATP, adenosine and nitric oxide in mediating muscle vasodilatation induced in the rat by acute systemic hypoxia.

    PubMed

    Skinner, M R; Marshall, J M

    1996-09-01

    1. In Saffan-anaesthetized rats, we have further investigated the mechanisms underlying the vasodilatation induced by adenosine in skeletal muscle by acute systemic hypoxia (breathing 8% O2 for 5 min). 2. In eleven rats the nitric oxide (NO) synthesis inhibitor nitro-L-arginine methyl ester (L-NAME, 10 mg kg-1, i.v.) reduced the increase in femoral vascular conductance (FVC) induced by hypoxia by approximately 50%. L-NAME had similar effects on the increase in FVC induced by intra-arterial (I.A.) infusion of adenosine (at 1.2 mg kg-1 min-1 for 5 min via the tail artery) and by ATP (I.A., 1 mg kg-1 min-1 for 5 min). Subsequent administration of the adenosine receptor antagonist 8-sulphophenyl theophylline (8-SPT, 20 mg kg-1, i.v.) virtually abolished the adenosine- and ATP-induced increase in FVC. 3. In a further nine rats, 8-SPT reduced the increase in FVC induced by hypoxia by approximately 50%. This remaining increase in FVC was substantially reduced by L-NAME. 4. In an additional nine rats, alpha,beta-methyleneADP (160 micrograms kg-1, i.v.) which inhibits the 5'-ectonucleotidase that degrades AMP to adenosine, reduced the peripheral vasodilatation (fall in arterial blood pressure, ABP) induced by ATP infusion, but had no effect on the increase in FVC or decrease in ABP evoked by systemic hypoxia. 5. These results provide the first evidence that the muscle vasodilatation induced by adenosine during systemic hypoxia is mainly dependent on NO synthesis. They also suggest that adenosine is released as such rather than being formed extracellularly from AMP. Given evidence that extraluminal adenosine acts in an NO-independent fashion we propose that hypoxia releases adenosine from the endothelium. Our results also indicate that hypoxia induces muscle vasodilatation that is adenosine independent but NO dependent: they allow the possibility that this is partly mediated by ATP released from the endothelium. PMID:8887765

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

    PubMed

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

    2016-07-01

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

  9. 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

  10. The role of adenosine in the early respiratory and cardiovascular changes evoked by chronic hypoxia in the rat

    PubMed Central

    Walsh, Martin P; Marshall, Janice M

    2006-01-01

    Experiments were performed on anaesthetized normoxic (N) rats and chronically hypoxic rats that had been exposed to 12% O2 for 1, 3 or 7 days (1, 3 or 7CH rats). The adenosine A1 receptor antagonist DPCPX did not affect the resting hyperventilation of 1–7CH rats breathing 12% O2 and increased resting heart rate (HR) in 1CH rats only. DPCPX partially restored the decreased baseline arterial pressure (ABP) and increased femoral vascular conductance (FVC) of 1 and 3CH rats, but had no effect in N or 7CH rats. DPCPX also attenuated the decrease in arterial blood pressure (ABP) and increase in FVC evoked by acute hypoxia in N and 1–7CH rats. The non-selective adenosine receptor antagonist 8-SPT had no further effect on baselines or cardiovascular responses to acute hypoxia, but attenuated the hypoxia-evoked increase in respiratory frequency in 1–7CH rats. In N, and 1 and 3CH rats, the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine had no effect on baselines or increases in FVC evoked by acetylcholine. We propose: (i) that tonically released adenosine acting on A1 receptors reduces HR in 1CH rats and stimulates endothelial NOS in 1 and 3CH rats to decrease ABP and increase FVC, the remaining NO-dependent tonic vasodilatation being independent of iNOS activity; (ii) that in 7CH rats, tonic adenosine release has waned; (iii) that in 1–7CH rats, adenosine released by acute hypoxia stimulates A1 but not A2 receptors to produce muscle vasodilatation, and stimulates carotid body A2 receptors to increase respiration. PMID:16690710

  11. Postsynaptic Adenosine A2A Receptors Modulate Intrinsic Excitability of Pyramidal Cells in the Rat Basolateral Amygdala

    PubMed Central

    Rau, Andrew R.; Ariwodola, Olusegun J.

    2015-01-01

    Background: The basolateral amygdala plays a critical role in the etiology of anxiety disorders and addiction. Pyramidal neurons, the primary output cells of this region, display increased firing following exposure to stressors, and it is thought that this increase in excitability contributes to stress responsivity and the expression of anxiety-like behaviors. However, much remains unknown about the underlying mechanisms that regulate the intrinsic excitability of basolateral amygdala pyramidal neurons. Methods: Ex vivo gramicidin perforated patch recordings were conducted in current clamp mode where hyper- and depolarizing current steps were applied to basolateral amygdala pyramidal neurons to assess the effects of adenosine A2A receptor modulation on intrinsic excitability. Results: Activation of adenosine A2A receptors with the selective A2A receptor agonist CGS-21680 significantly increased the firing rate of basolateral amygdala pyramidal neurons in rat amygdala brain slices, likely via inhibition of the slow afterhyperpolarization potential. Both of these A2A receptor-mediated effects were blocked by preapplication of a selective A2A receptor antagonist (ZM-241385) or by intra-pipette infusion of a protein kinase A inhibitor, suggesting a postsynaptic locus of A2A receptors on basolateral amygdala pyramidal neurons. Interestingly, bath application of the A2A receptor antagonist alone significantly attenuated basolateral amygdala pyramidal cell firing, consistent with a role for tonic adenosine in the regulation of the intrinsic excitability of these neurons. Conclusions: Collectively, these data suggest that adenosine, via activation of A2A receptors, may directly facilitate basolateral amygdala pyramidal cell output, providing a possible balance for the recently described inhibitory effects of adenosine A1 receptor activation on glutamatergic excitation of basolateral amygdala pyramidal cells. PMID:25716780

  12. Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

    PubMed Central

    Robles-Flores, M; Allende, G; Piña, E; García-Sáinz, J A

    1995-01-01

    The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity. Images Figure 2 PMID:8554517

  13. The effect of native and ACC deaminase-containing Azospirillum brasilense Cd1843 on the rooting of carnation cuttings.

    PubMed

    Li, Qiaosi; Saleh-Lakha, Saleema; Glick, Bernard R

    2005-06-01

    Carnation cuttings treated with non-transformed and 1-aminocyclopropane (ACC) deaminase-containing Azospirillum brasilense Cd1843 produced significantly more roots than untreated controls and fewer roots than cuttings treated with 0.1% indolebutyric acid (IBA). The roots produced by cuttings treated with ACC deaminase-containing Azospirillum brasilense Cd1843 were the longest roots resulting from any of the treatments, followed by non-transformed Azospirillum brasilense Cd1843, 0.1% IBA, and treatment with water. The results are interpreted in terms of a previously proposed model of bacterial promotion of plant growth by ACC deaminase and indoleacetic acid, and may have implications for the use of plant growth-promoting bacteria in the flower industry. PMID:16121231

  14. Yin and yang of cytidine deaminase roles in clinical response to azacitidine in the elderly: a pharmacogenetics tale.

    PubMed

    Fanciullino, Raphaelle; Mercier, Cédric; Serdjebi, Cindy; Venton, Geoffroy; Colle, Julien; Fina, Frédéric; Ouafik, L'Houcine; Lacarelle, Bruno; Ciccolini, Joseph; Costello, Régis

    2015-11-01

    Azacitidine is a mainstay for treating hematological disorders. Azacitidine is metabolized by cytidine deaminase, coded by a highly polymorphic gene. Here, we present two elderly patients with opposite clinical outcomes after azacitidine treatment. First, an acute myeloid leukemia patient showed life-threatening toxicities, but outstanding complete remission, after a single round of azacitidine. Further investigations showed that this patient was cytidine deaminase 79A>C (rs2072671) homozygous with a marked deficient phenotype. Next, a chronic myelomonocytic leukemia patient displayed complete lack of response despite several cycles of azacitidine. This patient had a rapid-deaminator phenotype linked to the -31delC deletion (rs3215400). These polymorphisms lead to opposite clinical outcomes in patients with myelodysplastic syndromes treated with azacitidine, thus suggesting that determining cytidine deaminase status could help to forecast clinical outcome.

  15. Biochemistry and genetics of ACC deaminase: a weapon to “stress ethylene” produced in plants

    PubMed Central

    Singh, Rajnish P.; Shelke, Ganesh M.; Kumar, Anil; Jha, Prabhat N.

    2015-01-01

    1-aminocyclopropane-1-carboxylate deaminase (ACCD), a pyridoxal phosphate-dependent enzyme, is widespread in diverse bacterial and fungal species. Owing to ACCD activity, certain plant associated bacteria help plant to grow under biotic and abiotic stresses by decreasing the level of “stress ethylene” which is inhibitory to plant growth. ACCD breaks down ACC, an immediate precursor of ethylene, to ammonia and α-ketobutyrate, which can be further metabolized by bacteria for their growth. ACC deaminase is an inducible enzyme whose synthesis is induced in the presence of its substrate ACC. This enzyme encoded by gene AcdS is under tight regulation and regulated differentially under different environmental conditions. Regulatory elements of gene AcdS are comprised of the regulatory gene encoding LRP protein and other regulatory elements which are activated differentially under aerobic and anaerobic conditions. The role of some additional regulatory genes such as AcdB or LysR may also be required for expression of AcdS. Phylogenetic analysis of AcdS has revealed that distribution of this gene among different bacteria might have resulted from vertical gene transfer with occasional horizontal gene transfer (HGT). Application of bacterial AcdS gene has been extended by developing transgenic plants with ACCD gene which showed increased tolerance to biotic and abiotic stresses in plants. Moreover, distribution of ACCD gene or its homolog's in a wide range of species belonging to all three domains indicate an alternative role of ACCD in the physiology of an organism. Therefore, this review is an attempt to explore current knowledge of bacterial ACC deaminase mediated physiological effects in plants, mode of enzyme action, genetics, distribution among different species, ecological role of ACCD and, future research avenues to develop transgenic plants expressing foreign AcdS gene to cope with biotic and abiotic stressors. Systemic identification of regulatory circuits

  16. 2-Alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation.

    PubMed

    Cristalli, G; Volpini, R; Vittori, S; Camaioni, E; Monopoli, A; Conti, A; Dionisotti, S; Zocchi, C; Ongini, E

    1994-05-27

    A series of new 2-alkynyl and 2-cycloalkynyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D- ribofuranuronamide (1, HE-NECA), bearing hydroxy, amino, chloro, and cyano groups in the side chain, were synthesized. The compounds were studied in binding and functional assays to assess their potency for the A2 compared to A1 adenosine receptor. The presence of an alpha-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2 agonist potency, leading to compounds endowed with sub-nanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2 selectivity. From functional experiments the 4-hydroxy-1-butynyl (6) and the 4-(2-tetrahydro-2H-pyranyloxy)-1-butynyl (16) derivatives appear to be very potent in inducing vasorelaxation without appreciable effect on heart rate. The new compounds were also tested as inhibitors of platelet aggregation induced by ADP. Introduction of an alpha-hydroxyl group in the alkynyl side chain caused a greater increase in antiaggregatory activity than either NECA or HE-NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. The presence of an alpha-quaternary carbon such as the 3-hydroxy-3,5-dimethyl-1-hexynyl (12) and the 3-hydroxy-3-phenyl-1-butynyl (15) derivatives markedly reduced the antiaggregatory potency without affecting the A2 affinity. The hydrophobicity index (k') of the new nucleosides barely correlated with the binding data, whereas high k' values were associated with increased A2 vs A1 selectivity but with reduced activity in all functional assays. Some of the compounds synthesized possess interesting pharmacological properties. Compounds having an appropriate balance between vasorelaxation and antiplatelet activity, if confirmed in vivo, deserve further development for the treatments of cardiovascular disorders.

  17. 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.

  18. Direct deamination of AMP, ADP, ATP and NADH by non-specific adenylate deaminase in the foot muscle of the snail Helix pomatia.

    PubMed Central

    Stankiewicz, A J

    1983-01-01

    Homogeneous adenylate deaminase from snail foot muscle deaminated 5'-AMP, 5'-ADP, 5'-ATP and NADH with similar velocity and affinity to all substrates. At millimolar concentration NAD+ was also deaminated to a comparable extent, but NADP+, NADPH and FAD were not substrates for the snail enzyme. The amount of deaminase activity per g of fresh tissue is 5-10 times greater than in the muscle of any other species studied. The activity of the snail deaminase is regulated by pH, KCl and buffer concentrations, and Pi; however, regulation seems to be very poor in comparison with that of muscle deaminases from other species, specific to 5'-AMP. Snail enzyme appears as the first animal deaminase so far described that has such characteristics. It offers also some opportunities as an analytical tool as a consequence of its very high affinity toward adenylates. PMID:6626180

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

    PubMed Central

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

    2016-01-01

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

  20. Modulation of AMP deaminase in rat hearts subjected to ischemia and reperfusion by purine riboside.

    PubMed

    Borkowski, T; Lipinski, M; Kaminski, R; Krzyminska-Stasiuk, E; Langowska, M; Raczak, G; Slominska, E M; Smolenski, R T

    2008-06-01

    Changes in AMP deaminase (AMPD) activity influence heart function and progression of heart disease, but the underlying mechanism is unknown. We evaluated the effect of purine riboside (Purr) on the activity of AMPD in perfused rat hearts and in isolated rat cardiomyocytes. Brief perfusion of the pre-ischemic heart with 200 micro M Purr resulted in activation of AMPD, more pronounced degradation of the adenine nucleotides, and reduced recovery of the adenine nucleotide pool during reperfusion. Brief incubation of rat cardiomyocytes with 200 micro M Purr also activated AMPD, while prolonged exposure resulted in enzyme inhibition. We conclude that Purr activates AMPD, whereas metabolites of this compound may inhibit the enzyme.

  1. Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

    NASA Technical Reports Server (NTRS)

    Lieberman, M. M.; Lanyi, J. K.

    1972-01-01

    The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

  2. Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.

    PubMed

    Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

    2013-12-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection.

  3. Significance of the d-Serine-Deaminase and d-Serine Metabolism of Staphylococcus saprophyticus for Virulence

    PubMed Central

    Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V.; Hultgren, Scott; Gatermann, Sören G.

    2013-01-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a d-serine-deaminase (DsdA). As d-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the d-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that d-serine-deaminase or the ability to respond to or to metabolize d-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular d-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high d-serine concentrations; however, its d-serine metabolism has not been described. The activity of the d-serine-deaminase was verified by analyzing the formation of pyruvate from d-serine in different strains with and without d-serine-deaminase. Cocultivation experiments were performed to show that d-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of d-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of d-serine. In addition, we show that S. saprophyticus is able to use d-serine as the sole carbon source, but interestingly, d-serine had a negative effect on growth when glucose was also present. Taken together, d-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of d-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection. PMID:24082071

  4. Developmental forms of human skeletal-muscle AMP deaminase. The kinetic and regulatory properties of the enzyme.

    PubMed Central

    Kaletha, K; Nowak, G

    1988-01-01

    AMP deaminase isoforms from human skeletal muscle can be separated chromatographically [Kaletha, Spychała & Nowak (1987) Experientia 43, 440-443]. In adult tissue nearly all the AMP deaminase activity was eluted from phosphocellulose with 0.75 M-KCl ('adult' isoform), and the remaining activity could be eluted with 2.0 M-KCl. Conversely, most of the AMP deaminase activity from 11-week-old fetal tissue was eluted from phosphocellulose with 2.0 M-KCl ('fetal' isoform). In the present paper the kinetic and regulatory properties of AMP deaminase extracted from 11- and 16-week-old fetal skeletal muscle are reported. The two isoforms from 11-week-old human fetus differed distinctly in these properties. The 'fetal' isoform had about 5-fold higher half-saturation constant (S0.5) value than the 'adult' form. It was also more sensitive to the influence of some important regulatory ligands (ADP, ATP and Pi), and exhibited a different pH/activity profile. The 'adult' isoform of AMP deaminase from fetal muscle and the enzyme from mature muscle possessed similar kinetic and regulatory properties. This isoform seems not to be subject to any major modifications during further ontogenesis. This is not true, however, for the 'fetal' isoform. In the muscle of 16-week-old human fetus, the 'fetal' isoform showed a peculiar, biphasic, type of substrate-saturation kinetics. This phenomenon may reflect appearance of the next, developmentally programmed, isoform of human skeletal-muscle AMP deaminase. PMID:3342010

  5. 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

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

    PubMed Central

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

    2015-01-01

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

  7. Selective Phosphodiesterase 4B Inhibitors: A Review

    PubMed Central

    Azam, Mohammed Afzal; Tripuraneni, Naga Srinivas

    2014-01-01

    Abstract Phosphodiesterase 4B (PDE4B) is a member of the phosphodiesterase family of proteins that plays a critical role in regulating intracellular levels of cyclic adenosine monophosphate (cAMP) by controlling its rate of degradation. It has been demonstrated that this isoform is involved in the orchestra of events which includes inflammation, schizophrenia, cancers, chronic obstructive pulmonary disease, contractility of the myocardium, and psoriatic arthritis. Phosphodiesterase 4B has constituted an interesting target for drug development. In recent years, a number of PDE4B inhibitors have been developed for their use as therapeutic agents. In this review, an up-to-date status of the inhibitors investigated for the inhibition of PDE4B has been given so that this rich source of structural information of presently known PDE4B inhibitors could be helpful in generating a selective and potent inhibitor of PDE4B. PMID:25853062

  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 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

  10. 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.

  11. 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

  12. Ab Initio ONIOM-Molecular Dynamics (MD) Study on the Deamination Reaction by Cytidine Deaminase

    SciTech Connect

    Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

    2007-08-23

    We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determing step is the release of the NH3 molecule. TM and MA were supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.

  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. Heat shock protein 70 (Hsp70)-stimulated deoxycytidine deaminases from a human lymphoma cell but not the activation-induced cytidine deaminase (AID) from Ramos 6.4 human Burkitt's lymphoma cells.

    PubMed

    Bases, Robert

    2011-01-01

    Deoxycytidine deaminase enzyme activity was reduced in lysates of human leukemic THP1 cells 24 h after transfection with siRNA designed to inhibit cell synthesis of heat shock protein 70 (Hsp70)1a and Hsp701b. The cytidine deaminase enzyme activity from the cell lysates was purified from an affinity column which contained bound single-stranded oligodeoxycytidylic acid. Deficient enzyme activity in certain elution fractions from the siRNA-transfected cells was restored by including recombinant HSP 70 in the assays. Enzyme activity in some other fractions was increased after siRNA transfection. Activation-induced cytidine deaminase (AID) is a central factor in the immune response. A more specific assay for AID was used to study the influence of Hsp70 on AID activity. Unlike Hsp70's ability to stimulate certain enzymes of DNA base excision repair and other cytidine deaminases, it had little effect on AID activity in vitro, or was weakly inhibitory.

  15. 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

  16. 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.

  17. Transfected adenosine A1 receptor-mediated modulation of thrombin-stimulated phospholipase C and phospholipase A2 activity in CHO cells.

    PubMed

    Dickenson, J M; Hill, S J

    1997-02-19

    Thrombin receptor activation in Chinese hamster ovary (CHO) cells stimulates the hydrolysis of inositol phospholipids and the release of arachidonic acid. Our previous studies have shown that activation of the human transfected adenosine A1 receptor in CHO cells (CHO-A1) potentiates the accumulation of inositol phosphates elicited by endogenous P2U purinoceptors and CCKA receptors. In this study we have investigated whether adenosine A1 receptor activation can modulate thrombin-stimulated arachidonic acid release and/or inositol phospholipid hydrolysis in CHO-A1 cells. Thrombin stimulated [3H]arachidonic acid release and total [3H]inositol phosphate accumulation in CHO-A1 cells. Both these responses to thrombin were were insensitive to pertussis toxin. The protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), potentiated thrombin-stimulated [3H]arachidonic acid. In marked contrast, PMA inhibited thrombin-stimulated [3H]inositol phosphate accumulation. The selective protein kinase C inhibitor Ro 31-8220 (3-¿1-[3-(2-isothioureido)propyl] indol-3-yl¿-4-(1-methylindol-3-yl)-3-pyrrolin-2,5-dione) had no effect on thrombin-stimulated [3H]arachidonic acid release but reversed the potentiation of thrombin-stimulated [3H]arachidonic acid release elicited by PMA. The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) augmented the release of [3H]arachidonic acid produced by thrombin. Co-activation of the adenosine A1 receptor also potentiated thrombin-stimulated [3H]inositol phosphate accumulation. The synergistic interactions between the adenosine A1 receptor and thrombin were abolished in pertussis-toxin-treated cells. The potentiation of [3H]arachidonic acid release by CPA was blocked by the protein kinase C inhibitors Ro 31-8220 and GF 109203X (3-[1-[3-(dimethylamino)propyl]-1 H-indol-3-yl]-4-(1 H-indol-3-yl)- 1H-pyrrole-2,5-dione). In conclusion, thrombin receptor activation in CHO-A1 cells stimulates the accumulation of [3H

  18. Uracil DNA Glycosylase Is Dispensable for Human Immunodeficiency Virus Type 1 Replication and Does Not Contribute to the Antiviral Effects of the Cytidine Deaminase Apobec3G

    PubMed Central

    Kaiser, Shari M.; Emerman, Michael

    2006-01-01

    It is well established that many host factors are involved in the replication of human immunodeficiency virus (HIV) type 1. One host protein, uracil DNA glycosylase 2 (UNG2), binds to multiple viral proteins and is packaged into HIV type 1 virions. UNG initiates the removal of uracils from DNA, and this has been proposed to be important both for reverse transcription and as a mediator to the antiviral effect of virion-incorporated Apobec3G, a cytidine deaminase that generates numerous uracils in the viral DNA during virus replication. We used a natural human UNG−/− cell line as well as cells that express a potent catalytic active-site inhibitor of UNG to assess the effects of removing UNG activity on HIV infectivity. In both cases, we find UNG2 activity and protein to be completely dispensable for virus replication. Moreover, we find that virion-associated UNG2 does not affect the loss of infectivity caused by Apobec3G. PMID:16378989

  19. ATTENUATION OF ISCHEMIC LIVER INJURY BY AUGMENTATION OF ENDOGENOUS ADENOSINE1,2

    PubMed Central

    Todo, Satoru; Zhu, Yue; Zhang, Shimin; Jin, Maeng Bong; Ishizaki, Naoki; Tanaka, Hiromu; Subbotin, Vladimir; Starzl, Thomas E.

    2010-01-01

    Hepatic grafts from non-heartbeating donors may alleviate the organ shortage, but they inherently suffer from warm ischemia. In the present study, we tested our hypothesis that augmentation of endogenous adenosine by inhibition of nucleoside transport with R75231 attenuates ischemic liver injury. Adult female beagle dogs underwent 2-hr hepatic vascular exclusion with venovenous bypass. R75231 was given to the animals by continuous intravenous infusion for 30 min before ischemia at a dose of 0.1 mg/kg (Group 2, n=6), 0.05 mg/kg (Group 3, n=6), or 0.025 mg/kg (Group 4, n=6). Nontreated animals were used as the control (Group 1, n=10). Animal survival, hepatic tissue blood flow, liver function, and histopathology were analyzed. Two-week animal survival was 30% in Group 1, 88% in Group 2, 100% in Group 3, and 100% in Group 4. Postreperfusion hepatic tissue blood flow was markedly improved by the treatment. Treatment significantly attenuated liver enzyme release, lipid peroxidation, and changes in adenine nucleotides and purine catabolites. Structural abnormality of the liver after reperfusion was markedly improved by R75231 treatment, showing better architecture and less neutrophil infiltration. Preischemic administration of a nucleoside transport inhibitor ameliorated ischemic liver injury due to the positive effects of augmented endogenous adenosine, and is applicable clinically when the liver is procured from a controlled non-heartbeating donor. PMID:9020320

  20. Increased 1-aminocyclopropane-1-carboxylate deaminase activity enhances Agrobacterium tumefaciens-mediated gene delivery into plant cells

    PubMed Central

    Someya, Tatsuhiko; Nonaka, Satoko; Nakamura, Kouji; Ezura, Hiroshi

    2013-01-01

    Agrobacterium-mediated transformation is a useful tool for the genetic modification in plants, although its efficiency is low for several plant species. Agrobacterium-mediated transformation has three major steps in laboratory-controlled experiments: the delivery of T-DNA into plant cells, the selection of transformed plant cells, and the regeneration of whole plants from the selected cells. Each of these steps must be optimized to improve the efficiency of Agrobacterium-mediated plant transformation. It has been reported that increasing the number of cells transformed by T-DNA delivery can improve the frequency of stable transformation. Previously, we demonstrated that a reduction in ethylene production by plant cells during cocultivation with A. tumefaciens-expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase resulted in increased T-DNA delivery into the plant cells. In this study, to further improve T-DNA delivery by A. tumefaciens, we modified the expression cassette of the ACC deaminase gene using vir gene promoter sequences. The ACC deaminase gene driven by the virD1 promoter was expressed at a higher level, resulting in a higher ACC deaminase activity in this A. tumefaciens strain than in the strain with the lac promoter used in a previous study. The newly developed A. tumefaciens strain improves the delivery of T-DNA into Solanum lycopersicum (tomato) and Erianthus ravennae plants and thus may be a powerful tool for the Agrobacterium-mediated genetic engineering of plants. PMID:24000136

  1. Presymptomatic and symptomatic ALS SOD1(G93A) mice differ in adenosine A1 and A2A receptor-mediated tonic modulation of neuromuscular transmission.

    PubMed

    Nascimento, Filipe; Sebastião, Ana M; Ribeiro, Joaquim A

    2015-12-01

    Amyotrophic lateral sclerosis (ALS) is a disease leading to neuromuscular transmission impairment. A2A adenosine receptor (A2AR) function changes with disease stage, but the role of the A(1) receptors (A1Rs) is unknown and may have a functional cross-talk with A2AR. The role of A1R in the SOD1(G93A) mouse model of ALS in presymptomatic (4-6 weeks old) and symptomatic (12-14 weeks old) phases was investigated by recording endplate potentials (EPPs), miniature endplate potentials (MEPPs), and quantal content (q.c.) of EPPs, from Mg(2+) paralyzed hemidiaphragm preparations. In presymptomatic mice, the A1R agonist, N (6)-cyclopentyladenosine (CPA) (50 nM), decreased mean EPP amplitude, MEPP frequency, and q.c. of EPPs, an effect quantitatively similar to that in age-matched wild-type (WT) mice. However, coactivation of A2AR with CGS 21680 (5 nM) prevented the effects of CPA in WT mice but not in presymptomatic SOD1(G93A) mice, suggestive of A1R/A2AR cross-talk disruption in this phase of ALS. DPCPX (50 nM) impaired CGS 21680 facilitatory action on neuromuscular transmission in WT but not in presymptomatic mice. In symptomatic animals, CPA only inhibited transmission if added in the presence of adenosine deaminase (ADA, 1 U/mL). ADA and DPCPX enhanced more transmission in symptomatic mice than in age-matched WT mice, suggestive of increase in extracellular adenosine during the symptomatic phase of ALS. The data documents that at the neuromuscular junction of presymptomatic SOD1(G93A) mice, there is a loss of A1R-A2AR functional cross-talk, while in symptomatic mice there is increased A1R tonic activation, and that with disease progression, changes in A1R-mediated adenosine modulation may act as aggravating factors during the symptomatic phase of ALS.

  2. Role of A3 adenosine receptor in diabetic neuropathy.

    PubMed

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

    2016-10-01

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

  3. Sesamin protects against renal ischemia reperfusion injury by promoting CD39-adenosine-A2AR signal pathway in mice.

    PubMed

    Li, Ke; Gong, Xia; Kuang, Ge; Jiang, Rong; Wan, Jingyuan; Wang, Bin

    2016-01-01

    Ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury with high morbidity and mortality due to limited therapy. Here, we examine whether sesamin attenuates renal IRI in an animal model and explore the underlying mechanisms. Male mice were subjected to right renal ischemia for 30 min followed by reperfusion for 24 h with sesamin (100 mg/kg) during which the left kidney was removed. Renal damage and function were assessed subsequently. The results showed that sesamin reduced kidney ischemia reperfusion injury, as assessed by decreased serum creatinine (Scr) and Blood urea nitrogen (BUN), alleviated tubular damage and apoptosis. In addition, sesamin inhibited neutrophils infiltration and pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β production in IR-preformed kidney. Notably, sesamin promoted the expression of CD39, A2A adenosine receptor (A2AAR), and A2BAR mRNA and protein as well as adenosine production. Furthermore, CD39 inhibitor or A2AR antagonist abolished partly the protection of sesamin in kidney IRI. In conclusion, sesamin could effectively protect kidney from IRI by inhibiting inflammatory responses, which might be associated with promoting the adenosine-CD39-A2AR signaling pathway. PMID:27347331

  4. [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

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

    PubMed

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

    2007-09-01

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

  6. 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.

  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.

  8. 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

  9. 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

  10. 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

  11. Activation of multiple sites by adenosine analogues in the rat isolated aorta.

    PubMed Central

    Prentice, D. J.; Hourani, S. M.

    1996-01-01

    1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100

  12. Thymine-based molecular beacon for sensing adenosine based on the inhibition of S-adenosylhomocysteine hydrolase activity.

    PubMed

    Nieh, Chih-Chun; Tseng, Wei-Lung

    2014-11-15

    This study presents a thymine (T)-based molecular beacon (MB) used for probing S-adenosylhomocysteine hydrolase (SAHH)-catalyzed hydrolysis of S-adenosylhomocysteine (SAH) and for sensing adenosine based on the inhibition of SAHH activity. The designed MB (T8-MB-T8) contained a 15-mer loop and a stem that consisted of a pair of 8-mer T bases, a fluorophore unit at the 5'-end, and a quencher unit at the 3'-end. In the presence of Hg(2+), a change in the conformation of T8-MB-T8 placed the fluorophore unit and the quencher in proximity to each other and caused collisional quenching of fluorescence between them. The Hg(2+)-induced fluorescence quenching of T8-MB-T8 occurred because the Hg(2+) induced T-T mismatches to form stable T-Hg(2+)-T coordination in the MB stem. SAHH catalyzed the hydrolysis of SAH to produce homocysteine. The generated homocysteine enabled the Hg(2+) to be removed from a hairpin-shaped T8-MB-T8 through the formation of a strong Hg(2+)-S bond, leading to the restoration of its fluorescence. The T8-MB-T8 · Hg(2+) probe showed a limit of detection for SAHH of 4 units L(-1) (approximately 0.24 nM) and was reusable for detecting the SAHH/SAH system. Because adenosine was an effective SAHH activity inhibitor, the T8-MB-T8 · Hg(2+) probe combining the SAHH and SAH systems was used for sensitive and selective detection of adenosine in urine without the interference of other adenosine analogs.

  13. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  14. Epigenetic Function of Activation-Induced Cytidine Deaminase and Its Link to Lymphomagenesis

    PubMed Central

    Dominguez, Pilar M.; Shaknovich, Rita

    2014-01-01

    Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of immunoglobulin (Ig) genes during B cell maturation and immune response. Expression of AID is tightly regulated due to its mutagenic and recombinogenic potential, which is known to target not only Ig genes, but also non-Ig genes, contributing to lymphomagenesis. In recent years, a new epigenetic function of AID and its link to DNA demethylation came to light in several developmental systems. In this review, we summarize existing evidence linking deamination of unmodified and modified cytidine by AID to base-excision repair and mismatch repair machinery resulting in passive or active removal of DNA methylation mark, with the focus on B cell biology. We also discuss potential contribution of AID-dependent DNA hypomethylation to lymphomagenesis. PMID:25566255

  15. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes

    PubMed Central

    Kato, Lucia; Begum, Nasim A.; Burroughs, A. Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O.; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-01-01

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites. PMID:22308462

  16. Markerless Gene Deletion with Cytosine Deaminase in Thermus thermophilus Strain HB27

    PubMed Central

    Wang, Lei; Hoffmann, Jana; Watzlawick, Hildegard

    2015-01-01

    We developed a counterselectable deletion system for Thermus thermophilus HB27 based on cytosine deaminase (encoded by codA) from Thermaerobacter marianensis DSM 12885 and the sensitivity of T. thermophilus HB27 to the antimetabolite 5-fluorocytosine (5-FC). The deletion vector comprises the pUC18 origin of replication, a thermostable kanamycin resistance marker functional in T. thermophilus HB27, and codA under the control of a constitutive putative trehalose promoter from T. thermophilus HB27. The functionality of the system was demonstrated by deletion of the bglT gene, encoding a β-glycosidase, and three carotenoid biosynthesis genes, CYP175A1, crtY, and crtI, from the genome of T. thermophilus HB27. PMID:26655764

  17. Nonimmunoglobulin target loci of activation-induced cytidine deaminase (AID) share unique features with immunoglobulin genes.

    PubMed

    Kato, Lucia; Begum, Nasim A; Burroughs, A Maxwell; Doi, Tomomitsu; Kawai, Jun; Daub, Carsten O; Kawaguchi, Takahisa; Matsuda, Fumihiko; Hayashizaki, Yoshihide; Honjo, Tasuku

    2012-02-14

    Activation-induced cytidine deaminase (AID) is required for both somatic hypermutation and class-switch recombination in activated B cells. AID is also known to target nonimmunoglobulin genes and introduce mutations or chromosomal translocations, eventually causing tumors. To identify as-yet-unknown AID targets, we screened early AID-induced DNA breaks by using two independent genome-wide approaches. Along with known AID targets, this screen identified a set of unique genes (SNHG3, MALAT1, BCL7A, and CUX1) and confirmed that these loci accumulated mutations as frequently as Ig locus after AID activation. Moreover, these genes share three important characteristics with the Ig gene: translocations in tumors, repetitive sequences, and the epigenetic modification of chromatin by H3K4 trimethylation in the vicinity of cleavage sites.

  18. Activation-Induced Cytidine Deaminase Links Ovulation-Induced Inflammation and Serous Carcinogenesis.

    PubMed

    Sapoznik, Stav; Bahar-Shany, Keren; Brand, Hadar; Pinto, Yishay; Gabay, Orshay; Glick-Saar, Efrat; Dor, Chen; Zadok, Oranit; Barshack, Iris; Zundelevich, Adi; Gal-Yam, Einav Nili; Yung, Yuval; Hourvitz, Ariel; Korach, Jacob; Beiner, Mario; Jacob, Jasmine; Levanon, Erez Y; Barak, Michal; Aviel-Ronen, Sarit; Levanon, Keren

    2016-02-01

    In recent years, the notion that ovarian carcinoma results from ovulation-induced inflammation of the fallopian tube epithelial cells (FTECs) has gained evidence. However, the mechanistic pathway for this process has not been revealed yet. In the current study, we propose the mutator protein activation-induced cytidine deaminase (AID) as a link between ovulation-induced inflammation in FTECs and genotoxic damage leading to ovarian carcinogenesis. We show that AID, previously shown to be functional only in B lymphocytes, is expressed in FTECs under physiological conditions, and is induced in vitro upon ovulatory-like stimulation and in vivo in carcinoma-associated FTECs. We also report that AID activity results in epigenetic, genetic and genomic damage in FTECs. Overall, our data provides new insights into the etiology of ovarian carcinogenesis and may set the ground for innovative approaches aimed at prevention and early detection. PMID:26936395

  19. Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability.

    PubMed

    Zanna, H; Nok, A J; Ibrahim, S; Inuwa, H M

    2013-12-01

    Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10 h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K(M) values of 0.60 mM and 0.65 mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12 h with a half-life of 5.80 h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.

  20. Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.

    PubMed

    Shibu, Marthandam Asokan; Yang, Hsueh-Hui; Lo, Chaur-Tsuen; Lin, Hong-Shin; Liu, Shu-Ying; Peng, Kou-Cheng

    2014-02-26

    Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine β-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 μM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 μM and 2.072 mM for cytosine β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.

  1. 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

  2. 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

  3. Platelet Inhibitors.

    PubMed

    Shifrin, Megan M; Widmar, S Brian

    2016-03-01

    Antithrombotic medications have become standard of care for management of acute coronary syndrome. Platelet adhesion, activation, and aggregation are essential components of platelet function; platelet-inhibiting medications interfere with these components and reduce incidence of thrombosis. Active bleeding is a contraindication for administration of platelet inhibitors. There is currently no reversal agent for platelet inhibitors, although platelet transfusion may be used to correct active bleeding after administration of platelet inhibitors. PMID:26897422

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Corrosion inhibitor

    SciTech Connect

    Wisotsky, M.J.; Metro, S.J.

    1989-10-31

    A corrosion inhibitor for use in synthetic ester lubricating oils is disclosed. It comprises an effective amount of: at least one aromatic amide; and at least one hydroxy substituted aromatic compound. The corrosion inhibitor thus formed is particularly useful in synthetic ester turbo lubricating oils.

  9. Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants

    SciTech Connect

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong; Wei, Shi-Cheng; Liang, Yu-He Su, Xiao-Dong

    2007-09-01

    A glucosamine 6-phosphate deaminase homologue from S. mutans was expressed, purified and crystallized. Diffraction data have been collected to 2.4 Å resolution. The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 Å resolution and belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å.

  10. Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203.

    PubMed

    Viterbo, Ada; Landau, Udi; Kim, Sofia; Chernin, Leonid; Chet, Ilan

    2010-04-01

    1-aminocyclopropane-1-carboxylate (ACC) deaminase activity was evaluated in the biocontrol and plant growth-promoting fungus Trichoderma asperellum T203. Fungal cultures grown with ACC as the sole nitrogen source showed high enzymatic activity. The enzyme encoding gene (Tas-acdS) was isolated, and an average 3.5-fold induction of the gene by 3 mM ACC was detected by real-time PCR. Escherichia coli bacteria carrying the intron-free cDNA of Tas-acdS cloned into the vector pAlter-EX1 under the control of the tac promoter revealed specific ACC deaminase (ACCD) activity and the ability to promote canola (Brassica napus) root elongation in pouch assays. RNAi silencing of the ACCD gene in T. asperellum showed decreased ability of the mutants to promote root elongation of canola seedlings. These data suggest a role for ACCD in the plant root growth-promotion effect by T. asperellum.

  11. Recent developments in use of 1-aminocyclopropane-1-carboxylate (ACC) deaminase for conferring tolerance to biotic and abiotic stress.

    PubMed

    Gontia-Mishra, Iti; Sasidharan, Shaly; Tiwari, Sharad

    2014-05-01

    Ethylene is an essential plant hormone also known as a stress hormone because its synthesis is accelerated by induction of a variety of biotic and abiotic stress. The plant growth promoting bacteria containing the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase enhances plant growth by decreasing plant ethylene levels under stress conditions. The expression of ACC deaminase (acdS) gene in transgenic plants is an alternative approach to overcome the ethylene-induced stress. Several transgenic plants have been engineered to express both bacterial/plant acdS genes which then lowers the stress-induced ethylene levels, thus efficiently combating the deleterious effects of environmental stresses. This review summarizes the current knowledge of various transgenic plants overexpressing microbial and plant acdS genes and their potential under diverse biotic and abiotic stresses. Transcription regulation mechanism of acdS gene from different bacteria, with special emphasis to nitrogen fixing bacteria is also discussed in this review.

  12. 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

  13. 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 §...

  14. 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 §...

  15. 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 §...

  16. 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 §...

  17. 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

  18. 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.

  19. NTP pattern of avian embryonic red cells: role of RNA degradation and AMP deaminase/5'-nucleotidase activity.

    PubMed

    Baumann, Rosemarie; Gotz, Robert; Dragon, Stefanie

    2003-03-01

    During terminal erythroid differentiation, degradation of RNA is a potential source for nucleotide triphosphates (NTPs) that act as allosteric effectors of hemoglobin. In this investigation, we assessed the developmental profile of RNA and purine/pyrimidine trinucleotides in circulating embryonic chick red blood cells (RBC). Extensive changes of the NTP pattern are observed which differ significantly from what is observed for adult RBC. The biochemical mechanisms have not been identified yet. Therefore, we studied the role of AMP deaminase and IMP/GMP 5'-nucleotidase, which are key enzymes for the regulation of the purine nucleotide pool. Finally, we tested the effect of major NTPs on the oxygen affinity of embryonic/adult hemoglobin. The results are as follows. 1) Together with ATP, UTP and CTP serve as allosteric effectors of hemoglobin. 2) Degradation of erythroid RNA is apparently a major source for NTPs. 3) Developmental changes of nucleotide content depend on the activities of key enzymes (AMP deaminase, IMP/GMP 5'-nucleotidase, and pyrimidine 5'-nucleotidase). 4) Oxygen-dependent hormonal regulation of AMP deaminase adjusts the red cell ATP concentration and therefore the hemoglobin oxygen affinity.

  20. Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

    PubMed Central

    Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

    2014-01-01

    Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

  1. 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

  2. Inhibition of muscarinic K+ current in guinea-pig atrial myocytes by PD 81,723, an allosteric enhancer of adenosine binding to A1 receptors

    PubMed Central

    Brandts, B; Bünemann, M; Hluchy, J; Sabin, G V; Pott, L

    1997-01-01

    PD 81,723 has been shown to enhance binding of adenosine to A1 receptors by stabilizing G protein-receptor coupling (‘allosteric enhancement'). Evidence has been provided that in the perfused hearts and isolated atria PD 81,723 causes a sensitization to adenosine via this mechanism. We have studied the effect of PD 81,723 in guinea-pig isolated atrial myocytes by use of whole-cell measurement of the muscarinic K+ current (IK(ACh)) activated by different Gi-coupled receptors (A1, M2, sphingolipid). PD 81,273 caused inhibition of IK(ACh) (IC50≃5 μM) activated by either of the three receptors. Receptor-independent IK(ACh) in cells loaded with GTP-γ-S and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to PD 81,723. At no combination of concentrations of adenosine and PD 81,723 could an enhancing effect be detected. The compound was active from the outside only. Loading of the cells with PD 81,723 (50 μM) via the patch pipette did not affect either IK(ACh) or its sensitivity to adenosine. We suggest that PD 81,723 acts as an inhibitor of inward rectifying K+ channels; this is supported by the finding that ventricular IK1, which shares a large degree of homology with the proteins (GIRK1/GIRK4) forming IK(ACh) but is not G protein-gated, was also blocked by this compound. It is concluded that the functional effects of PD 81,723 described in the literature are not mediated by the A1 adenosine receptor-Gi-IK(ACh) pathway. PMID:9249260

  3. The role of cyclic AMP and its protein kinase in mediating acetylcholine release and the action of adenosine at frog motor nerve endings.

    PubMed Central

    Hirsh, J. K.; Silinsky, E. M.; Solsona, C. S.

    1990-01-01

    1. The importance of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and its protein kinase (protein kinase A, PKA) in promoting acetylcholine (ACh) release was studied at frog motor nerve endings. The effects of cyclic AMP-dependent protein phosphorylation on the action of adenosine receptor agonists were also investigated. 2. Cyclic AMP was delivered to a local region of the cytoplasm just beneath the plasma membrane of motor nerve endings using phospholipid vesicles (liposomes) as a vehicle. Cyclic AMP in liposomes produced a parallel reduction in the mean level of evoked ACh release (m) and spontaneous ACh release (miniature endplate potential frequency; m.e.p.p.f) in most experiments. These inhibitory effects of cyclic AMP on quantal ACh release resemble the action of adenosine. 3. The effects of global increases in cytoplasmic cyclic AMP concentrations using lipophilic cyclic AMP analogues were generally different from those observed with cyclic AMP. 8-(4-Chlorophenylthio) cyclic AMP (CPT cyclic AMP) produced approximately two fold increases in m and m.e.p.p.f. Dibutyryl cyclic AMP (db cyclic AMP) also increased m and m.e.p.p.f, with the effect on m being smaller and more variable. 4. All three cyclic AMP analogues reduced the effects of adenosine receptor agonists on spontaneous and evoked ACh release. 5. The roles of protein phosphorylation in mediating ACh release and the inhibitory effects of adenosine were studied with the protein kinase inhibitor H7. H7 (30-100 microM) produced no consistent effect on evoked or spontaneous ACh release. At these concentrations, however, H7 exerted an unfortunate inhibitory action on the nicotinic ACh receptor/ion channel.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2175231

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Synergistic effect of therapeutic stem cells expressing cytosine deaminase and interferon-beta via apoptotic pathway in the metastatic mouse model of breast cancer.

    PubMed

    Yi, Bo-Rim; Kim, Seung U; Choi, Kyung-Chul

    2016-02-01

    As an approach to improve treatment of breast cancer metastasis to the brain, we employed genetically engineered stem cells (GESTECs, HB1.F3 cells) consisting of neural stem cells (NSCs) expressing cytosine deaminase and the interferon-beta genes, HB1.F3.CD and HB1.F3.CD.IFN-β. In this model, MDA-MB-231/Luc breast cancer cells were implanted in the right hemisphere of the mouse brain, while pre-stained GESTECs with redfluorescence were implanted in the contralateral brain. Two days after stem cells injection, 5-fluorocytosine (5-FC) was administrated via intraperitoneal injection. Histological analysis of extracted brain confirmed the therapeutic efficacy of GESTECs in the presence of 5-FC based on reductions in density and aggressive tendency of breast cancer cells, as well as pyknosis, karyorrhexis, and karyolysis relative to a negative control. Additionally, expression of PCNA decreased in the stem cells treated group. Treatment of breast cancer cells with 5-fluorouracil (5-FU) increased the expression of pro-apoptotic and anti-proliferative factor, BAX and p21 protein through phosphorylation of p53 and p38. Moreover, analysis of stem cell migratory ability revealed that MDA-MB-231 cells endogenously secreted VEGF, and stem cells expressed their receptor (VEGFR2). To confirm the role of VEGF/VEGFR2 signaling in tumor tropism of stem cells, samples were treated with the VEGFR2 inhibitor, KRN633. The number of migrated stem cells decreased significantly in response to KRN633 due to Erk1/2 activation and PI3K/Akt inhibition. Taken together, these results indicate that treatment with GESTECs, particularly HB1.F3.CD.IFN-β co-expressing CD.IFN-β, may be a useful strategy for treating breast cancer metastasis to the brain in the presence of a prodrug.

  9. The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion.

    PubMed

    Chin, A; Svejda, B; Gustafsson, B I; Granlund, A B; Sandvik, A K; Timberlake, A; Sumpio, B; Pfragner, R; Modlin, I M; Kidd, M

    2012-02-01

    Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98-99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10-6 M; IC50 = 3.7 × 10-8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT₁ (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT₁ transcription was regulated by PKA/MAPK and PI₃K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease. PMID:22038827

  10. The role of mechanical forces and adenosine in the regulation of intestinal enterochromaffin cell serotonin secretion

    PubMed Central

    Chin, A.; Svejda, B.; Gustafsson, B. I.; Granlund, A. B.; Sandvik, A. K.; Timberlake, A.; Sumpio, B.; Pfragner, R.; Modlin, I. M.

    2012-01-01

    Enterochromaffin (EC) cells of the diffuse neuroendocrine cell system secrete serotonin (5-HT) with activation of gut motility, secretion, and pain. These cells express adenosine (ADORA) receptors and are considered to function as mechanosensors. Physiological pathways mediating mechanosensitivity and adenosine responsiveness remain to be fully elucidated, as do their roles in inflammatory bowel disease (IBD) and neoplasia. Pure (98–99%) FACS-sorted normal and IBD human EC cells and neoplastic EC cells (KRJ-I) were studied. IBD-EC cells and KRJ-I overexpressed ADORA2B. NECA, a general ADORA receptor agonist, stimulated, whereas the A2B receptor antagonist MRS1754 inhibited, 5-HT release (EC50 = 1.8 × 10−6 M; IC50 = 3.7 × 10−8 M), which was associated with corresponding alterations in intracellular cAMP levels and pCREB (Ser133). Mechanical stimulation using a rhythmic flex model induced transcription and activation of Tph1 (tryptophan hydroxylase) and VMAT1 (vesicular monoamine transporter 1) and the release of 5-HT, which could be inhibited by MRS1754 and amplified by NECA. Secretion was also inhibited by H-89 (PKA inhibitor) while Tph1 and VMAT1 transcription was regulated by PKA/MAPK and PI3K-mediated signaling. Normal and IBD-EC cells also responded to NECA and mechanical stimulation with PKA activation, cAMP production, and 5-HT release, effects reversible by MRS1754. EC cells express stimulatory ADORA2B, and rhythmic stretch induces A2B activation, PKA/MAPK/IP3-dependent transcription, and PKA-dependent secretion of 5-HT synthesis and secretion. Receptor expression is amplified in IBD and neoplasia, and 5-HT release is increased. Determination of factors that regulate EC cell function are necessary for understanding its role as a mechanosensory cell and to facilitate the development of agents that can selectively target cell function in EC cell-associated disease. PMID:22038827

  11. 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).

  12. Hemodynamic and neurohumoral effects of various grades of selective adenosine transport inhibition in humans. Implications for its future role in cardioprotection.

    PubMed

    Rongen, G A; Smits, P; Ver Donck, K; Willemsen, J J; De Abreu, R A; Van Belle, H; Thien, T

    1995-02-01

    In 12 healthy male volunteers (27-53 yr), a placebo-controlled randomized double blind cross-over trial was performed to study the effect of the intravenous injection of 0.25, 0.5, 1, 2, 4, and 6 mg draflazine (a selective nucleoside transport inhibitor) on hemodynamic and neurohumoral parameters and ex vivo nucleoside transport inhibition. We hypothesized that an intravenous draflazine dosage without effect on hemodynamic and neurohumoral parameters would still be able to augment the forearm vasodilator response to intraarterially infused adenosine. Heart rate (electrocardiography), systolic blood pressure (Dinamap 1846 SX; Critikon, Portanje Electronica BV, Utrecht, The Netherlands) plasma norepinephrine and epinephrine increased dose-dependently and could almost totally be abolished by caffeine pretreatment indicating the involvement of adenosine receptors. Draflazine did not affect forearm blood flow (venous occlusion plethysmography). Intravenous injection of 0.5 mg draflazine did not affect any of the measured hemodynamic parameters but still induced a significant ex vivo nucleoside-transport inhibition of 31.5 +/- 4.1% (P < 0.05 vs placebo). In a subgroup of 10 subjects the brachial artery was cannulated to infuse adenosine (0.15, 0.5, 1.5, 5, 15, and 50 micrograms/100 ml forearm per min) before and after intravenous injection of 0.5 mg draflazine. Forearm blood flow amounted 1.9 +/- 0.3 ml/100 ml forearm per min for placebo and 1.8 +/- 0.2, 2.0 +/- 0.3, 3.8 +/- 0.9, 6.3 +/- 1.2, 11.3 +/- 2.2, and 19.3 +/- 3.9 ml/100 ml forearm per min for the six incremental adenosine dosages, respectively. After the intravenous draflazine infusion, these values were 1.6 +/- 0.2 ml/100 ml forearm per min for placebo and 2.1 +/- 0.3, 3.3 +/- 0.6, 5.8 +/- 1.1, 6.9 +/- 1.4, 14.4 +/- 2.9, and 23.5 +/- 4.0 ml/100 ml forearm per min, respectively (Friedman ANOVA: P < 0.05 before vs after draflazine infusion). In conclusion, a 30-50% inhibition of adenosine transport significantly

  13. Action of troxacitabine on cells transduced with human cytidine deaminase cDNA.

    PubMed

    Boivin, Anne-Julie; Gourdeau, Henriette; Momparler, Richard L

    2004-01-01

    Troxacitabine (beta-L-Dioxolane-cytidine; Troxatyl) is a beta-L-nucleoside analog, which has shown preclinical antitumor activity in human xenograft tumor models and antileukemic response in patients with relapsed myeloid leukemia. Troxacitabine is activated by cellular kinases and incorporated into DNA, inhibiting its replication. In contrast to other cytosine nucleoside analogs, troxacitabine is resistant to inactivation by cytidine deaminase (CD). In this study we have investigated the effects of increased intracellular levels of CD on the antineoplastic action of troxacitabine and the related antileukemic drug, cytosine arabinoside (ARA-C). Retroviral transduction of the human CD gene in A549 lung carcinoma cells (A549-CD cells) markedly increased the expression of this gene. The A549-CD cells were more resistant to the cytotoxic action of ARA-C than the wild type A549 cells as determined by clonogenic assays. In contrast, the CD-transduced cells were as or more sensitive to the cytotoxic action of troxacitabine than the wild type cells. These results suggest that troxacitabine may be an effective antineoplastic agent against tumors with high levels of CD that show drug resistance to cytosine nucleoside analogs.

  14. Endothelial Progenitor Cells Combined with Cytosine Deaminase-Endostatin for Suppression of Liver Carcinoma.

    PubMed

    Chen, Rong; Yu, Hui; An, Yan-Li; Chen, Hua-Jun; Jia, ZhenYu; Teng, Gao-Jun

    2016-06-01

    Transplantation of gene transfected endothelial progenitor cells (EPCs) provides a novel method for treatment of human tumors. To study treatment of hepatocellular carcinoma using cytosine deaminase (CD)- and endostatin (ES)-transfected endothelial progenitor cells (EPCs), mouse bone marrow-derived EPCs were cultured and transfected with Lenti6.3-CD-EGFP and Lenti6.3-ES-Monomer-DsRed labeled with superparamagnetic iron oxide (SPIO) nanoparticles. DiD (lipophilic fluorescent dye)-labeled EPCs were injected into normal mice and mice with liver carcinoma. The EPCs loaded with CD-ES were infused into the mice through caudal veins and tumor volumes were measured. The tumor volumes in the EPC + SPIO + CD/5-Fc + ES group were found to be smaller as a result and grew more slowly than those from the EPC + SPIO + LV (lentivirus, empty vector control) group. Survival times were also measured after infusion of the cells into the mice. The median survival time was found to be longer in the EPC + SPIO + CD/5-Fc + ES group than in the others. In conclusion, the EPCs transfected with CD-ES suppressed the liver carcinoma cells in vitro, migrated primarily to the carcinoma, inhibited tumor growth, and also extended the median survival time for the mice with liver carcinoma. PMID:27319212

  15. Adenoviral-mediated imaging of gene transfer using a somatostatin receptor-cytosine deaminase fusion protein.

    PubMed

    Lears, K A; Parry, J J; Andrews, R; Nguyen, K; Wadas, T J; Rogers, B E

    2015-03-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy owing to the enzyme's ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that both the SSTR2 and yCD were functional in binding assays, conversion assays and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  16. Characterization of immunoglobulin gene somatic hypermutation in the absence of activation-induced cytidine deaminase

    PubMed Central

    Longo, Nancy S.; Satorius, Colleen L.; Plebani, Alessandro; Durandy, Anne; Lipsky, Peter E.

    2008-01-01

    Somatic hypermutation (SHM) of Ig genes depends upon the deamination of C nucleotides in WRCY (W=A/T, R=A/G, Y=C/T) motifs by activation-induced cytidine deaminase (AICDA). Despite this, a large number of mutations occur in WA motifs that can be accounted for by the activity of polymerase eta (POL η). To determine whether there are AICDA-independent mutations and to characterize the relationship between AICDA- and POL η-mediated mutations, 1,470 H chain and 1,313 kappa and lambda chain rearrangements from three AICDA−/− patients were analyzed. The Ig mutation frequency of all VH genes from AICDA−/− patients was 40-fold less than that of normal donors whereas the mutation frequency of mutated VH sequences from AICDA−/− patients was 6.8-fold less than normal donors. AICDA−/− B cells lack mutations in WRCY/RGYW motifs as well as replacement mutations and mutational targeting in complementarity determining regions. A significantly reduced mutation frequency in WA motifs compared to normal donors and an increased percentage of transitions, which may relate to reduced uracil DNA-glycosylase (UNG) activity, suggest a role for AICDA in regulating POL η and UNG activity. Similar results were observed in VL rearrangements. The residual mutations were predominantly G:C substitutions, indicating that AICDA-independent cytidine deamination was a likely, yet inefficient, mechanism for mutating Ig genes. PMID:18606684

  17. Adaptive evolution of threonine deaminase in plant defense against insect herbivores

    SciTech Connect

    Gonzales-Vigil, Eliana; Bianchetti, Christopher M.; Phillips, Jr., George N.; Howe, Gregg A.

    2011-11-07

    Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to {alpha}-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

  18. Activation-induced cytidine deaminase (AID) is localized to subnuclear domains enriched in splicing factors

    SciTech Connect

    Hu, Yi Ericsson, Ida Doseth, Berit Liabakk, Nina B. Krokan, Hans E. Kavli, Bodil

    2014-03-10

    Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation. - Highlights: • AID and its interaction partner CTNNBL1 localize to Cajal bodies and nuclear speckles. • AID associates with its activating kinase PKA in nuclear speckles. • AID is linked to the splicing machinery in switching B-cells. • Our findings suggest a transcription-coupled splicing associated mechanism for AID targeting and activation.

  19. Involvement of activation-induced cytidine deaminase in skin cancer development

    PubMed Central

    Toda, Yoshinobu; Hiai, Hiroshi; Uemura, Munehiro; Nakamura, Motonobu; Hattori, Yukari; Bessho, Kazuhisa; Minato, Nagahiro

    2016-01-01

    Most skin cancers develop as the result of UV light–induced DNA damage; however, a substantial number of cases appear to occur independently of UV damage. A causal link between UV-independent skin cancers and chronic inflammation has been suspected, although the precise mechanism underlying this association is unclear. Here, we have proposed that activation-induced cytidine deaminase (AID, encoded by AICDA) links chronic inflammation and skin cancer. We demonstrated that Tg mice expressing AID in the skin spontaneously developed skin squamous cell carcinoma with Hras and Trp53 mutations. Furthermore, genetic deletion of Aicda reduced tumor incidence in a murine model of chemical-induced skin carcinogenesis. AID was expressed in human primary keratinocytes in an inflammatory stimulus–dependent manner and was detectable in human skin cancers. Together, the results of this study indicate that inflammation-induced AID expression promotes skin cancer development independently of UV damage and suggest AID as a potential target for skin cancer therapeutics. PMID:26974156

  20. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution

    NASA Astrophysics Data System (ADS)

    Senavirathne, Gayan; Bertram, Jeffrey G.; Jaszczur, Malgorzata; Chaurasiya, Kathy R.; Pham, Phuong; Mak, Chi H.; Goodman, Myron F.; Rueda, David

    2015-12-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ~5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer.

  1. LINE-1 retroelements complexed and inhibited by activation induced cytidine deaminase.

    PubMed

    Metzner, Mirjam; Jäck, Hans-Martin; Wabl, Matthias

    2012-01-01

    LINE-1 (abbreviated L1) is a major class of retroelements in humans and mice. If unrestricted, retroelements accumulate in the cytoplasm and insert their DNA into the host genome, with the potential to cause autoimmune disease and cancer. Retroviruses and other retroelements are inhibited by proteins of the APOBEC family, of which activation-induced cytidine deaminase (AID) is a member. Although AID is mainly known for being a DNA mutator shaping the antibody repertoire in B lymphocytes, we found that AID also restricts de novo L1 integrations in B- and non-B-cell lines. It does so by decreasing the protein level of open reading frame 1 (ORF1) of both exogenous and endogenous L1. In activated B lymphocytes, AID deficiency increased L1 mRNA 1.6-fold and murine leukemia virus (MLV) mRNA 2.7-fold. In cell lines and activated B lymphocytes, AID forms cytoplasmic high-molecular-mass complexes with L1 mRNA, which may contribute to L1 restriction. Because AID-deficient activated B lymphocytes do not express ORF1 protein, we suggest that ORF1 protein expression is inhibited by additional restriction factors in these cells. The greater increase in MLV compared to L1 mRNA in AID-deficient activated B lymphocytes may indicate less strict surveillance of retrovirus. PMID:23133680

  2. A Role for Host Activation-Induced Cytidine Deaminase in Innate Immune Defense against KSHV

    PubMed Central

    Bekerman, Elena; Jeon, Diana; Ardolino, Michele; Coscoy, Laurent

    2013-01-01

    Activation-induced cytidine deaminase (AID) is specifically induced in germinal center B cells to carry out somatic hypermutation and class-switch recombination, two processes responsible for antibody diversification. Because of its mutagenic potential, AID expression and activity are tightly regulated to minimize unwanted DNA damage. Surprisingly, AID expression has been observed ectopically during pathogenic infections. However, the function of AID outside of the germinal centers remains largely uncharacterized. In this study, we demonstrate that infection of human primary naïve B cells with Kaposi's sarcoma-associated herpesvirus (KSHV) rapidly induces AID expression in a cell intrinsic manner. We find that infected cells are marked for elimination by Natural Killer cells through upregulation of NKG2D ligands via the DNA damage pathway, a pathway triggered by AID. Moreover, without having a measurable effect on KSHV latency, AID impinges directly on the viral fitness by inhibiting lytic reactivation and reducing infectivity of KSHV virions. Importantly, we uncover two KSHV-encoded microRNAs that directly regulate AID abundance, further reinforcing the role for AID in the antiviral response. Together our findings reveal additional functions for AID in innate immune defense against KSHV with implications for a broader involvement in innate immunity to other pathogens. PMID:24244169

  3. Critical role of activation induced cytidine deaminase in experimental autoimmune encephalomyelitis.

    PubMed

    Sun, Yonglian; Peng, Ivan; Senger, Kate; Hamidzadeh, Kajal; Reichelt, Mike; Baca, Miriam; Yeh, Ronald; Lorenzo, Maria N; Sebrell, Andrew; Dela Cruz, Christopher; Tam, Lucinda; Corpuz, Racquel; Wu, Jiansheng; Sai, Tao; Roose-Girma, Merone; Warming, Søren; Balazs, Mercedesz; Gonzalez, Lino C; Caplazi, Patrick; Martin, Flavius; Devoss, Jason; Zarrin, Ali A

    2013-03-01

    Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE. PMID:23167594

  4. Critical role of activation induced cytidine deaminase in Experimental Autoimmune Encephalomyelitis

    PubMed Central

    2013-01-01

    Multiple Sclerosis (MS) is a neurodegenerative autoimmune disorder caused by chronic inflammation and demyelination within the central nervous system (CNS). Clinical studies in MS patients have demonstrated efficacy with B cell targeted therapies such as anti-CD20. However, the exact role that B cells play in the disease process is unclear. Activation Induced cytidine deaminase (AID) is an essential enzyme for the processes of antibody affinity maturation and isotype switching. To evaluate the impact of affinity maturation and isotype switching, we have interrogated the effect of AID-deficiency in an animal model of MS. Here, we show that the severity of experimental autoimmune encephalomyelitis (EAE) induced by the extracellular domain of human myelin oligodendrocyte glycoprotein (MOG1-125) is significantly reduced in Aicda deficient mice, which, unlike wild-type mice, lack serum IgG to myelin associated antigens. MOG specific T cell responses are comparable between wild-type and Aicda knockout mice suggesting an active role for antigen experienced B cells. Thus affinity maturation and/or class switching are critical processes in the pathogenesis of EAE. PMID:23167594

  5. Adenoviral-Mediated Imaging of Gene Transfer Using a Somatostatin Receptor-Cytosine Deaminase Fusion Protein

    PubMed Central

    Lears, Kimberly A.; Parry, Jesse J.; Andrews, Rebecca; Nguyen, Kim; Wadas, Thaddeus J.; Rogers, Buck E.

    2015-01-01

    Suicide gene therapy is a process by which cells are administered a gene that encodes a protein capable of converting a nontoxic prodrug into an active toxin. Cytosine deaminase (CD) has been widely investigated as a means of suicide gene therapy due to the enzyme’s ability to convert the prodrug 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil (5-FU). However, the extent of gene transfer is a limiting factor in predicting therapeutic outcome. The ability to monitor gene transfer, non-invasively, would strengthen the efficiency of therapy. In this regard, we have constructed and evaluated a replication-deficient adenovirus (Ad) containing the human somatostatin receptor subtype 2 (SSTR2) fused with a C-terminal yeast CD gene for the non-invasive monitoring of gene transfer and therapy. The resulting Ad (AdSSTR2-yCD) was evaluated in vitro in breast cancer cells to determine the function of the fusion protein. These studies demonstrated that the both the SSTR2 and yCD were functional in binding assays, conversion assays, and cytotoxicity assays. In vivo studies similarly demonstrated the functionality using conversion assays, biodistribution studies, and small animal positron-emission tomography (PET) imaging studies. In conclusion, the fusion protein has been validated as useful for the non-invasive imaging of yCD expression and will be evaluated in the future for monitoring yCD-based therapy. PMID:25837665

  6. Involvement of activation-induced cytidine deaminase in skin cancer development.

    PubMed

    Nonaka, Taichiro; Toda, Yoshinobu; Hiai, Hiroshi; Uemura, Munehiro; Nakamura, Motonobu; Yamamoto, Norio; Asato, Ryo; Hattori, Yukari; Bessho, Kazuhisa; Minato, Nagahiro; Kinoshita, Kazuo

    2016-04-01

    Most skin cancers develop as the result of UV light-induced DNA damage; however, a substantial number of cases appear to occur independently of UV damage. A causal link between UV-independent skin cancers and chronic inflammation has been suspected, although the precise mechanism underlying this association is unclear. Here, we have proposed that activation-induced cytidine deaminase (AID, encoded by AICDA) links chronic inflammation and skin cancer. We demonstrated that Tg mice expressing AID in the skin spontaneously developed skin squamous cell carcinoma with Hras and Trp53 mutations. Furthermore, genetic deletion of Aicda reduced tumor incidence in a murine model of chemical-induced skin carcinogenesis. AID was expressed in human primary keratinocytes in an inflammatory stimulus-dependent manner and was detectable in human skin cancers. Together, the results of this study indicate that inflammation-induced AID expression promotes skin cancer development independently of UV damage and suggest AID as a potential target for skin cancer therapeutics.

  7. Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif.

    PubMed

    Land, Allison M; Wang, Jiayi; Law, Emily K; Aberle, Ryan; Kirmaier, Andrea; Krupp, Annabel; Johnson, Welkin E; Harris, Reuben S

    2015-11-24

    APOBEC3B is a newly identified source of mutation in many cancers, including breast, head/neck, lung, bladder, cervical, and ovarian. APOBEC3B is a member of the APOBEC3 family of enzymes that deaminate DNA cytosine to produce the pro-mutagenic lesion, uracil. Several APOBEC3 family members function to restrict virus replication. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H combine to restrict HIV-1 in human lymphocytes. HIV-1 counteracts these APOBEC3s with the viral protein Vif, which targets the relevant APOBEC3s for proteasomal degradation. While APOBEC3B does not restrict HIV-1 and is not targeted by HIV-1 Vif in CD4-positive T cells, we asked whether related lentiviral Vif proteins could degrade APOBEC3B. Interestingly, several SIV Vif proteins are capable of promoting APOBEC3B degradation, with SIVmac239 Vif proving the most potent. This likely occurs through the canonical polyubiquitination mechanism as APOBEC3B protein levels are restored by MG132 treatment and by altering a conserved E3 ligase-binding motif. We further show that SIVmac239 Vif can prevent APOBEC3B mediated geno/cytotoxicity and degrade endogenous APOBEC3B in several cancer cell lines. Our data indicate that the APOBEC3B degradation potential of SIV Vif is an effective tool for neutralizing the cancer genomic DNA deaminase APOBEC3B. Further optimization of this natural APOBEC3 antagonist may benefit cancer therapy.

  8. Activation-induced deoxycytidine deaminase (AID) co-transcriptional scanning at single-molecule resolution.

    PubMed

    Senavirathne, Gayan; Bertram, Jeffrey G; Jaszczur, Malgorzata; Chaurasiya, Kathy R; Pham, Phuong; Mak, Chi H; Goodman, Myron F; Rueda, David

    2015-01-01

    Activation-induced deoxycytidine deaminase (AID) generates antibody diversity in B cells by initiating somatic hypermutation (SHM) and class-switch recombination (CSR) during transcription of immunoglobulin variable (IgV) and switch region (IgS) DNA. Using single-molecule FRET, we show that AID binds to transcribed dsDNA and translocates unidirectionally in concert with RNA polymerase (RNAP) on moving transcription bubbles, while increasing the fraction of stalled bubbles. AID scans randomly when constrained in an 8 nt model bubble. When unconstrained on single-stranded (ss) DNA, AID moves in random bidirectional short slides/hops over the entire molecule while remaining bound for ∼ 5 min. Our analysis distinguishes dynamic scanning from static ssDNA creasing. That AID alone can track along with RNAP during transcription and scan within stalled transcription bubbles suggests a mechanism by which AID can initiate SHM and CSR when properly regulated, yet when unregulated can access non-Ig genes and cause cancer. PMID:26681117

  9. Evolution of class switch recombination function in fish activation-induced cytidine deaminase, AID.

    PubMed

    Wakae, Koshou; Magor, Brad G; Saunders, Holly; Nagaoka, Hitoshi; Kawamura, Akemi; Kinoshita, Kazuo; Honjo, Tasuku; Muramatsu, Masamichi

    2006-01-01

    Following activation of mammalian B cells, class switch recombination (CSR) and somatic hypermutation (SHM) of the Ig heavy chain (IgH) gene can improve the functions of the expressed antibodies. Activation-induced cytidine deaminase (AID) is the only known B cell-specific protein required for inducing CSR and SHM in mammals. Lower vertebrates have an AID homologue, and there is some evidence of SHM in vivo. However there is no evidence of CSR in the cartilaginous or bony fishes, and this may be due in part to a lack of cis-elements in the IgH gene that are the normal targets of AID-mediated recombination. We have tested whether bony fish (zebrafish and catfish) AID can mediate CSR and SHM in mammalian cells. As expected, ectopic expression of fish AID in mouse fibroblasts resulted in mutations in an introduced SHM reporter gene, indicating that fish AID can mediate SHM. Unexpectedly, expression of fish AID in mouse AID-/- B cells induced surface IgG expression as well as switched transcripts from Ig gene loci, clearly indicating that the fish AID protein can mediate CSR, at least in mouse cells. These results suggest that the AID protein acquired the ability to mediate CSR before the IgH locus evolved the additional exon clusters and switch regions that are the targets of recombination. We discuss how pleiotropic functions of specific domains within the AID protein may have facilitated the early evolution of CSR in lower vertebrates.

  10. Target DNA sequence directly regulates the frequency of activation-induced deaminase-dependent mutations.

    PubMed

    Chen, Zhangguo; Viboolsittiseri, Sawanee S; O'Connor, Brian P; Wang, Jing H

    2012-10-15

    Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.

  11. Glucose metabolism during fasting is altered in experimental porphobilinogen deaminase deficiency.

    PubMed

    Collantes, María; Serrano-Mendioroz, Irantzu; Benito, Marina; Molinet-Dronda, Francisco; Delgado, Mercedes; Vinaixa, María; Sampedro, Ana; Enríquez de Salamanca, Rafael; Prieto, Elena; Pozo, Miguel A; Peñuelas, Iván; Corrales, Fernando J; Barajas, Miguel; Fontanellas, Antonio

    2016-04-01

    Porphobilinogen deaminase (PBGD) haploinsufficiency (acute intermittent porphyria, AIP) is characterized by neurovisceral attacks when hepatic heme synthesis is activated by endogenous or environmental factors including fasting. While the molecular mechanisms underlying the nutritional regulation of hepatic heme synthesis have been described, glucose homeostasis during fasting is poorly understood in porphyria. Our study aimed to analyse glucose homeostasis and hepatic carbohydrate metabolism during fasting in PBGD-deficient mice. To determine the contribution of hepatic PBGD deficiency to carbohydrate metabolism, AIP mice injected with a PBGD-liver gene delivery vector were included. After a 14 h fasting period, serum and liver metabolomics analyses showed that wild-type mice stimulated hepatic glycogen degradation to maintain glucose homeostasis while AIP livers activated gluconeogenesis and ketogenesis due to their inability to use stored glycogen. The serum of fasted AIP mice showed increased concentrations of insulin and reduced glucagon levels. Specific over-expression of the PBGD protein in the liver tended to normalize circulating insulin and glucagon levels, stimulated hepatic glycogen catabolism and blocked ketone body production. Reduced glucose uptake was observed in the primary somatosensorial brain cortex of fasted AIP mice, which could be reversed by PBGD-liver gene delivery. In conclusion, AIP mice showed a different response to fasting as measured by altered carbohydrate metabolism in the liver and modified glucose consumption in the brain cortex. Glucose homeostasis in fasted AIP mice was efficiently normalized after restoration of PBGD gene expression in the liver. PMID:26908609

  12. The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

    PubMed Central

    Ranieri-Raggi, Maria; Moir, Arthur J. G.; Raggi, Antonio

    2014-01-01

    Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner. HPRG is a major plasma protein. Recent investigations have reported that skeletal muscle cells do not synthesize HPRG but instead actively internalize plasma HPRG. X-ray absorption spectroscopy (XAS) performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (μ-aqua)(μ-carboxylato)dizinc(II) core with two histidine residues at each metal site. XAS on HPRG isolated from the AMPD complex showed that zinc is bound to the protein in a dinuclear cluster where each Zn2+ ion is coordinated by three histidine and one heavier ligand, likely sulfur from cysteine. We describe the existence in mammalian HPRG of a specific zinc binding site distinct from the His-Pro-rich region. The participation of HPRG in the assembly and maintenance of skeletal muscle AMPD by acting as a zinc chaperone is also demonstrated. PMID:24970226

  13. Targeting Na⁺/K⁺ -translocating adenosine triphosphatase in cancer treatment.

    PubMed

    Durlacher, Cameron T; Chow, Kevin; Chen, Xiao-Wu; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Zhou, Shu-Feng

    2015-05-01

    The Na(+) /K(+) -translocating adenosine triphosphatase (ATPase) transports sodium and potassium across the plasma membrane and represents a potential target in cancer chemotherapy. Na(+) /K(+) -ATPase belongs to the P-type ATPase family (also known as E1-E2 ATPase), which is involved in transporting certain ions, metals, and lipids across the plasma membrane of mammalian cells. In humans, the Na(+) /K(+) -ATPase is a binary complex of an α-subunit that has four isoforms (α1 -α4 ) and a β-subunit that has three isoforms (β1 -β3 ). This review aims to update our knowledge on the role of Na(+) /K(+) -ATPase in cancer development and metastasis, as well as on how Na(+) /K(+) -ATPase inhibitors kill tumour cells. The Na(+) /K(+) -ATPase has been found to be associated with cancer initiation, growth, development, and metastasis. Cardiac glycosides have exhibited anticancer effects in cell-based and mouse studies via inhibition of the Na(+) /K(+) -ATPase and other mechanisms. Na(+) /K(+) -ATPase inhibitors may kill cancer cells via induction of apoptosis and autophagy, radical oxygen species production, and cell cycle arrest. They also modulate multiple signalling pathways that regulate cancer cell survival and death, which contributes to their antiproliferative activities in cancer cells. The clinical evidence supporting the use of Na(+) /K(+) -ATPase inhibitors as anticancer drugs is weak. Several phase I and phase II clinical trials with digoxin, Anvirzel, and huachansu (an intravenous formulated extract of the venom of the wild toad), either alone or more often in combination with other anticancer agents, have shown acceptable safety profiles but limited efficacy in cancer patients. Well-designed randomized clinical trials with reasonable sample sizes are certainly warranted to confirm the efficacy and safety of cardiac glycosides for the treatment of cancer.

  14. 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

  15. 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

  16. 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

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

    PubMed

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

    2016-03-15

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

  18. 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

  19. 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

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

    PubMed Central

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

    2016-01-01

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

  1. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

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

    2013-01-24

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

  2. 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

  3. Adenosine 5′-monophosphate ameliorates D-galactosamine/lipopolysaccharide-induced liver injury through an adenosine receptor-independent mechanism in mice

    PubMed Central

    Zhan, Y; Wang, Z; Yang, P; Wang, T; Xia, L; Zhou, M; Wang, Y; Wang, S; Hua, Z; Zhang, J

    2014-01-01

    D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced lethality and acute liver failure is dependent on endogenously produced inflammatory cytokines. Adenosine has been proven to be a central role in the regulation of inflammatory response. It is not entirely clear that which adenosine action is actually crucial to limiting inflammatory tissue destruction. Here we showed that GalN/LPS challenge elevated hepatic adenosine and induced lethality in adenosine receptor-deficient mice with equal efficiency as wild-type mice. In GalN/LPS-treated mice, pretreatment with adenosine 5′-monophosphate (5′-AMP) significantly elevated hepatic adenosine level and reduced mortality through decreasing cytokine and chemokine production. In RAW264.7 cells, 5′-AMP treatment inhibited the production of inflammatory cytokines, which is not mediated through adenosine receptors. 5′-AMP failed to attenuate LPS-induced nuclear factor-κB (NF-κB) p65 nuclear translocation, but reduced LPS-induced recruitment of NF-κB p65 to inflammatory gene promoters and decreased LPS-induced enrichment of H3K4 dimethylation at the tumor necrosis factor-α (TNF-α) promoter, which was involved in 5′-AMP-induced elevation of cellular adenosine and a decline of methylation potential. In vitro biochemical analysis revealed that adenosine directly attenuated recruitment of NF-κB to the TNF-α and interleukin-6 promoters. Our findings demonstrate that 5′-AMP-inhibiting inflammatory response is not mediated by adenosine receptors and it may represent a potential protective agent for amelioration of LPS-induced liver injury. PMID:24407238

  4. Adenosine kinase inhibitors: polar 7-substitutent of pyridopyrimidine derivatives improving their locomotor selectivity.

    PubMed

    Zheng, Guo Zhu; Mao, Yue; Lee, Chih-Hung; Pratt, John K; Koenig, John R; Perner, Richard J; Cowart, Marlon D; Gfesser, Gregory A; McGaraughty, Steve; Chu, Katharine L; Zhu, Chang; Yu, Haixia; Kohlhaas, Kathy; Alexander, Karen M; Wismer, Carol T; Mikusa, Joseph; Jarvis, Michael F; Kowaluk, Elizabeth A; Stewart, Andrew O

    2003-09-15

    We have discovered that polar 7-substituents of pyridopyrimidine derivatives affect not only whole cell AK inhibitory potency, but also selectivity in causing locomotor side effects in vivo animal models. We have identified compound, 1o, which has potent whole cell AK inhibitory potency, analgesic activity and minimal reduction of locomotor activity.

  5. Effect of forskolin on alterations of vascular permeability induced with bradykinin, prostaglandin E1, adenosine, histamine and carrageenin in rats.

    PubMed

    Sugio, K; Daly, J W

    1983-07-01

    The effect of the diterpene forskolin on vascular permeability alone and in combination with bradykinin, prostaglandin E1, adenosine or histamine has been investigated in rats. Vascular permeability in rat skin was measured using [125I]-labelled bovine serum albumin ([125I]BSA) as a tracer. In addition, the effect of forskolin on footpad edema induced by the injection of a mixture of 2% carrageenin was determined. Forskolin caused a marked potentiation of the increase in vascular permeability in rat skin elicited by the intradermal injection of histamine or bradykinin. However, forskolin caused a significant suppression of the prostaglandin E1-induced vascular permeability response and at a low concentration suppressed the response to adenosine. Forskolin greatly potentiated the footpad edema induced with carrageenin in rats. Intravenous administration of the enzyme bromelain, which reduces plasma kininogen levels, inhibited the footpad edema induced with carrageenin or with a mixture of carrageenin and forskolin. Parenteral administration of a prostaglandin synthetase inhibitor, indomethacin, suppressed the footpad edema induced with carrageenin, but did not inhibit the footpad edema induced with a mixture of carrageenin and forskolin. An antihistamine, cyproheptadine, had no effect on carrageenin-induced footpad edema either in the presence or absence of forskolin. These results suggest that both bradykinin and prostaglandins are essential for the development of carrageenin-induced footpad edema and that bradykinin plays an important role in the potentiative effect of forskolin on footpad edema induced with carrageenin in rats.

  6. Crystal structure of a membrane-bound l-amino acid deaminase from Proteus vulgaris.

    PubMed

    Ju, Yingchen; Tong, Shuilong; Gao, Yongxiang; Zhao, Wei; Liu, Qi; Gu, Qiong; Xu, Jun; Niu, Liwen; Teng, Maikun; Zhou, Huihao

    2016-09-01

    l-amino acid oxidases/deaminases (LAAOs/LAADs) are a class of oxidoreductases catalyzing the oxidative deamination of l-amino acids to α-keto acids. They are widely distributed in eukaryotic and prokaryotic organisms, and exhibit diverse substrate specificity, post-translational modifications and cellular localization. While LAAOs isolated from snake venom have been extensively characterized, the structures and functions of LAAOs from other species are largely unknown. Here, we reported crystal structure of a bacterial membrane-bound LAAD from Proteus vulgaris (pvLAAD) in complex with flavin adenine dinucleotide (FAD). We found that the overall fold of pvLAAD does not resemble typical LAAOs. Instead it, is similar to d-amino acid oxidases (DAAOs) with an additional hydrophobic insertion module on protein surface. Structural analysis and liposome-binding assays suggested that the hydrophobic module serves as an extra membrane-binding site for LAADs. Bacteria from genera Proteus and Providencia were found to encode two classes of membrane-bound LAADs. Based on our structure, the key roles of residues Q278 and L317 in substrate selectivity were proposed and biochemically analyzed. While LAADs on the membrane were proposed to transfer electrons to respiratory chain for FAD re-oxidization, we observed that the purified pvLAAD could generate a significant amount of hydrogen peroxide in vitro, suggesting it could use dioxygen to directly re-oxidize FADH2 as what typical LAAOs usually do. These findings provide a novel insights for a better understanding this class of enzymes and will help developing biocatalysts for industrial applications. PMID:27422658

  7. Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

    SciTech Connect

    Chen, Jennifer K.; Hu, Lily J.; Wang Dongfang; Lamborn, Kathleen R.; Deen, Dennis F. . E-mail: dennisdeen@juno.com

    2007-04-01

    Purpose: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. Methods and Materials: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Results: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Conclusion: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.