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Sample records for human adenosine deaminase

  1. Distribution of adenosine deaminase complexing protein (ADCP) in human tissues.

    Science.gov (United States)

    Dinjens, W N; ten Kate, J; van der Linden, E P; Wijnen, J T; Khan, P M; Bosman, F T

    1989-12-01

    The normal distribution of adenosine deaminase complexing protein (ADCP) in the human body was investigated quantitatively by ADCP-specific radioimmunoassay (RIA) and qualitatively by immunohistochemistry. In these studies we used a specific rabbit anti-human ADCP antiserum. In all 19 investigated tissues, except erythrocytes, ADCP was found by RIA in the soluble and membrane fractions. From all tissues the membrane fractions contained more ADCP (expressed per mg protein) than the soluble fractions. High membrane ADCP concentrations were found in skin, renal cortex, gastrointestinal tract, and prostate. Immunoperoxidase staining confirmed the predominant membrane-associated localization of the protein. In serous sweat glands, convoluted tubules of renal cortex, bile canaliculi, gastrointestinal tract, lung, pancreas, prostate gland, salivary gland, gallbladder, mammary gland, and uterus, ADCP immunoreactivity was found confined to the luminal membranes of the epithelial cells. These data demonstrate that ADCP is present predominantly in exocrine glands and absorptive epithelia. The localization of ADCP at the secretory or absorptive apex of the cells suggests that the function of ADCP is related to the secretory and/or absorptive process.

  2. Human adenosine deaminase: properties and turnover in cultured T and B lymphoblasts

    International Nuclear Information System (INIS)

    Daddona, P.E.

    1981-01-01

    In this study, the properties and rate of turnover of adenosine deaminase are compared in cultured human T and B lymphoblast cell lines. 1) Relative to B lymphoblasts, the level of adenosine deaminase activity in extracts of T lymphoblast cell lines (MOLT-4, RPMI-8402, CCRF-CEM, and CCRF-HSB-2) is elevated 7-14-fold and differs by 2-fold between the C cell lines. 2) In both T and B lymphoblast extracts, the enzyme is apparently identical, based on K/sub m/ for adenosine and deoxyadenosine, K/sub i/ for inosine, V/sub max/ for adenosine, /sub S20,w/, isoelectric pH, and heat stability. Furthermore, by radioimmunoassay, the quantity of adenosine deaminase-immunocreative protein is proportional to the level of enzyme activity in all cell lines studies. 3) Using a purification and selective immunoprecipitation technique, the enzyme turnover could be assessed in cell lines labeled with [ 35 S]methionine. The apparent rate of adenosine deaminase synthesis, relative to total protein, is 2-fold faster in both T cell lines (RPMI-8402 and CCRF-CEM) than in the B cell lines (MGL-8 and GM-130). The apparent half-life (tsub1/2) for the enzyme degradation is 19 and 39 h, respectively, in CCFR-CEM and RPMI-8402, while the tsub1/2 in both B cell lines is 7-9 h. From the net rate of synthesis and degradation, the T cell lines, respectively, exhibit approximately a 6- and 12-fold difference in adenosine deaminase turnover relative to B cells, consistent with the observed differences in enzyme activity. This study suggests that while adenosine deaminase is apparently identical in both T and B lymphoblast cell lines, alterations in both the rate of enzyme synthesis and degradation contribute to its high steady state level in T cells

  3. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells

    International Nuclear Information System (INIS)

    Wilson, J.M.; Danos, O.; Grossman, M.; Raulet, D.H.; Mulligan, R.C.

    1990-01-01

    Recombinant retroviruses encoding human adenosine deaminase have been used to infect murine hematopoietic stem cells. In bone marrow transplant recipients reconstituted with the genetically modified cells, human ADA was detected in peripheral blood mononuclear cells of the recipients for at least 6 months after transplantation. In animals analyzed in detail 4 months after transplantation, human ADA and proviral sequences were detected in all hematopoietic lineages; in several cases, human ADA activity exceeded the endogenous activity. These studies demonstrate the feasibility of introducing a functional human ADA gene into hematopoietic stem cells and obtaining expression in multiple hematopoietic lineages long after transplantation. This approach should be helpful in designing effective gene therapies for severe combined immunodeficiency syndromes in humans

  4. A Phenotypic Screen for Functional Mutants of Human Adenosine Deaminase Acting on RNA 1.

    Science.gov (United States)

    Wang, Yuru; Havel, Jocelyn; Beal, Peter A

    2015-11-20

    Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes responsible for the conversion of adenosine to inosine at specific locations in cellular RNAs. ADAR1 and ADAR2 are two members of the family that have been shown to be catalytically active. Earlier, we reported a phenotypic screen for the study of human ADAR2 using budding yeast S. cerevisiae as the host system. While this screen has been successfully applied to the study of ADAR2, it failed with ADAR1. Here, we report a new reporter that uses a novel editing substrate and is suitable for the study of ADAR1. We screened plasmid libraries with randomized codons for two important residues in human ADAR1 (G1007 and E1008). The screening results combined with in vitro deamination assays led to the identification of mutants that are more active than the wild type protein. Furthermore, a screen of the ADAR1 E1008X library with a reporter construct bearing an A•G mismatch at the editing site suggests one role for the residue at position 1008 is to sense the identity of the base pairing partner for the editing site adenosine. This work has provided a starting point for future in vitro evolution studies of ADAR1 and led to new insight into ADAR's editing site selectivity.

  5. Restoration of adenosine deaminase-deficient human thymocyte development in vitro by inhibition of deoxynucleoside kinases.

    Science.gov (United States)

    Joachims, Michelle L; Marble, Patrick A; Laurent, Aletha B; Pastuszko, Peter; Paliotta, Marco; Blackburn, Michael R; Thompson, Linda F

    2008-12-01

    Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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

  7. Adenosine deaminase activity of erythrocytes in hyperuricemia

    International Nuclear Information System (INIS)

    Krueger, W.; Richter, V.; Beenken, O.; Weinhold, D.; Hirschberg, K.; Rotzsch, W.; Akademie der Wissenschaften der DDR, Leipzig. Zentralinstitut fuer Isotopen- und Strahlenforschung)

    1982-01-01

    Erythrocytic adenosine deaminase (ADA) activity was determined in 55 patients with primary hyperuricemia and in 37 healthy control persons. Unlike the controls, the ADA activity in the patient group showed a two-peak response. Hyperuricemia patients with high ADA activity also exhibited increased uric acid excretion and elevated 15 N incorporation into uric acid. High activity values of erythrocytic ADA can be interpreted as an uric acid overproduction, giving hints for a therapeutic plan. (author)

  8. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells

    International Nuclear Information System (INIS)

    Lim, B.; Apperley, J.F.; Orkin, S.H.; Williams, D.A.

    1989-01-01

    Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. The authors have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. They report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy

  9. Primary adenosine monophosphate (AMP) deaminase deficiency in a hypotonic infant.

    Science.gov (United States)

    Castro-Gago, Manuel; Gómez-Lado, Carmen; Pérez-Gay, Laura; Eirís-Puñal, Jesús; Martínez, Elena Pintos; García-Consuegra, Inés; Martín, Miguel Angel

    2011-06-01

    The spectrum of the adenosine monophosphate (AMP) deaminase deficiency ranges from asymptomatic carriers to patients who manifest exercise-induced muscle pain, occasionally rhabdomyolysis, and idiopathic hyperCKemia. However, previous to the introduction of molecular techniques, rare cases with congenital weakness and hypotonia have also been reported. We report a 6-month-old girl with the association of congenital muscle weakness and hypotonia, muscle deficiency of adenosine monophosphate deaminase, and the homozygous C to T mutation at nucleotide 34 of the adenosine monophosphate deaminase-1 gene. This observation indicates the possible existence of a primary adenosine monophosphate deaminase deficiency manifested by congenital muscle weakness and hypotonia.

  10. Adenosine-deaminase (ADA activity in Psoriasis (A Preliminary Study

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    S D Chaudhry

    1988-01-01

    Full Text Available Study of adenosine-deaminase activity ′in 23 patients hav-mg psoriasis compared with an equal number of healthy controls revealed significantly high ADA-activity in the psotiatic patients.

  11. Assignment of adenosine deaminase complexing protein (ADCP) gene(s) to human chromosome 2 in rodent-human somatic cell hybrids.

    Science.gov (United States)

    Herbschleb-Voogt, E; Grzeschik, K H; Pearson, P L; Meera Khan, P

    1981-01-01

    The experiments reported in this paper indicate that the expression of human adenosine deaminase complexing protein (ADCP) in the human-rodent somatic cell hybrids is influenced by the state of confluency of the cells and the background rodent genome. Thus, the complement of the L-cell derived A9 or B82 mouse parent apparently prevents the expression of human ADCP in the interspecific somatic cell hybrids. In the a3, E36, or RAG hybrids the human ADCP expression was not prevented by the rodent genome and was found to be proportional to the degree of confluency of the cell in the culture as in the case of primary human fibroblasts. An analysis of human chromosomes, chromosome specific enzyme markers, and ADCP in a panel of rodent-human somatic cell hybrids optimally maintained and harvested at full confluency has shown that the expression of human ADCP in the mouse (RAG)-human as well as in the hamster (E36 or a3)-human hybrids is determined by a gene(s) in human chromosome 2 and that neither chromosome 6 nor any other of the chromosomes of man carry any gene(s) involved in the formation of human ADCP at least in the Chinese hamster-human hybrids. A series of rodent-human hybrid clones exhibiting a mitotic separation of IDH1 and MDH1 indicated that ADCP is most probably situated between corresponding loci in human chromosome 2.

  12. Adenosine deaminase organic effect in normal and abnormal cerebrospinal fluid

    International Nuclear Information System (INIS)

    Hamad, A.M.; Samarai, M.A.

    2007-01-01

    To study the effect of the organic substances on adenosine deaminase (ADA) activity in normal and abnormal cerebrospinal fluid (CSF). Various concentrations of 2-mercaptopurine, Ame-tycine, Adenosine analogues (Guanine, Thymine) and ATP were tested to see their effect on ADA activity in normal and abnormal CSF. ADA activity in normal and abnormal CSF was remarkably decreased with the increasing of concentrations of substances tested. These effects may have important therapeutic implications. (author)

  13. Plasma Adenosine Deaminase Enzyme Reduces with Treatment of ...

    African Journals Online (AJOL)

    olayemitoyin

    Plasma Adenosine Deaminase Enzyme Reduces with Treatment of Pulmonary Tuberculosis in Nigerian Patients: Indication for. Diagnosis and Treatment Monitoring. Ige O.a, Edem V.F.b and Arinola O.G.b,*. aDepartment of Medicine, University of Ibadan, Ibadan, Nigeria b Department of Chemical Pathology,. University of ...

  14. Contributory role of adenosine deaminase in metabolic syndrome ...

    African Journals Online (AJOL)

    Adenosine deaminase (ADA) is an enzyme of purine metabolism commonly associated with severe combined immunodeficiency disease and believed to modulate bioactivity of insulin. Its contributory role in patients with metabolic syndrome (having features such as obesity, insulin resistance, fasting hyperglycaemia, lipid ...

  15. Contributory role of adenosine deaminase in metabolic syndrome

    African Journals Online (AJOL)

    olayemitoyin

    Cytokine balance was also changed in diet induced obese mice (Mito and Hiyosin, 2002). Although Mito et al (2000) ... immunity in man (Sadasivudu et al, 1982) adenosine deaminase modulates cell growth (Lelieuve et al, .... Colgiuri, S. (2002) The Carnivore Connection- evolution aspect of insulin resistance. Eur. J. Clin.

  16. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.)

    International Nuclear Information System (INIS)

    Chivot, J.J.; Depernet, D.; Caen, J.

    1970-01-01

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 μM adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors) [fr

  17. The emerging role of adenosine deaminases in insects

    Czech Academy of Sciences Publication Activity Database

    Doleželová, Eva; Žurovec, Michal; Doležal, T.; Šimek, Petr; Bryant, P. J.

    2005-01-01

    Roč. 35, č. 5 (2005), s. 381-389 ISSN 0965-1748 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant - others:United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : adenosine deaminase * ADA * growth factor Subject RIV: ED - Physiology Impact factor: 2.733, year: 2005

  18. ADENOSINE DEAMINASE ACTIVITY IN TYPE 2 DIABETES MELLITUS

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    Farija Peruvankuzhiyil

    2017-01-01

    Full Text Available BACKGROUND Altered blood levels of adenosine deaminase may help in predicting immunological dysfunction in diabetic individuals. But very few studies exist on ADA activity in type 2 diabetes mellitus. Aim of this study is to compare serum adenosine deaminase activity in type 2 diabetic patients with non-diabetic control. MATERIALS AND METHODS A comparative study design was used in data collection process. The study was conducted in 40 type 2 diabetes mellitus patients attending diabetic clinic or admitted in the medicine ward for metabolic control of diabetes in medical college, Calicut from January 2011 to January 2012. The adenosine deaminase (ADA level in the serum is measured by endpoint method in these patients. The results were expressed as mean and standard deviation. The statistical significance of the differences between the values was assessed by ANOVA. RESULTS Among 40 diabetic patients, mean ADA level in the serum is 38.56, SD±6.72 (min 30, max 53. Mean ADA level in the serum in the control group is 22.04±4.625 (min 13, max 29. CONCLUSION ADA level in the serum is found to be increased indicating its role as an important immunoenzyme marker in the aetiopathology of type 2 diabetes mellitus.

  19. Adenosine deaminase complexing protein (ADCP) immunoreactivity in colorectal adenocarcinoma.

    Science.gov (United States)

    ten Kate, J; van den Ingh, H F; Khan, P M; Bosman, F T

    1986-04-15

    Immunoreactive adenosine deaminase complexing protein (ADCP) was studied in 91 human colorectal adenocarcinomas. The expression of ADCP was correlated with that of secretory component (SC) and carcinoembryonic antigen (CEA), with the histological grade and the Dukes' stage of the carcinomas. The histological grade was scored semi-quantitatively according to 5 structural and 4 cytological variables. ADCP expression was observed in 3 different staining patterns, namely: (1) diffuse cytoplasmic (77% of the carcinomas); (2) granular cytoplasmic (13%); and (3) membrane-associated (66%). These patterns were observed alone or in combination. Eleven percent of the carcinomas exhibited no ADCP immunoreactivity. Linear regression analysis showed that the expression of ADCP correlates with that of SC and CEA. However, no significant correlation emerged between the histological parameters or the Dukes' stage and any of the immunohistological parameters. Comparison of the histological characteristics of carcinomas exhibiting little or no ADCP immunoreactivity with those showing extensive immunoreactivity, showed that membranous ADCP immunoreactivity occurs more frequently in well-differentiated carcinomas. Structural parameters showed a better correlation with membranous ADCP expression than the cytological variables. It is concluded that membranous expression of ADCP and CEA are indicators of a high level of differentiation as reflected primarily in the structural characteristics of the tumor.

  20. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

    Science.gov (United States)

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A 2A R present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A 2A R and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A 2A R involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A 2A R-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A 2A R). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

  1. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    Directory of Open Access Journals (Sweden)

    Estefanía Moreno

    2018-02-01

    Full Text Available Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26 and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET, we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26 and dendritic cells (expressing A2AR. This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector without partitioning these functions in different subunits.

  2. Adenosine deaminase complexing protein (ADCP): a transformation sensitive protein with potentials of a cancer marker.

    Science.gov (United States)

    Herbschleb-Voogt, E; Ten Kate, J; Meera Khan, P

    1983-01-01

    Several observations by independent investigators in the past have indicated that adenosine deaminase complexing protein (ADCP), present in considerable quantities in certain human tissues, was absent or decreased in the cancers originated from them. During the present study, electrophoretic analysis of adenosine deaminase (ADA) isozymes and radioimmunoassay for ADCP in the primary fibroblasts and the transformed as well as certain tumor derived cell lines have demonstrated that ADCP present in large quantities in the primary cells was absent or nearly absent in the transformed or tumor-derived cell lines. Though the mechanisms involved are not yet clear, the above observations indicate that ADCP has the potentials of a useful marker in the studies on transformed cells and cancer tissues.

  3. Evaluation of usefulness of pleural fluid adenosine deaminase in diagnosing tuberculous pleural effusion from empyema

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    Vijetha Shenoy

    2014-02-01

    Full Text Available Objective: To evaluate the utility of adenosine deaminase activity in the pleural fluid for the diagnosis of tuberculous pleural effusion from empyema of non-tubercular origin. Method: A retrospective analysis of data was performed on patients who were diagnosed to have tuberculous pleural effusion and empyema of non tubercular origin. Among 46 patients at Kasturba Hospital, Manipal University, Manipal, Karnataka, India, from November 201 2 to February 2013 who underwent pleural fluid adenosine deaminase estimation, 25 patients with tuberculous pleural effusion and 21 patients with empyema were diagnosed respectively. Adenosine deaminase in pleural fluid is estimated using colorimetric, Galanti and Guisti method. Results: Pleural fluid Adenosine Deaminase levels among tuberculous pleural effusion(109.38依 53.83 , empyema (141.20依71.69 with P=0.27. Conclusion: Pleural fluid adenosine deaminase alone cannot be used as a marker for the diagnosis of tuberculous pleural effusion.

  4. Diagnostic significance of adenosine deaminase in pleural tuberculosis

    International Nuclear Information System (INIS)

    Khurshid, R.; Shore, N.; Saleem, M.; Zameer, N.

    2009-01-01

    Tuberculosis (TB) is a major cause of pleural effusion, which in TB usually has lymphocytic and exudative characteristics. Analysis of adenosine deaminase (ADA) activity is a very useful diagnostic approach to achieve a more rapid and precise diagnosis in cases of Pleural TB (pTB). Fifty male and fifty female patients presenting with tuberculosis pleural effusion was included in the study. The patients were taken from the medical ward of Sir Ganga Ram Hospital between September 2001 and September 2002. Activity of Adenosine Deaminase (ADA) was estimated by the technique of Sodium dodecyl sulphate electrophoresis (SDS-EF) using 10% polyacrylamide gel. Mean age of males was 45.72+-19.22 years and of female was 43.74+-16.09 years. Mean protein level was 3.39+-0.24 g/dl in males, and it was 3.02+-0.26 g/dl in females. Mean specific gravity both in males and females was 1.020+-0.01. The results show an increased level of enzyme ADA in patients as compared to normal subjects. Estimation of ADA activity may provide basis for rapid and efficient diagnosis of pleural TB in different clinical settings. However study should be extended to larger number of patients to reach a better conclusion. (author)

  5. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

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    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  6. Moonlighting adenosine deaminase: a target protein for drug development.

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

    2015-01-01

    Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest. © 2014 Wiley Periodicals, Inc.

  7. Radio-chromatographic determination of plasmatic adenosine deaminase (A.D.); Determination radiochromatographique de l'adenosine deaminase (A.D.)

    Energy Technology Data Exchange (ETDEWEB)

    Chivot, J J; Depernet, D; Caen, J [Commissariat a l' Energie Atomique, Bruyeres-le-Chatel (France). Centre d' Etudes

    1970-07-01

    We were able, by using a radio-chromatographic method, to measure an adenosine deaminase activity in normal human heparinized platelet-poor plasma, which can degrade 0.016 {mu}M adenosine. This activity suppressed by heating 56 C for 30 minutes is inhibited by high concentrations of urea and is proportional to the amount of plasma, source of enzyme, in the systems. (authors) [French] Nous avons pu, en utilisant une methode radiochromatographique, mesurer une activite adenosine deaminasique dans le plasma humain pauvre en plaquettes heparine qui peut degrader 0,016 {mu}M d'adenosine. Cette activite qui est supprimee par chauffage a 56 degres pendant 30 minutes, est reduite par conservation a -20 C pendant une semaine, est inhibee par d'importantes concentrations d'uree et ne l'est pas, ni par le dipyridamol, ni par le pHMB. Cette activite est proportionnelle a la quantite de plasma, source d'enzyme, mise dans les differents systemes reactifs. (auteur)

  8. Hydrolytic cleavage of N-6-substituted adenine derivatives by eukaryotic adenine and adenosine deaminases

    Czech Academy of Sciences Publication Activity Database

    Pospíšilová, H.; Šebela, M.; Novák, Ondřej; Frébort, I.

    2008-01-01

    Roč. 28, č. 6 (2008), s. 335-347 ISSN 0144-8463 R&D Projects: GA ČR(CZ) GA522/06/0022 Institutional research plan: CEZ:AV0Z50380511 Keywords : adenine deaminase * adenosine deaminase (ADA) * aminohydrolase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.525, year: 2008

  9. Regulation of adenosine deaminase (ADA) on induced mouse experimental autoimmune uveitis (EAU) ?

    OpenAIRE

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

    2016-01-01

    Adenosine is an important regulator of the immune response and adenosine deaminase (ADA) inhibits this regulatory effect by converting adenosine into functionally inactive molecules. Studies have shown that adenosine receptor (AR) agonists can be either anti- or pro-inflammatory. Clarification of the mechanisms that cause these opposing effects should provide a better guide for therapeutic intervention. In this study, we investigated the effect of ADA on the development of experimental autoim...

  10. Adenosine Deaminase Inhibitor EHNA Exhibits a Potent Anticancer Effect Against Malignant Pleural Mesothelioma

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    Yasuhiro Nakajima

    2015-01-01

    Full Text Available Background/Aims: Malignant pleural mesothelioma (MPM is an aggressive malignant tumor and an effective therapy has been little provided as yet. The present study investigated the possibility for the adenosine deaminase (ADA inhibitor EHNA as a target of MPM treatment. Methods: MTT assay, TUNEL staining, monitoring of intracellular adenosine concentrations, and Western blotting were carried out in cultured human MPM cell lines without and with knocking-down ADA. The in vivo effect of EHNA was assessed in mice inoculated with NCI-H2052 MPM cells. Results: EHNA induced apoptosis of human MPM cell lines in a concentration (0.01-1 mM- and treatment time (24-48 h-dependent manner, but such effect was not obtained with another ADA inhibitor pentostatin. EHNA increased intracellular adenosine concentrations in a treatment time (3-9 h-dependent manner. EHNA-induced apoptosis of MPM cells was mimicked by knocking-down ADA, and the effect was neutralized by the adenosine kinase inhibitor ABT-702. EHNA clearly suppressed tumor growth in mice inoculated with NCI-H2052 MPM cells. Conclusion: The results of the present study show that EHNA induces apoptosis of MPM cells by increasing intracellular adenosine concentrations, to convert to AMP, and effectively prevents MPM cell proliferation. This suggests that EHNA may be useful for treatment of the tragic neoplasm MPM.

  11. How We Manage Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA SCID).

    Science.gov (United States)

    Kohn, Donald B; Gaspar, H Bobby

    2017-05-01

    Adenosine deaminase-deficient severe combined immune deficiency (ADA SCID) accounts for 10-15% of cases of human SCID. From what was once a uniformly fatal disease, the prognosis for infants with ADA SCID has improved greatly based on the development of multiple therapeutic options, coupled with more frequent early diagnosis due to implementation of newborn screening for SCID. We review the various treatment approaches for ADA SCID including allogeneic hematopoietic stem cell transplantation (HSCT) from a human leukocyte antigen-matched sibling or family member or from a matched unrelated donor or a haplo-identical donor, autologous HSCT with gene correction of the hematopoietic stem cells (gene therapy-GT), and enzyme replacement therapy (ERT) with polyethylene glycol-conjugated adenosine deaminase. Based on growing evidence of safety and efficacy from GT, we propose a treatment algorithm for patients with ADA SCID that recommends HSCT from a matched family donor, when available, as a first choice, followed by GT as the next option, with allogeneic HSCT from an unrelated or haplo-identical donor or long-term ERT as other options.

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

    Science.gov (United States)

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

    2016-11-01

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

  13. Adenosine deaminase production by an endophytic bacterium (Lysinibacillus sp.) from Avicennia marina.

    Science.gov (United States)

    Kathiresan, Kandasamy; Saravanakumar, Kandasamy; Sahu, Sunil Kumar; Sivasankaran, Muthu

    2014-06-01

    The present study was carried out with the following objectives: (1) to isolate the endophytic bacilli strains from the leaves of mangrove plant Avicennia marina, (2) to screen the potential strains for the production of adenosine deaminase, (3) to statistically optimize the factors that influence the enzyme activity in the potent strain, and (4) to identify the potent strain using 16S rRNA sequence and construct its phylogenetic tree. The bacterial strains isolated from the fresh leaves of a mangrove A. marina were assessed for adenosine deaminase activity by plating method. Optimization of reaction process was carried out using response surface methodology of central composite design. The potent strain was identified based on 16S rRNA sequencing and phylogeny. Of five endophytic strains, EMLK1 showed a significant deaminase activity over other four strains. The conditions for maximum activity of the isolated adenosine deaminase are described. The potent strain EMLK1 was identified as Lysinibacillus sp. (JQ710723) being the first report as a mangrove endophyte. Mangrove-derived endophytic bacillus strain Lysinibacillus sp. EMLK1 is proved to be a promising source for the production of adenosine deaminase and this enzyme deserves further studies for purification and its application in disease diagnosis.

  14. Radiochromatographic determination of activity of adenosine deaminase and purine nucleoside phosphorylase in blood cells

    International Nuclear Information System (INIS)

    Pechan, I.; Rendekova, V.; Pechanova, E.; Krizko, J.

    1982-01-01

    Expeditious and sensitive methods are described for determining the activities of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in human lymphocytes and erythrocytes. ADA and PNP activity is determined on the basis of the reaction of (U- 14 C)adenosine or (8- 14 C)inosine with the lysate of human blood cells. Reaction products are separated using paper chromatography. Following the measurement of the radioactivity of spots of adenosine, inosine and hypoxanthine, a calculation is made of ADA and PNP activity from the results of the said measurements. On a sample of 52 clinically healthy people average ADA and PNP activity in isolated lymphocytes was found to be (51.6+-18.8) and (185.6+-94.7) pcat/10 6 cells and in erythrocytes (9.8+-2.98) and (17.1+-3.19) pcat/mg of proteins, respectively. The advantage of the method is the small amount of sample needed (1 to 2 ml) which allows its application in pediatrics. (Ha)

  15. Urtica dioica inhibits cell growth and induces apoptosis by targeting Ornithine decarboxylase and Adenosine deaminase as key regulatory enzymes in adenosine and polyamines homeostasis in human breast cancer cell lines.

    Science.gov (United States)

    Fattahi, Sadegh; Ghadami, Elham; Asouri, Mohsen; Motevalizadeh Ardekanid, Ali; Akhavan-Niaki, Haleh

    2018-02-28

    Breast cancer is a heterogeneous and multifactorial disease with variable disease progression risk, and treatment response. Urtica dioica is a traditional herb used as an adjuvant therapeutic agent in cancer. In the present study, we have evaluated the effects of the aqueous extract of Urtica dioica on Adenosine deaminase (ADA) and Ornithine decarboxylase (ODC1) gene expression in MCF-7, MDA-MB-231, two breast cancer cell lines being estrogen receptor positive and estrogen receptor negative, respectively.  Cell lines were cultured in suitable media. After 24 h, different concentrations of the extract were added and after 72 h, ADA and ODC1 gene expression as well as BCL2 and BAX apoptotic genes were assessed by Taqman real time PCR assay. Cells viability was assessed by MTT assay, and apoptosis was also evaluated at cellular level. The intra and extracellular levels of ODC1 and ADA enzymes were evaluated by ELISA. Results showed differential expression of ADA and ODC1 genes in cancer cell lines. In MCF-7 cell line, the expression level of ADA was upregulated in a dose-dependent manner but its expression did not change in MDA-MB cell line. ODC1 expression was increased in both examined cell lines. Also, increased level of the apoptotic BAX/BCL-2 ratio was detected in MCF-7 cells. These results demonstrated that Urtica dioica induces apoptosis in breast cancer cells by influencing ODC1 and ADA genes expression, and estrogen receptors. The different responses observed with these cell lines could be due to the interaction of Urtica dioica as a phytoestrogen with the estrogen receptor.

  16. Nitric oxide - an activating factor of adenosine deaminase 2 in vitro.

    Science.gov (United States)

    Sargisova, Ye G; Andreasyan, N A; Hayrapetyan, H L; Harutyunyan, H A

    2012-01-01

    In this study we have investigated the effect of reactive oxygen species produced by some chemicals in aqueous solutions on activity of adenosine deaminase 2 (ADA2) purified from human blood plasma. An activating effect on ADA2 was observed in vitro with sodium nitroprusside (SNP), the source of NO (nitrosonium ions NO(-) in aqueous solutions). Not SH-groups of cysteine but other amino acid residues sensitive to NO were responsible for ADA2 activation. The SNP-derived activation was more pronounced when purified ADA2 was preincubated with heparin and different proteins as an experimental model of the protein environment in vivo. The most effective was heparin, which is known for its ability to regulate enzyme and protein functions in extracellular matrix. We conclude that ADA2 is a protein with flexible conformation that is affected by the protein environment, and it changes its activity under oxidative (nitrosative) stress.

  17. Adenosine Deaminase (ADA)-Deficient Severe Combined Immune Deficiency (SCID): Molecular Pathogenesis and Clinical Manifestations.

    Science.gov (United States)

    Bradford, Kathryn L; Moretti, Federico A; Carbonaro-Sarracino, Denise A; Gaspar, Hubert B; Kohn, Donald B

    2017-10-01

    Deficiency of adenosine deaminase (ADA, EC3.5.4.4), a housekeeping enzyme of purine metabolism encoded by the Ada gene, is a cause of human severe combined immune deficiency (SCID). Numerous deleterious mutations occurring in the ADA gene have been found in patients with profound lymphopenia (T - B - NK - ), thus underscoring the importance of functional purine metabolism for the development of the immune defense. While untreated ADA SCID is a fatal disorder, there are multiple life-saving therapeutic modalities to restore ADA activity and reconstitute protective immunity, including enzyme replacement therapy (ERT), allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) with autologous gene-corrected hematopoietic stem cells (HSC). We review the pathogenic mechanisms and clinical manifestations of ADA SCID.

  18. Adenosine deaminase complexing protein (ADCP) expression and metastatic potential in prostatic adenocarcinomas.

    Science.gov (United States)

    Dinjens, W N; Ten Kate, J; Kirch, J A; Tanke, H J; Van der Linden, E P; Van den Ingh, H F; Van Steenbrugge, G J; Meera Khan, P; Bosman, F T

    1990-03-01

    The expression of the adenosine deaminase complexing protein (ADCP) was investigated by immunohistochemistry in the normal and hyperplastic human prostate, in 30 prostatic adenocarcinomas, and in seven human prostatic adenocarcinoma cell lines grown as xenografts in athymic nude mice. In the normal and hyperplastic prostate, ADCP was localized exclusively in the apical membrane and the apical cytoplasm of the glandular epithelial cells. In prostatic adenocarcinomas, four distinct ADCP expression patterns were observed: diffuse cytoplasmic, membranous, both cytoplasmic and membranous, and no ADCP expression. The expression patterns were compared with the presence of metastases. We found an inverse correlation between membranous ADCP immunoreactivity and metastatic propensity. Exclusively membranous ADCP immunoreactivity occurred only in non-metastatic tumours. In contrast, the metastatic tumours showed no or diffuse cytoplasmic ADCP immunoreactivity. This suggests that immunohistochemical detection of ADCP might predict the biological behaviour of prostatic cancer. However, the occurrence of membranous ADCP immunoreactivity in the xenograft of a cell line (PC-EW), derived from a prostatic carcinoma metastasis, indicates that not only the tendency to metastasize modulates ADCP expression.

  19. Identification of a New Uncompetitive Inhibitor of Adenosine Deaminase from Endophyte Aspergillus niger sp.

    Science.gov (United States)

    Zhang, Xin-Guo; Liu, Jin-Wen; Tang, Peng; Liu, Zi-Yu; Guo, Guang-Jun; Sun, Qiao-Yun; Yin, Jian-Jun

    2018-05-01

    Adenosine deaminase (ADA) is an enzyme widely distributed from bacteria to humans. ADA is known as a potential therapeutic target for the treatment of lymphoproliferative disorders and cancer. Endophytes are endosymbionts, often bacteria or fungi, which live within plant tissues and internal organs or intercellular space. Endophytes have a broad variety of bioactive metabolites that are used for the identification of novel natural compounds. Here, 54 morphologically distinct endophyte strains were isolated from six plants such as Peganum harmala Linn., Rheum officinale Baill., Gentiana macrophylla Pall., Radix stephaniae tetrandrae, Myrrha, and Equisetum hyemale Linn. The isolated strains were used for the search of ADA inhibitors that resulted in the identification of the strain with the highest inhibition activity, Aspergillus niger sp. Four compounds were isolated from this strain using three-step chromatography procedure, and compound 2 was determined as the compound with the highest inhibition activity of ADA. Based on the results of 1 H and 13 C NMR spectroscopies, compound 2 was identified as 3-(4-nitrophenyl)-5-phenyl isoxazole. We showed that compound 2 was a new uncompetitive inhibitor of ADA with high cytotoxic effect on HepG2 and SMCC-7721 cells (the IC 50 values were 0.347 and 0.380 mM, respectively). These results suggest that endophyte strains serve as promising sources for the identification of ADA inhibitors, and compound 2 could be an effective drug in the cancer treatment.

  20. Curcumin inhibits adenosine deaminase and arginase activities in cadmium-induced renal toxicity in rat kidney

    Directory of Open Access Journals (Sweden)

    Ayodele Jacob Akinyemi

    2017-04-01

    Full Text Available In this study, the effect of enzymes involved in degradation of renal adenosine and l-arginine was investigated in rats exposed to cadmium (Cd and treated with curcumin, the principal active phytochemical in turmeric rhizome. Animals were divided into six groups (n = 6: saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. The results of this study revealed that the activities of renal adenosine deaminase and arginase were significantly increased in Cd-treated rats when compared with the control (p < 0.05. However, co-treatment with curcumin inhibits the activities of these enzymes compared with Cd-treated rats. Furthermore, Cd intoxication increased the levels of some renal biomarkers (serum urea, creatinine, and electrolytes and malondialdehyde level with a concomitant decrease in functional sulfhydryl group and nitric oxide (NO. However, co-treatment with curcumin at 12.5 mg/kg and 25 mg/kg, respectively, increases the nonenzymatic antioxidant status and NO in the kidney, with a concomitant decrease in the levels of malondialdehyde and renal biomarkers. Therefore, our results reinforce the importance of adenosine deaminase and arginase activities in Cd poisoning conditions and suggest some possible mechanisms of action by which curcumin prevent Cd-induced renal toxicity in rats.

  1. Investigations into the origin of the molecular recognition of several adenosine deaminase inhibitors.

    Science.gov (United States)

    Gillerman, Irina; Fischer, Bilha

    2011-01-13

    Inhibitors of adenosine deaminase (ADA, EC 3.5.4.4) are potential therapeutic agents for the treatment of various health disorders. Several highly potent inhibitors were previously identified, yet they exhibit unacceptable toxicities. We performed a SAR study involving a series of C2 or C8 substituted purine-riboside analogues with a view to discover less potent inhibitors with a lesser toxicity. We found that any substitution at C8 position of nebularine resulted in total loss of activity toward calf intestinal ADA. However, several 2-substituted-adenosine, 8-aza-adenosine, and nebularine analogues exhibited inhibitory activity. Specifically, 2-Cl-purine riboside, 8-aza-2-thiohexyl adenosine, 2-thiohexyl adenosine, and 2-MeS-purine riboside were found to be competitive inhibitors of ADA with K(i) values of 25, 22, 6, and 3 μM, respectively. We concluded that electronic parameters are not major recognition determinants of ADA but rather steric parameters. A C2 substituent which fits ADA hydrophobic pocket and improves H-bonding with the enzyme makes a good inhibitor. In addition, a gg rotamer about C4'-C5' bond is apparently an important recognition determinant.

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

    Science.gov (United States)

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

    2013-08-01

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

  3. Conformational change of adenosine deaminase during ligand-exchange in a crystal.

    Science.gov (United States)

    Kinoshita, Takayoshi; Tada, Toshiji; Nakanishi, Isao

    2008-08-15

    Adenosine deaminase (ADA) perpetuates chronic inflammation by degrading extracellular adenosine which is toxic for lymphocytes. ADA has two distinct conformations: open form and closed form. From the crystal structures with various ligands, the non-nucleoside type inhibitors bind to the active site occupying the critical water-binding-position and sustain the open form of apo-ADA. In contrast, substrate mimics do not occupy the critical position, and induce the large conformational change to the closed form. However, it is difficult to predict the binding of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), as it possesses characteristic parts of both the substrate and the non-nucleoside inhibitors. The crystal structure shows that EHNA binds to the open form through a novel recognition of the adenine base accompanying conformational change from the closed form of the PR-ADA complex in crystalline state.

  4. Optimising the utility of pleural fluid adenosine deaminase for the diagnosis of adult tuberculous pleural effusion in Hong Kong.

    Science.gov (United States)

    Chang, K C; Chan, M C; Leung, W M; Kong, F Y; Mak, C M; Chen, S Pl; Yu, W C

    2018-02-01

    Pleural fluid adenosine deaminase level can be applied to rapidly detect tuberculous pleural effusion. We aimed to establish a local diagnostic cut-off value for pleural fluid adenosine deaminase to identify patients with tuberculous pleural effusion, and optimise its utility. We retrospectively reviewed the medical records of consecutive adults with pleural fluid adenosine deaminase level measured by the Diazyme commercial kit (Diazyme Laboratories, San Diego [CA], United States) during 1 January to 31 December 2011 in a cluster of public hospitals in Hong Kong. We considered its level alongside early (within 2 weeks) findings in pleural fluid and pleural biopsy, with and without applying Light's criteria in multiple scenarios. For each scenario, we used the receiver operating characteristic curve to identify a diagnostic cut-off value for pleural fluid adenosine deaminase, and estimated its positive and negative predictive values. A total of 860 medical records were reviewed. Pleural effusion was caused by congestive heart failure, chronic renal failure, or hypoalbuminaemia caused by liver or kidney diseases in 246 (28.6%) patients, malignancy in 198 (23.0%), non-tuberculous infection in 168 (19.5%), tuberculous pleural effusion in 157 (18.3%), and miscellaneous causes in 91 (10.6%). All those with tuberculous pleural effusion had a pleural fluid adenosine deaminase level of ≤100 U/L. When analysis was restricted to 689 patients with pleural fluid adenosine deaminase level of ≤100 U/L and early negative findings for malignancy and non-tuberculous infection in pleural fluid, the positive predictive value was significantly increased and the negative predictive value non-significantly reduced. Using this approach, neither additionally restricting analysis to exudates by Light's criteria nor adding closed pleural biopsy would further enhance predictive values. As such, the diagnostic cut-off value for pleural fluid adenosine deaminase is 26.5 U/L, with a

  5. Effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

    International Nuclear Information System (INIS)

    Barton, R.W.

    1985-01-01

    Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease and inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2'-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20-40% and 60-80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF

  6. Clinical efficacy of gene-modified stem cells in adenosine deaminase-deficient immunodeficiency.

    Science.gov (United States)

    Shaw, Kit L; Garabedian, Elizabeth; Mishra, Suparna; Barman, Provaboti; Davila, Alejandra; Carbonaro, Denise; Shupien, Sally; Silvin, Christopher; Geiger, Sabine; Nowicki, Barbara; Smogorzewska, E Monika; Brown, Berkley; Wang, Xiaoyan; de Oliveira, Satiro; Choi, Yeong; Ikeda, Alan; Terrazas, Dayna; Fu, Pei-Yu; Yu, Allen; Fernandez, Beatriz Campo; Cooper, Aaron R; Engel, Barbara; Podsakoff, Greg; Balamurugan, Arumugam; Anderson, Stacie; Muul, Linda; Jagadeesh, G Jayashree; Kapoor, Neena; Tse, John; Moore, Theodore B; Purdy, Ken; Rishi, Radha; Mohan, Kathey; Skoda-Smith, Suzanne; Buchbinder, David; Abraham, Roshini S; Scharenberg, Andrew; Yang, Otto O; Cornetta, Kenneth; Gjertson, David; Hershfield, Michael; Sokolic, Rob; Candotti, Fabio; Kohn, Donald B

    2017-05-01

    Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase-deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) γ-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1-2.6) and granulocytes (VCN = 0.01-0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. ClinicalTrials.gov NCT00794508. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA

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

    Directory of Open Access Journals (Sweden)

    Cátia Vieira

    2014-01-01

    Full Text Available 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.

  8. Diagnostic Value of Serum Adenosine Deaminase (ADA) Level for Pulmonary Tuberculosis.

    Science.gov (United States)

    Salmanzadeh, Shokrollah; Tavakkol, Heshmatollah; Bavieh, Khalid; Alavi, Seyed Mohammad

    2015-03-01

    Diagnosis of tuberculosis (TB) is not always easy, thus employing methods with a short duration and acceptable sensitivity and specificity is necessary to diagnose TB. The aim of this study was to investigate the diagnostic value of serum adenosine deaminase (ADA) level for diagnosis of pulmonary tuberculosis. A total of 160 sex and age-matched subjects were included in this study, and were divided to four groups; forty patients with pulmonary tuberculosis (PTB) diagnosed based on the national TB program (NTP), forty patients with non-tuberculosis bacterial pneumonia, forty patients with lung cancer and forty people who were healthy in every respect. Serum adenosine deaminase activity in patients of each group was measured by the Giusti and Galanti calorimetry method using a commercial kit (Diazyme, USA). The ANOVA analysis was used to compare groups for quantitative variables. Mean serum ADA level in the PTB group was clearly higher than the mean serum ADA in the other three groups. Mean serum ADA was 26 IU/L in PTB patients, 19.48 IU/L in patients with pneumonia, 15.8 IU/L in patients with lung cancer, and 10.7 IU/L in the control group (P ADA in patients with PTB sensitivity and specificity was defined as 35% and 91%, respectively. Serum ADA activity with high specificity percentage may be a useful alternative test in restricted resource areas to rule out diagnosis of PTB. However, serum ADA activity is not a useful tool for TB diagnosis.

  9. Development of gene therapy: potential in severe combined immunodeficiency due to adenosine deaminase deficiency

    Directory of Open Access Journals (Sweden)

    Claudia A Montiel-Equihua

    2009-12-01

    Full Text Available Claudia A Montiel-Equihua, Adrian J Thrasher, H Bobby GasparCentre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, London, UKAbstract: The history of stem cell gene therapy is strongly linked to the development of gene therapy for severe combined immunodeficiencies (SCID and especially adenosine deaminase (ADA-deficient SCID. Here we discuss the developments achieved in over two decades of clinical and laboratory research that led to the establishment of a protocol for the autologous transplant of retroviral vector-mediated gene-modified hematopoietic stem cells, which has proved to be both successful and, to date, safe. Patients in trials in three different countries have shown long-term immunological and metabolic correction. Nevertheless, improvements to the safety profile of viral vectors are underway and will undoubtedly reinforce the position of stem cell gene therapy as a treatment option for ADA-SCID.Keywords: adenosine deaminase, severe combined immunodeficiency, gene therapy, hematopoietic stem cell, retrovirus, clinical trial

  10. Pleural Fluid Adenosine Deaminase (ADA) Predicts Survival in Patients with Malignant Pleural Effusion.

    Science.gov (United States)

    Terra, Ricardo Mingarini; Antonangelo, Leila; Mariani, Alessandro Wasum; de Oliveira, Ricardo Lopes Moraes; Teixeira, Lisete Ribeiro; Pego-Fernandes, Paulo Manuel

    2016-08-01

    Systemic and local inflammations have been described as relevant prognostic factors in patients with cancer. However, parameters that stand for immune activity in the pleural space have not been tested as predictors of survival in patients with malignant pleural effusion. The objective of this study was to evaluate pleural lymphocytes and Adenosine Deaminase (ADA) as predictors of survival in patients with recurrent malignant pleural effusion. Retrospective cohort study includes patients who underwent pleurodesis for malignant pleural effusion in a tertiary center. Pleural fluid protein concentration, lactate dehydrogenase, glucose, oncotic cytology, cell count, and ADA were collected before pleurodesis and analyzed. Survival analysis was performed considering pleurodesis as time origin, and death as the event. Backwards stepwise Cox regression was used to find predictors of survival. 156 patients (out of 196 potentially eligible) were included in this study. Most were female (72 %) and breast cancer was the most common underlying malignancy (53 %). Pleural fluid ADA level was stratified as low (Pleural fluid cell count and lymphocytes number and percentage did not correlate with survival. Pleural fluid Adenosine Deaminase levels (pleural effusion who undergo pleurodesis.

  11. Regulation of adenosine deaminase (ADA) on induced mouse experimental autoimmune uveitis (EAU) ‡

    Science.gov (United States)

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

    2016-01-01

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

  12. Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

    Czech Academy of Sciences Publication Activity Database

    Žurovec, Michal; Doležal, Tomáš; Gaži, Michal; Pavlová, Eva; Bryant, P. J.

    2002-01-01

    Roč. 99, č. 7 (2002), s. 4403-4408 ISSN 0027-8424 R&D Projects: GA ČR GA204/01/1022; GA AV ČR IAA5007107 Institutional research plan: CEZ:AV0Z5007907 Keywords : adenosine daminase * minimal medium Subject RIV: CE - Biochemistry Impact factor: 10.701, year: 2002

  13. Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

    Science.gov (United States)

    Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

    2002-01-01

    The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID

  14. Serum adenosine deaminase activity and its isoenzyme in patients treated for tuberculosis

    International Nuclear Information System (INIS)

    Rokayan, S.A

    2003-01-01

    Objective: Increased serum adenosine deaminase (ADA) activity, mainly associated with tuberculosis can also occur in a number of other diseases thus negatively affecting the diagnostic utility of ADA measurements in tuberculosis. The aim of the study was to determine whether or not the combined use of the activity of ADA, its isoenzymes and differential cell counts would provide a more efficient means of diagnosing tuberculosis than the use of ADA levels alone. Results: Data suggested significant (p 0.75) of ADA/sub 2/ADA was found to be better indicator of tuberculosis. Lymphocyte neutrophil ratio (L/N)> 0.69 gave additional benefit to increase the sensitivity and specificity for the use of ADA as marker in diagnosing tuberculosis. Conclusion: The combined use of activity of ADA, its isoenzymes and total and differential cell counts is a better indicator and gives better understanding to diagnose and evaluate tuberculosis and response to therapy. (author)

  15. IgG4-related Pleuritis with Elevated Adenosine Deaminase in Pleural Effusion: A Case Report.

    Science.gov (United States)

    Nagayasu, Atsushi; Kubo, Satoshi; Nakano, Kazuhisa; Nakayamada, Shingo; Iwata, Shigeru; Miyagawa, Ippei; Fukuyo, Shunsuke; Saito, Kazuyoshi; Tanaka, Yoshiya

    2018-03-09

    An 81-year-old man was admitted with bilateral pleural effusion. A clinical examination showed lymphocytic pleura effusion and elevated serum IgG4 levels, so that IgG4-related disease was suggested, whereas tuberculous pleurisy was suspected because of high adenosine deaminase (ADA) levels in the pleural effusion. A surgical pleural biopsy revealed that there were large numbers of IgG4-positive cells and IgG4/IgG positive cell ratio exceeded 40% in several sites. Accordingly, we diagnosed IgG4-related pleuritis and treated with the patient with glucocorticoid therapy. The ADA levels in pleural effusion can increase in IgG4-related pleuritis, and it is therefore important to perform a pleural biopsy.

  16. One adenosine deaminase allele in a patient with severe combined immunodeficiency contains a point mutation abolishing enzyme activity.

    OpenAIRE

    Valerio, D; Dekker, B M; Duyvesteyn, M G; van der Voorn, L; Berkvens, T M; van Ormondt, H; van der Eb, A J

    1986-01-01

    We have cloned and sequenced an adenosine deaminase (ADA) gene from a patient with severe combined immunodeficiency (SCID) caused by inherited ADA deficiency. Two point mutations were found, resulting in amino acid substitutions at positions 80 (Lys to Arg) and 304 (Leu to Arg) of the protein. Hybridization experiments with synthetic oligonucleotide probes showed that the determined mutations are present in both DNA and RNA from the ADA-SCID patient. In addition, wild-type sequences could be ...

  17. Cerebrospinal fluid adenosine deaminase activity: A complimentary tool in the early diagnosis of tuberculous meningitis

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    Taori Girdhar M

    2006-03-01

    Full Text Available Abstract Background Tuberculous meningitis (TBM is the commonest form of neurotuberculosis caused by Mycobacterium tuberculosis bacilli (MTB. The diagnosis of TBM is often difficult. A reliable, cost-effective and rapid diagnostic test, which can be performed in any standard pathology laboratory, could be of help in the diagnosis of TBM. In the present study we measured the adenosine deaminase (ADA activity in cerebrospinal fluid (CSF of TBM and non-TBM patients. Method ADA activity in CSF was determined according to a method based on the Berthlot reaction, which is the formation of a colored indophenol complex from ammonia liberated from adenosine, and quantified spectrophotometrically. Results The CSF ADA activity from TBM patients was compared with CSF ADA from non-TBM infectious meningitis patients, and from patients with non-infectious neurological disorders. The mean CSF ADA activity was found to be significantly higher in CSF of TBM patients, 14.31 ± 3.87 (2.99–26.94, mean ± SD with range, than in the CSF from non-TBM infectious meningitis, 9.25 ± 2.14 (4.99–13.96 and from the non-infectious neurological disorders group, 2.71 ± 1.96 (0.00–7.68, P Conclusion This study demonstrated that ADA activity in the CSF of TBM patients, using a cut-off value 11.39 U/L/min, can be useful for the early differential diagnosis of TBM. This test can be performed in any pathology laboratory where more sophisticated methods are not available.

  18. Circulating type 1 vaccine-derived poliovirus may evolve under the pressure of adenosine deaminases acting on RNA.

    Science.gov (United States)

    Liu, Yanhan; Ma, Tengfei; Liu, Jianzhu; Zhao, Xiaona; Cheng, Ziqiang; Guo, Huijun; Xu, Ruixue; Wang, Shujing

    2015-01-01

    Poliovirus, the causative agent of poliomyelitis, is a human enterovirus and member of the Picornaviridae family. An effective live-attenuated poliovirus vaccine strain (Sabin 1) has been developed and has protected humans from polio. However, a few cases of vaccine virulence reversion have been documented in several countries. For instance, circulating type 1 vaccine-derived poliovirus is a highly pathogenic poliovirus that evolved from an avirulent strain, but the mechanism by which vaccine strains undergo reversion remains unclear. In this study, vaccine strains exhibited A to G/U to C and G to A/C to U hypermutations in the reversed evolution of Sabin 1. Furthermore, the mutation ratios of U to C and C to U were higher than those of other mutation types. Dinucleotide editing context was then analyzed. Results showed that A to G and U to C mutations exhibited preferences similar to adenosine deaminases acting on RNA (ADAR). Hence, ADARs may participate in poliovirus vaccine evolution.

  19. The diagnostic value of procalcitonin, adenosine deaminase for tuberculous pleural effusions

    International Nuclear Information System (INIS)

    Sun Jia; Jing Xiufeng; Hui Fuxin

    2010-01-01

    Objective: To explore differential diagnostic value of procalcitonin (PCT), adenosine deaminase (ADA) in pleural fluid and serum for tuberculous pleural effusions. Methods: The concentrations of PCT and ADA both in serum and pleural fluid in one hundred and twenty-eight patients with pleural effusion were detected. These patients were divided into three groups. Fifty-two patients with tuberculous plueral effusion were composed of the tuberculous group. Twenty-two patients with parapneumonic effusion composed the pneumonic group and forty patients with malignant pleural effusion and fourteen patients with heart faliure composed of the control group. Results: There were no statistically significant differences in serum PCT among the three groups (P > 0.05). PCT of pleural fluid was significantly increased in tuberculous and parapneumonic groups compared to the control group (P < 0.05). ADA activities in tuberculous serum and pleural fluid were both higher than those in the parapneumonic and the control groups (P < 0.01). The ratio of ADA in pleural fluid and serum (P /S) was calculated. The diagnostic sensitivity and specificity of P /S (cut-off value 1.27) were 92.3% and 100% respectively for tuberculous pleural effusions calcuted by receiver operating curve. Conclusion: Combined measurements of PCT and ADA in pleural fluid are useful in diagnosing tuberculous pleural effusions. (authors)

  20. Raised Serum Adenosine Deaminase Level in Nonobese Type 2 Diabetes Mellitus

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    Vineet Kumar Khemka

    2013-01-01

    Full Text Available The role of inflammation being minimal in the pathogenesis of type 2 diabetes mellitus (T2DM in nonobese patients; the aim of the study was to investigate the role of adenosine deaminase (ADA and see its association with diabetes mellitus. The preliminary case control study comprised of 56 cases and 45 healthy controls which were age and sex matched. 3 mL venous blood samples were obtained from the patients as well as controls after 8–10 hours of fasting. Serum ADA and routine biochemical parameters were analyzed. Serum ADA level was found significantly higher among nonobese T2DM subjects with respect to controls (38.77±14.29 versus 17.02±5.74 U/L; P<0.0001. Serum ADA level showed a significant positive correlation with fasting plasma glucose (r=0.657; P<0.0001 level among nonobese T2DM subjects, but no significant correlation was observed in controls (r=-0.203; P=0.180. However, no correlation was observed between serum ADA level compared to BMI and HbA1c levels. Our study shows higher serum ADA, triglycerides (TG and fasting plasma glucose (FPG levels in nonobese T2DM patients, and a strong correlation between ADA and FPG which suggests an association between ADA and nonobese T2DM subjects.

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

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    Iván Darío BRAVO-TOBAR

    2015-10-01

    Full Text Available 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.

  2. Diagnostic value of adenosine deaminase in ascites for tuberculosis ascites: a meta-analysis.

    Science.gov (United States)

    Tao, Lin; Ning, Hong-Jian; Nie, Hai-Ming; Guo, Xiao-Yun; Qin, Shan-Yu; Jiang, Hai-Xing

    2014-05-01

    The diagnosis of tuberculosis (TB) ascites using standard diagnostic tools is difficult. The aim of the present meta-analysis was to establish the overall diagnostic accuracy of adenosine deaminase (ADA) levels in ascites for diagnosing TB ascites. A systematic review was performed of English language publications prior to April 2013. Sensitivity, specificity, and other measures of the accuracy of ADA for the diagnosis of TB ascites using ascites fluid were summarized using a random-effects model or a fixed-effects model. Receiver operating characteristic curves were used to summarize overall test performance. Seventeen studies involving 1797 subjects were eligible for the analysis. The summary estimates of sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and the area under cure of overall analysis were: 0.93, 0.94, 13.55, 0.11, 169.83, and 0.976, respectively; the results of sensitivity analysis of studies that used Giusti method were 0.94, 0.94, 12.99, 0.08, 183.18, and 0.977, respectively. Our results suggest that ADA in the ascites can be a sensitive and specific target and a critical criterion for the diagnosis of TB ascites. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Assessment of adenosine deaminase (ADA) activity and oxidative stress in patients with chronic tonsillitis.

    Science.gov (United States)

    Garca, Mehmet Fatih; Demir, Halit; Turan, Mahfuz; Bozan, Nazım; Kozan, Ahmet; Belli, Şeyda Bayel; Arslan, Ayşe; Cankaya, Hakan

    2014-06-01

    To emphasize the effectiveness of adenosine deaminase (ADA) enzyme, which has important roles in the differentiation of lymphoid cells, and oxidative stress in patients with chronic tonsillitis. Serum and tissue samples were obtained from 25 patients who underwent tonsillectomy due to recurrent episodes of acute tonsillitis. In the control group, which also had 25 subjects, only serum samples were taken as obtaining tissue samples would not have been ethically appropriate. ADA enzyme activity, catalase (CAT), carbonic anhydrase (CA), nitric oxide (NO) and malondialdehyde (MDA) were measured in the serum and tissue samples of patients and control group subjects. The serum values of both groups were compared. In addition, the tissue and serum values of patients were compared. Serum ADA activity and the oxidant enzymes MDA and NO values of the patient group were significantly higher than those of the control group (p ADA activity (p > 0.05). Elevated ADA activity may be effective in the pathogenesis of chronic tonsillitis both by impairing tissue structure and contributing to SOR formation.

  4. Diagnostic Utility of Pleural Fluid Adenosine Deaminase Level in Tuberculousis Pleural Effusion

    International Nuclear Information System (INIS)

    Suleman, A.; Abbasi, M. A.; Anwar, S. A.; Kamal, M.; Khan, H.

    2016-01-01

    Background: Early diagnosis and management of tuberculosis is essential for decreasing the disease burden. Pakistan is one of the few countries of world with a very high burden of tuberculosis. Many diagnostic tests are available for detection of tuberculosis but each is fraught with certain limitations of its own. Methods: This study was a cross sectional validation study that sought to determine the validity of pleural fluid adenosine deaminase levels for diagnosis of tuberculous pleural effusion. Results: A total of 160 patients with exudative lymphocytic pleural effusions were enrolled in this study. The mean pleural fluid ADA level was 52.18±1.98 U/L. The mean pleural fluid ADA level in patients diagnosed to have tuberculosis on pleural biopsy/histopathology was higher as compared to patients who did not have tuberculous pleural effusion 52.16±2.4 U/L vs 38.6±3.14 U/L. The difference was found to be statistically significant between the two groups (p<0.05). The sensitivity, specificity, ppv and npv of pleural fluid ADA level were 88.88 percent, 77.04 percent, 86.28 percent and 81.04 percent respectively. Conclusion: Despite wide variations in the reported sensitivity and specificity of pleural fluid ADA level, it can be used as a surrogate for pleural biopsy when the latter is not feasible. (author)

  5. Diagnostic value of pleural fluid adenosine deaminase activity in tuberculosis pleurisy

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    Abbas ali Niazi

    2009-09-01

    Full Text Available Background: Diagnosis of tuberculosis pleurisies is difficult because of its nonspecific clinical presentation and insufficient traditional diagnostic methods. We investigated the use of adenosine deaminase (ADA activity in tuberculosis pleurisies. Methods: A number of 85 patients were analyzed with exudative pleural effusions. Using the ROC curve, we determined the optimal cutoff for TB pleurisy. Results: A number of 58 exudative samples were nontuberculous (non-TB and 27 were tuberculosis (TB. There was statistically significant difference (p<0.0001 between the means of pleural fluid ADA levels among the TB and non-TB populations. The prevalence of TB pleurisy in the studied population was 31%. Using the cutoff point equal to 35 for diagnosing TB effusions the sensitivity and specificity 70.3% and 91.3%, respectively. The positive predictive value (PPV was 79.1% and the negative predictive value (NPV was 86.8%. A pleural fluid ADA value <19 IU/L suggests that a tuberculosis effusion is highly unlikely. Conclusion: Pleural fluid total ADA assay is a sensitive and specific test suitable for rapid diagnosis of TB pleurisy.

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

    International Nuclear Information System (INIS)

    Sadat Hayatshahi, Sayyed Hamed; Abdolmaleki, Parviz; Safarian, Shahrokh; Khajeh, Khosro

    2005-01-01

    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 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%

  7. Evaluation of the adenosine deaminase (ADA) G22A gene polymorphism with recurrent spontaneous abortion among Egyptian patients

    Science.gov (United States)

    Abu-Gabal, Khadiga; Katta, Maha; Ibrahim, Raghda

    2017-01-01

    Introduction Adenosine and deoxyadenosine metabolism is influenced by adenosine deaminase (ADA) enzyme. ADA increases in different diseases and is considered as one of the markers for cell-mediated immunity. Pregnancy is associated with depressed cell-mediated immunity. The level of ADA expression, which seems to play a key role in maintaining pregnancy, is influenced by adenosine deaminase G22A gene polymorphism. We aimed in our study to evaluate the association of ADA G22A gene polymorphism with recurrent spontaneous abortion (RSA) in Egyptian women. Material and methods Adenosine deaminase G22A gene polymorphism was genotyped in 40 patients (age range 22-39 years) with a history of RSA, selected from those attending the Gynaecology and Obstetrics Clinic of Beni-Suef University Hospital, and 20 age-matched healthy women as a control group, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. Results In our study, no statistically significant difference was found between RSA patients and control group as regards ADA G22A genotypes (p = 0.653) and alleles (p = 0.697). A comparison of the frequencies of ADA alleles in RSA patients as regards the below-35-years-old age group revealed that ADA 2(A) allele was associated with a low risk for RSA in patients aged 35 years old or younger (p = 0.008). Conclusions In conclusion, our study revealed an age-dependent protective value of ADA 2(A) allele in recurrent spontaneous abortions among the Egyptian population. PMID:29204093

  8. Diagnostic significance of pleural fluid adenosine deaminase activity in tuberculous pleurisy

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    Sharmeen Ahmed

    2011-01-01

    Full Text Available Diagnosis of tuberculous pleural effusion (TPE is difficult because of its non-specific clinical presentation and insufficient efficiency of conventional diagnostic methods. The study was carried out to evaluate the utility of adenosine deaminase (ADA activity in pleural fluid for the diagnosis of TPE. ADA activity was measured in pleural fluid of 103 pleural effusion patients by colorimetric method using a commercial ADA assay kit. The diagnosis of TPE was made from pleural fluid examinations (including cytology, biochemistry, and bacteriology and pleural biopsy. Patient with negative result of this methods were diagnosed by response of empirical treatment. Out of 130 cases, 62 (61.1% had TPE and the remaining 41 (39.8% had pleural effusion due to non tuberculous diseases. There was statistically significant difference (p < 0.001 between the mean of pleural fluid ADA levels (70.82±22.54 U/L in TPE group and (30.07±22.93 U/L in non-TPE group. Of 62 TPE cases, microscopy for AFB and culture for M.tuberculosis in pleural fluid revealed positivity in 9.6% and 22.5% cases respectively, and biopsy of pleura showed typical epithelioid granuloma in only 43.5% cases. The cut-off value of ADA for diagnosing TPE was 40 U/L using a ROC curve, with a sensitivity of 94% and specificity of 88%. Positive and negative predictive value of ADA assay were 92% and 90% respectively. The overall test accuracy was 90%. Pleural fluid ADA assay is therefore a simple, rapid, highly sensitive and specific adjunct test for diagnosis of TPE. Ibrahim Med. Coll. J. 2011; 5(1: 1-5

  9. Cerebrospinal fluid adenosine deaminase levels as a diagnostic marker in tuberculous meningitis in adult Nepalese patients

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    Anil Chander

    2013-02-01

    Full Text Available Objective: To study the cerebrospinal fluid (CSF adenosine deaminase (ADA levels in tuberculous meningitis (TBM and non-TBM -viral meningitis cases and to determine its diagnostic significance as a biochemical marker of TBM infection.Methods: The study population comprised two different patient groups. TBM - group I - 28 cases and non-TBM-viral meningitis - 22 cases. These were enrolled consecutively in the study and CSF specimens were collected from them. ADA estimation was carried out by spectrophotometry.Results: ADA levels (mean依 SD in the TBM and non-TBM groups were 16.46依6.24 U/L and 5.13依2.96 U/L, respectively (highly significant P10 IU/L, the test showed a good sensitivity of 82.14% (95% CI 64.41-92.12 and a high specificity of 90.91% (95% CI 72.19-97.47. Positive and negative predictive value and positive and negative likelihood ratios and accuracy of the test in TBM cases were 92% (95% CI 75.03-97.77, 80% (95% CI 60.86-91.13, 9.03 (95% CI 2.38- 34.25, 0.19 (95% CI 0.09-0.44 and 86%, respectively.Conclusion: CSF ADA levels are elevated in the TBM cases as compared to the non-TBM - viral meningitis cases with a good sensitivity and a high specificity. It is a simple and inexpensive diagnostic adjunctive test in the rapid and early diagnosis of TBM.

  10. Autoimmune dysregulation and purine metabolism in adenosine deaminase (ADA-deficiency

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    Aisha Vanessa Sauer

    2012-08-01

    Full Text Available Genetic defects in the adenosine deaminase (ADA gene are among the most common causes for severe combined immunodeficiency (SCID. ADA-SCID patients suffer from lymphopenia, severely impaired cellular and humoral immunity, failure to thrive and recurrent infections. Currently available therapeutic options for this otherwise fatal disorder include bone marrow transplantation (BMT, enzyme replacement therapy with bovine ADA (PEG-ADA or hematopoietic stem cell gene therapy (HSC-GT. Although varying degrees of immune reconstitution can be achieved by these treatments, breakdown of tolerance is a major concern in ADA-SCID. Immune dysregulation such as autoimmune hypothyroidism, diabetes mellitus, hemolytic anemia, and immune thrombocytopenia are frequently observed in milder forms of the disease. However, several reports document similar complications also in patients on long-term PEG-ADA and after BMT or GT treatment.A skewed repertoire and decreased immune functions have been implicated in autoimmunity observed in certain B-cell and/or T-cell immunodeficiencies, but it remains unclear to what extent specific mechanisms of tolerance are affected in ADA deficiency. Herein we provide an overview about ADA-SCID and the autoimmune manifestations reported in these patients before and after treatment. We also assess the value of the ADA-deficient mouse model as a useful tool to study both immune and metabolic disease mechanisms. With focus on regulatory T and B cells we discuss the lymphocyte subpopulations particularly prone to contribute to the loss of self-tolerance and onset of autoimmunity in ADA deficiency. Moreover we address which aspects of immune dysregulation are specifically related to alterations in purine metabolism caused by the lack of ADA and the subsequent accumulation of metabolites with immunomodulatory properties.

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

    Science.gov (United States)

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

    2016-06-01

    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. 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. 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. 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.

  12. Diagnosis of tuberculosis pleurisy with adenosine deaminase (ADA): a systematic review and meta-analysis.

    Science.gov (United States)

    Gui, Xuwei; Xiao, Heping

    2014-01-01

    This systematic review and meta-analysis was performed to determine accuracy and usefulness of adenosine deaminase (ADA) in diagnosis of tuberculosis pleurisy. Medline, Google scholar and Web of Science databases were searched to identify related studies until 2014. Two reviewers independently assessed quality of studies included according to standard Quality Assessment of Diagnosis Accuracy Studies (QUADAS) criteria. The sensitivity, specificity, diagnostic odds ratio and other parameters of ADA in diagnosis of tuberculosis pleurisy were analyzed with Meta-DiSC1.4 software, and pooled using the random effects model. Twelve studies including 865 tuberculosis pleurisy patients and 1379 non-tuberculosis pleurisy subjects were identified from 110 studies for this meta-analysis. The sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnosis odds ratio (DOR) of ADA in the diagnosis of tuberculosis pleurisy were 45.25 (95% CI 27.63-74.08), 0.86 (95% CI 0.84-0.88), 0.88 (95% CI 0.86-0.90), 6.32 (95% CI 4.83-8.26) and 0.15 (95% 0.11-0.22), respectively. The area under the summary receiver operating characteristic curve (SROC) was 0.9340. Our results demonstrate that the sensitivity and specificity of ADA are high in the diagnosis of tuberculosis pleurisy especially when ADA≥50 (U/L). Thus, ADA is a relatively sensitive and specific marker for tuberculosis pleurisy diagnosis. However, it is cautious to apply these results due to the heterogeneity in study design of these studies. Further studies are required to confirm the optimal cut-off value of ADA.

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

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    Ruolin Li

    2016-05-01

    Full Text Available 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

  14. Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

    Science.gov (United States)

    Carriglio, Nicola; Klapwijk, Jan; Hernandez, Raisa Jofra; Vezzoli, Michela; Chanut, Franck; Lowe, Rhiannon; Draghici, Elena; Nord, Melanie; Albertini, Paola; Cristofori, Patrizia; Richards, Jane; Staton, Hazel; Appleby, Jonathan; Aiuti, Alessandro; Sauer, Aisha V

    2017-03-01

    GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients. Ex vivo gene therapy of patient bone-marrow CD34+ cells is an alternative treatment. In order to prepare for a marketing authorization application in the European Union, preclinical safety studies in mice were requested by the European Medicines Agency (EMA). A pilot study and a main biodistribution study were performed according to Good Laboratory Practice (GLP) at the San Raffaele Telethon Institute for Gene Therapy test facility. In the main study, human umbilical cord blood (UCB)-derived CD34+ cells were transduced with gamma-retroviral vector used in the production of GSK2696273. Groups of 10 male and 10 female NOD-SCID gamma (NSG) mice were injected intravenously with a single dose of transduced- or mock-transduced UCB CD34+ cells, and they were observed for 4 months. Engraftment and multilineage differentiation of blood cells was observed in the majority of animals in both groups. There was no significant difference in the level of chimerism between the two groups. In the gene therapy group, vector was detectable in lymphohemopoietic and nonlymphohemopoietic tissues, consistent with the presence of gene-modified human hematopoietic donor cells. Given the absence of relevant safety concerns in the data, the nonclinical studies and the clinical experience with GSK2696273 supported a successful application for market authorization in the European Union for the treatment of ADA-SCID patients, for whom no suitable human leukocyte

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

    Energy Technology Data Exchange (ETDEWEB)

    Ku, Min-Je [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Won-Ho [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Nam, Ki-hyun; Rhee, Kyeong-hee [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Ki-Seog [Biotechnology and Genetic Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Kim, Eunice EunKyung [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Yu, Myung-Hee [Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Hwang, Kwang Yeon, E-mail: hwangky@kist.re.kr [Biomedical Research Center, Life Science Division, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Functional Proteomics Center, Korea Institute of Science and Technology, 39-1 Hawolgok-dong, Seongbuk-gu, Seoul 136-791 (Korea, Republic of)

    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%.

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

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    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. © Georg Thieme Verlag KG Stuttgart · New York.

  18. Molecular characterization of adenosine 5'-monophosphate deaminase--the key enzyme responsible for the umami taste of nori (Porphyra yezoensis Ueda, Rhodophyta).

    Science.gov (United States)

    Minami, Seiko; Sato, Minoru; Shiraiwa, Yoshihiro; Iwamoto, Koji

    2011-12-01

    The enzyme adenosine 5'-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5'-monophosphate to inosine 5'-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.

  19. The genetics of feto-placental development: A study of acid phosphatase locus 1 and adenosine deaminase polymorphisms in a consecutive series of newborn infants

    Directory of Open Access Journals (Sweden)

    Bergamaschi Antonio

    2008-09-01

    Full Text Available Abstract Background Acid phosphatase locus 1 and adenosine deaminase locus 1 polymorphisms show cooperative effects on glucose metabolism and immunological functions. The recent observation of cooperation between the two systems on susceptibility to repeated spontaneous miscarriage prompted us to search for possible interactional effects between these genes and the correlation between birth weight and placental weight. Deviation from a balanced development of the feto-placental unit has been found to be associated with perinatal morbidity and mortality and with cardiovascular diseases in adulthood. Methods We examined 400 consecutive newborns from the Caucasian population of Rome. Birth weight, placental weight, and gestational length were registered. Acid phosphatase locus 1 and adenosine deaminase locus 1 phenotypes were determined by starch gel electrophoresis and correlation analysis was performed by SPSS programs. Informed verbal consent to participate in the study was obtained from the mothers. Results Highly significant differences in birth weight-placental weight correlations were observed among acid phosphatase locus 1 phenotypes (p = 0.005. The correlation between birth weight and placental weight was markedly elevated in subjects carrying acid phosphatase locus 1 phenotypes with medium-low F isoform concentration (A, CA and CB phenotypes compared to those carrying acid phosphatase locus 1 phenotypes with medium-high F isoform concentration (BA and B phenotypes (p = 0.002. Environmental and developmental variables were found to exert a significant effect on birth weight-placental weight correlation in subjects with medium-high F isoform concentrations, but only a marginal effect was observed in those with medium-low F isoform concentrations. The correlation between birth weight and placental weight is higher among carriers of the adenosine deaminase locus 1 allele*2, which is associated with low activity, than in homozygous adenosine

  20. A case of red-cell adenosine deaminase overproduction associated with hereditary hemolytic anemia found in Japan.

    Science.gov (United States)

    Miwa, S; Fujii, H; Matsumoto, N; Nakatsuji, T; Oda, S; Asano, H; Asano, S

    1978-01-01

    A case of red cell adenosine deaminase (ADA) overproduction associated with hereditary hemolytic anemia is reported here. This appears to be the second report. Proband is a 38-year-old Japanese male who had hemoglobin, 15.8 g/100 ml; reticulocyte count, 4.5%; serum indirect bilirubin, 4.9 mg/100 ml; 51Cr-labeled red cell half-life, 12 days; red cells showed moderate stomatocytosis. His red cell ADA activity showed 40-fold increase while that of the mother showed 4-fold increase. The mother was hematologically normal. The father had a normal enzyme activity. The proband and the mother showed slightly high serum uric acid levels. The proband's red cell showed: ATP, 628 nmoles/ml (normal, 1,010--1,550); adenine nucleotide pool, 46% of the normal mean; 2,3-diphosphoglycerate content, 3,782 nmoles/ml (normal 4,170--5,300); increased oxygen affinity of hemoglobin, P50 of intact erythrocytes being 21.8 mmHg (normal, 24.1--26.1). Red cell glycolytic intermediates in the proband were low in general, and the rate of lactate production was low. Kinetic studies using crude hemolysate revealed a normal Km for adenosine, normal electrophoretic mobility but slightly abnormal pH curve and slightly low utilization of 2-deoxyadenosine. The ADA activity of lymphocytes was nearly normal.

  1. Partial resolution of bone lesions. A child with severe combined immunodeficiency disease and adenosine deaminase deficiency after enzyme-replacement therapy

    International Nuclear Information System (INIS)

    Yulish, B.S.; Stern, R.C.; Polmar, S.H.

    1980-01-01

    A child with severe combined immunodeficiency disease and adenosine deaminase deficiency, with characteristic bone dysplasia, was treated with transfusions of frozen irradiated RBCs as a means of enzyme replacement. This therapy resulted in restoration of immunologic competence and partial resolution of the bone lesions. Although the natural history of these lesions without therapy is not known, enzyme-replacement therapy may have played a role in the resolution of this patient's bone lesions

  2. First Occurrence of Plasmablastic Lymphoma in Adenosine Deaminase-Deficient Severe Combined Immunodeficiency Disease Patient and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Maddalena Migliavacca

    2018-02-01

    Full Text Available Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein–Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%. The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.

  3. Can pleural adenosine deaminase (ADA) levels in pleural tuberculosis predict the presence of pulmonary tuberculosis? A CT analysis

    International Nuclear Information System (INIS)

    Koh, Myung Je; Lee, In Jae; Kim, Joo-Hee

    2016-01-01

    Aim: To assess the relationship between imaging features of pulmonary tuberculosis at computed tomography (CT) and adenosine deaminase (ADA) values via pleural fluid analysis in patients with pleural tuberculosis. Materials and methods: This retrospective study enrolled 60 patients who underwent fluid analysis for ADA and chest CT and were diagnosed with tuberculosis by culture or polymerase chain reaction of pleural fluid and sputum. The presence of centrilobular nodules, consolidation, cavitation, and mediastinal lymphadenopathy at CT were evaluated. The relationship between ADA values and the pattern of pulmonary involvement of tuberculosis was analysed. Results: Pulmonary involvement was seen in 42 of the 60 patients. A centrilobular nodular pattern was seen in 37 and consolidation in 22. In 17 patients, both findings were identified. A centrilobular nodular pattern was more common than consolidation or cavitary lesions. When ADA values were high, pulmonary involvement was more frequent (p=0.002). Comparing low and high ADA groups using an obtained cut-off value of 80 IU/l, the high group had more frequent pulmonary involvement (p<0.001). Conclusion: Patients with tuberculous pleurisy who had high ADA values had a higher probability of manifesting pulmonary tuberculosis. High ADA values may help predict contagious pleuroparenchymal tuberculosis. The most common pulmonary involvement of tuberculous pleurisy showed a centrilobular nodular pattern. - Highlights: • To know the relationship of ADA values and pulmonary involvement pattern of pleural tuberculosis. • To help exact diagnosis of pleuroparenchymal tuberculosis in clinical setting. • The imaging findings of pleuroparenchymal tuberculosis.

  4. Utility of adenosine deaminase (ADA), PCR & thoracoscopy in differentiating tuberculous & non-tuberculous pleural effusion complicating chronic kidney disease.

    Science.gov (United States)

    Kumar, Sravan; Agarwal, Ritesh; Bal, Amanjit; Sharma, Kusum; Singh, Navneet; Aggarwal, Ashutosh N; Verma, Indu; Rana, Satyawati V; Jha, Vivekanand

    2015-03-01

    Pleural effusion is a common occurrence in patients with late-stage chronic kidney disease (CKD). In developing countries, many effusions remain undiagnosed after pleural fluid analysis (PFA) and patients are empirically treated with antitubercular therapy. The aim of this study was to evaluate the role of adenosine deaminase (ADA), nucleic acid amplification tests (NAAT) and medical thoracoscopy in distinguishing tubercular and non-tubercular aetiologies in exudative pleural effusions complicating CKD. Consecutive stage 4 and 5 CKD patients with pleural effusions underwent PFA including ADA and PCR [65 kDa gene; multiplex (IS6110, protein antigen b, MPB64)]. Patients with exudative pleural effusion undiagnosed after PFA underwent medical thoracoscopy. All 107 patients underwent thoracocentesis with 45 and 62 patients diagnosed as transudative and exudative pleural effusions, respectively. Twenty six of the 62 patients underwent medical thoracoscopy. Tuberculous pleurisy was diagnosed in six while uraemic pleuritis was diagnosed in 20 subjects. The sensitivity and specificity of pleural fluid ADA, 65 kDa gene PCR, and multiplex PCR were 66.7 and 90 per cent, 100 and 50 per cent, and 100 and 100 per cent, respectively. Thoracoscopy was associated with five complications in three patients. Uraemia remains the most common cause of pleural effusion in CKD even in high TB prevalence country. Multiplex PCR and thoracoscopy are useful investigations in the diagnostic work-up of pleural effusions complicating CKD while the sensitivity and/or specificity of ADA and 65 kDa gene PCR is poor.

  5. Can pleural adenosine deaminase (ADA) levels in pleural tuberculosis predict the presence of pulmonary tuberculosis? A CT analysis.

    Science.gov (United States)

    Koh, Myung Je; Lee, In Jae; Kim, Joo-Hee

    2016-06-01

    To assess the relationship between imaging features of pulmonary tuberculosis at computed tomography (CT) and adenosine deaminase (ADA) values via pleural fluid analysis in patients with pleural tuberculosis. This retrospective study enrolled 60 patients who underwent fluid analysis for ADA and chest CT and were diagnosed with tuberculosis by culture or polymerase chain reaction of pleural fluid and sputum. The presence of centrilobular nodules, consolidation, cavitation, and mediastinal lymphadenopathy at CT were evaluated. The relationship between ADA values and the pattern of pulmonary involvement of tuberculosis was analysed. Pulmonary involvement was seen in 42 of the 60 patients. A centrilobular nodular pattern was seen in 37 and consolidation in 22. In 17 patients, both findings were identified. A centrilobular nodular pattern was more common than consolidation or cavitary lesions. When ADA values were high, pulmonary involvement was more frequent (p=0.002). Comparing low and high ADA groups using an obtained cut-off value of 80 IU/l, the high group had more frequent pulmonary involvement (pADA values had a higher probability of manifesting pulmonary tuberculosis. High ADA values may help predict contagious pleuroparenchymal tuberculosis. The most common pulmonary involvement of tuberculous pleurisy showed a centrilobular nodular pattern. Copyright © 2016 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

  6. Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans.

    Science.gov (United States)

    Candotti, Fabio; Shaw, Kit L; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F; Weinberg, Kenneth I; Crooks, Gay M; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S; Rosenblatt, Howard M; Davis, Carla M; Hanson, Celine; Rishi, Radha G; Wang, Xiaoyan; Gjertson, David; Yang, Otto O; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A; Engel, Barbara C; Podsakoff, Gregory M; Hershfield, Michael S; Blaese, R Michael; Parkman, Robertson; Kohn, Donald B

    2012-11-01

    We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

  7. Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica.

    Science.gov (United States)

    Dieni, Christopher A; Storey, Kenneth B

    2008-04-22

    The wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65-70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle. Wood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to approximately 1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg.ATP and Mg.ADP and inhibited by Mg.GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5 degrees C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing

  8. Crystallization and preliminary X-ray crystallographic analysis of adenosine 5′-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5′-phosphate

    International Nuclear Information System (INIS)

    Han, Byung Woo; Bingman, Craig A.; Mahnke, Donna K.; Sabina, Richard L.; Phillips, George N. Jr

    2005-01-01

    Adenosine 5′-monophosphate deaminase from A. thaliana has been crystallized in complex with coformycin 5′-phosphate. Diffraction data have been collected to 3.34 Å resolution. Adenosine 5′-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5′-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5′-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6 2 22, with unit-cell parameters a = b = 131.325, c = 208.254 Å, α = β = 90, γ = 120°. Diffraction data were collected to 3.34 Å resolution from a crystal in complex with coformycin 5′-phosphate and to 4.05 Å resolution from a crystal of a mercury derivative

  9. Crystallization and preliminary X-ray crystallographic analysis of adenosine 5′-monophosphate deaminase (AMPD) from Arabidopsis thaliana in complex with coformycin 5′-phosphate

    Energy Technology Data Exchange (ETDEWEB)

    Han, Byung Woo [Department of Biochemistry, University of Wisconsin-Madison, WI 53706-1544 (United States); Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States); Bingman, Craig A. [Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States); Mahnke, Donna K.; Sabina, Richard L. [Department of Biochemistry, The Medical College of Wisconsin, Milwaukee, WI 53226-4801 (United States); Phillips, George N. Jr, E-mail: phillips@biochem.wisc.edu [Department of Biochemistry, University of Wisconsin-Madison, WI 53706-1544 (United States); Center for Eukaryotic Structural Genomics (CESG), University of Wisconsin-Madison, WI 53706-1549 (United States)

    2005-08-01

    Adenosine 5′-monophosphate deaminase from A. thaliana has been crystallized in complex with coformycin 5′-phosphate. Diffraction data have been collected to 3.34 Å resolution. Adenosine 5′-monophosphate deaminase (AMPD) is a eukaryotic enzyme that converts adenosine 5′-monophosphate (AMP) to inosine 5′-monophosphate (IMP) and ammonia. AMPD from Arabidopsis thaliana (AtAMPD) was cloned into the baculoviral transfer vector p2Bac and co-transfected along with a modified baculoviral genome into Spodoptera frugiperda (Sf9) cells. The resulting recombinant baculovirus were plaque-purified, amplified and used to overexpress recombinant AtAMPD. Crystals of purified AtAMPD have been obtained to which coformycin 5′-phosphate, a transition-state inhibitor, is bound. Crystals belong to space group P6{sub 2}22, with unit-cell parameters a = b = 131.325, c = 208.254 Å, α = β = 90, γ = 120°. Diffraction data were collected to 3.34 Å resolution from a crystal in complex with coformycin 5′-phosphate and to 4.05 Å resolution from a crystal of a mercury derivative.

  10. A decision tree for the genetic diagnosis of deficiency of adenosine deaminase 2 (DADA2): a French reference centres experience.

    Science.gov (United States)

    Rama, Mélanie; Duflos, Claire; Melki, Isabelle; Bessis, Didier; Bonhomme, Axelle; Martin, Hélène; Doummar, Diane; Valence, Stéphanie; Rodriguez, Diana; Carme, Emilie; Genevieve, David; Heimdal, Ketil; Insalaco, Antonella; Franck, Nathalie; Queyrel-Moranne, Viviane; Tieulie, Nathalie; London, Jonathan; Uettwiller, Florence; Georgin-Lavialle, Sophie; Belot, Alexandre; Koné-Paut, Isabelle; Hentgen, Véronique; Boursier, Guilaine; Touitou, Isabelle; Sarrabay, Guillaume

    2018-04-23

    Deficiency of adenosine deaminase 2 (DADA2) is a recently described autoinflammatory disorder. Genetic analysis is required to confirm the diagnosis. We aimed to describe the identifying symptoms and genotypes of patients referred to our reference centres and to improve the indications for genetic testing. DNA from 66 patients with clinically suspected DADA2 were sequenced by Sanger or next-generation sequencing. Detailed epidemiological, clinical and biological features were collected by use of a questionnaire and were compared between patients with and without genetic confirmation of DADA2. We identified 13 patients (19.6%) carrying recessively inherited mutations in ADA2 that were predicted to be deleterious. Eight patients were compound heterozygous for mutations. Seven mutations were novel (4 missense variants, 2 predicted to affect mRNA splicing and 1 frameshift). The mean age of the 13 patients with genetic confirmation was 12.7 years at disease onset and 20.8 years at diagnosis. Phenotypic manifestations included fever (85%), vasculitis (85%) and neurological disorders (54%). Features best associated with a confirmatory genotype included fever with neurologic or cutaneous attacks (odds ratio [OR] 10.71, p = 0.003 and OR 10.9, p < 0.001), fever alone (OR 8.1, p = 0.01), and elevated C-reactive protein (CRP) level with neurologic involvement (OR 6.63, p = 0.017). Our proposed decision tree may help improve obtaining genetic confirmation of DADA2 in the context of autoinflammatory symptoms. Prerequisites for quick and low-cost Sanger analysis include one typical cutaneous or neurological sign, one marker of inflammation (fever or elevated CRP level), and recurrent or chronic attacks in adults.

  11. A Hospital Based Study on Estimation of Adenosine Deaminase Activity (ADA) in Cerebrospinal Fluid (CSF) in Various Types of Meningitis.

    Science.gov (United States)

    Agarwal, Ashok Kumar; Bansal, Sonia; Nand, Vidya

    2014-02-01

    Tuberculosis kills 3.70 lakh patients in India every year,out of which 7-12 % are meningeal involvement. Delay in its diagnosis and initiation of treatment results in poor prognosis and squeal in up to 25% of cases. The aim of the present study is to look for a simple, rapid, cost effective, and fairly specific test in differentiating tubercular aetiology from other causes of meningitis. In the present study we measured the adenosine deaminase activity (ADA) in Cerebrospinal Fluid (CSF) of Tubercular Meningitis (TBM) and non-TBM patients. Fifty six patients attending hospital with symptoms and signs of meningitis were selected and divided into three groups: tubercular, pyogenic, and aseptic meningitis, depending upon the accepted criteria. CSF was drawn and ADA estimated. Out of 32 tubercular patients, 28 had CSF-ADA at or above the cut-off value while four had below. Out of 24 non-tuberculous patients (pyogenic and aseptic meningitis), two aseptic meningitis (AM) patient had ADA levels at or above the cut-off value while 22 had below this value. RESULTS of our study indicate that ADA level estimation in CSF is not only of considerable value in the diagnosis of TBM, CSF, and ADA level 10 U/L as a cut-off value with sensitivity 87.5% and specificity 83.33% and positive predictive value of the test was 87.5%.and 83.3% negative predictive value. It can be concluded that ADA estimation in CSF is not only simple, inexpensive and rapid but also fairly specific method for making a diagnosis of tuberculous aetiology in TBM, especially when there is a dilemma of differentiating the tuberculous aetiology from non-tuberculous ones. For this reason ADA estimation in TBM may find a place as a routine investigation.

  12. Mixed Inhibition of Adenosine Deaminase Activity by 1,3-Dinitrobenzene: A Model for Understanding Cell-Selective Neurotoxicity in Chemically-Induced Energy Deprivation Syndromes in Brain

    Science.gov (United States)

    Wang, Yipei; Liu, Xin; Schneider, Brandon; Zverina, Elaina A.; Russ, Kristen; Wijeyesakere, Sanjeeva J.; Fierke, Carol A.; Richardson, Rudy J.; Philbert, Martin A.

    2012-01-01

    Astrocytes are acutely sensitive to 1,3-dinitrobenzene (1,3-DNB) while adjacent neurons are relatively unaffected, consistent with other chemically-induced energy deprivation syndromes. Previous studies have investigated the role of astrocytes in protecting neurons from hypoxia and chemical injury via adenosine release. Adenosine is considered neuroprotective, but it is rapidly removed by extracellular deaminases such as adenosine deaminase (ADA). The present study tested the hypothesis that ADA is inhibited by 1,3-DNB as a substrate mimic, thereby preventing adenosine catabolism. ADA was inhibited by 1,3-DNB with an IC50 of 284μM, Hill slope, n = 4.8 ± 0.4. Native gel electrophoresis showed that 1,3-DNB did not denature ADA. Furthermore, adding Triton X-100 (0.01–0.05%, wt/vol), Nonidet P-40 (0.0015–0.0036%, wt/vol), or bovine serum albumin (0.05 mg/ml or changing [ADA] (0.2 and 2nM) did not substantially alter the 1,3-DNB IC50 value. Likewise, dynamic light scattering showed no particle formation over a (1,3-DNB) range of 149–1043μM. Kinetics revealed mixed inhibition with 1,3-DNB binding to ADA (KI = 520 ± 100μM, n = 1 ± 0.6) and the ADA-adenosine complex (KIS = 262 ± 7μM, n = 6 ± 0.6, indicating positive cooperativity). In accord with the kinetics, docking predicted binding of 1,3-DNB to the active site and three peripheral sites. In addition, exposure of DI TNC-1 astrocytes to 10–500μM 1,3-DNB produced concentration-dependent increases in extracellular adenosine at 24 h. Overall, the results demonstrate that 1,3-DNB is a mixed inhibitor of ADA and may thus lead to increases in extracellular adenosine. The finding may provide insights to guide future work on chemically-induced energy deprivation. PMID:22106038

  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.

    Science.gov (United States)

    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.

  14. Analysis of serum adenosine deaminase (ADA) and ADA1 and ADA2 isoenzyme activities in HIV positive and HIV-HBV co-infected patients.

    Science.gov (United States)

    Khodadadi, Iraj; Abdi, Mohammad; Ahmadi, Abbas; Wahedi, Mohammad Saleh; Menbari, Shahoo; Lahoorpour, Fariba; Rahbari, Rezgar

    2011-08-01

    To determine adenosine deaminase (ADA) activity as a possible diagnostic marker in HIV and HIV-HBV co-infected patients. Blood samples were collected from 72 healthy, 33 HIV positive and 30 HIV-HBV co-infected subjects. Blood CD4+ cell count was recorded and serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total ADA, and ADA1 and ADA2 isoenzyme activities were determined. Serum ALT, AST, total ADA and ADA2 isoenzyme activities were significantly higher in HIV positive and HIV-HBV co-infected groups compare to the control (pADA activities (R(2)=0.589, pADA was significantly increased in HIV and HIV-HBV co-infections. Therefore, because of its low cost and simplicity to perform, ADA activity might be considered as a useful diagnostic tool among the other markers in these diseases. Copyright © 2011 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  15. Ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) activities in prostate cancer patients: influence of Gleason score, treatment and bone metastasis.

    Science.gov (United States)

    Battisti, Vanessa; Maders, Liési D K; Bagatini, Margarete D; Battisti, Iara E; Bellé, Luziane P; Santos, Karen F; Maldonado, Paula A; Thomé, Gustavo R; Schetinger, Maria R C; Morsch, Vera M

    2013-04-01

    The relation between adenine nucleotides and cancer has already been described in literature. Considering that the enzymes ectonucleotide pyrophosphatase/phosphodiesterase (E-NPP) and adenosine deaminase (ADA) act together to control nucleotide levels, we aimed to investigate the role of these enzymes in prostate cancer (PCa). E-NPP and ADA activities were determined in serum and platelets of PCa patients and controls. We also verified the influence of the Gleason score, bone metastasis and treatment in the enzyme activities. Platelets and serum E-NPP activity increased, whereas ADA activity in serum decreased in PCa patients. In addition, Gleason score, metastasis and treatment influenced E-NPP and ADA activities. We may propose that E-NPP and ADA are involved in the development of PCa. Moreover, E-NPP and ADA activities are modified in PCa patients with distinct Gleason score, with bone metastasis, as well as in patients under treatment. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  16. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    Science.gov (United States)

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  17. Tandem mass spectrometry, but not T-cell receptor excision circle analysis, identifies newborns with late-onset adenosine deaminase deficiency.

    Science.gov (United States)

    la Marca, Giancarlo; Canessa, Clementina; Giocaliere, Elisa; Romano, Francesca; Duse, Marzia; Malvagia, Sabrina; Lippi, Francesca; Funghini, Silvia; Bianchi, Leila; Della Bona, Maria Luisa; Valleriani, Claudia; Ombrone, Daniela; Moriondo, Maria; Villanelli, Fabio; Speckmann, Carsten; Adams, Stuart; Gaspar, Bobby H; Hershfield, Michael; Santisteban, Ines; Fairbanks, Lynette; Ragusa, Giovanni; Resti, Massimo; de Martino, Maurizio; Guerrini, Renzo; Azzari, Chiara

    2013-06-01

    Adenosine deaminase (ADA)-severe combined immunodeficiency (SCID) is caused by genetic variants that disrupt the function of ADA. In its early-onset form, it is rapidly fatal to infants. Delayed or late-onset ADA-SCID is characterized by insidious progressive immunodeficiency that leads to permanent organ damage or death. Quantification of T-cell receptor excision circles (TRECs) or tandem mass spectrometry (tandem-MS) analysis of dried blood spots (DBSs) collected at birth can identify newborns with early-onset ADA-SCID and are used in screening programs. However, it is not clear whether these analyses can identify newborns who will have delayed or late-onset ADA-SCID before symptoms appear. We performed a retrospective study to evaluate whether tandem-MS and quantitative TREC analyses of DBSs could identify newborns who had delayed-onset ADA-SCID later in life. We tested stored DBSs collected at birth from 3 patients with delayed-onset ADA-SCID using tandem-MS (PCT EP2010/070517) to evaluate levels of adenosine and 2'-deoxyadenosine and real-time PCR to quantify TREC levels. We also analyzed DBSs from 3 newborns with early-onset ADA-SCID and 2 healthy newborn carriers of ADA deficiency. The DBSs taken at birth from the 3 patients with delayed-onset ADA-SCID had adenosine levels of 10, 25, and 19 μmol/L (normal value, <1.5 μmol/L) and 2'-deoxyadenosine levels of 0.7, 2.7, and 2.4 μmol/L (normal value, <0.07 μmol/L); the mean levels of adenosine and 2'-deoxyadenosine were respectively 12.0- and 27.6-fold higher than normal values. DBSs taken at birth from all 3 patients with delayed-onset ADA deficiency had normal TREC levels, but TRECs were undetectable in blood samples taken from the same patients at the time of diagnosis. Tandem-MS but not TREC quantification identifies newborns with delayed- or late-onset ADA deficiency. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  18. Increased Number of Circulating CD8/CD26 T Cells in the Blood of Duchenne Muscular Dystrophy Patients Is Associated with Augmented Binding of Adenosine Deaminase and Higher Muscular Strength Scores

    Directory of Open Access Journals (Sweden)

    Jonathan H. Soslow

    2017-12-01

    Full Text Available Duchenne muscular dystrophy (DMD is an X-linked disorder that leads to cardiac and skeletal myopathy. The complex immune activation in boys with DMD is incompletely understood. To better understand the contribution of the immune system into the progression of DMD, we performed a systematic characterization of immune cell subpopulations obtained from peripheral blood of DMD subjects and control donors. We found that the number of CD8 cells expressing CD26 (also known as adenosine deaminase complexing protein 2 was increased in DMD subjects compared to control. No differences, however, were found in the levels of circulating factors associated with pro-inflammatory activation of CD8/CD26 cells, such as tumor necrosis factor-α (TNFα, granzyme B, and interferon-γ (IFNγ. The number of CD8/CD26 cells correlated directly with quantitative muscle testing (QMT in DMD subjects. Since CD26 mediates binding of adenosine deaminase (ADA to the T cell surface, we tested ADA-binding capacity of CD8/CD26 cells and the activity of bound ADA. We found that mononuclear cells (MNC obtained from DMD subjects with an increased number of CD8/CD26 T cells had a greater capacity to bind ADA. In addition, these MNC demonstrated increased hydrolytic deamination of adenosine to inosine. Altogether, our data demonstrated that (1 an increased number of circulating CD8/CD26 T cells is associated with preservation of muscle strength in DMD subjects, and (2 CD8/CD26 T cells from DMD subjects mediated degradation of adenosine by adenosine deaminase. These results support a role for T cells in slowing the decline in skeletal muscle function, and a need for further investigation into contribution of CD8/CD26 T cells in the regulation of chronic inflammation associated with DMD.

  19. G22A Polymorphism of Adenosine Deaminase and its Association with Biochemical Characteristics of Gestational Diabetes Mellitus in an Iranian Population

    Directory of Open Access Journals (Sweden)

    Mohammad Ali Takhshid

    2015-03-01

    Full Text Available Adenosine deaminase (ADA is an important regulator of insulin action. The single nucleotide polymorphism (SNP G22A in the ADA gene decreases enzymatic activity of ADA. The aim of this study was to investigate the relationship between the SNP G22A and blood glycemic control, insulin resistance, and obesity of gestational diabetes mellitus (GDM patients in an Iranian population. SNP G22A was determined in women with GDM (N=70 and healthy pregnant women (control, N=70 using polymerase chain reaction-restriction fragment length polymorphism. Fasting plasma glucose (FPG, hemoglobin A1C (HbA1c, plasma insulin levels and plasma lipids were measured using commercial kits. Homeostasis model of assessment for insulin resistance (HOMA-IR was calculated. The distribution of genotypes and alleles among GDM patients was similar to that of the control group. FPG and HbA1c were significantly higher in GDM patients with GG genotype compared with GDM patients with GA+AA genotype and non-GDM patients. The frequency of GG genotype was significantly higher in obese GDM patients compared to lean GDM patients. The SNP G22A in the ADA gene was not associated with the risk of GDM in our population. GG genotype was associated with poor glycemic control and obesity in GDM patients.

  20. The Role of G22 A Adenosine Deaminase 1 Gene Polymorphism and the Activities of ADA Isoenzymes in Fertile and Infertile Men.

    Science.gov (United States)

    Fattahi, Amir; Khodadadi, Iraj; Amiri, Iraj; Latifi, Zeinab; Ghorbani, Marzieh; Tavilani, Heidar

    2015-10-01

    To evaluate frequency distribution of adenosine deaminase 1 (ADA1) G22 A alleles and genotypes in fertile and infertile men. In this study we evaluate frequency distribution of ADA1 G22 A alleles and genotypes in 200 fertile and 200 infertile men. The polymerase chain reaction-restriction fragment length polymorphism technique was used for determining ADA1 G22 A variants. In addition, ADA isoenzymes activities (ADA1 and ADA2) were measured using colorimetric method. The frequency of GG genotype was significantly higher and GA genotype was lower in infertile males compared with fertile men (P = .048 and P = .045, respectively). However, there was not any noticeable difference in allele distribution between groups (P >.05). Based on logistic regression analysis, the GA genotype has a protective role and can decrease the risk of male infertility 1.7 times (P = .046). There were significantly higher activities of ADAT and its isoenzymes in infertile males compared with fertile men (P ADA1 activity with GG genotype was higher than GA carriers in all population (P = .001). Our results revealed that the activity of ADA isoenzymes and distribution of ADA1 G22 A genotypes were different among fertile and infertile men and more likely the GA genotype, which had lower ADA1 activity and was higher in fertile men is a protective factor against infertility. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Serum Adenosine Deaminase (ADA) Activity: A Novel Screening Test to Differentiate HIV Monoinfection From HIV-HBV and HIV-HCV Coinfections.

    Science.gov (United States)

    Abdi, Mohammad; Rahbari, Rizgar; Khatooni, Zahed; Naseri, Nima; Najafi, Adel; Khodadadi, Iraj

    2016-05-01

    CD4(+) cell count, the common HIV infection screening test, is costly and unable to differentiate HIV monoinfection from its concurrent infection with hepatitis B or C virus. We aimed to ascertain diagnostic value of serum adenosine deaminase (ADA) activity as a useful tool to differentiate HIV mono- and co-infection. Blood samples were collected from 30 HIV-HBV and 30 HIV-HCV coinfected patients, 33 HIV positive subjects, and 72 controls. CD4(+) cell count, serum total ADA (tADA), and ADA1, and ADA2 isoenzyme activities were determined and their sensitivity and specificity were computed. tADA and ADA2 activities were significantly higher and CD4(+) counts were markedly lower in all patients compared with controls. Strong inverse agreements between CD4(+) cell counts and both tADA and ADA2 activities were observed. Serum tADA and ADA1 activities showed the highest specificity and the highest sensitivity, respectively, for differentiating HIV monoinfection from HIV-HBV and HIV-HCV coinfections. We showed strong agreement and correlation between CD4(+) cell count and ADA enzyme activity. Based on high ADA sensitivity and specificity, it is concluded that determination of ADA activity might be a novel diagnostic tool to distinguish of HIV monoinfection from its coinfection with HBV or HCV. © 2015 Wiley Periodicals, Inc.

  2. Exploiting the pyrazolo[3,4-d]pyrimidin-4-one ring system as a useful template to obtain potent adenosine deaminase inhibitors.

    Science.gov (United States)

    La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Salerno, Silvia; Simorini, Francesca; Taliani, Sabrina; Marini, Anna Maria; Da Settimo, Federico; Lavecchia, Antonio; Novellino, Ettore; Antonioli, Luca; Fornai, Matteo; Blandizzi, Corrado; Del Tacca, Mario

    2009-03-26

    A number of pyrazolo[3,4-d]pyrimidin-4-ones bearing either alkyl or arylalkyl substituents in position 2 of the nucleus were synthesized and tested for their ability to inhibit adenosine deaminase (ADA) from bovine spleen. The 2-arylalkyl derivatives exhibited excellent inhibitory activity, showing Ki values in the nanomolar/subnanomolar range. The most active compound, 1-(4-((4-oxo-4,5-dihydropyrazolo[3,4-d]pyrimidin-2-yl)methyl)phenyl)-3-(4-(trifluoromethyl)phenyl)urea, 14d, was tested in rats with colitis induced by 2,4-dinitrobenzenesulfonic acid to assess its efficacy to attenuate bowel inflammation. The treatment with 14d induced a significant amelioration of both systemic and intestinal inflammatory alterations in animals with experimental colitis. Docking simulations of the synthesized compounds into the ADA catalytic site were also performed to rationalize the structure-activity relationships observed and to highlight the key pharmacophoric elements of these products, thus prospectively guiding the design of novel ADA inhibitors.

  3. Rapid, reliable, and sensitive detection of adenosine deaminase activity by UHPLC-Q-Orbitrap HRMS and its application to inhibitory activity evaluation of traditional Chinese medicines.

    Science.gov (United States)

    Qi, Shenglan; Guan, Huida; Deng, Gang; Yang, Tao; Cheng, Xuemei; Liu, Wei; Liu, Ping; Wang, Changhong

    2018-05-10

    Adenosine deaminase (ADA), which is a key enzyme in the metabolism of purine nucleosides, plays important roles in diverse disorders, such as tuberculosis, diabetes, liver disorders, and cancer. Determination of the activities of ADA and its isoenzymes in body fluids has received considerable attention in the diagnosis and treatment of relative diseases. Ultraviolet spectroscopy with adenosine (AD) as a substrate is a classical approach for screening potential ADA inhibitors by measuring the decrease in substrate (AD) at 265 nm or increase in the product (inosine) at 248 nm. However, AD and inosine share a very close maximum absorption wavelength, and the reaction is uncertain and is frequently interfered by the background color of matrix compounds or plant extracts. Thus, the method usually yields false positive or negative results. In this study, a novel, rapid, sensitive, and accurate ultra-high-performance liquid chromatography-Q exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometric (UHPLC-Q-Orbitrap HRMS) method was developed for determining and screening ADA inhibitors by directly determining the deamination product of AD, inosine. A proper separation was achieved for inosine and chlormequat (internal standard) within 2 min via isocratic elution (0.1% formic acid:methanol = 85:15, v/v) at a flow rate of 0.3 mL min -1 on a Waters ACQUITY HSS T3 column (2.1 mm × 100 mm, 1.8 μm) following a simple precipitation of proteins. The intra- and inter-day precisions of the developed method were below 7.17% and 8.99%, respectively. The method exhibited advantages of small total reaction volume (60 μL), short running time (2 min), high sensitivity (lowest limit of quantification of 0.02 μM for inosine), and low cost (small enzyme consumption of 0.007 unit mL -1 for ADA and substrate of 3.74 μM for AD in individual inhibition), and no matrix effects (101.64%-107.12%). Stability results showed that all

  4. Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity

    Science.gov (United States)

    Lee, Jae-Geun; Kang, Dong Gu; Yu, Jung Re; Kim, Youngree; Kim, Jinsoek; Koh, Gwanpyo

    2011-01-01

    Background Dipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents. Methods Fasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects. Results ADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; PADA activity was correlated with fasting plasma glucose (r=0.258, P9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; PADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; PADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect. PMID:21738897

  5. Curcumin improves episodic memory in cadmium induced memory impairment through inhibition of acetylcholinesterase and adenosine deaminase activities in a rat model.

    Science.gov (United States)

    Akinyemi, Ayodele Jacob; Okonkwo, Princess Kamsy; Faboya, Opeyemi Ayodeji; Onikanni, Sunday Amos; Fadaka, Adewale; Olayide, Israel; Akinyemi, Elizabeth Olufisayo; Oboh, Ganiyu

    2017-02-01

    Curcumin, the main polyphenolic component of turmeric (Curcuma longa) rhizomes has been reported to exert cognitive enhancing potential with limited scientific basis. Hence, this study sought to evaluate the effect of curcumin on cerebral cortex acetylcholinesterase (AChE) and adenosine deaminase (ADA) activities in cadmium (Cd)-induced memory impairment in rats. Animals were divided into six groups (n = 6): saline/vehicle, saline/curcumin 12.5 mg/kg, saline/curcumin 25 mg/kg, Cd/vehicle, Cd/curcumin 12.5 mg/kg, and Cd/curcumin 25 mg/kg. Rats received Cd (2.5 mg/kg) and curcumin (12.5 and 25 mg/kg, respectively) by gavage for 7 days. The results of this study revealed that cerebral cortex AChE and ADA activities were increased in Cd-poisoned rats, and curcumin co-treatment reversed these activities to the control levels. Furthermore, Cd intoxication increased the level of lipid peroxidation in cerebral cortex with a concomitant decreased in functional sulfuhydryl (-SH) group and nitric oxide (NO), a potent neurotransmitter and neuromodulatory agent. However, the co-treatment with curcumin at 12.5 and 25 mg/kg, respectively increased the non-enzymatic antioxidant status and NO in cerebral cortex with a decreased in malondialdehyde (MDA) level. Therefore, inhibition of AChE and ADA activities as well as increased antioxidant status by curcumin in Cd-induced memory dysfunction could suggest some possible mechanism of action for their cognitive enhancing properties.

  6. Evaluation of a diagnostic flow chart applying medical thoracoscopy, adenosine deaminase and T-SPOT.TB in diagnosis of tuberculous pleural effusion.

    Science.gov (United States)

    He, Y; Zhang, W; Huang, T; Wang, X; Wang, M

    2015-10-01

    To evaluate a diagnostic flow chart applying medical thoracoscoy (MT), adenosine deaminase (ADA) and T-SPOT.TB in diagnosis of tuberculous pleural effusion (TPE) at a high TB burden country. 136 patients with pleural effusion (PE) were enrolled and divided into TPE and Non-TPE group. MT (histology), PE ADA and T-SPOT.TB were conducted on all patients. ROC analysis was performed for the best cut-off value of PE ADA in detection of TPE. The diagnostic flow chart applying MT, ADA and T-SPOT.TB was evaluated for improving the limitations of each diagnostic method. ROC analysis showed that the best cut-off value of PE ADA was 30U/L. The sensitivity and specificity of these tests were calculated respectively to be: 71.4% (58.5%-81.6%) and 100% (95.4-100.0%) for MT, 92.9% (83.0-97.2%) and 68.8% (57.9-77.9%) for T-SPOT.TB, and 80.0% (69.6-88.1%) and 92.9% (82.7-98.0%) for PE ADA. The sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, positive predictive value and negative predictive value of the diagnostic flow chart were 96.4% (87.9-99.0%), 96.3% (89.6-98.7%), 25.714, 0.037, 97.4 and 94.9, respectively. The diagnostic flow chart applying MT, ADA and T-SPOT.TB is an accurate and rapid diagnostic method in detection of TPE.

  7. Changes in Adenosine Deaminase Activity in Patients with Type 2 Diabetes Mellitus and Effect of DPP-4 Inhibitor Treatment on ADA Activity.

    Science.gov (United States)

    Lee, Jae-Geun; Kang, Dong Gu; Yu, Jung Re; Kim, Youngree; Kim, Jinsoek; Koh, Gwanpyo; Lee, Daeho

    2011-04-01

    Dipeptidyl peptidase 4 (DPP-4, also known as CD26) binds with adenosine deaminase (ADA) to activate T lymphocytes. Here, we investigated whether ADA activity is specifically affected by treatment with DPP-4 inhibitor (DPP4I) compared with other anti-diabetic agents. Fasting ADA activity, in addition to various metabolic and biochemical parameters, were measured in 262 type 2 diabetes mellitus (T2DM) patients taking various anti-diabetic agents and in 46 non-diabetic control subjects. ADA activity was increased in T2DM patients compared with that in non-diabetic control subjects (mean±standard error, 23.1±0.6 U/L vs. 18.6±0.8 U/L; PADA activity was correlated with fasting plasma glucose (r=0.258, P9%) showed significantly increased ADA activity (21.1±0.8 U/L vs. 25.4±1.6 U/L; PADA activity in T2DM patients did not differ from those of other oral anti-diabetic agents or insulin. T2DM patients on metformin monotherapy showed a lower ADA activity (20.9±1.0 U/L vs. 28.1±2.8 U/L; PADA activity is increased in T2DM patients compared to that in non-diabetic patients, is positively correlated with blood glucose level, and that DPP4I has no additional specific effect on ADA activity, except for a glycemic control- or HbA1c-dependent effect.

  8. Identification of pleural effusion with low levels of adenosine deaminase but without signs of acute inflammation or pleural thickening to diagnose early malignant pleural mesothelioma

    International Nuclear Information System (INIS)

    Moriyama, Satoru; Tanahashi, Masayuki; Suzuki, Eriko

    2012-01-01

    We reviewed the clinical findings and diagnostic methods used in the diagnosis of malignant pleural mesothelioma (MPM) in patients with pleural effusion with low levels of adenosine deaminase (ADA), but without signs of acute inflammation or pleural thickening. The hospital records of 40 patients with pleural effusion of unknown origin or pleural thickening were retrospectively investigated. In all of those studies, pleural effusion was exudative, lymphocyte-dominant, no mycobacteria or other bacteria, and low levels of ADA. There were 30 men and 10 women with an average age of 62.8 years old. The diagnosis of MPM was obtained by cytology of the pleural effusion in 3 patients and by core-needle biopsy of the thickened pleura in 3. Thoracoscopic pleural biopsy under general anesthesia was performed for the other 34 patients. The pathologic diagnosis of pleural biopsy was MPM in 20 patients, inflammatory change in 12, and pleural dissemination of cancer in 2. All of the 7 patients with more than 100 μg/ml of hyaluronic acid in their pleural effusion received a diagnosis of MPM. A total of 20 of 23 patients with irregular or nodular pleural thickening on computed tomography findings were confirmed to have MPM. Moreover, 6 of 17 patients with smooth pleural thickening were confirmed to have MPM. In patients with nodular pleural thickening it was easy to make the diagnosis. However, in those with smooth thickening, careful observation was required to select the appropriate biopsy site and resection margins of full-thickness pleura. As the rate of MPM in the patients with pleural effusion with low levels of ADA, but without signs of acute inflammation or pleural thickening is high (65%), an early thoracoscopic pleural biopsy is strongly recommended. (author)

  9. Kinetics of adenylate metabolism in human and rat myocardium

    OpenAIRE

    Tavenier, M.; Skladanowski, A.C.; Abreu, R.A. de; Jong, J.W. de

    1995-01-01

    textabstractPathways producing and converting adenosine have hardly been investigated in human heart, contrasting work in other species. We compared the kinetics of enzymes associated with purine degradation and salvage in human and rat heart cytoplasm assaying for adenosine deaminase, nucleoside phosphorylase, xanthine oxidoreductase, AMP deaminase, AMP- and IMP-specific 5′-nucleotidases, adenosine kinase and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Xanthine oxidoreductase was...

  10. [Diagnostic performance of T-SPOT.TB on peripheral blood in combination with adenosine deaminase on pleural fluid for the diagnosis of tuberculous pleurisy within different age group].

    Science.gov (United States)

    Xu, H Y; Zhang, D Q; Ye, J R; Su, S S; Xie, Y P; Chen, C S; Li, Y P

    2017-06-27

    Objective: To evaluate the performance of T cell enzyme-linked immuno-spot assay (T-SPOT) on peripheral blood in combination with adenosine deaminase (ADA) on pleural fluid for diagnosis of tuberculous (TB) pleurisy within different age groups. Methods: The data of patients with pleural effusion from the Department of Pulmonary and Critical Care Medicine of the First Affiliated Hospital of Wenzhou Medical University from April 2012 to November 2016 were retrospectively analyzed, and the diagnoses of these patients were histopathologically confirmed through medical thoracoscopy. The cases who had confirmed diagnosis, in the same time, received peripheral blood T-SPOT.TB were enrolled. The performance of peripheral blood T-SPOT.TB in combination with pleural fluid ADA on diagnosing TB pleurisy in the younger patients (16-59 years old) and elderly patients (≥60 years old) were analyzed respectively. The sensitivity, specificity and the receiver operating characteristic (ROC) curve were adopted for statistical analysis. Results: A total of 448 cases were finally enrolled, 341(76.1%) confirmed with TB pleurisy, 224 males, 117 females, (47±19) years old; and 107 (23.9%) classified as non-TB pleurisy, 65 males, 42 females, (61±14) years old. There were 285 cases who were classified as younger group, and the other 163 cases were classified as elderly group. The sensitivity and specificity of peripheral blood T-SPOT.TB were 85.4% (204/239) and 71.7% (33/46) in the younger patients, 76.5% (78/102) and 59.0% (36/61) respectively in the elderly patients. The sensitivity of peripheral blood T-SPOT.TB in the younger patients was significantly higher than that in the elderly patients ( P =0.047). The sensitivity and specificity were 99.2% and 95.7% in combination with peripheral blood T-SPOT.TB and pleural fluid ADA respectively in the younger patients. The area under ROC curve (AUC) of T-SPOT.TB in the younger patients was 0.833, AUC of T-SPOT.TB combined with ADA was 0

  11. Value of combined detection of interferon-γ, vascular endothelial growth factor, C-reactive protein and adenosine deaminase in differential diagnosis of tuberculous and malignant pleural effusion

    International Nuclear Information System (INIS)

    Li Guo'an; Han Sugui; Zhou Xiuyan; He Weishe; Sun Fangchu

    2012-01-01

    Objective: To explore the value of interferon II, vascular endothelial growth factor, C-reactive protein and adenosine deaminase in differential diagnosis of tuberculous and malignant pleural effusion. Methods: 122 cases with tuberculous pleurisy, 56 cases of malignant pleural effusion, 48 cases of tuberculous pleural effusion, 18 cases of inflammatory and other pleural fluid were studied. The serum and pleural fluid levels of IFN-γ, VEGF-C, CRP and ADA serum in those patients were detected. Results: The IFN-γ, CRP and ADA levels in tuberculous pleural effusion were higher than in malignant pleural effusion(P<0.01). According to the receiver operator characteristic (ROC) curve, when 100 ng/L was regarded as critical value of IFN-γ, the sensitivity and specificity of IFN-γ in diagnosing tuberculous pleural effusion were 83.1% and 92.3% respectively. When 45 U/L ADA was regarded as critical value of ADA, the sensitivity and specificity of ADA in diagnosing tuberculous pleural effusion were 85.6% and 96.3% respectively. When 110 mg/L was regarded as critical value of CRP, the sensitivity and specificity of CRP were 79.1% and 84.2% respectively. When combine detection of three markers, the diagnosis sensitivity and specificity were 87.8% and 86.0% respectively. The VEGF-C concentration in malignant pleural effusion was higher than that in tuberculous pleural effusion and inflammatory and other pleural effusion (P<0.01). When the ratio of VEGF-C to ADA≥8, the sensitivity and specificity in diagnosis of malignant pleural effusion were 86.3% and 82.6% respectively, and the ration VEGF-C to ADA≤3, the sensitivity and specificity in diagnosis of tuberculous pleural effusion were 85.1% and 87.1% respectively. Conclusion: The combined detection of IFN-γ, CRP and ADA could improve sensitivity and specificity in diagnosing tuberculous pleurisy. The concentration ratios of VEGF-C to ADA have clinical value in differential diagnosis of pleural effusions. (authors)

  12. Clinical value of jointly detection serum lactate dehydrogenase/pleural fluid adenosine deaminase and pleural fluid carcinoembryonic antigen in the identification of malignant pleural effusion.

    Science.gov (United States)

    Zhang, Fan; Hu, Lijuan; Wang, Junjun; Chen, Jian; Chen, Jie; Wang, Yumin

    2017-09-01

    Limited data are available for the diagnostic value, and for the diagnostic sensitivity and specificity of joint detection of serum lactate dehydrogenase (sLDH)/pleural fluid adenosine deaminase (pADA) and pleural fluid carcinoembryonic antigen (pCEA) in malignant pleural effusion (MPE). We collected 987 pleural effusion specimens (of which 318 were malignant pleural effusion, 374 were tubercular pleural effusion, and 295 were parapneumonic effusion specimens) from the First Affiliated Hospital of Wenzhou Medical University from July 2012 to March 2016. The pADA, sLDH, pleural fluid LDH (pLDH), serum C-reactive protein (sCRP), pleural fluid protein, pCEA, white blood cell (WBC), and red blood cell (RBC) were analyzed, and the clinical data of each group were collected for statistical analysis. The level of sLDH/pADA, pCEA, and RBC from the MPE group was markedly higher than the tuberculosis pleural effusion (TB) group (Mann-Whitney U=28422.000, 9278.000, 30518, P=.000, .000, .000) and the parapneumonic pleural fluid group (Mann-Whitney U=5972.500, 7113.000, 36750.500, P=.000, .000, .000). The receiver operating characteristic curve ROC showed that the area under the ROC curve (AUC) (=0.924, 0.841) of pCEA and sLDH/pADA (cutoff=4.9, 10.6) were significantly higher than other markers for the diagnosis of MPE. Thus, joint detection of pCEA and sLDH/pADA suggested that the sensitivity, specificity, and AUC was 0.94, 81.70, and 94.32 at the cutoff 0.16 and diagnostic performance was higher than pCEA or sLDH/pADA. Joint detection of sLDH/pADA and pCEA can be used as a good indicator for the identification of benign and MPE with higher sensitivity and specificity than pCEA or sLDH/pADA. © 2016 Wiley Periodicals, Inc.

  13. Gene expression profiles in adenosine-treated human mast cells ...

    African Journals Online (AJOL)

    Gene expression profiles in adenosine-treated human mast cells. ... SW Kang, JE Jeong, CH Kim, SH Choi, SH Chae, SA Jun, HJ Cha, JH Kim, YM Lee, YS ... beta 4, ring finger protein, high-mobility group, calmodulin 2, RAN binding protein, ...

  14. Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

    Science.gov (United States)

    Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

    2008-01-01

    Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

  15. On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells.

    Science.gov (United States)

    Costa, M Adelina; Barbosa, A; Neto, E; Sá-e-Sousa, A; Freitas, R; Neves, J M; Magalhães-Cardoso, T; Ferreirinha, F; Correia-de-Sá, P

    2011-05-01

    Purines are important modulators of bone cell biology. ATP is metabolized into adenosine by human primary osteoblast cells (HPOC); due to very low activity of adenosine deaminase, the nucleoside is the end product of the ecto-nucleotidase cascade. We, therefore, investigated the expression and function of adenosine receptor subtypes (A(1) , A(2A) , A(2B) , and A(3) ) during proliferation and osteogenic differentiation of HPOC. Adenosine A(1) (CPA), A(2A) (CGS21680C), A(2B) (NECA), and A(3) (2-Cl-IB-MECA) receptor agonists concentration-dependently increased HPOC proliferation. Agonist-induced HPOC proliferation was prevented by their selective antagonists, DPCPX, SCH442416, PSB603, and MRS1191. CPA and NECA facilitated osteogenic differentiation measured by increases in alkaline phosphatase (ALP) activity. This contrasts with the effect of CGS21680C which delayed HPOC differentiation; 2-Cl-IB-MECA was devoid of effect. Blockade of the A(2B) receptor with PSB603 prevented osteogenic differentiation by NECA. In the presence of the A(1) antagonist, DPCPX, CPA reduced ALP activity at 21 and 28 days in culture. At the same time points, blockade of A(2A) receptors with SCH442416 transformed the inhibitory effect of CGS21680C into facilitation. Inhibition of adenosine uptake with dipyridamole caused a net increase in osteogenic differentiation. The presence of all subtypes of adenosine receptors on HPOC was confirmed by immunocytochemistry. Data show that adenosine is an important regulator of osteogenic cell differentiation through the activation of subtype-specific receptors. The most abundant A(2B) receptor seems to have a consistent role in cell differentiation, which may be balanced through the relative strengths of A(1) or A(2A) receptors determining whether osteoblasts are driven into proliferation or differentiation. Copyright © 2010 Wiley-Liss, Inc.

  16. Avaliação da adenosina desaminase no diagnóstico da tuberculose pleural: uma metanálise brasileira Evaluation of adenosine deaminase in the diagnosis of pleural tuberculosis: a Brazilian meta-analysis

    Directory of Open Access Journals (Sweden)

    Patrizio Morisson

    2008-04-01

    Full Text Available OBJETIVO: Avaliar trabalhos brasileiros resumindo a acurácia da adenosina desaminase no diagnóstico da tuberculose pleural, com o intuito de contribuir para a concretização do exame como rotina na investigação dos derrames pleurais. MÉTODOS: Depois de realizada uma busca por trabalhos brasileiros referentes à dosagem da adenosina desaminase no líquido pleural, estes foram avaliados e incluídos no estudo. A análise dos dados foi feita por meio da curva summary receiver operating characteristic (SROC que possibilitou a reunião dos estudos quanto a acurácia para o diagnóstico. Com os valores globais de sensibilidade e especificidade foi aplicado o teorema de Bayes para calcular as probabilidades pós-teste em diferentes prevalências da doença. RESULTADOS: Entre 1987 e 2005 foram encontrados 25 estudos contendo informações suficientes que poderiam ser utilizadas na metanálise. Após avaliação, foram incluídos nove estudos, totalizando 1.674 pacientes. De acordo com a curva SROC, foi encontrada uma sensibilidade de 91,8% (IC95%: 89,8-93,6% e uma especificidade de 88,4% (IC95%: 86,0-90,5%, com uma área abaixo da curva de 0,969. O odds ratio global foi de 112,0 (IC95%: 51,6-243,2. Considerando uma prevalência da tuberculose pleural de 50% (considerada neutra, a probabilidade do diagnóstico pós-teste positivo é de 88,7% e sua exclusão após resultado negativo de 91,5%. CONCLUSÕES: Apesar das diferenças encontradas entre os estudos, é possível concluir que a adenosina desaminase possui alta acurácia no diagnóstico da tuberculose pleural, devendo ser utilizada de rotina em sua investigação.OBJECTIVE: To evaluate Brazilian studies by summarizing the accuracy of adenosine deaminase in the diagnosis of pleural tuberculosis, with the objective of lending support to the movement to make the test part of the routine investigation of pleural effusions. METHODS: A search for Brazilian studies related to the determination of

  17. 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

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Monaghan, Gemma; Børsting, Claus

    2007-01-01

    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...

  18. Functional coupling between adenosine A1 receptors and G-proteins in rat and postmortem human brain membranes determined with conventional guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding or [35S]GTPγS/immunoprecipitation assay.

    Science.gov (United States)

    Odagaki, Yuji; Kinoshita, Masakazu; Ota, Toshio; Meana, J Javier; Callado, Luis F; Matsuoka, Isao; García-Sevilla, Jesús A

    2018-06-01

    Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A 1 receptor and G-protein was assessed by means of two [ 35 S]GTPγS binding assays, i.e., conventional filtration method and [ 35 S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A 1 receptor-mediated Gα i-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [ 35 S]GTPγS binding determined with conventional assay derives from functional activation of Gα i/o proteins (not restricted only to Gα i-3 ) coupled to adenosine A 1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [ 35 S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %E max values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A 1 receptor/G-protein interaction in postmortem human brain tissue.

  19. Turnover of adenosine in plasma of human and dog blood

    International Nuclear Information System (INIS)

    Moeser, G.H.S.; Schrader, J.; Deussen, A.

    1989-01-01

    To determine half-life and turnover of plasma adenosine, heparinized blood from healthy volunteers was incubated with radiolabeled adenosine in the physiological concentration range of 0.1-1 microM. Plasma levels of adenosine in vitro were 82 +/- 14 nM and were similar to those determined immediately after blood collection with a ''stopping solution.'' Dipyridamole (83 microM) and erythro-9(2-hydroxynon-3yl)-adenine (EHNA) (8 microM) did not measurably alter basal adenosine levels but completely blocked the uptake of added adenosine. Inhibition of ecto-5'-nucleotidase with 100 microM alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP) reduced plasma adenosine to 22 +/- 6 nM. For the determination of adenosine turnover, the decrease in specific radioactivity of added [ 3 H]adenosine was measured using a dipyridamole-containing stopping solution. Without altering basal adenosine levels, the half-life was estimated to be 0.6 s. Similar experiments were carried out with washed erythrocytes or in the presence of AOPCP, yielding half-lives of 0.7 and 0.9 s, respectively. When the initial adenosine concentration was 1 microM, its specific activity decreased by only 11% within 5 s, whereas total plasma adenosine exponentially decreased with a half-life of 1.5 s. Venous plasma concentrations were measured after relief of a 3-min forearm ischemia. Changes in plasma adenosine did not correlate well with changes in blood flow but were augmented in the presence of dipyridamole

  20. Characteristics of high affinity and low affinity adenosine binding sites in human cerebral cortex

    International Nuclear Information System (INIS)

    John, D.; Fox, I.V.

    1986-01-01

    The binding characteristics of human brain cortical membrane fractions were evaluated to test the hypothesis that there are A 1 and A 2 adenosine binding sites. The ligands used were 2-chloro(8- 3 H) adenosine and N 6 -(adenine-2, 8- 3 H) cyclohexayladenosine. Binding of chloroadenosine to human brain cortical membranes was time dependent, reversible and concentration dependent. The kinetic constant determinations from binding studies of the adenosine receptor are presented. Utilizing tritium-cyclohexyladenosine as ligand the authors observed evidence for a high affinity binding site in human brain cortical membranes with a kd of 5 nM

  1. Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

    DEFF Research Database (Denmark)

    Hanisch, Frank; Hellsten, Ylva; Zierz, Stephan

    2006-01-01

    homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities...... with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different...... AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57...

  2. Discovery of a cAMP Deaminase That Quenches Cyclic AMP-Dependent Regulation

    Science.gov (United States)

    Goble, Alissa M.; Feng, Youjun; Raushel, Frank M.; Cronan, John E.

    2013-01-01

    An enzyme of unknown function within the amidohydrolase superfamily was discovered to catalyze the hydrolysis of the universal second messenger, cyclic-3’, 5’-adenosine monophosphate (cAMP). The enzyme, which we have named CadD, is encoded by the human pathogenic bacterium Leptospira interrogans. Although CadD is annotated as an adenosine deaminase, the protein specifically deaminates cAMP to cyclic-3’, 5’-inosine monophosphate (cIMP) with a kcat/Km of 2.7 ± 0.4 × 105 M−1 s−1 and has no activity on adenosine, adenine, or 5’-adenosine monophosphate (AMP). This is the first identification of a deaminase specific for cAMP. Expression of CadD in Escherichia coli mimics the loss of adenylate cyclase in that it blocks growth on carbon sources that require the cAMP-CRP transcriptional activator complex for expression of the cognate genes. The cIMP reaction product cannot replace cAMP as the ligand for CRP binding to DNA in vitro and cIMP is a very poor competitor of cAMP activation of CRP for DNA binding. Transcriptional analyses indicate that CadD expression represses expression of several cAMP-CRP dependent genes. CadD adds a new activity to the cAMP metabolic network and may be a useful tool in intracellular study of cAMP-dependent processes. PMID:24074367

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

    Science.gov (United States)

    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.

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

    International Nuclear Information System (INIS)

    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 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 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 45 Ca uptake at the early time points

  5. Adenosine receptors in rat and human pancreatic ducts stimulate chloride transport

    DEFF Research Database (Denmark)

    Novak, Ivana; Hede, Susanne; Hansen, Mette

    2007-01-01

    , it was found that 58% of PANC-1 cells responded to adenosine, whereas only 9% of CFPAC-1 cells responded. Adenosine elicited Ca(2+) signals only in a few rat and human duct cells, which did not seem to correlate with Cl(-) signals. A(2A) receptors were localized in the luminal membranes of rat pancreatic ducts......, plasma membrane of many PANC-1 cells, but only a few CFPAC-1 cells. Taken together, our data indicate that A(2A) receptors open Cl(-) channels in pancreatic ducts cells with functional CFTR. We propose that adenosine can stimulate pancreatic secretion and, thereby, is an active player in the acini...

  6. Insulin and adenosine regulate the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes.

    Science.gov (United States)

    Kiechle, F L; Sykes, E; Artiss, J D

    1995-01-01

    Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

  7. Genetics Home Reference: adenosine deaminase deficiency

    Science.gov (United States)

    ... Combined Immunodeficiency (SCID) and Conditions Associated with T Cell Lymphoneia (PDF) Genetic Testing (1 link) Genetic Testing Registry: Severe ... Diseases Immune Deficiency Foundation Jeffrey Modell Foundation National Organization for Rare ... OMIM (1 link) SEVERE COMBINED IMMUNODEFICIENCY, AUTOSOMAL RECESSIVE, T CELL-NEGATIVE, B CELL-NEGATIVE, NK CELL-NEGATIVE, DUE ...

  8. Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells.

    Science.gov (United States)

    Kreuzer, J; Denger, S; Reifers, F; Beisel, C; Haack, K; Gebert, J; Kübler, W

    1996-07-01

    Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and

  9. 胸腔积液腺苷脱氨酶对内科胸腔镜检查临床病例选择的意义%Clinical significance of adenosine deaminase in selection patient for medical thoracoscopy

    Institute of Scientific and Technical Information of China (English)

    李玺; 黄小艳; 刘斌; 荣福

    2017-01-01

    Objective To evaluate clinical significance of adenosine deaminase (ADA) in selection patient for medical thoracoscopy.Methods A retrospective analysis of January 2013 to April 2016,198 cases unexplained pleural effusion patients were divided into youth group,middle-aged and elderly groups.To determine the sensitivity and specificity of ADA≥45 U/L or ADA≥45 U/L combine percentage of lymphocytes in white blood cells ≥50% as the diagnosis criteria of tuberculous pleurisy,and analysis gender and age influence on ADA.Results The diagnosis of medical thoracoscopy in unexplained pleural effusion was 98.9 %.The sensitivity and specificity of ADA≥45 U/L in diagnosis of tuberculous pleurisy were 73.3% and 89.2%;The sensitivity and specificity of ADA≥ 45 U/L combine percentage of lymphocytes in white blood cells≥ 50% in diagnosis of tuberculous pleurisy were 71% and 98.5%.Especially in youth group,the sensitivity and specificity of ADA ≥ 45 U/L combine percentage of lymphocytes in white blood cells≥50% in diagnosis of tuberculous pleurisy was 100%.Conclusions If young patients have typical clinical manifestations of tuberculous pleurisy and ADA≥45 U/L percentage of lymphocytes in white blood cells ≥ 50% in pleural effusion,anti-TB treatment without further thoracoscopy is appropriate;For middle-aged and elderly patients of unexplained pleural liquid,thoracoscopy is recommended to avoid misdiagnosis.%目的 探讨胸腔积液腺苷脱氨酶(ADA)对内科胸腔镜检查临床病例选择的意义.方法 回顾性分析2013年1月至2016年4月经内科胸腔镜检查的不明原因胸腔积液患者198例,分为青年组、中年组和老年组,以胸腔积液ADA≥45 U/L或ADA≥45 U/L联合淋巴细胞占白细胞比例≥50%作为诊断结核性胸膜炎的标准,确定其敏感度和特异度,并分析性别、年龄对ADA的影响.结果 内科胸腔镜对不明原因胸腔积液的诊断率为98.9%.胸腔积液ADA≥45 U/L诊

  10. Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2 in schizophrenic patients Diminuição da frequência da variante alélica de baixa atividade da adenosina desaminase (ADA1*2 em pacientes esquizofrênicos

    Directory of Open Access Journals (Sweden)

    Gustavo Pimentel Dutra

    2010-09-01

    Full Text Available OBJECTIVE: Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A (ADA1*2 exhibits 20-30% lower enzymatic activity in individuals with the G/A genotype than individuals with the G/G genotype. The aim of this study was to evaluate the ADA polymorphism 22G→A (ADA1*2 in schizophrenic patients and healthy controls. METHOD: The genotypes of the ADA 22G→A were identified with allele-specific PCR strategy in 152 schizophrenic patients and 111 healthy individuals. RESULTS: A significant decrease in the frequency of the G/A genotype was seen in schizophrenic patients (7/152 - 4.6% relative to controls (13/111 - 11.7%, p = 0.032, OR = 2.6. CONCLUSION: These results suggest that the G/A genotype associated with low adenosine deaminase activity and, supposingly, with higher adenosine levels is less frequent among schizophrenic patients.OBJETIVO: A adenosina pode ter um papel importante na fisiopatologia da esquizofrenia, uma vez que modula a liberação de vários neurotransmissores, tais como glutamato, dopamina, serotonina e acetilcolina, diminui a atividade neuronal por hiperpolarização pós-sináptica e inibe a atividade dopaminérgica. A adenosina desaminase participa do metabolismo das purinas pela conversão de adenosina em inosina. O mais frequente polimorfismo funcional da adenosina desaminase (22G →A (ADA1*2 exibe uma diminuição de 20-30% da atividade funcional em indivíduos com genótipo G/A quando comparados com indivíduos com o genótipo G/G. O objetivo deste estudo foi avaliar o polimorfismo 22G→A (ADA1*2 em pacientes esquizofrênicos e em

  11. Adenosine concentrations in the interstitium of resting and contracting human skeletal muscle

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Maclean, D.; Rådegran, G.

    1998-01-01

    BACKGROUND: Adenosine has been proposed to be a locally produced regulator of blood flow in skeletal muscle. However, the fundamental questions of to what extent adenosine is formed in skeletal muscle tissue of humans, whether it is present in the interstitium, and where it exerts its vasodilatory...... rest (0.13+/-0.03, 0.07+/-0.03, and 0.07+/-0.02 micromol/L, respectively) to exercise (10 W; 2.00+/-1.32, 2.08+/-1.23, and 1.65+/-0.50 micromol/L, respectively; Pskeletal muscle...... and demonstrates that adenosine and its precursors increase in the exercising muscle interstitium, at a rate associated with intensity of muscle contraction and the magnitude of muscle blood flow....

  12. The adenosine A2B receptor is involved in anion secretion in human pancreatic duct Capan-1 epithelial cells

    DEFF Research Database (Denmark)

    Hayashi, M.; Inagaki, A.; Novak, Ivana

    2016-01-01

    Adenosine modulates a wide variety of biological processes via adenosine receptors. In the exocrine pancreas, adenosine regulates transepithelial anion secretion in duct cells and is considered to play a role in acini-to-duct signaling. To identify the functional adenosine receptors and Cl......− channels important for anion secretion, we herein performed experiments on Capan-1, a human pancreatic duct cell line, using open-circuit Ussing chamber and gramicidin-perforated patch-clamp techniques. The luminal addition of adenosine increased the negative transepithelial potential difference (Vte......) in Capan-1 monolayers with a half-maximal effective concentration value of approximately 10 μM, which corresponded to the value obtained on whole-cell Cl− currents in Capan-1 single cells. The effects of adenosine on Vte, an equivalent short-circuit current (Isc), and whole-cell Cl− currents were inhibited...

  13. Investigation on the diagnosis significance of C reactive protein and adenosine deaminase in cerebrospinal fluid among children with meningitis%脑脊液C反应蛋白和腺苷脱氨酶检测在小儿脑膜炎中的诊断价值探讨

    Institute of Scientific and Technical Information of China (English)

    徐仁荣; 张慧华; 朱华丽

    2015-01-01

    Objective To determine C reactive protein and adenosine deaminase in cerebrospinal fluid,and to investigate the clinical diagnosis significance for tuberculosis meningitis,purulent meningitis and viral meningitis. Methods A total of 31 5 children with meningitis (1 02 cases of tuberculosis meningitis,1 08 cases of purulent meningitis and 1 05 cases of viral meningitis)were enrolled,96 children undergoing operation without meningitis were enrolled as control group,and their cerebrospinal fluid samples were collected.The levels of C reactive protein and adenosine deaminase were determined,and the results were compared.Results C reactive protein and adenosine deaminase in control and viral meningitis groups were significantly lower than those in tuberculosis and purulent meningitis groups (P 0.05 ).C reactive protein in purulent meningitis group was higher than that in tuberculosis meningitis group(P <0.05 ),and adenosine deaminase was lower than that in tuberculosis meningitis group (P <0.05).C reactive protein in purulent meningitis group was positive,and the positive rate was 1 00%.There were 63 positive cases in tuberculosis meningitis group,and the positive rate was 61 .76%.That in viral meningtis was negative.Conclusions In cerebrospinal fluid,C reactive protein and adenosine deaminase determinations have important reference significance for the differential diagnosis of bacterial meningitis (purulent meningitis and tuberculosis meningitis)and viral meningitis.Adenosine deaminase may be a good indicator for the diagnosis of tuberculosis meningitis,in order to provide the reference for the early diagnosis of various types of meningitis.%目的:探讨小儿脑脊液C反应蛋白和腺苷脱氨酶在结核性脑膜炎、化脓性脑膜炎和病毒性脑膜炎早期诊断中的临床意义。方法分别检测315例小儿脑膜炎患儿(包括结核性脑膜炎102例、化脓性脑膜炎108例、病毒性脑膜炎105例)及96例非脑膜炎外科手术

  14. Involvement of A1 adenosine receptors and neural pathways in adenosine-induced bronchoconstriction in mice.

    Science.gov (United States)

    Hua, Xiaoyang; Erikson, Christopher J; Chason, Kelly D; Rosebrock, Craig N; Deshpande, Deepak A; Penn, Raymond B; Tilley, Stephen L

    2007-07-01

    High levels of adenosine can be measured from the lungs of asthmatics, and it is well recognized that aerosolized 5'AMP, the precursor of adenosine, elicits robust bronchoconstriction in patients with this disease. Characterization of mice with elevated adenosine levels secondary to the loss of adenosine deaminase (ADA) expression, the primary metabolic enzyme for adenosine, further support a role for this ubiquitous mediator in the pathogenesis of asthma. To begin to identify pathways by which adenosine can alter airway tone, we examined adenosine-induced bronchoconstriction in four mouse lines, each lacking one of the receptors for this nucleoside. We show, using direct measures of airway mechanics, that adenosine can increase airway resistance and that this increase in resistance is mediated by binding the A(1) receptor. Further examination of this response using pharmacologically, surgically, and genetically manipulated mice supports a model in which adenosine-induced bronchoconstriction occurs indirectly through the activation of sensory neurons.

  15. Role of adenosine in regulating the heterogeneity of skeletal muscle blood flow during exercise in humans

    DEFF Research Database (Denmark)

    Heinonen, Ilkka; Nesterov, Sergey V; Kemppainen, Jukka

    2007-01-01

    receptor blockade. BF heterogeneity within muscles was calculated from 16-mm(3) voxels in BF images and heterogeneity among the muscles from the mean values of the four QF compartments. Mean BF in the whole QF and its four parts increased, and heterogeneity decreased with workload both without......Evidence from both animal and human studies suggests that adenosine plays a role in the regulation of exercise hyperemia in skeletal muscle. We tested whether adenosine also plays a role in the regulation of blood flow (BF) distribution and heterogeneity among and within quadriceps femoris (QF...... and with theophylline (P heterogeneity among the QF muscles, yet blockade increased within-muscle BF heterogeneity in all four QF muscles (P = 0.03). Taken together, these results show that BF becomes less heterogeneous with increasing...

  16. Human Adenosine A2A Receptor: Molecular Mechanism of Ligand Binding and Activation

    Directory of Open Access Journals (Sweden)

    Byron Carpenter

    2017-12-01

    Full Text Available Adenosine receptors (ARs comprise the P1 class of purinergic receptors and belong to the largest family of integral membrane proteins in the human genome, the G protein-coupled receptors (GPCRs. ARs are classified into four subtypes, A1, A2A, A2B, and A3, which are all activated by extracellular adenosine, and play central roles in a broad range of physiological processes, including sleep regulation, angiogenesis and modulation of the immune system. ARs are potential therapeutic targets in a variety of pathophysiological conditions, including sleep disorders, cancer, and dementia, which has made them important targets for structural biology. Over a decade of research and innovation has culminated with the publication of more than 30 crystal structures of the human adenosine A2A receptor (A2AR, making it one of the best structurally characterized GPCRs at the atomic level. In this review we analyze the structural data reported for A2AR that described for the first time the binding of mode of antagonists, including newly developed drug candidates, synthetic and endogenous agonists, sodium ions and an engineered G protein. These structures have revealed the key conformational changes induced upon agonist and G protein binding that are central to signal transduction by A2AR, and have highlighted both similarities and differences in the activation mechanism of this receptor compared to other class A GPCRs. Finally, comparison of A2AR with the recently solved structures of A1R has provided the first structural insight into the molecular determinants of ligand binding specificity in different AR subtypes.

  17. Adenosine A2b receptor promotes progression of human oral cancer

    International Nuclear Information System (INIS)

    Kasama, Hiroki; Sakamoto, Yosuke; Kasamatsu, Atsushi; Okamoto, Atsushi; Koyama, Tomoyoshi; Minakawa, Yasuyuki; Ogawara, Katsunori; Yokoe, Hidetaka; Shiiba, Masashi; Tanzawa, Hideki; Uzawa, Katsuhiro

    2015-01-01

    Adenosine A2b receptor (ADORA2B) encodes an adenosine receptor that is a member of the G protein-coupled receptor superfamily. This integral membrane protein stimulates adenylate cyclase activity in the presence of adenosine. Little is known about the relevance of ADORA2B to human malignancy including oral squamous cell carcinoma (OSCC). We aimed to characterize the expression state and function of ADORA2B in OSCC. The ADORA2B expression levels in nine OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using an ADORA2B knockdown model, we assessed cellular proliferation and expression of hypoxia-inducible factor1α (HIF-1α). We examined the adenosine receptor expression profile under both normoxic and hypoxic conditions in the OSCC-derived cells. In addition to in vitro data, the clinical correlation between the ADORA2B expression levels in primary OSCCs (n = 100 patients) and the clinicopathological status by immunohistochemistry (IHC) also was evaluated. ADORA2B mRNA and protein were up-regulated significantly (p < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. Suppression of ADORA2B expression with shRNA significantly (p < 0.05) inhibited cellular proliferation compared with the control cells. HIF-1α also was down-regulated in ADORA2B knockdown OSCC cells. During hypoxia, ADORA2B expression was induced significantly (p < 0.05) in the mRNA and protein after 24 hours of incubation in OSCC-derived cells. IHC showed that ADORA2B expression in primary OSCCs was significantly (p < 0.05) greater than in the normal oral counterparts and that ADORA2B-positive OSCCs were correlated closely (p < 0.05) with tumoral size. Our results suggested that ADORA2B controls cellular proliferation via HIF-1α activation, indicating that ADORA2B may be a key regulator of tumoral progression in OSCCs. The online version of this article (doi:10.1186/s12885-015-1577-2) contains

  18. Caffeine May Reduce Perceived Sweet Taste in Humans, Supporting Evidence That Adenosine Receptors Modulate Taste.

    Science.gov (United States)

    Choo, Ezen; Picket, Benjamin; Dando, Robin

    2017-09-01

    Multiple recent reports have detailed the presence of adenosine receptors in sweet sensitive taste cells of mice. These receptors are activated by endogenous adenosine in the plasma to enhance sweet signals within the taste bud, before reporting to the primary afferent. As we commonly consume caffeine, a powerful antagonist for such receptors, in our daily lives, an intriguing question we sought to answer was whether the caffeine we habitually consume in coffee can inhibit the perception of sweet taste in humans. 107 panelists were randomly assigned to 2 groups, sampling decaffeinated coffee supplemented with either 200 mg of caffeine, about the level found in a strong cup of coffee, or an equally bitter concentration of quinine. Participants subsequently performed sensory testing, with the session repeated in the alternative condition in a second session on a separate day. Panelists rated both the sweetened coffee itself and subsequent sucrose solutions as less sweet in the caffeine condition, despite the treatment having no effect on bitter, sour, salty, or umami perception. Panelists were also unable to discern whether they had consumed the caffeinated or noncaffeinated coffee, with ratings of alertness increased equally, but no significant improvement in reaction times, highlighting coffee's powerful placebo effect. This work validates earlier observations in rodents in a human population. © 2017 Institute of Food Technologists®.

  19. Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy.

    Science.gov (United States)

    Aronica, Eleonora; Zurolo, Emanuele; Iyer, Anand; de Groot, Marjolein; Anink, Jasper; Carbonell, Caterina; van Vliet, Erwin A; Baayen, Johannes C; Boison, Detlev; Gorter, Jan A

    2011-09-01

    Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphologic feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathologic hallmark of TLE. Using immunocytochemistry and Western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up-regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3-4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of patients with TLE. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several proinflammatory molecules (interleukin-1β and lipopolysaccharide). These results suggest that dysregulation of ADK in astrocytes is a common pathologic hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte-based adenosine cycle and inflammation. Whether this interaction also can play a role in vivo needs to be further investigated. Wiley Periodicals, Inc. © 2011 International League Against Epilepsy.

  20. Traditional Acupuncture Triggers a Local Increase in Adenosine in Human Subjects

    OpenAIRE

    Takano, Takahiro; Chen, Xiaolin; Luo, Fang; Fujita, Takumi; Ren, Zeguang; Goldman, Nanna; Zhao, Yuanli; Markman, John D.; Nedergaard, Maiken

    2012-01-01

    Acupuncture is a form of Eastern medicine that has been practiced for centuries. Despite its long history and worldwide application, the biological mechanisms of acupuncture in relieving pain have been poorly defined. Recent studies in mice, however, demonstrate that acupuncture triggers increases in interstitial adenosine, which reduces the severity of chronic pain through adenosine A1 receptors, suggesting that adenosine-mediated antinociception contributes to the clinical benefits of acupu...

  1. Selective in vivo radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells transduced with the E. coli cytosine deaminase (CD) gene

    International Nuclear Information System (INIS)

    Gabel, M.; Kim, J.H.; Kolozsvary, A.; Khil, M.; Freytag, S.

    1998-01-01

    Purpose: The E. coli cytosine deaminase (CD) gene encodes an enzyme capable of converting the nontoxic prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a known radiosensitizer. Having previously shown that combined CD suicide gene therapy and radiation (RT) results in pronounced radiosensitization in vitro, we progressed to in vivo studies of combined therapy. Methods and Materials: WiDr human colon cancer cells were transduced in vitro with the CD gene and cells expressing CD were selected for use as xenografts in a nude mouse model. After administration of 5-FC, tumors received 10-30 Gy local field radiation (RT) and tumor growth delay was compared to control animals receiving either 5-FU, 5-FC, or RT alone. Results: Maximal growth delay was seen in mice treated with 5-FC for 6 consecutive days prior to RT. Combined treatment with 15 Gy radiation resulted in a dose-modifying factor (DMF) of 1.50, and a greater DMF was observed with higher doses of radiation. There was no appreciable toxicity using this new approach. In contrast, a similar treatment of combined 5-FU and radiation resulted in considerable toxicity and no appreciable radiosensitization. Conclusion: The present results show that combined suicide gene therapy and RT results in pronounced antitumor effect without any notable toxicity. This indicates that the CD gene may be useful in the development of novel treatment strategies combining radiation and gene therapy in the treatment of locally advanced cancers

  2. Comparison of exogenous adenosine and voluntary exercise on human skeletal muscle perfusion and perfusion heterogeneity

    DEFF Research Database (Denmark)

    Heinonen, Ilkka H.A.; Kemppainen, Jukka; Kaskinoro, Kimmo

    2010-01-01

    Adenosine is a widely used pharmacological agent to induce a 'high flow' control condition to study the mechanisms of exercise hyperemia, but it is not known how well adenosine infusion depicts exercise-induced hyperemia especially in terms of blood flow distribution at the capillary level in hum...

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

    Science.gov (United States)

    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

  4. High pleural fluid adenosine deaminase levels: A valuable tool for ...

    African Journals Online (AJOL)

    To determine the positive predictive value (PPV) of FADA, the frequent causes of FPs in our laboratory and the demographic characteristics of tuberculous pleural effusions (TPEs) and non-tuberculous pleural effusions (NTPEs). Methods. High FADA results generated in the past year were extracted with corresponding TB ...

  5. Validity of serum Adenosine deaminase in diagnosis of tuberculosis

    African Journals Online (AJOL)

    abp

    2013-08-14

    Aug 14, 2013 ... culture methods take several weeks to become positive where the diagnosis of .... Indian. Pediatr. 1995; 32 (10): 1087-8. PubMed | Google Scholar. 12. Mishra OP ... patients. Southeast Asian J Trop Med Public Health. 2007.

  6. A role for adenosine deaminase in Drosophila larval development

    Czech Academy of Sciences Publication Activity Database

    Doležal, T.; Doleželová, Eva; Žurovec, Michal; Bryant, P. J.

    2005-01-01

    Roč. 3, č. 7 (2005), s. 1213-1224 ISSN 1544-9173 R&D Projects: GA ČR(CZ) GA204/04/1205; GA AV ČR(CZ) IAA5007107 Grant - others:United States National Science Foundation(US) 440860-21565 Institutional research plan: CEZ:AV0Z50070508 Keywords : Drosophila Subject RIV: ED - Physiology Impact factor: 14.672, year: 2005

  7. Insulin-increased L-arginine transport requires A(2A adenosine receptors activation in human umbilical vein endothelium.

    Directory of Open Access Journals (Sweden)

    Enrique Guzmán-Gutiérrez

    Full Text Available Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1. This process involves the activation of A(2A adenosine receptors (A(2AAR in human umbilical vein endothelial cells (HUVECs. Insulin increases hCAT-1 activity and expression in HUVECs, and A(2AAR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A(2AAR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR, and SLC7A1 (for hCAT-1 reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K(m for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1(-1606 or pGL3-hCAT-1(-650 constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1(-1606, and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A(2AAR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes.

  8. Molecular mimicry between Helicobacter pylori antigens and H+, K+ --adenosine triphosphatase in human gastric autoimmunity

    NARCIS (Netherlands)

    Amedei, Amedeo; Bergman, Mathijs P.; Appelmelk, Ben J.; Azzurri, Annalisa; Benagiano, Marisa; Tamburini, Carlo; van der Zee, Ruurd; Telford, John L.; Vandenbroucke-Grauls, Christina M. J. E.; D'Elios, Mario M.; del Prete, Gianfranco

    2003-01-01

    Autoimmune gastritis and Helicobacter pylori-associated gastric atrophy develop through similar mechanisms involving the proton pump H+,K+-adenosine triphosphatase as autoantigen. Here, we report that H. pylori-infected patients with gastric autoimmunity harbor in vivo-activated gastric CD4+ T cells

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

    Czech Academy of Sciences Publication Activity Database

    Štreitová, Denisa; Weiterová, Lenka; Hofer, Michal; Holá, Jiřina; Horváth, Viktor; Kozubík, Alois; Znojil, V.

    2007-01-01

    Roč. 25, č. 6 (2007), s. 419-426 ISSN 0735-7907 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : MOLT-4 leukemia cell s * cell growth * adenosine Subject RIV: BO - Biophysics Impact factor: 2.106, year: 2007

  10. Adenosine A2B receptor: from cell biology to human diseases

    Science.gov (United States)

    Sun, Ying; Huang, Pingbo

    2016-08-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR’s functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases.

  11. Adenosine A1 receptors in human sleep regulation studied by electroencephalography (EEG) and positron emission tomography (PET)

    International Nuclear Information System (INIS)

    Geissler, E.

    2007-01-01

    Sleep is an essential physiological process. However, the functions of sleep and the endogenous mechanisms involved in sleep regulation are only partially understood. Convergent lines of evidence support the hypothesis that the build-up of sleep propensity during wakefulness and its decline during sleep are associated with alterations in brain adenosine levels and adenosine receptor concentrations. The non-selective A 1 and A 2A adenosine receptor antagonist caffeine stimulates alertness and is known to attenuate changes in the waking and sleep electroencephalogram (EEG) typically observed after prolonged waking. Several findings point to an important function of the adenosine A 1 receptor (A 1 AR) in the modulation of vigilance states. The A 1 AR is densely expressed in brain regions involved in sleep regulation, and pharmacological manipulations affecting the A 1 AR were shown to influence sleep propensity and sleep depth. However, an involvement of the A 2A adenosine receptor (A 2A AR) is also assumed. The distinct functions of the A 1 and A 2A receptor subtypes in sleep-wake regulation and in mediating the effects of caffeine have not been identified so far. The selective adenosine A 1 receptor antagonist, 8-cyclopentyl-3-(3- 18 Ffluoropropyl)- 1-propylxanthine ( 18 F-CPFPX), offers the opportunity to get further insights into adenosinergic mechanisms by in vivo imaging of the A 1 AR subtype with positron emission tomography (PET). The aim of this thesis was to elucidate the role of adenosine A 1 receptors in human sleep regulation, combining 18 F-CPFPX PET brain imaging and EEG recordings, the gold standard in sleep research. It was hypothesized that sleep deprivation would induce adenosine accumulation and/or changes in A 1 AR density. Thus, the question was addressed whether these effects of prolonged wakefulness can be visualized by altered 18 F-CPFPX binding. Moreover, it was investigated whether radioligand uptake might be influenced by caffeine, since

  12. A heterodimer of human 3'-phospho-adenosine-5'-phosphosulphate (PAPS) synthases is a new sulphate activating complex

    International Nuclear Information System (INIS)

    Grum, Daniel; Boom, Johannes van den; Neumann, Daniel; Matena, Anja; Link, Nina M.; Mueller, Jonathan W.

    2010-01-01

    3'-Phospho-adenosine-5'-phosphosulphate (PAPS) synthases are fundamental to mammalian sulphate metabolism. These enzymes have recently been linked to a rising number of human diseases. Despite many studies, it is not yet understood how the mammalian PAPS synthases 1 and 2 interact with each other. We provide first evidence for heterodimerisation of these two enzymes by pull-down assays and Foerster resonance energy transfer (FRET) measurements. Kinetics of dimer dissociation/association indicates that these heterodimers form as soon as PAPSS1 and -S2 encounter each other in solution. Affinity of the homo- and heterodimers were found to be in the low nanomolar range using anisotropy measurements employing proteins labelled with the fluorescent dye IAEDANS that - in spite of its low quantum yield - is well suited for anisotropy due to its large Stokes shift. Within its kinase domain, the PAPS synthase heterodimer displays similar substrate inhibition by adenosine-5'-phosphosulphate (APS) as the homodimers. Due to divergent catalytic efficacies of PAPSS1 and -S2, the heterodimer might be a way of regulating PAPS synthase function within mammalian cells.

  13. β-Nicotinamide adenine dinucleotide acts at prejunctional adenosine A1 receptors to suppress inhibitory musculomotor neurotransmission in guinea pig colon and human jejunum

    Science.gov (United States)

    Wang, Guo-Du; Wang, Xi-Yu; Liu, Sumei; Xia, Yun; Zou, Fei; Qu, Meihua; Needleman, Bradley J.; Mikami, Dean J.

    2015-01-01

    Intracellular microelectrodes were used to record neurogenic inhibitory junction potentials in the intestinal circular muscle coat. Electrical field stimulation was used to stimulate intramural neurons and evoke contraction of the smooth musculature. Exposure to β-nicotinamide adenine dinucleotide (β-NAD) did not alter smooth muscle membrane potential in guinea pig colon or human jejunum. ATP, ADP, β-NAD, and adenosine, as well as the purinergic P2Y1 receptor antagonists MRS 2179 and MRS 2500 and the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine, each suppressed inhibitory junction potentials in guinea pig and human preparations. β-NAD suppressed contractile force of twitch-like contractions evoked by electrical field stimulation in guinea pig and human preparations. P2Y1 receptor antagonists did not reverse this action. Stimulation of adenosine A1 receptors with 2-chloro-N6-cyclopentyladenosine suppressed the force of twitch contractions evoked by electrical field stimulation in like manner to the action of β-NAD. Blockade of adenosine A1 receptors with 8-cyclopentyl-1,3-dipropylxanthine suppressed the inhibitory action of β-NAD on the force of electrically evoked contractions. The results do not support an inhibitory neurotransmitter role for β-NAD at intestinal neuromuscular junctions. The data suggest that β-NAD is a ligand for the adenosine A1 receptor subtype expressed by neurons in the enteric nervous system. The influence of β-NAD on intestinal motility emerges from adenosine A1 receptor-mediated suppression of neurotransmitter release at inhibitory neuromuscular junctions. PMID:25813057

  14. Human mitochondrial Hsp70 (mortalin): shedding light on ATPase activity, interaction with adenosine nucleotides, solution structure and domain organization.

    Science.gov (United States)

    Dores-Silva, Paulo R; Barbosa, Leandro R S; Ramos, Carlos H I; Borges, Júlio C

    2015-01-01

    The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

  15. ATP induced vasodilatation and purinergic receptors in the human leg: roles of nitric oxide, prostaglandins and adenosine

    DEFF Research Database (Denmark)

    Mortensen, Stefan P; Gonzalez-Alonso, Jose; Bune, Laurids

    2009-01-01

    .05) and was associated with a parallel lowering in leg vascular conductance and cardiac output and a compensatory increase in leg O2 extraction. Infusion of theophylline did not alter the ATP induced leg hyperemia or systemic variables. Real time PCR analysis of the mRNA content from the vastus lateralus muscle of 8...... subjects showed the highest expression of P2Y2 receptors of the 10 investigated P2 receptor subtypes. Immunohistochemistry showed that P2Y2 receptors were located in the endothelium of microvessels and smooth muscle cells, whereas P2X1 receptors were located in the endothelium and the sacrolemma....... Collectively, these results indicate that NO and prostaglandins, but not adenosine, play a role in ATP induced vasodilation in human skeletal muscle. The localization of the P2Y2 and P2X1 receptors suggest that these receptors may mediate ATP induced vasodilation in skeletal muscle. Key words: Skeletal Muscle...

  16. Circadian variations of adenosine and of its metabolism. Could adenosine be a molecular oscillator for circadian rhythms?

    Science.gov (United States)

    Chagoya de Sánchez, V

    1995-03-01

    The present review describes the biological implications of the periodic changes of adenosine concentrations in different tissues of the rat. Adenosine is a purine molecule that could have been formed in the prebiotic chemical evolution and has been preserved. The rhythmicity of this molecule, as well as its metabolism and even the presence of specific receptors, suggests a regulatory role in eukaryotic cells and in multicellular organisms. Adenosine may be considered a chemical messenger and its action could take place at the level of the same cell (autocrine), the same tissue (paracrine), or on separate organs (endocrine). Exploration of the circadian variations of adenosine was planned considering the liver as an important tissue for purine formation, the blood as a vehicle among tissues, and the brain as the possible acceptor for hepatic adenosine or its metabolites. The rats used in these studies were adapted to a dark-light cycle of 12 h with an unrestrained feeding and drinking schedule. The metabolic control of adenosine concentration in the different tissues studied through the 24-h cycle is related to the activity of adenosine-metabolizing enzyme: 5'-nucleotidase adenosine deaminase, adenosine kinase, and S-adenosylhomocysteine hydrolase. Some possibilities of the factors modulating the activity of these enzymes are commented upon. The multiphysiological action of adenosine could be mediated by several actions: (i) by interaction with extracellular and intracellular receptors and (ii) through its metabolism modulating the methylation pathway, possibly inducing physiological lipoperoxidation, or participating in the energetic homeostasis of the cell. The physiological meaning of the circadian variations of adenosine and its metabolism was focused on: maintenance of the energetic homeostasis of the tissues, modulation of membrane structure and function, regulation of fasting and feeding metabolic pattern, and its participation in the sleep-wake cycle. From

  17. 脑脊液免疫球蛋白、乳酸脱氢酶及腺苷脱氨酶检测在成人颅内感染鉴别诊断中的临床意义%Clinical significance of immunoglobulins, lactatedehydrogenase ,and adenosine deaminase detection in differential diagnosis of intracranial infections in adults

    Institute of Scientific and Technical Information of China (English)

    张丽琴

    2012-01-01

    OBJECTIVE To investigate the changes of immunoglobulin (Ig), lactate dehydrogenase(LDH), and adenosine deaminase(ADA) in cerebrospinal fluid (CSF)of the adults with intracranial infections and explore the clinical significance.METHODS Totally 17 patients with tuberculous meningitis, 19 patients with purulent meningitis, and 21 patients with viral meningitis were recruited in this study.The levels of LDH, ADA, and Ig in CSF from.each patient were measured and compared with the control group.RESULTS The concentrations of LDH, ADA, IgM, IgG.and IgA in CSF of the patients with purulent meningitis were(85.60±18.11)U/L,(2.59 ±0.52)U/L,(63.85±13.44)mg/L,(92.55±35.18)mg/L,and (20.60±8.75)mg/L,respectively ; the levels of LDA, ADA, IgM, IgG, and IgA in CSF of the patients with tuberculous meningitis were(78.80±22.38)U/L, (11.83±2.65)U/L, (18.90±8.62)mg/L, (180.69±42.28)mg/L, and(41.63±ll.42)mg/L, respectively) the levels of LDA, ADA, IgM, IgG, and IgA in CSF of the patients with viral meningitis were(22.43±9.56)U/L, (2.66±0.64)U/L, (5.83±1.34)mg/L,(20.90±10.35)mg/L,and(7.20±2.30)mg/L, respectively; of the three groups of adults with intracranial infectious diseases, the levels of IgM, IgG, IgA, and LDH in CSF of the patients with tuberculous meningitis or purulent meningitis were significantly higher than those in the patients with viral meningitis(P<0.01), the levels of IgG and IgA in CSF of the patients with tuberculous meningitis increased most significantly, the level of IgM in CSF of the patients with purulent meningitis increased most significantly, the activity of ADA of the tuberculous meningitis group was significantly higher than control group and other two groups(P<0.01 ); no statistical significance was found when between the indexes of viral meningitis group and control group.CONCLUSION The blood brain barriers of adult patients have been injured in varying degreest the detection of Ig, LDH in CSF is helpful to clinical diagnosis and

  18. Sustained Elevated Adenosine via ADORA2B Promotes Chronic Pain through Neuro-immune Interaction

    Directory of Open Access Journals (Sweden)

    Xia Hu

    2016-06-01

    Full Text Available The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA, an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada−/− mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund’s adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets.

  19. AID/APOBEC cytosine deaminase induces genome-wide kataegis

    Directory of Open Access Journals (Sweden)

    Lada Artem G

    2012-12-01

    Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

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

    International Nuclear Information System (INIS)

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

    1990-01-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[ 3 H]ethylcarboxamidoadenosine [( 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 [ 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

  1. A3 Adenosine Receptors Modulate Hypoxia-inducible Factor-1a Expression in Human A375 Melanoma Cells

    Directory of Open Access Journals (Sweden)

    Stefania Merighi

    2005-10-01

    Full Text Available Hypoxia-inducible factor-1 (HIF-1 is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% 02, adenosine upregulates HIF-1α protein expression in a dose-dependent and timedependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2 protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1a and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.

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

    Science.gov (United States)

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

    2014-11-01

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

  3. Binding of the Antagonist Caffeine to the Human Adenosine Receptor hA2AR in Nearly Physiological Conditions.

    Directory of Open Access Journals (Sweden)

    Ruyin Cao

    Full Text Available Lipid composition may significantly affect membrane proteins function, yet its impact on the protein structural determinants is not well understood. Here we present a comparative molecular dynamics (MD study of the human adenosine receptor type 2A (hA(2AR in complex with caffeine--a system of high neuro-pharmacological relevance--within different membrane types. These are POPC, mixed POPC/POPE and cholesterol-rich membranes. 0.8-μs MD simulations unambiguously show that the helical folding of the amphipathic helix 8 depends on membrane contents. Most importantly, the distinct cholesterol binding into the cleft between helix 1 and 2 stabilizes a specific caffeine-binding pose against others visited during the simulation. Hence, cholesterol presence (~33%-50% in synaptic membrane in central nervous system, often neglected in X-ray determination of membrane proteins, affects the population of the ligand binding poses. We conclude that including a correct description of neuronal membranes may be very important for computer-aided design of ligands targeting hA(2AR and possibly other GPCRs.

  4. Exposure of Human Lung Cancer Cells to 8-Chloro-Adenosine Induces G2/M Arrest and Mitotic Catastrophe

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    Hong-Yu Zhang

    2004-11-01

    Full Text Available 8-Chloro-adenosine (8-CI-Ado is a potent chemotherapeutic agent whose cytotoxicity in a variety of tumor cell lines has been widely investigated. However, the molecular mechanisms are uncertain. In this study, we found that exposure of human lung cancer cell lines A549 (p53-wt and H1299 (p53-depleted to 8-CI-Ado induced cell arrest in the G2/M phase, which was accompanied by accumulation of binucleated and polymorphonucleated cells resulting from aberrant mitosis and failed cytokinesis. Western blotting showed the loss of phosphorylated forms of Cdc2 and Cdc25C that allowed progression into mitosis. Furthermore, the increase in Ser10-phosphorylated histone H3-positive cells revealed by fluorescence-activated cell sorting suggested that the agent-targeted cells were able to exit the G2 phase and enter the M phase. Immunocytochemistry showed that microtubule and microfilament arrays were changed in exposed cells, indicating that the dynamic instability of microtubules and microfilaments was lost, which may correlate with mitotic dividing failure. Aberrant mitosis resulted in mitotic catastrophe followed by varying degrees of apoptosis, depending on the cell lines. Thus, 8-CI-Ado appears to exert its cytotoxicity toward cells in culture by inducing mitotic catastrophe.

  5. Release of Periplasmic Nucleotidase Induced by Human Antimicrobial Peptide in E. coli Causes Accumulation of the Immunomodulator Adenosine.

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    Andreia Bergamo Estrela

    Full Text Available Previous work by our group described that human β-defensin-2 induces accumulation of extracellular adenosine (Ado in E. coli cultures through a non-lytic mechanism causing severe plasmolysis. Here, we investigate the presence of AMP as a direct precursor and the involvement of a bacterial enzyme in the generation of extracellular Ado by treated bacteria. Following hBD-2 treatment, metabolites were quantified in the supernatants using targeted HPLC-MS/MS analysis. Microbial growth was monitored by optical density and cell viability was determined by colony forming units counts. Phosphatase activity was measured using chromogenic substrate pNPP. The results demonstrate that defensin-treated E. coli strain W releases AMP in the extracellular space, where it is converted to Ado by a bacterial soluble factor. An increase in phosphatase activity in the supernatant was observed after peptide treatment, similar to the effect of sucrose-induced osmotic stress, suggesting that the periplasmic 5'nucleotidase (5'-NT is released following the plasmolysis event triggered by the peptide. Ado accumulation was enhanced in the presence of Co2+ ion and inhibited by EDTA, further supporting the involvement of a metallo-phosphatase such as 5'-NT in extracellular AMP conversion into Ado. The comparative analysis of hBD-induced Ado accumulation in different E. coli strains and in Pseudomonas aeruginosa revealed that the response is not correlated to the peptide's effect on cell viability, but indicates it might be dependent on the subcellular distribution of the nucleotidase. Taken together, these data shed light on a yet undescribed mechanism of host-microbial interaction: a human antimicrobial peptide inducing selective release of a bacterial enzyme (E. coli 5'-NT, leading to the formation of a potent immunomodulator metabolite (Ado.

  6. Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors

    DEFF Research Database (Denmark)

    Gnad, Thorsten; Scheibler, Saskia; von Kügelgen, Ivar

    2014-01-01

    hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A...

  7. Quantification of adenosine A2A receptors in the human brain using [11C]TMSX and positron emission tomography

    International Nuclear Information System (INIS)

    Naganawa, Mika; Kimura, Yuichi; Oda, Keiichi; Ishii, Kenji; Ishiwata, Kiichi; Mishina, Masahiro; Manabe, Yoshitsugu; Chihara, Kunihiro

    2007-01-01

    [7-methyl- 11 C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([ 11 C]TMSX) is a positron-emitting adenosine A 2A receptor (A2AR) antagonist for visualisation of A2AR distribution by positron emission tomography (PET). The aims of this paper were to use a kinetic model to analyse the behaviour of [ 11 C]TMSX in the brain and to examine the applicability of the Logan plot. We also studied the applicability of a simplified Logan plot by omitting metabolite correction and arterial blood sampling. The centrum semiovale was used as a reference region on the basis of a post-mortem study showing that it has a negligibly low density of A2ARs. Compartmental analysis was performed in five normal subjects. Parametric images of A2AR binding potential (BP) were also generated using a Logan plot with or without metabolite correction and with or without arterial blood sampling. To omit arterial blood sampling, we applied a method to extract the plasma-related information using independent component analysis (EPICA). The estimated K 1 /k 2 was confirmed to be common in the centrum semiovale and main cortices. The three-compartment model was well fitted to the other regions using the fixed value of K 1 /k 2 estimated from the centrum semiovale. The estimated BPs using the Logan plot matched those derived from compartment analysis. Without the metabolite correction, the estimate of BP underestimated the true value by 5%. The estimated BPs agreed regardless of arterial blood sampling. A three-compartment model with a reference region, the centrum semiovale, describes the kinetic behaviour of [ 11 C]TMSX PET images. A2ARs in the human brain can be visualised as a BP image using [ 11 C]TMSX PET without arterial blood sampling. (orig.)

  8. Comparison of Effects on Gene Expression Activity of Low-Molecular-Weight Lychee Fruit Polyphenol (Oligonol®, Adenosine, and Minoxidil in Human Dermal Papilla Cells

    Directory of Open Access Journals (Sweden)

    Koji Wakame

    2017-06-01

    Full Text Available Background: Oligonol® (OLG is a functional food product and ingredient for cosmetics derived from a lychee fruit polyphenol. It has been reported to act on the skin as an anti-inflammatory and prevent UVB-induced skin damage. Aim: In this study, with the aim of exploring new functionalities of OLG on the scalp, we investigated the effect of OLG on human dermal papilla cells by comparing with adenosine and minoxidil at the genetic level. Method: OLG, adenosine, and minoxidil were applied to human dermal papilla cell lines for 24 h, after which VEGF, FGF-7, WNT5a, and WNT10a mRNA expressions were measured by real-time PCR analysis. Additionally, using DNA microarrays, we investigated the effect on 205 inflammation-related genes. Result: Consequently, in human dermal papilla cell lines, FGF-7 and WNT10a mRNA expression were observed in 100 µg/mL OLG-supplemented cells. The results of the DNA microarray analysis showed that 10 genes were suppressed by OLG. Conclusions: OLG may be expected to affect function of human dermal papilla cell by regulating the expression of genes related to cell proliferation and inflammation.

  9. A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

    Science.gov (United States)

    Dong, Huina; Zu, Xin; Zheng, Ping; Zhang, Dawei

    2015-01-01

    Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH3, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples. PMID:25580842

  10. The polydeoxyadenylate tract of Alu repetitive elements is polymorphic in the human genome

    International Nuclear Information System (INIS)

    Economou, E.P.; Bergen, A.W.; Warren, A.C.; Antonarakis, S.E.

    1990-01-01

    To identify DNA polymorphisms that are abundant in the human genome and are detectable by polymerase chain reaction amplification of genomic DNA, the authors hypothesize that the polydeoxyadenylate tract of the Alu family of repetitive elements is polymorphic among human chromosomes. Analysis of the 3' ends of three specific Alu sequences showed two occurrences, one in the adenosine deaminase gene and other in the β-globin pseudogene, were polymorphic. This novel class of polymorphism, termed AluVpA [Alu variable poly(A)] may represent one of the most useful and informative group of DNA markers in the human genome

  11. QSAR of adenosine receptor antagonists. Part 3: Exploring physicochemical requirements for selective binding of 1,2,4-triazolo[5,1-i]purine derivatives with human adenosine A3 receptor subtype.

    Science.gov (United States)

    Roy, Kunal; Leonard, J Thomas; Sengupta, Chandana

    2004-07-16

    Considering potential of selective adenosine A3 receptor antagonists in the development of prospective therapeutic agents, an attempt has been made to explore selectivity requirements of 1,2,4-triazolo[5,1-i]purine derivatives for binding with cloned human adenosine A3 receptor subtype. In this study, partition coefficient (logP) values of the molecules (calculated by Crippen's fragmentation method) and Wang-Ford charges of the common atoms of the triazolopurine nucleus (calculated from molecular electrostatic potential surface of energy minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best equation describing A3 binding affinity [n=29, Q2=0.796, Ra2=0.853, R2=0.874, R=0.935, s=0.342, F=41.5 (df 4,24), SDEP=0.396] showed parabolic relation with logP (optimum value being 4.134). Further, it was found that an aromatic substituent conjugated with the triazole nucleus should be present at R2 position for A3 binding affinity. Again, high negative charges on N2 and N4 are conducive to the binding affinity. While exploring selectivity requirements of the compounds for binding with A3 receptor over that with A2A receptor, the selectivity relation [n=23, Q2=0.909, Ra2=0.918, R2=0.933, R=0.966, s=0.401, F=62.4 (df 4,18), SDEP=0.412] showed that an aromatic R2 substituent conjugated with the triazole nucleus contributes significantly to the selectivity. Again, presence of a 4-substituted-phenyl ring (except 4-OH-phenyl and 4-CH3-phenyl) at R2 position also increases selectivity. Further, charge difference between N2 and N11 (negative charge on the former should be higher and that on the latter should be less) contributes significantly to the selectivity. In addition, negative charge on N7 is conducive while presence of substituents like propyl, butyl, pentyl or phenyl at R1 position is detrimental for the A3 selectivity.

  12. AMP is an adenosine A1 receptor agonist.

    Science.gov (United States)

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

    2012-02-17

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

  13. Wireless Instantaneous Neurotransmitter Concentration System-based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring.

    Science.gov (United States)

    Agnesi, Filippo; Tye, Susannah J; Bledsoe, Jonathan M; Griessenauer, Christoph J; Kimble, Christopher J; Sieck, Gary C; Bennet, Kevin E; Garris, Paul A; Blaha, Charles D; Lee, Kendall H

    2009-10-01

    In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig. The

  14. Wireless Instantaneous Neurotransmitter Concentration System–based amperometric detection of dopamine, adenosine, and glutamate for intraoperative neurochemical monitoring

    Science.gov (United States)

    Agnesi, Filippo; Tye, Susannah J.; Bledsoe, Jonathan M.; Griessenauer, Christoph J.; Kimble, Christopher J.; Sieck, Gary C.; Bennet, Kevin E.; Garris, Paul A.; Blaha, Charles D.; Lee, Kendall H.

    2009-01-01

    Object In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. Methods The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme-linked electrode to measure glutamate; and 3) a multiple enzyme-linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal

  15. Adenosine and preeclampsia.

    Science.gov (United States)

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

    2017-06-01

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

  16. Structural Basis for Inhibition of Mycobacterial and Human Adenosine Kinase by 7-Substituted 7-(Het)aryl-7-deazaadenine Ribonucleosides

    Czech Academy of Sciences Publication Activity Database

    Snášel, Jan; Nauš, Petr; Dostál, Jiří; Hnízda, Aleš; Fanfrlík, Jindřich; Brynda, Jiří; Bourderioux, Aurelie; Dušek, Michal; Dvořáková, H.; Stolaříková, J.; Zábranská, Helena; Pohl, Radek; Konečný, P.; Džubák, P.; Votruba, Ivan; Hajdúch, M.; Řezáčová, Pavlína; Veverka, Václav; Hocek, Michal; Pichová, Iva

    2014-01-01

    Roč. 57, č. 20 (2014), s. 8268-8279 ISSN 0022-2623 R&D Projects: GA ČR GAP207/11/0344; GA MŠk LO1302; GA MŠk(CZ) LK11205 EU Projects: European Commission(XE) 241587 - SYSTEMTB Grant - others:GA MŠk(CZ) LM2011020 Institutional support: RVO:61388963 ; RVO:68378271 Keywords : 7-(het)aryl-7-deazaadenine ribonucleosides * enzyme inhibition * adenosine kinase * cytostatic activity Subject RIV: CC - Organic Chemistry Impact factor: 5.447, year: 2014

  17. Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts

    International Nuclear Information System (INIS)

    Kaplan, J.H.; Forbush, B. III; Hoffman, J.F.

    1978-01-01

    2-Nitrobenzyl phosphate and 1-(2-nitro)phenylethyl phosphate have been synthesized and demonstrated to be suitable as photolabile sources of inorganic phosphate. The same protecting groups were attached to the terminal phosphate of adenosine 5'-triphosphate. These caged ATP compounds released adenosine 5'-triphosphate on illumination at 340 nm in aqueous solution and P 3 -1-(2-nitro)phenylethyl-ATP gave about a 70 percent yield in under 30 s. The unphotolyzed caged ATP was neither a substrate nor inhibitor of purified renal Na,K-ATPase (EC 3.61.3). Following photolysis in the presence of the enzyme, the liberated ATP was hydrolyzed but at an inhibited rate. The photo-dependent inhibition could be eliminated by prior addition of glutathione or bisulfite to the irradiated solution. Caged ATP was incorporated into resealed human erythrocyte ghosts prepared from red blood cells depleted of internal energy stores. While the NA : K pump was unable to use incorporated caged ATP as a substrate, the ATP liberated by photolysis activated the pump as evidenced by measurements of K-dependent, ouabain-sensitive Na efflux. Thus the caged ATP can be used as a stable source of ATP unmetabolizable by intracellular ATPases until the ATP is released following photolytic irradiation

  18. Replication protein A (RPA) hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

    Science.gov (United States)

    Lada, Artem G; Waisertreiger, Irina S-R; Grabow, Corinn E; Prakash, Aishwarya; Borgstahl, Gloria E O; Rogozin, Igor B; Pavlov, Youri I

    2011-01-01

    Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G. We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast. Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

  19. Replication protein A (RPA hampers the processive action of APOBEC3G cytosine deaminase on single-stranded DNA.

    Directory of Open Access Journals (Sweden)

    Artem G Lada

    Full Text Available Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G, restricts retroviruses, and Activation Induced Deaminase (AID generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA, the eukaryotic single-stranded DNA (ssDNA binding protein, severely inhibits the deamination activity and processivity of A3G.We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the "hit and run" single base substitution events observed in yeast.Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.

  20. Exercise-induced increase in interstitial bradykinin and adenosine concentrations in skeletal muscle and peritendinous tissue in humans

    DEFF Research Database (Denmark)

    Langberg, H; Bjørn, C; Boushel, Robert Christopher

    2002-01-01

    been established. Microdialysis (molecular mass cut-off 5 kDa) was performed simultaneously in calf muscle and peritendinous Achilles tissue at rest and during 10 min periods of incremental (0.75 W, 2 W, 3.5 W and 4.75 W) dynamic plantar flexion exercise in 10 healthy individuals (mean age 27 years...... increased both in muscle (from 0.48 +/- 0.07 micromol l(-1) to 1.59 +/- 0.35 micromol l(-1); P increases the interstitial concentrations......Bradykinin is known to cause vasodilatation in resistance vessels and may, together with adenosine, be an important regulator of tissue blood flow during exercise. Whether tissue concentrations of bradykinin change with exercise in skeletal muscle and tendon-related connective tissue has not yet...

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

    Science.gov (United States)

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

    1978-01-01

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

  2. Cellular radiosensitivity in human severe-combined-immunodeficiency (SCID) syndromes

    International Nuclear Information System (INIS)

    Sproston, Anthony R.M.; West, Catharine M.L.; Hendry, Jolyon H.

    1997-01-01

    Purpose: The aim of the work was to establish to what extent a variety of human severe-combined-immunodeficiency (SCID) disorders are associated with in vitro cellular hypersensitivity to ionizing radiation. Materials and methods: A study was made of fibroblast strains established from individuals with adenosine deaminase deficiency, T(-)B(-) SCID, Omenn's syndrome and a SCID heterozygote. For comparison, an assessment was also made of the radiosensitivity of a series of fibroblast strains derived from: normal donors, a patient with ataxia-telangiectasia (A-T) and an A-T heterozygote. Radiosensitivity was determined using a clonogenic assay following both high (HDR) and low (LDR) dose-rate irradiation. Results: Following HDR irradiation, the fibroblast strains derived from the different human SCID disorders displayed a wide range of radiosensitivity: the adenosine deaminase deficiency cells were similar in radiosensitivity to normal fibroblasts, T(-)B(-) cells were as hypersensitive to radiation as A-T cells and the Omenn's syndrome cells showed intermediate radiosensitivity. However, whereas all four normal cell strains studied showed significant LDR sparing, none of the SCID fibroblasts did. Conclusions: These data indicate that human SCID is variable in terms of radiosensitivity depending on the particular defect. In addition, the lack of LDR sparing of radiation-induced damage suggests the involvement of some form(s) of DNA repair defect in all the human SCID syndromes

  3. Adenosine A{sub 1} receptors in human sleep regulation studied by electroencephalography (EEG) and positron emission tomography (PET)[Dissertation 17227

    Energy Technology Data Exchange (ETDEWEB)

    Geissler, E

    2007-07-01

    Sleep is an essential physiological process. However, the functions of sleep and the endogenous mechanisms involved in sleep regulation are only partially understood. Convergent lines of evidence support the hypothesis that the build-up of sleep propensity during wakefulness and its decline during sleep are associated with alterations in brain adenosine levels and adenosine receptor concentrations. The non-selective A{sub 1} and A{sub 2A} adenosine receptor antagonist caffeine stimulates alertness and is known to attenuate changes in the waking and sleep electroencephalogram (EEG) typically observed after prolonged waking. Several findings point to an important function of the adenosine A{sub 1} receptor (A{sub 1}AR) in the modulation of vigilance states. The A{sub 1}AR is densely expressed in brain regions involved in sleep regulation, and pharmacological manipulations affecting the A{sub 1}AR were shown to influence sleep propensity and sleep depth. However, an involvement of the A{sub 2A} adenosine receptor (A{sub 2A}AR) is also assumed. The distinct functions of the A{sub 1} and A{sub 2A} receptor subtypes in sleep-wake regulation and in mediating the effects of caffeine have not been identified so far. The selective adenosine A{sub 1} receptor antagonist, 8-cyclopentyl-3-(3-{sup 18}Ffluoropropyl)- 1-propylxanthine ({sup 18}F-CPFPX), offers the opportunity to get further insights into adenosinergic mechanisms by in vivo imaging of the A{sub 1}AR subtype with positron emission tomography (PET). The aim of this thesis was to elucidate the role of adenosine A{sub 1} receptors in human sleep regulation, combining {sup 18}F-CPFPX PET brain imaging and EEG recordings, the gold standard in sleep research. It was hypothesized that sleep deprivation would induce adenosine accumulation and/or changes in A{sub 1}AR density. Thus, the question was addressed whether these effects of prolonged wakefulness can be visualized by altered {sup 18}F-CPFPX binding. Moreover, it was

  4. Effects of tofacitinib on nucleic acid metabolism in human articular chondrocytes.

    Science.gov (United States)

    Koizumi, Hideki; Arito, Mitsumi; Endo, Wataru; Kurokawa, Manae S; Okamoto, Kazuki; Omoteyama, Kazuki; Suematsu, Naoya; Beppu, Moroe; Kato, Tomohiro

    2015-07-01

    In our previous screening of chondrocyte protein profiles, the amount of adenosine monophosphate deaminase (AMPD) 2 was found to be decreased by tofacitinib. Extending the study, here we confirmed the decrease of AMPD2 by tofacitinib and further investigated effects of tofacitinib on purine nucleotide metabolism. Human articular chondrocytes and a chondrosarcoma cell line: OUMS-27 were stimulated with tofacitinib. Then the levels of AMPD2 and its related enzymes were investigated by Western blot. The levels of AMP and adenosine were assessed by mass spectrometry. We confirmed the significant decrease of AMPD2 by tofacitinib in chondrocytes (p = 0.025). The levels of adenosine kinase and 5'-nucleotidase were decreased in chondrocytes, although they did not meet statistical significance (p = 0.067 and p = 0.074, respectively). The results from OUMS-27 were similar to those from the chondrocytes. The cellular adenosine levels were significantly decreased by tofacitinib in OUMS-27 (p = 0.014). The cellular AMP levels were increased, although they did not meet statistical significance in OUMS-27 (p = 0.066). Our data indicate that tofacitinib increases the cellular levels of adenosine, which is known to have anti-inflammatory activity, through the downregulation of AMPD2. This would be a novel functional aspect of tofacitinib.

  5. Myoadenylate deaminase deficiency, hypertrophic cardiomyopathy and gigantism syndrome.

    Science.gov (United States)

    Skyllouriotis, M L; Marx, M; Bittner, R E; Skyllouriotis, P; Gross, M; Wimmer, M

    1997-07-01

    We report a 20-year-old man with gigantism syndrome, hypertrophic cardiomyopathy, muscle weakness, exercise intolerance, and severe psychomotor retardation since childhood. Histochemical and biochemical analysis of skeletal muscle biopsy revealed myoadenylate deaminase deficiency; molecular genetic analysis confirmed the diagnosis of primary (inherited) myoadenylate deaminase deficiency. Plasma, urine, and muscle carnitine concentrations were reduced. L-Carnitine treatment led to gradual improvement in exercise tolerance and cognitive performance; plasma and tissue carnitine levels returned to normal, and echocardiographic evidence of left ventricular hypertrophy disappeared. The combination of inherited myoadenylate deaminase deficiency, gigantism syndrome and carnitine deficiency has not previously been described.

  6. Discovery of a Bacterial 5-Methylcytosine Deaminase

    Science.gov (United States)

    2015-01-01

    5-Methylcytosine is found in all domains of life, but the bacterial cytosine deaminase from Escherichia coli (CodA) will not accept 5-methylcytosine as a substrate. Since significant amounts of 5-methylcytosine are produced in both prokaryotes and eukaryotes, this compound must eventually be catabolized and the fragments recycled by enzymes that have yet to be identified. We therefore initiated a comprehensive phylogenetic screen for enzymes that may be capable of deaminating 5-methylcytosine to thymine. From a systematic analysis of sequence homologues of CodA from thousands of bacterial species, we identified putative cytosine deaminases where a “discriminating” residue in the active site, corresponding to Asp-314 in CodA from E. coli, was no longer conserved. Representative examples from Klebsiella pneumoniae (locus tag: Kpn00632), Rhodobacter sphaeroides (locus tag: Rsp0341), and Corynebacterium glutamicum (locus tag: NCgl0075) were demonstrated to efficiently deaminate 5-methylcytosine to thymine with values of kcat/Km of 1.4 × 105, 2.9 × 104, and 1.1 × 103 M–1 s–1, respectively. These three enzymes also catalyze the deamination of 5-fluorocytosine to 5-fluorouracil with values of kcat/Km of 1.2 × 105, 6.8 × 104, and 2.0 × 102 M–1 s–1, respectively. The three-dimensional structure of Kpn00632 was determined by X-ray diffraction methods with 5-methylcytosine (PDB id: 4R85), 5-fluorocytosine (PDB id: 4R88), and phosphonocytosine (PDB id: 4R7W) bound in the active site. When thymine auxotrophs of E. coli express these enzymes, they are capable of growth in media lacking thymine when supplemented with 5-methylcytosine. Expression of these enzymes in E. coli is toxic in the presence of 5-fluorocytosine, due to the efficient transformation to 5-fluorouracil. PMID:25384249

  7. AMP Is an Adenosine A1 Receptor Agonist*

    Science.gov (United States)

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

    2012-01-01

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

  8. The effect of nucleotides and adenosine on stimulus-evoked glutamate release from rat brain cortical slices.

    Science.gov (United States)

    Bennett, G C; Boarder, M R

    2000-10-01

    Evidence has previously been presented that P1 receptors for adenosine, and P2 receptors for nucleotides such as ATP, regulate stimulus-evoked release of biogenic amines from nerve terminals in the brain. Here we investigated whether adenosine and nucleotides exert presynaptic control over depolarisation-elicited glutamate release. Slices of rat brain cortex were perfused and stimulated with pulses of 46 mM K(+) in the presence of the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (0.2 mM). High K(+) substantially increased efflux of glutamate from the slices. Basal glutamate release was unchanged by the presence of nucleotides or adenosine at concentrations of 300 microM. Adenosine, ATP, ADP and adenosine 5'-O-(3-thiotriphoshate) at 300 microM attenuated depolarisation-evoked release of glutamate. However UTP, 2-methylthio ATP, 2-methylthio ADP, and alpha,beta-methylene ATP at 300 microM had no effect on stimulated glutamate efflux. Adenosine deaminase blocked the effect of adenosine, but left the response to ATP unchanged. The A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine antagonised the inhibitory effect of both adenosine and ATP. Cibacron blue 3GA inhibited stimulus-evoked glutamate release when applied alone. When cibacron blue 3GA was present with ATP, stimulus-evoked glutamate release was almost eliminated. However, this P2 antagonist had no effect on the inhibition by adenosine. These results show that the release of glutamate from depolarised nerve terminals of the rat cerebral cortex is inhibited by adenosine and ATP. ATP appears to act directly and not through conversion to adenosine.

  9. [Features of influence adenosine, AMP and hyperadrenalinemiya on the immune status, metabolic enzymes of purine nucleotides and the antioxidant defense system].

    Science.gov (United States)

    Tapbergenov, S O; Sovetov, B S; Tapbergenov, A T

    2016-11-01

    Administration of a large dose of adrenaline (4 mg/kg 60 min before analysis) increased blood levels of total leukocytes, lymphocytes, decreased T-cell suppressors, leukocyte migration inhibition reaction (LMIR) and NBT test, but increased the level of conjugated dienes (CD). Administration of AMPand adenosine increased levels of total leukocytes, lymphocytes, T- lymphocytes, T-helpers, decreased the level of malondialdehyde (MDA), LMIR, and T-cell suppressors. Sympathetic hyperactivation induced by administration of a large dose of adrenaline (4 mg/kg 60 min before analysis) was accompanied by an increase in heart and liver activities of glutathione peroxidase (GPx), catalase, AMP deaminase (AMPD), and adenosine deaminase (AD). Administration of AMP or adenosine caused a decrease in activities of glutathione reductase (GR), GPx, catalase, a decrease in the MDA level and an increase in activities of AMPD and AD in the heart. In the liver AMP and adenosine also caused a decrease in activities of glutathione reductase (GR), GPx, a decrease in the MDA level and an increase in activities of AMPD and AD. The data obtained suggest that administration of adrenaline, AMP, and adenosine influences activity of enzymes involved in purine nucleotide metabolism. However, in contrast to adrenaline, administration of AMP or adenosine does not provoke stress reaction.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-09-17

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

  11. Two Cases of Severe Combined Immunodeficiency Caused By Adenosine Deaminase Deficiency

    Directory of Open Access Journals (Sweden)

    Turkan Patiroglu

    2014-08-01

    Full Text Available Severe Combined Immune Deficiency (SCID is a primary immune deficiency disorder manifested with severe infections upon first months of life, which is characterized by diverse genetic defects in T and B lymphocyte functions and occasionally in NK cells. ADA deficiency is a form of SCID progressing with severe lymphopenia and immune deficiency caused by toxic metabolites of ADA. Bone marrow transplantation (BMT is the only curative treatment although prophylactic anti-microbial therapy, intravenous immunoglobulin (IVIG and enzyme replacement can achieve transient improvements. Early diagnosis before development of severe infections and organ injury and referral to pediatric immunology clinics will make considerable contributions to prognosis. Here, we presented 2 cousins with SCID who had positive family history with deceased sibling; presented with tanning at skin, severe neonatal infections and Q246X (c736C>T non-sense mutation in exon 8 in ADA gene  in order to emphasize this rare mutation and pediatric emergencies associated with this disorder.

  12. Synthesis of [5-14C]pentostatin, an antileukemic agent and potent adenosine deaminase inhibitor

    International Nuclear Information System (INIS)

    Woo, P.W.K.; Lee, H.T.

    1990-01-01

    Reaction of triethyl ortho[ 14 C]formate (2) with 2-amino-1-(5-amino-1H-imidazol-4-yl)ethanone dihydrochloride (1) in the presence of molecular sieves 4A gave 6,7-dihydro[5- 14 C]imidazo[4,5-d]{1,3]diazepin-8(3H)-one hydrochloride monodimethyl sulfoxide (3) (radiochemical yield, 60%). The latter was persilylated with bis(trimethylsilyl)trifluroacetamide (4) and glycosylated with 2-deoxy-3,5-di-0-p-toluoyl-α-D-erythro-pentufuranosyl chloride (6) to give a mixture from which the 3-N-β-glycosylated product 8 was isolated by chromatography and crystallization (13%). Deprotective saponification with methanolic sodium methoxide and subsequent sodium borohydride reduction of the 8-keto function gave a (R,S)-mixture from which the desired (R)-isomer, [5- 14 C]pentostatin (11), was isolated by preparative HPLC over a C18 column, desalting with Diaion-HP20, and subsequent crystallization (39%). (author)

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

    Directory of Open Access Journals (Sweden)

    Biswajit Biswas

    2016-01-01

    Full Text Available 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.

  14. The effect of cyclophosphamide and gamma irradiation on adenosine deaminase and purine nucleoside phosphorylase in mice

    International Nuclear Information System (INIS)

    Hosek, B.; Bohaecek, J.; Sikulova, J.

    1991-01-01

    Changes in ADA and PNP activities in the spleens and thymuses of mice were studied after a single administration of cyclophosphamide and after whole-body gamma irradiation, applied alone or three days after CY application, In the first days after the treatment the enzyme activities were significantly depressed with the exception of ADA in the spleen, where a high elevation in relation to controls was observed. During the regeneration period a pronounced rise of PNP activity in the spleen occurred mainly after a combined application of CY and irradiation. In the thymus the regeneration was manifested by a mild increase of both ADA and PNP activities towards control values. The findings suggest that the expressive changes of ADA and PNP activities, participating in the purine salvage pathway, may, after a cytotoxic treatment, influence the nucleotide pool and DNA synthesis in lymphoid organs

  15. AMP and adenosine are both ligands for adenosine 2B receptor signaling.

    Science.gov (United States)

    Holien, Jessica K; Seibt, Benjamin; Roberts, Veena; Salvaris, Evelyn; Parker, Michael W; Cowan, Peter J; Dwyer, Karen M

    2018-01-15

    Adenosine is considered the canonical ligand for the adenosine 2B receptor (A 2B R). A 2B R is upregulated following kidney ischemia augmenting post ischemic blood flow and limiting tubular injury. In this context the beneficial effect of A 2B R signaling has been attributed to an increase in the pericellular concentration of adenosine. However, following renal ischemia both kidney adenosine monophosphate (AMP) and adenosine levels are substantially increased. Using computational modeling and calcium mobilization assays, we investigated whether AMP could also be a ligand for A 2B R. The computational modeling suggested that AMP interacts with more favorable energy to A 2B R compared with adenosine. Furthermore, AMPαS, a non-hydrolyzable form of AMP, increased calcium uptake by Chinese hamster ovary (CHO) cells expressing the human A 2B R, indicating preferential signaling via the G q pathway. Therefore, a putative AMP-A 2B R interaction is supported by the computational modeling data and the biological results suggest this interaction involves preferential G q activation. These data provide further insights into the role of purinergic signaling in the pathophysiology of renal IRI. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Adenosine and sleep

    International Nuclear Information System (INIS)

    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 1 receptors, 3 H-L-PIA binding was measured. The Bmax values for 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 3 H-L-PIA binding resulted from REM sleep deprivation and not from stress

  17. The catalase activity of diiron adenine deaminase

    Energy Technology Data Exchange (ETDEWEB)

    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. Elevated placental adenosine signaling contributes to the pathogenesis of preeclampsia.

    Science.gov (United States)

    Iriyama, Takayuki; Sun, Kaiqi; Parchim, Nicholas F; Li, Jessica; Zhao, Cheng; Song, Anren; Hart, Laura A; Blackwell, Sean C; Sibai, Baha M; Chan, Lee-Nien L; Chan, Teh-Sheng; Hicks, M John; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2015-02-24

    Preeclampsia is a prevalent hypertensive disorder of pregnancy and a leading cause of maternal and neonatal morbidity and mortality worldwide. This pathogenic condition is speculated to be caused by placental abnormalities that contribute to the maternal syndrome. However, the specific factors and signaling pathways that lead to impaired placentas and maternal disease development remain elusive. Using 2 independent animal models of preeclampsia (genetically engineered pregnant mice with elevated adenosine exclusively in placentas and a pathogenic autoantibody-induced preeclampsia mouse model), we demonstrated that chronically elevated placental adenosine was sufficient to induce hallmark features of preeclampsia, including hypertension, proteinuria, small fetuses, and impaired placental vasculature. Genetic and pharmacological approaches revealed that elevated placental adenosine coupled with excessive A₂B adenosine receptor (ADORA2B) signaling contributed to the development of these features of preeclampsia. Mechanistically, we provided both human and mouse evidence that elevated placental CD73 is a key enzyme causing increased placental adenosine, thereby contributing to preeclampsia. We determined that elevated placental adenosine signaling is a previously unrecognized pathogenic factor for preeclampsia. Moreover, our findings revealed the molecular basis underlying the elevation of placental adenosine and the detrimental role of excess placental adenosine in the pathophysiology of preeclampsia, and thereby, we highlight novel therapeutic targets. © 2014 American Heart Association, Inc.

  19. Quantification of adenosine A{sub 2A} receptors in the human brain using [{sup 11}C]TMSX and positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Naganawa, Mika [Tokyo Metropolitan Institute of Gerontology, Positron Medical Center, Tokyo (Japan); Japan Society for the Promotion of Science, Tokyo (Japan); Kimura, Yuichi; Oda, Keiichi; Ishii, Kenji; Ishiwata, Kiichi [Tokyo Metropolitan Institute of Gerontology, Positron Medical Center, Tokyo (Japan); Mishina, Masahiro [Nippon Medical School Chiba-Hokusoh Hospital, Neurological Institute, Chiba (Japan); Manabe, Yoshitsugu; Chihara, Kunihiro [Nara Institute of Science and Technology, Graduate School of Information Science, Nara (Japan)

    2007-05-15

    [7-methyl-{sup 11}C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([{sup 11}C]TMSX) is a positron-emitting adenosine A{sub 2A} receptor (A2AR) antagonist for visualisation of A2AR distribution by positron emission tomography (PET). The aims of this paper were to use a kinetic model to analyse the behaviour of [{sup 11}C]TMSX in the brain and to examine the applicability of the Logan plot. We also studied the applicability of a simplified Logan plot by omitting metabolite correction and arterial blood sampling. The centrum semiovale was used as a reference region on the basis of a post-mortem study showing that it has a negligibly low density of A2ARs. Compartmental analysis was performed in five normal subjects. Parametric images of A2AR binding potential (BP) were also generated using a Logan plot with or without metabolite correction and with or without arterial blood sampling. To omit arterial blood sampling, we applied a method to extract the plasma-related information using independent component analysis (EPICA). The estimated K{sub 1}/k{sub 2} was confirmed to be common in the centrum semiovale and main cortices. The three-compartment model was well fitted to the other regions using the fixed value of K{sub 1}/k{sub 2} estimated from the centrum semiovale. The estimated BPs using the Logan plot matched those derived from compartment analysis. Without the metabolite correction, the estimate of BP underestimated the true value by 5%. The estimated BPs agreed regardless of arterial blood sampling. A three-compartment model with a reference region, the centrum semiovale, describes the kinetic behaviour of [{sup 11}C]TMSX PET images. A2ARs in the human brain can be visualised as a BP image using [{sup 11}C]TMSX PET without arterial blood sampling. (orig.)

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

    Science.gov (United States)

    Rubinstein, D; Warrendorf, E

    1975-06-01

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

  1. Leptin interferes with 3',5'-Cyclic Adenosine Monophosphate (cAMP signaling to inhibit steroidogenesis in human granulosa cells

    Directory of Open Access Journals (Sweden)

    HoYuen Basil

    2009-10-01

    Full Text Available Abstract Background Obesity has been linked to an increased risk of female infertility. Leptin, an adipocytokine which is elevated during obesity, may influence gonadal function through modulating steroidogenesis in granulosa cells. Methods The effect of leptin on progesterone production in simian virus 40 immortalized granulosa (SVOG cells was examined by Enzyme linked immunosorbent assay (ELISA. The effect of leptin on the expression of the steroidogenic enzymes (StAR, P450scc, 3betaHSD in SVOG cells was examined by real-time PCR and Western blotting. The mRNA expression of leptin receptor isoforms in SVOG cells were examined by using PCR. SVOG cells were co-treated with leptin and specific pharmacological inhibitors to identify the signaling pathways involved in leptin-reduced progesterone production. Silencing RNA against leptin receptor was used to determine that the inhibition of leptin on cAMP-induced steroidogenesis acts in a leptin receptor-dependent manner. Results and Conclusion In the present study, we investigated the cellular mechanisms underlying leptin-regulated steroidogenesis in human granulosa cells. We show that leptin inhibits 8-bromo cAMP-stimulated progesterone production in a concentration-dependent manner. Furthermore, we show that leptin inhibits expression of the cAMP-stimulated steroidogenic acute regulatory (StAR protein, the rate limiting de novo protein in progesterone synthesis. Leptin induces the activation of ERK1/2, p38 and JNK but only the ERK1/2 (PD98059 and p38 (SB203580 inhibitors attenuate the leptin-induced inhibition of cAMP-stimulated StAR protein expression and progesterone production. These data suggest that the leptin-induced MAPK signal transduction pathway interferes with cAMP/PKA-stimulated steroidogenesis in human granulosa cells. Moreover, siRNA mediated knock-down of the endogenous leptin receptor attenuates the effect of leptin on cAMP-induced StAR protein expression and progesterone

  2. Characterization of a cultured human T-cell line with genetically altered ribonucleotide reductase activity. Model for immunodeficiency.

    Science.gov (United States)

    Waddell, D; Ullman, B

    1983-04-10

    From human CCRF-CEM T-cells growing in continuous culture, we have selected, isolated, and characterized a clonal cell line, APHID-D2, with altered ribonucleotide reductase activity. In comparative growth rate experiments, the APHID-D2 cell line is less sensitive than the parental cell line to growth inhibition by deoxyadenosine in the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of adenosine deaminase. The APHID-D2 cell line has elevated levels of all four dNTPs. The resistance of the APHID-D2 cell line to growth inhibition by deoxyadenosine and the abnormal dNTP levels can be explained by the fact that the APHID-D2 ribonucleotide reductase, unlike the parental ribonucleotide reductase, is not normally sensitive to inhibition by dATP. These results suggest that the allosteric site of ribonucleotide reductase which binds both dATP and ATP is altered in the APHID-D2 line. The isolation of a mutant clone of human T-cells which contains a ribonucleotide reductase that has lost its normal sensitivity to dATP and which is resistant to deoxyadenosine-mediated growth inhibition suggests that a primary pathogenic target of accumulated dATP in lymphocytes from patients with adenosine deaminase deficiency may be the cellular ribonucleotide reductase.

  3. A High Affinity Adenosine Kinase from Anopheles gambiae

    Science.gov (United States)

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

    2011-01-01

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (Km 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap4A (2.0 Å resolution) reveals interactions for adenosine, ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg2+ ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layered α/β/α sandwich, and a small cap domain in contact with adenosine. The specificity and tight-binding for adenosine arises from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168 and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64 and Asn68 and the ribosyl 2′- and 3′-hydroxyl groups. The structure is more similar to human adenosine kinase (48% identity) than to AK from Toxoplasma gondii (31% identity). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role of this enzyme to maintain the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects. PMID:21247194

  4. Adenosine: a putative mediator of bronchoconstriction in asthma

    Energy Technology Data Exchange (ETDEWEB)

    Mann, J.S.

    1987-01-01

    The protective effect of a muscarinic cholinergic antagonists, ipratropium bromide (IB) from inhaled adenosine- and methacholine-induced bronchoconstriction in asthma was studied. Inhaled IB protected from methacholine- but not adenosine-induced bronchoconstriction. Parasympathetically mediated bronchoconstriction is therefore unlikely to account for adenosine's airway effect in asthma. The capacity of theophylline, a bronchodilator and a competitive antagonist of adenosine at its cell surface receptors, to protect asthmatic subjects from adenosine- and histamine-induced bronchoconstriction was determined. Asthmatic airways are infiltrated with inflammatory cells. Human leucocytes prelabeled with (/sup 3/H)-adenine when activated with the calcium ionophore A23187 released labelled hypoxanthine, inosine and adenosine which was associated with a dose-related release of histamine. The chemotactic peptide f-MLP while inducing histamine release had an inconstant effect on release of label. In four of five experiments f-MLP produced a transient early increase in label release but in the remaining experiment no significant release was observed. Anti-human IgE failed to induce significant label release despite releasing histamine. Activated leucocytes are therefore a potential source of adenosine in asthma.

  5. Characterization of pterin deaminase from Mucor indicus MTCC 3513

    Science.gov (United States)

    Thandeeswaran, M.; Karthika, P.; Mahendran, R.; Palaniswamy, M.; Angayarkanni, J.

    2018-03-01

    Pterin deaminase is an amidohydrolase enzyme which hydrolyses pteridines to produce lumazine derivatives and ammonia. Even though the enzyme was shown as early as 1959 for its anticancer efficacy there was a long gap in the communique after that which was in 2013. In our study we have chosen Mucor indicus MTCC 3513 which was a promising strain for production of different industrial products.The pterin deaminase enzyme was harvested and extracellular from M. indicus. The extracellular sample was partially purified by using ethanol precipitation and ion exchange column (Hi-Trap QFF) in Fast Protein Liquid Chromatography. The molecular weight of the purified pterin deaminase enzyme was apparently determined by SDS-PAGE. The purified enzyme was further biochemically characterized. Molecular docking studies with the predicted sequence showed higher binding affinity towards folic acid interaction. The structure of this protein may open the windows for new drug targets for cancer therapy.

  6. Characterization of ectonucleotidases in human medulloblastoma cell lines: ecto-5'NT/CD73 in metastasis as potential prognostic factor.

    Directory of Open Access Journals (Sweden)

    Angélica Regina Cappellari

    Full Text Available Medulloblastoma (MB is the most common malignant brain tumor in children and occurs mainly in the cerebellum. Important intracellular signaling molecules, such those present in the Sonic Hedgehog and Wnt pathways, are involved in its development and can also be employed to determine tumor grade and prognosis. Ectonucleotidases, particularly ecto-5'NT/CD73, are important enzymes in the malignant process of different tumor types regulating extracellular ATP and adenosine levels. Here, we investigated the activity of ectonucleotidases in three malignant human cell lines: Daoy and ONS76, being representative of primary MB, and the D283 cell line, derived from a metastatic MB. All cell lines secreted ATP into the extracellular medium while hydrolyze poorly this nucleotide, which is in agreement with the low expression and activity of pyrophosphate/phosphodiesterase, NTPDases and alkaline phosphatase. The analysis of AMP hydrolysis showed that Daoy and ONS76 completely hydrolyzed AMP, with parallel adenosine production (Daoy and inosine accumulation (ONS76. On the other hand, D283 cell line did not hydrolyze AMP. Moreover, primary MB tumor cells, Daoy and ONS76 express the ecto-5'NT/CD73 while D283 representative of a metastatic tumor, revealed poor expression of this enzyme, while the ecto-adenosine deaminase showed higher expression in D283 compared to Daoy and ONS76 cells. Nuclear beta-catenin has been suggested as a marker for MB prognosis. Further it can promotes expression of ecto-5'NT/CD73 and suppression of adenosine deaminase. It was observed that Daoy and ONS76 showed greater nuclear beta-catenin immunoreactivity than D283, which presented mainly cytoplasmic immunoreactivity. In summary, the absence of ecto-5'NT/CD73 in the D283 cell line, a metastatic MB phenotype, suggests that high expression levels of this ectonucleotidase could be correlated with a poor prognosis in patients with MB.

  7. Adenosine Receptors and Wound Healing

    Directory of Open Access Journals (Sweden)

    Bruce N. Cronstein

    2004-01-01

    Full Text Available Recent studies have demonstrated that application of topical adenosine A2A receptor agonists promotes more rapid wound closure and clinical studies are currently underway to determine the utility of topical A2A adenosine receptor agonists in the therapy of diabetic foot ulcers. The effects of adenosine A2A receptors on the cells and tissues of healing wounds have only recently been explored. We review here the known effects of adenosine A2A receptor occupancy on the cells involved in wound healing.

  8. Adenosine receptor desensitization and trafficking.

    Science.gov (United States)

    Mundell, Stuart; Kelly, Eamonn

    2011-05-01

    As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    First, Eric A

    2015-08-15

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

  10. AMP Deaminase 3 Deficiency Enhanced 5′-AMP Induction of Hypometabolism

    Science.gov (United States)

    Daniels, Isadora Susan; O′Brien, William G.; Nath, Vinay; Zhao, Zhaoyang; Lee, Cheng Chi

    2013-01-01

    A hypometabolic state can be induced in mice by 5′-AMP administration. Previously we proposed that an underlying mechanism for this hypometabolism is linked to reduced erythrocyte oxygen transport function due to 5′-AMP uptake altering the cellular adenylate equilibrium. To test this hypothesis, we generated mice deficient in adenosine monophosphate deaminase 3 (AMPD3), the key catabolic enzyme for 5′-AMP in erythrocytes. Mice deficient in AMPD3 maintained AMPD activities in all tissues except erythrocytes. Developmentally and morphologically, the Ampd3−/− mice were indistinguishable from their wild type siblings. The levels of ATP, ADP but not 5′-AMP in erythrocytes of Ampd3−/− mice were significantly elevated. Fasting blood glucose levels of the Ampd3−/− mice were comparable to wild type siblings. In comparison to wild type mice, the Ampd3−/− mice displayed a deeper hypometabolism with a significantly delayed average arousal time in response to 5′-AMP administration. Together, these findings demonstrate a central role of AMPD3 in the regulation of 5′-AMP mediated hypometabolism and further implicate erythrocytes in this behavioral response. PMID:24066180

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

    Science.gov (United States)

    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.

  12. Detrimental effects of adenosine signaling in sickle cell disease

    Science.gov (United States)

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

    2016-01-01

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

  13. Mechanism-specific effects of adenosine on ventricular tachycardia.

    Science.gov (United States)

    Lerman, Bruce B; Ip, James E; Shah, Bindi K; Thomas, George; Liu, Christopher F; Ciaccio, Edward J; Wit, Andrew L; Cheung, Jim W; Markowitz, Steven M

    2014-12-01

    There is no universally accepted method by which to diagnose clinical ventricular tachycardia (VT) due to cAMP-mediated triggered activity. Based on cellular and clinical data, adenosine termination of VT is thought to be consistent with a diagnosis of triggered activity. However, a major gap in evidence mitigates the validity of this proposal, namely, defining the specificity of adenosine response in well-delineated reentrant VT circuits. To this end, we systematically studied the effects of adenosine in a model of canine reentrant VT and in human reentrant VT, confirmed by 3-dimensional, pace- and substrate mapping. Adenosine (12 mg [IQR 12-24]) failed to terminate VT in 31 of 31 patients with reentrant VT due to structural heart disease, and had no effect on VT cycle length (age, 67 years [IQR 53-74]); ejection fraction, 35% [IQR 20-55]). In contrast, adenosine terminated VT in 45 of 50 (90%) patients with sustained focal right or left outflow tract tachycardia. The sensitivity of adenosine for identifying VT due to triggered activity was 90% (95% CI, 0.78-0.97) and its specificity was 100% (95% CI, 0.89-1.0). Additionally, reentrant circuits were mapped in the epicardial border zone of 4-day-old infarcts in mongrel dogs. Adenosine (300-400 μg/kg) did not terminate sustained VT or have any effect on VT cycle length. These data support the concept that adenosine's effects on ventricular myocardium are mechanism specific, such that termination of VT in response to adenosine is diagnostic of cAMP-mediated triggered activity. © 2014 Wiley Periodicals, Inc.

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

    Science.gov (United States)

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

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

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

    Science.gov (United States)

    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

  16. Contraction induced secretion of VEGF from skeletal muscle cells is mediated by adenosine

    DEFF Research Database (Denmark)

    Høier, Birgitte; Olsen, Karina; Nyberg, Michael Permin

    2010-01-01

    and that the contraction induced secretion of VEGF is partially mediated via adenosine acting on A(2B) adenosine receptors. Moreover, the contraction induced secretion of VEGF protein from muscle is dependent on both PKA and MAPK activation, but only the MAPK pathway appears to be adenosine dependent.......The role of adenosine and contraction for secretion of VEGF in skeletal muscle was investigated in human subjects and rat primary skeletal muscle cells. Microdialysis probes were inserted into the thigh muscle of seven male subjects and dialysate was collected at rest, during infusion of adenosine...... and contraction caused secretion of VEGF (pcontraction induced secretion of VEGF protein was abolished by the A(2B) antagonist enprofyllin and markedly reduced by inhibition of PKA or MAPK. The results demonstrate that adenosine causes secretion of VEGF from human skeletal muscle cells...

  17. Quantitative analysis of adenosine A1 receptors in human brain using positron emission tomography and [1 -methyl-11C]8-dicyclopropylmethyl-1-methyl-3-propylxanthine

    International Nuclear Information System (INIS)

    Kimura, Yuichi; Ishii, Kenji; Fukumitsu, Nobuyoshi; Oda, Keiichi; Sasaki, Toru; Kawamura, Kazunori; Ishiwata, Kiichi

    2004-01-01

    Fully quantitative analysis of the adenosine A 1 receptor (A1R) in the brain with 11 C-MPDX and positron emission tomography is reported. The kinetics is described using a two-tissue three-compartment model, and estimated binding potentials correspond well with the estimates made by Logan plot. The image of the binding potential of the MPDX is physiologically reasonable. We conclude that MPDX is applicable to the visualization of the A1Rs in the brain with Logan plot

  18. Extracellular adenosine production by ecto-5′-nucleotidase (CD73) enhances radiation-induced lung fibrosis

    Science.gov (United States)

    Wirsdörfer, Florian; de Leve, Simone; Cappuccini, Federica; Eldh, Therese; Meyer, Alina V.; Gau, Eva; Thompson, Linda F.; Chen, Ning-Yuan; Karmouty-Quintana, Harry; Fischer, Ute; Kasper, Michael; Klein, Diana; Ritchey, Jerry W.; Blackburn, Michael R.; Westendorf, Astrid M.; Stuschke, Martin; Jendrossek, Verena

    2016-01-01

    Radiation-induced pulmonary fibrosis is a severe side effect of thoracic irradiation, but its pathogenesis remains poorly understood and no effective treatment is available. In this study, we investigated the role of the extracellular adenosine as generated by the ecto-5'-nucleotidase CD73 in fibrosis development after thoracic irradiation. Exposure of wild-type C57BL/6 mice to a single dose (15 Gray) of whole thorax irradiation triggered a progressive increase in CD73 activity in the lung between 3 and 30 weeks post-irradiation. In parallel, adenosine levels in bronchoalveolar lavage fluid (BALF) were increased by approximately three-fold. Histological evidence of lung fibrosis was observed by 25 weeks after irradiation. Conversely, CD73-deficient mice failed to accumulate adenosine in BALF and exhibited significantly less radiation-induced lung fibrosis (P<0.010). Furthermore, treatment of wild-type mice with pegylated adenosine deaminase (PEG-ADA) or CD73 antibodies also significantly reduced radiation-induced lung fibrosis. Taken together, our findings demonstrate that CD73 potentiates radiation-induced lung fibrosis, suggesting that existing pharmacological strategies for modulating adenosine may be effective in limiting lung toxicities associated with the treatment of thoracic malignancies. PMID:26921334

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Science.gov (United States)

    2011-01-01

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

  2. Enzymatic conformational fluctuations along the reaction coordinate of cytidine deaminase

    OpenAIRE

    Noonan, Ryan C.; Carter, Charles W.; Bagdassarian, Carey K.

    2002-01-01

    Analysis of the crystal structures for cytidine deaminase complexed with substrate analog 3-deazacytidine, transition-state analog zebularine 3,4-hydrate, and product uridine establishes significant changes in the magnitude of atomic-scale fluctuations along the (approximate) reaction coordinate of this enzyme. Differences in fluctuations between the substrate analog complex, transition-state analog complex, and product complex are monitored via changes in corresponding crystallographic tempe...

  3. Gender-specific association of ADA genetic polymorphism with human longevity.

    Science.gov (United States)

    Napolioni, Valerio; Lucarini, Nazzareno

    2010-08-01

    Aim of this study was to investigate whether the polymorphic ADA (Adenosine Deaminase, EC 3.5.4.4) gene, which determines the cellular level of adenosine and plays a crucial role in the regulation of the immune system and in the control of metabolic rates, is involved in longevity. 884 unrelated healthy individuals (age range 10-106 years, 400 males and 484 females) from central Italy were studied. ADA genotyping was performed by RFLP-PCR. Frequency distributions were compared using the chi-square test and a three-way contingency table analysis by a log linear model was applied to test independence between the variables. We found that ADA influences human life-span in a sex and age specific way. An increased frequency of ADA*2 carriers was found in males aged 80-85, and a decreased frequency in males over 85 (chi(2) = 13.93; df = 3; P = 0.003); significant differences among the age groups was not found in females. A strong interaction among age groups, ADA genotype and sex (G = 15.086; df = 3; P = 0.0017) was found. Males aged 80-85 could be protected from ischemic stroke by higher levels of adenosine (determined by the ADA*2 allele). The decrease of ADA*2 carriers in males over 85 may depend essentially on immunological factors; reduced levels of adenosine protect from asthma and other pulmonary diseases and lead to a reduced activation of inflammatory cells and pro-inflammatory cytokines production. Moreover, the low level of adenosine may potentiate the activity of NK and other cellular effectors against tumor cells. The negligible effect of ADA genetic polymorphism in females suggest a marginal influence of genetic factors in determining longevity in this sex, confirming previous reports.

  4. TOR induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease

    Science.gov (United States)

    Detke, Siegfried

    2007-01-01

    TOR is an atypical multidrug resistance protein present in the human protozoan parasite, Leishmania. Resistance to the toxic adenosine analog tubercidin was brought about by redirecting the adenosine permease from the plasma membrane to the multivesicular tubule lysosome. The cells became resistant to tubercidin because they were unable to take up and accumulate this toxic purine. The domain which was recognized by TOR in this internalization pathway was identified by expressing portions of this transporter in Leishmania and assessing whether they were capable of hindering the multidrug resistance capability of TOR. This approach identified the adenosine permease region spanning Met289 to Trp305. This region was also the epitope recognized by the internalization mechanism. An internal deletion mutant lacking Met289-Trp305 was functionally active but could no longer be internalized in cells with high TOR levels. The internalization and altered trafficking of the adenosine permease by TOR was observed in yeast and human embryonic kidney cells co-expressing these two Leishmania proteins indicating that the internalization process was conserved in evolutionary diverse organisms. The inability of Saccharomyces with a temperature sensitive ubiquitin ligase to internalize adenosine permease suggested that ubiquitination was involved in this altered trafficking. PMID:17428463

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

    Science.gov (United States)

    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...

  6. Genotype-specific enrichment of ACC deaminase-positive bacteria in winter wheat rhizospheres

    Science.gov (United States)

    Bacteria that produce ACC deaminase promote plant growth and development by lowering levels of the stress hormone ethylene through deamination of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene. Therefore, it is hypothesized that ACC deaminase positive (ACC+) bacteri...

  7. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Science.gov (United States)

    2010-04-01

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

  8. Deficits in Endogenous Adenosine Formation by Ecto-5′-Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

    Directory of Open Access Journals (Sweden)

    Laura Oliveira

    2015-01-01

    Full Text Available AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO from released adenine nucleotides. ADO, via A2A receptors (A2ARs, is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG. Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.

  9. Increased extracellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death

    Czech Academy of Sciences Publication Activity Database

    Zuberová, M.; Fencková, M.; Šimek, Petr; Janečková, L.; Doležal, T.

    2010-01-01

    Roč. 3, 11/12 (2010), s. 773-784 ISSN 1754-8403 R&D Projects: GA ČR(CZ) GD204/09/H058 Grant - others:AV ČR(CZ) KJB501410602; GA ČR(CZ) GA204/09/1463 Institutional research plan: CEZ:AV0Z50070508 Keywords : central nervous system * glycogen phosphorylase * immune response Subject RIV: CE - Biochemistry Impact factor: 4.584, year: 2010

  10. Role of ascitic fluid adenosine deaminase (ADA) and serum CA-125 in the diagnosis of tuberculous peritonitis.

    Science.gov (United States)

    Ali, N; Nath, N C; Parvin, R; Rahman, A; Bhuiyan, T M; Rahman, M; Mohsin M N

    2014-12-01

    This cross sectional study was carried out in the department of gastroenterology, BIRDEM, Dhaka from January 2010 to May 2011 to determine the role of ascitic fluid ADA and serum CA-125 in the diagnosis of clinically suspected tubercular peritonitis. Total 30 patients (age 39.69 ± 21.26, 18M/12F) with clinical suspicion of tuberculosis peritonitis were included in this study after analyzing selection criteria. Laparoscopic peritoneal biopsy with 'histopathological diagnosis' was considered gold standard against which accuracics of two biomarkers (ADA & CA-125) were compared. Cut off value of ADA and CA-125 are 24 u/l, 35 U/ml respectively. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of ADA as a diagnostic modality in tuberculos peritonitis were 87.5%, 83.33%, 95.45%, 62.5% and 86.67% respectively where as CA-125 was found to have 83.33% sensitivity, 50% specificity, 86.9% positive predictive value, 42.85% negative predictive value and 76.6% accuracy. Both biomarkers are simple, non-invasive, rapid and relatively cheap diagnostic test where as laparoscopy is an invasive procedure, costly & requires trained staff and not without risk and also not feasible in all the centre in our country. So ascitic fluid ADA and serum CA-125 are important diagnostic test for peritoneal tuberculosis.

  11. [(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

    Science.gov (United States)

    Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

    2002-02-11

    This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

  12. Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao, E-mail: li@sri.org [Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Alabama 35205 (United States)

    2005-06-01

    Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3{sub 1}21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

  13. Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

    International Nuclear Information System (INIS)

    Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao

    2005-01-01

    Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3 1 21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme

  14. The A2B Adenosine Receptor Modulates the Epithelial– Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells

    Science.gov (United States)

    Giacomelli, Chiara; Daniele, Simona; Romei, Chiara; Tavanti, Laura; Neri, Tommaso; Piano, Ilaria; Celi, Alessandro; Martini, Claudia; Trincavelli, Maria L.

    2018-01-01

    The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different

  15. The A2B Adenosine Receptor Modulates the Epithelial– Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells

    Directory of Open Access Journals (Sweden)

    Chiara Giacomelli

    2018-01-01

    Full Text Available The epithelial-mesenchymal transition (EMT is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1, which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin and the mesenchymal one (Vimentin, N-cadherin, respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to

  16. Adenosine for postoperative analgesia: A systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Xin Jin

    Full Text Available Perioperative infusion of adenosine has been suggested to reduce the requirement for inhalation anesthetics, without causing serious adverse effects in humans. We conducted a meta-analysis of randomized controlled trials evaluating the effect of adenosine on postoperative analgesia.We retrieved articles in computerized searches of Scopus, Web of Science, PubMed, EMBASE, and Cochrane Library databases, up to July 2016. We used adenosine, postoperative analgesia, and postoperative pain(s as key words, with humans, RCT, and CCT as filters. Data of eligible studies were extracted, which included pain scores, cumulative opioid consumption, adverse reactions, and vital signs. Overall incidence rates, relative risk (RR, and 95% confidence intervals (CI were calculated employing fixed-effects or random-effects models, depending on the heterogeneity of the included trials.In total, 757 patients from 9 studies were included. The overall effect of adenosine on postoperative VAS/VRS scores and postoperative opioid consumption was not significantly different from that of controls (P >0.1. The occurrence of PONV and pruritus was not statistically significantly different between an adenosine and nonremifentanil subgroup (P >0.1, but the rate of PONV occurrence was greater in the remifentanil subgroup (P 0.1.Adenosine has no analgesic effect or prophylactic effect against PONV, but reduce systolic blood pressure and heart rates. Adenosine may benefit patients with hypertension, ischemic heart disease, and tachyarrhythmia, thereby improving cardiac function.

  17. Hypermutation by intersegmental transfer of APOBEC3G cytidine deaminase.

    Science.gov (United States)

    Nowarski, Roni; Britan-Rosich, Elena; Shiloach, Tamar; Kotler, Moshe

    2008-10-01

    Deamination of cytidine residues in single-stranded DNA (ssDNA) is an important mechanism by which apolipoprotein B mRNA-editing, catalytic polypeptide-like (APOBEC) enzymes restrict endogenous and exogenous viruses. The dynamic process underlying APOBEC-induced hypermutation is not fully understood. Here we show that enzymatically active APOBEC3G can be detected in wild-type Vif(+) HIV-1 virions, albeit at low levels. In vitro studies showed that single enzyme-DNA encounters result in distributive deamination of adjacent cytidines. Nonlinear translocation of APOBEC3G, however, directed scattered deamination of numerous targets along the DNA. Increased ssDNA concentrations abolished enzyme processivity in the case of short, but not long, DNA substrates, emphasizing the key role of rapid intersegmental transfer in targeting the deaminase. Our data support a model by which APOBEC3G intersegmental transfer via monomeric binding to two ssDNA segments results in dispersed hypermutation of viral genomes.

  18. Regulatory factors governing adenosine-to-inosine (A-to-I) RNA editing.

    Science.gov (United States)

    Hong, HuiQi; Lin, Jaymie Siqi; Chen, Leilei

    2015-03-31

    Adenosine-to-inosine (A-to-I) RNA editing, the most prevalent mode of transcript modification in higher eukaryotes, is catalysed by the adenosine deaminases acting on RNA (ADARs). A-to-I editing imposes an additional layer of gene regulation as it dictates various aspects of RNA metabolism, including RNA folding, processing, localization and degradation. Furthermore, editing events in exonic regions contribute to proteome diversity as translational machinery decodes inosine as guanosine. Although it has been demonstrated that dysregulated A-to-I editing contributes to various diseases, the precise regulatory mechanisms governing this critical cellular process have yet to be fully elucidated. However, integration of previous studies revealed that regulation of A-to-I editing is multifaceted, weaving an intricate network of auto- and transregulations, including the involvement of virus-originated factors like adenovirus-associated RNA. Taken together, it is apparent that tipping of any regulatory components will have profound effects on A-to-I editing, which in turn contributes to both normal and aberrant physiological conditions. A complete understanding of this intricate regulatory network may ultimately be translated into new therapeutic strategies against diseases driven by perturbed RNA editing events. Herein, we review the current state of knowledge on the regulatory mechanisms governing A-to-I editing and propose the role of other co-factors that may be involved in this complex regulatory process.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

    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

  20. Alterations in the brain adenosine metabolism cause behavioral and neurological impairment in ADA-deficient mice and patients

    Science.gov (United States)

    Sauer, Aisha V.; Hernandez, Raisa Jofra; Fumagalli, Francesca; Bianchi, Veronica; Poliani, Pietro L.; Dallatomasina, Chiara; Riboni, Elisa; Politi, Letterio S.; Tabucchi, Antonella; Carlucci, Filippo; Casiraghi, Miriam; Carriglio, Nicola; Cominelli, Manuela; Forcellini, Carlo Alberto; Barzaghi, Federica; Ferrua, Francesca; Minicucci, Fabio; Medaglini, Stefania; Leocani, Letizia; la Marca, Giancarlo; Notarangelo, Lucia D.; Azzari, Chiara; Comi, Giancarlo; Baldoli, Cristina; Canale, Sabrina; Sessa, Maria; D’Adamo, Patrizia; Aiuti, Alessandro

    2017-01-01

    Adenosine Deaminase (ADA) deficiency is an autosomal recessive variant of severe combined immunodeficiency (SCID) caused by systemic accumulation of ADA substrates. Neurological and behavioral abnormalities observed in ADA-SCID patients surviving after stem cell transplantation or gene therapy represent an unresolved enigma in the field. We found significant neurological and cognitive alterations in untreated ADA-SCID patients as well as in two groups of patients after short- and long-term enzyme replacement therapy with PEG-ADA. These included motor dysfunction, EEG alterations, sensorineural hypoacusia, white matter and ventricular alterations in MRI as well as a low mental development index or IQ. Ada-deficient mice were significantly less active and showed anxiety-like behavior. Molecular and metabolic analyses showed that this phenotype coincides with metabolic alterations and aberrant adenosine receptor signaling. PEG-ADA treatment corrected metabolic adenosine-based alterations, but not cellular and signaling defects, indicating an intrinsic nature of the neurological and behavioral phenotype in ADA deficiency. PMID:28074903

  1. Adenosine inhibits neutrophil vascular endothelial growth factor release and transendothelial migration via A2B receptor activation.

    LENUS (Irish Health Repository)

    Wakai, A

    2012-02-03

    The effects of adenosine on neutrophil (polymorphonuclear neutrophils; PMN)-directed changes in vascular permeability are poorly characterized. This study investigated whether adenosine modulates activated PMN vascular endothelial growth factor (vascular permeability factor; VEGF) release and transendothelial migration. PMN activated with tumour necrosis factor-alpha (TNF-alpha, 10 ng\\/mL) were incubated with adenosine and its receptor-specific analogues. Culture supernatants were assayed for VEGF. PMN transendothelial migration across human umbilical vein endothelial cell (HUVEC) monolayers was assessed in vitro. Adhesion molecule receptor expression was assessed flow cytometrically. Adenosine and some of its receptor-specific analogues dose-dependently inhibited activated PMN VEGF release. The rank order of potency was consistent with the affinity profile of human A2B receptors. The inhibitory effect of adenosine was reversed by 3,7-dimethyl-1-propargylxanthine, an A2 receptor antagonist. Adenosine (100 microM) or the A2B receptor agonist 5\\'-N-ethylcarboxamidoadenosine (NECA, 100 microM) significantly reduced PMN transendothelial migration. However, expression of activated PMN beta2 integrins and HUVEC ICAM-1 were not significantly altered by adenosine or NECA. Adenosine attenuates human PMN VEGF release and transendothelial migration via the A2B receptor. This provides a novel target for the modulation of PMN-directed vascular hyperpermeability in conditions such as the capillary leak syndrome.

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

    Science.gov (United States)

    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.

  3. Nitric oxide synthesis-promoting effects of valsartan in human umbilical vein endothelial cells via the Akt/adenosine monophosphate-activated protein kinase/endothelial nitric oxide synthase pathway

    Directory of Open Access Journals (Sweden)

    Yingshuai Zhao

    2017-05-01

    Full Text Available Valsartan (VAL, an antagonist of angiotensin II receptor type 1, has antihypertensive and multiple cardiovascular protective effects. The pleiotropic functions of VAL are related to the increased synthesis and biological activity of intravascular nitric oxide (NO. In this study, the role and mechanisms of VAL in the synthesis of NO were examined in human umbilical vein endothelial cells (HUVECs. Ten µmol/L of VAL was used to treat EA.hy926 cells for 30 minutes, 1, 3, 6, 12, and 24 hours, and three concentrations of VAL (i.e., 10, 1, and 0.1 µmol/L were used to treat EA.hy926 cells for 24 hours. The cells were divided into five groups: control, VAL, VAL + Compound C (adenosine monophosphate-activated protein kinase [AMPK] inhibitor, 1 µmol/L, VAL + LY294002 (Akt [protein kinase B] inhibitor, 10 µmol/L, and VAL + L-nitro-arginine methyl ester (L-NAME, endothelial NO synthase [eNOS] inhibitor, 500 µmol/L groups. The NO content in the VAL-treated HUVEC line (EA.hy926 was detected using the nitrate reductase method, and western blot was used to detect the phosphorylation of Akt, AMPK, and eNOS, as well as the changes in total protein levels. VAL increased NO synthesis in EA.hy926 cells in time- and dose-dependent manners (p < 0.05 and the intracellular phosphorylation levels of Akt, AMPK, and eNOS at the corresponding time points. LY294002, Compound C, and L-NAME could inhibit the VAL-promoted NO synthesis. VAL activated Akt, AMPK, and eNOS, thus promoting NO synthesis and playing a protective role in endothelial cells. These results partially explained the mechanisms underlying the cardiovascular protective effects of VAL.

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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. Growth inhibition of human breast cancer cells and down-regulation of ODC1 and ADA genes by Nepeta binaloudensis

    Directory of Open Access Journals (Sweden)

    Akbar Safipour Afshar

    Full Text Available ABSTRACT Nepeta binaloudensis Jamzad, Lamiaceae, is a rare medicinal plant endemic to Iran. In spite of many studies about the chemical constituents and antibacterial effects of this species, no report has been provided about its cytotoxic and anticancer activities. In this study we have evaluated the effects of EtOH 70%, hexane and aqueous extracts of N. binaloudensis on the cell proliferation and n-hexane extract on the expression of adenosine deaminase and ornithine decarboxylase 1 genes in breast cancer cell lines (MCF-7, MDA-MB-231 compared to non-cancer line (MCF-10A. The cell lines were subjected to increasing doses of the extracts ranging from 10 to 320 µg/ml. Cell viability was quantified by MTS assay. Expression of adenosine deaminase and ornithine decarboxylase 1 genes was analyzed by real time PCR. N. binaloudensis inhibited the growth of malignant cells in a time and dose-dependent manner. Among extracts of N. binaloudensis, the hexane extract was found to be more toxic compared to other extracts. Results showed a marked decrease in the expression of ornithine decarboxylase 1 and adenosine deaminase genes in cancer cell lines. At 60 µg/ml concentration of N. binaloudensis hexane extract ornithine decarboxylase 1 and adenosine deaminase mRNA expression were reduced 4.9 fold and 3.5 fold in MCF-7 cell line and 3.6 fold and 2.6 fold in MDA-MB-231 cell line compared to control, respectively. The result of our study highlights the potential influences of N. binaloudensis hexane extract on ornithine decarboxylase 1 and adenosine deaminase genes expression in breast cancer cells and its relation to inhibition of cancer cell growth.

  7. Mechanish of dTTP Inhibition of the Bifunctional dCTP Deaminase:dUTPase Encoded by Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Helt, Signe Smedegaard; Thymark, Majbritt; Harris, Pernille

    2008-01-01

    Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be cha......Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme...

  8. Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID

    Science.gov (United States)

    Sauer, Aisha V.; Brigida, Immacolata; Carriglio, Nicola; Jofra Hernandez, Raisa; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L.; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna

    2012-01-01

    Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)–mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA–treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA−/− Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA–treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA–treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID. Trials were registered at www.clinicaltrials.gov as NCT00598481/NCT00599781. PMID:22184407

  9. Alterations in the adenosine metabolism and CD39/CD73 adenosinergic machinery cause loss of Treg cell function and autoimmunity in ADA-deficient SCID.

    Science.gov (United States)

    Sauer, Aisha V; Brigida, Immacolata; Carriglio, Nicola; Hernandez, Raisa Jofra; Scaramuzza, Samantha; Clavenna, Daniela; Sanvito, Francesca; Poliani, Pietro L; Gagliani, Nicola; Carlucci, Filippo; Tabucchi, Antonella; Roncarolo, Maria Grazia; Traggiai, Elisabetta; Villa, Anna; Aiuti, Alessandro

    2012-02-09

    Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

  10. Adenosine derived from Staphylococcus aureus-engulfed macrophages functions as a potent stimulant for the induction of inflammatory cytokines in mast cells

    DEFF Research Database (Denmark)

    Ma, Ying Jie; Kim, Chan-Hee; Ryu, Kyoung-Hwa

    2011-01-01

    In this study, we attempted to isolate novel mast cell-stimulating molecules from Staphylococcus aureus. Water-soluble extract of S. aureus cell lysate strongly induced human interleukin- 8 in human mast cell line-1 and mouse interleukin-6 in mouse bone marrow-derived mast cells. The active...... adenosine receptor blocker, verified that purified adenosine can induce interleukin-8 production via adenosine receptors on mast cells. Moreover, adenosine was purified from S. aureusengulfed RAW264.7 cells, a murine macrophage cell line, used to induce phagocytosis of S. aureus. These results show a novel...

  11. Extracellular adenosine-induced Rac1 activation in pulmonary endothelium: Molecular mechanisms and barrier-protective role.

    Science.gov (United States)

    Kovacs-Kasa, Anita; Kim, Kyung Mi; Cherian-Shaw, Mary; Black, Stephen M; Fulton, David J; Verin, Alexander D

    2018-08-01

    We have previously shown that Gs-coupled adenosine receptors (A2a) are primarily involved in adenosine-induced human pulmonary artery endothelial cell (HPAEC) barrier enhancement. However, the downstream events that mediate the strengthening of the endothelial cell (EC) barrier via adenosine signaling are largely unknown. In the current study, we tested the overall hypothesis that adenosine-induced Rac1 activation and EC barrier enhancement is mediated by Gs-dependent stimulation of cAMP-dependent Epac1-mediated signaling cascades. Adenoviral transduction of HPAEC with constitutively-active (C/A) Rac1 (V12Rac1) significantly increases transendothelial electrical resistance (TER) reflecting an enhancement of the EC barrier. Conversely, expression of an inactive Rac1 mutant (N17Rac1) decreases TER reflecting a compromised EC barrier. The adenosine-induced increase in TER was accompanied by activation of Rac1, decrease in contractility (MLC dephosphorylation), but not Rho inhibition. Conversely, inhibition of Rac1 activity attenuates adenosine-induced increase in TER. We next examined the role of cAMP-activated Epac1 and its putative downstream targets Rac1, Vav2, Rap1, and Tiam1. Depletion of Epac1 attenuated the adenosine-induced Rac1 activation and the increase in TER. Furthermore, silencing of Rac1 specific guanine nucleotide exchange factors (GEFs), Vav2 and Rap1a expression significantly attenuated adenosine-induced increases in TER and activation of Rac1. Depletion of Rap1b only modestly impacted adenosine-induced increases in TER and Tiam1 depletion had no effect on adenosine-induced Rac1 activation and TER. Together these data strongly suggest that Rac1 activity is required for adenosine-induced EC barrier enhancement and that the activation of Rac1 and ability to strengthen the EC barrier depends, at least in part, on cAMP-dependent Epac1/Vav2/Rap1-mediated signaling. © 2017 Wiley Periodicals, Inc.

  12. Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy

    Directory of Open Access Journals (Sweden)

    Nico Lachmann

    2013-03-01

    Full Text Available Gene transfer of drug resistance (CTX-R genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O6-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C, gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. Overproduction, Purification and Characterization of Adenylate Deaminase from Aspergillus oryzae.

    Science.gov (United States)

    Li, Shubo; Qian, Yi; Liang, Yunlong; Chen, Xinkuan; Zhao, Mouming; Guo, Yuan; Pang, Zongwen

    2016-12-01

    Adenylate deaminase (AMPD, EC 3.5.4.6) is an aminohydrolase that widely used in the food and medicine industries. In this study, the gene encoding Aspergillus oryzae AMPD was cloned and expressed in Escherichia coli. Induction with 0.75 mM isopropyl β-D-l-thiogalactopyranoside resulted in an enzyme activity of 1773.9 U/mL. Recombinant AMPD was purified to electrophoretic homogeneity using nickel affinity chromatography, and its molecular weight was calculated as 78.6 kDa. Purified AMPD exhibited maximal activity at 35 °C, pH 6.0 and 30 mM K + , with apparent K m and V max values of 2.7 × 10 -4  M and 77.5 μmol/mg/min under these conditions. HPLC revealed that recombinant AMPD could effectively catalyse the synthesis of inosine-5'-monophosphate (IMP) with minimal by-products, indicating high specificity and suggesting that it could prove useful for IMP production.

  15. Time Window Is Important for Adenosine Preventing Cold-induced Injury to the Endothelium.

    Science.gov (United States)

    Li, Yan; Hu, Xiao-Xia; Fu, Li; Chen, Jing; Lu, Li-He; Liu, Xiang; Xu, Zhe; Zhou, Li; Wang, Zhi-Ping; Zhang, Xi; Ou, Zhi-Jun; Ou, Jing-Song

    2017-06-01

    Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.

  16. Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease.

    Directory of Open Access Journals (Sweden)

    Koji Hase

    Full Text Available Activation-induced cytidine deaminase (AID expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM and class switch recombination (CSR. Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2 associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID-/- mice spontaneously develop tertiary lymphoid organs (TLOs in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

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

    Science.gov (United States)

    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. Functional ADA polymorphism increases sleep depth and reduces vigilant attention in humans.

    Science.gov (United States)

    Bachmann, Valérie; Klaus, Federica; Bodenmann, Sereina; Schäfer, Nikolaus; Brugger, Peter; Huber, Susanne; Berger, Wolfgang; Landolt, Hans-Peter

    2012-04-01

    Homeostatically regulated slow-wave oscillations in non-rapid eye movement (REM) sleep may reflect synaptic changes across the sleep-wake continuum and the restorative function of sleep. The nonsynonymous c.22G>A polymorphism (rs73598374) of adenosine deaminase (ADA) reduces the conversion of adenosine to inosine and predicts baseline differences in sleep slow-wave oscillations. We hypothesized that this polymorphism affects cognitive functions, and investigated whether it modulates electroencephalogram (EEG), behavioral, subjective, and biochemical responses to sleep deprivation. Attention, learning, memory, and executive functioning were quantified in healthy adults. Right-handed carriers of the variant allele (G/A genotype, n = 29) performed worse on the d2 attention task than G/G homozygotes (n = 191). To test whether this difference reflects elevated homeostatic sleep pressure, sleep and sleep EEG before and after sleep deprivation were studied in 2 prospectively matched groups of G/A and G/G genotype subjects. Deep sleep and EEG 0.75- to 1.5-Hz oscillations in non-REM sleep were significantly higher in G/A than in G/G genotype. Moreover, attention and vigor were reduced, whereas waking EEG alpha activity (8.5-12 Hz), sleepiness, fatigue, and α-amylase in saliva were enhanced. These convergent data demonstrate that genetic reduction of ADA activity elevates sleep pressure and plays a key role in sleep and waking quality in humans.

  19. [Isolation, identification and characterization of ACC deaminase-containing endophytic bacteria from halophyte Suaeda salsa].

    Science.gov (United States)

    Teng, Songshan; Liu, Yanping; Zhao, Lei

    2010-11-01

    We Isolated and characterized 1-aminocyclopropane-1-carboxylate (ACC) deaminase-containing endophytic bacteria from halophyte Suaeda salsa to understand the interactions between endophytes and halophyte. ACC deaminase-containing endophytic bacteria were isolated from root, stalk and leaf of Suaeda salsa and were identified based on morphological, physiological-biochemical properties, API and 16S rRNA sequence analysis. Isolates were evaluated for their ACC deaminase, antifungal, protease activity, siderophores and phytohormones, such as indole-3-acetic acid, gibberellic acid and abscisic acid production, as well as atmospheric nitrogen fixation and phosphate solubilization. Four ACC deaminase-containing endophytic bacteria strains named as LP11, SS12, TW1 and TW2 were isolated and identified as Pseudomonas oryzihabitans, Pseudomonas sp., Pantoea agglomerans and Pseudomonas putida respectively. All the strains possessed the phosphate-solubilizing ability and could produce siderophores and phytohormones more or less. None of them could fix atmospheric nitrogen or produce protease. Only strain SS12 showed antagonism against two phytopathogenic fungi viz Fusarium oxysporum f. sp. conglutinans and F. oxysporum f. sp. cucumerinum. ACC deaminase-containing endophytic bacteria of Pseudomonas sp. and Pantoea sp. isolated from halophyte Suaeda salsa have abundant biological characteristics related to plant growth promotion, stress homeostasis regulation and biocontrol activity.

  20. In vivo effects of adenosine 5´-triphosphate on rat preneoplastic liver

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    Ana V. Frontini

    2011-04-01

    Full Text Available The utilization of adenosine 5´-triphosphate (ATP infusions to inhibit the growth of some human and animals tumors was based on the anticancer activity observed in in vitro and in vivo experiments, but contradictory results make the use of ATP in clinical practice rather controversial. Moreover, there is no literature regarding the use of ATP infusions to treat hepatocarcinomas. The purpose of this study was to investigate whether ATP prevents in vivo oncogenesis in very-early-stage cancer cells in a well characterized two-stage model of hepatocarcinogenesis in the rat. As we could not preclude the possible effect due to the intrinsic properties of adenosine, a known tumorigenic product of ATP hydrolysis, the effect of the administration of adenosine was also studied. Animals were divided in groups: rats submitted to the two stage preneoplasia initiation/promotion model of hepatocarcinogenesis, rats treated with intraperitoneal ATP or adenosine during the two phases of the model and appropriate control groups. The number and volume of preneoplastic foci per liver identified by the expression of glutathione S-transferase placental type and the number of proliferating nuclear antigen positive cells significantly increased in ATP and adenosine treated groups. Taken together, these results indicate that in this preneoplastic liver model, ATP as well as adenosine disturb the balance between apoptosis and proliferation contributing to malignant transformation.

  1. Mutation Processes in 293-Based Clones Overexpressing the DNA Cytosine Deaminase APOBEC3B.

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    Monica K Akre

    Full Text Available Molecular, cellular, and clinical studies have combined to demonstrate a contribution from the DNA cytosine deaminase APOBEC3B (A3B to the overall mutation load in breast, head/neck, lung, bladder, cervical, ovarian, and other cancer types. However, the complete landscape of mutations attributable to this enzyme has yet to be determined in a controlled human cell system. We report a conditional and isogenic system for A3B induction, genomic DNA deamination, and mutagenesis. Human 293-derived cells were engineered to express doxycycline-inducible A3B-eGFP or eGFP constructs. Cells were subjected to 10 rounds of A3B-eGFP exposure that each caused 80-90% cell death. Control pools were subjected to parallel rounds of non-toxic eGFP exposure, and dilutions were done each round to mimic A3B-eGFP induced population fluctuations. Targeted sequencing of portions of TP53 and MYC demonstrated greater mutation accumulation in the A3B-eGFP exposed pools. Clones were generated and microarray analyses were used to identify those with the greatest number of SNP alterations for whole genome sequencing. A3B-eGFP exposed clones showed global increases in C-to-T transition mutations, enrichments for cytosine mutations within A3B-preferred trinucleotide motifs, and more copy number aberrations. Surprisingly, both control and A3B-eGFP clones also elicited strong mutator phenotypes characteristic of defective mismatch repair. Despite this additional mutational process, the 293-based system characterized here still yielded a genome-wide view of A3B-catalyzed mutagenesis in human cells and a system for additional studies on the compounded effects of simultaneous mutation mechanisms in cancer cells.

  2. Effects of adenosine on the organ injury and dysfunction caused by hemorrhagic shock

    International Nuclear Information System (INIS)

    Soliman, M.M.

    2009-01-01

    Objectives: Adenosine has been shown in animal and human studies to decrease the post-ischemic myocardial injury by lowering the levels of tumor necrosis factor-a. The objectives of the study was to examine the protective effects of adenosine on the organ injury (liver, kidney, pancreas) associated with hemorrhagic shock in rats. Methodology: The study was conducted at Cardiovascular Physiology laboratory, King Saud University, Riyadh in 2007-2008. Anesthetized male Sprague- Dawley rats were assigned to hemorrhage and resuscitation treated with 20mM adenosine , untreated, or similar time matched control groups (n=6 per group). Rats were hemorrhaged for one hour using a reservoir model. Arterial blood pressure was monitored for one hour, and maintained at a mean arterial blood pressure of 40 mmHg. Adenosine 20mM was injected intra-arterially, before resuscitation in the adenosine treated group. Resuscitation was performed by re infusion of the sheded blood for 30 minutes. Arterial blood samples were analyzed for biochemical indicators of multiple organ injury: 1) liver function: aspartate aminotransferase (AST), alanine aminotransferase (ALT), 2) renal function: urea and creatinine, 3) pancreatic function: amylase. Results: In the control group there was no significant rise in the serum levels of (i) urea and creatinine, (ii) aspartate aminotransferase (AST) and alanine aminotransferase (ALT), (iii) amylase. While in the adenosine treated group, resuscitation from one hour of hemorrhagic shock resulted in significant rises in the serum levels of (i) urea and creatinine, (ii) aspartate aminotransferase (AST) and alanine aminotransferase (ALT), (iii) amylase. Treatment of rats with 20mM adenosine before resuscitation following one hour of hemorrhagic shock decreased the multiple organ injury and dysfunction caused by hemorrhagic shock. Conclusion: Adenosine attenuated the renal, liver and pancreatic injury caused by hemorrhagic shock and resuscitation in rats. Thus

  3. Biological function of activation-induced cytidine deaminase (AID

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    Ritu Kumar

    2014-10-01

    Full Text Available 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.

  4. Pulsed electromagnetic fields increased the anti-inflammatory effect of A₂A and A₃ adenosine receptors in human T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts.

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    Fabrizio Vincenzi

    Full Text Available Adenosine receptors (ARs have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2, an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint

  5. Role of adenosine receptors in caffeine tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Holtzman, S.G.; Mante, S.; Minneman, K.P. (Emory Univ. School of Medicine, Atlanta, GA (USA))

    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.

  6. Role of adenosine receptors in caffeine tolerance

    International Nuclear Information System (INIS)

    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

  7. Activation of adenosine low-affinity A3 receptors inhibits the enteric short interplexus neural circuit triggered by histamine.

    Science.gov (United States)

    Bozarov, Andrey; Wang, Yu-Zhong; Yu, Jun Ge; Wunderlich, Jacqueline; Hassanain, Hamdy H; Alhaj, Mazin; Cooke, Helen J; Grants, Iveta; Ren, Tianhua; Christofi, Fievos L

    2009-12-01

    We tested the novel hypothesis that endogenous adenosine (eADO) activates low-affinity A3 receptors in a model of neurogenic diarrhea in the guinea pig colon. Dimaprit activation of H2 receptors was used to trigger a cyclic coordinated response of contraction and Cl(-) secretion. Contraction-relaxation was monitored by sonomicrometry (via intracrystal distance) simultaneously with short-circuit current (I(sc), Cl(-) secretion). The short interplexus reflex coordinated response was attenuated or abolished by antagonists at H2 (cimetidine), 5-hydroxytryptamine 4 receptor (RS39604), neurokinin-1 receptor (GR82334), or nicotinic (mecamylamine) receptors. The A1 agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) abolished coordinated responses, and A1 antagonists could restore normal responses. A1-selective antagonists alone [8-cyclopentyltheophylline (CPT), 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX), or 8-cyclopentyl-N(3)-[3-(4-(fluorosulfonyl)benzoyloxy)propyl]-xanthine (FSCPX)] caused a concentration-dependent augmentation of crypt cell secretion or contraction and acted at nanomolar concentrations. The A3 agonist N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) abolished coordinated responses and the A3 antagonist 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191) could restore and further augment responses. The IB-MECA effect was resistant to knockdown of adenosine A1 receptor with the irreversible antagonist FSCPX; the IC(50) for IB-MECA was 0.8 microM. MRS1191 alone could augment or unmask coordinated responses to dimaprit, and IB-MECA suppressed them. MRS1191 augmented distension-evoked reflex I(sc) responses. Adenosine deaminase mimicked actions of adenosine receptor antagonists. A3 receptor immunoreactivity was differentially expressed in enteric neurons of different parts of colon. After tetrodotoxin, IB-MECA caused circular muscle relaxation. The data support the novel concept that

  8. Targeted inhibition of osteosarcoma tumor growth by bone marrow-derived mesenchymal stem cells expressing cytosine deaminase/5-fluorocytosine in tumor-bearing mice.

    Science.gov (United States)

    NguyenThai, Quynh-Anh; Sharma, Neelesh; Luong, Do Huynh; Sodhi, Simrinder Singh; Kim, Jeong-Hyun; Kim, Nameun; Oh, Sung-Jong; Jeong, Dong Kee

    2015-01-01

    Mesenchymal stem cells (MSCs) are considered as an attractive approach for gene or drug delivery in cancer therapy. In the present study, the ability of human bone marrow-derived MSCs expressing the cytosine deaminase/5-fluorocytosine prodrug (CD/5-FC MSCs) to target the human osteosarcoma cell line Cal72 was evaluated. The stable CD/5-FC MSC cell line was established by transfection of pEGFP containing the cytosine deaminase gene into MSCs with G418 selection. The anti-tumor effect was verified by a bystander effect assay in vitro and co-injection of Cal72 and CD/5-FC MSCs in cancer-bearing mice. The therapeutic CD/5-FC MSCs retained the characteristics of multipotent cells, such as differentiation into adipocytes/osteocytes and expression of mesenchymal markers (CD90 and CD44), and showed migration toward Cal72 cells to a greater extent than the native MSCs. The bystander effect assay showed that the CD/5-FC MSCs significantly augmented Cal72 cytotoxicity in direct co-culture and in the presence of 5-FC through the application of conditioned medium. In osteosarcoma-bearing mice, the CD/5-FC MSCs inhibited tumor growth compared to control mice subcutaneously injected with only Cal72 cells. Taken together, these findings suggest that CD/5-FC MSCs may be suitable for targeting human osteosarcoma. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Aberrant Bone Density in Aging Mice Lacking the Adenosine Transporter ENT1

    Science.gov (United States)

    Hinton, David J.; McGee-Lawrence, Meghan E.; Lee, Moonnoh R.; Kwong, Hoi K.; Westendorf, Jennifer J.; Choi, Doo-Sup

    2014-01-01

    Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1) is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months) compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP), an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density. PMID:24586402

  10. Aberrant bone density in aging mice lacking the adenosine transporter ENT1.

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    David J Hinton

    Full Text Available Adenosine is known to regulate bone production and resorption in humans and mice. Type 1 equilibrative nucleoside transporter (ENT1 is responsible for the majority of adenosine transport across the plasma membrane and is ubiquitously expressed in both humans and mice. However, the contribution of ENT1-mediated adenosine levels has not been studied in bone remodeling. With the recent identification of the importance of adenosine signaling in bone homeostasis, it is essential to understand the role of ENT1 to develop novel therapeutic compounds for bone disorders. Here we examined the effect of ENT1 deletion on bone density using X-ray, dual energy X-ray absorptiometry and micro-computerized tomography analysis. Our results show that bone density and bone mineral density is reduced in the lower thoracic and lumbar spine as well as the femur of old ENT1 null mice (>7 months compared to wild-type littermates. Furthermore, we found increased mRNA expression of tartrate-resistant acid phosphatase (TRAP, an osteoclast marker, in isolated long bones from 10 month old ENT1 null mice compared to wild-type mice. In addition, aged ENT1 null mice displayed severe deficit in motor coordination and locomotor activity, which might be attributed to dysregulated bone density. Overall, our study suggests that ENT1-regulated adenosine signaling plays an essential role in lumbar spine and femur bone density.

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

    International Nuclear Information System (INIS)

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

    1986-01-01

    The authors have previously demonstrated that 3 H-purine release was enhanced during synaptic activation of the prelabelled frog sympathetic ganglion. In addition, during orthodromic stimulation, there is an increased 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 μl 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

  12. Quantitative analysis of adenosine A{sub 1} receptors in human brain using positron emission tomography and [1 -methyl-{sup 11}C]8-dicyclopropylmethyl-1-methyl-3-propylxanthine

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, Yuichi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan)]. E-mail: ukimura@ieee.org; Ishii, Kenji [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan); Fukumitsu, Nobuyoshi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan); Department of Radiation Oncology, University of Tsukuba Hospital, Amakubo, 2-1-1, Tsukuba, 305-8576 (Japan); Oda, Keiichi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan); Sasaki, Toru [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan); Kawamura, Kazunori [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan); SHI Accelerator Service Ltd., 1-17-6, Ohsaki, Shinagawa, Tokyo, 141-0032 (Japan); Ishiwata, Kiichi [Positron Medical Center, Tokyo Metropolitan Institute of Gerontology, 1-1, Naka, Itabashi, Tokyo, 173-0022 (Japan)

    2004-11-01

    Fully quantitative analysis of the adenosine A{sub 1} receptor (A1R) in the brain with {sup 11}C-MPDX and positron emission tomography is reported. The kinetics is described using a two-tissue three-compartment model, and estimated binding potentials correspond well with the estimates made by Logan plot. The image of the binding potential of the MPDX is physiologically reasonable. We conclude that MPDX is applicable to the visualization of the A1Rs in the brain with Logan plot.

  13. Activation of adenosine receptors and inhibition of cyclooxygenases: two recent pharmacological approaches to modulation of radiation suppressed hematopoiesis

    International Nuclear Information System (INIS)

    Hofer, M.; Pospisil, M.; Vacek, A.; Hola, J.; Weiterova, L.; Streitova, D.; Znojil, V.

    2008-01-01

    Searching for drugs conforming to requirements for protection and/or treatment of radiation-induced damage belongs to the most important tasks of current radiobiology. In the Laboratory of Experimental Hematology, Institute of Biophysics, v.v.i., Academy of Sciences of the Czech Republic, Brno, Czech Republic, two original approaches for stimulation of radiation-suppressed hematopoiesis have been tested in recent years, namely activation of adenosine receptors and inhibition of cyclooxygenases. Non-selective activation of adenosine receptors, induced by combined administration of dipyridamole, a drug preventing adenosine uptake and supporting thus its extracellular receptor-mediated action, and adenosine monophosphate, an adenosine prodrug, has been found to stimulate hematopoiesis when the drugs were given either pre- or post-irradiation. When synthetic adenosine receptor agonists selective for individual adenosine receptor subtypes were tested, stimulatory effects in myelosuppressed mice have been found after administration of IB-MECA, a selective adenosine A3 receptor agonist. Non-selective cyclooxygenase inhibitors, inhibiting both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), indomethacin, diclofenac, or flurbiprofen, have been observed to act positively on radiation-perturbed hematopoiesis in sublethally irradiated mice. However, their undesirable gastrointestinal side effects have been found to negatively influence survival of lethally irradiated animals. Recently tested selective COX-2 inhibitor meloxicam, preserving protective action of COX-1-synthesized prostaglandins in the gastrointestinal tissues, has been observed to retain the hematopoiesis-stimulating effects of non-selective cyclooxygenase inhibitors and to improve the survival of animals exposed to lethal radiation doses. These findings bear evidence for the possibility to use selective adenosine A3 receptor agonists and selective COX-2 inhibitors in human practice for treatment of

  14. Adenosine induced ventricular arrhythmias in the emergency room

    NARCIS (Netherlands)

    Tan, H. L.; Spekhorst, H. H.; Peters, R. J.; Wilde, A. A.

    2001-01-01

    While adenosine effectively terminates most supraventricular tachycardias (SVT), rare case reports have demonstrated its proarrhythmic potential, including induction of ventricular tachycardia (VT). The aim of this study was to define the proarrhythmic effects of adenosine in a large, unselected

  15. Caffeine and Selective Adenosine Receptor Antagonists as New Therapeutic Tools for the Motivational Symptoms of Depression

    Directory of Open Access Journals (Sweden)

    Laura López-Cruz

    2018-06-01

    Full Text Available Major depressive disorder is one of the most common and debilitating psychiatric disorders. Some of the motivational symptoms of depression, such anergia (lack of self-reported energy and fatigue are relatively resistant to traditional treatments such as serotonin uptake inhibitors. Thus, new pharmacological targets are being investigated. Epidemiological data suggest that caffeine consumption can have an impact on aspects of depressive symptomatology. Caffeine is a non-selective adenosine antagonist for A1/A2A receptors, and has been demonstrated to modulate behavior in classical animal models of depression. Moreover, selective adenosine receptor antagonists are being assessed for their antidepressant effects in animal studies. This review focuses on how caffeine and selective adenosine antagonists can improve different aspects of depression in humans, as well as in animal models. The effects on motivational symptoms of depression such as anergia, fatigue, and psychomotor slowing receive particular attention. Thus, the ability of adenosine receptor antagonists to reverse the anergia induced by dopamine antagonism or depletion is of special interest. In conclusion, although further studies are needed, it appears that caffeine and selective adenosine receptor antagonists could be therapeutic agents for the treatment of motivational dysfunction in depression.

  16. Structural Mapping of Adenosine Receptor Mutations

    DEFF Research Database (Denmark)

    Jespers, Willem; Schiedel, Anke C; Heitman, Laura H

    2018-01-01

    The four adenosine receptors (ARs), A1, A2A, A2B, and A3, constitute a subfamily of G protein-coupled receptors (GPCRs) with exceptional foundations for structure-based ligand design. The vast amount of mutagenesis data, accumulated in the literature since the 1990s, has been recently supplemente...

  17. The Role of Adenosine Receptors in Psychostimulant Addiction

    Directory of Open Access Journals (Sweden)

    Inmaculada Ballesteros-Yáñez

    2018-01-01

    Full Text Available Adenosine receptors (AR are a family of G-protein coupled receptors, comprised of four members, named A1, A2A, A2B, and A3 receptors, found widely distributed in almost all human body tissues and organs. To date, they are known to participate in a large variety of physiopathological responses, which include vasodilation, pain, and inflammation. In particular, in the central nervous system (CNS, adenosine acts as a neuromodulator, exerting different functions depending on the type of AR and consequent cellular signaling involved. In terms of molecular pathways and second messengers involved, A1 and A3 receptors inhibit adenylyl cyclase (AC, through Gi/o proteins, while A2A and A2B receptors stimulate it through Gs proteins. In the CNS, A1 receptors are widely distributed in the cortex, hippocampus, and cerebellum, A2A receptors are localized mainly in the striatum and olfactory bulb, while A2B and A3 receptors are found at low levels of expression. In addition, AR are able to form heteromers, both among themselves (e.g., A1/A2A, as well as with other subtypes (e.g., A2A/D2, opening a whole range of possibilities in the field of the pharmacology of AR. Nowadays, we know that adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission and therefore reward systems, being A1 receptors colocalized in heteromeric complexes with D1 receptors, and A2A receptors with D2 receptors. This review documents the present state of knowledge of the contribution of AR, particularly A1 and A2A, to psychostimulants-mediated effects, including locomotor activity, discrimination, seeking and reward, and discuss their therapeutic relevance to psychostimulant addiction. Studies presented in this review reinforce the potential of A1 agonists as an effective strategy to counteract psychostimulant-induced effects. Furthermore, different experimental data support the hypothesis that A2A/D2 heterodimers are

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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, [ 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

  19. APOBEC3 cytidine deaminases in double-strand DNA break repair and cancer promotion.

    Science.gov (United States)

    Nowarski, Roni; Kotler, Moshe

    2013-06-15

    High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR. ©2013 AACR.

  20. Carbamazepine and oxcarbazepine, but not eslicarbazepine, enhance excitatory synaptic transmission onto hippocampal CA1 pyramidal cells through an antagonist action at adenosine A1 receptors.

    Science.gov (United States)

    Booker, Sam A; Pires, Nuno; Cobb, Stuart; Soares-da-Silva, Patrício; Vida, Imre

    2015-06-01

    This study assessed the anticonvulsant and seizure generation effects of carbamazepine (CBZ), oxcarbazepine (OXC) and eslicarbazepine (S-Lic) in wild-type mice. Electrophysiological recordings were made to discriminate potential cellular and synaptic mechanisms underlying anti- and pro-epileptic actions. The anticonvulsant and pro-convulsant effects were evaluated in the MES, the 6-Hz and the Irwin tests. Whole-cell patch-clamp recordings were used to investigate the effects on fast excitatory and inhibitory synaptic transmission in hippocampal area CA1. The safety window for CBZ, OXC and eslicarbazepine (ED50 value against the MES test and the dose that produces grade 5 convulsions in all mice), was 6.3, 6.0 and 12.5, respectively. At high concentrations the three drugs reduced synaptic transmission. CBZ and OXC enhanced excitatory postsynaptic currents (EPSCs) at low, therapeutically-relevant concentrations. These effects were associated with no change in inhibitory postsynaptic currents (IPSCs) resulting in altered balance between excitation and inhibition. S-Lic had no effect on EPSC or IPSC amplitudes over the same concentration range. The CBZ mediated enhancement of EPSCs was blocked by DPCPX, a selective antagonist, and occluded by CCPA, a selective agonist of the adenosine A1 receptor. Furthermore, reduction of endogenous adenosine by application of the enzyme adenosine deaminase also abolished the CBZ- and OXC-induced increase of EPSCs, indicating that the two drugs act as antagonists at native adenosine receptors. In conclusion, CBZ and OXC possess pro-epileptic actions at clinically-relevant concentrations through the enhancement of excitatory synaptic transmission. S-Lic by comparison has no such effect on synaptic transmission, explaining its lack of seizure exacerbation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Inducing salt tolerance in maize through ACC-deaminase biotechnology (abstract)

    International Nuclear Information System (INIS)

    Shahroona, B.; Arshad, M.; Zahir, Z.A.

    2005-01-01

    Ethylene is one of the five established classes of phytohormones. Its involvement in evoking physiological responses in plants exposed to any kind of environmental stresses (such as salinity, drought and metal toxicity stresses) is well established, thus it has also been designated as 'stress' hormone. It is generally believed that stress induces accelerated synthesis of ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), which subsequently results in the out burst of ethylene production and plant responds to this higher level of ethylene. Thus any check on this accelerated ethylene production in plants exposed to salinity stress could help in minimizing the negative impact of this stress and plants might survive better. There are some soil bacteria which carry ACC-deaminase enzyme and their presence on root surface results in lowering the accelerated production of ethylene. Trials were conducted under axenic condition to study the effect of inoculation with ACC-deaminase containing rhizobacteria to ameliorate the effect of salinity on seedling growth. Maize seedlings were exposed to different salinity levels and results indicated that inoculation with ACC-deaminase containing rhizobacteria significantly increased root elongation, shoot length, fresh and dry weight of seedlings at all the salinity levels. (author)

  2. A halotolerant Enterobacter sp. displaying ACC deaminase activity promotes rice seedling growth under salt stress.

    Science.gov (United States)

    Sarkar, Anumita; Ghosh, Pallab Kumar; Pramanik, Krishnendu; Mitra, Soumik; Soren, Tithi; Pandey, Sanjeev; Mondal, Monohar Hossain; Maiti, Tushar Kanti

    2018-01-01

    Agricultural productivity is proven to be hampered by the synthesis of reactive oxygen species (ROS) and production of stress-induced ethylene under salinity stress. One-aminocyclopropane-1-carboxylic acid (ACC) is the direct precursor of ethylene synthesized by plants. Bacteria possessing ACC deaminase activity can use ACC as a nitrogen source preventing ethylene production. Several salt-tolerant bacterial strains displaying ACC deaminase activity were isolated from rice fields, and their plant growth-promoting (PGP) properties were determined. Among them, strain P23, identified as an Enterobacter sp. based on phenotypic characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry data and the 16S rDNA sequence, was selected as the best-performing isolate for several PGP traits, including phosphate solubilization, IAA production, siderophore production, HCN production, etc. Enterobacter sp. P23 was shown to promote rice seedling growth under salt stress, and this effect was correlated with a decrease in antioxidant enzymes and stress-induced ethylene. Isolation of an acdS mutant strain enabled concluding that the reduction in stress-induced ethylene content after inoculation of strain P23 was linked to ACC deaminase activity. Copyright © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. An enzyme-free strategy for ultrasensitive detection of adenosine using a multipurpose aptamer probe and malachite green.

    Science.gov (United States)

    Zhao, Hui; Wang, Yong-Sheng; Tang, Xian; Zhou, Bin; Xue, Jin-Hua; Liu, Hui; Liu, Shan-Du; Cao, Jin-Xiu; Li, Ming-Hui; Chen, Si-Han

    2015-08-05

    We report on an enzyme-free and label-free strategy for the ultrasensitive determination of adenosine. A novel multipurpose adenosine aptamer (MAAP) is designed, which serves as an effective target recognition probe and a capture probe for malachite green. In the presence of adenosine, the conformation of the MAAP is converted from a hairpin structure to a G-quadruplex. Upon addition of malachite green into this solution, a noticeable enhancement of resonance light scattering was observed. The signal response is directly proportional to the concentration of adenosine ranging from 75 pM to 2.2 nM with a detection limit of 23 pM, which was 100-10,000 folds lower than those obtained by previous reported methods. Moreover, this strategy has been applied successfully for detecting adenosine in human urine and blood samples, further proving its reliability. The mechanism of adenosine inducing MAAP to form a G-quadruplex was demonstrated by a series of control experiments. Such a MAAP probe can also be used to other strategies such as fluorescence or spectrophotometric ones. We suppose that this strategy can be expanded to develop a universal analytical platform for various target molecules in the biomedical field and clinical diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Crystal structures of T. b. rhodesiense adenosine kinase complexed with inhibitor and activator: implications for catalysis and hyperactivation.

    Directory of Open Access Journals (Sweden)

    Sabine Kuettel

    2011-05-01

    Full Text Available BACKGROUND: The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP utilizing adenosine triphosphate (ATP as the preferred phosphoryl donor. METHODS AND FINDINGS: Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK: the structure of TbrAK in complex with the bisubstrate inhibitor P(1,P(5-di(adenosine-5'-pentaphosphate (AP5A at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl-2H-pyrazol-3-yl]morpholine (compound 1 at 2.8 Å resolution. CONCLUSIONS: The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.

  5. Restriction of Equine Infectious Anemia Virus by Equine APOBEC3 Cytidine Deaminases ▿ †

    Science.gov (United States)

    Zielonka, Jörg; Bravo, Ignacio G.; Marino, Daniela; Conrad, Elea; Perković, Mario; Battenberg, Marion; Cichutek, Klaus; Münk, Carsten

    2009-01-01

    The mammalian APOBEC3 (A3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. The A3 locus on the chromosome 28 of the horse genome contains multiple A3 genes: two copies of A3Z1, five copies of A3Z2, and a single copy of A3Z3, indicating a complex evolution of multiple gene duplications. We have cloned and analyzed for expression the different equine A3 genes and examined as well the subcellular distribution of the corresponding proteins. Additionally, we have tested the functional antiretroviral activity of the equine and of several of the human and nonprimate A3 proteins against the Equine infectious anemia virus (EIAV), the Simian immunodeficiency virus (SIV), and the Adeno-associated virus type 2 (AAV-2). Hematopoietic cells of horses express at least five different A3s: A3Z1b, A3Z2a-Z2b, A3Z2c-Z2d, A3Z2e, and A3Z3, whereas circulating macrophages, the natural target of EIAV, express only part of the A3 repertoire. The five A3Z2 tandem copies arose after three consecutive, recent duplication events in the horse lineage, after the split between Equidae and Carnivora. The duplicated genes show different antiviral activities against different viruses: equine A3Z3 and A3Z2c-Z2d are potent inhibitors of EIAV while equine A3Z1b, A3Z2a-Z2b, A3Z2e showed only weak anti-EIAV activity. Equine A3Z1b and A3Z3 restricted AAV and all equine A3s, except A3Z1b, inhibited SIV. We hypothesize that the horse A3 genes are undergoing a process of subfunctionalization in their respective viral specificities, which might provide the evolutionary advantage for keeping five copies of the original gene. PMID:19458006

  6. Distribution of ADAT-Dependent Codons in the Human Transcriptome

    Directory of Open Access Journals (Sweden)

    Àlbert Rafels-Ybern

    2015-07-01

    Full Text Available Nucleotide modifications in the anticodons of transfer RNAs (tRNA play a central role in translation efficiency, fidelity, and regulation of translation, but, for most of these modifications, the details of their function remain unknown. The heterodimeric adenosine deaminases acting on tRNAs (ADAT2-ADAT3, or ADAT are enzymes present in eukaryotes that convert adenine (A to inosine (I in the first anticodon base (position 34 by hydrolytic deamination. To explore the influence of ADAT activity on mammalian translation, we have characterized the human transcriptome and proteome in terms of frequency and distribution of ADAT-related codons. Eight different tRNAs can be modified by ADAT and, once modified, these tRNAs will recognize NNC, NNU and NNA codons, but not NNG codons. We find that transcripts coding for proteins highly enriched in these eight amino acids (ADAT-aa are specifically enriched in NNC, NNU and NNA codons. We also show that the proteins most enriched in ADAT-aa are composed preferentially of threonine, alanine, proline, and serine (TAPS. We propose that the enrichment in ADAT-codons in these proteins is due to the similarities in the codons that correspond to TAPS.

  7. Amyotrophic Lateral Sclerosis (ALS and Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    Ana M. Sebastião

    2018-04-01

    Full Text Available In the present review we discuss the potential involvement of adenosinergic signaling, in particular the role of adenosine receptors, in amyotrophic lateral sclerosis (ALS. Though the literature on this topic is not abundant, the information so far available on adenosine receptors in animal models of ALS highlights the interest to continue to explore the role of these receptors in this neurodegenerative disease. Indeed, all motor neurons affected in ALS are responsive to adenosine receptor ligands but interestingly, there are alterations in pre-symptomatic or early symptomatic stages that mirror those in advanced disease stages. Information starts to emerge pointing toward a beneficial role of A2A receptors (A2AR, most probably at early disease states, and a detrimental role of caffeine, in clear contrast with what occurs in other neurodegenerative diseases. However, some evidence also exists on a beneficial action of A2AR antagonists. It may happen that there are time windows where A2AR prove beneficial and others where their blockade is required. Furthermore, the same changes may not occur simultaneously at the different synapses. In line with this, it is not fully understood if ALS is a dying back disease or if it propagates in a centrifugal way. It thus seems crucial to understand how motor neuron dysfunction occurs, how adenosine receptors are involved in those dysfunctions and whether the early changes in purinergic signaling are compensatory or triggers for the disease. Getting this information is crucial before starting the design of purinergic based strategies to halt or delay disease progression.

  8. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS 2, 3 and 4 in Astrocyte-Like Cells.

    Directory of Open Access Journals (Sweden)

    Till Nicolas Eusemann

    Full Text Available The "regulators of g-protein signalling" (RGS comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

  9. Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo.

    Science.gov (United States)

    Shevidi, Saba; Uchida, Alicia; Schudrowitz, Natalie; Wessel, Gary M; Yajima, Mamiko

    2017-12-01

    A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9-DA), which induces a single nucleotide conversion in the genome. Cas9-DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh, or SpPks, each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9-DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9-DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036-1046, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 [ 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 [ 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

  11. Maintaining Genome Stability: The Role of Helicases and Deaminases

    Science.gov (United States)

    2008-07-01

    Errors in duplicating DNA can result in genomic instability, leading to various human diseases, such as cancer, immune system disorder, muscle dystrophy ...as cancer, immune system disorder, muscle dystrophy , and neurodegenerations. Thus, maintaining genomic integrity is vital to the normal growth of...31–38. Eberharter, A., R. Ferreira and P. Becker , 2005 Dynamic chro- matin: concerted nucleosome remodelling and acetylation. Biol. Chem. 386: 745

  12. Adenosine contribution to normal renal physiology and chronic kidney disease.

    Science.gov (United States)

    Oyarzún, Carlos; Garrido, Wallys; Alarcón, Sebastián; Yáñez, Alejandro; Sobrevia, Luis; Quezada, Claudia; San Martín, Rody

    2017-06-01

    Adenosine is a nucleoside that is particularly interesting to many scientific and clinical communities as it has important physiological and pathophysiological roles in the kidney. The distribution of adenosine receptors has only recently been elucidated; therefore it is likely that more biological roles of this nucleoside will be unveiled in the near future. Since the discovery of the involvement of adenosine in renal vasoconstriction and regulation of local renin production, further evidence has shown that adenosine signaling is also involved in the tubuloglomerular feedback mechanism, sodium reabsorption and the adaptive response to acute insults, such as ischemia. However, the most interesting finding was the increased adenosine levels in chronic kidney diseases such as diabetic nephropathy and also in non-diabetic animal models of renal fibrosis. When adenosine is chronically increased its signaling via the adenosine receptors may change, switching to a state that induces renal damage and produces phenotypic changes in resident cells. This review discusses the physiological and pathophysiological roles of adenosine and pays special attention to the mechanisms associated with switching homeostatic nucleoside levels to increased adenosine production in kidneys affected by CKD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Diagnostic value of combined determination of serum and chest fluid adenosine deaminase (ADA), IL-2, IL-6, IL-10 contents for differentiation of tuberculous from malignant pleural effusion

    International Nuclear Information System (INIS)

    Wu Jiaming; Wang Limin

    2005-01-01

    Objective: To investigate the possible diagnostic value of combined determination of serum and chest fluid contents of ADA, IL-2, IL-6, IL-10 in patients with tuberculous and malignant pleural effusion. Methods: Serum and chest fluid ADA (with biochemical method), IL-2, IL-6, IL-10 (with ELISA) contents were measured in 56 patients with tuberculosis pleural effusion, 53 patients with malignant effusion and 30 controls (in serum only). The receiving operative characteristic (ROC) curve for each parameter was analyzed for study of respective area under curse (Auc). Results: The serum IL-6 levels in both groups of patients were significantly higher than those in the controls (P<0.05). The chest fluid contents of ADA, IL-2, IL-6 and IL-10 in patients with tuberculous effusion were all significantly higher than those in patients with malignancies (P<0.05). The Auc in the ROC was largest in the case of ADA, followed by IL-10, IL-6 with IL-2 the least. Conclusion: Determination of chest fluid ADA, IL-2, IL-6, IL-10 contents was helpful in the differentiation of tuberculous from malignant pleural effusion. Combined determination of chest fluid ADA and IL-10 provided the highest accuracy rate for differentional diagnosis. (authors)

  14. Study on the clinical value of determination of serum adenosine deaminase (ADA) and HA, PIIIP levels for diagnosis of chronic hepatitis

    International Nuclear Information System (INIS)

    Ma Yun

    2007-01-01

    Objective: To explore the clinical value of determination of serum ADA and HA, PIIIP levels for diagnosis of chronic hepatitis. Methods: Serum ADA levels were determined with biochemistry and HA, PIIIP levels were detected with RIA in 36 patients with chronic hepatitis and 35 controls. Results: The levels of serum ADA, HA and PIIIP were significantly higher in the patients than those in controls (P<0.01). Serum ADA, HA, PIIIP levelers were mutually positively correlated (r=0.6117, 0.7134, P<0.01). Conclusion: Detection of serum ADA levels could reflect the liver reserve function and status of fibrosis. (authors)

  15. Regulation of Expression of the prb-1b / ACC Deaminase gene by UV-B in Transgenic tomatoes

    International Nuclear Information System (INIS)

    Tamot, B.K.; Pauls, K.P.; Glick, R.

    2003-01-01

    Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UWA4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns of the transgene. No ACC deaminase RNA or protein was detected bu RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with alpha-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid , ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots of transformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-B light

  16. Effects of high doses of intracoronary adenosine on the assessment of fractional flow reserve

    Directory of Open Access Journals (Sweden)

    Ahmed Khashaba

    2014-03-01

    Conclusions: Intracoronary adenosine, at doses higher than currently suggested, lows obtaining FFR values similar to IV adenosine. Intravenous adenosine, which remains the gold standard, might thus be reserved for those lesions with equivocal FFR values.

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

    Science.gov (United States)

    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.

  18. Adenosine 5'-Monophosphate Aerosol Challenge Does Not Provoke Airflow Limitation in Healthy Cats

    Directory of Open Access Journals (Sweden)

    K. Vondráková

    2006-01-01

    Full Text Available The purpose of our study was to investigate the effects of nebulized adenosine 5'- monophosphate on airflow limitation in healthy cats determined by barometric whole body plethysmography (BWBP, in comparison to the effects of carbachol. Ten healthy 4- to 6-year-old domestic shorthair cats were included in the study. Each cat was placed in a BWBP plexiglass chamber (volume 38 l. Changes in box pressure were measured at baseline and after nebulization of vehicle and increasing concentrations of carbachol and adenosine 5'- monophosphate. Airway responsiveness was monitored as increases in enhanced pause (PENH, a unitless variable derived from dose-response curves estimating airflow limitation. The chosen endpoint was the agonist concentration which increased PENH to 300% of the value obtained after saline nebulization (PCPENH 300. Inter-day repeatability of measurements was assessed by repeated bronchoprovocations with both agonists 2-3 days apart. For carbachol, PCPENH300 was reached in all cats and correlated significantly between days (mean ± SD; 0.54 ± 0.42 mg/ml and 0.64 ± 0.45 mg/ml respectively; r = 0.58, p < 0.05 In contrast, we found no reaction to adenosine 5'- monophosphate even with the highest concentration nebulized during both measurements. At baseline, mean ± SD PENH was 0.47 ± 0.18 and 0.58 ± 0.24 (measurements 1 and 2, whereas PENH after 500 mg/ml adenosine 5'- monophosphate was 0.46 ± 0.20 and 0.71 ± 0.37. All bronchoprovocation tests were well tolerated by the cats. We conclude that healthy airways in cats do not demonstrate airway responsiveness to inhaled adenosine 5'- monophosphate. This is in agreement with observations in humans as well as our previous findings in dogs, where adenosine 5'- monophosphate had no effect on healthy canine airways, but caused significant airflow limitation after induction of acute bronchitis. To define the value of bronchoprovocation testing with adenosine 5'- monophosphate in the feline

  19. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    International Nuclear Information System (INIS)

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

    1989-01-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

  20. Application of the newly developed Japanese adenosine normal database for adenosine stress myocardial scintigraphy.

    Science.gov (United States)

    Harata, Shingo; Isobe, Satoshi; Morishima, Itsuro; Suzuki, Susumu; Tsuboi, Hideyuki; Sone, Takahito; Ishii, Hideki; Murohara, Toyoaki

    2015-10-01

    The currently available Japanese normal database (NDB) in stress myocardial perfusion scintigraphy recommended by the Japanese Society of Nuclear Medicine (JSNM-NDB) is created based on the data from exercise tests. The newly developed adenosine normal database (ADS-NDB) remains to be validated for patients undergoing adenosine stress test. We tested whether the diagnostic accuracy of adenosine stress test is improved by the use of ADS-NDB (Kanazawa University). Of 233 consecutive patients undergoing (99m)Tc-MIBI adenosine stress test, 112 patients were tested. The stress/rest myocardial (99m)Tc-MIBI single-photon emission computed tomography (SPECT) images were analyzed by AutoQUANT 7.2 with both ADS-NDB and JSNM-NDB. The summed stress score (SSS) and summed difference score (SDS) were calculated. The agreements of the post-stress defect severity between ADS-NDB and JSNM-NDB were assessed using a weighted kappa statistic. In all patients, mean SSSs of all, right coronary artery (RCA), left anterior descending (LAD), and left circumflex (LCx) territories were significantly lower with ADS-NDB than those with JSNM-NDB. Mean SDSs in all, RCA, and LAD territories were significantly lower with ADS-NDB than those with JSNM-NDB. In 28 patients with significant coronary stenosis, the mean SSS in the RCA territory was significantly lower with ADS-NDB than that with JSNM-NDB. In 84 patients without ischemia, both mean SSSs and SDSs in all, RCA, LAD, and LCx territories were significantly lower with ADS-NDB than those with JSNM-NDB. Weighted kappa values of all patients, patients with significant stenosis, and patients without ischemia were 0.89, 0.83, and 0.92, respectively. Differences were observed between results from ADS-NDB and JSNM-NDB. The diagnostic accuracy of adenosine stress myocardial perfusion scintigraphy may be improved by reducing false-positive results.

  1. Gene therapy for adenosine deaminase–deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans

    Science.gov (United States)

    Candotti, Fabio; Shaw, Kit L.; Muul, Linda; Carbonaro, Denise; Sokolic, Robert; Choi, Christopher; Schurman, Shepherd H.; Garabedian, Elizabeth; Kesserwan, Chimene; Jagadeesh, G. Jayashree; Fu, Pei-Yu; Gschweng, Eric; Cooper, Aaron; Tisdale, John F.; Weinberg, Kenneth I.; Crooks, Gay M.; Kapoor, Neena; Shah, Ami; Abdel-Azim, Hisham; Yu, Xiao-Jin; Smogorzewska, Monika; Wayne, Alan S.; Rosenblatt, Howard M.; Davis, Carla M.; Hanson, Celine; Rishi, Radha G.; Wang, Xiaoyan; Gjertson, David; Yang, Otto O.; Balamurugan, Arumugam; Bauer, Gerhard; Ireland, Joanna A.; Engel, Barbara C.; Podsakoff, Gregory M.; Hershfield, Michael S.; Blaese, R. Michael; Parkman, Robertson

    2012-01-01

    We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)–deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34+ cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m2). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency. PMID:22968453

  2. Modulation of microRNA editing, expression and processing by ADAR2 deaminase in glioblastoma.

    Science.gov (United States)

    Tomaselli, Sara; Galeano, Federica; Alon, Shahar; Raho, Susanna; Galardi, Silvia; Polito, Vinicia Assunta; Presutti, Carlo; Vincenti, Sara; Eisenberg, Eli; Locatelli, Franco; Gallo, Angela

    2015-01-13

    ADAR enzymes convert adenosines to inosines within double-stranded RNAs, including microRNA (miRNA) precursors, with important consequences on miRNA retargeting and expression. ADAR2 activity is impaired in glioblastoma and its rescue has anti-tumoral effects. However, how ADAR2 activity may impact the miRNome and the progression of glioblastoma is not known. By integrating deep-sequencing and array approaches with bioinformatics analyses and molecular studies, we show that ADAR2 is essential to edit a small number of mature miRNAs and to significantly modulate the expression of about 90 miRNAs in glioblastoma cells. Specifically, the rescue of ADAR2 activity in cancer cells recovers the edited miRNA population lost in glioblastoma cell lines and tissues, and rebalances expression of onco-miRNAs and tumor suppressor miRNAs to the levels observed in normal human brain. We report that the major effect of ADAR2 is to reduce the expression of a large number of miRNAs, most of which act as onco-miRNAs. ADAR2 can edit miR-222/221 and miR-21 precursors and decrease the expression of the corresponding mature onco-miRNAs in vivo and in vitro, with important effects on cell proliferation and migration. Our findings disclose an additional layer of complexity in miRNome regulation and provide information to better understand the impact of ADAR2 editing enzyme in glioblastoma. We propose that ADAR2 is a key factor for maintaining edited-miRNA population and balancing the expression of several essential miRNAs involved in cancer.

  3. Involvement of adenosine in the antiinflammatory action of ketamine.

    Science.gov (United States)

    Mazar, Julia; Rogachev, Boris; Shaked, Gad; Ziv, Nadav Y; Czeiger, David; Chaimovitz, Cidio; Zlotnik, Moshe; Mukmenev, Igor; Byk, Gerardo; Douvdevani, Amos

    2005-06-01

    Ketamine is an anesthetic drug. Subanesthetic doses of ketamine have been shown to reduce interleukin-6 concentrations after surgery and to reduce mortality and the production of tumor necrosis factor alpha and interleukin 6 in septic animals. Similarly, adenosine was shown to reduce tumor necrosis factor alpha and mortality of septic animals. The aim of this study was to determine whether adenosine mediates the antiinflammatory effects of ketamine. Sepsis was induced in mice by lipopolysaccharide or Escherichia coli inoculation. Leukocyte recruitment and cytokine concentrations were used as inflammation markers. Adenosine concentrations were assayed by high-performance liquid chromatography, and the involvement of adenosine in the effects of ketamine was demonstrated by adenosine receptor agonists and antagonists. Ketamine markedly reduced mortality from sepsis, leukocyte recruitment, and tumor necrosis factor-alpha and interleukin-6 concentrations. Ketamine administration in mice and rats was associated with a surge at 20-35 min of adenosine in serum (up to 5 microm) and peritoneal fluid. The adenosine A2A receptor agonist CGS-21680 mimicked the effect of ketamine in peritonitis, whereas the A2A receptor antagonists DMPX and ZM 241385 blocked its antiinflammatory effects. In contrast, A1 and A3 receptor antagonists had no effect. ZM 241385 reversed the beneficial effect of ketamine on survival from bacterial sepsis. The current data suggest that the sepsis-protective antiinflammatory effects of ketamine are mediated by the release of adenosine acting through the A2A receptor.

  4. Basal and adenosine receptor-stimulated levels of cAMP are reduced in lymphocytes from alcoholic patients

    International Nuclear Information System (INIS)

    Diamond, I.; Wrubel, B.; Estrin, W.; Gordon, A.

    1987-01-01

    Alcoholism causes serious neurologic disease that may be due, in part, to the ability of ethanol to interact with neural cell membranes and change neuronal function. Adenosine receptors are membrane-bound proteins that appear to mediate some of the effects of ethanol in the brain. Human lymphocytes also have adenosine receptors, and their activation causes increases in cAMP levels. To test the hypothesis that basal and adenosine receptor-stimulated cAMP levels in lymphocytes might be abnormal in alcoholism, the authors studied lymphocytes from 10 alcoholic subjects, 10 age- and sex-matched normal individuals, and 10 patients with nonalcoholic liver disease. Basal and adenosine receptor-stimulated cAMP levels were reduced 75% in lymphocytes from alcoholic subjects. Also, there was a 76% reduction in ethanol stimulation of cAMP accumulation in lymphocytes from alcoholics. Similar results were demonstrable in isolated T cells. Unlike other laboratory tests examined, these measurements appeared to distinguish alcoholics from normal subjects and from patients with nonalcoholic liver disease. Reduced basal and adenosine receptor-stimulated levels of cAMP in lymphocytes from alcoholics may reflect a change in cell membranes due either to chronic alcohol abuse or to a genetic predisposition unique to alcoholic subjects

  5. A Novel Regulator of Activation-Induced Cytidine Deaminase/APOBECs in Immunity and Cancer: Schrödinger’s CATalytic Pocket

    Directory of Open Access Journals (Sweden)

    Mani Larijani

    2017-04-01

    Full Text Available Activation-induced cytidine deaminase (AID and its relative APOBEC3 cytidine deaminases boost immune response by mutating immune or viral genes. Because of their genome-mutating activities, AID/APOBECs are also drivers of tumorigenesis. Due to highly charged surfaces, extensive non-specific protein–protein/nucleic acid interactions, formation of polydisperse oligomers, and general insolubility, structure elucidation of these proteins by X-ray crystallography and NMR has been challenging. Hence, almost all available AID/APOBEC structures are of mutated and/or truncated versions. In 2015, we reported a functional structure for AID using a combined computational–biochemical approach. In so doing, we described a new regulatory mechanism that is a first for human DNA/RNA-editing enzymes. This mechanism involves dynamic closure of the catalytic pocket. Subsequent X-ray and NMR studies confirmed our discovery by showing that other APOBEC3s also close their catalytic pockets. Here, we highlight catalytic pocket closure as an emerging and important regulatory mechanism of AID/APOBEC3s. We focus on three sub-topics: first, we propose that variable pocket closure rates across AID/APOBEC3s underlie differential activity in immunity and cancer and review supporting evidence. Second, we discuss dynamic pocket closure as an ever-present internal regulator, in contrast to other proposed regulatory mechanisms that involve extrinsic binding partners. Third, we compare the merits of classical approaches of X-ray and NMR, with that of emerging computational–biochemical approaches, for structural elucidation specifically for AID/APOBEC3s.

  6. APOBEC3G enhances lymphoma cell radioresistance by promoting cytidine deaminase-dependent DNA repair.

    Science.gov (United States)

    Nowarski, Roni; Wilner, Ofer I; Cheshin, Ori; Shahar, Or D; Kenig, Edan; Baraz, Leah; Britan-Rosich, Elena; Nagler, Arnon; Harris, Reuben S; Goldberg, Michal; Willner, Itamar; Kotler, Moshe

    2012-07-12

    APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.

  7. Reentry Tachycardia in Children: Adenosine Can Make It Worse.

    Science.gov (United States)

    Hien, Maximilian D; Benito Castro, Fernando; Fournier, Philippe; Filleron, Anne; Tran, Tu-Anh

    2016-10-08

    We report on a rare but severe complication of adenosine use in a child with reentry tachycardia. Treatment with adenosine, which is the standard medical therapy of atrioventricular reentry tachycardia, led to the development of an irregular wide complex tachycardia, caused by rapid ventricular response to atrial fibrillation. The girl was finally stabilized with electrical cardioversion. We analyze the pathomechanism and discuss possible treatment options. Atrial fibrillation, as well as its conduction to the ventricles, can be caused by adenosine. Rapid ventricular response in children with Wolff-Parkinson-White syndrome is more frequent than previously believed. A patient history of atrial fibrillation is a contraindication for cardioversion with adenosine and needs to be assessed in children with reentry tachycardia. High-risk patients may potentially profit from prophylactic comedication with antiarrhythmic agents, such as flecainide, ibutilide, or vernakalant, before adenosine administration.

  8. Characterization of cardiac adenosine receptors using N6-phenyladenosines and a new radioligand, [125I]-(m-aminophenyl)adenosine

    International Nuclear Information System (INIS)

    Kwatra, M.M.; Hosey, M.M.; Green, R.

    1986-01-01

    The chick heart contains adenosine receptors with characteristics similar to the R adenosine receptors found in the CNS. They have synthesized several N 6 -phenyladenosines and tested their potencies for inhibiting the binding of [ 125 I](p-aminobenzyl)adenosine {[ 125 I]ABA) to chick heart membranes. Of the 12 compounds tested, N 6 -(p-aminobenzyl) adenosine (ABA) was the least potent (IC 50 ∼ 40 nM) while N 6 -(m-nitrophenyl)adenosine(MNPA) was the most potent (IC 50 ∼ 1 nM). The IC 50 of N 6 -(m-aminophenyl)adenosine(MAPA) was greater than that of N 6 -phenyladenosine(PA) while that of MNPA was less than that of PA. The effects of these electron-releasing (-NH 2 ) and electron-withdrawing (-NO 2 ) groups along with data obtained with other phenyl-substituted N 6 -phenyladenosines suggest that the electron density of the N 6 -nitrogen may affect the affinities of these compounds for the cardiac adenosine receptor. MAPA can be iodinated to produce a new ligand, [ 125 I]MAPA. This iodination, like that of ABA, increases the affinity of the compound and produces a ligand with good affinity and low nonspecific binding suitable for studies on tissues with low concentrations of adenosine receptors

  9. Regulated production and anti-HIV type 1 activities of cytidine deaminases APOBEC3B, 3F, and 3G.

    Science.gov (United States)

    Rose, Kristine M; Marin, Mariana; Kozak, Susan L; Kabat, David

    2005-07-01

    APOBEC3G and 3F (A3G and A3F) cytidine deaminases incorporate into retroviral cores where they lethally hypermutate nascent DNA reverse transcripts. As substantiated here, the viral infectivity factor (Vif) encoded by human immunodeficiency virus type-1 (HIV-1) binds A3G and A3F and induces their degradation, thereby precluding their incorporation into viral progeny. Previous evidence suggested that A3G is expressed in H9 and other nonpermissive cells that contain this antiviral defense but not in several permissive cells, and that overexpression of A3G or A3F makes permissive cells nonpermissive. Using a broader panel of cell lines, we confirmed a correlation between A3G and cellular abilities to inactivate HIV-1(Deltavif). However, there was a quantitative discrepancy because several cells with weak antiviral activities had similar amounts of wild-type A3G mRNA and protein compared to H9 cells. Antiviral activity of H9 cells was also attenuated in some conditions. These quantitative discrepancies could not be explained by the presence of A3F or other A3G paralogs in some of the cell lines. Thus, A3A, A3B, and A3C had weak but significant anti-HIV-1 activities and did not dominantly interfere with A3G or A3F antiviral functions. Control of A3G synthesis by the protein kinase C/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathway was also similar in permissive and nonpermissive cells. A3G in highly permissive cells is degraded by Vif, suggesting that it is not in a sequestered site, and is specifically incorporated in low amounts into HIV-1(Deltavif). Although A3G and/or A3F inactivate HIV-1(Deltavif) and are neutralized by Vif, the antiviral properties of cell lines are also influenced by other cellular and viral factors.

  10. [Isolation and identification of hydrogen-oxidizing bacteria producing 1-aminocyclopropane-1-carboxylate deaminase and the determination of enzymatic activity].

    Science.gov (United States)

    Fu, Bo; Wang, Weiwei; Tang, Ming; Chen, Xingdu

    2009-03-01

    We used Medicago sativa rhizosphere in Shaanxi province of China to isolate and identify hydrogen-oxidizing bacteria that produced ACC (1-aminocyclopropane-1-carboxylate) deaminase, and then studied the mechanism why they can promote the growth of plants. Hydrogen-oxidizing bacteria were isolated by gas-cycle incubation system. We studied the morphological character, physiological characteristics, 16S rDNA sequence analysis and built the phylogenic tree. Thin layer chromatography was used to isolate the strain that produced ACC deaminase. Ninhydrin reaction was used to test the enzyme activity. In total 37 strains were isolated, 8 of which could oxidize H2 strongly and grow chemolithoautotrophically. We initially identified them as hydrogen-oxidizing bacteria. Only strain WMQ-7 produced ACC deaminase among these 8 strains. Morphological and physiological characteristics analysis showed that strain WMQ-7 was essentially consistent with Pseudomonas putida. The 16S rDNA sequence analysis (GenBank accession number EU807744) suggested that strain WMQ-7 was clustered together with Pseudomonas putida in phylogenetic tree, with the sequence identity of 99%. Based on all these results, strain WMQ-7 was identified as Pseudomonas putida. The enzyme activity of strain WMQ-7 was 0.671 U/microg. A strain producing ACC deaminase was identified and tested.

  11. Activation-induced cytidine deaminase induces reproducible DNA breaks at many non-Ig Loci in activated B cells

    NARCIS (Netherlands)

    Staszewski, Ori; Baker, Richard E.; Ucher, Anna J.; Martier, Raygene; Stavnezer, Janet; Guikema, Jeroen E. J.

    2011-01-01

    After immunization or infection, activation-induced cytidine deaminase (AID) initiates diversification of immunoglobulin (Ig) genes in B cells, introducing mutations within the antigen-binding V regions (somatic hypermutation, SHM) and double-strand DNA breaks (DSBs) into switch (S) regions, leading

  12. Characterization of 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase-Containing Pseudomonas spp. in the Rhizosphere of Salt-Stressed Canola

    NARCIS (Netherlands)

    Akhgar, A.; Arzanlou, M.; Bakker, Peter; Hamidpour, M.

    2014-01-01

    When exposed to biotic or abiotic stress conditions, plants produce ethylene from its immediate precursor 1-aminocyclopropane-1- carboxylate (ACC), leading to retarded root growth and senescence. Many plant growth-promoting rhizobacteria contain the enzyme ACC deaminase and this enzyme can cleave

  13. [The influence of fasting, of a hyperprotein diet and of nicotinamide on hepatic L-threonine deaminase].

    Science.gov (United States)

    Aleo, M F; Casella, A; Marinello, E

    1981-09-15

    The induction of L-threonine deaminase, following nicotinamide injection has been studied: the effect of fasting and of hyperproteic diet have been also taken in consideration. Maximal induction is observed after 5 days hyperproteic diet, and is additional only with nicotinamide treatment. Results are interpreted assuming a different hepatic content and behavior of multiple forms of the enzyme.

  14. Isolation and characterization of novel bacteria containing acc deaminase from the rhizosphere resource on dry-farming lands

    International Nuclear Information System (INIS)

    Zheng, P.; Tian, L.; Chen, F.; Cui, Z.

    2014-01-01

    Soil-microbe-plant interactions are known to be intricate and they can greatly influence the crop vigor and yield. Plant growth promoting rhizobacteria (PGPR) containing ACC deaminase can markedly affect plant metabolic processes under stress conditions. In the present study, we isolated 300 bacterial strains from the rhizosphere of maize or apple grown in drought-hit soil including four different locations of the Loess Plateau, China. Of all isolated strains, four with ACC deaminase activity (ranging from 28.88 to 155.12 nmol alpha-ketobutyrate mg-1 h-1) were further studied by determining their biological characters and sequencing the 16S rRNA gene. All four strains showed positive performance in terms of arabinose, citrate utilization, urease, indol, glucose and melibiose. In connection with the results of biochemical characters and phylogenetic analysis, these strains commonly belong to three different genera: Klebsiella, Pseudomonas and Raoultella and four different species: Klebsiella oxytoca, Klebsiella variicola, Pseudomonas fluorescens and Raoultella planticola. Although some researchers have reported their performance under stress conditions, we are the first to report Klebsiella oxytoca, Klebsiella variicola and Raoultella planticola containing ACC deaminase under drought stress. These findings are a reasonable explanation to their superb ability of causing stress-resistance in maize (Zea mays) or apple (Malus domestica) plants. The presence of diverse PGPR possessing potential ACC deaminase activity may be beneficial for enhancing crop production under different stress conditions. (author)

  15. Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

    Science.gov (United States)

    Funaro, Michael G; Nemani, Krishnamurthy V; Chen, Zhihang; Bhujwalla, Zaver M; Griswold, Karl E; Gimi, Barjor

    2016-02-01

    Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

  16. Adenosine receptors and caffeine in retinopathy of prematurity.

    Science.gov (United States)

    Chen, Jiang-Fan; Zhang, Shuya; Zhou, Rong; Lin, Zhenlang; Cai, Xiaohong; Lin, Jing; Huo, Yuqing; Liu, Xiaoling

    2017-06-01

    Retinopathy of prematurity (ROP) is a major cause of childhood blindness in the world and is caused by oxygen-induced damage to the developing retinal vasculature, resulting in hyperoxia-induced vaso-obliteration and subsequent delayed retinal vascularization and hypoxia-induced pathological neovascularization driven by vascular endothelial growth factor (VEGF) signaling pathway in retina. Current anti-VEGF therapy has shown some effective in a clinical trial, but is associated with the unintended effects on delayed eye growth and retinal vasculature development of preterm infants. Notably, cellular responses to hypoxia are characterized by robust increases in extracellular adenosine production and the markedly induced adenosine receptors, which provide a novel target for preferential control of pathological angiogenesis without affecting normal vascular development. Here, we review the experimental evidence in support of adenosine receptor-based therapeutic strategy for ROP, including the aberrant adenosine signaling in oxygen-induced retinopathy and the role of three adenosine receptor subtypes (A 1 R, A 2A R, A 2B R) in development and treatment of ROP using oxygen-induced retinopathy models. The clinical and initial animal evidence that implicate the therapeutic effect of caffeine (a non-selective adenosine receptor antagonist) in treatment of ROP are highlighted. Lastly, we discussed the translational potential as well therapeutic advantage of adenosine receptor- and caffeine-based therapy for ROR and possibly other proliferative retinopathy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Characteristic molecular vibrations of adenosine receptor ligands.

    Science.gov (United States)

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

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  18. Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III

    International Nuclear Information System (INIS)

    Rose, S.; Frydman, R.B.; de los Santos, C.; Sburlati, A.; Valasinas, A.; Frydman, B.

    1988-01-01

    Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer to total PBG consumption resulted in low yields of uroporphyrinogen I(uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane but was different from that of a 2-(hydroxymethyl)bilane. The 13 C NMR spectra of incubations carried out with [11- 13 C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11- 13 C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time

  19. Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction.

    Science.gov (United States)

    De Lorenzo, Silvana; Veggetti, Mariela; Muchnik, Salomón; Losavio, Adriana

    2004-05-01

    inhibit the increases in mepp frequency evoked by 15 and 20 mm K(+). We demonstrated that, at high K(+) concentrations, endogenous AD occupies A1 receptors, impairing the action of CCPA, since incubation with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, an A(1) receptor antagonist) and adenosine deaminase (ADA), which degrades AD into the inactive metabolite inosine, increased mepp frequency compared with that obtained in 15 and 20 mm K(+) in the absence of the drugs. Moreover, CCPA was able to induce presynaptic inhibition in the presence of ADA. It is concluded that, at high K(+) concentrations, the activation of A(1) receptors by endogenous AD prevents excessive neurotransmitter release.

  20. Synthesis of carbon-11 labelled cyclopentyltheophylline: A radioligand for PET studies of adenosine receptors

    International Nuclear Information System (INIS)

    Yorke, J.C.; Prenant, C.; Crouzel, C.

    1990-01-01

    Adenosine is presently considered as a neuromodulator, and an adenosine system has been described including secretory neurons, with a diffused distribution, specific receptors and a re-uptake system distributed heterogeneously in different anatomic areas. In order to localize the adenosine receptors in vivo by PET, the authors have synthesized the carbon-11 labelled 8-cyclopentyltheophylline, a known adenosine antagonist of A 1 receptors

  1. Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

    Directory of Open Access Journals (Sweden)

    A.R.M. Sabbatini

    2011-02-01

    Full Text Available Histidine-rich glycoprotein (HRG is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD. We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

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

    Science.gov (United States)

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

    2017-07-15

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

  3. Acquisition of Genetic Aberrations by Activation-Induced Cytidine Deaminase (AID) during Inflammation-Associated Carcinogenesis

    International Nuclear Information System (INIS)

    Takai, Atsushi; Marusawa, Hiroyuki; Chiba, Tsutomu

    2011-01-01

    Genetic abnormalities such as nucleotide alterations and chromosomal disorders that accumulate in various tumor-related genes have an important role in cancer development. The precise mechanism of the acquisition of genetic aberrations, however, remains unclear. Activation-induced cytidine deaminase (AID), a nucleotide editing enzyme, is essential for the diversification of antibody production. AID is expressed only in activated B lymphocytes under physiologic conditions and induces somatic hypermutation and class switch recombination in immunoglobulin genes. Inflammation leads to aberrant AID expression in various gastrointestinal organs and increased AID expression contributes to cancer development by inducing genetic alterations in epithelial cells. Studies of how AID induces genetic disorders are expected to elucidate the mechanism of inflammation-associated carcinogenesis

  4. ACC deaminase and IAA producing growth promoting bacteria from the rhizosphere soil of tropical rice plants.

    Science.gov (United States)

    Bal, Himadri Bhusan; Das, Subhasis; Dangar, Tushar K; Adhya, Tapan K

    2013-12-01

    Beneficial plant-associated bacteria play a key role in supporting and/or promoting plant growth and health. Plant growth promoting bacteria present in the rhizosphere of crop plants can directly affect plant metabolism or modulate phytohormone production or degradation. We isolated 355 bacteria from the rhizosphere of rice plants grown in the farmers' fields in the coastal rice field soil from five different locations of the Ganjam district of Odisha, India. Six bacteria producing both ACC deaminase (ranging from 603.94 to 1350.02 nmol α-ketobutyrate mg(-1)  h(-1) ) and indole acetic acid (IAA; ranging from 10.54 to 37.65 μM ml(-1) ) in pure cultures were further identified using polyphasic taxonomy including BIOLOG((R)) , FAME analysis and the 16S rRNA gene sequencing. Phylogenetic analyses of the isolates resulted into five major clusters to include members of the genera Bacillus, Microbacterium, Methylophaga, Agromyces, and Paenibacillus. Seed inoculation of rice (cv. Naveen) by the six individual PGPR isolates had a considerable impact on different growth parameters including root elongation that was positively correlated with ACC deaminase activity and IAA production. The cultures also had other plant growth attributes including ammonia production and at least two isolates produced siderophores. Study indicates that presence of diverse rhizobacteria with effective growth-promoting traits, in the rice rhizosphere, may be exploited for a sustainable crop management under field conditions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. A putative antiviral role of plant cytidine deaminases [version 2; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Susana Martín

    2017-06-01

    Full Text Available Background: A mechanism of innate antiviral immunity operating against viruses infecting mammalian cells has been described during the last decade.  Host cytidine deaminases (e.g., APOBEC3 proteins edit viral genomes, giving rise to hypermutated nonfunctional viruses; consequently, viral fitness is reduced through lethal mutagenesis.  By contrast, sub-lethal hypermutagenesis may contribute to virus evolvability by increasing population diversity.  To prevent genome editing, some viruses have evolved proteins that mediate APOBEC3 degradation.  The model plant Arabidopsis thaliana genome encodes nine cytidine deaminases (AtCDAs, raising the question of whether deamination is an antiviral mechanism in plants as well. Methods: Here we tested the effects of expression of AtCDAs on the pararetrovirus Cauliflower mosaic virus (CaMV. Two different experiments were carried out. First, we transiently overexpressed each one of the nine A. thaliana AtCDA genes in Nicotiana bigelovii plants infected with CaMV, and characterized the resulting mutational spectra, comparing them with those generated under normal conditions.  Secondly, we created A. thaliana transgenic plants expressing an artificial microRNA designed to knock-out the expression of up to six AtCDA genes.  This and control plants were then infected with CaMV.  Virus accumulation and mutational spectra where characterized in both types of plants. Results:  We have shown that the A. thaliana AtCDA1 gene product exerts a mutagenic activity, significantly increasing the number of G to A mutations in vivo, with a concomitant reduction in the amount of CaMV genomes accumulated.  Furthermore, the magnitude of this mutagenic effect on CaMV accumulation is positively correlated with the level of AtCDA1 mRNA expression in the plant. Conclusions: Our results suggest that deamination of viral genomes may also work as an antiviral mechanism in plants.

  6. Assay for mutagenesis in heterozygous diploid human lymphoblasts

    Science.gov (United States)

    Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

    1981-01-01

    An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

  7. Cordycepin Increases Nonrapid Eye Movement Sleep via Adenosine Receptors in Rats.

    Science.gov (United States)

    Hu, Zhenzhen; Lee, Chung-Il; Shah, Vikash Kumar; Oh, Eun-Hye; Han, Jin-Yi; Bae, Jae-Ryong; Lee, Kinam; Chong, Myong-Soo; Hong, Jin Tae; Oh, Ki-Wan

    2013-01-01

    Cordycepin (3'-deoxyadenosine) is a naturally occurring adenosine analogue and one of the bioactive constituents isolated from Cordyceps militaris/Cordyceps sinensis, species of the fungal genus Cordyceps. It has traditionally been a prized Chinese folk medicine for the human well-being. Because of similarity of chemical structure of adenosine, cordycepin has been focused on the diverse effects of the central nervous systems (CNSs), like sleep regulation. Therefore, this study was undertaken to know whether cordycepin increases the natural sleep in rats, and its effect is mediated by adenosine receptors (ARs). Sleep was recorded using electroencephalogram (EEG) for 4 hours after oral administration of cordycepin in rats. Sleep architecture and EEG power spectra were analyzed. Cordycepin reduced sleep-wake cycles and increased nonrapid eye movement (NREM) sleep. Interestingly, cordycepin increased θ (theta) waves power density during NREM sleep. In addition, the protein levels of AR subtypes (A1, A2A, and A2B) were increased after the administration of cordycepin, especially in the rat hypothalamus which plays an important role in sleep regulation. Therefore, we suggest that cordycepin increases theta waves power density during NREM sleep via nonspecific AR in rats. In addition, this experiment can provide basic evidence that cordycepin may be helpful for sleep-disturbed subjects.

  8. Cordycepin Increases Nonrapid Eye Movement Sleep via Adenosine Receptors in Rats

    Directory of Open Access Journals (Sweden)

    Zhenzhen Hu

    2013-01-01

    Full Text Available Cordycepin (3′-deoxyadenosine is a naturally occurring adenosine analogue and one of the bioactive constituents isolated from Cordyceps militaris/Cordyceps sinensis, species of the fungal genus Cordyceps. It has traditionally been a prized Chinese folk medicine for the human well-being. Because of similarity of chemical structure of adenosine, cordycepin has been focused on the diverse effects of the central nervous systems (CNSs, like sleep regulation. Therefore, this study was undertaken to know whether cordycepin increases the natural sleep in rats, and its effect is mediated by adenosine receptors (ARs. Sleep was recorded using electroencephalogram (EEG for 4 hours after oral administration of cordycepin in rats. Sleep architecture and EEG power spectra were analyzed. Cordycepin reduced sleep-wake cycles and increased nonrapid eye movement (NREM sleep. Interestingly, cordycepin increased θ (theta waves power density during NREM sleep. In addition, the protein levels of AR subtypes (A1, A2A, and A2B were increased after the administration of cordycepin, especially in the rat hypothalamus which plays an important role in sleep regulation. Therefore, we suggest that cordycepin increases theta waves power density during NREM sleep via nonspecific AR in rats. In addition, this experiment can provide basic evidence that cordycepin may be helpful for sleep-disturbed subjects.

  9. Photoreaction of 4,5',8-trimethylpsoralen with adenosine

    International Nuclear Information System (INIS)

    Sangchul Shim; Seungju Choi

    1990-01-01

    The near-UV induced photoreaction of 4,5',8-trimethylpsoralen (TMP) with adenosine was investigated in a dry film state. Four major photoadducts were isolated and purified by reverse-phase liquid chromatography. The structures of the photoproducts were elucidated on the basis of spectroscopic methods, including UV, FT-IR, mass spectrometry (FAB and EI methods) and 1 H-NMR analysis. These photoproducts were characterized to be TMP-adenosine 1:1 adducts, which resulted from the covalent bond formation between the carbon C(4) of TMP and ribose 1' or 5' carbon of adenosine. Of the photoadducts, one photoadduct (V) was the major product, reflecting some selectivity in the photoreaction of TMP with adenosine in the solid state. (author)

  10. Mechanism of protection of adenosine from sulphate radical anion ...

    Indian Academy of Sciences (India)

    Unknown

    Keywords. Repair by caffeic acid; repair of adenosine radicals; oxidation by sulphate radical anions. ... known that hydroxycinnamic acids are natural anti- oxidants ... acid. 2. Experimental ..... ously and independently under kinetic conditions at.

  11. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

    Science.gov (United States)

    Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

    2010-01-01

    Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the presence of colony stimulating factor-1 and receptor activator of NF-κB ligand in the presence or absence of the A1R antagonist 1,3-dipropyl-8-cyclopentyl xanthine (DPCPX). Osteoclast morphology was analyzed in tartrate-resistant acid phosphatase or F-actin-stained samples, and bone resorption was evaluated by toluidine blue staining of dentin. BMCs from A1R-knockout mice form fewer osteoclasts than BMCs from WT mice, and the A1R antagonist DPCPX inhibits osteoclast formation (IC50=1 nM), with altered morphology and reduced ability to resorb bone. A1R blockade increased ubiquitination and degradation of TRAF6 in RAW264.7 cells induced to differentiate into osteoclasts. These studies suggest a critical role for adenosine in bone homeostasis via interaction with adenosine A1R and further suggest that A1R may be a novel pharmacologic target to prevent the bone loss associated with inflammatory diseases and menopause.—Kara, F. M., Chitu, V., Sloane, J., Axelrod, M., Fredholm, B. B., Stanley, R., Cronstein, B. N. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function. PMID:20181934

  12. Interstitial adenosine concentration is increased by dipyridamole

    International Nuclear Information System (INIS)

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

    1986-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Guan-Jing; Li, Lan-Fen; Li, Dan; Liu, Cong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China); Wei, Shi-Cheng, E-mail: kqsc-wei@bjmu.edu.cn [Peking University School of Stomatology, Beijing 100081 (China); Liang, Yu-He, E-mail: kqsc-wei@bjmu.edu.cn; Su, Xiao-Dong [National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871 (China)

    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.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

    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 1 2 1 2 1 , with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 Å

  15. Crystallization and preliminary X-ray characterization of the tetrapyrrole-biosynthetic enzyme porphobilinogen deaminase from Bacillus megaterium

    International Nuclear Information System (INIS)

    Azim, N.; Deery, E.; Warren, M. J.; Erskine, P.; Cooper, J. B.; Wood, S. P.; Akhtar, M.

    2013-01-01

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step in the biosynthesis of tetrapyrroles in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. PBGD from B. megaterium was expressed and the enzyme was crystallized in a form which diffracts synchrotron radiation to high resolution. The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Expression in Escherichia coli of a His-tagged form of Bacillus megaterium PBGD permitted the crystallization and preliminary X-ray analysis of the enzyme from this species at high resolution

  16. Unfolding Role of a Danger Molecule Adenosine Signaling in Modulation of Microbial Infection and Host Cell Response

    Directory of Open Access Journals (Sweden)

    Jaden S. Lee

    2018-01-01

    Full Text Available Ectonucleotidases CD39 and CD73, specific nucleotide metabolizing enzymes located on the surface of the host, can convert a pro-inflammatory environment driven by a danger molecule extracellular-ATP to an adenosine-mediated anti-inflammatory milieu. Accordingly, CD39/CD73 signaling has been strongly implicated in modulating the intensity, duration, and composition of purinergic danger signals delivered to host. Recent studies have eluted potential roles for CD39 and CD73 in selective triggering of a variety of host immune cells and molecules in the presence of pathogenic microorganisms or microbial virulence molecules. Growing evidence also suggests that CD39 and CD73 present complimentary, but likely differential, actions against pathogens to shape the course and severity of microbial infection as well as the associated immune response. Similarly, adenosine receptors A2A and A2B have been proposed to be major immunomodulators of adenosine signaling during chronic inflammatory conditions induced by opportunistic pathogens, such as oral colonizer Porphyromonas gingivalis. Therefore, we here review the recent studies that demonstrate how complex network of molecules in the extracellular adenosine signaling machinery and their interactions can reshape immune responses and may also be targeted by opportunistic pathogens to establish successful colonization in human mucosal tissues and modulate the host immune response.

  17. Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

    Science.gov (United States)

    Chiavegatti, T; Costa, V L; Araújo, M S; Godinho, R O

    2007-01-01

    Background and purpose: cAMP is a key intracellular signalling molecule that regulates multiple processes of the vertebrate skeletal muscle. We have shown that cAMP can be actively pumped out from the skeletal muscle cell. Since in other tissues, cAMP efflux had been associated with extracellular generation of adenosine, in the present study we have assessed the fate of interstitial cAMP and the existence of an extracellular cAMP-adenosine signalling pathway in skeletal muscle. Experimental approach: cAMP efflux and/or its extracellular degradation were analysed by incubating rat cultured skeletal muscle with exogenous cAMP, forskolin or isoprenaline. cAMP and its metabolites were quantified by radioassay or HPLC, respectively. Key results: Incubation of cells with exogenous cAMP was followed by interstitial accumulation of 5′-AMP and adenosine, a phenomenon inhibited by selective inhibitors of ecto-phosphodiesterase (DPSPX) and ecto-nucleotidase (AMPCP). Activation of adenylyl cyclase (AC) in cultured cells with forskolin or isoprenaline increased cAMP efflux and extracellular generation of 5′-AMP and adenosine. Extracellular cAMP-adenosine pathway was also observed after direct and receptor-dependent stimulation of AC in rat extensor muscle ex vivo. These events were attenuated by probenecid, an inhibitor of ATP binding cassette family transporters. Conclusions and implications: Our results show the existence of an extracellular biochemical cascade that converts cAMP into adenosine. The functional relevance of this extracellular signalling system may involve a feedback modulation of cellular response initiated by several G protein-coupled receptor ligands, amplifying cAMP influence to a paracrine mode, through its metabolite, adenosine. PMID:18157164

  18. Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

    Directory of Open Access Journals (Sweden)

    Thomas Pleli

    2018-03-01

    Full Text Available Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2 inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors. In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

  19. Extracellular adenosine controls NKT-cell-dependent hepatitis induction.

    Science.gov (United States)

    Subramanian, Meenakshi; Kini, Radhika; Madasu, Manasa; Ohta, Akiko; Nowak, Michael; Exley, Mark; Sitkovsky, Michail; Ohta, Akio

    2014-04-01

    Extracellular adenosine regulates inflammatory responses via the A2A adenosine receptor (A2AR). A2AR deficiency results in much exaggerated acute hepatitis, indicating nonredundancy of adenosine-A2AR pathway in inhibiting immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. An A2AR agonist abolished NKT-cell-dependent induction of acute hepatitis by concanavalin A (Con A) or α-galactosylceramide in mice, corresponding to downregulation of activation markers and cytokines in NKT cells and of NK-cell co-activation. These results show that A2AR signaling can downregulate NKT-cell activation and suppress NKT-cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-galactosylceramide induced more severe hepatitis in A2AR-deficient mice than in WT controls. Transfer of A2AR-deficient NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT-cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT-cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression

    Science.gov (United States)

    Headrick, John P; Willems, Laura; Ashton, Kevin J; Holmgren, Kirsten; Peart, Jason; Matherne, G Paul

    2003-01-01

    The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2–4 months) and moderately aged (16–18 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 ± 2 vs. 18 ± 2 mmHg in young) and reduced developed (37 ± 3 vs. 83 ± 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 ± 2 vs. 16 ± 2 IU g−1in young) whereas the incidence of tachyarrhythmias was similar in young (15 ± 1 %) and aged hearts (16 ± 1 %). Functional analysis confirmed equipotent effects of 50 μm adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 μm adenosine improved diastolic (5 ± 1 mmHg) and developed pressures (134 ± 7 mmHg) and LDH loss (6 ± 2 IU g−1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to ∼10 %). In contrast to adenosine, 50 μm diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 μm 5-hydroxydecanoate (5-HD) and 3 μm chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 μm 8-sulfophenyltheophylline) and A1-selective (150 nm 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (∼8 fmol mg−1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring

  1. Neuropharmacology of purinergic receptors in human submucous plexus: Involvement of P2X₁, P2X₂, P2X₃ channels, P2Y and A₃ metabotropic receptors in neurotransmission.

    Science.gov (United States)

    Liñán-Rico, A; Wunderlich, J E; Enneking, J T; Tso, D R; Grants, I; Williams, K C; Otey, A; Michel, K; Schemann, M; Needleman, B; Harzman, A; Christofi, F L

    2015-08-01

    The role of purinergic signaling in human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. LSCM-Fluo-4/(Ca(2+))-imaging of postsynaptic Ca(2+) transients (PSCaTs) was used as a reporter of synaptic transmission evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons (identified by HuC/D-immunoreactivity) in 235 ganglia from 107 patients; P2XR-immunoreactivity was evaluated in 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging tested effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines: Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50 = 25 µM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50 = 111 nM) in neurons without stimulatory ADPbS responses (EC50 = 960 nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca(2+) transients or Ca(2+) oscillations (ATP,EC50 = 400 mM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20 nM) or high (5 µM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-immunoreactivity follow the order P2X2 > P2X1 > P2X3; P2X1 + P2X2 and P2X3 + P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7, P2Y1,2,12-14R. Purines are critical regulators of neurotransmission in human ENS. Purinergic signaling involves P2X1, P2X2, P2X3 channels, P2X1 + P2X2 co-localization and inhibitory P2Y or A3 receptors. These are

  2. Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function

    OpenAIRE

    Kara, Firas M.; Chitu, Violeta; Sloane, Jennifer; Axelrod, Matthew; Fredholm, Bertil B.; Stanley, E. Richard; Cronstein, Bruce N.

    2010-01-01

    Adenosine regulates a wide variety of physiological processes via interaction with one or more G-protein-coupled receptors (A1R, A2AR, A2BR, and A3R). Because A1R occupancy promotes fusion of human monocytes to form giant cells in vitro, we determined whether A1R occupancy similarly promotes osteoclast function and formation. Bone marrow cells (BMCs) were harvested from C57Bl/6 female mice or A1R-knockout mice and their wild-type (WT) littermates and differentiated into osteoclasts in the pre...

  3. Regioselective 1-N-Alkylation and Rearrangement of Adenosine Derivatives.

    Science.gov (United States)

    Oslovsky, Vladimir E; Drenichev, Mikhail S; Mikhailov, Sergey N

    2015-01-01

    Several methods for the preparation of some N(6)-substituted adenosines based on selective 1-N-alkylation with subsequent Dimroth rearrangement were developed. The proposed methods seem to be effective for the preparation of natural N(6)-isopentenyl- and N(6)-benzyladenosines, which are known to possess pronounced biological activities. Direct 1-N-alkylation of 2',3',5'-tri-O-acetyladenosine and 3',5'-di-O-acetyl-2'-deoxyadenosine with alkyl halides in N,N-dimethylformamide (DMF) in the presence of BaCO3 and KI gave 1-N-substituted derivatives with quantitative yields, whereas 1-N-alkylation of adenosine was accompanied by significant O-alkylation. Moreover, the reaction of trimethylsilyl derivatives of N(6)-acetyl-2',3',5'-tri-O-acetyladenosine and N(6)-acetyl-3',5'-di-O-acetyl-2'-deoxyadenosine with alkyl halides leads to the formation of the stable 1-N-substituted adenosines. Dimroth rearrangement of 1-N-substituted adenosines in aqueous ammonia yields pure N(6)-substituted adenosines.

  4. Preclinical studies on [{sup 11}C]MPDX for mapping adenosine A{sub 1} receptors by positron emission tomography

    Energy Technology Data Exchange (ETDEWEB)

    Ishiwata, Kiichi; Kimura, Yuichi; Oda, Keiichi; Kawamura, Kazunori; Ishii, Kenji; Senda, Michio [Tokyo Metropolitan Inst. of Gerontology (Japan). Positron Medical Center; Nariai, Tadashi; Wakabayashi, Shinichi [Tokyo Medical and Dental Univ. (Japan). School of Medicine; Shimada, Junichi [Kyowa Hakko Kogyo Co. Ltd., Tokyo (Japan). Pharmaceutical Research Inst.

    2002-09-01

    In previous in vivo studies with mice, rats and cats, we have demonstrated that [{sup 11}C]MPDX ([1-methyl-{sup 11}C]8-dicyclopropylmethyl-1-methyl-3-propylxanthine) is a potential radioligand for mapping adenosine A{sub 1} receptors of the brain by positron emission tomography (PET). In the present study, we performed a preclinical study. The radiation absorbed-dose by [{sup 11}C]MPDX in humans estimated from the tissue distribution in mice was low enough for clinical use, and the acute toxicity and mutagenicity of MPDX were not found. The monkey brain was clearly visualized by PET with [{sup 11}C]MPDX. We have concluded that [{sup 11}C]MPDX is suitable for mapping adenosine A{sub 1} receptors in the human brain by PET. (author)

  5. Importance of tissue perfusion in ST segment elevation myocardial infarction patients undergoing reperfusion strategies: role of adenosine.

    Science.gov (United States)

    Forman, Mervyn B; Jackson, Edwin K

    2007-11-01

    High risk ST segment elevation myocardial infarction (STEMI) patients undergoing reperfusion therapy continue to exhibit significant morbidity and mortality due in part to myocardial reperfusion injury. Importantly, preclinical studies demonstrate that progressive microcirculatory failure (the "no-reflow" phenomenon) contributes significantly to myocardial reperfusion injury. Diagnostic techniques to measure tissue perfusion have validated this concept in humans, and it is now clear that abnormal tissue perfusion occurs frequently in STEMI patients undergoing reperfusion therapy. Moreover, because tissue perfusion correlates poorly with epicardial blood flow (TIMI flow grade), clinical studies show that tissue perfusion is an independent predictor of early and late mortality in STEMI patients and is associated with infarct size, ventricular function, CHF and ventricular arrhythmias. The mechanisms responsible for abnormal tissue perfusion are multifactorial and include both mechanical obstruction and vasoconstrictor humoral factors. Adenosine, an endogenous nucleoside, maintains microcirculatory flow following reperfusion by activating four well-characterized extracellular receptors. Because activation of adenosine receptors attenuates the mechanical and functional mechanisms leading to the "no reflow" phenomenon and activates other cardioprotective pathways as well, it is not surprising that both experimental and clinical studies show striking myocardial salvage with intravenous infusions of adenosine administered in the peri-reperfusion period. For example, a post hoc analysis of the AMISTAD II trial indicates a significant reduction in 1 and 6-month mortality in STEMI patients undergoing reperfusion therapy who are treated with adenosine within 3 hours of symptoms. In conclusion, adenosine's numerous cardioprotective effects, including attenuation of the "no-reflow" phenomenon, support its use in high risk STEMI undergoing reperfusion.

  6. Chronic sleep restriction induces long-lasting changes in adenosine and noradrenaline receptor density in the rat brain.

    Science.gov (United States)

    Kim, Youngsoo; Elmenhorst, David; Weisshaupt, Angela; Wedekind, Franziska; Kroll, Tina; McCarley, Robert W; Strecker, Robert E; Bauer, Andreas

    2015-10-01

    Although chronic sleep restriction frequently produces long-lasting behavioural and physiological impairments in humans, the underlying neural mechanisms are unknown. Here we used a rat model of chronic sleep restriction to investigate the role of brain adenosine and noradrenaline systems, known to regulate sleep and wakefulness, respectively. The density of adenosine A1 and A2a receptors and β-adrenergic receptors before, during and following 5 days of sleep restriction was assessed with autoradiography. Rats (n = 48) were sleep-deprived for 18 h day(-1) for 5 consecutive days (SR1-SR5), followed by 3 unrestricted recovery sleep days (R1-R3). Brains were collected at the beginning of the light period, which was immediately after the end of sleep deprivation on sleep restriction days. Chronic sleep restriction increased adenosine A1 receptor density significantly in nine of the 13 brain areas analysed with elevations also observed on R3 (+18 to +32%). In contrast, chronic sleep restriction reduced adenosine A2a receptor density significantly in one of the three brain areas analysed (olfactory tubercle which declined 26-31% from SR1 to R1). A decrease in β-adrenergic receptors density was seen in substantia innominata and ventral pallidum which remained reduced on R3, but no changes were found in the anterior cingulate cortex. These data suggest that chronic sleep restriction can induce long-term changes in the brain adenosine and noradrenaline receptors, which may underlie the long-lasting neurocognitive impairments observed in chronic sleep restriction. © 2015 European Sleep Research Society.

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

    Science.gov (United States)

    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.

  8. Significance of determination of serum cytidine deaminase (CD) levels for diagnosis of active rheumatoid arthritis (RA)

    International Nuclear Information System (INIS)

    Xiao Chuangqing; Jang Xiaogong; He Yunnan

    2005-01-01

    Objective: To determine the clinical value of measurement of serum cytidine deaminase (CD) levels in patients with active rheumatoid arthritis (RA). Methods: Serum levels of CD were detected with spectrophotometry, in 33 patients with active RA and 60 controls. The erythrocyte sedimentation rate (ESR) and CRP content were also determined in both groups. Results: The ser- um CD contents in patients with active RA(14.80 ± 2.11U/ml) were significantly higher than those in controls(4.86±1.86 U/ml,P<0.01). The CRP contents (51.46 ± 20.43mg/L) and ESR readings(85.03 ± 27.6mm/h) in the patients were also significantly higher than those in the controls(3.40 ± 2.21mg/L and 13.04 ± 4.89mm/h respectively, all P<0.01). In the patients, the serum CD contents were linearly positively correlated with the ESR contents and CRP readings (r=0.6324 and 0.8013 respectively, P <0.01). Conclusion: Serum CD is an early biochemical marker for diagnosis of active rheumatoid arthritis and is also of prognostic value. (authors)

  9. Drought response of Mucuna pruriens (L. DC. inoculated with ACC deaminase and IAA producing rhizobacteria.

    Directory of Open Access Journals (Sweden)

    Aansa Rukya Saleem

    Full Text Available Drought is one of the major constraints limiting agricultural production worldwide and is expected to increase in the future. Limited water availability causes significant effects to plant growth and physiology. Plants have evolved different traits to mitigate the stress imposed by drought. The presence of plant growth-promoting rhizobacteria (PGPR could play an important role in improving plant performances and productivity under drought. These beneficial microorganisms colonize the rhizosphere of plants and increase drought tolerance by lowering ethylene formation. In the present study, we demonstrate the potential to improve the growth of velvet bean under water deficit conditions of two different strains of PGPR with ACCd (1-Aminocyclopropane-1-Carboxylate deaminase activity isolated from rainfed farming system. We compared uninoculated and inoculated plants with PGPR to assess: a photosynthetic performance and biomass; b ACC content and ethylene emission from leaves and roots; c leaf isoprene emission. Our results provided evidence that under drought conditions inoculation with PGPR containing the ACCd enzyme could improve plant growth compared to untreated plants. Ethylene emission from roots and leaves of inoculated velvet bean plants was significantly lower than uninoculated plants. Moreover, isoprene emission increased with drought stress progression and was higher in inoculated plants compared to uninoculated counterparts. These findings clearly illustrate that selected PGPR strains isolated from rainfed areas could be highly effective in promoting plant growth under drought conditions by decreasing ACC and ethylene levels in plants.

  10. Drought response of Mucuna pruriens (L.) DC. inoculated with ACC deaminase and IAA producing rhizobacteria.

    Science.gov (United States)

    Saleem, Aansa Rukya; Brunetti, Cecilia; Khalid, Azeem; Della Rocca, Gianni; Raio, Aida; Emiliani, Giovanni; De Carlo, Anna; Mahmood, Tariq; Centritto, Mauro

    2018-01-01

    Drought is one of the major constraints limiting agricultural production worldwide and is expected to increase in the future. Limited water availability causes significant effects to plant growth and physiology. Plants have evolved different traits to mitigate the stress imposed by drought. The presence of plant growth-promoting rhizobacteria (PGPR) could play an important role in improving plant performances and productivity under drought. These beneficial microorganisms colonize the rhizosphere of plants and increase drought tolerance by lowering ethylene formation. In the present study, we demonstrate the potential to improve the growth of velvet bean under water deficit conditions of two different strains of PGPR with ACCd (1-Aminocyclopropane-1-Carboxylate deaminase) activity isolated from rainfed farming system. We compared uninoculated and inoculated plants with PGPR to assess: a) photosynthetic performance and biomass; b) ACC content and ethylene emission from leaves and roots; c) leaf isoprene emission. Our results provided evidence that under drought conditions inoculation with PGPR containing the ACCd enzyme could improve plant growth compared to untreated plants. Ethylene emission from roots and leaves of inoculated velvet bean plants was significantly lower than uninoculated plants. Moreover, isoprene emission increased with drought stress progression and was higher in inoculated plants compared to uninoculated counterparts. These findings clearly illustrate that selected PGPR strains isolated from rainfed areas could be highly effective in promoting plant growth under drought conditions by decreasing ACC and ethylene levels in plants.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Yi, E-mail: yihooyi@gmail.com; Ericsson, Ida, E-mail: ida.ericsson@ntnu.no; Doseth, Berit, E-mail: berit.doseth@ntnu.no; Liabakk, Nina B., E-mail: nina.beate.liabakk@ntnu.no; Krokan, Hans E., E-mail: hans.krokan@ntnu.no; Kavli, Bodil, E-mail: bodil.kavli@ntnu.no

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  13. High frequencies of chromatid aberrations produced during G/sub 2/ in human lymphocytes by very low doses (0. 025-0. 4 Gy) of X-rays in combination with inhibitors of DNA synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, H.C.; Kihlman, B.A. (Uppsala Univ. (Sweden). Dept. of Genetics)

    1984-09-01

    Whole-blood cultures of human lymphocytes were exposed in the G/sub 2/-phase (3.5 h before harvesting) to various doses of X-rays and post-treated for 3 h with inhibitors of DNA synthesis. The inhibitors used were 2'-deoxyadenosine (dAdo), hydroxyurea (HU) and 1-..beta..-D-arabinofuranosylcytosine (ara-C). To prevent deamination of dAdo by adenosine deaminase (ADA), the dAdo treatments were carried out in the presence of the ADA inhibitor coformycin. HU and ara-C were used either alone or in combination. After the 3-h inhibitor treatments, the cultures were harvested and slides prepared and analyzed for chromatid aberrations in metaphase. When the inhibitors were used at concentrations high enough to cause marked chromosome damage by themselves, very low doses of X-rays (0.025-0.2 Gy) were sufficient to produce a dramatic increase in the frequency of chromatid aberrations. High frequencies of chromatid aberrations were also obtained when cultures that had received moderate doses of X-rays (0.4-0.8 Gy) were post-treated with low inhibitor concentrations that produce no or only a few aberrations by themselves.

  14. Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson's Disease

    National Research Council Canada - National Science Library

    Schwarzschild, Michael A; Xu, Kui

    2008-01-01

    ...) that are leading candidate modulators of PD risk. In Year 4 we have obtained and reported evidence that the adenosine receptor blocker caffeine as well as specific genetic depletion of the A2A subtype of adenosine receptor...

  15. Fractional Flow Reserve: Intracoronary versus intravenous adenosine induced maximal coronary hyperemia

    Directory of Open Access Journals (Sweden)

    P.S. Sandhu

    2013-03-01

    Conclusions: This study suggests that IC adenosine is equivalent to IV infusion for the determination of FFR. The administration of IC adenosine is easy to use, cost effective, safe and associated with fewer systemic events.

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

    Science.gov (United States)

    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.

  17. Purification and properties of adenosine kinase from rat brain.

    Science.gov (United States)

    Yamada, Y; Goto, H; Ogasawara, N

    1980-12-04

    Adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) has been purified to apparent homogeneity from rat brain by (NH4)2SO4 fractionation, affinity chromatography on AMP-Sepharose 4B, gel filtration with Sephadex G-100, and DE-52 cellulose column chromatography. The yield was 56% of the initial activity with a final specific activity of 7.8 mumol/min per mg protein. The molecular weight was estimated as 38 000 by gel filtration with Sephadex G-100 and 41 000 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). The enzyme catalyzed the phosphorylation of adenosine, deoxyadenosine, arabinoadenosine, inosine and ribavirin. The activity of deoxyadenosine phosphorylation was 20% that of adenosine phosphorylation. The pH optimum profile was biphasic; a sharp pH optimum at pH 5.5 and a broad pH optimum at pH 7.5-8.5. The Km value for adenosine was 0.2 microM and the maximum activity was observed at 0.5 microM. At higher concentrations of adenosine, the activity was strongly inhibited. The Km value for ATP was 0.02 mM and that for Mg2+ was 0.1 mM. GTP, dGTP, dATP and UTP were also proved to be effective phosphate donors. Co2+ was as effective as Mg2+, and Ca2+, Mn2+ or Ni2+ showed about 50% of the activity for Mg2+. The kinase is quite unstable, but stable in the presence of a high concentration of salt; e.g., 0.15 M KCl.

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

    Energy Technology Data Exchange (ETDEWEB)

    Hung, Szu-Ying; Shih, Ya-Chen [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); Tseng, Wei-Lung, E-mail: tsengwl@mail.nsysu.edu.tw [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan (China); Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Taiwan (China); Center for Stem Cell Research, Kaohsiung Medical University, Taiwan (China)

    2015-02-01

    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

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

    Science.gov (United States)

    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

  1. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    Science.gov (United States)

    2016-03-01

    diabetic retinopathy . Life Sci. 2013 Jul 30;93(2-3):78-88. doi: 10.1016/j.lfs.2013.05.024. Epub 2013 Jun 12.PMID:23770229 7 AIMS: This study was...undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling...reported recently that adenosine kinase upregulated in retinal tissue of diabetic retinopathy (Elsherbiny et al., 2013). Adenosine kinase (ADK) converts

  2. A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase.

    Science.gov (United States)

    Li, Z; Chang, S; Lin, L; Li, Y; An, Q

    2011-08-01

    1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates. © 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.

  3. Gene expression profiles in adenosine-treated human mast cells

    African Journals Online (AJOL)

    ajl yemi

    2011-10-26

    Oct 26, 2011 ... assignment (Ewing and Green, 1998a; Ewing et al., 1998b). The trace files were trimmed with trim-alt 0.05 (P-score>20). In addition, vector trimming was conducted with cross-match software. Each gene expression pattern was analyzed by clustering. (30 bp or more 94% homology) and assembly.

  4. Novel ligands for the human adenosine A1 receptor

    NARCIS (Netherlands)

    Chang, Lisa Chung Wai

    2005-01-01

    This research describes the quest to create 'super-caffeines', substances that only produce the desired effects of caffeine, and unlike caffeine, substances that should only have to be taken in measured, minute, controlled amounts to achieve these effects. Unless particular steps are taken to avoid

  5. DMPD: Shaping of monocyte and macrophage function by adenosine receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17056121 Shaping of monocyte and macrophage function by adenosine receptors. Hasko ...tml) (.csml) Show Shaping of monocyte and macrophage function by adenosine receptors. PubmedID 17056121 Titl...e Shaping of monocyte and macrophage function by adenosine receptors. Authors Has

  6. The role of glial adenosine receptors in neural resilience and the neurobiology of mood disorders

    NARCIS (Netherlands)

    Calker, D; Biber, K

    2005-01-01

    Adenosine receptors were classified into A(1)- and A(2)-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that

  7. Adenosine kinase deficiency disrupts the methionine cycle and causes hypermethioninemia, encephalopathy, and abnormal liver function.

    Science.gov (United States)

    Bjursell, Magnus K; Blom, Henk J; Cayuela, Jordi Asin; Engvall, Martin L; Lesko, Nicole; Balasubramaniam, Shanti; Brandberg, Göran; Halldin, Maria; Falkenberg, Maria; Jakobs, Cornelis; Smith, Desiree; Struys, Eduard; von Döbeln, Ulrika; Gustafsson, Claes M; Lundeberg, Joakim; Wedell, Anna

    2011-10-07

    Four inborn errors of metabolism (IEMs) are known to cause hypermethioninemia by directly interfering with the methionine cycle. Hypermethioninemia is occasionally discovered incidentally, but it is often disregarded as an unspecific finding, particularly if liver disease is involved. In many individuals the hypermethioninemia resolves without further deterioration, but it can also represent an early sign of a severe, progressive neurodevelopmental disorder. Further investigation of unclear hypermethioninemia is therefore important. We studied two siblings affected by severe developmental delay and liver dysfunction. Biochemical analysis revealed increased plasma levels of methionine, S-adenosylmethionine (AdoMet), and S-adenosylhomocysteine (AdoHcy) but normal or mildly elevated homocysteine (Hcy) levels, indicating a block in the methionine cycle. We excluded S-adenosylhomocysteine hydrolase (SAHH) deficiency, which causes a similar biochemical phenotype, by using genetic and biochemical techniques and hypothesized that there was a functional block in the SAHH enzyme as a result of a recessive mutation in a different gene. Using exome sequencing, we identified a homozygous c.902C>A (p.Ala301Glu) missense mutation in the adenosine kinase gene (ADK), the function of which fits perfectly with this hypothesis. Increased urinary adenosine excretion confirmed ADK deficiency in the siblings. Four additional individuals from two unrelated families with a similar presentation were identified and shown to have a homozygous c.653A>C (p.Asp218Ala) and c.38G>A (p.Gly13Glu) mutation, respectively, in the same gene. All three missense mutations were deleterious, as shown by activity measurements on recombinant enzymes. ADK deficiency is a previously undescribed, severe IEM shedding light on a functional link between the methionine cycle and adenosine metabolism. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  8. Diagnostic Algorithm for Glycogenoses and Myoadenylate Deaminase Deficiency Based on Exercise Testing Parameters: A Prospective Study.

    Directory of Open Access Journals (Sweden)

    Fabrice Rannou

    Full Text Available Our aim was to evaluate the accuracy of aerobic exercise testing to diagnose metabolic myopathies.From December 2008 to September 2012, all the consecutive patients that underwent both metabolic exercise testing and a muscle biopsy were prospectively enrolled. Subjects performed an incremental and maximal exercise testing on a cycle ergometer. Lactate, pyruvate, and ammonia concentrations were determined from venous blood samples drawn at rest, during exercise (50% predicted maximal power, peak exercise, and recovery (2, 5, 10, and 15 min. Biopsies from vastus lateralis or deltoid muscles were analysed using standard techniques (reference test. Myoadenylate deaminase (MAD activity was determined using p-nitro blue tetrazolium staining in muscle cryostat sections. Glycogen storage was assessed using periodic acid-Schiff staining. The diagnostic accuracy of plasma metabolite levels to identify absent and decreased MAD activity was assessed using Receiver Operating Characteristic (ROC curve analysis.The study involved 51 patients. Omitting patients with glycogenoses (n = 3, MAD staining was absent in 5, decreased in 6, and normal in 37 subjects. Lactate/pyruvate at the 10th minute of recovery provided the greatest area under the ROC curves (AUC, 0.893 ± 0.067 to differentiate Abnormal from Normal MAD activity. The lactate/rest ratio at the 10th minute of recovery from exercise displayed the best AUC (1.0 for discriminating between Decreased and Absent MAD activities. The resulting decision tree achieved a diagnostic accuracy of 86.3%.The present algorithm provides a non-invasive test to accurately predict absent and decreased MAD activity, facilitating the selection of patients for muscle biopsy and target appropriate histochemical analysis.

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

    Science.gov (United States)

    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. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. PET imaging of adenosine A2A receptors

    NARCIS (Netherlands)

    Zhou, Xiaoyun

    2017-01-01

    This thesis describes the development and evaluation of [11C]preladenant as a novel radioligand for in vivo imaging of adenosine A2A receptors in the brain with positron-emission tomography (PET). The 11C-labeled drug [11C]preladenant was produced with high radiochemical yield and specific activity.

  11. Spectral studies of lanthanide-nucleic acid component interaction: complexes of adenine, adenosine, adenosine 5'-mono-, adenosine 5'-di- and adenosine 5' tri-phosphates with praseodymium(III)

    International Nuclear Information System (INIS)

    Joseph, George; Anjaiah, K.; Misra, S.N.

    1990-01-01

    The interactions of adenine, adenosine, adenosine 5'-mono-, adenosine 5'-di-and adenosine 5'-tri-phosphates with praseodymium(III) have been studied in different stoichiometries and at varying hydrogen ion concentrations by absorption spectral studies. The sharp bands in the spectra have been individually analysed by Gaussian curve analysis, and various spectral parameters have been computed using partial and multiple regression methods on an HP-1000/45 computer. The changes in and the magnitudes of these parameters have been correlated with the degrees of outer- and inner-sphere coordination around praseodymium(III). Crystalline complexes of the type: Pr(nucleotide) 2 (H 2 O) 2 (where nucleotide = AMP, ADP and ATP) have been characterized on the basis of analytical, IR and 1 H NMR spectral data. These studies indicate that the binding of the nucleotide is through phosphoric oxygen. These complexes in aqueous medium show significant ionization which supports the observed weak 4f-4f bands, lower values of nephelauxetic effect and the parameters derived from coulombic and spin-orbit interactions. (author). 3 t abs., 28 refs

  12. Adenosine receptor modulation of seizure susceptibility in rats

    International Nuclear Information System (INIS)

    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 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 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 1 adenosine receptors in the cerebral cortex

  13. Cerebral adenosine A1 receptors are upregulated in rodent encephalitis

    NARCIS (Netherlands)

    Paul, Souman; Khanapur, Shivashankar; Boersma, Wytske; Sijbesma, Jurgen W.; Ishiwata, Kiichi; Elsinga, Philip H.; Meerlo, Peter; Doorduin, Janine; Dierckx, Rudi A.; van Waarde, Aren

    2014-01-01

    Adenosine A(1) receptors (A(1) Rs) are implied in the modulation of neuroinflammation. Activation of cerebral A(1) Rs acts as a brake on the microglial response after traumatic brain injury and has neuroprotective properties in animal models of Parkinson's disease and multiple sclerosis.

  14. Adenosine Receptor Heteromers and their Integrative Role in Striatal Function

    Directory of Open Access Journals (Sweden)

    Sergi Ferré

    2007-01-01

    Full Text Available By analyzing the functional role of adenosine receptor heteromers, we review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS. Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a “biochemical fingerprint” to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as “processors” of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the “local module” (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit, where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module.

  15. The role of adenosine receptor agonists in regulation of hematopoiesis

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Weiterová, Lenka; Hoferová, Zuzana

    2011-01-01

    Roč. 16, č. 1 (2011), s. 675-685 ISSN 1420-3049 R&D Projects: GA MO OVBIOFYZ20101; GA ČR(CZ) GA305/08/0158 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : adenosine receptors * hematopoiesis * myelosuppression Subject RIV: BO - Biophysics Impact factor: 2.386, year: 2011

  16. A comparison of adenosine and arbutamine for myocardial perfusion imaging

    International Nuclear Information System (INIS)

    Anagnostopoulos, C.; Pennell, D.; Francis, J.; Serup-Hansen, K.; Davies, G.; Underwood, R.

    1998-01-01

    We have compared our standard stress protocol (adenosine combined with exercise) with the new stress agent arbutamine, for thallium-201 myocardial perfusion imaging (MPI) in order to assess the comparative value of arbutamine. We studied 23 patients referred for MPI, and each patient had two studies (18 males, median age 66 years, five with previous myocardial infarction). Uptake scores were assigned to each of nine segments, and the extent and severity of defects were measured using a polar plot. Haemodynamic changes were greater with arbutamine (rate-pressure product increase 78% vs 51%, P = 0.003). Symptoms were experienced by 21 patients with arbutamine and 16 with adenosine (P = 0.07). Agreement between the techniques for classification of patients as normal or as having reversible, fixed or mixed defects was good (19 of 23 studies, 83%, κ = 0.76). Agreement for similar classification of segments was also good (82%, κ = 0.71). Segmental agreement for stress scores was good (86%, κ = 0.77). However, mean size of stress defect was larger with adenosine (83±52 pixels vs 65±48 pixels, P<0.05), though severity and reversibility were similar (P = NS). We conclude that arbutamine provides comparable results to those obtained with adenosine and exercise and that the observed differences are not clinically significant. (orig.)

  17. Safety of adenosine in stress cerebral perfusion imaging

    International Nuclear Information System (INIS)

    Hu Pengcheng; Gu Yushen; Liu Wenguan; Xiu Yan; Zhu Weimin; Chen Shuguang; Shi Hongcheng

    2009-01-01

    Objective: To evaluate the safety of adenosine as pharmacological stress agents in stress cerebral perfusion imaging. Methods: Eighty patients under investigation for suspected cerebral vessel disease were recruited. Each had a resting scan and a stress scan on different days. The adenosine stress protocol was as same as the protocol used in adenosine stress myocardial perfusion imaging. Subjective and objective side-effects were investigated during pharmacological stress procedure. Results: All patients completed the 6 min infusion protocol without premature termination on safety criteria or due to intolerable symptoms. 46 patients had mild side effects. 20 patients (25%) had dizziness, 12 patients (15%) had palpitation, 1 patient (1%) was hypotensive, 7 patients (9%) had dyspnoea, 4 patients (5%) felt hot, 3 patients (4%) had sweat, 4 patients (5%) had nausea, 6 patients (8%) had flushing, 19 patients (24%) had chest pain, 6 patients (8%) had abdomen pain, 3 patients (4%) had abnormal taste and 1 patient (1%) were thirsty. Transient ST change occurred in only 1 patient. Conclusion: Adenosine stress cerebral perfusion imaging is a safe diagnostic method with mild side effects. (authors)

  18. Diversification of AID/APOBEC-like deaminases in metazoa: multiplicity of clades and widespread roles in immunity.

    Science.gov (United States)

    Krishnan, Arunkumar; Iyer, Lakshminarayan M; Holland, Stephen J; Boehm, Thomas; Aravind, L

    2018-04-03

    AID/APOBEC deaminases (AADs) convert cytidine to uridine in single-stranded nucleic acids. They are involved in numerous mutagenic processes, including those underpinning vertebrate innate and adaptive immunity. Using a multipronged sequence analysis strategy, we uncover several AADs across metazoa, dictyosteliida, and algae, including multiple previously unreported vertebrate clades, and versions from urochordates, nematodes, echinoderms, arthropods, lophotrochozoans, cnidarians, and porifera. Evolutionary analysis suggests a fundamental division of AADs early in metazoan evolution into secreted deaminases (SNADs) and classical AADs, followed by diversification into several clades driven by rapid-sequence evolution, gene loss, lineage-specific expansions, and lateral transfer to various algae. Most vertebrate AADs, including AID and APOBECs1-3, diversified in the vertebrates, whereas the APOBEC4-like clade has a deeper origin in metazoa. Positional entropy analysis suggests that several AAD clades are diversifying rapidly, especially in the positions predicted to interact with the nucleic acid target motif, and with potential viral inhibitors. Further, several AADs have evolved neomorphic metal-binding inserts, especially within loops predicted to interact with the target nucleic acid. We also observe polymorphisms, driven by alternative splicing, gene loss, and possibly intergenic recombination between paralogs. We propose that biological conflicts of AADs with viruses and genomic retroelements are drivers of rapid AAD evolution, suggesting a widespread presence of mutagenesis-based immune-defense systems. Deaminases like AID represent versions "institutionalized" from the broader array of AADs pitted in such arms races for mutagenesis of self-DNA, and similar recruitment might have independently occurred elsewhere in metazoa. Copyright © 2018 the Author(s). Published by PNAS.

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

    Science.gov (United States)

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

    2015-01-01

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

  20. Adenosine metabolism in Toxoplasma gondii: potential targets for chemotherapy.

    Science.gov (United States)

    el Kouni, Mahmoud H

    2007-01-01

    Toxoplasma gondii is an intracellular parasitic protozoan that infects approximately a billion people worldwide. Infection with T. gondii represents a major health problem for immunocompromised individuals, such as AIDS patients, organ transplant recipients, and the unborn children of infected mothers. Currently available drugs usually do not eradicate infection and as many as 50% of the patients do not respond to this therapy. Furthermore, they are ineffective against T. gondii tissue cysts. In addition, prolonged exposure to these drugs induces serious host toxicity forcing the discontinuation of the therapy. Finally, there is no effective vaccine currently available for the treatment of toxoplasmosis. Therefore, it is necessary to develop new and effective drugs for the treatment and management of toxoplasmosis. The rational design of a drug depends on the exploitation of fundamental biochemical or physiological differences between pathogens and their host. Some of the most striking differences between T. gondii and their mammalian host are found in purine metabolism. T. gondii, like most parasites studied, lack the ability to synthesize purines do novo and depend on the salvage of purines from their host to satisfy their requirements of purines. In this respect, the salvage of adenosine is the major source of purines in T. gondii. Therefore, interference with adenosine uptake and metabolism in T. gondii can be selectively detrimental to the parasite. The host cells, on the other hand, can still obtain their purine requirements by their de novo pathways. This review will focus on the broad aspects of the adenosine transport and the enzyme adenosine kinase (EC 2.7.1.20) which are the two primary routes for adenosine utilization in T. gondii, in an attempt to illustrate their potentials as targets for chemotherapy against this parasite.

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

    Science.gov (United States)

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

    2004-12-15

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

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

    Science.gov (United States)

    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.

  3. Structural Probing and Molecular Modeling of the A₃ Adenosine Receptor: A Focus on Agonist Binding.

    Science.gov (United States)

    Ciancetta, Antonella; Jacobson, Kenneth A

    2017-03-11

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A₁, A 2A , A 2B and A₃, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A₃AR (hA₃AR) subtype is implicated in several cytoprotective functions. Therefore, hA₃AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A₃AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A₃AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  4. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    Science.gov (United States)

    Ciancetta, Antonella; Jacobson, Kenneth A.

    2017-01-01

    Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR) superfamily. The human A3AR (hA3AR) subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure-activity relationships (SARs) of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM) data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates. PMID:28287473

  5. Structural Probing and Molecular Modeling of the A3 Adenosine Receptor: A Focus on Agonist Binding

    Directory of Open Access Journals (Sweden)

    Antonella Ciancetta

    2017-03-01

    Full Text Available Adenosine is an endogenous modulator exerting its functions through the activation of four adenosine receptor (AR subtypes, termed A1, A2A, A2B and A3, which belong to the G protein-coupled receptor (GPCR superfamily. The human A3AR (hA3AR subtype is implicated in several cytoprotective functions. Therefore, hA3AR modulators, and in particular agonists, are sought for their potential application as anti-inflammatory, anticancer, and cardioprotective agents. Structure-based molecular modeling techniques have been applied over the years to rationalize the structure–activity relationships (SARs of newly emerged A3AR ligands, guide the subsequent lead optimization, and interpret site-directed mutagenesis (SDM data from a molecular perspective. In this review, we showcase selected modeling-based and guided strategies that were applied to elucidate the binding of agonists to the A3AR and discuss the challenges associated with an accurate prediction of the receptor extracellular vestibule through homology modeling from the available X-ray templates.

  6. Effects of 2 adenosine antagonists, quercetin and caffeine, on vigilance and mood.

    Science.gov (United States)

    Olson, Craig A; Thornton, Jennifer A; Adam, Gina E; Lieberman, Harris R

    2010-10-01

    Quercetin, a phenolic flavonoid found in small quantities in some fruits and vegetables, is an adenosine receptor antagonist in vitro marketed as a dietary supplement for purported caffeine-like effects. A double-blind, placebo-controlled, between-subjects study was conducted to compare the behavioral effects of quercetin to a central adenosine receptor antagonist, caffeine. Fifty-seven volunteers received either 2000 mg of quercetin dihydrate (a dose estimated based on in vitro receptor binding to be equivalent in potency to 200 mg of caffeine), placebo, or 200 mg of caffeine. One hour later, a 45-minute visual vigilance task was administered. The Profile of Mood States questionnaire was completed before treatment and immediately after vigilance testing. On the vigilance task, caffeine increased the number of stimuli detected (P mood disturbance Profile of Mood States scores compared with placebo. Quercetin did not significantly alter any parameter, but values were typically intermediate between caffeine and placebo on those tests affected by caffeine. Quercetin is unlikely to have any effects when consumed by humans in quantities present in the diet or in dietary supplements. Caffeine (200 mg) administration resulted in the expected effects on vigilance and mood.

  7. Selection of efficient salt-tolerant bacteria containing ACC deaminase for promotion of tomato growth under salinity stress

    Directory of Open Access Journals (Sweden)

    Kannika Chookietwattana* and Kedsukon Maneewan

    2012-05-01

    Full Text Available For successful application of plant growth promoting bacteria (PGPB in salt-affected soil, bioinoculant with salt-tolerant property is required in order to provide better survival and perform well in the field. The present study aimed to select the most efficient salt-tolerant bacterium containing 1-aminocyclopropane-1-carboxylic acid (ACC deaminase from eighty four bacterial strains and to investigate the effects of the selected bacterium on the germination and growth of tomato (Licopersicon esculentum Mill. cv. Seeda under saline conditions. The Bacillus licheniformis B2r was selected for its ability to utilize ACC as a sole nitrogen source under salinity stress. It also showed a high ACC deaminase activity at 0.6 M NaCl salinity. Tomato plants inoculated with the selected bacterium under various saline conditions (0, 30, 60, 90 and 120 mM NaCl revealed a significant increase in the germination percentage, germination index, root length, and seedling dry weight especially at salinity levels ranging from 30-90 mM NaCl. The work described in this report is an important step in developing an efficient salt-tolerant bioinoculant to facilitate plant growth in saline soil.

  8. The Role of Adenosine A2BR in Metastatic Melanoma

    Science.gov (United States)

    2017-07-01

    burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense...would like to interrogate the role of adenosine receptor (A2BR) in regulating primary tumor growth and metastasis in experimental models of melanoma...The positive control was a triple negative breast cancer cell line, E0771. To interrogate the role of A2BR in aiding tumor metastasis, we used VeCad

  9. Synthesis of adenosine triphosphate tritiated in position 2 and 8

    International Nuclear Information System (INIS)

    Cossery, Jean-Michel

    1986-01-01

    Adenosine triphosphate or ATP is an important molecule present at the cellular level in many fundamental biochemical mechanism, and the study of its metabolism is therefore of particular interest. In this thesis for pharmacy graduation, the author first describes the different steps of synthesis and purification leading to chloride-2-ATP, a precursor of the final tritiated molecule. Then, the author explains the tritiation of this molecule to obtain an ATP tritiated in position 2 and in position 8 [fr

  10. Expression of activation-induced cytidine deaminase is confined to B-cell non-Hodgkin's lymphomas of germinal-center phenotype

    NARCIS (Netherlands)

    Smit, Laura A.; Bende, Richard J.; Aten, Jan; Guikema, Jeroen E. J.; Aarts, Wilhelmina M.; van Noesel, Carel J. M.

    2003-01-01

    Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation and class switch recombination of the immunoglobulin (IG) genes in B cells. It has recently been proposed that AID, as the newly identified DNA mutator in man, may be instrumental in initiation and progression of

  11. 5' adenosine monophosphate-activated protein kinase, metabolism and exercise.

    Science.gov (United States)

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

    2004-01-01

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

  12. The impact of adenosine pharmacologic stress combined with low-level exercise in patients undergoing myocardial perfusion imaging (BIWAKO adenosine-Ex trial)

    International Nuclear Information System (INIS)

    Monzen, Hajime; Hara, Masatake; Hirata, Makoto

    2011-01-01

    The combination of adenosine infusion with low-level exercise has become a common approach for inducing stress during stress myocardial perfusion imaging (MPI). We investigated stress MPI performed by combined low-level exercise and adenosine infusion. This combined protocol can decrease adverse reactions and reduce the effect of scattered rays from the liver. Subjects were clinically referred for a 53-min rest-stress Tc-99m Sestamibi MPI procedure using BIWAKO PROTOCOL. Ninety-eight patients (44.5%) underwent adenosine infusion with ergometer exercise testing and 122 patients (55.5%) underwent adenosine infusion without exercise testing. We evaluated the liver/heart (L/H) uptake ratio, background activity in the upper mediastinum, and adverse reactions. The L/H ratio and background activity were lower in the adenosine-exercise group than in the adenosine-non-exercise group (1.8±0.54 vs. 2.1±0.62, P<0.0056; 43.1±12.2 vs. 61.5±15.4, P<0.0001). The adenosine-exercise group had fewer adverse reactions than the adenosine-non-exercise group (11.2 vs. 19.7%). All of the adverse reactions were minor, with the exception of severe back pain in one case. The incidence of adverse reactions in our study was lower than that in previous studies for unknown reason. Adenosine infusion in combination with low-level exercise seems to result in higher-quality images and fewer adverse reactions than adenosine infusion without exercise. The combined protocol decreases adverse reactions and improves the quality of myocardial perfusion images by decreasing background activity. (author)

  13. Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

    Science.gov (United States)

    Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

    2006-10-01

    To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

  14. Acute hyperammonemia and systemic inflammation is associated with increased extracellular brain adenosine in rats

    DEFF Research Database (Denmark)

    Bjerring, Peter Nissen; Dale, Nicholas; Larsen, Fin Stolze

    2015-01-01

    ) and cerebral blood flow (CBF). We measured the adenosine concentration with biosensors in rat brain slices exposed to ammonia and in a rat model with hyperammonemia and systemic inflammation. Exposure to ammonia in concentrations from 0.15-10 mM led to increases in the cortical adenosine concentration up to 18......Acute liver failure (ALF) can lead to brain edema, cerebral hyperperfusion and intracranial hypertension. These complications are thought to be mediated by hyperammonemia and inflammation leading to altered brain metabolism. As increased levels of adenosine degradation products have been found...... in brain tissue of patients with ALF we investigated whether hyperammonemia could induce adenosine release in brain tissue. Since adenosine is a potent vasodilator and modulator of cerebral metabolism we furthermore studied the effect of adenosine receptor ligands on intracranial pressure (ICP...

  15. Semisynthesis, Characterization and Evaluation of New Adenosine Derivatives as Antiproliferative Agents

    Directory of Open Access Journals (Sweden)

    Francisco Valdés Zurita

    2018-05-01

    Full Text Available We describe the semisynthesis and biological effects of adenosine derivatives, which were anticipated to function as agonists for the A3 receptor. Molecular docking was used to select candidate compounds. Fifteen nucleoside derivatives were obtained through nucleophilic substitutions of the N6-position of the nucleoside precursor 6-chloropurine riboside by amines of different origin. All compounds were purified by column chromatography and further characterized by spectroscopic and spectrometric techniques, showing moderate yield. These molecules were then evaluated for their antiproliferative activity in human gastric cancer cells expressing the A3 receptor. We found that the compounds obtained have antiproliferative activity and that new structural modifications can enhance their biological activity. The ADME (Absorption, Distribution, Metabolism and Excretion properties of the most active compounds were also evaluated theoretically.

  16. Cyclic adenosine 3:5-monophosphate binding proteins in Hartmannella culbertsoni

    International Nuclear Information System (INIS)

    Verma, A.K.; Krishna Murti, C.R.

    1976-01-01

    When 100, 000 g supernatant fractions of homogenates of Hartmannella culbertsoni were incubated with ('- 3 H)-cyclic adenosine 3 : 5 monophosphate and passed through a sephadex G-100 column, radioactivity appeared with protein fractions eluted after the void colume. About 75% radioactivity bound to these fractions was recovered as cyclic adenosine 3 : 5 monophosphate. Unlabelled cAMP diluted the amount of radioactivity bound. Adenosine, deoxyadenosine, 5-AMP, 3-AMP, ADP and ATP did not inhibit binding. (author)

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

    OpenAIRE

    Chee, Hyun Keun; Oh, S. June

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine ...

  18. Extracellular Nucleotide Hydrolysis in Dermal and Limbal Mesenchymal Stem Cells: A Source of Adenosine Production.

    Science.gov (United States)

    Naasani, Liliana I Sous; Rodrigues, Cristiano; de Campos, Rafael Paschoal; Beckenkamp, Liziane Raquel; Iser, Isabele C; Bertoni, Ana Paula Santin; Wink, Márcia R

    2017-08-01

    Human Limbal (L-MSCs) and Dermal Mesenchymal Stem Cell (D-MSCs) possess many properties that increase their therapeutic potential in ophthalmology and dermatology. It is known that purinergic signaling plays a role in many aspects of mesenchymal stem cells physiology. They release and respond to purinergic ligands, altering proliferation, migration, differentiation, and apoptosis. Therefore, more information on these processes would be crucial for establishing future clinical applications using their differentiation potential, but without undesirable side effects. This study evaluated and compared the expression of ecto-nucleotidases, the enzymatic activity of degradation of extracellular nucleotides and the metabolism of extracellular ATP in D-MSCs and L-MSCs, isolated from discard tissues of human skin and sclerocorneal rims. The D-MSCs and L-MSCs showed a differentiation potential into osteogenic, adipogenic, and chondrogenic lineages and the expression of markers CD105 + , CD44 + , CD14 - , CD34 - , CD45 - , as expected. Both cells hydrolyzed low levels of extracellular ATP and high levels of AMP, leading to adenosine accumulation that can regulate inflammation and tissue repair. These cells expressed mRNA for ENTPD1, 2, 3, 5 and 6, and CD73 that corresponded to the observed enzymatic activities. Thus, considering the degradation of ATP and adenosine production, limbal MSCs are very similar to dermal MSCs, indicating that from the aspect of extracellular nucleotide metabolism L-MSCs are very similar to the characterized D-MSCs. J. Cell. Biochem. 118: 2430-2442, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. Adenosine: an activity-dependent axonal signal regulating MAP kinase and proliferation in developing Schwann cells.

    Science.gov (United States)

    Stevens, Beth; Ishibashi, Tomoko; Chen, Jiang-Fan; Fields, R Douglas

    2004-02-01

    Nonsynaptic release of ATP from electrically stimulated dorsal root gangion (DRG) axons inhibits Schwann cell (SC) proliferation and arrests SC development at the premyelinating stage, but the specific types of purinergic receptor(s) and intracellular signaling pathways involved in this form of neuron-glia communication are not known. Recent research shows that adenosine is a neuron-glial transmitter between axons and myelinating glia of the CNS. The present study investigates the possibility that adenosine might have a similar function in communicating between axons and premyelinating SCs. Using a combination of pharmacological and molecular approaches, we found that mouse SCs in culture express functional adenosine receptors and ATP receptors, a far more complex array of purinergic receptors than thought previously. Adenosine, but not ATP, activates ERK/MAPK through stimulation of cAMP-linked A2(A) adenosine receptors. Both ATP and adenosine inhibit proliferation of SCs induced by platelet-derived growth factor (PDGF), via mechanisms that are partly independent. In contrast to ATP, adenosine failed to inhibit the differentiation of SCs to the O4+ stage. This indicates that, in addition to ATP, adenosine is an activity-dependent signaling molecule between axons and premyelinating Schwann cells, but that electrical activity, acting through adenosine, has opposite effects on the differentiation of myelinating glia in the PNS and CNS.

  20. Adenosine A(2A) receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC.

    Science.gov (United States)

    Brand, Frank; Klutz, Athena M; Jacobson, Kenneth A; Fredholm, Bertil B; Schulte, Gunnar

    2008-08-20

    G protein-coupled receptors, such as the adenosine A(2A) receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A(2A) receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A(2A) (K(i)=149+/-27 nM) as well as A(3) receptors (K(i)=240+/-160 nM) but not to adenosine A(1) receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand's functionality at adenosine A(2A) but not A(2B) receptors. In live-cell imaging studies, Alexa488-APEC-induced adenosine A(2A) receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A(2A) receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A(2A) receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC described here showed that it provides a useful tool for tracing adenosine A(2A) receptors in vitro.

  1. Molecular vibration-activity relationship in the agonism of adenosine receptors.

    Science.gov (United States)

    Chee, Hyun Keun; Oh, S June

    2013-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Hyun Keun Chee

    2013-12-01

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

  3. An Escherichia coli strain deficient for both exonuclease 5 and deoxycytidine triphosphate deaminase shows enhanced sensitivity to ionizing radiation

    International Nuclear Information System (INIS)

    Estevenon, A.M.; Kooistra, J.; Sicard, N.

    1995-01-01

    An Escherichia coli mutant lacking deoxycytidine triphosphate deaminase (Dcd) activity and an unknown function encoded by a gene designated ior exhibits sensitivity to ionizing radiation whereas dcd mutants themselves are not sensitive. A DNA fragment from an E. coli genomic library that restores the wild type level of UV and gamma ray resistance to this mutant has been cloned in the multicopy vector pBR322. Comparison of its restriction map with the physical map of the E. coli chromosome revealed complete identity to the recBD genes. ior affects ATP-dependent exonuclease activity, suggesting that it is an allele of recB. This mutation alone does not confer sensitivity to UV and gamma radiation, indicating that lack of Dcd activity is also required for expression of radiation sensitivity

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Chun eYang

    2013-06-01

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

  6. Age-dependent changes of presynaptic neuromodulation via A1-adenosine receptors in rat hippocampal slices.

    Science.gov (United States)

    Sperlágh, B; Zsilla, G; Baranyi, M; Kékes-Szabó, A; Vizi, E S

    1997-10-01

    The presynaptic neuromodulation of stimulation-evoked release of [3H]-acetylcholine by endogenous adenosine, via A1-adenosine receptors, was studied in superfused hippocampal slices taken from 4-, 12- and 24-month-old rats. 8-Cyclopentyl-1,3-dimethylxanthine (0.25 microM), a selective A1-receptor antagonist, increased significantly the electrical field stimulation-induced release of [3H]-acetylcholine in slices prepared from 4- and 12-month-old rats, showing a tonic inhibitory action of endogenous adenosine via stimulation of presynaptic A1-adenosine receptors. In contrast, 8-cyclopentyl-1,3-dimethylxanthine had no effect in 24-month-old rats. 2-Chloroadenosine (10 microM), an adenosine receptor agonist decreased the release of [3H]-acetylcholine in slices taken from 4- and 12-month-old rats, and no significant change was observed in slices taken from 24-month-old rats. In order to show whether the number/or affinity of the A1-receptors was affected in aged rats, [3H]-8-cyclopentyl-1,3-dimethylxanthine binding was studied in hippocampal membranes prepared from rats of different ages. Whereas the Bmax value was significantly lower in 2-year-old rats than in younger counterparts, the dissociation constant (Kd) was not affected by aging, indicating that the density rather than the affinity of adenosine receptors was altered. Endogenous adenosine levels present in the extracellular space were also measured in the superfusate by high performance liquid chromatography (HPLC) coupled with ultraviolet detection, and an age-related increase in the adenosine level was found. In summary, our results indicate that during aging the level of adenosine in the extracellular fluid is increased in the hippocampus. There is a downregulation and reduced responsiveness of presynaptic adenosine A1-receptors, and it seems likely that these changes are due to the enhanced adenosine level in the extracellular space.

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

    Science.gov (United States)

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

    2016-03-30

    Sleep homeostasis reflects a centrally mediated drive for sleep, which increases during waking and resolves during subsequent sleep. Here we demonstrate that mice deficient for glial adenosine kinase (AdK), the primary metabolizing enzyme for adenosine (Ado), exhibit enhanced expression of this homeostatic drive by three independent measures: (1) increased rebound of slow-wave activity; (2) increased consolidation of slow-wave sleep; and (3) increased time constant of slow-wave activity decay during an average slow-wave sleep episode, proposed and validated here as a new index for homeostatic sleep drive. Conversely, mice deficient for the neuronal adenosine A1 receptor exhibit significantly decreased sleep drive as judged by these same indices. Neuronal knock-out of AdK did not influence homeostatic sleep need. Together, these findings implicate a glial-neuronal circuit mediated by intercellular Ado, controlling expression of homeostatic sleep drive. Because AdK is tightly regulated by glial metabolic state, our findings suggest a functional link between cellular metabolism and sleep homeostasis. The work presented here provides evidence for an adenosine-mediated regulation of sleep in response to waking (i.e., homeostatic sleep need), requiring activation of neuronal adenosine A1 receptors and controlled by glial adenosine kinase. Adenosine kinase acts as a highly sensitive and important metabolic sensor of the glial ATP/ADP and AMP ratio directly controlling intracellular adenosine concentration. Glial equilibrative adenosine transporters reflect the intracellular concentration to the extracellular milieu to activate neuronal adenosine receptors. Thus, adenosine mediates a glial-neuronal circuit linking glial metabolic state to neural-expressed sleep homeostasis. This indicates a metabolically related function(s) for this glial-neuronal circuit in the buildup and resolution of our need to sleep and suggests potential therapeutic targets more directly related to

  8. Activation of Adenosine Receptor A2A Increases HSC Proliferation and Inhibits Death and Senescence by Down-regulation of p53 and Rb

    Directory of Open Access Journals (Sweden)

    Md. Kaimul eAhsan

    2014-04-01

    Full Text Available Background & Aims: During fibrosis hepatic stellate cells (HSC undergo activation, proliferation and senescence but the regulation of these important processes is poorly understood. The adenosine A2A receptor (A2A is known to be present on HSC, and its activation results in liver fibrosis. In this study, we tested if A2A has a role in the regulation of HSC proliferation, apoptosis, senescence, and the relevant molecular mechanism.Methods: The ability of adenosine to regulate p53 and Rb protein levels, proliferation, apoptosis and senescence was tested in the human HSC cell line LX-2 and rat primary HSC.Results: Adenosine receptor activation down-regulates p53 and Rb protein levels, increases BrdU incorporation and increases cell survival in LX-2 cells and in primary rat HSC. These effects of NECA were reproduced by an adenosine A2A receptor specific agonist (CGS21680 and blocked by a specific antagonist (ZM241385. By day twenty-one of culture primary rat HSC entered senescence and expressed -gal which was significantly inhibited by NECA. Furthermore, NECA induced down regulation of p53 and Rb and Rac1, and decreased phosphorylation of p44-42 MAP Kinase in LX-2 cells and primary rat HSC. These effects were reproduced by the cAMP analog 8-Bromo-cAMP, and the adenylyl cyclase activator forskolin, and were blocked by PKA inhibitors.Conclusions: These results demonstrate that A2A receptor regulates a number of HSC fate decisions and induces greater HSC proliferation, reduces apoptosis and senescence by decreasing p53 and Rb through cAMP-PKA/Rac1/p38 MAPK pathway. This provides a mechanism for adenosine induced HSC regulation and liver fibrosis.

  9. Differential response of Drosophila cell lines to extracellular adenosine

    Czech Academy of Sciences Publication Activity Database

    Fleischmannová, J.; Kučerová, Lucie; Šandová, Kateřina; Steinbauerová, Veronika; Brož, Václav; Šimek, Petr; Žurovec, Michal

    2012-01-01

    Roč. 42, č. 5 (2012), s. 321-331 ISSN 0965-1748 R&D Projects: GA MŠk(CZ) LC06077 Grant - others:AV ČR(CZ) KJB501410801; European Community´s Seventh Framwork Programme (FP7/2007-2013)(CZ) 229518 Institutional research plan: CEZ:AV0Z50070508 Institutional support: RVO:60077344 Keywords : adenosine recycling * nucleoside transport * Mbn2 Subject RIV: CE - Biochemistry Impact factor: 3.234, year: 2012 http://www.sciencedirect.com/science/article/pii/S0965174812000033

  10. Stemcell Information: SKIP000184 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available negtive, due to adenosine deaminase deficiency adenosine deaminase 欠損患者線維芽細胞(GM01390) 由来。常染...色体劣性adenosine deaminase 欠失。患者は重度の免疫不全、T細胞、B細胞、NK細胞陰性。 ... human ES-like -- Retroviru...deficiency 102700 ... 0-9 Male ... Yes No iPSCs derived from GM01390 Fibroblast. T cell negtive, B cell negative, NK cell...Collections/NIGMS/ipsc_list.aspx?PgId=696 ... 18691744 10.1016/j.cell.2008.07.041 Disease-Specific Induced P

  11. The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

    Science.gov (United States)

    Kim, Mihwa; Ham, Ahrom; Kim, Katelyn Yu-Mi; Brown, Kevin M.; Lee, H. Thomas

    2014-01-01

    Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation. PMID:24945528

  12. Overexpression of adenosine A2A receptors in rats: effects on depression, locomotion and anxiety

    Directory of Open Access Journals (Sweden)

    Joana E Coelho

    2014-06-01

    Full Text Available Adenosine A2A receptors (A2AR are a sub-type of receptors enriched in basal ganglia, activated by the neuromodulator adenosine, which interact with dopamine D2 receptors. Although this reciprocal antagonistic interaction is well established in motor function, the outcome in dopamine-related behaviors remains uncertain, in particular in depression and anxiety. We have demonstrated an upsurge of A2AR associated to aging and chronic stress. Furthermore, Alzheimer’s disease patients present A2AR accumulation in cortical areas together with depressive signs. We now tested the impact of overexpressing A2AR in forebrain neurons on dopamine related behavior, namely depression. Adult male rats overexpressing human A2AR under the control of CaMKII promoter [Tg(CaMKII-hA2AR] and aged-matched wild-types (WT of the same strain (Sprague-Dawley were studied. The forced swimming test (FST, sucrose preference test (SPT and the open-field test (OFT were performed to evaluate behavioral despair, anhedonia, locomotion and anxiety. Tg(CaMKII-hA2AR animals spent more time floating and less time swimming in the FST and presented a decreased sucrose preference at 48h in the SPT. They also covered higher distances in the OFT and spent more time in the central zone than the WT. The results indicate that Tg(CaMKII-hA2AR rats exhibit depressive-like behavior, hyperlocomotion and altered exploratory behavior. This A2AR overexpression may explain the depressive signs found in aging, chronic stress and Alzheimer’s disease.

  13. Overexpression of Adenosine A2A Receptors in Rats: Effects on Depression, Locomotion, and Anxiety.

    Science.gov (United States)

    Coelho, Joana E; Alves, Pedro; Canas, Paula M; Valadas, Jorge S; Shmidt, Tatiana; Batalha, Vânia L; Ferreira, Diana G; Ribeiro, Joaquim A; Bader, Michael; Cunha, Rodrigo A; do Couto, Frederico Simões; Lopes, Luísa V

    2014-01-01

    Adenosine A2A receptors (A2AR) are a sub-type of receptors enriched in basal ganglia, activated by the neuromodulator adenosine, which interact with dopamine D2 receptors. Although this reciprocal antagonistic interaction is well-established in motor function, the outcome in dopamine-related behaviors remains uncertain, in particular in depression and anxiety. We have demonstrated an upsurge of A2AR associated to aging and chronic stress. Furthermore, Alzheimer's disease patients present A2AR accumulation in cortical areas together with depressive signs. We now tested the impact of overexpressing A2AR in forebrain neurons on dopamine-related behavior, namely depression. Adult male rats overexpressing human A2AR under the control of CaMKII promoter [Tg(CaMKII-hA2AR)] and aged-matched wild-types (WT) of the same strain (Sprague-Dawley) were studied. The forced swimming test (FST), sucrose preference test (SPT), and the open-field test (OFT) were performed to evaluate behavioral despair, anhedonia, locomotion, and anxiety. Tg(CaMKII-hA2AR) animals spent more time floating and less time swimming in the FST and presented a decreased sucrose preference at 48 h in the SPT. They also covered higher distances in the OFT and spent more time in the central zone than the WT. The results indicate that Tg(CaMKII-hA2AR) rats exhibit depressive-like behavior, hyperlocomotion, and altered exploratory behavior. This A2AR overexpression may explain the depressive signs found in aging, chronic stress, and Alzheimer's disease.

  14. 125I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors

    International Nuclear Information System (INIS)

    Linden, J.; Patel, A.; Earl, C.Q.; Craig, R.H.; Daluge, S.M.

    1988-01-01

    A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A2 binding affinity. Two acidic 3-(arylamino)-8-phenylxanthine derivatives were labeled with 125 I and evaluated as A1 receptor radioligands. The new radioligands bound to A1 receptors with KD values of 1-1.25 nM. Specific binding represented over 80% of total binding. High concentrations of NaCl or other salts increased the binding affinity of acidic but not neutral antagonists, suggesting that interactions between ionized xanthines and receptors may be affected significantly by changes in ionic strength. On the basis of binding studies with these antagonists and isotope dilution with the agonist [ 125 I]N6-(4-amino-3-iodobenzyl)adenosine, multiple agonist affinity states of A1 receptors have been identified

  15. Lack of adenosine A(3) receptors causes defects in mouse peripheral blood parameters

    Czech Academy of Sciences Publication Activity Database

    Hofer, Michal; Pospíšil, Milan; Dušek, L.; Hoferová, Zuzana; Komůrková, Denisa

    2014-01-01

    Roč. 10, č. 3 (2014), s. 509-514 ISSN 1573-9538 R&D Projects: GA ČR(CZ) GAP303/11/0128 Institutional support: RVO:68081707 Keywords : Adenosine A(3) receptor * Adenosine A(3) receptor knockout mice * Hematopoiesis Subject RIV: BO - Biophysics Impact factor: 3.886, year: 2014

  16. Adenosine A2A Receptor Modulates the Activity of Globus Pallidus Neurons in Rats

    Directory of Open Access Journals (Sweden)

    Hui-Ling Diao

    2017-11-01

    Full Text Available The globus pallidus is a central nucleus in the basal ganglia motor control circuit. Morphological studies have revealed the expression of adenosine A2A receptors in the globus pallidus. To determine the modulation of adenosine A2A receptors on the activity of pallidal neurons in both normal and parkinsonian rats, in vivo electrophysiological and behavioral tests were performed in the present study. The extracellular single unit recordings showed that micro-pressure administration of adenosine A2A receptor agonist, CGS21680, regulated the pallidal firing activity. GABAergic neurotransmission was involved in CGS21680-induced modulation of pallidal neurons via a PKA pathway. Furthermore, application of two adenosine A2A receptor antagonists, KW6002 or SCH442416, mainly increased the spontaneous firing of pallidal neurons, suggesting that endogenous adenosine system modulates the activity of pallidal neurons through adenosine A2A receptors. Finally, elevated body swing test (EBST showed that intrapallidal microinjection of adenosine A2A receptor agonist/antagonist induced ipsilateral/contralateral-biased swing, respectively. In addition, the electrophysiological and behavioral findings also revealed that activation of dopamine D2 receptors by quinpirole strengthened KW6002/SCH442416-induced excitation of pallidal activity. Co-application of quinpirole with KW6002 or SCH442416 alleviated biased swing in hemi-parkinsonian rats. Based on the present findings, we concluded that pallidal adenosine A2A receptors may be potentially useful in the treatment of Parkinson's disease.

  17. Transient Delivery of Adenosine as a Novel Therapy to Prevent Epileptogenesis

    Science.gov (United States)

    2015-10-01

    Chemother 6:98–101. Bukoski RD, Sparks HV, and Mela -Riker LM (1986) A role for mitochondria in myocardial adenosine production. Adv Exp Med Biol 194:157–167...Bukoski RD, Sparks HV, and Mela LM (1983) Rat heart mitochondria release adenosine. Biochem Biophys Res Commun 113:990–995. Burnstock G, Fredholm BB

  18. Small-animal PET study of adenosine A(1) receptors in rat brain: blocking receptors and raising extracellular adenosine.

    Science.gov (United States)

    Paul, Soumen; Khanapur, Shivashankar; Rybczynska, Anna A; Kwizera, Chantal; Sijbesma, Jurgen W A; Ishiwata, Kiichi; Willemsen, Antoon T M; Elsinga, Philip H; Dierckx, Rudi A J O; van Waarde, Aren

    2011-08-01

    Activation of adenosine A(1) receptors (A(1)R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A(1)R can be visualized using 8-dicyclopropylmethyl-1-(11)C-methyl-3-propyl-xanthine ((11)C-MPDX) and PET. This study aims to test whether (11)C-MPDX can be used for quantitative studies of cerebral A(1)R in rodents. (11)C-MPDX was injected (intravenously) into isoflurane-anesthetized male Wistar rats (300 g). A dynamic scan of the central nervous system was obtained, using a small-animal PET camera. A cannula in a femoral artery was used for blood sampling. Three groups of animals were studied: group 1, controls (saline-treated); group 2, animals pretreated with the A(1)R antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 mg, intraperitoneally); and group 3, animals pretreated (intraperitoneally) with a 20% solution of ethanol in saline (2 mL) plus the adenosine kinase inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d] pyrimidine dihydrochloride (ABT-702) (1 mg). DPCPX is known to occupy cerebral A(1)R, whereas ethanol and ABT-702 increase extracellular adenosine. In groups 1 and 3, the brain was clearly visualized. High uptake of (11)C-MPDX was noted in striatum, hippocampus, and cerebellum. In group 2, tracer uptake was strongly suppressed and regional differences were abolished. The treatment of group 3 resulted in an unexpected 40%-45% increase of the cerebral uptake of radioactivity as indicated by increases of PET standardized uptake value, distribution volume from Logan plot, nondisplaceable binding potential from 2-tissue-compartment model fit, and standardized uptake value from a biodistribution study performed after the PET scan. The partition coefficient of the tracer (K(1)/k(2) from the model fit) was not altered under the study conditions. (11)C-MPDX shows a regional distribution in rat brain consistent with binding to A(1)R. Tracer binding is blocked by the selective A

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

    Science.gov (United States)

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

    2010-09-01

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

  20. Presynaptic inhibition of GABAergic synaptic transmission by adenosine in mouse hypothalamic hypocretin neurons.

    Science.gov (United States)

    Xia, J X; Xiong, J X; Wang, H K; Duan, S M; Ye, J N; Hu, Z A

    2012-01-10

    Hypocretin neurons in the lateral hypothalamus, a new wakefulness-promoting center, have been recently regarded as an important target involved in endogenous adenosine-regulating sleep homeostasis. The GABAergic synaptic transmissions are the main inhibitory afferents to hypocretin neurons, which play an important role in the regulation of excitability of these neurons. The inhibitory effect of adenosine, a homeostatic sleep-promoting factor, on the excitatory glutamatergic synaptic transmissions in hypocretin neurons has been well documented, whether adenosine also modulates these inhibitory GABAergic synaptic transmissions in these neurons has not been investigated. In this study, the effect of adenosine on inhibitory postsynaptic currents (IPSCs) in hypocretin neurons was examined by using perforated patch-clamp recordings in the acute hypothalamic slices. The findings demonstrated that adenosine suppressed the amplitude of evoked IPSCs in a dose-dependent manner, which was completely abolished by 8-cyclopentyltheophylline (CPT), a selective antagonist of adenosine A1 receptor but not adenosine A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl) xanthine. A presynaptic origin was suggested as following: adenosine increased paired-pulse ratio as well as reduced GABAergic miniature IPSC frequency without affecting the miniature IPSC amplitude. Further findings demonstrated that when the frequency of electrical stimulation was raised to 10 Hz, but not 1 Hz, a time-dependent depression of evoked IPSC amplitude was detected in hypocretin neurons, which could be partially blocked by CPT. However, under a higher frequency at 100 Hz stimulation, CPT had no action on the depressed GABAergic synaptic transmission induced by such tetanic stimulation in these hypocretin neurons. These results suggest that endogenous adenosine generated under certain stronger activities of synaptic transmissions exerts an inhibitory effect on GABAergic synaptic transmission in hypocretin

  1. Adenosine-loaded dissolving microneedle patches to improve skin wrinkles, dermal density, elasticity and hydration.

    Science.gov (United States)

    Kang, G; Tu, T N T; Kim, S; Yang, H; Jang, M; Jo, D; Ryu, J; Baek, J; Jung, H

    2018-04-01

    Although dissolving microneedle patches have been widely studied in the cosmetics field, no comparisons have been drawn with the topical applications available for routine use. In this study, two wrinkle-improving products, adenosine-loaded dissolving microneedle patches and an adenosine cream, were evaluated for efficacy, with respect to skin wrinkling, dermal density, elasticity, and hydration, and safety in a clinical test on the crow's feet area. Clinical efficacy and safety tests were performed for 10 weeks on 22 female subjects with wrinkles around their eyes. The adenosine-loaded dissolving microneedle patch was applied once every 3 days, in the evening, for 8 weeks to the designated crow's feet area. The adenosine cream was applied two times per day, in the morning and evening, for 8 weeks to the other crow's feet area. Skin wrinkling, dermal density, elasticity, and hydration were measured by using PRIMOS ® premium, Dermascan ® C, Cutometer ® MPA580, and Corneometer ® CM 825, respectively. In addition, subjective skin irritation was evaluated by self-observation, and objective skin irritation was assessed through expert interviews. The adenosine-loaded dissolving microneedle patches had a similar or better efficacy than the adenosine cream. Both groups showed statistically significant efficacy for almost all parameters (P hydration efficacy (P skin-improvement parameters, adenosine-loaded dissolving microneedle patches showed the same or better effect than the adenosine cream, although the weekly adenosine dose was 140 times lower. The dissolving microneedle patches caused no adverse reactions. These adenosine-loaded dissolving microneedle patches are expected to be safe, effective, and novel cosmetics for skin improvement. © 2018 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  2. Hide and seek: a comparative autoradiographic in vitro investigation of the adenosine A3 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Haeusler, D.; Fuchshuber, F.; Girschele, F.; Hacker, M.; Wadsak, W.; Mitterhauser, Markus [Medical University of Vienna, Department of Biomedical Imaging and Image-guided Therapy, Vienna (Austria); Grassinger, L. [University of Applied Sciences Wiener Neustadt, Department of Biomedical Analytics, Wiener Neustadt (Austria); Hoerleinsberger, W.J. [Medical University of Vienna, Department of Biomedical Imaging and Image-guided Therapy, Vienna (Austria); University of Vienna, Cognitive Science Research Platform, Vienna (Austria); Hoeftberger, R.; Leisser, I. [Medical University of Vienna, Institute of Neurology, Vienna (Austria); Shanab, K.; Spreitzer, H. [University of Vienna, Department of Drug and Natural Product Synthesis, Vienna (Austria); Gerdenitsch, W. [Medical University of Vienna, Institute of Biomedicinal Research, Vienna (Austria)

    2015-05-01

    Since the adenosine A3 receptor (A3R) is considered to be of high clinical importance in the diagnosis and treatment of ischaemic conditions (heart and brain), glaucoma, asthma, arthritis, cancer and inflammation, a suitable and selective A3R PET tracer such as [{sup 18}F]FE rate at SUPPY would be of high clinical value for clinicians as well as patients. A3R was discovered in the late 1990s, but there is still little known regarding its distribution in the CNS and periphery. Hence, in autoradiographic experiments the distribution of A3R in human brain and rat tissues was investigated and the specific binding of the A3R antagonist FE rate at SUPPY and MRS1523 compared. Immunohistochemical staining (IHC) experiments were also performed to validate the autoradiographic findings. For autoradiographic competition experiments human post-mortem brain and rat tissues were incubated with [{sup 125}I]AB-MECA and highly selective compounds to block the other adenosine receptor subtypes. Additionally, IHC was performed with an A3 antibody. Specific A3R binding of MRS1523 and FE rate at SUPPY was found in all rat peripheral tissues examined with the highest amounts in the spleen (44.0 % and 46.4 %), lung (44.5 % and 45.0 %), heart (39.9 % and 42.9 %) and testes (27.4 % and 29.5 %, respectively). Low amounts of A3R were found in rat brain tissues (5.9 % and 5.6 %, respectively) and human brain tissues (thalamus 8.0 % and 9.1 %, putamen 7.8 % and 8.2 %, cerebellum 6.0 % and 7.8 %, hippocampus 5.7 % and 5.6 %, caudate nucleus 4.9 % and 6.4 %, cortex 4.9 % and 6.3 %, respectively). The outcome of the A3 antibody staining experiments complemented the results of the autoradiographic experiments. The presence of A3R protein was verified in central and peripheral tissues by autoradiography and IHC. The specificity and selectivity of FE rate at SUPPY was confirmed by direct comparison with MRS1523, providing further evidence that [{sup 18}F]FE rate at SUPPY may be a suitable A3 PET

  3. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.

    Science.gov (United States)

    Burton, Peter; Adams, David R; Abraham, Achamma; Allcock, Robert W; Jiang, Zhong; McCahill, Angela; Gilmour, Jane; McAbney, John; Kaupisch, Alexandra; Kane, Nicole M; Baillie, George S; Baker, Andrew H; Milligan, Graeme; Houslay, Miles D; Mountford, Joanne C

    2010-12-15

    hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.

  4. Mechanism of adenylate kinase. Dose adenosine 5'-triphosphate bind to the adenosine 5'-monophosphate site

    Energy Technology Data Exchange (ETDEWEB)

    Shyy, Y.J.; Tian, G.; Tsai, M.D.

    1987-10-06

    Although the subtrate binding properties of adenylate kinase (AK) have been studied extensively by various biochemical and biophysical techniques, it remains controversial whether uncomplexed adenosine 5'-triphosphate (ATP) binds to the adenosine 5'-monophosphate (AMP) site of AK. The authors present two sets of experiments which argue against binding of ATP to the AMP site. (a) /sup 31/P nuclear magnetic resonance titration of ATP with AK indicated a 1:1 stoichiometry on the basis of changes in coupling constants and line widths. This ruled out binding of ATP to both sites. (b) ATP and MgATP were found to behave similarly by protecting AK from spontaneous inactivation while AMP showed only a small degree of protection. Such inactivation could also be protected or reversed by dithioerythritol and is most likely due to oxidation of sulfhydryl groups, one of which (cysteine-25) is located near the MgATP site. The results support binding of ATP to the MgATP site predominantly, instead of the AMP site, in the absence of Mg/sup 2 +/.

  5. A Bulky Rhodium Complex Bound to an Adenosine-Adenosine DNA Mismatch: General Architecture of the Metalloinsertion Binding Mode†

    Science.gov (United States)

    Zeglis, Brian M.; Pierre, Valérie C.; Kaiser, Jens T.; Barton, Jacqueline K.

    2009-01-01

    Two crystal structures are determined for Δ-Rh(bpy)2(chrysi)3+ (chrysi = 5,6-chrysenequinone diimine) bound to the oligonucleotide duplex 5′-CGGAAATTACCG-3′ containing two adenosine-adenosine mismatches (italics) through metalloinsertion. Diffraction quality crystals with two different space groups (P3221 and P43212) were obtained under very similar crystallization conditions. In both structures, the bulky rhodium complex inserts into the two mismatched sites from the minor groove side, ejecting the mismatched bases into the major groove. The conformational changes are localized to the mismatched site; the metal complex replaces the mismatched base pair without an increase in base pair rise. The expansive metal complex is accommodated in the duplex by a slight opening in the phosphodiester backbone; all sugars retain a C2′-endo puckering, and flanking base pairs neither stretch nor shear. The structures differ, however, in that in one of the structures, an additional metal complex is bound by intercalation from the major groove at the central 5′-AT-3′ step. We conclude that this additional metal complex is intercalated into this central step because of crystal packing forces. The structures described here of Δ-Rh(bpy)2(chrysi)3+ bound to thermodynamically destabilized AA mismatches share critical features with binding by metalloinsertion in two other oligonucleotides containing different single base mismatches. These results underscore the generality of the metalloinsertion as a new mode of non-covalent binding by small molecules with a DNA duplex. PMID:19374348

  6. Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription.

    Science.gov (United States)

    Nair, Smita; Zlotnick, Adam

    2018-05-15

    Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive. IMPORTANCE Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription

  7. Extracellular adenosine generation in the regulation of pro-inflammatory responses and pathogen colonization.

    Science.gov (United States)

    Alam, M Samiul; Costales, Matthew G; Cavanaugh, Christopher; Williams, Kristina

    2015-05-05

    Adenosine, an immunomodulatory biomolecule, is produced by the ecto-enzymes CD39 (nucleoside triphosphate dephosphorylase) and CD73 (ecto-5'-nucleotidase) by dephosphorylation of extracellular ATP. CD73 is expressed by many cell types during injury, infection and during steady-state conditions. Besides host cells, many bacteria also have CD39-CD73-like machinery, which helps the pathogen subvert the host inflammatory response. The major function for adenosine is anti-inflammatory, and most recent research has focused on adenosine's control of inflammatory mechanisms underlying various autoimmune diseases (e.g., colitis, arthritis). Although adenosine generated through CD73 provides a feedback to control tissue damage mediated by a host immune response, it can also contribute to immunosuppression. Thus, inflammation can be a double-edged sword: it may harm the host but eventually helps by killing the invading pathogen. The role of adenosine in dampening inflammation has been an area of active research, but the relevance of the CD39/CD73-axis and adenosine receptor signaling in host defense against infection has received less attention. Here, we review our recent knowledge regarding CD73 expression during murine Salmonellosis and Helicobacter-induced gastric infection and its role in disease pathogenesis and bacterial persistence. We also explored a possible role for the CD73/adenosine pathway in regulating innate host defense function during infection.

  8. Effects of adenosine on pressure-flow relationships in an in vitro model of compartment syndrome.

    Science.gov (United States)

    Shrier, I; Baratz, A; Magder, S

    1997-03-01

    Blood flow through skeletal muscle is best modeled with a vascular waterfall at the arteriolar level. Under these conditions, flow is determined by the difference between perfusion pressure (Pper) and the waterfall pressure (Pcrit), divided by the arterial resistance (Ra). By pump perfusing an isolated canine gastrocnemius muscle (n = 6) after it was placed within an airtight box, with and without adenosine infusion, we observed an interaction between the pressure surrounding a muscle (as occurs in compartment syndrome) and baseline vascular tone. We titrated adenosine concentration to double baseline flow. We measured Pcrit and Ra at box pressures (Pbox), which resulted in 100 (Pbox = 0), 90, 75, and 50% flow without adenosine; and 200, 180, 150, 100, and 50% flow with adenosine. Without adenosine, each 10% decline in flow was associated with a 5.7 mmHg increase in Pcrit (P 0.9). We conclude that increases in pressure surrounding a muscle limit flow primarily through changes in Pcrit with and without adenosine-induced vasodilation. The interaction between Pbox and adenosine with respect to Pcrit but not Ra suggests that Pbox affects the tone of the vessels responsible for Pcrit but not Ra.

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

    Energy Technology Data Exchange (ETDEWEB)

    Andersson, Olov, E-mail: olov.andersson@ki.se

    2014-02-01

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

  10. Polymorphisms in adenosine receptor genes are associated with infarct size in patients with ischemic cardiomyopathy.

    Science.gov (United States)

    Tang, Z; Diamond, M A; Chen, J-M; Holly, T A; Bonow, R O; Dasgupta, A; Hyslop, T; Purzycki, A; Wagner, J; McNamara, D M; Kukulski, T; Wos, S; Velazquez, E J; Ardlie, K; Feldman, A M

    2007-10-01

    The goal of this experiment was to identify the presence of genetic variants in the adenosine receptor genes and assess their relationship to infarct size in a population of patients with ischemic cardiomyopathy. Adenosine receptors play an important role in protecting the heart during ischemia and in mediating the effects of ischemic preconditioning. We sequenced DNA samples from 273 individuals with ischemic cardiomyopathy and from 203 normal controls to identify the presence of genetic variants in the adenosine receptor genes. Subsequently, we analyzed the relationship between the identified genetic variants and infarct size, left ventricular size, and left ventricular function. Three variants in the 3'-untranslated region of the A(1)-adenosine gene (nt 1689 C/A, nt 2206 Tdel, nt 2683del36) and an informative polymorphism in the coding region of the A3-adenosine gene (nt 1509 A/C I248L) were associated with changes in infarct size. These results suggest that genetic variants in the adenosine receptor genes may predict the heart's response to ischemia or injury and might also influence an individual's response to adenosine therapy.

  11. Evidence for evoked release of adenosine and glutamate from cultured cerebellar granule cells

    International Nuclear Information System (INIS)

    Schousboe, A.; Frandsen, A.; Drejer, J.

    1989-01-01

    Evoked release of [ 3 H]-D-aspartate which labels the neurotransmitter glutamate pool in cultured cerebellar granule cells was compared with evoked release of adenosine from similar cultures. It was found that both adenosine and [3H]-D-aspartate could be released from the neurons in a calcium dependent manner after depolarization of the cells with either 10-100 microM glutamate or 50 mM KCl. Cultures of cerebellar granule cells treated with 50 microM kainate to eliminate GABAergic neurons behaved in the same way. This together with the observation that cultured astrocytes did not exhibit a calcium dependent, potassium stimulated adenosine release strongly suggest that cerebellar granule cells release adenosine in a neurotransmitter-like fashion together with glutamate which is the classical neurotransmitter of these neurons. Studies of the metabolism of adenosine showed that in the granule cells adenosine is rapidly metabolized to ATP, ADP, and AMP, but in spite of this, adenosine was found to be released preferential to ATP

  12. Tolerance and diagnostic accuracy of an abbreviated adenosine infusion for myocardial scintigraphy: a randomized, prospective study.

    Science.gov (United States)

    Treuth, M G; Reyes, G A; He, Z X; Cwajg, E; Mahmarian, J J; Verani, M S

    2001-01-01

    The objectives of this study were 2-fold: (1) to determine the tolerance of adenosine perfusion tomography with the use of an abbreviated (3-minute) infusion in comparison to the standard (6-minute) infusion, and (2) to assess the relative diagnostic accuracy of a 3-minute adenosine infusion in patients referred for arteriography. An abbreviated adenosine infusion may decrease the frequency and duration of side effects and be a more cost-effective alternative. We prospectively randomized 599 patients undergoing adenosine myocardial perfusion tomography to either a 3-minute or 6-minute adenosine infusion at 140 microg/kg per minute. Among the 599 enrolled patients, 142 subsequently underwent coronary angiography. Patients randomized to the 3-minute adenosine infusion tolerated the procedure better than those randomized to the standard infusion (P <.01). Flushing, headache, neck pain, and atrioventricular block were all significantly less frequent (P <.01) with the abbreviated infusion. Moreover, patients receiving the abbreviated infusion had less hypotension and tachycardia (P <.05). The sensitivity of the test for detection of coronary artery disease was 88% for both the 3- and 6-minute infusions. In patients with abnormal scan results, perfusion defect size was slightly larger in those receiving a 6-minute infusion versus those receiving a 3-minute infusion (P =.05). An abbreviated 3-minute adenosine infusion, in combination with perfusion tomography, has similar sensitivity for detection of coronary artery disease and is better tolerated than the standard 6-minute infusion.

  13. Alterations in electrocardiogram of adenosine test for 99Tcm-MIBI myocardial perfusion imaging

    International Nuclear Information System (INIS)

    Xie Boqia; Tian Yueqin; Zheng Lihui

    2011-01-01

    Objective: To analyze alterations in electrocardiogram (ECG) of adenosine test in 99 Tc m -MIBI myocardial perfusion imaging(MPI)SPECT study. Methods: A total of 641 patients were included in the study. The patients each underwent 99 Tc m -MIBI MPI with adenosine test. The ECGs were taken before, during, and after adenosine infusion. Results: In all, abnormal ECGs were found in 205(32.0%) patients. During adenosine infusion, 20.6%(132/641) of patients suffered from arrhythmia, 29.5%(39/132) had atrial premature beats, 34.1% (45/132) had premature ventricular beats, and 6.1% (8/132) had sinoatrial block. In addition, 5.3% (7/132) had first-, 24.2% (32/132) had second-, and 0.8% (1/132) had third-degree atrioventricular block (AVB). After adenosine infusion, 4.4%( 28/641) of patients suffered from arrhythmia, 57.1% (16/28) had atrial premature beats, 39.3% (11/28) had premature ventricular beats, and 3.6% (1/28) had sinoatrial block. The perfusion images showed ischemia in 36 patients and infarction in 8 patients. Adenosine infusion was terminated in 39 patients (6.1%) because of poorly tolerated side effects. However, no death or acute myocardial infarction occurred in the study. Conclusions: Adenosine pharmacologic test for 99 Tc m -MIBI MPI may result in relatively high incidence of arrhythmia in ECG monitoring. (authors)

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

    International Nuclear Information System (INIS)

    Andersson, Olov

    2014-01-01

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

  15. A simplified radioimmunoassay of adenosine-3':5'-monophosphate

    International Nuclear Information System (INIS)

    Katoh, Yoshiki; Takezawa, Junichi; Suzuki, Morio; Kuninaka, Akira; Yoshino, Hiroshi

    1975-01-01

    Dextran-coated charcoal was proved to be able to separate free adenosine-3':5'monophosphate (cAMP) from antibody-bound cAMP. Only free cAMO was adsorbed on dextran-coated charcoal within 1 min after contacting the charcoal. In a reaction mixture of cAMP and anti-cAMP-plasma, most of antibody-bound cAMP had not been adsorbed 4 min after contacting. The data obtained were found to be almost the same as the data of another experiment using cellulose ester filter separation technique. Thus, dextran-coated charcoal could be employed to simplify the radioimmunoassay of cAMP. (author)

  16. N6-adenosine methylation in MiRNAs.

    Directory of Open Access Journals (Sweden)

    Tea Berulava

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

  17. The Adverse Events and Hemodynamic Effects of Adenosine-Based Cardiac MRI

    International Nuclear Information System (INIS)

    Voigtlander, Thomas; Magedanz, Annett; Schmermund, Axel; Bramlage, Peter; Elsaesser, Amelie; Kauczor, Hans-Ulrich; Mohrs, Oliver K.

    2011-01-01

    We wanted to prospectively assess the adverse events and hemodynamic effects associated with an intravenous adenosine infusion in patients with suspected or known coronary artery disease and who were undergoing cardiac MRI. One hundred and sixty-eight patients (64 ± 9 years) received adenosine (140 μg/kg/min) during cardiac MRI. Before and during the administration, the heart rate, systemic blood pressure, and oxygen saturation were monitored using a MRI-compatible system. We documented any signs and symptoms of potential adverse events. In total, 47 out of 168 patients (28%) experienced adverse effects, which were mostly mild or moderate. In 13 patients (8%), the adenosine infusion was discontinued due to intolerable dyspnea or chest pain. No high grade atrioventricular block, bronchospasm or other life-threatening adverse events occurred. The hemodynamic measurements showed a significant increase in the heart rate during adenosine infusion (69.3 ± 11.7 versus 82.4 ± 13.0 beats/min, respectively; p < 0.001). A significant but clinically irrelevant increase in oxygen saturation occurred during adenosine infusion (96 ± 1.9% versus 97 ± 1.3%, respectively; p < 0.001). The blood pressure did not significantly change during adenosine infusion (systolic: 142.8 ± 24.0 versus 140.9 ± 25.7 mmHg; diastolic: 80.2 ± 12.5 mmHg versus 78.9 ± 15.6, respectively). This study confirms the safety of adenosine infusion during cardiac MRI. A considerable proportion of all patients will experience minor adverse effects and some patients will not tolerate adenosine infusion. However, all adverse events can be successfully managed by a radiologist. The increased heart rate during adenosine infusion highlights the need to individually adjust the settings according to the patient, e.g., the number of slices of myocardial perfusion imaging.

  18. Novel approaches for targeting the adenosine A2A receptor.

    Science.gov (United States)

    Yuan, Gengyang; Gedeon, Nicholas G; Jankins, Tanner C; Jones, Graham B

    2015-01-01

    The adenosine A2A receptor (A2AR) represents a drug target for a wide spectrum of diseases. Approaches for targeting this membrane-bound protein have been greatly advanced by new stabilization techniques. The resulting X-ray crystal structures and subsequent analyses provide deep insight to the A2AR from both static and dynamic perspectives. Application of this, along with other biophysical methods combined with fragment-based drug design (FBDD), has become a standard approach in targeting A2AR. Complementarities of in silico screening based- and biophysical screening assisted- FBDD are likely to feature in future approaches in identifying novel ligands against this key receptor. This review describes evolution of the above approaches for targeting A2AR and highlights key modulators identified. It includes a review of: adenosine receptor structures, homology modeling, X-ray structural analysis, rational drug design, biophysical methods, FBDD and in silico screening. As a drug target, the A2AR is attractive as its function plays a role in a wide spectrum of diseases including oncologic, inflammatory, Parkinson's and cardiovascular diseases. Although traditional approaches such as high-throughput screening and homology model-based virtual screening (VS) have played a role in targeting A2AR, numerous shortcomings have generally restricted their applications to specific ligand families. Using stabilization methods for crystallization, X-ray structures of A2AR have greatly accelerated drug discovery and influenced development of biophysical-in silico hybrid screening methods. Application of these new methods to other ARs and G-protein-coupled receptors is anticipated in the future.

  19. SU-C-303-01: Activation-Induced Cytidine Deaminase Confers Cancer Resistance to Radiation Therapy

    International Nuclear Information System (INIS)

    Yi, S; La Count, S; Liu, J; Bai, X; Lu, L

    2015-01-01

    Purpose: To study the role of activation-induced cytidine deaminase (AID) in malignant cell resistance to radiation therapy. Methods: We first developed several small devices that could be used to adopt radiation beams from clinical high dose rate brachy therapy (HDR) or linac-based megavoltage machines to perform pre-clinical cell and mouse experiments. Then we used these devices to deliver radiation to AID-positive and AID-silenced cancer cells or tumors formed by these cells in mice. Cells and mice bearing tumors received the same dose under the same experimental conditions. For cells, we observed the apoptosis and the cell survival rate over time. For mice bearing tumors, we measured and recorded the tumor sizes every other day for 4 weeks. Results: For cell experiments, we found that the AID-positive cells underwent much less apoptosis compared with AID-silenced cells upon radiation. And for mouse experiments, we found that AID-positive tumors grew significantly faster than the AID-silenced tumors despite of receiving the same doses of radiation. Conclusion: Our study suggests that AID may confer cancer resistance to radiation therapy, and AID may be a significant biomarker predicting cancer resistance to radiation therapy for certain cancer types

  20. Adenosine concentration in the porcine coronary artery wall and A2A receptor involvement in hypoxia-induced vasodilatation.

    Science.gov (United States)

    Frøbert, Ole; Haink, Gesine; Simonsen, Ulf; Gravholt, Claus H; Levin, Max; Deussen, Andreas

    2006-01-15

    We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F(2alpha) (10(-5) M), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [(14)C]adenosine over the microdialysis membrane increased from 0.32 +/- 0.02 to 0.46 +/- 0.02 (n = 4, P lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilatation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at the A(2A) receptor level.

  1. Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Friis, Ulla Glenert; Uhrenholt, Torben Rene

    2007-01-01

    of calcium from the sarcoplasmic reticulum (SR), stimulated presumably by IP(3), is involved in the adenosine contraction mechanism of the afferent arteriole. In agreement with this notion is the observation that 2 aminoethoxydiphenyl borate (100 microM) blocked the adenosine-induced constriction whereas...... was abolished by IAA-94. Furthermore, the vasoconstriction caused by adenosine was significantly inhibited by 5 microM nifedipine (control 8.3 +/- 0.2 microM, ado 3.6 +/- 0.6 microM, ado + nifedipine 6.8 +/- 0.2 microM) suggesting involvement of voltage-dependent calcium channels. CONCLUSION: We conclude...

  2. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Frandsen, Ulrik

    1997-01-01

    1. The present study examined the capacity for adenosine formation, uptake and metabolism in contracting primary rat muscle cells and in microvascular endothelial cells in culture. 2. Strong and moderate electrical simulation of skeletal muscle cells led to a significantly greater increase....... 3. Addition of microvascular endothelial cells to the cultured skeletal muscle cells enhanced the contraction-induced accumulation of extracellular adenosine (P Skeletal muscle cells were...... in the extracellular adenosine concentration (421 +/- 91 and 235 +/- 30 nmol (g protein)-1, respectively; P muscle cells (161 +/- 20 nmol (g protein)-1). The ATP concentration was lower (18%; P contracted, but not in the moderately contracted muscle cells...

  3. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    International Nuclear Information System (INIS)

    Delamere, N.A.; Socci, R.R.; King, K.L.

    1990-01-01

    The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin

  4. Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Vincenzetti, Silvia; Cambi, Alessandra; Neuhard, Jan

    1999-01-01

    -optical emission spectroscopy analysis indicated that the enzyme contains 1 mol of zinc atom per mole of subunit. The kinetic properties of AT-CDA1 both toward the natural substrates and with analogs indicated that the catalytic mechanism of the plant enzyme is probably very similar to that of the human the...

  5. Caffeine, Adenosine Receptors and Estrogen in Toxin Models of Parkinson's Disease

    National Research Council Canada - National Science Library

    Schwarzschild, Michael A; Xu, Kui

    2008-01-01

    Continued progress has been made toward each of the Specific Aims (SAs) 1 and 2 (SA 3 completed) of our research project, Caffeine, adenosine receptors and estrogen in toxin models of Parkinson's disease...

  6. Catalytic dephosphorylation of adenosine monophosphate (AMP) to form supramolecular nanofibers/hydrogels.

    Science.gov (United States)

    Du, Xuewen; Li, Junfeng; Gao, Yuan; Kuang, Yi; Xu, Bing

    2012-02-18

    The use of enzyme to instruct the self-assembly of the nucleoside of adenosine in water provides a new class of molecular nanofibers/hydrogels as functional soft materials. This journal is © The Royal Society of Chemistry 2012

  7. Injections of the selective adenosine A2A antagonist MSX-3 into the nucleus accumbens core attenuate the locomotor suppression induced by haloperidol in rats.

    Science.gov (United States)

    Ishiwari, Keita; Madson, Lisa J; Farrar, Andrew M; Mingote, Susana M; Valenta, John P; DiGianvittorio, Michael D; Frank, Lauren E; Correa, Merce; Hockemeyer, Jörg; Müller, Christa; Salamone, John D

    2007-03-28

    There is considerable evidence of interactions between adenosine A2A receptors and dopamine D2 receptors in striatal areas, and antagonists of the A2A receptor have been shown to reverse the motor effects of DA antagonists in animal models. The D2 antagonist haloperidol produces parkinsonism in humans, and also induces motor effects in rats, such as suppression of locomotion. The present experiments were conducted to study the ability of the adenosine A2A antagonist MSX-3 to reverse the locomotor effects of acute or subchronic administration of haloperidol in rats. Systemic (i.p.) injections of MSX-3 (2.5-10.0 mg/kg) were capable of attenuating the suppression of locomotion induced by either acute or repeated (i.e., 14 day) administration of 0.5 mg/kg haloperidol. Bilateral infusions of MSX-3 directly into the nucleus accumbens core (2.5 microg or 5.0 microg in 0.5 microl per side) produced a dose-related increase in locomotor activity in rats treated with 0.5 mg/kg haloperidol either acutely or repeatedly. There were no overall significant effects of MSX-3 infused directly into the dorsomedial nucleus accumbens shell or the ventrolateral neostriatum. These results indicate that antagonism of adenosine A2A receptors can attenuate the locomotor suppression produced by DA antagonism, and that this effect may be at least partially mediated by A2A receptors in the nucleus accumbens core. These studies suggest that adenosine and dopamine systems interact to modulate the locomotor and behavioral activation functions of nucleus accumbens core.

  8. Discovery of indolylpiperazinylpyrimidines with dual-target profiles at adenosine A2A and dopamine D2 receptors for Parkinson's disease treatment.

    Directory of Open Access Journals (Sweden)

    Yi-Ming Shao

    Full Text Available Parkinson's disease (PD is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7-11.2 μM as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5-40.2 μM. Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM, hepatotoxicity (up to 30 μM or cardiotoxicity (up to 30 μM.

  9. Adenosine A1 receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury

    OpenAIRE

    Winerdal, Max; Winerdal, Malin E.; Wang, Ying-Qing; Fredholm, Bertil B.; Winqvist, Ola; Ådén, Ulrika

    2015-01-01

    Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A1 receptor deficiency (A1R−/−) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction siz...

  10. Treatment of out-of-hospital supraventricular tachycardia: adenosine vs verapamil.

    Science.gov (United States)

    Brady, W J; DeBehnke, D J; Wickman, L L; Lindbeck, G

    1996-06-01

    To compare the use of adenosine and the use of verapamil as out-of-hospital therapy for supraventricular tachycardia (SVT). A period of prospective adenosine use (March 1993 to February 1994) was compared with a historical control period of verapamil use (March 1990 to February 1991) for SVT. Data were obtained for SVT patients treated in a metropolitan, fire-department-based paramedic system serving a population of approximately 1 million persons. Standard drug protocols were used and patient outcomes (i.e., conversion rates, complications, and recurrences) were monitored. During the adenosine treatment period, 105 patients had SVT; 87 (83%) received adenosine, of whom 60 (69%) converted to a sinus rhythm (SR). Vagal maneuvers (VM) resulted in restoration of SR in 8 patients (7.6%). Some patients received adenosine for non-SVT rhythms: 7 sinus tachycardia, 18 atrial fibrilation, 7 wide-complex tachycardia (WCT), and 2 ventricular tachycardia; no non-SVT rhythm converted to SR and none of these patients experienced an adverse effect. Twenty-five patients were hemodynamically unstable (systolic blood pressure fibrillation). Recurrence of SVT was noted in 2 adenosine patients and 2 verapamil patients in the out-of-hospital setting and in 23 adenosine patients and 15 verapamil patients after ED arrival, necessitating additional therapy (p = 0.48 and 0.88, for recurrence rates and types of additional therapies, respectively). Hospital diagnoses, outcomes, and ED dispositions were similar for the 2 groups. Adenosine and verapamil were equally successful in converting out-of-hospital SVT in patients with similar etiologies responsible for the SVT. Recurrence of SVT occurred at similar rates for the 2 medications. Rhythm misidentification remains a common issue in out-of-hospital cardiac care in this emergency medical services system.

  11. No Effect of Nutritional Adenosine Receptor Antagonists on Exercise Performance in the Heat

    Science.gov (United States)

    2008-11-01

    358–363, 1996. 11. Cook NC, Samman S. Flavonoids —chemistry, metabolism, cardiopro- tective effects, and dietary sources. Nutr Biochem 7: 66–76, 1996...metabolism and health effects of dietary flavonoids in man. Biomed Pharmacother 51: 305–310, 1997. R400 ADENOSINE RECEPTOR ANTAGONISM AND EXERCISE IN THE HEAT...Interactions of flavonoids with adenosine receptors. J Med Chem 39: 781–788, 1996. 35. MacRae HS, Mefferd KM. Dietary antioxidant supplementation com

  12. The Use of Adenosine Agonists to Treat Nerve Agent-Induced Seizure and Neuropathology

    Science.gov (United States)

    2016-09-01

    not be cited for purposes of advertisement . REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting burden for this...survivability. That was an important step to clinical relevancy as it was feared that ADO’s depression of cardiovascular output would exacerbate...kainate, adenosine and neuropeptide Y receptors. Neurochemical Research. 28: 1501-1515. 23. Bjorness, T. E. & R. W. Greene . 2009. Adenosine and sleep

  13. The Neuroprotective Benefits of Central Adenosine Receptor Stimulation in a Soman Nerve Agent Rat Model

    Science.gov (United States)

    2014-04-01

    where treatment is delayed and nerve agent-induced status epilepticus develops. New therapeutic targets are required to improve survivability and...Exp Ther 304(3): 1307-1313. Compton, J. R. (2004). Adenosine Receptor Agonist Pd 81,723 Protects Against Seizure/ Status Epilepticus and...Dragunow (1994). " Status epilepticus may be caused by loss of adenosine anticonvulsant mechanisms." Neuroscience 58(2): 245-261. Youssef, A. F. and B. W

  14. Actinides and rare earths complexation with adenosine phosphate nucleotides

    International Nuclear Information System (INIS)

    Mostapha, Sarah

    2013-01-01

    Organophosphorus compounds are important molecules in both nuclear industry and living systems fields. Indeed, several extractants of organophosphorus compounds (such as TBP, HDEHP) are used in the nuclear fuel cycle reprocessing and in the biological field. For instance, the nucleotides are organophosphates which play a very important role in various metabolic processes. Although the literature on the interactions of actinides with inorganic phosphate is abundant, published studies with organophosphate compounds are generally limited to macroscopic and / or physiological approaches. The objective of this thesis is to study the structure of several organophosphorus compounds with actinides to reach a better understanding and develop new specific buildings blocks. The family of the chosen molecules for this procedure consists of three adenine nucleotides mono, bi and triphosphate (AMP, adenosine monophosphate - ADP, adenosine diphosphate - ATP, adenosine triphosphate) and an amino-alkylphosphate (AEP O-phosphoryl-ethanolamine). Complexes synthesis was conducted in aqueous and weakly acidic medium (2.8-4) for several lanthanides (III) (Lu, Yb, Eu) and actinides (U (VI), Th (IV) and Am (III)). Several analytical and spectroscopic techniques have been used to describe the organization of the synthesized complexes: spectrometric analysis performed by FTIR and NMR were used to identify the functional groups involved in the complexation, analysis by ESI-MS and pH-metric titration were used to determine the solution speciation and EXAFS analyzes were performed on Mars beamline of the SOLEIL synchrotron, have described the local cation environment, for both solution and solid compounds. Some theoretical approaches of DFT were conducted to identify stable structures in purpose of completing the experimental studies. All solid complexes (AMP, ADP, ATP and AEP) have polynuclear structures, while soluble ATP complexes are mononuclear. For all synthesized complexes, it has been

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  16. Suppression of adenosine-activated chloride transport by ethanol in airway epithelia.

    Directory of Open Access Journals (Sweden)

    Sammeta V Raju

    Full Text Available Alcohol abuse is associated with increased lung infections. Molecular understanding of the underlying mechanisms is not complete. Airway epithelial ion transport regulates the homeostasis of airway surface liquid, essential for airway mucosal immunity and lung host defense. Here, air-liquid interface cultures of Calu-3 epithelial cells were basolaterally exposed to physiologically relevant concentrations of ethanol (0, 25, 50 and 100 mM for 24 hours and adenosine-stimulated ion transport was measured by Ussing chamber. The ethanol exposure reduced the epithelial short-circuit currents (I(SC in a dose-dependent manner. The ion currents activated by adenosine were chloride conductance mediated by cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP-activated chloride channel. Alloxazine, a specific inhibitor for A(2B adenosine receptor (A(2BAR, largely abolished the adenosine-stimulated chloride transport, suggesting that A(2BAR is a major receptor responsible for regulating the chloride transport of the cells. Ethanol significantly reduced intracellular cAMP production upon adenosine stimulation. Moreover, ethanol-suppression of the chloride secretion was able to be restored by cAMP analogs or by inhibitors to block cAMP degradation. These results imply that ethanol exposure dysregulates CFTR-mediated chloride transport in airways by suppression of adenosine-A(2BAR-cAMP signaling pathway, which might contribute to alcohol-associated lung infections.

  17. Topical adenosine increases thick hair ratio in Japanese men with androgenetic alopecia.

    Science.gov (United States)

    Watanabe, Y; Nagashima, T; Hanzawa, N; Ishino, A; Nakazawa, Y; Ogo, M; Iwabuchi, T; Tajima, M

    2015-12-01

    Hair thickness is more important than hair density in the appearance of baldness in male with androgenetic alopecia (AGA). Adenosine improves hair loss by stimulating hair growth and by thickening hair shafts in women. The objective of this study was to evaluate the hair growth efficacy and safety of topical adenosine in men with AGA. A lotion containing either adenosine or niacinamide was administered to the scalps of 102 Japanese men twice daily for 6 months in a double-blind, randomized study. Efficacy was evaluated by dermatologists who assessed the quality of the hair and by calculating the percentages of vellus-like and thick hairs among the vertex hairs, as well as hair density. Adenosine was significantly (P < 0.05) superior to niacinamide in terms of global improvement of AGA, increase in the percentage of thick hairs (at least 60 μm) and self-assessment of hair thickness by the study participants. No causal adverse event due to the adenosine lotion was observed. These data indicate that adenosine increases thick hair ratio in Japanese men with AGA, and this compound is useful for the improvement of AGA. © 2015 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  18. Safety of adenosine stress myocardial perfusion imaging by a one-route infusion protocol

    International Nuclear Information System (INIS)

    Kawai, Yuko; Kishino, Koh

    2006-01-01

    When adenosine stress testing is performed, a vein is generally accessed in each arm. To determine whether the one-route infusion protocol, that is, infusion via one upper arm vein, is safe, myocardial perfusion imaging was performed during adenosine stress testing in patients with angina pectoris. Sixty-six consecutive patients (43 men, 68±11 years of age) with suspected coronary artery disease were enrolled in this study. For the stress test, adenosine was injected at 120 μg/kg/min for 6 minutes. Systolic blood pressure, diastolic blood pressure, and heart rate did not show any significant changes after injection of the adenosine and radioisotope (RI) tracer. Adverse events during infusion of the adenosine were seen in 42 (64%) patients and included chest discomfort/oppression in 17 (26%) and dyspnea/throat discomfort in 15 (23%). On the other hand, adverse events just after infusion of the RI tracer occurred in 5 (8%) patients and included chest oppression in 2 (3%) and dyspnea in 1 (2%). Almost all adverse events disappeared quickly without treatment. Therefore, we concluded that adenosine stress myocardial perfusion imaging using a one-route infusion protocol is safe and useful to do for patients unable to secure veins in both arms. (author)

  19. Protection against methamphetamine-induced neurotoxicity to neostriatal dopaminergic neurons by adenosine receptor activation.

    Science.gov (United States)

    Delle Donne, K T; Sonsalla, P K

    1994-12-01

    Methamphetamine (METH)-induced neurotoxicity to nigrostriatal dopaminergic neurons in experimental animals appears to have a glutamatergic component because blockade of N-methyl-D-aspartate receptors prevents the neuropathologic consequences. Because adenosine affords neuroprotection against various forms of glutamate-mediated neuronal damage, the present studies were performed to investigate whether adenosine plays a protective role in METH-induced toxicity. METH-induced decrements in neostriatal dopamine content and tyrosine hydroxylase activity in mice were potentiated by concurrent treatment with caffeine, a nonselective adenosine antagonist that blocks both A1 and A2 adenosine receptors. In contrast, chronic treatment of mice with caffeine through their drinking water for 4 weeks, which increased the number of adenosine A1 receptors in the neostriatum and frontal cortex, followed by drug washout, prevented the neurochemical changes produced by the treatment of mice with METH treatment. In contrast, this treatment did not prevent 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine-induced dopaminergic neurotoxicity. Furthermore, concurrent administration of cyclopentyladenosine, an adenosine A1 receptor agonist, attenuated the METH-induced neurochemical changes. This protection by cyclopentyladenosine was blocked by cyclopentyltheophylline, an A1 receptor antagonist. These results indicate that activation of A1 receptors can protect against METH-induced neurotoxicity in mice.

  20. Oral sucrose for heel lance enhances adenosine triphosphate use in preterm neonates with respiratory distress.

    Science.gov (United States)

    Angeles, Danilyn M; Asmerom, Yayesh; Boskovic, Danilo S; Slater, Laurel; Bacot-Carter, Sharon; Bahjri, Khaled; Mukasa, Joseph; Holden, Megan; Fayard, Elba

    2015-01-01

    To examine the effects of oral sucrose on procedural pain, and on biochemical markers of adenosine triphosphate utilization and oxidative stress in preterm neonates with mild to moderate respiratory distress. Preterm neonates with a clinically required heel lance that met study criteria (n = 49) were randomized into three groups: (1) control (n = 24), (2) heel lance treated with placebo and non-nutritive sucking (n = 15) and (3) heel lance treated with sucrose and non-nutritive sucking (n = 10). Plasma markers of adenosine triphosphate degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile. Data were analyzed using repeated measures analysis of variance, chi-square and one-way analysis of variance. We found that in preterm neonates who were intubated and/or were receiving ⩾30% FiO2, a single dose of oral sucrose given before a heel lance significantly increased markers of adenosine triphosphate use. We found that oral sucrose enhanced adenosine triphosphate use in neonates who were intubated and/or were receiving ⩾30% FiO2. Although oral sucrose decreased pain scores, our data suggest that it also increased energy use as evidenced by increased plasma markers of adenosine triphosphate utilization. These effects of sucrose, specifically the fructose component, on adenosine triphosphate metabolism warrant further investigation.

  1. Adenosine signaling promotes regeneration of pancreatic β-cells in vivo

    Science.gov (United States)

    Andersson, Olov; Adams, Bruce A.; Yoo, Daniel; Ellis, Gregory C.; Gut, Philipp; Anderson, Ryan M.; German, Michael S.; Stainier, Didier Y. R.

    2012-01-01

    Diabetes can be controlled with insulin injections, but a curative approach that restores the number of insulin-producing β-cells is still needed. Using a zebrafish model of diabetes, we screened ~7000 small molecules to identify enhancers of β-cell regeneration. The compounds we identified converge on the adenosine signaling pathway and include exogenous agonists and compounds that inhibit degradation of endogenously produced adenosine. The most potent enhancer of β-cell regeneration was the adenosine agonist 5′-N-Ethylcarboxamidoadenosine (NECA), which acting through the adenosine receptor A2aa increased β-cell proliferation and accelerated restoration of normoglycemia in zebrafish. Despite markedly stimulating β-cell proliferation during regeneration, NECA had only a modest effect during development. The proliferative and glucose-lowering effect of NECA was confirmed in diabetic mice, suggesting an evolutionarily conserved role for adenosine in β-cell regeneration. With this whole-organism screen, we identified components of the adenosine pathway that could be therapeutically targeted for the treatment of diabetes. PMID:22608007

  2. Enhanced A3 adenosine receptor selectivity of multivalent nucleoside-dendrimer conjugates

    Directory of Open Access Journals (Sweden)

    Shainberg Asher

    2008-10-01

    Full Text Available Abstract Background An approach to use multivalent dendrimer carriers for delivery of nucleoside signaling molecules to their cell surface G protein-coupled receptors (GPCRs was recently introduced. Results A known adenosine receptor (AR agonist was conjugated to polyamidoamine (PAMAM dendrimer carriers for delivery of the intact covalent conjugate to on the cell surface. Depending on the linking moiety, multivalent conjugates of the N6-chain elongated functionalized congener ADAC (N6-[4-[[[4-[[[(2-aminoethylamino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl]-adenosine achieved unanticipated high selectivity in binding to the cytoprotective human A3 AR, a class A GPCR. The key to this selectivity of > 100-fold in both radioreceptor binding (Ki app = 2.4 nM and functional assays (EC50 = 1.6 nM in inhibition of adenylate cyclase was maintaining a free amino group (secondary in an amide-linked chain. Attachment of neutral amide-linked chains or thiourea-containing chains preserved the moderate affinity and efficacy at the A1 AR subtype, but there was no selectivity for the A3 AR. Since residual amino groups on dendrimers are associated with cytotoxicity, the unreacted terminal positions of this A3 AR-selective G2.5 dendrimer were present as carboxylate groups, which had the further benefit of increasing water-solubility. The A3 AR selective G2.5 dendrimer was also visualized binding the membrane of cells expressing the A3 receptor but did not bind cells that did not express the receptor. Conclusion This is the first example showing that it is feasible to modulate and even enhance the pharmacological profile of a ligand of a GPCR based on conjugation to a nanocarrier and the precise structure of the linking group, which was designed to interact with distal extracellular regions of the 7 transmembrane-spanning receptor. This ligand tool can now be used in pharmacological models of tissue rescue from ischemia and to probe the existence of A3 AR

  3. Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

    Directory of Open Access Journals (Sweden)

    Simon Gemble

    2015-07-01

    Full Text Available Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at "difficult-to-replicate" sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS, a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3'-5' DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

  4. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio [Kanazawa Univ. (Japan). School of Medicine

    2002-10-01

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 {mu}Ci. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  5. Potential benefits of combining cytosine deaminase/5-fluorocytosine gene therapy and irradiation for prostate cancer. Experimental study

    International Nuclear Information System (INIS)

    Kato, Hiroaki; Koshida, Kiyoshi; Yokoyama, Kunihiko; Mizokami, Atsushi; Namiki, Mikio

    2002-01-01

    The purpose of this study was to investigate the potential of combining cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and radiation therapy (either external beam radiation or radioimmunotherapy [RIT]), for the treatment of prostate cancer. Tumor xenografts of CD-transduced LNCaP cells grown in the testes of severe combined immunodeficiency (SCID) mice were used to evaluate antitumor effect. The mice were injected intraperitoneally with 500 mg/kg of 5-FC, or with 5, 15 or 30 mg/kg of 5-fluorouracil (5-FU), for 9 days. The tumors were treated with fractionated radiation at a dose of 1 or 3 Gy/day for 3 days, or I-131 labelled anti-prostate specific antigen (anti-PSA) monoclonal antibody (mAb) administration at a subtherapeutic dose of 20 or 80 μCi. Intratumoral and serum concentrations of 5-FU were measured using high performance liquid chromatography. Mice treated with CD/5-FC gene therapy presented a significant tumor growth inhibition comparable to that obtained with 15 mg/kg, 5-FU systemic administration without marked weight loss. Treatment with CD/5-FC gene therapy resulted in higher tumor but lower serum concentrations of 5-FU than treatment with systemic 5-FU chemotherapy. An additive antitumor effect was obtained when CD/5-FC therapy was combined with 1 Gy irradiation, which by itself did not produce a significant antitumor effect. However, the efficacy of CD/5-FC therapy was not enhanced when combined with RIT, probably due to poor accumulation of the mAb as the tumor/blood ratio never exceeded 1. These findings indicate that CD/5-FC gene therapy for prostate cancer may function with enhanced antitumor effect when combined with external beam radiation. However, combining CD/5-FC gene therapy and RIT using an anti-PSA mAb may not be effective because of insufficient accumulation of the mAb at the target tumors. (author)

  6. Expression of activation-induced cytidine deaminase gene in B lymphocytes of patients with common variable immunodeficiency.

    Science.gov (United States)

    Abolhassani, Hassan; Farrokhi, Amir Salek; Pourhamdi, Shabnam; Mohammadinejad, Payam; Sadeghi, Bamdad; Moazzeni, Seyed-Mohammad; Aghamohammadi, Asghar

    2013-08-01

    Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by reduced serum level of IgG, IgA or IgM and recurrent bacterial infections. Class switch recombination (CSR) as a critical process in immunoglobulin production is defective in a group of CVID patients. Activation-induced cytidine deaminase (AID) protein is an important molecule involving CSR process. The aim of this study was to investigate the AID gene mRNA production in a group of CVID patients indicating possible role of this molecule in this disorder. Peripheral blood mononuclear cells (PBMC) of 29 CVID patients and 21 healthy controls were isolated and stimulated by CD40L and IL-4 to induce AID gene expression. After 5 days AID gene mRNA production was investigated by real time polymerase chain reaction. AID gene was expressed in all of the studied patients. However the mean density of extracted AID mRNA showed higher level in CVID patients (230.95±103.04 ng/ml) rather than controls (210.00±44.72 ng/ml; P=0.5). CVID cases with lower level of AID had decreased total level of IgE (P=0.04) and stimulated IgE production (P=0.02); while cases with increased level of AID presented higher level of IgA (P=0.04) and numbers of B cells (P=0.02) and autoimmune disease (P=0.02). Different levels of AID gene expression may have important roles in dysregulation of immune system and final clinical presentation in CVID patients. Therefore investigating the expression of AID gene can help in classifying CVID patients.

  7. Autosomal recessive hyper IgM syndrome associated with activation-induced cytidine deaminase gene in three Turkish siblings presented with tuberculosis lymphadenitis - Case report.