Science.gov

Sample records for adenosine diphosphate-ribose polymerase

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

    PubMed

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

    2008-05-15

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

  2. Inhibition of poly(adenosine diphosphate-ribose) polymerase by the active form of vitamin D

    PubMed Central

    MABLEY, JON G.; WALLACE, REBECCA; PACHER, PÁL; MURPHY, KANNEGANTI; SZABÓ, CSABA

    2008-01-01

    Vitamin D is well characterized for its role in mineral homeostasis and maintenance of normal skeletal architecture. Vitamin D has been demonstrated to exert anti-inflammatory effects in a variety of disease states including diabetes, arthritis and inflammatory bowel disease. In these diseases poly[adenosine diphosphate (ADP)-ribose] polymerase (PARP) inhibitors have also proved effective as anti-inflammatory agents. Here we present data demonstrating that the active metabolite of vitamin D, 1α,25-dihydroxyvitamin D3, is a PARP inhibitor. UV irradiation-mediated PARP activation in human keratinocytes can be inhibited by treatment with vitamin D, 7-dehydrocholesterol or 1α,25-dihydroxyvitamin D3. Inhibition of cytochrome P450 reversed the PARP inhibitory action of vitamin D and 7-dehydrocholesterol, indicating that conversion to 1α,25-dihydroxyvitamin D3 mediates their PARP inhibitory action. Vitamin D may protect keratinocytes against over-activation of PARP resulting from exposure to sunlight. PARP inhibition may contribute to the pharmacological and anti-inflammatory effects of vitamin D. PMID:17487428

  3. Prognostic and clinicopathological value of poly (adenosine diphosphate-ribose) polymerase expression in breast cancer: A meta-analysis

    PubMed Central

    Qiao, Weiqiang; Pan, Linlin; Kou, Changgui; Li, Ke; Yang, Ming

    2017-01-01

    Background Previous studies have shown that the poly (adenosine diphosphate-ribose) polymerase (PARP) level is a promising indicator of breast cancer. However, its prognostic value remains controversial. The present meta-analysis evaluated the prognostic value of PARP expression in breast cancer. Materials and methods Eligible studies were retrieved from the PubMed, Web of Science, Embase, and Cochrane Library databases through July 20, 2016. Studies investigating PARP expression as well as reporting survival data in breast cancer were included. Two independent reviewers carried out all literature searches. The pooled relative risk (RR) and hazard ratio (HR) with 95% confidence interval (95% CI) were applied to assess the association between PARP expression and the clinicopathological features and survival outcome in breast cancer. Results A total of 3506 patients from eight eligible studies were included. We found that higher PARP expression indicated a worse clinical outcome in early stage breast cancer, with a HR of 3.08 (95% CI, 1.14–8.29, P = 0.03) for disease-free survival and a HR of 1.82 (95% CI, 1.20–2.76; P = 0.005) for overall survival. Moreover, increased PARP expression was significantly associated with higher nuclear grade (RR, 1.51; 95% CI, 1.12–2.04; P = 0.008) in breast cancer. A similar correlation was detected in triple-negative breast cancer (TNBC; RR, 1.81; 95% CI, 1.04–3.17; P = 0.04). Conclusions Our findings indicated that elevated PARP expression correlated with worse prognosis in early stage breast cancer. Furthermore, high PARP expression was associated with higher nuclear grade and TNBC. PMID:28212434

  4. Reduced nicotinamide adenine dinucleotide fluorescence lifetime detected poly(adenosine-5'-diphosphate-ribose) polymerase-1-mediated cell death and therapeutic effect of pyruvate

    NASA Astrophysics Data System (ADS)

    Guo, Han-Wen; Wei, Yau-Huei; Wang, Hsing-Wen

    2011-06-01

    Noninvasive detection of cell death has the potential for definitive diagnosis and monitoring treatment outcomes in real time. Reduced nicotinamide adenine dinucleotide (NADH) fluorescence intensity has long been used as a noninvasive optical probe of metabolic states. NADH fluorescence lifetime has recently been studied for its potential as an alternative optical probe of cellular metabolic states and cell death. In this study, we investigated the potential using NADH fluorescence intensity and/or lifetime to detect poly(adenosine-5'-diphosphate-ribose) polymerase-1 (PARP-1)-mediated cell death in HeLa cells. We also examined if NADH signals respond to treatment by pyruvate. The mechanism of PARP-1-mediated cell death has been well studied that extensive PARP-1 activation leads to cytosolic nicotinamide adenine dinucleotide depletion resulting in glycolytic inhibition, mitochondrial failure, and death. Pyruvate could restore electron transport chain to prevent energy failure and death. Our results show that NADH fluorescence lifetime, not intensity, responded to PARP-1-mediated cell death and the rescue effect of pyruvate. This lifetime change of NADH fluorescence happened before the collapse of mitochondrial membrane potential and mitochondrial uncoupling. Together with our previous findings in staurosporine-induced cell death, we suggest that NADH fluorescence lifetime increase during cell death is mainly due to increased protein-protein interactions but not the intracellular NADH content.

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

    SciTech Connect

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

    2014-02-01

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

  6. HPLC analysis of cyclic adenosine diphosphate ribose and adenosine diphosphate ribose: determination of NAD+ metabolites in hippocampal membranes.

    PubMed

    Casabona, G; Sturiale, L; L'Episcopo, M R; Raciti, G; Fazzio, A; Sarpietro, M G; Genazzani, A A; Cambria, A; Nicoletti, F

    1995-01-01

    Cyclic adenosine diphosphate-ribose (cADPR) and ADPR were separated by high-performance liquid chromatography (HPLC) on a CarboPac PA-1 column at strong basic pH and quantitated by a pulsed amperometric detector. Although this HPLC method was quite sensitive and highly reproducible, it did not allow the separation of cADPR from guanosine monophosphate (GMP) which, when present, could be removed by ion-affinity chromatography, using gel-immobilized Fe3+ columns. Crude synaptic membranes from rat hippocampi were incubated with nicotinamide adenine dinucleotide (NAD) and acidic extracts were subject to HPLC analysis after neutralization. Incubation led to a time-dependent formation of ADPR, which was amplified when membranes were incubated in the presence of guanosine trisphosphate (GTP), guanosine-5'-0-(3-thiotrisphosphate) (GTP-gamma-S) or AlF3. cADPR did not accumulate in detectable amounts and only a minimal proportion (< 5%) of radioactivity originating from [3H]NAD co-eluted with authentic cADPR in extracts from hippocampal membranes. The simultaneous detection of cADPR and ADPR we have described may help the search for inhibitors of cADPR metabolism, which will allow to measure the cADPR that accumulates under basal conditions or in response to extracellular signals.

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

    PubMed Central

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

    2016-01-01

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

  8. Characterization of polymers of adenosine diphosphate ribose generated in vitro and in vivo.

    PubMed

    Alvarez-Gonzalez, R; Jacobson, M K

    1987-06-02

    Methods have been developed and applied to determine the size and branching frequency of polymers of ADP-ribose synthesized in nucleotide-permeable cultured mouse cells and in intact cultured cells. Polymers were purified by affinity chromatography with a boronate resin and were fractionated according to size molecular sieve high-performance liquid chromatography. Fractions were enzymatically digested to nucleotides, which were separated by strong anion exchange high-performance liquid chromatography. From these data, average polymer size and branching frequency were calculated. A wide range of polymer sizes was observed. Polymers as large as 190 residues with at least five points of branching per molecule were generated in vitro. Polymers of up to 67 residues containing up to two points of branching per molecule were isolated from intact cells following treatment with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Cells treated with hyperthermia prior to DNA damage contained polymers of an average maximum size of 244 residues containing up to six points of branching per molecule. The detection of large polymers of ADP-ribose in intact cells suggests that alterations in chromatin organization effected by poly(ADP-ribosylation) may extend beyond the covalently modified proteins and very likely involve noncovalent interactions of poly(ADP-ribose) with other components of chromatin.

  9. Covalent binding of 4-carbamoylbenzenediazonium chloride to deoxyguanine bases of DNA resulting in apparent irreversible inhibition of Poly(adenosine diphosphoribose) polymerase at the nicotinamide binding site

    SciTech Connect

    McLick, J.; Bauer, P.I.; Hakam, A.; Kun, E.

    1987-04-21

    The poly(adenosine diphosphoribose) polymerase activity of isolated liver nuclei was inhibited by 4-carbamoylbenzenediazonium chloride, referred to as 4-diazoniobenzamide, an effect that was dependent on the time of incubation and the concentration of the diazonium compound, with inhibition following first-order kinetics. The inhibition was not reversed by reisolation of nuclie and centrifugal washing, whereas the inhibition by benzamide or 4-aminobenzamide was completely reversible under these conditions. Simultaneous incubation of 4-diazoniobenzamide with benzamide prevented enzyme inhibition. The 4-diazoniobenzoic acid analogue was not inhibitory. The mechanism of action of 4-diazoniobenzamide was traced to a specific covalent binding to (/sup 14/C) dGMP of DNA to form N/sup 2/-((4-carbamoylphenyl)azo)-2'-deoxyguanosine 5'-monophosphate. Coenzymic DNA, by tight association with the polymerase protein, fixes the -C(O)NH/sub 2/ moiety of the adduct at the (/sup 32/P) nicotinamide-binding site of the enzyme.

  10. Discovery of β-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

    PubMed

    Clarke, Michael O; Mackman, Richard; Byun, Daniel; Hui, Hon; Barauskas, Ona; Birkus, Gabriel; Chun, Byoung-Kwon; Doerffler, Edward; Feng, Joy; Karki, Kapil; Lee, Gary; Perron, Michel; Siegel, Dustin; Swaminathan, Swami; Lee, William

    2015-06-15

    Novel 4'-substituted β-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of β-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

  11. Core-Scaffold-Inspired Asymmetric Synthesis of Polysubstituted Chiral Hexahydropyridazines that Potently Inhibit Breast Cancer Cell Proliferation by Inducing Apoptosis.

    PubMed

    Leng, Hai-Jun; Peng, Fu; Zingales, Sarah; Huang, Wei; Wang, Biao; Zhao, Qian; Zhou, Rui; He, Gu; Peng, Cheng; Han, Bo

    2015-12-07

    The highly enantioselective preparation of pharmacologically interesting hexahydropyridazine derivatives based on a multicomponent cascade reaction is described. This one-pot approach utilizes an organocatalytic Michael reaction followed by intermolecular α-amination and intramolecular hemiaminalization to yield a chiral pyridazine backbone with contiguous stereogenic centers and multiple functional groups in good yield and with high stereoselectivity. Compounds synthesized by this method potently inhibited proliferation of MCF-7 breast cancer cells. Mechanistic studies suggest that compound 5 c exerts these anticancer effects by inducing apoptosis through extracellular signal related kinase (ERK)- and poly(adenosine diphosphate ribose) polymerase (PARP)-regulated pathways, as well as mitochondrial pathways.

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

    PubMed

    Verdin, Eric

    2015-12-04

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

  13. Effect of minocycline on cerebral ischemia-reperfusion injury★

    PubMed Central

    Zheng, Yuanyin; Xu, Lijuan; Yin, Jinbao; Zhong, Zhichao; Fan, Hongling; Li, Xi; Chang, Quanzhong

    2013-01-01

    Minocylcine, a tetracycline derivate, has been shown to cross the blood-brain barrier and enter the central nervous system. In this study, cerebral ischemia-reperfusion injury models were established using the suture method, and minocycline was immediately injected intraperitoneally after cerebral ischemia-reperfusion (22.5 mg/kg, initially 45 mg/kg) at a 12-hour interval. Results showed that after minocycline treatment, the volume of cerebral infarction was significantly reduced, the number of surviving cell in the hippocampal CA1 region increased, the number of apoptotic cells decreased, the expression of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 protein was down-regulated, and the escape latency in the water maze test was significantly shortened compared with the ischemia-reperfusion group. Our experimental findings indicate that minocycline can protect against neuronal injury induced by focal ischemia-reperfusion, which may be mediated by the inhibition of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 protein expression. PMID:25206381

  14. Extracellular adenosine triphosphate and adenosine in cancer.

    PubMed

    Stagg, J; Smyth, M J

    2010-09-30

    Adenosine triphosphate (ATP) is actively released in the extracellular environment in response to tissue damage and cellular stress. Through the activation of P2X and P2Y receptors, extracellular ATP enhances tissue repair, promotes the recruitment of immune phagocytes and dendritic cells, and acts as a co-activator of NLR family, pyrin domain-containing 3 (NLRP3) inflammasomes. The conversion of extracellular ATP to adenosine, in contrast, essentially through the enzymatic activity of the ecto-nucleotidases CD39 and CD73, acts as a negative-feedback mechanism to prevent excessive immune responses. Here we review the effects of extracellular ATP and adenosine on tumorigenesis. First, we summarize the functions of extracellular ATP and adenosine in the context of tumor immunity. Second, we present an overview of the immunosuppressive and pro-angiogenic effects of extracellular adenosine. Third, we present experimental evidence that extracellular ATP and adenosine receptors are expressed by tumor cells and enhance tumor growth. Finally, we discuss recent studies, including our own work, which suggest that therapeutic approaches that promote ATP-mediated activation of inflammasomes, or inhibit the accumulation of tumor-derived extracellular adenosine, may constitute effective new means to induce anticancer activity.

  15. Adenosine receptor neurobiology: overview.

    PubMed

    Chen, Jiang-Fan; Lee, Chien-fei; Chern, Yijuang

    2014-01-01

    Adenosine is a naturally occurring nucleoside that is distributed ubiquitously throughout the body as a metabolic intermediary. In the brain, adenosine functions as an important upstream neuromodulator of a broad spectrum of neurotransmitters, receptors, and signaling pathways. By acting through four G-protein-coupled receptors, adenosine contributes critically to homeostasis and neuromodulatory control of a variety of normal and abnormal brain functions, ranging from synaptic plasticity, to cognition, to sleep, to motor activity to neuroinflammation, and cell death. This review begun with an overview of the gene and genome structure and the expression pattern of adenosine receptors (ARs). We feature several new developments over the past decade in our understanding of AR functions in the brain, with special focus on the identification and characterization of canonical and noncanonical signaling pathways of ARs. We provide an update on functional insights from complementary genetic-knockout and pharmacological studies on the AR control of various brain functions. We also highlight several novel and recent developments of AR neurobiology, including (i) recent breakthrough in high resolution of three-dimension structure of adenosine A2A receptors (A2ARs) in several functional status, (ii) receptor-receptor heterodimerization, (iii) AR function in glial cells, and (iv) the druggability of AR. We concluded the review with the contention that these new developments extend and strengthen the support for A1 and A2ARs in brain as therapeutic targets for neurologic and psychiatric diseases.

  16. Does prolonged biliary obstructive jaundice sensitize the liver to endotoxemia?

    PubMed

    Iida, Ayako; Yoshidome, Hiroyuki; Shida, Takashi; Kimura, Fumio; Shimizu, Hiroaki; Ohtsuka, Masayuki; Morita, Yasuhiro; Takeuchi, Dan; Miyazaki, Masaru

    2009-04-01

    Biliary obstructive jaundice (OJ) is an important clinical consideration concerning high bacteremic risk. Hepatocyte apoptosis is one of the causes of cholestatic liver injury. The aim of the current study was to examine the precise pathway and time course of hepatocyte apoptosis during OJ with LPS administration and to determine if OJ sensitizes the liver to endotoxemia. Male C57BL/6 mice were subjected to bile duct ligation and division and were administered with LPS at 3 (OJ3) or 14 (OJ14) days after surgery. Fas ligand expression, poly (adenosine diphosphate-ribose) polymerase p85 fragment immunohistochemistry, activation of caspases 3, 8, and 9, serum alanine aminotransferase levels, and hepatic adenosine triphosphate (ATP) contents were examined. Survival after LPS administration in male C57BL/6 or gld/gld (Fas ligand-deficient) mice was determined. The expression of Fas ligand increased during OJ. After LPS administration, the expression of cleaved caspases 3 and 8 increased in Sham3, Sham14, OJ3, and OJ14 mice, and it significantly increased in OJ14 compared with other mice. Poly (adenosine diphosphate-ribose) polymerase p85 immunohistochemistry showed significant hepatocyte apoptosis after LPS administration in OJ14 mice relative to OJ3. In OJ14 with LPS administration, ATP contents significantly decreased and alanine aminotransferase levels increased. Hepatocyte apoptosis was decreased in gld/gld OJ14 mice compared with C57BL/6 OJ14. All C57BL/6 OJ14 mice with LPS died, but survival in gld/gld OJ14 significantly ameliorated. In prolonged OJ with LPS administration, hepatocyte apoptosis depending on Fas ligand expression significantly increased in association with a decrease in ATP contents, thus resulting in liver necrapoptosis.

  17. Adenosine and sleep

    SciTech Connect

    Yanik, G.M. Jr.

    1987-01-01

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

  18. Nicotinamide in dermatology and photoprotection.

    PubMed

    Surjana, Devita; Damian, Diona L

    2011-01-01

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

  19. A conserved NAD(+) binding pocket that regulates protein-protein interactions during aging.

    PubMed

    Li, Jun; Bonkowski, Michael S; Moniot, Sébastien; Zhang, Dapeng; Hubbard, Basil P; Ling, Alvin J Y; Rajman, Luis A; Qin, Bo; Lou, Zhenkun; Gorbunova, Vera; Aravind, L; Steegborn, Clemens; Sinclair, David A

    2017-03-24

    DNA repair is essential for life, yet its efficiency declines with age for reasons that are unclear. Numerous proteins possess Nudix homology domains (NHDs) that have no known function. We show that NHDs are NAD(+) (oxidized form of nicotinamide adenine dinucleotide) binding domains that regulate protein-protein interactions. The binding of NAD(+) to the NHD domain of DBC1 (deleted in breast cancer 1) prevents it from inhibiting PARP1 [poly(adenosine diphosphate-ribose) polymerase], a critical DNA repair protein. As mice age and NAD(+) concentrations decline, DBC1 is increasingly bound to PARP1, causing DNA damage to accumulate, a process rapidly reversed by restoring the abundance of NAD(+) Thus, NAD(+) directly regulates protein-protein interactions, the modulation of which may protect against cancer, radiation, and aging.

  20. Defective DNA repair mechanisms in prostate cancer: impact of olaparib

    PubMed Central

    De Felice, Francesca; Tombolini, Vincenzo; Marampon, Francesco; Musella, Angela; Marchetti, Claudia

    2017-01-01

    The field of prostate oncology has continued to change dramatically. It has truly become a field that is intensely linked to molecular genetic alterations, especially DNA-repair defects. Germline breast cancer 1 gene (BRCA1) and breast cancer 2 gene (BRCA2) mutations are implicated in the highest risk of prostate cancer (PC) predisposition and aggressiveness. Poly adenosine diphosphate ribose polymerase (PARP) proteins play a key role in DNA repair mechanisms and represent a valid target for new therapies. Olaparib is an oral PARP inhibitor that blocks DNA repair pathway and coupled with BRCA mutated-disease results in tumor cell death. In phase II clinical trials, including patients with advanced castration-resistant PC, olaparib seems to be efficacious and well tolerated. Waiting for randomized phase III trials, olaparib should be considered as a promising treatment option for PC. PMID:28280302

  1. Biosensor reveals multiple sources for mitochondrial NAD⁺.

    PubMed

    Cambronne, Xiaolu A; Stewart, Melissa L; Kim, DongHo; Jones-Brunette, Amber M; Morgan, Rory K; Farrens, David L; Cohen, Michael S; Goodman, Richard H

    2016-06-17

    Nicotinamide adenine dinucleotide (NAD(+)) is an essential substrate for sirtuins and poly(adenosine diphosphate-ribose) polymerases (PARPs), which are NAD(+)-consuming enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD(+) concentrations within these subcellular compartments are thought to regulate the activity of NAD(+)-consuming enzymes; however, the challenge in measuring compartmentalized NAD(+) in cells has precluded direct evidence for this type of regulation. We describe the development of a genetically encoded fluorescent biosensor for directly monitoring free NAD(+) concentrations in subcellular compartments. We found that the concentrations of free NAD(+) in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD(+) biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD(+) concentrations.

  2. Effects of acute doxorubicin treatment on hepatic proteome lysine acetylation status and the apoptotic environment

    PubMed Central

    Dirks-Naylor, Amie J; Kouzi, Samir A; Bero, Joseph D; Tran, Ngan TK; Yang, Sendra; Mabolo, Raean

    2014-01-01

    AIM: To determine if doxorubicin (Dox) alters hepatic proteome acetylation status and if acetylation status was associated with an apoptotic environment. METHODS: Doxorubicin (20 mg/kg; Sigma, Saint Louis, MO; n = 8) or NaCl (0.9%; n = 7) was administered as an intraperitoneal injection to male F344 rats, 6-wk of age. Once animals were treated with Dox or saline, all animals were fasted until sacrifice 24 h later. RESULTS: Dox treatment decreased proteome lysine acetylation likely due to a decrease in histone acetyltransferase activity. Proteome deacetylation may likely not be associated with a proapoptotic environment. Dox did not increase caspase-9, -8, or -3 activation nor poly (adenosine diphosphate-ribose) polymerase-1 cleavage. Dox did stimulate caspase-12 activation, however, it likely did not play a role in apoptosis induction. CONCLUSION: Early effects of Dox involve hepatic proteome lysine deacetylation and caspase-12 activation under these experimental conditions. PMID:25225604

  3. Rat cardiac myocyte adenosine transport and metabolism

    SciTech Connect

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

    1987-01-01

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

  4. Purine metabolism in adenosine deaminase deficiency.

    PubMed Central

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

    1976-01-01

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

  5. Adenosine-Associated Delivery Systems

    PubMed Central

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

    2016-01-01

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

  6. Adenosine receptor targets for pain.

    PubMed

    Sawynok, J

    2016-12-03

    The main focus for the development of adenosine targets as analgesics to date has been A1Rs due to its antinociceptive profile in various preclinical pain models. The usefulness of systemic A1R agonists may be limited by other effects (cardiovascular, motor), but enhanced selectivity for pain might occur with partial agonists, potent and highly selective agonists, or allosteric modulators. A2AR agonists exhibit some peripheral pronociceptive effects, but also act on immune cells to suppress inflammation and on spinal glia to suppress pain signaling and may be useful for inflammatory and neuropathic pain. A2BR agonists exhibit peripheral proinflammatory effects on immune cells, but also spinal antinociceptive effects similar to A2AR agonists. A3Rs are now demonstrated to produce antinociception in several preclinical neuropathic pain models, with mechanistic actions on glial cells, and may be useful for neuropathic pain. Endogenous adenosine levels can be augmented by inhibition of metabolism (via adenosine kinase) or increased generation (via nucleotidases), and these approaches have implications for pain. Endogenous adenosine contributes to antinociception by several pharmacological agents, herbal remedies, acupuncture, transcutaneous electrical nerve stimulation, exercise, joint mobilization, and water immersion via spinal and/or peripheral effects, such that this system appears to constitute a major pain regulatory system. Finally, caffeine inhibits A1-, A2A- and A3Rs with similar potency, and dietary caffeine intake will need attention in trials of: (a) agonists and/or modulators acting at these receptors, (b) some pharmacological and herbal analgesics, and (c) manipulations that enhance endogenous adenosine levels, all of which are inhibited by caffeine and/or A1R antagonists in preclinical studies. All adenosine receptors have effects on spinal glial cells in regulating nociception, and gender differences in the involvement of such cells in chronic

  7. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

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

  8. Pathological overproduction: the bad side of adenosine.

    PubMed

    Borea, Pier Andrea; Gessi, Stefania; Merighi, Stefania; Vincenzi, Fabrizio; Varani, Katia

    2017-03-02

    Adenosine is an endogenous ubiquitous purine nucleoside, which is increased by hypoxia, ischaemia and tissue damage and mediates a number of physiopathological effects by interacting with four GPCRs, identified as A1 , A2A , A2B and A3 . Physiological and acutely increased adenosine is mostly associated with beneficial effects that include vasodilatation and a decrease in inflammation. In contrast, chronic overproduction of adenosine occurs in important pathological states, where long-lasting increases in the nucleoside levels are responsible for the bad side of adenosine associated with chronic inflammation, fibrosis and organ damage. In this review, we describe and critically discuss the pathological overproduction of adenosine and analyse when, where and how adenosine exerts its detrimental effects throughout the body.

  9. Torsades de pointes after adenosine administration.

    PubMed

    Teodorovich, Nicholay; Margolin, Elena; Kogan, Yonatan; Paz, Ofir; Swissa, Moshe

    2016-01-01

    Adenosine can produce arrhythmias, which are generally short living. It may induce PACs and PVCs, sinus bradycardia, and atrial fibrillation. There have been reports of transient polymorphic VT (torsades de pointes) in patients with LQTS and others in people with normal QT interval. We report a case of a long episode of polymorphic VT induced by adenosine. A 27 year old woman received 6 mg adenosine for PSVT, which terminated and torsades de pointes developed, persisting for 17 seconds and terminated spontaneously. This is the longest described duration of the torsades after adenosine administration in patients with normal QT interval.

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

    SciTech Connect

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

    1990-04-01

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

  11. Modulation of N-type Ca2+ currents by A1-adenosine receptor activation in male rat pelvic ganglion neurons.

    PubMed

    Park, K S; Jeong, S W; Cha, S K; Lee, B S; Kong, I D; Ikeda, S R; Lee, J W

    2001-11-01

    Modulation of voltage-activated Ca2+ channels by adenosine was investigated in male rat major pelvic ganglion (MPG) neurons by using the whole-cell variant of the patch-clamp technique. Adenosine inhibited high voltage-activated (HVA) Ca2+ currents in a concentration-dependent manner with an EC50 of 313 nM and a maximal inhibition of 36%, respectively. Inhibition of HVA Ca2+ currents in adrenergic and cholinergic MPG neurons was similar. Adenosine did not modulate T-type Ca2+ channels present in adrenergic MPG neurons. Reverse transcription-polymerase chain reaction analysis indicated that MPG neurons express mRNAs encoding A1 and A2a receptors. Ca2+ current inhibition by adenosine was mimicked by N6-cyclopentyladenosine, an A1-selective agonist (EC50 = 63 nM) and prevented by 100 nM 8-cyclopentyl-1,3-dipropylxanthine, an A1-selective antagonist. Conversely, CGS 21680, an A2a-selective agonist, displayed a relatively low potency (EC50 = 2200 nM) for inhibiting Ca2+ currents. The action of adenosine was significantly attenuated by 2 mM guanosine-5'-thiodiphosphate or 500 ng/ml pertussis toxin. The voltage dependence of adenosine-induced current inhibition was evident by 1) a bell-shaped profile between the current inhibition and test potentials, 2) kinetic slowing in the presence of agonist, and 3) relief of the current inhibition by a conditioning prepulse to +80 mV. Finally, 1 microM omega-conotoxin GVIA occluded adenosine-induced current inhibition. Taken together, we concluded that adenosine inhibits N-type Ca2+ currents by activation of A1 receptors via a voltage-dependent and PTX-sensitive pathway in rat MPG neurons. Our data may explain how adenosine acts as an inhibitory modulator of ganglionic and neuromuscular transmission in the pelvic plexus.

  12. Adenosine modulates cell growth in the human breast cancer cells via adenosine receptors.

    PubMed

    Panjehpour, Mojtaba; Karami-Tehrani, Fatemeh

    2007-01-01

    Adenosine modulates the proliferation, survival, and apoptosis of many different cell types. The present study was performed to investigate the role of adenosine receptors in the human breast cancer cell lines MCF-7 and MDA-MB468. The biological effects of adenosine on the cells were analyzed by adenylyl cyclase and cell viability assay as well as RT-PCR of adenosine receptors. RT-PCR results show the expression of the transcript of all adenosine receptors in both cell lines. By using adenosine and selective adenosine receptor agonists or antagonists, we found that A3 stimulation reduced cell viability, which was abolished by pretreatment with A3 receptor antagonist. Moreover, we demonstrated that adenosine (natural agonist) triggers a cytotoxic signal via A3 receptor activation that was not seen for other subclasses of adenosine receptors. Intracellular cAMP concentration was changed significantly only for A3 and A2B receptor-selective agonists, which indicates the functional form of these receptors on the cell surface. In conclusion, our findings revealed the role of adenosine receptors in breast cancer cell lines on growth modulation role of A3 and functional form of A2B, although its involvement in cell growth modulation was not seen. Theses findings as well as data by others may provide a possible application of adenosine receptor agonists/antagonists in breast malignancies.

  13. Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina

    SciTech Connect

    Braas, K.M.; Zarbin, M.A.; Snyder, S.H.

    1987-06-01

    Using specific sensitive antisera against adenosine, we have immunocytochemically localized endogenous adenosine to specific layers of rat, guinea pig, monkey, and human retina. Highest adenosine immunoreactivity was observed in ganglion cells and their processes in the optic nerve fiber layer. Substantial staining was also found throughout the inner plexiform layer and in select cells in the inner nuclear layer. Adenosine A1 receptors, labeled with the agonists L-(/sup 3/H)phenylisopropyladenosine and /sup 125/I-labeled hydroxy-phenylisopropyladenosine, were autoradiographically localized. The highest levels of binding sites occurred in the nerve fiber, ganglion cell, and inner plexiform layers of the retina in all the species examined. The distribution of adenosine A1 receptor sites closely parallels that of retinal neurons and fibers containing immunoreactive adenosine. These results suggest a role for endogenous adenosine as a coneurotransmitter in ganglion cells and their fibers in the optic nerve.

  14. Enzymatic regeneration of adenosine triphosphate cofactor

    NASA Technical Reports Server (NTRS)

    Marshall, D. L.

    1974-01-01

    Regenerating adenosine triphosphate (ATP) from adenosine diphosphate (ADP) by enzymatic process which utilizes carbamyl phosphate as phosphoryl donor is technique used to regenerate expensive cofactors. Process allows complex enzymatic reactions to be considered as candidates for large-scale continuous processes.

  15. Directed Polymerase Evolution

    PubMed Central

    Chen, Tingjian; Romesberg, Floyd E.

    2014-01-01

    Polymerases evolved in nature to synthesize DNA and RNA, and they underlie the storage and flow of genetic information in all cells. The availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. To circumvent these limitations, researchers have turned to directed evolution to tailor the properties and/or substrate repertoire of polymerases for different applications, and several systems have been developed for this purpose. These systems draw on different methods of creating a pool of randomly mutated polymerases and are differentiated by the process used to isolate the most fit members. A variety of polymerases have been evolved, providing new or improved functionality, as well as interesting new insight into the factors governing activity. PMID:24211837

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

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  17. Halogenated pyrrolopyrimidine analogues of adenosine from marine organisms: pharmacological activities and potent inhibition of adenosine kinase.

    PubMed

    Davies, L P; Jamieson, D D; Baird-Lambert, J A; Kazlauskas, R

    1984-02-01

    Two novel halogenated pyrrolopyrimidine analogues of adenosine, isolated from marine sources, have been examined for pharmacological and biochemical activities. 4-Amino-5-bromo-pyrrolo[2,3-d]pyrimidine, from a sponge of the genus Echinodictyum, had bronchodilator activity at least as potent as theophylline but with a different biochemical profile; unlike theophylline it had no antagonist activity at CNS adenosine receptors and it was quite a potent inhibitor of adenosine uptake and adenosine kinase in brain tissue. 5'-Deoxy-5-iodotubercidin, isolated from the red alga Hypnea valentiae, caused potent muscle relaxation and hypothermia when injected into mice. This compound was a very potent inhibitor of adenosine uptake into rat and guinea-pig brain slices and an extremely potent inhibitor of adenosine kinase from guinea-pig brain and rat brain and liver. Neither of these two pyrrolopyrimidine analogues was a substrate for, or an inhibitor of, adenosine deaminase. Neither compound appeared to have any direct agonist activity on guinea-pig brain adenosine-stimulated adenylate cyclase (A2 adenosine receptors). 5'-Deoxy-5-iodotubercidin is unique in two respects: it appears to be the first naturally-occurring example of a 5'-deoxyribosyl nucleoside and is the first example of a specifically iodinated nucleoside from natural sources. It may be the most potent adenosine kinase inhibitor yet described and, by virtue of its structure, may prove to be the most specific.

  18. Homeostatic control of synaptic activity by endogenous adenosine is mediated by adenosine kinase.

    PubMed

    Diógenes, Maria José; Neves-Tomé, Raquel; Fucile, Sergio; Martinello, Katiuscia; Scianni, Maria; Theofilas, Panos; Lopatár, Jan; Ribeiro, Joaquim A; Maggi, Laura; Frenguelli, Bruno G; Limatola, Cristina; Boison, Detlev; Sebastião, Ana M

    2014-01-01

    Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.

  19. Optical Aptasensors for Adenosine Triphosphate

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1991-07-01

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

  1. Adenosine Kinase: Exploitation for Therapeutic Gain

    PubMed Central

    2013-01-01

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

  2. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  3. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  4. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenosine triphosphate release assay. 864.7040... Adenosine triphosphate release assay. (a) Identification. An adenosine triphosphate release assay is a device that measures the release of adenosine triphosphate (ATP) from platelets following...

  5. Repeated administration of adenosine increases its cardiovascular effects in rats.

    PubMed

    Vidrio, H; García-Márquez, F; Magos, G A

    1987-01-20

    Hypotensive and negative chronotropic responses to adenosine in anesthetized rats increased after previous administration of the nucleoside. Bradycardia after adenosine in the isolated perfused rat heart was also potentiated after repeated administration at short intervals. This self-potentiation could be due to extracellular accumulation of adenosine and persistent stimulation of receptors caused by saturation or inhibition of cellular uptake of adenosine.

  6. Some aspects of adenosine triphosphate synthesis from adenine and adenosine in human red blood cells

    PubMed Central

    Whittam, R.; Wiley, J. S.

    1968-01-01

    1. The synthesis of ATP has been studied in human erythrocytes. Fresh cells showed no net synthesis of ATP when incubated with adenine or adenosine, although labelled adenine was incorporated into ATP in small amounts. 2. Cold-stored cells (3-6 weeks old) became progressively depleted of adenine nucleotides but incubation with adenosine or adenine plus inosine restored the ATP concentration to normal within 4 hr. Incorporation of labelled adenine or adenosine into the ATP of incubated stored cells corresponded to net ATP synthesis by these cells. 3. Synthesis of ATP from adenosine plus adenine together was 75% derived from adenine and only 25% from adenosine, indicating that nucleotide synthesis from adenine inhibits the simultaneous synthesis of nucleotide from adenosine. PMID:5723519

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

    PubMed Central

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

    2014-01-01

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

  8. The expanding polymerase universe.

    PubMed

    Goodman, M F; Tippin, B

    2000-11-01

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

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

    PubMed

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

    2015-09-01

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

  10. Genetics Home Reference: adenosine deaminase 2 deficiency

    MedlinePlus

    ... This Page Bras J, Guerreiro R, Santo GC. Mutant ADA2 in vasculopathies. N Engl J Med. 2014 ... M, Anikster Y, King MC, Levy-Lahad E. Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy. ...

  11. Role of adenosine receptors in caffeine tolerance

    SciTech Connect

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

    1991-01-01

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

  12. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium

    SciTech Connect

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

    1986-08-01

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

  13. The polymerase chain reaction.

    PubMed

    Welch, Hazel M

    2012-01-01

    The polymerase chain reaction (PCR) has had a significant impact on all aspects of the molecular biosciences, from cancer research to forensic science. The sensitivity and specificity inherent in the technique allow minute quantities of genetic material to be detected while the unique properties of thermostable DNA polymerase ensure that abundant copies are reliably reproduced to levels that can be visualized and/or used for further applications. This chapter describes applications of PCR and PCR-RT to investigate primary cancer and metastatic disease at both the DNA and mRNA expression levels.

  14. Neurabin scaffolding of adenosine receptor and RGS4 regulates anti-seizure effect of endogenous adenosine.

    PubMed

    Chen, Yunjia; Liu, Yin; Cottingham, Christopher; McMahon, Lori; Jiao, Kai; Greengard, Paul; Wang, Qin

    2012-02-22

    Endogenous adenosine is an essential protective agent against neural damage by various insults to the brain. However, the therapeutic potential of adenosine receptor-directed ligands for neuroprotection is offset by side effects in peripheral tissues and organs. An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine-evoked responses by a neural tissue-specific protein, neurabin. Neurabin attenuated adenosine A(1) receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G-protein signaling 4 (RGS4), a protein known to turn off G-protein signaling. Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. Notably, the dose of kainate capable of inducing an ∼50% rate of death in wild-type (WT) mice did not affect neurabin-null mice or WT mice cotreated with an RGS4 inhibitor. The enhanced anti-seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on-site and on-demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin-RGS4 as a novel tissue-selective regulatory mechanism for fine-tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine.

  15. Polymerase chain reaction system

    DOEpatents

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

    2004-03-02

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

  16. MOLECULAR PROBES FOR EXTRACELLULAR ADENOSINE RECEPTORS

    PubMed Central

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

    2012-01-01

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

  17. Radioimmunochemical quantitation of human adenosine deaminase.

    PubMed Central

    Daddona, P E; Frohman, M A; Kelley, W N

    1979-01-01

    Markedly reduced or absent adenosine deaminase activity in man is associated with an autosomal recesive form of severe conbined immunodeficiency disease. To further define the genetic nature of this enzyme defect, we have quantitated immunologically active adenosine deaminase (CRM) in the hemolysate of homozygous deficient patients and their heterozygous parents. A highly specific radioimmunoassay was developed capable of detecting 0.05% of normal erythrocyte adenosine deaminase. Hemolysates from nine heterozygotes (five families) showed a wide range in CRM (32--100% of normal) and variable absolute specific activities with several being at least 1 SD BELOW THE NORMAL MEAN. Hemolysates from four unrelated patients showed less than 0.09% adenosine deaminase activity with CRM ranging from less than 0.06 to 5.6% of the normal mean. In conclusion, heterozygote and homozygote hemolysates from five of the eight families analyzed revealed variable levels of CRM suggesting heterogeneous genetic alteration or expression of the silent or defective allele(s) of adenosine deaminase. PMID:468994

  18. The adenosine kinase hypothesis of epileptogenesis

    PubMed Central

    Boison, Detlev

    2008-01-01

    Current therapies for epilepsy are largely symptomatic and do not affect the underlying mechanisms of disease progression, i.e. epileptogenesis. Given the large percentage of pharmacoresistant chronic epilepsies, novel approaches are needed to understand and modify the underlying pathogenetic mechanisms. Although different types of brain injury (e.g. status epilepticus, traumatic brain injury, stroke) can trigger epileptogenesis, astrogliosis appears to be a homotypic response and hallmark of epilepsy. Indeed, recent findings indicate that epilepsy might be a disease of astrocyte dysfunction. This review focuses on the inhibitory neuromodulator and endogenous anticonvulsant adenosine, which is largely regulated by astrocytes and its key metabolic enzyme adenosine kinase (ADK). Recent findings support the “ADK hypothesis of epileptogenesis”: (i) Mouse models of epileptogenesis suggest a sequence of events leading from initial downregulation of ADK and elevation of ambient adenosine as an acute protective response, to changes in astrocytic adenosine receptor expression, to astrocyte proliferation and hypertrophy (i.e. astrogliosis), to consequential overexpression of ADK, reduced adenosine and – finally – to spontaneous focal seizure activity restricted to regions of astrogliotic overexpression of ADK. (ii) Transgenic mice overexpressing ADK display increased sensitivity to brain injury and seizures. (iii) Inhibition of ADK prevents seizures in a mouse model of pharmacoresistant epilepsy. (iv) Intrahippocampal implants of stem cells engineered to lack ADK prevent epileptogenesis. Thus, ADK emerges both as a diagnostic marker to predict, as well as a prime therapeutic target to prevent, epileptogenesis. PMID:18249058

  19. Caffeine, adenosine receptors, and synaptic plasticity.

    PubMed

    Costenla, Ana Rita; Cunha, Rodrigo A; de Mendonça, Alexandre

    2010-01-01

    Few studies to date have looked at the effects of caffeine on synaptic plasticity, and those that did used very high concentrations of caffeine, whereas the brain concentrations attained by regular coffee consumption in humans should be in the low micromolar range, where caffeine exerts pharmacological actions mainly by antagonizing adenosine receptors. Accordingly, rats drinking caffeine (1 g/L) for 3 weeks, displayed a concentration of caffeine of circa 22 microM in the hippocampus. It is known that selective adenosine A1 receptor antagonists facilitate, whereas selective adenosine A2A receptor antagonists attenuate, long term potentiation (LTP) in the hippocampus. Although caffeine is a non-selective antagonist of adenosine receptors, it attenuates frequency-induced LTP in hippocampal slices in a manner similar to selective adenosine A2A receptor antagonists. These effects of low micromolar concentration of caffeine (30 microM) are maintained in aged animals, which is important when a possible beneficial effect for caffeine in age-related cognitive decline is proposed. Future studies will still be required to confirm and detail the involvement of A1 and A2A receptors in the effects of caffeine on hippocampal synaptic plasticity, using both pharmacological and genetic approaches.

  20. Adenosine Deaminases Acting on RNA, RNA Editing, and Interferon Action

    PubMed Central

    George, Cyril X.; Gan, Zhenji; Liu, Yong

    2011-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze adenosine (A) to inosine (I) editing of RNA that possesses double-stranded (ds) structure. A-to-I RNA editing results in nucleotide substitution, because I is recognized as G instead of A both by ribosomes and by RNA polymerases. A-to-I substitution can also cause dsRNA destabilization, as I:U mismatch base pairs are less stable than A:U base pairs. Three mammalian ADAR genes are known, of which two encode active deaminases (ADAR1 and ADAR2). Alternative promoters together with alternative splicing give rise to two protein size forms of ADAR1: an interferon-inducible ADAR1-p150 deaminase that binds dsRNA and Z-DNA, and a constitutively expressed ADAR1-p110 deaminase. ADAR2, like ADAR1-p110, is constitutively expressed and binds dsRNA. A-to-I editing occurs with both viral and cellular RNAs, and affects a broad range of biological processes. These include virus growth and persistence, apoptosis and embryogenesis, neurotransmitter receptor and ion channel function, pancreatic cell function, and post-transcriptional gene regulation by microRNAs. Biochemical processes that provide a framework for understanding the physiologic changes following ADAR-catalyzed A-to-I ( = G) editing events include mRNA translation by changing codons and hence the amino acid sequence of proteins; pre-mRNA splicing by altering splice site recognition sequences; RNA stability by changing sequences involved in nuclease recognition; genetic stability in the case of RNA virus genomes by changing sequences during viral RNA replication; and RNA-structure-dependent activities such as microRNA production or targeting or protein–RNA interactions. PMID:21182352

  1. Adenine and adenosine salvage in Leishmania donovani.

    PubMed

    Boitz, Jan M; Ullman, Buddy

    2013-08-01

    6-aminopurine metabolism in Leishmania is unique among trypanosomatid pathogens since this genus expresses two distinct routes for adenine salvage: adenine phosphoribosyltransferase (APRT) and adenine deaminase (AAH). To evaluate the relative contributions of APRT and AAH, adenine salvage was evaluated in Δaprt, Δaah, and Δaprt/Δaah null mutants of L. donovani. The data confirm that AAH plays the dominant role in adenine metabolism in L. donovani, although either enzyme alone is sufficient for salvage. Adenosine salvage was also evaluated in a cohort of null mutants. Adenosine is also primarily converted to hypoxanthine, either intracellularly or extracellularly, but can also be phosphorylated to the nucleotide level by adenosine kinase when the predominant pathways are genetically or pharmacologically blocked. These data provide genetic verification for the relative contributions of 6-aminopurine metabolizing pathways in L. donovani and demonstrate that all of the pathways can function under appropriate conditions of genetic or pharmacologic perturbation.

  2. Human adenosine deaminase. Distribution and properties.

    PubMed

    Van der Weyden, M B; Kelley, W N

    1976-09-25

    Adenosine deaminase exists in multiple molecular forms in human tissue. One form of the enzyme appears to be "particulate". Three forms of the enzyme are soluble and interconvertible with apparent molecular weights of approximately 36,000, 114,000, and 298,000 (designated small, intermediate, and large, respectively). The small form of adenosine deaminase is convertible to the large form only in the presence of a protein, which has an apparent molecular weight of 200,000 and has no adenosine deaminase activity. This conversion of the small form of the enzyme to the large form occurs at 4 degrees, exhibits a pH optimum of 5.0 to 8.0, and is associated with a loss of conversion activity. The small form of the enzyme predominates in tissue preparations exhibiting the higher enzyme-specific activities and no detectable conversion activity. The large form of adenosine deaminase predominates in tissue extracts exhibiting the lower enzyme specific activities and abundant conversion activity. The small form of adenosine deaminase shows several electrophoretic variants by isoelectric focusing. The electrophoretic heterogeneity observed with the large form of the enzyme is similar to that observed with the small form, with the exception that several additional electrophoretic variants are uniformly identified. No organ specificity is demonstrable for the different electrophoretic forms. The kinetic characteristics of the three soluble molecular species of adenosine deaminase are identical except for pH optimum, which is 5.5 for the intermediate species and 7.0 to 7.4 for the large and small forms.

  3. A new class of adenosine receptors in brain: Characterization by 2-chloro( sup 3 H)adenosine binding

    SciTech Connect

    Chin, Jerome Hsicheng.

    1988-01-01

    Considerable evidence has accumulated in recent years to support a role for adenosine as an important physiological modulator in many mammalian tissues. In brain, adenosine is a potent depressant of neuronal firing and synaptic transmission. The exact mechanisms by which adenosine analogs depress nerve cell activity in the brain are not clear. Despite considerable investigation, neither the A1 nor the A2 adenosine receptors associated with adenylate cyclase have been able to account adequately for the actions of adenosine in brain. It has been proposed that additional adenosine receptors, possibly linked to calcium channels, are present in the central nervous system and are responsible for the physiological actions of adenosine. In this thesis, evidence is provided for the existence of a novel class of adenosine receptors in rat brain. The methods used to identify this new class of receptors involved radioligand binding techniques which have been successfully employed to characterize the properties of many neurotransmitter and drug receptors. 2-Chloro({sup 3}H)adenosine (Cl({sup 3}H)Ado) was selected as the ligand for these experiments since is a water-soluble, metabolically-stable analog of adenosine and a potent depressant of synaptic transmission in brain. The results demonstrate the presence of a distinct class of 2-chloro({sup 3}H)adenosine binding sites in rat forebrain membranes with an apparent K{sub D} of about 10 {mu}M and a B{sub max} of about 60 pmol per mg of protein. Specific 2-chloro ({sup 3}H)adenosine binding is highly specific for adenosine agonists and antagonists. Inhibition of binding by adenosine agonists exhibits an order of potency 2-chloroadenosine > 5{prime}-N-ethylcarboxamide adenosine > ({minus})-N{sup 6}-(R-phenylisopropyl)adenosine, which differs from that of both A1 and A2 adenosine receptors.

  4. Regulation of adenosine transport by acute and chronic ethanol exposure

    SciTech Connect

    Nagy, L.E.; Casso, D.; Diamond, I.; Gordon, A.S. )

    1989-02-09

    Chronic exposure to ethanol results in a desensitization of adenosine receptor-stimulated cAMP production. Since adenosine is released by cells and is known to desensitize its own as well as other receptors, it may be involved in ethanol-induced desensitization of adenosine receptor function. Therefore, we have examine the acute and chronic effects of ethanol on the transport of adenosine via the nucleoside transport. Acute exposure to ethanol caused an inhibition of adenosine uptake in S49 lymphoma cells. This decrease in uptake resulted in accumulation of extracellular adenosine after ethanol exposure. The effect of ethanol was specific to nucleoside transport. Uptake of uridine, also transported by the nucleoside transporter, was inhibited by ethanol to the same degree as adenosine uptake, while neither isoleucine nor deoxyglucose uptake was altered by ethanol treatment. Inhibition of adenosine uptake by ethanol was non-competitive and dependent on the concentration of ethanol. After chronic exposure to ethanol, cells became tolerant to the acute effects of ethanol. There was no longer an acute inhibition of adenosine uptake, nor was these accumulation of extracellular adenosine. Chronic ethanol exposure also resulted in a decrease in the absolute rate of adenosine uptake. Binding studies using a high affinity lignad for the nucleoside transporter, nitrobenzylthioinosine (NBMPR), indicate that this decreased uptake was due to a decrease in the maximal number of binding sites. These ethanol-induced changes in adenosine transport may be important for the acute and chronic effects of ethanol.

  5. The role of adenosine in Alzheimer's disease.

    PubMed

    Rahman, Anisur

    2009-09-01

    Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system manifested by cognitive and memory deterioration, a variety of neuropsychiatric symptoms, behavioral disturbances, and progressive impairment of daily life activities. Current pharmacotherapies are restricted to symptomatic interventions but do not prevent progressive neuronal degeneration. Therefore, new therapeutic strategies are needed to intervene with these progressive pathological processes. In the past several years adenosine, a ubiquitously released purine ribonucleoside, has become important for its neuromodulating capability and its emerging positive experimental effects in neurodegenerative diseases. Recent research suggests that adenosine receptors play important roles in the modulation of cognitive function. The present paper attempts to review published reports and data from different studies showing the evidence of a relationship between adenosinergic function and AD-related cognitive deficits. Epidemiological studies have found an association between coffee (a nonselective adenosine receptor antagonist) consumption and improved cognitive function in AD patients and in the elderly. Long-term administration of caffeine in transgenic animal models showed a reduced amyloid burden in brain with better cognitive performance. Antagonists of adenosine A2A receptors mimic these beneficial effects of caffeine on cognitive function. Neuronal cell cultures with amyloid beta in the presence of an A2A receptor antagonist completely prevented amyloid beta-induced neurotoxicity. These findings suggest that the adenosinergic system constitutes a new therapeutic target for AD, and caffeine and A2A receptor antagonists may have promise to manage cognitive dysfunction in AD.

  6. A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development

    PubMed Central

    Jones, Karlie R.; Choi, Uimook; Gao, Ji-Liang; Thompson, Robert D.; Rodman, Larry E.; Malech, Harry L.; Kang, Elizabeth M.

    2017-01-01

    Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds. PMID:28317879

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

    PubMed

    Chen, Xiao-Meng; Zhang, Meng; Fan, Peng-Li; Qin, Yu-Hua; Zhao, Hong-Wei

    2016-06-01

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

  8. Kolaviron, a biflavonoid of Garcinia kola seed mitigates ischemic/reperfusion injury by modulation of pro-survival and apoptotic signaling pathways

    PubMed Central

    Oyagbemi, Ademola Adetokunbo; Bester, Dirk; Esterhuyse, Johan; Farombi, Ebenezer Olatunde

    2017-01-01

    Objective: The study was designed to investigate the ameliorative effect of Kolaviron (KV) on ischemic/reperfusion injury in experimental animal models. Materials and Methods: Male Wistar rats were randomly divided into two groups: Group 1 received corn oil as a vehicle and rats in Group 2 were administered KV at 200 mg/kg for 4 weeks. The rats were fed with rat standard chow pellet and water administered ad libitum. After 4 weeks of KV administration, hearts were excised and mounted on the working heart perfusion system. Western blot analysis for protein expression was carried out on frozen heart samples. Results: There was significant (P < 0.05) reduction in the activity of catalase, superoxide dismutase, and glutathione peroxidase with concomitant reduction in oxygen radical absorbance capacity in ischemic rat heart of control compared to group pre-treated with KV, respectively. Similarly, intracellular reactive oxygen species and malondialdehyde were significantly elevated in control compared to KV pre-treated rats. KV significantly increased total Akt/protein kinase B (PKB), phosphorylated Akt/PKB at serine 473 and also caused a significant reduction in p38 mitogen-activated protein kinase, Caspase 3, and cleaved poly adenosine diphosphate ribose polymerase. Conclusion: Taken together, KV offered significant cardioprotection via free radical scavenging activity and upregulation of pro-survival pathway. PMID:28163959

  9. Hydroxychavicol, a Piper betle leaf component, induces apoptosis of CML cells through mitochondrial reactive oxygen species-dependent JNK and endothelial nitric oxide synthase activation and overrides imatinib resistance.

    PubMed

    Chakraborty, Jayashree B; Mahato, Sanjit K; Joshi, Kalpana; Shinde, Vaibhav; Rakshit, Srabanti; Biswas, Nabendu; Choudhury Mukherjee, Indrani; Mandal, Labanya; Ganguly, Dipyaman; Chowdhury, Avik A; Chaudhuri, Jaydeep; Paul, Kausik; Pal, Bikas C; Vinayagam, Jayaraman; Pal, Churala; Manna, Anirban; Jaisankar, Parasuraman; Chaudhuri, Utpal; Konar, Aditya; Roy, Siddhartha; Bandyopadhyay, Santu

    2012-01-01

    Alcoholic extract of Piper betle (Piper betle L.) leaves was recently found to induce apoptosis of CML cells expressing wild type and mutated Bcr-Abl with imatinib resistance phenotype. Hydroxy-chavicol (HCH), a constituent of the alcoholic extract of Piper betle leaves, was evaluated for anti-CML activity. Here, we report that HCH and its analogues induce killing of primary cells in CML patients and leukemic cell lines expressing wild type and mutated Bcr-Abl, including the T315I mutation, with minimal toxicity to normal human peripheral blood mononuclear cells. HCH causes early but transient increase of mitochondria-derived reactive oxygen species. Reactive oxygen species-dependent persistent activation of JNK leads to an increase in endothelial nitric oxide synthase-mediated nitric oxide generation. This causes loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, cleavage of caspase 9, 3 and poly-adenosine diphosphate-ribose polymerase leading to apoptosis. One HCH analogue was also effective in vivo in SCID mice against grafts expressing the T315I mutation, although to a lesser extent than grafts expressing wild type Bcr-Abl, without showing significant bodyweight loss. Our data describe the role of JNK-dependent endothelial nitric oxide synthase-mediated nitric oxide for anti-CML activity of HCH and this molecule merits further testing in pre-clinical and clinical settings.

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

    PubMed

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

    2015-03-23

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

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

    PubMed Central

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

    2015-01-01

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

  12. Fermented wheat germ extract induced cell death and enhanced cytotoxicity of Cisplatin and 5-Fluorouracil on human hepatocellular carcinoma cells.

    PubMed

    Tai, Cheng-Jeng; Wang, Wen-Ching; Wang, Chien-Kai; Wu, Chih-Hsiung; Yang, Mei-Due; Chang, Yu-Jia; Jian, Jiun-Yu; Tai, Chen-Jei

    2013-01-01

    Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death worldwide. Due to the difficulties of early diagnosis, curative treatments are not available for most patients. Palliative treatments such as chemotherapy are often associated with low response rate, strong adverse effects and limited clinical benefits for patients. The alternative approaches such as fermented wheat germ extract (FWGE) with anti-tumor efficacy may provide improvements in the clinical outcome of current therapy for HCC. This study aimed to clarify antitumor efficacy of FWGE and the combination drug effect of FWGE with chemotherapeutic agents, cisplatin and 5-fluorouracil (5-Fu) in human HCC cells, HepG2, Hep3B, and HepJ5. The present study indicated that FWGE exhibited potential to suppress HepG2, Hep3B, and HepJ5 cells, with the half maximal inhibitory concentrations (IC50) of FWGE were 0.494, 0.371 and 1.524 mg/mL, respectively. FWGE also induced Poly (Adenosine diphosphate ribose) polymerase (PARP) associated cell death in Hep3B cells. Moreover, the FWGE treatment further enhanced the cytotoxicity of cisplatin in all tested HCC cells, and cytotoxicity of 5-Fu in a synergistic manner in HepJ5 cells. Collectively, the results identified the anti-tumor efficacy of FWGE in HCC cells and suggested that FWGE can be used as a supplement to effectively improve the tumor suppression efficiency of cisplatin and 5-Fu in HCC cells.

  13. Preclinical evaluation on the tumor suppression efficiency and combination drug effects of fermented wheat germ extract in human ovarian carcinoma cells.

    PubMed

    Wang, Chia-Woei; Wang, Chien-Kai; Chang, Yu-Jia; Choong, Chen-Yen; Lin, Chi-Shian; Tai, Cheng-Jeng; Tai, Chen-Jei

    2015-01-01

    Fermented wheat germ extract (FWGE) is a nutrient supplement and a potential antitumor ingredient for developing an integrated chemotherapy with standard chemotherapeutic drugs for treating ovarian cancer patients. In this study, we evaluated the tumor suppression efficiency of FWGE in human ovarian carcinoma cells, SKOV-3 and ES-2, and found the half-maximal inhibitory concentrations (IC50s) to be 643.76 μg/mL and 246.11 μg/mL after 48 h of FWGE treatment. FWGE treatment also induced programmed cell death by activating the caspase-7 cleavage in both SKOV-3 and ES-2 cells, but only caspase-3 and poly(adenosine diphosphate-ribose) polymerase cleavages were activated in SKOV-3 cells. Moreover, FWGE exhibited combination drug effects with cisplatin and docetaxel in SKOV-3 and ES-2 cells by enhancing the cytotoxicity of both drugs. In conclusion, we found that FWGE not only suppressed cell growth but also induced caspase-3-related and caspase-7-related cell death in human ovarian carcinoma cells. FWGE treatment further enhanced the cytotoxicity of cisplatin and docetaxel, suggesting that FWGE is a potential ingredient in the development of adjuvant chemotherapy with cisplatin or docetaxel for treating ovarian cancer patients.

  14. Nicotinamide adenine dinucleotide homeostasis and signalling in heart disease: Pathophysiological implications and therapeutic potential.

    PubMed

    Mericskay, Mathias

    2016-03-01

    Heart failure is a highly morbid syndrome generating enormous socio-economic costs. The failing heart is characterized by a state of deficient bioenergetics that is not currently addressed by classical clinical approaches. Nicotinamide adenine dinucleotide (NAD(+)/NADH) is a major coenzyme for oxidoreduction reactions in energy metabolism; it has recently emerged as a signalling molecule with a broad range of activities, ranging from calcium (Ca(2+)) signalling (CD38 ectoenzyme) to the epigenetic regulation of gene expression involved in the oxidative stress response, catabolic metabolism and mitochondrial biogenesis (sirtuins, poly[adenosine diphosphate-ribose] polymerases [PARPs]). Here, we review current knowledge regarding alterations to myocardial NAD homeostasis that have been observed in various models of heart failure, and their effect on mitochondrial functions, Ca(2+), sirtuin and PARP signalling. We highlight the therapeutic approaches that are currently in use or in development, which inhibit or stimulate NAD(+)-consuming enzymes, and emerging approaches aimed at stimulating NAD biosynthesis in the failing heart.

  15. Proflavin suppresses the growth of human osteosarcoma MG63 cells through apoptosis and autophagy

    PubMed Central

    ZHANG, MAO-SHU; NIU, FU-WEN; LI, KUN

    2015-01-01

    Proflavin is one of the novel acridine derivatives that possess various pharmacological effects. Although numerous studies have been performed to investigate proflavin, its effects have not been investigated on the human osteosarcoma MG63 cell line. The core aim of the present study was to test the effects of proflavin on the viability of MG63 cells and the induction of apoptosis and autophagy in MG63 cells. The induction of apoptosis was examined by measuring the changes in the expression of the B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein mRNA and proteins. Apoptotic cell death was identified by the proteolytic cleavage of poly (adenosine diphosphate-ribose) polymerase and caspase-3 and caspase-9. In addition, the autophagic effects of proflavin were examined by the quantitation of the mRNA expression of autophagy protein 5 and Beclin 1, in addition to the identification of the accumulation of microtubule-associated protein 1 light chain 3-II. The present results revealed that proflavin inhibited the proliferation of MG63 cells in a dose-dependent manner. Proflavin-induced cell death was attributed to apoptosis and autophagy. Overall, the present results indicated that the antiseptic agent proflavin exerts anticancer potential through the synergistic activity of apoptosis and autophagy. PMID:26171052

  16. Induction of apoptosis in human promyelocytic leukemia HL60 cells by panaxynol and panaxydol.

    PubMed

    Yan, Zhonghong; Yang, Ruolin; Jiang, Yi; Yang, Zhihui; Yang, Junrui; Zhao, Qian; Lu, Yang

    2011-06-29

    Panaxynol and panaxydol are naturally occurring polyacetylenes, isolated from the lipophilic fractions of Panax notoginseng, that exert anti-proliferative effects against malignant cells. However, to the best of our knowledge, no study concerning the inhibitory effects of the two polyacetylenes on cell growth of human promyelocytic leukemia cells has been reported. In this paper, we examined the antiproliferation and proapoptotic effects of panaxynol and panaxydol on HL60 cells and investigated their mechanism of action. Cell growth inhibition of panaxynol and panaxydol were determined by trypan blue dye exclusion assays. Apoptosis of cells was revealed by morphological observation, analysis for nuclear DNA distribution and by annexin V-FITC/ PI staining using flow cytometry. It was found that panaxynol and panaxydol markedly inhibited proliferation of HL60 cells in a time- and dose-dependent manner via an apoptotic pathway. In concern with these findings, Western blot analysis showed proteolytic activation of PKCδ, caspase-3 activation and cleavage of poly (ADP [adenosine diphosphate]-ribose) polymerase in HL60 cells treated by panaxynol and panaxydol. In conclusion, panaxynol and panaxydol have profound effects on growth and apoptosis of HL60 cells, suggesting those substances are worthy of further exploration as potential anti-cancer agents.

  17. Adenosine receptors and the central nervous system.

    PubMed

    Sebastião, Ana M; Ribeiro, Joaquim A

    2009-01-01

    The adenosine receptors (ARs) in the nervous system act as a kind of "go-between" to regulate the release of neurotransmitters (this includes all known neurotransmitters) and the action of neuromodulators (e.g., neuropeptides, neurotrophic factors). Receptor-receptor interactions and AR-transporter interplay occur as part of the adenosine's attempt to control synaptic transmission. A(2A)ARs are more abundant in the striatum and A(1)ARs in the hippocampus, but both receptors interfere with the efficiency and plasticity-regulated synaptic transmission in most brain areas. The omnipresence of adenosine and A(2A) and A(1) ARs in all nervous system cells (neurons and glia), together with the intensive release of adenosine following insults, makes adenosine a kind of "maestro" of the tripartite synapse in the homeostatic coordination of the brain function. Under physiological conditions, both A(2A) and A(1) ARs play an important role in sleep and arousal, cognition, memory and learning, whereas under pathological conditions (e.g., Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, stroke, epilepsy, drug addiction, pain, schizophrenia, depression), ARs operate a time/circumstance window where in some circumstances A(1)AR agonists may predominate as early neuroprotectors, and in other circumstances A(2A)AR antagonists may alter the outcomes of some of the pathological deficiencies. In some circumstances, and depending on the therapeutic window, the use of A(2A)AR agonists may be initially beneficial; however, at later time points, the use of A(2A)AR antagonists proved beneficial in several pathologies. Since selective ligands for A(1) and A(2A) ARs are now entering clinical trials, the time has come to determine the role of these receptors in neurological and psychiatric diseases and identify therapies that will alter the outcomes of these diseases, therefore providing a hopeful future for the patients who suffer from these diseases.

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

    PubMed

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

    2009-04-01

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

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

    SciTech Connect

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

    1987-04-01

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

  20. Neuroprotective effects of adenosine deaminase in the striatum

    PubMed Central

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

    2016-01-01

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

  1. An adenosine nucleoside analogue NITD008 inhibits EV71 proliferation.

    PubMed

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

    2014-12-01

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

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

    DOE PAGES

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

    2014-01-01

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

  3. Adenosine thallium 201 myocardial perfusion scintigraphy

    SciTech Connect

    Verani, M.S. )

    1991-07-01

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

  4. Therapeutic epilepsy research: from pharmacological rationale to focal adenosine augmentation

    PubMed Central

    Boison, Detlev; Stewart, Kerry-Ann

    2009-01-01

    Epilepsy is a common seizure disorder affecting approximately 70 million people worldwide. Current pharmacotherapy is neuron-centered, frequently accompanied by intolerable side-effects, and fails to be effective in about one third of patients. Therefore, new therapeutic concepts are needed. Recent research suggests an astrocytic basis of epilepsy, presenting the possibility of novel therapeutic targets. In particular, dysfunction of the astrocyte-controlled, endogenous, adenosine-based seizure control system of the brain is implicated in seizure generation. Thus, astrogliosis – a pathological hallmark of the epileptic brain – is associated with upregulation of the adenosine-removing enzyme adenosine kinase (ADK), resulting in focal adenosine deficiency. Both astrogliotic upregulation of ADK in epilepsy and transgenic overexpression of ADK are associated with seizures, and inhibition of ADK prevents seizures in a mouse model of pharmacoresistant epilepsy. These findings link adenosine deficiency with seizures and predict that adenosine augmentation therapies (AATs) will likely be effective in preventing seizures. Given the widespread systemic and central side effects of systemically administered AATs, focal AATs (i.e., limited to the astrogliotic lesion) are a necessity. This Commentary will discuss the pharmacological rationale for the development of focal AATs. Additionally, several AAT strategies will be discussed: (1) adenosine released from silk-based brain implants; (2) adenosine released from locally implanted encapsulated cells; (3) adenosine released from stem cell-derived brain implants; and (4) adenosine augmenting gene therapies. Finally, new developments and therapeutic challenges in using focal AATs for epilepsy therapy will critically be evaluated. PMID:19682439

  5. Phosphorylation of Cytokinin by Adenosine Kinase from Wheat Germ 1

    PubMed Central

    Chen, Chong-Maw; Eckert, Richard L.

    1977-01-01

    Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N6-(δ2-isopentenyl)adenosine. Electrophoretic analysis indicated that only N6-(δ2-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine. The phosphorylation of the cytokinin and adenosine required ATP and Mg2+. The pH optimum was from 6.8 to 7.2 for both the cytokinin and adenosine. At pH 7 and 37 C the Km and Vmax for the cytokinin were 31 μm and 8.3 nmoles per mg protein per minute, and the values for adenosine were 8.7 μm and 46 nmoles per mg protein per minute. Crude enzyme preparations from tobacco callus tissue and wheat germ phosphorylated N6-(δ2-isopentenyl)adenosine. These preparations also phosphorylated N6-(δ2-isopentenyl)adenine when 5-phosphorylribose-1-pyrophosphate was present. PMID:16659870

  6. The Janus face of adenosine: antiarrhythmic and proarrhythmic actions.

    PubMed

    Szentmiklosi, A József; Galajda, Zoltán; Cseppento, Ágnes; Gesztelyi, Rudolf; Susán, Zsolt; Hegyi, Bence; Nánási, Péter P

    2015-01-01

    Adenosine is a ubiquitous, endogenous purine involved in a variety of physiological and pathophysiological regulatory mechanisms. Adenosine has been proposed as an endogenous antiarrhythmic substance to prevent hypoxia/ischemia-induced arrhythmias. Adenosine (and its precursor, ATP) has been used in the therapy of various cardiac arrhythmias over the past six decades. Its primary indication is treatment of paroxysmal supraventricular tachycardia, but it can be effective in other forms of supraventricular and ventricular arrhythmias, like sinus node reentry based tachycardia, triggered atrial tachycardia, atrioventricular nodal reentry tachycardia, or ventricular tachycardia based on a cAMP-mediated triggered activity. The main advantage is the rapid onset and the short half life (1- 10 sec). Adenosine exerts its antiarrhythmic actions by activation of A1 adenosine receptors located in the sinoatrial and atrioventricular nodes, as well as in activated ventricular myocardium. However, adenosine can also elicit A2A, A2B and A3 adenosine receptor-mediated global side reactions (flushing, dyspnea, chest discomfort), but it may display also proarrhythmic actions mediated by primarily A1 adenosine receptors (e.g. bradyarrhythmia or atrial fibrillation). To avoid the non-specific global adverse reactions, A1 adenosine receptor- selective full agonists (tecadenoson, selodenoson, trabodenoson) have been developed, which agents are currently under clinical trial. During long-term administration with orthosteric agonists, adenosine receptors can be internalized and desensitized. To avoid desensitization, proarrhythmic actions, or global adverse reactions, partial A1 adenosine receptor agonists, like CVT-2759, were developed. In addition, the pharmacologically "silent" site- and event specific adenosinergic drugs, such as adenosine regulating agents and allosteric modulators, might provide attractive opportunity to increase the effectiveness of beneficial actions of adenosine

  7. Chemoelectrical energy conversion of adenosine triphosphate

    NASA Astrophysics Data System (ADS)

    Sundaresan, Vishnu Baba; Sarles, Stephen Andrew; Leo, Donald J.

    2007-04-01

    Plant and animal cell membranes transport charged species, neutral molecules and water through ion pumps and channels. The energy required for moving species against established concentration and charge gradients is provided by the biological fuel - adenosine triphosphate (ATP) -synthesized within the cell. The adenosine triphosphatase (ATPases) in a plant cell membrane hydrolyze ATP in the cell cytoplasm to pump protons across the cell membrane. This establishes a proton gradient across the membrane from the cell exterior into the cell cytoplasm. This proton motive force stimulates ion channels that transport nutrients and other species into the cell. This article discusses a device that converts the chemical energy stored in adenosine triphosphate into electrical power using a transporter protein, ATPase. The V-type ATPase proteins used in our prototype are extracted from red beet(Beta vulgaris) tonoplast membranes and reconstituted in a bilayer lipid membrane or BLM formed from POPC and POPS lipids. A pH7 medium that can support ATP hydrolysis is provided on both sides of the membrane and ATP is dissolved in the pH7 buffer on one side of the membrane. Hydrolysis of ATP results in the formation of a phosphate ion and adenosine diphosphate. The energy from the reaction activates ATPase in the BLM and moves a proton across the membrane. The charge gradient established across the BLM due to the reaction and ion transport is converted into electrical current by half-cell reference electrodes. The prototype ATPase cell with an effective BLM area of 4.15 mm2 carrying 15 μl of ATPase proteins was observed to develop a steady state peak power output of 70 nW, which corresponds to a specific power of 1.69 μW/cm2 and a current density of 43.4 μA/cm2 of membrane area.

  8. Role of adenosine in oligodendrocyte precursor maturation

    PubMed Central

    Coppi, Elisabetta; Cellai, Lucrezia; Maraula, Giovanna; Dettori, Ilaria; Melani, Alessia; Pugliese, Anna Maria; Pedata, Felicita

    2015-01-01

    Differentiation and maturation of oligodendroglial cells are postnatal processes that involve specific morphological changes correlated with the expression of stage-specific surface antigens and functional voltage-gated ion channels. A small fraction of oligodendrocyte progenitor cells (OPCs) generated during development are maintained in an immature and slowly proliferative or quiescent state in the adult central nervous system (CNS) representing an endogenous reservoir of immature cells. Adenosine receptors are expressed by OPCs and a key role of adenosine in oligodendrocyte maturation has been recently recognized. As evaluated on OPC cultures, adenosine, by stimulating A1 receptors, promotes oligodendrocyte maturation and inhibits their proliferation; on the contrary, by stimulating A2A receptors, it inhibits oligodendrocyte maturation. A1 and A2A receptor-mediated effects are related to opposite modifications of outward delayed rectifying membrane K+ currents (IK) that are involved in the regulation of oligodendrocyte differentiation. Brain A1 and A2A receptors might represent new molecular targets for drugs useful in demyelinating pathologies, such as multiple sclerosis (MS), stroke and brain trauma. PMID:25964740

  9. Effects of adenosine perfusion on the metabolism and contractile activity of Rana ridibunda heart.

    PubMed

    Lazou, A; Beis, I

    1987-01-01

    The effects of adenosine were examined on the isolated perfused heart of the frog Rana ridibunda. Adenosine produced negative chronotropic and inotropic effects on frog ventricle in a concentration-dependent manner. The effects of adenosine on cardiac metabolism were also investigated by measuring the tissue content of adenine nucleotides, lactate, pyruvate, adenosine and inorganic phosphate, during adenosine perfusion. Adenosine had no effect on the tissue content of metabolites. No net synthesis of adenine nucleotides was observed during perfusion with increasing concentrations of adenosine. Lactate output from the heart decreased significantly with adenosine perfusion. Correlation of adenosine effects on cardiac muscle with the effects of hypoxia are discussed.

  10. Effect of adenosine and adenosine analogues on cyclic AMP accumulation in cultured mesangial cells and isolated glomeruli of the rat.

    PubMed Central

    Olivera, A.; Lopez-Novoa, J. M.

    1992-01-01

    1. Changes in intracellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were studied in rat isolated glomeruli and cultured glomerular mesangial cells exposed to adenosine and to the preferential A1 receptor agonist N6-R-1-methyl-2-phenylethyl adenosine (R-PIA), or the potent A2 adenosine receptor agonist 5-(N-ethylcarboxamide)adenosine (NECA). 2. Whereas NECA and adenosine triggered a dose-dependent increase in cyclic AMP values with EC50 values of approximately 10(-6) M and 3 x 10(-5) M respectively, R-PIA lowered cyclic AMP levels at concentrations of 10(-6) M or less and increased them at higher concentrations. 3. The time-course of the increase induced by 10(-6) M NECA was slower than that induced by 10(-4) M adenosine. Adenosine produced a maximal stimulation within the first minute, whereas the effect of NECA in both glomeruli and mesangial cells was noticeable only from the second minute of incubation. 4. The effects of the agonists R-PIA and NECA on the cyclic AMP system were blocked respectively by the A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthihe (DPCPX) at 10(-6) M and the A2 antagonist N-(2-dimethylaminoethyl)-N-methyl-4-(2, 3, 6, 7-tetrahydro-2,b-dioxo-1, 3-dipropyl-1H-purin-8-yl) benzene sulphonamide (PD115,199) at 10(-6) M. Theophylline, a known antagonist of adenosine receptors, inhibited the action of adenosine on cyclic AMP in mesangial cells. Dipyridamole, an inhibitor of the uptake of adenosine by the cells, enhanced the response to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1330173

  11. [The involvement of adenosine and adenosine deaminase in experimental myocardial infarct].

    PubMed

    Stratone, A; Busuioc, A; Roşca, V; Bazgan, L; Popa, M; Hăulică, I

    1989-01-01

    By the ligature of the left coronary artery in the rat anesthetized with nembutal (10 mg/100 i.p.) a significant increase of the 5'-nucleotidase activity (Wooton method) was noticed 10 minutes after the left ventricle infarction (from an average value of 1038.5 +/- 187 mU/g tissue to 1537 +/- 225 mU/g fresh tissue). The adenosine desaminase levels spectrophotometrically determined by Denstedt technique, do not appear significantly modified 10 or 30 minutes after the left ventricle infarction. The chromatographically determined adenosine levels, by HPLC technique, decrease from the average value of 11.63 +/- 1.4 micrograms/mg PT to 8.60 +/- 1.0 micrograms/mg PT 30 minutes after infarction. The observed changes are explained by the conditions of hypoxia in the infarcted ventricle which lead to the raise in adenosine levels by activating the 5'-nucleotidase and their depression by a very fast metabolism of the same substance.

  12. Adenosine and inflammation: what's new on the horizon?

    PubMed

    Antonioli, Luca; Csóka, Balázs; Fornai, Matteo; Colucci, Rocchina; Kókai, Endre; Blandizzi, Corrado; Haskó, György

    2014-08-01

    Adenosine contributes to the maintenance of tissue integrity by modulating the immune system. Encouraging results have emerged with adenosine receptor ligands for the management of several inflammatory conditions in preclinical and clinical settings. However, therapeutic applications of these drugs are sometimes complicated by the occurrence of serious adverse effects. The scientific community is making intensive efforts to design novel adenosine receptor ligands endowed with greater selectivity or to develop innovative compounds acting as allosteric receptor modulators. In parallel, research is focusing on novel pharmacological entities (designated as adenosine-regulating agents) that can increase, in a site- and event-specific manner, adenosine concentrations at the inflammatory site, thereby minimizing the adverse systemic effects of adenosine.

  13. Rhodium Complex and Enzyme Couple Mediated Electrochemical Detection of Adenosine.

    PubMed

    Han, Dawoon; Kim, Hyeong-Mook; Chand, Rohit; Kim, Gyumin; Shin, Ik-Soo; Kim, Yong-Sang

    2015-10-01

    Adenosine is one of the nucleoside which plays an important role in signal transduction and neuromodulation. This work proposes a simple electrochemical assay, comprising two enzymes and rhodium complex based electron transfer mediator, for the detection of adenosine. Sequential reaction of adenosine deaminase and L-glutamic dehydrogenase and the supporting cycle between β-NADH and mediator enable quantitative analysis of adenosine. Role of electron transfer mediator is the conveyance of proton from electrode to β-NAD(+) for regeneration of β-NADH. The electrochemical characteristics of electron transfer mediator were also studied. Real-time adenosine detection was carried out using this multiple enzyme based chronoamperometric assay. The analysis results show a low limit of detection (140 μM) and good correspondence between current signal and the adenosine concentration (R (2) = 0.997).

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

    PubMed

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

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2011-04-01

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

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

    SciTech Connect

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

    1989-04-01

    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 (/sup 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.

  18. Cardioprotection with adenosine: 'a riddle wrapped in a mystery'.

    PubMed

    Przyklenk, Karin; Whittaker, Peter

    2005-07-01

    Review of the published literature on adenosine and cardioprotection could lead one to paraphrase the famous words of Sir Winston Churchill (Radio broadcast, 1 October 1939 (in reference to Russia)) and conclude: 'I cannot forecast to you the action of adenosine. It is a riddle wrapped in a mystery inside an enigma'. That is, although it is well-established that adenosine can render cardiomyocytes resistant to lethal ischemia/reperfusion-induced injury, new and intriguing insights continue to emerge as to the mechanisms by which adenosine might limit myocardial infarct size.

  19. Adenosine modulates LPS-induced cytokine production in porcine monocytes.

    PubMed

    Ondrackova, Petra; Kovaru, Hana; Kovaru, Frantisek; Leva, Lenka; Faldyna, Martin

    2013-03-01

    Adenosine plays an important role during inflammation, particularly through modulation of monocyte function. The objective of the present study was to evaluate the effect of synthetic adenosine analogs on cytokine production by porcine monocytes. The LPS-stimulated cytokine production was measured by flow cytometry and quantitative real-time PCR. Adenosine receptor expression was measured by quantitative real-time PCR. The present study demonstrates that adenosine analog N-ethylcarboxyamidoadenosine (NECA) down-regulates TNF-α production and up-regulates IL-8 production by LPS-stimulated porcine monocytes. The effect was more pronounced in CD163(-) subset of monocytes compared to the CD163(+) subset. Although both monocyte subsets express mRNA for A1, A2A, A2B and A3 adenosine receptors, the treatment of monocytes with various adenosine receptor agonists and antagonists proved that the effect of adenosine is mediated preferentially via A2A adenosine receptor. Moreover, the study suggests that the effect of NECA on porcine monocytes alters the levels of the cytokines which could play a role in the differentiation of naive T cells into Th17 cells. The results suggest that adenosine plays an important role in modulation of cytokine production by porcine monocytes.

  20. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment

    PubMed Central

    Ohta, Akio

    2016-01-01

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

  1. The Role of Adenosine Signaling in Headache: A Review

    PubMed Central

    Fried, Nathan T.; Elliott, Melanie B.; Oshinsky, Michael L.

    2017-01-01

    Migraine is the third most prevalent disease on the planet, yet our understanding of its mechanisms and pathophysiology is surprisingly incomplete. Recent studies have built upon decades of evidence that adenosine, a purine nucleoside that can act as a neuromodulator, is involved in pain transmission and sensitization. Clinical evidence and rodent studies have suggested that adenosine signaling also plays a critical role in migraine headache. This is further supported by the widespread use of caffeine, an adenosine receptor antagonist, in several headache treatments. In this review, we highlight evidence that supports the involvement of adenosine signaling in different forms of headache, headache triggers, and basic headache physiology. This evidence supports adenosine A2A receptors as a critical adenosine receptor subtype involved in headache pain. Adenosine A2A receptor signaling may contribute to headache via the modulation of intracellular Cyclic adenosine monophosphate (cAMP) production or 5' AMP-activated protein kinase (AMPK) activity in neurons and glia to affect glutamatergic synaptic transmission within the brainstem. This evidence supports the further study of adenosine signaling in headache and potentially illuminates it as a novel therapeutic target for migraine. PMID:28335379

  2. An Essential Role for Adenosine Signaling in Alcohol Abuse

    PubMed Central

    Ruby, Christina L.; Adams, Chelsea; Knight, Emily J.; Nam, Hyung Wook; Choi, Doo-Sup

    2014-01-01

    In the central nervous system (CNS), adenosine plays an important role in regulating neuronal activity and modulates signaling by other neurotransmitters, including GABA, glutamate, and dopamine. Adenosine suppresses neurotransmitter release, reduces neuronal excitability, and regulates ion channel function through activation of four classes of G protein-coupled receptors, A1, A2A, A2B, and A3. Central adenosine levels are largely controlled by nucleoside transporters, which regulate adenosine levels across the plasma membrane. Adenosine has been shown to modulate cortical glutamate signaling and ventral-tegmental dopaminergic signaling, which are involved in several aspects of alcohol use disorders. Acute ethanol elevates extracellular adenosine levels by selectively inhibiting the type 1 equilibrative nucleoside transporter, ENT1. Raised adenosine levels mediate the ataxic and sedative/hypnotic effects of ethanol through activation of A1 receptors in the cerebellum, striatum, and cerebral cortex. Recently, we have shown that pharmacological inhibition or genetic deletion of ENT1 reduces the expression of excitatory amino acid transporter 2 (EAAT2), the primary regulator of extracellular glutamate, in astrocytes. These lines of evidence support a central role for adenosine-mediated glutamate signaling and the involvement of astrocytes in regulating ethanol intoxication and preference. In this paper, we discuss recent findings on the implication of adenosine signaling in alcohol use disorders. PMID:21054262

  3. Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay.

    PubMed

    Vassiliou, W; Epp, J B; Wang, B B; Del Vecchio, A M; Widlanski, T; Kao, C C

    2000-09-01

    We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs). These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis.

  4. Elevated adenosine signaling via adenosine A2B receptor induces normal and sickle erythrocyte sphingosine kinase 1 activity.

    PubMed

    Sun, Kaiqi; Zhang, Yujin; Bogdanov, Mikhail V; Wu, Hongyu; Song, Anren; Li, Jessica; Dowhan, William; Idowu, Modupe; Juneja, Harinder S; Molina, Jose G; Blackburn, Michael R; Kellems, Rodney E; Xia, Yang

    2015-03-05

    Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide in vivo genetic evidence that adenosine deaminase (ADA) deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

  5. Separation of adenosine diphosphate--adenosine triphosphate-exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase.

    PubMed

    Stahl, W L; Sattin, A; McIlwain, H

    1966-05-01

    1. A microsomal fraction from ox cerebral cortex catalysed [(14)C]ADP-ATP exchange at a speed similar to that at which it liberated P(i) from ATP in the presence of Na(+), K(+) and Mg(2+). 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na(+), K(+) or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide-cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na(+)-plus-K(+)-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP-ATP-exchange activity does not take part in the Na(+)-plus-K(+)-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP-ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na(+)-plus-K(+)-stimulated adenosine triphosphatase is indicated.

  6. Separation of adenosine diphosphate-adenosine triphosphate–exchange activity from the cerebral microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase

    PubMed Central

    Stahl, W. L.; Sattin, A.; McIlwain, H.

    1966-01-01

    1. A microsomal fraction from ox cerebral cortex catalysed [14C]ADP–ATP exchange at a speed similar to that at which it liberated Pi from ATP in the presence of Na+, K+ and Mg2+. 2. Repeated washing the fraction with MgATP solutions solubilized most of the exchange activity and left the adenosine triphosphatase insoluble and little changed in activity. The exchange activity was accompanied by negligible adenosine-triphosphatase activity and was enriched by precipitation at chosen pH and by DEAE-Sephadex. At no stage was its activity affected by Na+, K+ or ouabain. 3. The washed microsomal fraction was exposed to a variety of reagents; a sodium iodide–cysteine treatment increased both adenosine-triphosphatase and exchange activities, as also did a synthetic zeolite. Preparations were obtained with exchange activities less than 3% of their Na+-plus-K+-stimulated adenosine-triphosphatase activity. Some contribution to the residual exchange activity was made by an adenylate kinase. 4. Thus over 95% of the microsomal ADP–ATP-exchange activity does not take part in the Na+-plus-K+-stimulated adenosine-triphosphatase reaction. Participation of some of the residual 3% of the ADP–ATP-exchange activity has not been excluded, but there appears no firm evidence for its participation in the adenosine triphosphatase; the bearing of this conclusion on mechanisms proposed for the Na+-plus-K+-stimulated adenosine triphosphatase is indicated. PMID:4223577

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

    PubMed Central

    Robson, Simon C.; Schuppan, Detlef

    2010-01-01

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

  8. Adenosine signaling in normal and sickle erythrocytes and beyond

    PubMed Central

    Zhang, Yujin; Xia, Yang

    2012-01-01

    Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications. In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A2B receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O2 release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A2A receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD. Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and progression

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

    PubMed

    Boison, Detlev; Aronica, Eleonora

    2015-10-01

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

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

    SciTech Connect

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

    1986-03-01

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

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

    PubMed Central

    Boison, Detlev; Aronica, Eleonora

    2015-01-01

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

  12. Adenosine signaling promotes hematopoietic stem and progenitor cell emergence.

    PubMed

    Jing, Lili; Tamplin, Owen J; Chen, Michael J; Deng, Qing; Patterson, Shenia; Kim, Peter G; Durand, Ellen M; McNeil, Ashley; Green, Julie M; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K; Schlaeger, Thorsten M; Huttenlocher, Anna; Daley, George Q; Ravid, Katya; Zon, Leonard I

    2015-05-04

    Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1(+)/cmyb(+) HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl(+) hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP-protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

  13. Adenosine strongly potentiates pressor responses to nicotine in rats.

    PubMed Central

    von Borstel, R W; Renshaw, A A; Wurtman, R J

    1984-01-01

    Intravenous infusion of subhypotensive doses of adenosine strongly potentiates the pressor response of anesthetized rats to nicotine. A dose of nicotine (40 micrograms/kg, i.v.), which, given alone, elicits a peak increase in diastolic pressure of approximately equal to 15 mm Hg, increases pressure by approximately equal to 70 mm Hg when arterial plasma adenosine levels have been increased to 2 microM from a basal concentration of approximately equal to 1 microM. The pressor response to cigarette smoke applied to the lungs is also strongly potentiated during infusion of adenosine. Slightly higher adenosine concentrations (approximately equal to 4 microM) attenuate pressor responses to electrical stimulation of preganglionic sympathetic nerves, or to injections of the alpha-adrenergic agonist phenylephrine, but continue to potentiate pressor responses to nicotine. Low doses (0.25-5 micrograms/kg) of the synthetic adenosine receptor agonists 5'-N-cyclopropylcarboxamidoadenosine, 2-chloroadenosine, and N6-L-phenylisopropyladenosine also potentiate pressor responses to nicotine. Caffeine and theophylline (10 mg/kg) block the potentiating effect of adenosine, and also decrease basal responses to nicotine, suggesting that endogenous adenosine might normally potentiate some nicotine responses. The synergism between nicotine and adenosine appears to take place within sympathetic ganglia. PMID:6591207

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

    PubMed

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

    2015-05-01

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

  15. Adenosine receptors and asthma in humans.

    PubMed

    Wilson, C N

    2008-10-01

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

  16. Adenosine receptors and dyskinesia in pathophysiology.

    PubMed

    Tomiyama, Masahiko

    2014-01-01

    First, the recent progress in the pathogenesis of levodopa-induced dyskinesia was described. Serotonin neurons play an important role in conversion from levodopa to dopamine and in the release of converted dopamine into the striatum in the Parkinsonian state. Since serotonin neurons lack buffering effects on synaptic dopamine concentration, the synaptic dopamine markedly fluctuates depending on the fluctuating levodopa concentration in the serum after taking levodopa. The resultant pulsatile stimulation makes the striatal direct-pathway neurons get potential that releases excessive GABA into the output nuclei of the basal ganglia. When levodopa is administered, the stored GABA is released, the output nuclei become hypoactive, and then dyskinesias emerge. Second, effects of adenosine A2A receptor antagonists on dyskinesia were described. It has been demonstrated that the expression of adenosine A2A receptors is increased in Parkinson's disease (PD) patients with dyskinesias, suggesting that blockade of A2A receptors is beneficial for dyskinesias. Preclinical studies have shown that A2A receptor antagonists reduce liability of dyskinesias in PD models. Clinical trials have demonstrated that A2A antagonists increase functional ON-time (ON without troublesome dyskinesia) in PD patients suffering from wearing-off phenomenon, although they may increase dyskinesia in patients with advanced PD.

  17. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    SciTech Connect

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

    2011-12-31

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

  18. Adenosine reduces postbypass transfusion requirements in humans after heart surgery.

    PubMed Central

    Mentzer, R M; Rahko, P S; Canver, C C; Chopra, P S; Love, R B; Cook, T D; Hegge, M O; Lasley, R D

    1996-01-01

    OBJECTIVE: The objective of this study was to determine the effect, if any, of adenosine blood cardioplegia on blood component usage after heart surgery. SUMMARY BACKGROUND DATA: The most common cause of nonsurgical postcardiopulmonary bypass bleeding is platelet dysfunction. For this reason, pharmacologic agents are under investigation in an effort to reduce the need for transfusion in this setting. METHODS: A posthoc analysis of blood product usage was performed in data obtained from a Phase I, single center, open label, randomized study performed in 63 patients. The trial was designed to test the safety and tolerance of adenosine when added to blood cardioplegia in increasing doses to enhance myocardial protection. The database provided information regarding the effect of adenosine cardioplegia on venous plasma adenosine concentrations, the amount of platelets, fresh frozen plasma and packed erythrocytes used, and the association between the adenosine dose and postoperative thoracic drainage. RESULTS: The postoperative thoracic drainage at 6 hours, 24 hours, and at the time of chest tube removal in the high-dose adenosine cardioplegia group was 68%, 76%, and 75% of the placebo and low-dose adenosine cardioplegia group (p < 0.05). The highest dose of adenosine studied increased baseline adenosine venous plasma levels 360-fold, from 0.17 +/- 0.09 mumol/L to 42.30 +/- 11.20 mumol/L (p < 0.05). This marked increase was associated with a 68%, 56%, and 58% reduction in platelet, fresh frozen plasma, and packed erythrocyte usage, respectively (p < 0.05). CONCLUSIONS: In addition to enhancing the heart's tolerance to ischemia, adenosine-supplemented cardioplegic solution also may reduce bleeding after cardiopulmonary bypass. PMID:8857856

  19. Adenosine Deaminases Acting on RNA (ADARs) are both Antiviral and Proviral Dependent upon the Virus

    PubMed Central

    Samuel, Charles E.

    2010-01-01

    A-to-I RNA editing, the deamination of adenosine (A) to inosine (I) that occurs in regions of RNA with double-stranded character, is catalyzed by a family of Adenosine Deaminases Acting on RNA (ADARs). In mammals there are three ADAR genes. Two encode proteins that possess demonstrated deaminase activity: ADAR1, which is interferon-inducible, and ADAR2 which is constitutively expressed. ADAR3, by contrast, has not yet been shown to bean active enzyme. The specificity of the ADAR1 and ADAR2 deaminases ranges from highly site-selective to non-selective, dependent on the duplex structure of the substrate RNA. A-to-I editing is a form of nucleotide substitution editing, because I is decoded as guanosine (G) instead of A by ribosomes during translation and by polymerases during RNA-dependent RNA replication. Additionally, A-to-I editing can alter RNA structure stability as I:U mismatches are less stable than A:U base pairs. Both viral and cellular RNAs are edited by ADARs. A-to-I editing is of broad physiologic significance. Among the outcomes of A-to-I editing are biochemical changes that affect how viruses interact with their hosts, changes that can lead to either enhanced or reduced virus growth and persistence dependent upon the specific virus. PMID:21211811

  20. Fluorescence aptameric sensor for isothermal circular strand-displacement polymerization amplification detection of adenosine triphosphate.

    PubMed

    Song, Weiling; Zhang, Qiao; Xie, Xuxu; Zhang, Shusheng

    2014-11-15

    In this work, isothermal circular strand-displacement polymerization amplification assay is developed for highly specific and sensitive detection of adenosine triphosphate (ATP). The amplification process consists of circular common target molecule-displacement polymerization (CCDP) and circular nucleic acid strand-displacement polymerization (CNDP). In the presence of ATP, the complementary strand was released from the aptamer by the target recognition of ATP, and catalyzed the subsequent cycle reaction. With the polymerase and primer, the displaced target triggers the process of CCDP. With the involvement of nicking endonuclease, the released complementary strand triggers the CNDP. Combined CCDP with CNDP, the exponentially produced fluorescence probes are obtained, achieving a detection limit of ATP as low as 2.6 × 10(-10)M. Moreover, the proposed strategy exhibits an excellent specificity and is successfully applied in real sample assay which demonstrates potential application in practical samples.

  1. Adenosine receptors and diabetes: Focus on the A(2B) adenosine receptor subtype.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Gessi, Stefania

    2015-09-01

    Over the last two decades, diabetes mellitus has become one of the most challenging health problems worldwide. Diabetes mellitus, classified as type I and II, is a pathology concerning blood glucose level in the body. The nucleoside adenosine has long been known to affect insulin secretion, glucose homeostasis and lipid metabolism, through activation of four G protein coupled adenosine receptors (ARs), named A1, A2A, A2B and A3. Currently, the novel promising subtype to develop new drugs for diabetes treatment is the A2BAR subtype. The use of selective agonists and antagonists for A2BAR subtype in various diabetic animal models allowed us to identify several effects of A2BAR signaling in cell metabolism. In particular, the focus of this review is to summarize the studies on purinergic signaling associated with diabetes through A2BARs modulation.

  2. Ethanol Tolerance Affects Endogenous Adenosine Signaling in Mouse Hippocampus

    PubMed Central

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

    2016-01-01

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

  3. Adenosine 2A receptors in acute kidney injury.

    PubMed

    Vincent, I S; Okusa, M D

    2015-07-01

    Acute kidney injury (AKI) is an important clinical problem that may lead to death and for those who survive, the sequelae of AKI include loss of quality of life, chronic kidney disease and end-stage renal disease. The incidence of AKI continues to rise without clear successes in humans for the pharmacological prevention of AKI or treatment of established AKI. Dendritic cells and macrophages are critical early initiators of innate immunity in the kidney and orchestrate inflammation subsequent to ischaemia-reperfusion injury. These innate cells are the most abundant leucocytes present in the kidney, and they represent a heterogeneous population of cells that are capable of responding to cues from the microenvironment derived from pathogens or endogenous inflammatory mediators such as cytokines or anti-inflammatory mediators such as adenosine. Lymphocyte subsets such as natural killer T cells and Tregs also play roles in regulating ischaemic injury by promoting and suppressing inflammation respectively. Adenosine, produced in response to IR, is generally considered as a protective signalling molecule and elicits its physiological responses through four distinct adenosine receptors. However, its short half-life, lack of specificity and rapid metabolism limit the use of adenosine as a therapeutic agent. These adenosine receptors play various roles in regulating the activity of the aforementioned hematopoietic cells in elevated levels of adenosine such as during hypoxia. This review focuses on the importance of one receptor, the adenosine 2A subtype, in blocking inflammation associated with AKI.

  4. Temporal variations of adenosine metabolism in human blood.

    PubMed

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yáñez, L; Aguilar-Roblero, R; Oksenberg, A; Vega-González, A; Villalobos, L; Rosenthal, L; Fernández-Cancino, F; Drucker-Colín, R; Díaz-Muñoz, M

    1996-08-01

    Eight diurnally active (06:00-23:00 h) subjects were adapted for 2 days to the room conditions where the experiments were performed. Blood sampling for adenosine metabolites and metabolizing enzymes was done hourly during the activity span and every 30 min during sleep. The results showed that adenosine and its catabolites (inosine, hypoxanthine, and uric acid), adenosine synthesizing (S-adenosylhomocysteine hydrolase and 5'-nucleotidase), degrading (adenosine deaminase) and nucleotide-forming (adenosine kinase) enzymes as well as adenine nucleotides (AMP, ADP, and ATP) undergo statistically significant fluctuations (ANOVA) during the 24 h. However, energy charge was invariable. Glucose and lactate chronograms were determined as metabolic indicators. The same data analyzed by the chi-square periodogram and Fourier series indicated ultradian oscillatory periods for all the metabolites and enzymatic activities determined, and 24-h oscillatory components for inosine, hypoxanthine, adenine nucleotides, glucose, and the activities of SAH-hydrolase, 5'-nucleotidase, and adenosine kinase. The single cosinor method showed significant oscillatory components exclusively for lactate. As a whole, these results suggest that adenosine metabolism may play a role as a biological oscillator coordinating and/or modulating the energy homeostasis and physiological status of erythrocytes in vivo and could be an important factor in the distribution of purine rings for the rest of the organism.

  5. Characterization of P1 (adenosine) purinoceptors.

    PubMed

    Jarvis, Michael F

    2013-10-08

    The purine nucleoside adenosine (ADO) is an important modulator of cellular function in mammalian tissues, modulating cellular function and neuronal excitability via interactions with different cell surface receptor subtypes that are heterogeneously distributed in both the mammalian CNS and peripheral tissues. Four ADO receptor subtypes have been cloned and characterized. Described in this unit are three radioligand binding assays for pharmacological characterization of the high-affinity ADO receptor subtypes A1, A2A, and A3 receptors. Pharmacological characterization of the low-affinity A2B receptor has been enabled by the use of tritiated xanthine PSB-603. Because receptor localization is an important criterion for differentiation of receptor subtypes, a support protocol that describes the methodology for the localization of ADO receptors in rat brain tissue using autoradiography is also included.

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

    NASA Astrophysics Data System (ADS)

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

    2004-10-01

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

  7. Adenosine and Preexcitation Variants: Reappraisal of Electrocardiographic Changes.

    PubMed

    Ali, Hussam; Lupo, Pierpaolo; Foresti, Sara; De Ambroggi, Guido; Epicoco, Gianluca; Fundaliotis, Angelica; Cappato, Riccardo

    2016-07-01

    Intravenous adenosine is a short-acting blocker of the atrioventricular node that has been used to unmask subtle or latent preexcitation, and also to enable catheter ablation in selected patients with absent or intermittent preexcitation. Depending on the accessory pathway characteristics, intravenous adenosine may produce specific electrocardiographic changes highly suggestive of the preexcitation variant. Herein, we view different ECG responses to this pharmacological test in various preexcitation patterns that were confirmed by electrophysiological studies. Careful analysis of electrocardiographic changes during adenosine test, with emphasis on P-delta interval, preexcitation degree, and atrioventricular block, can be helpful to diagnose the preexcitation variant/pattern.

  8. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  9. Possible mechanism of adenosine protection in carbon tetrachloride acute hepatotoxicity. Role of adenosine by-products and glutathione peroxidase.

    PubMed

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Yáñez, L; Vidrio, S; Díaz-Muñoz, M

    1995-02-01

    Adenosine proved to be an effective hepatoprotector increasing the survival rate of rats receiving lethal doses of CCl4. Searching for the mechanism of action, we found that adenosine transiently prevents the necrotic liver damage associated to an acute CCl4 treatment. The antilipoperoxidative action of the nucleoside was evidenced by a decrease of TBA-reactive products and the diene conjugates elicited by the hepatotoxin. Adenosine's protective effect was demonstrated by reverting the decrease of cytochrome P-450 while preserved intact the activity of the microsomal enzyme glucose-6-phosphatase. CCl4 promoted an increase in the oxidant stress through an enhancement in oxidized glutathione levels. This action was also completely counteracted by the nucleoside. Adenosine was unable to prevent CCl4 activation and, even, increased .CCl3 formation in the presence of PBN in vivo. However, in the presence of the nucleoside, irreversible binding of 14CCl4 to the microsomal lipid fraction of the treated animals was decreased. These results suggest that adenosine protective action might be exerted at the level of the propagation reaction following CCl4 activation. Two possible mechanisms were associated to the nucleoside protection: (1) the peroxide-metabolyzed enzymes, GSH-per, showed a marked increase after 30 minutes of adenosine treatment, which was potentiated by the hepatotoxin, suggesting an important role of this enzyme in the nucleoside's action; (2) the adenosine catabolism induced an increase in uric acid level, and allopurinol, a purine metabolism inhibitor, prevented such elevation as well as the antilipoperoxidative action of adenosine and the increase of GSH-per associated with the nucleoside treatment. These facts strongly suggest that the protective effect elicited by adenosine is not a direct one, but rather is related to its catabolic products, such as uric acid, which has been recognized as a free radical scavenger.

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  12. Adenosine promotes vascular barrier function in hyperoxic lung injury

    PubMed Central

    Davies, Jonathan; Karmouty‐Quintana, Harry; Le, Thuy T.; Chen, Ning‐Yuan; Weng, Tingting; Luo, Fayong; Molina, Jose; Moorthy, Bhagavatula; Blackburn, Michael R.

    2014-01-01

    Abstract Hyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response in the lung with cellular infiltration and pulmonary edema. Adenosine is a signaling molecule that is generated extracellularly by CD73 in response to injury. Extracellular adenosine signals through cell surface receptors and has been found to be elevated and plays a protective role in acute injury situations. In particular, ADORA2B activation is protective in acute lung injury. However, little is known about the role of adenosine signaling in hyperoxic lung injury. We hypothesized that hyperoxia‐induced lung injury leads to CD73‐mediated increases in extracellular adenosine, which is protective through ADORA2B signaling pathways. To test this hypothesis, we exposed C57BL6, CD73−/−, and Adora2B−/− mice to 95% oxygen or room air and examined markers of pulmonary inflammation, edema, and monitored lung histology. Hyperoxic exposure caused pulmonary inflammation and edema in association with elevations in lung adenosine levels. Loss of CD73‐mediated extracellular adenosine production exacerbated pulmonary edema without affecting inflammatory cell counts. Furthermore, loss of the ADORA2B had similar results with worsening of pulmonary edema following hyperoxia exposure without affecting inflammatory cell infiltration. This loss of barrier function correlated with a decrease in occludin in pulmonary vasculature in CD73−/− and Adora2B−/− mice following hyperoxia exposure. These results demonstrate that exposure to a hyperoxic environment causes lung injury associated with an increase in adenosine concentration, and elevated adenosine levels protect vascular barrier function in hyperoxic lung injury through the ADORA2B‐dependent regulation of occludin. PMID:25263205

  13. Sequence requirements for transcriptional arrest in exon 1 of the human adenosine deaminase gene

    SciTech Connect

    Zhi Chen; Kellems, R.E.; Innis, J.W. ); Sun, Minghua; Wright, D.A. )

    1991-12-01

    The authors have previously demonstrated that a transcriptional arrest site exists in exon 1 of the human adenosine deaminase (ADA) gene and that this site may play a role in ADA gene expression. Sequences involved in this process are not known precisely. To further define the template requirements for transcriptional arrest within exon 1 of the human ADA gene, various ADA templates were constructed and their abilities to confer transcriptional arrest were determined following injection into Xenopus oocytes. The exon 1 transcriptional arrest signal functioned downstream of several RNA polymerase II promoters and an RNA polymerase II promoter, implying that the transcriptional arrest site in exon 1 of the ADA gene is promoter independent. They identified a 43-bp DNA fragment which functions as a transcriptional arrest signal. Additional studies showed that the transcriptional arrest site functioned only in the naturally occurring orientation. Therefore, they have identified a 43-bp DNA fragment which functions as a transcriptional arrest signal in an orientation-dependent and promoter-independent manner. On the basis of the authors findings, they hypothesize that tissue-specific expression of the ADA gene is governed by factors that function as antiterminators to promote transcriptional readthrough of the exon 1 transcriptional arrest site.

  14. The A3 adenosine receptor: history and perspectives.

    PubMed

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

    2015-01-01

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

  15. Adenosine deaminase 1 and concentrative nucleoside transporters 2 and 3 regulate adenosine on the apical surface of human airway epithelia: implications for inflammatory lung diseases.

    PubMed

    Hirsh, Andrew J; Stonebraker, Jaclyn R; van Heusden, Catja A; Lazarowski, Eduardo R; Boucher, Richard C; Picher, Maryse

    2007-09-11

    Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.

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

    PubMed Central

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

    1994-01-01

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

  17. Apigenin induces caspase-dependent apoptosis by inhibiting signal transducer and activator of transcription 3 signaling in HER2-overexpressing SKBR3 breast cancer cells.

    PubMed

    Seo, Hye-Sook; Ku, Jin Mo; Choi, Han-Seok; Woo, Jong-Kyu; Jang, Bo-Hyoung; Go, Hoyeon; Shin, Yong Cheol; Ko, Seong-Gyu

    2015-08-01

    Phytoestrogens have been demonstrated to inhibit tumor induction; however, their molecular mechanisms of action have remained elusive. The present study aimed to investigate the effects of a phytoestrogen, apigenin, on proliferation and apoptosis of the human epidermal growth factor receptor 2 (HER2)-expressing breast cancer cell line SKBR3. Proliferation assay, MTT assay, fluorescence-activated cell sorting analysis, western blot analysis, immunocytochemistry, reverse transcription-polymerase chain reaction and ELISA assay were used in the present study. The results of the present study indicated that apigenin inhibited the proliferation of SKBR3 cells in a dose-and time-dependent manner. This inhibition of growth was accompanied by an increase in the sub-G0/G1 apoptotic population. Furthermore, apigenin enhanced the expression levels of cleaved caspase-8 and -3, and induced the cleavage of poly(adenosine diphosphate ribose) polymerase in SKBR3 cells, confirming that apigenin promotes apoptosis via a caspase-dependent pathway. Apigenin additionally reduced the expression of phosphorylated (p)-janus kinase 2 and p-signal transducer and activator of transcription 3 (STAT3), inhibited CoCl2-induced vascular endothelial growth factor (VEGF) secretion and decreased the nuclear localization of STAT3. The STAT3 inhibitor S31-201 decreased the cellular proliferation rate and reduced the expression of p-STAT3 and VEGF. Therefore, these results suggested that apigenin induced apoptosis via the inhibition of STAT3 signaling in SKBR3 cells. In conclusion, the results of the present study indicated that apigenin may be a potentially useful compound for the prevention or treatment of HER2-overexpressing breast cancer.

  18. Inhibition of Nitro-Oxidative Stress Attenuates Pulmonary and Systemic Injury Induced by High-Tidal Volume Mechanical Ventilation.

    PubMed

    Martínez-Caro, Leticia; Nin, Nicolás; Sánchez-Rodríguez, Carolina; Ferruelo, Antonio; El Assar, Mariam; de Paula, Marta; Fernández-Segoviano, Pilar; Esteban, Andrés; Lorente, José A

    2015-07-01

    Mechanisms contributing to pulmonary and systemic injury induced by high tidal volume (VT) mechanical ventilation are not well known. We tested the hypothesis that increased peroxynitrite formation is involved in organ injury and dysfunction induced by mechanical ventilation. Male Sprague-Dawley rats were subject to low- (VT, 9 mL/kg; positive end-expiratory pressure, 5 cmH2O) or high- (VT, 25 mL/kg; positive end-expiratory pressure, 0 cmH2O) VT mechanical ventilation for 120 min, and received 1 of 3 treatments: 3-aminobenzamide (3-AB, 10 mg/kg, intravenous, a poly adenosine diphosphate ribose polymerase [PARP] inhibitor), or the metalloporphyrin manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 5 mg/kg intravenous, a peroxynitrite scavenger), or no treatment (control group), 30 min before starting the mechanical ventilation protocol (n = 8 per group, 6 treatment groups). We measured mean arterial pressure, peak inspiratory airway pressure, blood chemistry, and gas exchange. Oxidation (fluorescence for oxidized dihydroethidium), protein nitration (immunofluorescence and Western blot for 3-nitrotyrosine), PARP protein (Western blot) and gene expression of the nitric oxide (NO) synthase (NOS) isoforms (quantitative real-time reverse transcription polymerase chain reaction) were measured in lung and vascular tissue. Lung injury was quantified by light microscopy. High-VT mechanical ventilation was associated with hypotension, increased peak inspiratory airway pressure, worsened oxygenation; oxidation and protein nitration in lung and aortic tissue; increased PARP protein in lung; up-regulation of NOS isoforms in lung tissue; signs of diffuse alveolar damage at histological examination. Treatment with 3AB or MnTMPyP attenuated the high-VT mechanical ventilation-induced changes in pulmonary and cardiovascular function; down-regulated the expression of NOS1, NOS2, and NOS3; decreased oxidation and nitration in lung and aortic tissue; and attenuated

  19. [Gene therapy for adenosine deaminase deficiency].

    PubMed

    Sakiyama, Yukio; Ariga, Tadashi; Ohtsu, Makoto

    2005-03-01

    A four year-old boy with adenosine deaminase (ADA-) deficient severe combined immunodeficiency(SCID) receiving PEG-ADA was treated under a gene therapy protocol targeting peripheral blood lymphocytes (PBLs) in 1995. After eleven infusions of autologous PBLs transduced with retroviral vector LASN encoding ADAcDNA, he exhibited increased levels of the CD8+ T lymphocytes, serum immunoglobulin, specific antibodies and delayed type hypersensitivity skin tests. Follow-up studies also provided evidence of long-term persistence and function of transduced PBLs with improvement in the immune function. However, the therapeutic effect of this gene therapy has been difficult to assess because of the concomitant treatment of PEG-ADA. Two ADA-SCID patients have been currently treated with autologous bone marrow CD34+ cells engineered with a retroviral vector GCsapM-ADA after discontinuation of PEG-ADA. The restoration of intracellular ADA enzymatic activity in lymphocytes and granulocytes resulted in correction of the systemic toxicity and liver function in the absence of PEG-ADA treatment. Both patients are at home where they are clinically well, and they do not experience adversed effect, with follow up being 12 months after CD34+ cells gene therapy.

  20. Detecting adenosine triphosphate in the pericellular space.

    PubMed

    Falzoni, Simonetta; Donvito, Giovanna; Di Virgilio, Francesco

    2013-06-06

    Release of adenosine triphosphate (ATP) into the extracellular space occurs in response to a multiplicity of physiological and pathological stimuli in virtually all cells and tissues. A role for extracellular ATP has been identified in processes as different as neurotransmission, endocrine and exocrine secretion, smooth muscle contraction, bone metabolism, cell proliferation, immunity and inflammation. However, ATP measurement in the extracellular space has proved a daunting task until recently. To tackle this challenge, some years ago, we designed and engineered a novel luciferase probe targeted to and expressed on the outer aspect of the plasma membrane. This novel probe was constructed by appending to firefly luciferase the N-terminal leader sequence and the C-terminal glycophosphatidylinositol anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase, is targeted and localized to the outer side of the plasma membrane. With this probe, we have generated stably transfected HEK293 cell clones that act as an in vitro and in vivo sensor of the extracellular ATP concentration in several disease conditions, such as experimentally induced tumours and inflammation.

  1. Adenosine acts as an inhibitor of lymphoma cell growth: a major role for the A3 adenosine receptor.

    PubMed

    Fishman, P; Bar-Yehuda, S; Ohana, G; Pathak, S; Wasserman, L; Barer, F; Multani, A S

    2000-07-01

    In this study, we demonstrated several mechanisms exploring the inhibitory effect of low-dose adenosine on lymphoma cell growth. Adenosine, a purine nucleoside present in plasma and other extracellular fluids, acts as a regulatory molecule, by binding to G-protein associated cell-surface receptors, A1, A2 and A3. Recently we showed that low-dose adenosine released by muscle cells, inhibits tumour cell growth and thus attributes to the rarity of muscle metastases. In the present work, a cytostatic effect of adenosine on the proliferation of the Nb2-11C rat lymphoma cell line was demonstrated. This effect was mediated through the induction of cell cycle arrest in the G0/G1 phase and by decreasing the telomeric signal in these cells. Adenosine was found to exert its antiproliferative effect mainly through binding to its A3 receptor. The cytostatic anticancer activity, mediated through the A3 adenosine receptor, turns it into a potential target for the development of anticancer therapies.

  2. Drosophila factor 2, an RNA polymerase II transcript release factor, has DNA-dependent ATPase activity.

    PubMed

    Xie, Z; Price, D

    1997-12-12

    Drosophila factor 2 has been identified as a component of negative transcription elongation factor (N-TEF) that causes the release of RNA polymerase II transcripts in an ATP-dependent manner (Xie, Z. and Price D. H. (1996) J. Biol. Chem. 271, 11043-11046). We show here that the transcript release activity of factor 2 requires ATP or dATP and that adenosine 5'-O-(thiotriphosphate) (ATPgammaS), adenosine 5'-(beta,gamma-imino)triphosphate (AMP-PNP), or other NTPs do not support the activity. Factor 2 demonstrated a strong DNA-dependent ATPase activity that correlated with its transcript release activity. At 20 microg/ml DNA, the ATPase activity of factor 2 had an apparent Km(ATP) of 28 microM and an estimated Kcat of 140 min-1. Factor 2 caused the release of nascent transcripts associated with elongation complexes generated by RNA polymerase II on a dC-tailed template. Therefore, no other protein cofactors are required for the transcript release activity of factor 2. Using the dC-tailed template assay, it was found that renaturation of the template was required for factor 2 function.

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

    PubMed

    Pornbanlualap, Somchai; Chalopagorn, Pornchanok

    2011-08-01

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

  4. Role of A3 adenosine receptor in diabetic neuropathy.

    PubMed

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

    2016-10-01

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

  5. Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides.

    PubMed Central

    Cunha, R. A.; Correia-de-Sá, P.; Sebastião, A. M.; Ribeiro, J. A.

    1996-01-01

    1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings. PMID:8886406

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

    PubMed

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

    2007-09-01

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

  7. Adenosine and protection from acute kidney injury

    PubMed Central

    Yap, Steven C.; Lee, H. Thomas

    2012-01-01

    Purpose of Review Acute Kidney Injury (AKI) is a major clinical problem without effective therapy. Development of AKI among hospitalized patients drastically increases mortality, and morbidity. With increases in complex surgical procedures together with a growing elderly population, the incidence of AKI is rising. Renal adenosine receptor (AR) manipulation may have great therapeutic potential in mitigating AKI. In this review, we discuss renal AR biology and potential clinical therapies for AKI. Recent Findings The 4 AR subtypes (A1AR, A2AAR, A2BAR and A3AR) have diverse effects on the kidney. The pathophysiology of AKI may dictate the specific AR subtype activation needed to produce renal protection. The A1AR activation in renal tubules and endothelial cells produces beneficial effects against ischemia and reperfusion (IR) injury by modulating metabolic demand, decreasing necrosis, apoptosis and inflammation. The A2AAR protects against AKI by modulating leukocyte-mediated renal and systemic inflammation whereas the A2BAR activation protects by direct activation of renal parenchymal ARs. In contrast, the A1AR antagonism may play a protective role in nephrotoxic AKI and radiocontrast induced nephropathy by reversing vascular constriction and inducing naturesis and diuresis. Furthermore, as the A3AR-activation exacerbates apoptosis and tissue damage due to renal IR, selective A3AR antagonism may hold promise to attenuate renal IR injury. Finally, renal A1AR activation also protects against renal endothelial dysfunction caused by hepatic IR injury. Summary Despite the current lack of therapies for the treatment and prevention of AKI, recent research suggests that modulation of renal ARs holds promise in treating AKI and extrarenal injury. PMID:22080856

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

    PubMed

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

    2015-02-01

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

  9. Attenuation of exercise vasodilatation by adenosine deaminase in anaesthetized dogs.

    PubMed Central

    Goonewardene, I P; Karim, F

    1991-01-01

    1. In dogs anaesthetized with sodium pentobarbitone and artificially ventilated, the gracilis muscles were vascularly isolated and perfused at a constant flow of 28.4 +/- 4.6 ml min-1 (100 g muscle tissue)-1 (99.8 +/- 4.5% of maximum free flow, means +/- standard error of the mean (S.E.M.), n = 9). 2. Three to five minutes of electrical stimulation of the cut peripheral end of the obturator nerve (4 Hz, 6 V, 0.2 ms) resulted in muscle contraction (0.61 +/- 0.14 kg (100 g)-1 during solvent infusion and 0.56 +/- 0.10 kg (100 g)-1 during intra-arterial adenosine deaminase infusion (50 U min-1) and an immediate decrease in arterial perfusion pressure from 184.5 +/- 8.1 mmHg to 148.2 +/- 5.7 mmHg (18.7 +/- 3.4% decrease) during solvent infusion, and from 193.5 +/- 7.16 to 142.0 +/- 10.2 mmHg (25.4 +/- 6.1% decrease) during adenosine deaminase infusion 10 s after the commencement of muscle stimulation. After about 5 min of muscle contractions, the arterial perfusion pressure decreased to 120.8 +/- 7.8 mmHg (32.9 +/- 5.8% decrease) during solvent infusion, and to 152.8 +/- 11.2 mmHg (20.9 +/- 5.3% decrease) during adenosine deaminase infusion (i.e. 37.9 +/- 6.2% attenuation of the fall in arterial perfusion pressure). The time taken for 90% recovery of the arterial perfusion pressure was 72.1 +/- 10.9 s during solvent infusion, and 51.5 +/- 9.3 s during adenosine deaminase infusion (P less than 0.05). 3. Adenosine (2 x 10(-3) mol l-1) infusion in the resting muscle during solvent infusion (final concentration in arterial blood 1.3 x 10(-4) +/- 6.0 x 10(-5) mol l-1) resulted in a 34.8 +/- 7.2% fall in arterial perfusion pressure but a fall of only 7.2 +/- 1.8% during adenosine deaminase infusion (50 U min-1; P less than 0.05; n = 5) indicating that adenosine deaminase infused at 50 U min-1 was more than adequate to metabolize endogenous adenosine produced during muscle contractions. 4. These data suggest that adenosine contributes about 40% to the sustained

  10. Differential adenosine sensitivity of diaphragm and skeletal muscle arterioles.

    PubMed

    Aaker, Aaron; Laughlin, M H

    2002-09-01

    The hyperemic response in exercising skeletal muscle is dependent on muscle fiber-type composition and fiber recruitment patterns, but the vascular control mechanisms producing exercise hyperemia in skeletal muscle remain poorly understood. The purpose of this study was to test the hypothesis that arterioles from white, low-oxidative skeletal muscle are less responsive to adenosine-induced dilation than are arterioles from diaphragm (Dia) and red, high-oxidative skeletal muscle. Second-order arterioles (2As) were isolated from the white portion of gastrocnemius muscle (WG; low-oxidative, fast-twitch muscle tissue) and two types of high-oxidative skeletal muscle [Dia and red portion of gastrocnemius muscle (RG)] of rats. Results reveal that 2As from all three types of muscle dilated in response to the endothelium-dependent dilator acetylcholine (WG: 48 +/- 3%, Dia: 51 +/- 3%, RG: 74 +/- 3%). In contrast, adenosine dilated only 2As from WG (48 +/- 4%) and Dia (46 +/- 5%) but not those from RG (5 +/- 5%). Thus adenosine-induced dilator responses differed among 2As of these different types of muscle tissue. However, the results do not support our hypothesis because 2As from Dia and WG dilated in response to adenosine, whereas 2As from RG did not. We conclude that the adenosine responsiveness of 2As from rat skeletal muscle cannot be predicted only by the fiber-type composition or oxidative capacity of the skeletal muscle tissue wherein the arteriole lies.

  11. Adenosine hypothesis of schizophrenia –opportunities for pharmacotherapy

    PubMed Central

    Boison, Detlev; Singer, Philipp; Shen, Hai-Ying; Feldon, Joram; Yee, Benjamin K.

    2011-01-01

    Pharmacotherapy of schizophrenia based on the dopamine hypothesis remains unsatisfactory for the negative and cognitive symptoms of the disease. Enhancing N-methyl-d-aspartate receptors (NMDAR) function is expected to alleviate such persistent symptoms, but successful development of novel clinically effective compounds remains challenging. Adenosine is a homeostatic bioenergetic network modulator that is able to affect complex networks synergistically at different levels (receptor dependent pathways, biochemistry, bioenergetics, and epigenetics). By affecting brain dopamine and glutamate activities it represents a promising candidate for restoring the functional imbalance in these neurotransmitter systems believed to underlie the genesis of schizophrenia symptoms, as well as restoring homeostasis of bioenergetics. Suggestion of an adenosine hypothesis of schizophrenia further posits that adenosinergic dysfunction might contribute to the emergence of multiple neurotransmitter dysfunctionscharacteristic of schizophrenia via diverse mechanisms. Given the importance of adenosine in early brain development and regulation of brain immune response, it also bears direct relevance to the aetiology of schizophrenia. Here, we provide an overview of the rationale and evidence in support of the therapeutic potential of multiple adenosinergic targets, including the high-affinity adenosine receptors (A1R and A2AR), and the regulatory enzyme adenosine kinase (ADK). Key preliminary clinical data and preclinical findings are reviewed. PMID:21315743

  12. Regioselective 1-N-Alkylation and Rearrangement of Adenosine Derivatives.

    PubMed

    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.

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

    PubMed

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

    2016-05-01

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

  14. Identification and partial characterization of an adenosine(5')tetraphospho(5')adenosine hydrolase on intact bovine aortic endothelial cells.

    PubMed Central

    Ogilvie, A; Lüthje, J; Pohl, U; Busse, R

    1989-01-01

    The biologically active dinucleotides adenosine(5')tetraphospho(5')adenosine (Ap4A) and adenosine(5')-triphospho(5')adenosine (Ap3A), which are both releasable into the circulation from storage pools in thrombocytes, are catabolized by intact bovine aortic endothelial cells. 1. Compared with extracellular ATP and ADP, which are very rapidly hydrolysed, the degradation of Ap4A and Ap3A by endothelial ectohydrolases is relatively slow, resulting in a much longer half-life on the endothelial surface of the blood vessel. The products of hydrolysis are further degraded and finally taken up as adenosine. 2. Ap4A hydrolase has high affinity for its substrate (Km 10 microM). 3. ATP as well as AMP transiently accumulates in the extracellular fluid, suggesting an asymmetric split of Ap4A by the ectoenzyme. 4. Mg2+ or Mn2+ at millimolar concentration are needed for maximal activity; Zn2+ and Ca2+ are inhibitory. 5. The hydrolysis of Ap4A is retarded by other nucleotides, such as ATP and Ap3A, which are released from platelets simultaneously with Ap4A. PMID:2541689

  15. Targeting adenosine receptors to prevent inflammatory skin diseases.

    PubMed

    Gessi, Stefania; Merighi, Stefania; Borea, Pier Andrea

    2014-08-01

    Adenosine mediates its effects through activation of a family of four G-protein-coupled receptors, named A1 , A2A , A2B and A3 . This nucleoside plays an important role in immunity and inflammation, and the A2A adenosine receptor subtype has a key role in the inhibition of inflammatory processes besides promoting wound healing. In this issue of Experimental Dermatology, Arasa et al. show that the topical application of a selective A2A agonist, CGS 21680, to mouse skin reduced epidermal hyperplasia as well as skin inflammation, similarly to topical corticoids, without side effects like skin atrophy. Rigorously following up this work is important for the development of novel treatment strategies for chronic hyperproliferative inflammatory dermatoses, such as targeting the A2A adenosine receptor family.

  16. Release of Adenosine and ATP During Ischemia and Epilepsy

    PubMed Central

    Dale, Nicholas; Frenguelli, Bruno G

    2009-01-01

    Eighty years ago Drury & Szent-Györgyi described the actions of adenosine, AMP (adenylic acid) and ATP (pyrophosphoric or diphosphoric ester of adenylic acid) on the mammalian cardiovascular system, skeletal muscle, intestinal and urinary systems. Since then considerable insight has been gleaned on the means by which these compounds act, not least of which in the distinction between the two broad classes of their respective receptors, with their many subtypes, and the ensuing diversity in cellular consequences their activation invokes. These myriad actions are of course predicated on the release of the purines into the extracellular milieu, but, surprisingly, there is still considerable ambiguity as to how this occurs in various physiological and pathophysiological conditions. In this review we summarise the release of ATP and adenosine during seizures and cerebral ischemia and discuss mechanisms by which the purines adenosine and ATP may be released from cells in the CNS under these conditions. PMID:20190959

  17. Correlation between blood adenosine metabolism and sleep in humans.

    PubMed

    Díaz-Muñoz, M; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yááñez, L; Aguilar-Roblero, R; Rosenthal, L; Villalobos, L; Fernández-Cancino, F; Drucker-Colín, R; Chagoya De Sanchez, V

    1999-01-01

    Blood adenosine metabolism, including metabolites and metabolizing enzymes, was studied during the sleep period in human volunteers. Searching for significant correlations among biochemical parameters found: adenosine with state 1 of slow-wave sleep (SWS); activity of 5'-nucleotidase with state 2 of SWS; inosine and AMP with state 3-4 of SWS; and activity of 5'-nucleotidase and lactate with REM sleep. The correlations were detected in all of the subjects that presented normal hypnograms, but not in those who had fragmented sleep the night of the experiment. The data demonstrate that it is possible to obtain information of complex brain operations such as sleep by measuring biochemical parameters in blood. The results strengthen the notion of a role played by adenosine, its metabolites and metabolizing enzymes, during each of the stages that constitute the sleep process in humans.

  18. Alphavirus polymerase and RNA replication.

    PubMed

    Pietilä, Maija K; Hellström, Kirsi; Ahola, Tero

    2017-01-16

    Alphaviruses are typically arthropod-borne, and many are important pathogens such as chikungunya virus. Alphaviruses encode four nonstructural proteins (nsP1-4), initially produced as a polyprotein P1234. nsP4 is the core RNA-dependent RNA polymerase but all four nsPs are required for RNA synthesis. The early replication complex (RC) formed by the polyprotein P123 and nsP4 synthesizes minus RNA strands, and the late RC composed of fully processed nsP1-nsP4 is responsible for the production of genomic and subgenomic plus strands. Different parts of nsP4 recognize the promoters for minus and plus strands but the binding also requires the other nsPs. The alphavirus polymerase has been purified and is capable of de novo RNA synthesis only in the presence of the other nsPs. The purified nsP4 also has terminal adenylyltransferase activity, which may generate the poly(A) tail at the 3' end of the genome. Membrane association of the nsPs is vital for replication, and alphaviruses induce membrane invaginations called spherules, which form a microenvironment for RNA synthesis by concentrating replication components and protecting double-stranded RNA intermediates. The RCs isolated as crude membrane preparations are active in RNA synthesis in vitro, but high-resolution structure of the RC has not been achieved, and thus the arrangement of viral and possible host components remains unknown. For some alphaviruses, Ras-GTPase-activating protein (Src-homology 3 (SH3) domain)-binding proteins (G3BPs) and amphiphysins have been shown to be essential for RNA replication and are present in the RCs. Host factors offer an additional target for antivirals, as only few alphavirus polymerase inhibitors have been described.

  19. Role of multiplex polymerase chain reaction in diagnosing tubercular meningitis

    PubMed Central

    Berwal, Anupam; Chawla, Kiran; Vishwanath, Shashidhar; Shenoy, Vishnu Prasad

    2017-01-01

    Tuberculous meningitis (TBM) is one of the most serious manifestations of extrapulmonary tuberculosis. Timely and accurate diagnosis provides a favorable prognosis in patients with TBM. The study evaluated the use of multiplex polymerase chain reaction (PCR) in the diagnosis of TBM. A study was conducted on 74 patients clinically suspected with TBM. The cerebrospinal fluid (CSF) specimens were processed for smear microscopy, middle brook 7H9 culture, and multiplex PCR using primers directed against IS6110 gene and 38 kD protein for detection of Mycobacterium tuberculosis. The results were analyzed to assess the role of multiplex PCR in the diagnosis of TBM. A total of 26 (35.1%) patients were diagnosed with TBM. Microscopy was negative in all while culture was positive in two cases only. Comparing with clinical diagnosis and CSF adenosine deaminase levels of ≥10 U/L, multiplex PCR showed sensitivity, specificity, positive predictive value, and negative predictive value of 71.4%, 89.6%, 83.3%, and 81.2%, respectively, in the diagnosis of TBM. PMID:28367034

  20. Demonstration of adenosine deaminase activity in human fibroblast lysosomes.

    PubMed Central

    Lindley, E R; Pisoni, R L

    1993-01-01

    Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2'-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2'-3'-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase, which could assist in maintaining cellular energy requirements under conditions of nutritional deprivation. PMID:8452534

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

    PubMed

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

    2016-05-01

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

  2. Intravenous Adenosine for Surgical Management of Penetrating Heart Wounds

    PubMed Central

    Kokotsakis, John; Hountis, Panagiotis; Antonopoulos, Nikolaos; Skouteli, Elian; Athanasiou, Thanos; Lioulias, Achilleas

    2007-01-01

    Accurate suturing of penetrating cardiac injuries is difficult. Heart motion, ongoing blood loss, arrhythmias due to heart manipulation, and the near-death condition of the patient can all affect the outcome. Rapid intravenous injection of adenosine induces temporary asystole that enables placement of sutures in a motionless surgical field. Use of this technique improves surgical conditions, and it is faster than other methods. Herein, we describe our experience with the use of intravenous adenosine to successfully treat 3 patients who had penetrating heart wounds. PMID:17420798

  3. Computer-assisted analysis of adenosine triphosphate data.

    PubMed

    Erkenbrecher, C W; Crabtree, S J; Stevenson, L H

    1976-09-01

    A computer program has been written to assist in the analysis of adenosine 5'-triphosphate data. The program is designed to calculate a dilution curve and to correct sample and adenosine 5'-triphosphate standard data for background and dilution effects. In addition, basic statistical parameters and estimates of biomass carbon are also calculated for each group of samples and printed in a convenient format. The versatility of the program to analyze data from both qauatic and terrestrial samples is noted as well as its potential use with various types of instrumentation and extraction techniques.

  4. Development of Coronary Vasospasm during Adenosine-Stress Myocardial Perfusion CT Imaging.

    PubMed

    Nam, Jeong Gu; Choi, Seong Hoon; Kang, Byeong Seong; Bang, Min Seo; Kwon, Woon Jeong

    2015-01-01

    Adenosine is a short-acting coronary vasodilator, and it is widely used during pharmacological stress myocardial perfusion imaging. It has a well-established safety profile, and most of its side effects are known to be mild and transient. Until now, coronary vasospasm has been rarely reported as a side effect of adenosine during or after adenosine stress test. This study reports a case of coronary vasospasm which was documented on stress myocardial perfusion CT imaging during adenosine stress test.

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

    PubMed Central

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

    1994-01-01

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

  6. Synthesis of 1,N6-etheno-2-aza-adenosine (2-aza-ε-adenosine): a new cytotoxic fluorescent nucleoside

    PubMed Central

    Tsou, K.C.; Yip, K.F.; Miller, E.E.; Lo, K.W.

    1974-01-01

    1,N6-Etheno-2-aza-adenosine was synthesized by treating 1,N6-etheno-adenosine with alkali, followed by nitrosation. The mechanism of formation of this novel nucleoside was elucidated using adenosine tritiated at C-8 and C-2, and was found to deformylate exclusively at C-2. This new 2-aza nucleoside fluoresces at 494 nm when excited at 358 nm. Toxicity study showed the compound is active in a rat mammary tumor tissue culture line, but inactive in HeLa and Glioma 26 tissue culture lines. It was also found to selectively inhibit the thymidine incorporation into DNA in a rat mammary tumor, but exhibits no ill effect on normal proliferative tissue. The reactive intermediate 3-β-D-ribofuranosyl-4-amino-5-(imidazol-2-yl) imidazole was identified and was found to be an active agent in tissue culture. PMID:10793738

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

    PubMed

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

    2008-10-04

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

  8. In vivo assessment of coronary flow and cardiac function after bolus adenosine injection in adenosine receptor knockout mice.

    PubMed

    Teng, Bunyen; Tilley, Stephen L; Ledent, Catherine; Mustafa, S Jamal

    2016-06-01

    Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((π/4) × D(2) × VTI × HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states.

  9. Doxorubicin has a synergistic cytotoxicity with cucurbitacin B in anaplastic thyroid carcinoma cells.

    PubMed

    Kim, Si Hyoung; Kang, Jun Goo; Kim, Chul Sik; Ihm, Sung-Hee; Choi, Moon Gi; Yoo, Hyung Joon; Lee, Seong Jin

    2017-02-01

    In this study, the combined effect of doxorubicin with cucurbitacin B on survival of anaplastic thyroid carcinoma cells was evaluated. For experiments, 8505C and CAL62 human anaplastic thyroid carcinoma cells were used. Cell viability, the percentage of viable cells, and cytotoxic activity were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, multiplexed cytotoxicity assay, and cytotoxicity assay, respectively. Reactive oxygen species production was measured. In experiments, doxorubicin and cucurbitacin B reduced cell viability in a dose- and time-dependent manner. Cotreatment of doxorubicin and cucurbitacin B, compared with treatment of doxorubicin alone, decreased the percentage of viable cells and increased cytotoxic activity. All of the combination index values were lower than 1.0, suggesting the synergism between doxorubicin and cucurbitacin B in induction of cytotoxicity. In cells treated with both doxorubicin and cucurbitacin B, compared with doxorubicin alone, the protein levels of cleaved poly(adenosine diphosphate-ribose) polymerase and cyclooxygenase 2 and reactive oxygen species production were enhanced. In contrast, the protein levels of B-cell chronic lymphocytic leukemia/lymphoma 2 and survivin and B-cell chronic lymphocytic leukemia/lymphoma 2/B-cell chronic lymphocytic leukemia/lymphoma 2-associated x protein ratio were diminished. The protein levels of Janus kinase 2 and signal transducer and activator of transcription 3 were reduced, while phospho-extracellular signal-regulated kinase 1/2 protein levels were elevated without change in total extracellular signal-regulated kinase 1/2 protein levels. These results suggest that doxorubicin synergizes with cucurbitacin B in induction of cytotoxicity in anaplastic thyroid carcinoma cells. Moreover, synergistic cytotoxicity of doxorubicin with cucurbitacin B is mediated by B-cell chronic lymphocytic leukemia/lymphoma 2 family proteins, survivin, and reactive oxygen

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

    PubMed

    Frajese, Giovanni Vanni; Benvenuto, Monica; Fantini, Massimo; Ambrosin, Elena; Sacchetti, Pamela; Masuelli, Laura; Giganti, Maria Gabriella; Modesti, Andrea; Bei, Roberto

    2016-06-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. High PARP-1 expression is associated with tumor invasion and poor prognosis in gastric cancer

    PubMed Central

    Liu, Ying; Zhang, Yu; Zhao, Ying; Gao, Dongna; Xing, Jing; Liu, Hui

    2016-01-01

    Poly (adenosine diphosphate-ribose) polymerase 1 (PARP-1) was previously demonstrated to be overexpressed in numerous malignant tumors and associated with invasiveness and poor prognosis. However, the expression of the PARP-1 protein in gastric cancer and its association with clinical outcomes requires further investigation. In the present study, the expression of PARP-1 in 564 gastric cancer tissues and 335 tumor-adjacent control tissues is investigated, using tissue microarray-based immunohistochemistry. PARP-1 expression levels were demonstrated to be significantly higher in gastric cancer tissue samples, as compared with control tissue samples. In gastric cancer, high PARP-1 expression levels were significantly associated with Helicobacter pylori (H. pylori) infection (P=0.032), decreased differentiation (P<0.001), increased depth of invasion (P=0.037), presence of lymphatic invasion (P<0.001), presence of lymph node metastasis (P<0.001), and advanced tumor-node-metastasis (TNM) stage (P=0.015). High PARP-1 expression levels were associated with a significantly shorter overall survival rate (P<0.001) and disease-free survival rate (P=0.001) in patients with gastric cancer, particularly a subset of patients with H. pylori infection or an advanced TNM stage. In addition, univariate analysis indicated that PARP-1 high expression levels were significantly associated with a poor prognosis in gastric cancer. These results suggest that PARP-1 expression may be involved in the progression and prognosis of gastric cancer, particularly H. pylori-positive or advanced-stage gastric cancer. PMID:27895737

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

    PubMed

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

    2010-03-01

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

  14. Polyphenol-rich sweet potato greens extract inhibits proliferation and induces apoptosis in prostate cancer cells in vitro and in vivo

    PubMed Central

    Karna, Prasanthi; Gundala, Sushma R.; Gupta, Meenakshi V.; Shamsi, Shahab A.; Pace, Ralphenia D.; Yates, Clayton; Narayan, Satya; Aneja, Ritu

    2011-01-01

    Sweet potato (Ipomoea batatas) leaves or greens, extensively consumed as a vegetable in Africa and Asia, are an excellent source of dietary polyphenols such as anthocyanins and phenolic acids. Here, we show that sweet potato greens extract (SPGE) has the maximum polyphenol content compared with several commercial vegetables including spinach. The polyphenol-rich SPGE exerts significant antiproliferative activity in a panel of prostate cancer cell lines while sparing normal prostate epithelial cells. Mechanistically, SPGE perturbed cell cycle progression, reduced clonogenic survival, modulated cell cycle and apoptosis regulatory molecules and induced apoptosis in human prostate cancer PC-3 cells both in vitro and in vivo. SPGE-induced apoptosis has a mitochondrially mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis regulatory molecules such as inactivation of Bcl2, upregulation of BAX, cytochrome c release and activation of downstream apoptotic signaling. SPGE caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-nick-end labeling (TUNEL) staining of increased concentration of 3′-DNA ends. Furthermore, apoptotic induction was caspase dependent as shown by cleavage of caspase substrate, poly (adenosine diphosphate-ribose) polymerase. Oral administration of 400 mg/kg SPGE remarkably inhibited growth and progression of prostate tumor xenografts by ∼69% in nude mice, as shown by tumor volume measurements and non-invasive real-time bioluminescent imaging. Most importantly, SPGE did not cause any detectable toxicity to rapidly dividing normal tissues such as gut and bone marrow. This is the first report to demonstrate the in vitro and in vivo anticancer activity of sweet potato greens in prostate cancer. PMID:21948980

  15. Veliparib in combination with whole-brain radiation therapy for patients with brain metastases from non-small cell lung cancer: results of a randomized, global, placebo-controlled study.

    PubMed

    Chabot, Pierre; Hsia, Te-Chun; Ryu, Jeong-Seon; Gorbunova, Vera; Belda-Iniesta, Cristobal; Ball, David; Kio, Ebenezer; Mehta, Minesh; Papp, Katherine; Qin, Qin; Qian, Jane; Holen, Kyle D; Giranda, Vince; Suh, John H

    2017-01-01

    Veliparib is a potent, orally bioavailable, poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor that crosses the blood-brain barrier and has been shown to potentiate the effects of radiation in preclinical and early clinical studies. This phase 2, randomized, global study evaluated the efficacy and safety of veliparib in combination with whole-brain radiation therapy (WBRT) in patients with brain metastases from non-small cell lung cancer (NSCLC). Three-hundred and seven patients with brain metastases from NSCLC were randomized 1:1:1 to WBRT (30 Gy in 10 fractions) plus 50 mg veliparib twice daily (BID; n = 103), 200 mg veliparib BID (n = 102), or placebo BID (n = 102). Treatment began within 28 days of diagnosis. Tumor response and safety were assessed; the primary endpoint was overall survival (OS). Patients who received ≥1 dose of treatment were included in the safety analysis. All randomized patients were included in the efficacy endpoint analyses. Patient characteristics were well balanced between treatment arms. Median OS was 185 days for patients treated with WBRT plus placebo and 209 days for WBRT plus veliparib (50 or 200 mg). No statistically significant differences in OS, intracranial response rate, and time to clinical or radiographic progression between any of the treatment arms were noted. No differences were observed in adverse events (all grades) across treatment arms; nausea, fatigue, alopecia, and headache were the most commonly reported. No new safety signals were identified for veliparib. A significant unmet need for therapies that improve the outcomes of patients with brain metastases from NSCLC remains.

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

    SciTech Connect

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

    2009-07-15

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

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

    PubMed

    Tsuchiya, Ayako; Nishizaki, Tomoyuki

    2015-10-21

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

  18. Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5' -phosphoramidate from adenosine 5' -phosphosulphate and ammonia.

    PubMed

    Fankhauser, H; Schiff, J A; Garber, L J

    1981-06-01

    Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5'-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5'-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5'-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5'-phosphoramidate. The enzyme that catalyses the formation of adenosine 5'-phosphoramidate from ammonia and adenosine 5'-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH(4))(2)SO(4) precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2-agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000-65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3'-phosphate 5'-phosphosulphate will not replace adenosine 5'-phosphosulphate, and the apparent K(m) for the last-mentioned compound is 0.82mm. The apparent K(m) for ammonia (assuming NH(3) to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5'-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5'-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme

  19. Laser photobleaching leads to a fluorescence grade adenosine deaminase.

    PubMed

    Parola, A H; Caiolfa, V R; Bar, I; Rosenwaks, S

    1989-09-01

    The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.

  20. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  1. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  2. Quantitative changes in adenosine deaminase isoenzymes in human colorectal adenocarcinomas.

    PubMed

    ten Kate, J; Wijnen, J T; van der Goes, R G; Quadt, R; Griffioen, G; Bosman, F T; Khan, P M

    1984-10-01

    Several reports have suggested that a decrease or absence of adenosine deaminase complexing protein (ADCP) is consistently associated with cancer. However, in other studies, decreased as well as increased ADCP levels were found. In the present study, we investigated ADCP levels in 37 colorectal adenocarcinomas and correlated the results with clinicopathological characteristics in individual carcinomas. The levels of adenosine deaminase (EC 3.5.4.4) and soluble ADCP were determined in tissue samples by, respectively, a spectrophotometric assay and an ADCP specific radioimmunoassay. The values in the individual tumors were compared with their histological characteristics, such as degree of differentiation, nuclear grading, and the preoperative plasma carcinoembryonic antigen levels in the patients. It was found that ADCP was decreased in about a third of the tumors but unaltered or even increased in others. However, there was an overall 40% increase of the adenosine deaminase activity in the tumors compared to normal tissue. There seems to be no simple correlation between any of the clinicopathological parameters and the ADCP or adenosine deaminase levels. Methods detecting ADCP at single cell level might be helpful in exploring its potential use as a cancer-associated marker.

  3. Correlation of transient adenosine release and oxygen changes in the caudate-putamen.

    PubMed

    Wang, Ying; Venton, B Jill

    2017-01-01

    Adenosine is an endogenous nucleoside that modulates important physiological processes, such as vasodilation, in the central nervous system. A rapid, 2-4 s, mode of adenosine signaling has been recently discovered, but the relationship between this type of adenosine and blood flow change has not been characterized. In this study, adenosine and oxygen changes were simultaneously measured using fast-scan cyclic voltammetry. Oxygen changes occur when there is an increase in local cerebral blood flow and thus are a measure of vasodilation. About 34% of adenosine transients in the rat caudate-putamen are correlated with a subsequent transient change in oxygen. The amount of oxygen was correlated with the concentration of adenosine release and larger adenosine transients (over 0.4 μM) always had subsequent oxygen changes. The average duration of adenosine and oxygen transients was 3.2 and 3.5 s, respectively. On average, the adenosine release starts and peaks 0.2 s prior to the oxygen. The A2a antagonist, SCH442416, decreased the number of both adenosine and oxygen transient events by about 32%. However, the A1 antagonist, DPCPX, did not significantly affect simultaneous adenosine and oxygen release. The nitric oxide (NO) synthase inhibitor l-NAME also did not affect the concentration or number of adenosine and oxygen release events. These results demonstrate that both adenosine and oxygen release are modulated via A2a receptors. The correlation of transient concentrations, time delay between adenosine and oxygen peaks, and effect of A2a receptors suggests that adenosine modulates blood flow on a rapid, sub-second time scale. Read the Editorial Highlight for this article on page 10.

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

    PubMed

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

    2016-03-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  7. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release

    PubMed Central

    Nguyen, Michael D.; Venton, B. Jill

    2014-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future. PMID:26900429

  8. Fast-scan Cyclic Voltammetry for the Characterization of Rapid Adenosine Release.

    PubMed

    Nguyen, Michael D; Venton, B Jill

    2015-01-01

    Adenosine is a signaling molecule and downstream product of ATP that acts as a neuromodulator. Adenosine regulates physiological processes, such as neurotransmission and blood flow, on a time scale of minutes to hours. Recent developments in electrochemical techniques, including fast-scan cyclic voltammetry (FSCV), have allowed direct detection of adenosine with sub-second temporal resolution. FSCV studies have revealed a novel mode of rapid signaling that lasts only a few seconds. This rapid release of adenosine can be evoked by electrical or mechanical stimulations or it can be observed spontaneously without stimulation. Adenosine signaling on this time scale is activity dependent; however, the mode of release is not fully understood. Rapid adenosine release modulates oxygen levels and evoked dopamine release, indicating that adenosine may have a rapid modulatory role. In this review, we outline how FSCV can be used to detect adenosine release, compare FSCV with other techniques used to measure adenosine, and present an overview of adenosine signaling that has been characterized using FSCV. These studies point to a rapid mode of adenosine modulation, whose mechanism and function will continue to be characterized in the future.

  9. Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta

    SciTech Connect

    Kroll, K.; Kelm, M.K.; Buerrig, K.F.S.; Schrader, J.

    1989-06-01

    This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabeled with 3H-adenosine or 3H-inosine for 10 minutes at concentrations of 0.012-100 microM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic acid (0.75% for 15 seconds). In the physiological concentration range of adenosine (0.012-1 microM), fractional sequestration by endothelium was 90-92% of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 microM was 85%. At 100 microM adenosine or inosine, fractional sequestration by aortic endothelium was 33% and 39%, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nucleotides. Incorporation of inosine into endothelial ATP was approximately 15% that of adenosine. Inhibition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not influence sequestration of 0.1 microM adenosine, but resulted in a 49% reduction of total endothelial incorporation at 100 microM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to only 1%/hr of total endothelial radioactivity.

  10. Intracerebral adenosine infusion improves neurological outcome after transient focal ischemia in rats.

    PubMed

    Kitagawa, Hisashi; Mori, Atsushi; Shimada, Jun; Mitsumoto, Yasuhide; Kikuchi, Tetsuro

    2002-04-01

    Second Institute of New Drug Research, Otsuka Pharmaceutical Co., Ltd., Tokushima, Japan In order to elucidate the role of adenosine in brain ischemia, the possible protective effects of adenosine on ischemic brain injury were investigated in a rat model of brain ischemia both in vitro and in vivo. Exogenous adenosine dose-dependently rescued cortical neuronal cells from injury after glucose deprivation in vitro. Adenosine (1 mM) also significantly reduced hypoglycemia/hypoxia-induced glutamate release from the hippocampal slice. In a rat model of transient middle cerebral artery occlusion (MCAO), extracellular adenosine concentration was increased immediately after occlusion, and then returned to the baseline by 30 min after reperfusion. Adenosine infusion through a microdialysis probe into the ipsilateral striatum (1 mM adenosine, 2 microl min(-1), total 4.5 h from the occlusion to 3 h after reperfusion) showed a significant improvement in the neurological outcome, and about 25% reduction of infarct volume, although the effect did not reach statistical significance, compared with the vehicle-treated group at 20 h after 90 min of MCAO. These results demonstrated the neuroprotective effect of adenosine against ischemic brain injury both in vitro and in vivo, suggesting the possible therapeutic application of adenosine regulating agents, which inhibit adenosine uptake or metabolism to enhance or maintain extracellular endogenous adenosine levels, for stroke treatment.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

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

  12. Adenosine augmentation therapies (AATs) for epilepsy: prospect of cell and gene therapies

    PubMed Central

    Boison, Detlev

    2009-01-01

    Deficiencies in the brain’s own adenosine-based seizure control system contribute to seizure generation. Consequently, reconstitution of adenosinergic neuromodulation constitutes a rational approach for seizure control. This review will critically discuss focal adenosine augmentation strategies and their potential for antiepileptic and disease modifying therapy. Due to systemic side effects of adenosine focal adenosine augmentation – ideally targeted to an epileptic focus – becomes a therapeutic necessity. This has experimentally been achieved in kindled seizure models as well as in post status epilepticus models of spontaneous recurrent seizures using three different therapeutic strategies that will be discussed here: (i) Polymer-based brain implants that were loaded with adenosine; (ii) Brain implants comprised of cells engineered to release adenosine and embedded in a cell-encapsulation device; (iii) Direct transplantation of stem cells engineered to release adenosine. To meet the therapeutic goal of focal adenosine augmentation, genetic disruption of the adenosine metabolizing enzyme adenosine kinase (ADK) in rodent and human cells was used as a molecular strategy to induce adenosine release from cellular brain implants, which demonstrated antiepileptic and neuroprotective properties. New developments and therapeutic challenges in using AATs for epilepsy therapy will critically be evaluated. PMID:19428218

  13. Protective effect of adenosine against a calcium paradox in the isolated frog heart.

    PubMed

    Touraki, M; Lazou, A

    1992-01-01

    The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.

  14. Adenosine Signaling Increases Proinflammatory and Profibrotic Mediators through Activation of a Functional Adenosine 2B Receptor in Renal Fibroblasts.

    PubMed

    Wilkinson, Patrick F; Farrell, Francis X; Morel, Diane; Law, William; Murphy, Suzanne

    2016-07-01

    Interstitial renal fibrosis is a major pathophysiological manifestation of patients diagnosed with Chronic Kidney Disease (CKD), Diabetic Nephropathy (DN) and other inflammatory diseases. Adenosine signaling is an innate autocrine and paracrine cellular signaling pathway involving several key mediators that are elevated in the blood and kidneys of patients with DN. In these studies, we hypothesized that extracellular adenosine signals through one or more functional adenosine GPCRs on renal fibroblasts which increases profibrotic and proinflammatory mediators by inducing an activated fibroblast phenotype. Utilizing the renal fibroblast cell line NRK-49F, the presence and relative abundance of adenosine receptors (AR) A1, A2A, A2B, and A3 were quantified by RT-PCR. Under normal homeostatic conditions, only AR1 and AR2B were detected. The functionality of each receptor was then assessed by receptor specific pharmacological agonism and antagonism and assessed for modulation of the GPCR associated secondary messenger molecule, cyclic adenosine monophosphate (cAMP). Agonism of the AR2B receptor resulted in increased intracellular cAMP while agonism of the AR1 receptor inhibited cAMP modulation. Upon direct agonism of the AR2B receptor, transcripts for profibrotic and inflammatory mediators including SMA-α, IL-6, TGF-β, CTGF, and fibronectin were elevated between 2-4 fold. These data indicate that renal fibroblasts express a functional AR1 receptor that inhibits cAMP upon stimulation, leading to a functional AR2B receptor that increases cAMP upon stimulation and also induces an activated fibroblast phenotype resulting in increased fibrotic and inflammatory mediators.

  15. Adenosine transiently modulates stimulated dopamine release in the caudate-putamen via A1 receptors.

    PubMed

    Ross, Ashley E; Venton, B Jill

    2015-01-01

    Adenosine modulates dopamine in the brain via A1 and A2A receptors, but that modulation has only been characterized on a slow time scale. Recent studies have characterized a rapid signaling mode of adenosine that suggests a possible rapid modulatory role. Here, fast-scan cyclic voltammetry was used to characterize the extent to which transient adenosine changes modulate stimulated dopamine release (5 pulses at 60 Hz) in rat caudate-putamen brain slices. Exogenous adenosine was applied and dopamine concentration monitored. Adenosine only modulated dopamine when it was applied 2 or 5 s before stimulation. Longer time intervals and bath application of 5 μM adenosine did not decrease dopamine release. Mechanical stimulation of endogenous adenosine 2 s before dopamine stimulation also decreased stimulated dopamine release by 41 ± 7%, similar to the 54 ± 6% decrease in dopamine after exogenous adenosine application. Dopamine inhibition by transient adenosine was recovered within 10 min. The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the dopamine modulation, whereas dopamine modulation was unaffected by the A2A receptor antagonist SCH 442416. Thus, transient adenosine changes can transiently modulate phasic dopamine release via A1 receptors. These data demonstrate that adenosine has a rapid, but transient, modulatory role in the brain. Here, transient adenosine was shown to modulate phasic dopamine release on the order of seconds by acting at the A1 receptor. However, sustained increases in adenosine did not regulate phasic dopamine release. This study demonstrates for the first time a transient, neuromodulatory function of rapid adenosine to regulate rapid neurotransmitter release.

  16. Adenosine A2 receptors modulate haloperidol-induced catalepsy in rats.

    PubMed

    Mandhane, S N; Chopde, C T; Ghosh, A K

    1997-06-11

    The effect of adenosine A1 and A2 receptor agonists and antagonists was investigated on haloperidol-induced catalepsy in rats. Pretreatment (i.p.) with the non-selective adenosine receptor antagonist, theophylline, or the selective adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), significantly reversed haloperidol-induced catalepsy, whereas the selective adenosine A1 receptor antagonists, 8-phenyltheophylline and 8-cyclopentyl-1,3-dipropylxanthine produced no effect. Similar administration of the adenosine A2 receptor agonists, 5'-(N-cyclopropyl)-carboxamidoadenosine and 5'-N-ethylcarboxamidoadenosine (NECA), and the mixed agonists with predominantly A1 site of action, N6-(2-phenylisopropyl) adenosine or 2-chloroadenosine, potentiated haloperidol-induced catalepsy. Higher doses of the adenosine agonists produced catalepsy when given alone. However, N6-cyclopentyladenosine, a highly selective adenosine A1 receptor agonist, was ineffective in these respects. The per se cataleptic effect of adenosine agonists was blocked by DMPX and the centrally acting anticholinergic agent, scopolamine. Scopolamine also attenuated the potentiation of haloperidol-induced catalepsy by adenosine agonists. Further, i.c.v. administration of NECA and DMPX produced a similar effect as that produced after their systemic administration. These findings demonstrate the differential influence of adenosine A1 and A2 receptors on haloperidol-induced catalepsy and support the hypothesis that the functional interaction between adenosine and dopamine mechanisms might occur through adenosine A2 receptors at the level of cholinergic neurons. The results suggest that adenosine A2, but not A1, receptor antagonists may be of potential use in the treatment of Parkinson's disease.

  17. A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase

    PubMed Central

    2015-01-01

    Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation. PMID:25537790

  18. Polymerase Gamma Disease through the Ages

    ERIC Educational Resources Information Center

    Saneto, Russell P.; Naviaux, Robert K.

    2010-01-01

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

  19. Spinal serotonin 5-HT7 and adenosine A1 receptors, as well as peripheral adenosine A1 receptors, are involved in antinociception by systemically administered amitriptyline.

    PubMed

    Liu, Jean; Reid, Allison R; Sawynok, Jana

    2013-01-05

    The present study explored a link between spinal 5-HT(7) and adenosine A(1) receptors in antinociception by systemic amitriptyline in normal and adenosine A(1) receptor knock-out mice using the 2% formalin test. In normal mice, antinociception by systemic amitriptyline 3mg/kg was blocked by intrathecal administration of the selective adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) 10 nmol. Blockade was also seen in adenosine A(1) receptor +/+ mice, but not in -/- mice lacking these receptors. In both normal and adenosine A(1) receptor +/+ mice, the selective 5-HT(7) receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride (SB269970) 3 μg blocked antinociception by systemic amitriptyline, but it did not prevent antinociception in adenosine A(1) receptor -/- mice. In normal mice, flinching was unaltered when the selective 5-HT(7) receptor agonist (2S)-(+)-5-(1,3,5-trimethylpyrazol-4-yl)-2-(dimethylamino)tetralin (AS-19) 20 μg was administered alone, but increased when co-administered intrathecally with DPCPX 10 nmol or SB269970 3 μg. Intrathecal AS-19 decreased flinching in adenosine A(1) receptor +/+ mice compared to -/- mice. Systemic amitriptyline appears to reduce nociception by activating spinal adenosine A(1) receptors secondarily to 5-HT(7) receptors. Spinal actions constitute only one aspect of antinociception by amitriptyline, as intraplantar DPCPX 10 nmol blocked antinociception by systemic amitriptyline in normal and adenosine A(1) receptor +/+, but not -/- mice. Adenosine A(1) receptor interactions are worthy of attention, as chronic oral caffeine (0.1, 0.3g/L, doses considered relevant to human intake levels) blocked antinociception by systemic amitriptyline in normal mice. In conclusion, adenosine A(1) receptors contribute to antinociception by systemic amitriptyline in both spinal and peripheral compartments.

  20. Properties of enzyme fraction A from Chlorella and copurification of 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase, adenosine 5'phosphosulfate sulfohydrolase and adenosine-5'-phosphosulfate cyclase activities.

    PubMed

    Lik-Shing Tsang, M; Schiff, J A

    1976-05-17

    Enzyme fraction A from Chlorella which catalyzes the formation of adenosine 5'-phosphosulfate from adenosine 3'-phosphate 5'-phosphosulfate is further characterized. Fraction A is found to contain an Mg2+ -activated and Ca2+ -inhibited 3' (2')-nucleotidase specific for 3' (2'), 5'-biphosphonucleosides. This activity has been named 3' (2), 5'-biphosphonucleoside 3' (2')-phosphohydrolase. The A fraction is also found to contain an activity which catalyzes the formation of adenosine 3':5'-monophosphate (cyclic AMP) from adenosine 5'-phosphosulfate (adenosine 5'-phosphosulfate cyclase). Under the same conditions of assay, 5'-ATP and 5'-ADP are not substrated for cyclic AMP formation. Unlike the 3' (2'), 5'-biphosphonucleoside 3' (2')-phosphohydrolase activity, the adenosine 5'-phosphosulfate cyclase activity does not require Mg2+, requires NH+4 or Na+, and is not inhibited by Ca2+. The A fraction also contains an adenosine 5'-phospho sulfate sulfohydrolase activity which forms 5'-AMP and sulfate. The three activities remain together during purification and acrylamide gel electrophoresis of the purified preparation yields a pattern where only one protein band has all three activities. The phosphohydrolase can be separated from the other two activities by affinity chromatography on agarose-hexyl-adenosine 3'n5'-bisphosphate yielding a phosphohydrolase preparation showing a single band on gel electrophoresis. The adenosine 5'-phosphosulfate cyclase may provide an alternate route of cyclic AMP formation from sulfate via ATP sulfurylase, but its regulatory significance in Chlorella, if any, remains to be demonstrated. In sulfate reduction, the phosphohydrolase may serve to provide a readily utilized pool of adenosine 5'-phosphosulfate as needed by the adenosine 5'-phosphosulfate sulfotransferase. The cyclase and sulfohydrolase activities would be regarded as side reactions incidental to this pathway, but may be of importance in other metabolic and regulatory reactions.

  1. Adenosine receptor control of cognition in normal and disease.

    PubMed

    Chen, Jiang-Fan

    2014-01-01

    Adenosine and adenosine receptors (ARs) are increasingly recognized as important therapeutic targets for controlling cognition under normal and disease conditions for its dual roles of neuromodulation as well as of homeostatic function in the brain. This chapter first presents the unique ability of adenosine, by acting on the inhibitory A1 and facilitating A2A receptor, to integrate dopamine, glutamate, and BNDF signaling and to modulate synaptic plasticity (e.g., long-term potentiation and long-term depression) in brain regions relevant to learning and memory, providing the molecular and cellular bases for adenosine receptor (AR) control of cognition. This led to the demonstration of AR modulation of social recognition memory, working memory, reference memory, reversal learning, goal-directed behavior/habit formation, Pavlovian fear conditioning, and effort-related behavior. Furthermore, human and animal studies support that AR activity can also, through cognitive enhancement and neuroprotection, reverse cognitive impairments in animal models of Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and schizophrenia. Lastly, epidemiological evidence indicates that regular human consumption of caffeine, the most widely used psychoactive drug and nonselective AR antagonists, is associated with the reduced cognitive decline in aging and AD patients, and with the reduced risk in developing PD. Thus, there is a convergence of the molecular studies revealing AR as molecular targets for integrating neurotransmitter signaling and controlling synaptic plasticity, with animal studies demonstrating the strong procognitive impact upon AR antagonism in normal and disease brains and with epidemiological and clinical evidences in support of caffeine and AR drugs for therapeutic modulation of cognition. Since some of adenosine A2A receptor antagonists are already in phase III clinical trials for motor benefits in PD patients with remarkable safety profiles

  2. Thermally multiplexed polymerase chain reaction

    PubMed Central

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

    2015-01-01

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

  3. TRANSCRIPTION. Structures of the RNA polymerase-σ54 reveal new and conserved regulatory strategies.

    PubMed

    Yang, Yun; Darbari, Vidya C; Zhang, Nan; Lu, Duo; Glyde, Robert; Wang, Yi-Ping; Winkelman, Jared T; Gourse, Richard L; Murakami, Katsuhiko S; Buck, Martin; Zhang, Xiaodong

    2015-08-21

    Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ(54)) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate-dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ(54) holoenzyme at 3.8 angstroms reveals molecular details of σ(54) and its interactions with RNAP. The structure explains how σ(54) targets different regions in RNAP to exert its inhibitory function. Although σ(54) and the major σ factor, σ(70), have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.

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

    PubMed

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

    2015-10-01

    Initial velocity and product inhibition studies of Toxoplasma gondii adenosine kinase (TgAK, EC 2.7.1.20) 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.

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

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

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

  6. Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses

    NASA Astrophysics Data System (ADS)

    Moore, Kimberly A.; Nicoll, Roger A.; Schmitz, Dietmar

    2003-11-01

    The release properties of synapses in the central nervous system vary greatly, not only across anatomically distinct types of synapses but also among the same class of synapse. This variation manifests itself in large part by differences in the probability of transmitter release, which affects such activity-dependent presynaptic forms of plasticity as paired-pulse facilitation and frequency facilitation. This heterogeneity in presynaptic function reflects differences in the intrinsic properties of the synaptic terminal and the activation of presynaptic neurotransmitter receptors. Here we show that the unique presynaptic properties of the hippocampal mossy fiber synapse are largely imparted onto the synapse by the continuous local action of extracellular adenosine at presynaptic A1 adenosine receptors, which maintains a low basal probability of transmitter release.

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

    PubMed Central

    Festugato, Moira

    2015-01-01

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

  8. Expression of human adenosine deaminase in murine hematopoietic cells.

    PubMed Central

    Belmont, J W; MacGregor, G R; Wager-Smith, K; Fletcher, F A; Moore, K A; Hawkins, D; Villalon, D; Chang, S M; Caskey, C T

    1988-01-01

    Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells. Images PMID:3072474

  9. Evidence for an A1-adenosine receptor in the guinea-pig atrium.

    PubMed Central

    Collis, M. G.

    1983-01-01

    1 The purpose of this study was to determine whether the adenosine receptor that mediates a decrease in the force of contraction of the guinea-pig atrium is of the A1- or A2-sub-type. 2 Concentration-response curves to adenosine and a number of 5'- and N6-substituted analogues were constructed and the order of potency of the purines was: 5'-N-cyclopropylcarboxamide adenosine (NCPCA) = 5'-N-ethylcarboxamide adenosine (NECA) greater than N6cyclohexyladenosine (CHA) greater than L-N6-phenylisopropyl adenosine (L-PIA) = 2-chloroadenosine- greater than adenosine greater than D-N6-phenylisopropyl adenosine (D-PIA). 3 The difference in potency between the stereoisomers D- and L-PIA was over 100 fold. 4 The adenosine transport inhibitor, dipyridamole, potentiated submaximal responses to adenosine but had no significant effect on those evoked by the other purines. 5 Theophylline antagonized responses evoked by all purines, and with D-PIA revealed a positive inotropic effect that was abolished by atenolol. 6 The results indicate the existence of an adenosine A1-receptor in the guinea-pig atrium. PMID:6297647

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

    PubMed Central

    Witts, Emily C.; Nascimento, Filipe

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2016-03-15

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

  16. Mechanical stimulation evokes rapid increases in extracellular adenosine concentration in the prefrontal cortex.

    PubMed

    Ross, Ashley E; Nguyen, Michael D; Privman, Eve; Venton, B Jill

    2014-07-01

    Mechanical perturbations can release ATP, which is broken down to adenosine. In this work, we used carbon-fiber microelectrodes and fast-scan cyclic voltammetry to measure mechanically stimulated adenosine in the brain by lowering the electrode 50 μm. Mechanical stimulation evoked adenosine in vivo (average: 3.3 ± 0.6 μM) and in brain slices (average: 0.8 ± 0.1 μM) in the prefrontal cortex. The release was transient, lasting 18 ± 2 s. Lowering a 15-μm-diameter glass pipette near the carbon-fiber microelectrode produced similar results as lowering the actual microelectrode. However, applying a small puff of artificial cerebral spinal fluid was not sufficient to evoke adenosine. Multiple stimulations within a 50-μm region of a slice did not significantly change over time or damage cells. Chelating calcium with EDTA or blocking sodium channels with tetrodotoxin significantly decreased mechanically evoked adenosine, signifying that the release is activity dependent. An alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, did not affect mechanically stimulated adenosine; however, the nucleoside triphosphate diphosphohydrolase 1,2 and 3 (NTDPase) inhibitor POM-1 significantly reduced adenosine so a portion of adenosine is dependent on extracellular ATP metabolism. Thus, mechanical perturbations from inserting a probe in the brain cause rapid, transient adenosine signaling which might be neuroprotective. We have discovered immediate changes in adenosine concentration in the prefrontal cortex following mechanical stimulation. The adenosine increase lasts only about 20 s. Mechanically stimulated adenosine was activity dependent and mostly because of extracellular ATP metabolism. This rapid, transient increase in adenosine may help protect tissue and would occur during implantation of any electrode, such as during deep brain stimulation.

  17. Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise.

    PubMed

    Casey, Darren P; Madery, Brandon D; Pike, Tasha L; Eisenach, John H; Dietz, Niki M; Joyner, Michael J; Wilkins, Brad W

    2009-10-01

    We tested the hypothesis that adenosine contributes to augmented skeletal muscle vasodilation during hypoxic exercise. In separate protocols, subjects performed incremental rhythmic forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n = 8), subjects received an intra-arterial administration of saline (control) and aminophylline (adenosine receptor antagonist). In protocol 2 (n = 10), subjects received intra-arterial phentolamine (alpha-adrenoceptor antagonist) and combined phentolamine and aminophylline administration. Forearm vascular conductance (FVC; in ml x min(-1).100 mmHg(-1)) was calculated from forearm blood flow (in ml/min) and blood pressure (in mmHg). In protocol 1, the change in FVC (DeltaFVC; change from normoxic baseline) during hypoxic exercise with saline was 172 +/- 29 and 314 +/- 34 ml x min(-1) x 100 mmHg(-1) (10% and 20%, respectively). Aminophylline administration did not affect DeltaFVC during hypoxic exercise at 10% (190 +/- 29 ml x min(-1)x100 mmHg(-1), P = 0.4) or 20% (287 +/- 48 ml x min(-1) x 100 mmHg(-1), P = 0.3). In protocol 2, DeltaFVC due to hypoxic exercise with phentolamine infusion was 313 +/- 30 and 453 +/- 41 ml x min(-1) x 100 mmHg(-1) (10% and 20% respectively). DeltaFVC was similar at 10% (352 +/- 39 ml min(-1) x 100 mmHg(-1), P = 0.8) and 20% (528 +/- 45 ml x min(-1) x 100 mmHg(-1), P = 0.2) hypoxic exercise with combined phentolamine and aminophylline. In contrast, DeltaFVC to exogenous adenosine was reduced by aminophylline administration in both protocols (P < 0.05 for both). These observations suggest that adenosine receptor activation is not obligatory for the augmented hyperemia during hypoxic exercise in humans.

  18. Severe combined immunodeficiency due to adenosine deaminase deficiency.

    PubMed

    Hussain, Waqar; Batool, Asma; Ahmed, Tahir Aziz; Bashir, Muhammad Mukarram

    2012-03-01

    Severe Combined Immunodeficiency is the term applied to a group of rare genetic disorders characterised by defective or absent T and B cell functions. Patients usually present in first 6 months of life with respiratory/gastrointestinal tract infections and failure to thrive. Among the various types of severe combined immunodeficiency, enzyme deficiencies are relatively less common. We report the case of a 6 years old girl having severe combined immunodeficiency due to adenosine deaminase deficiency.

  19. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    SciTech Connect

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

    2013-01-24

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

  20. Agonist Derived Molecular Probes for A2A Adenosine Receptors

    PubMed Central

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

    2011-01-01

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

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

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

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

  2. RNA polymerase II transcription: structure and mechanism.

    PubMed

    Liu, Xin; Bushnell, David A; Kornberg, Roger D

    2013-01-01

    A minimal RNA polymerase II (pol II) transcription system comprises the polymerase and five general transcription factors (GTFs) TFIIB, -D, -E, -F, and -H. The addition of Mediator enables a response to regulatory factors. The GTFs are required for promoter recognition and the initiation of transcription. Following initiation, pol II alone is capable of RNA transcript elongation and of proofreading. Structural studies reviewed here reveal roles of GTFs in the initiation process and shed light on the transcription elongation mechanism. This article is part of a Special Issue entitled: RNA Polymerase II Transcript Elongation.

  3. Transcription by RNA polymerases I and III

    PubMed Central

    Paule, Marvin R.; White, Robert J.

    2000-01-01

    The task of transcribing nuclear genes is shared between three RNA polymerases in eukaryotes: RNA polymerase (pol) I synthesises the large rRNA, pol II synthesises mRNA and pol III synthesises tRNA and 5S rRNA. Although pol II has received most attention, pol I and pol III are together responsible for the bulk of transcriptional activity. This survey will summarise what is known about the process of transcription by pol I and pol III, how it happens and the proteins involved. Attention will be drawn to the similarities between the three nuclear RNA polymerase systems and also to their differences. PMID:10684922

  4. Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reactions and a novel bioluminescent pyrophosphate assay.

    PubMed

    Arakawa, Hidetoshi; Karasawa, Koji; Igarashi, Takeshi; Suzuki, Shigeya; Goto, Nobuichi; Maeda, Masako

    2004-10-15

    We developed a novel bioluminescent assay for detection of pyrophosphate in polymerase chain reaction (PCR) product. The principle of this method is as follows: pyrophosphate released by PCR is converted to adenosine 5'-triphosphate (ATP) by pyruvate phosphate dikinase in the presence of the substrate pyruvate phosphate and the coenzyme adenosine 5'-monophosphate; subsequently, ATP concentration is determined by firefly luciferase reaction. The detection limit of pyrophosphate is 1.56 x 10(-15)mol/assay. Additionally, luminescent intensity reached a maximum at approximately 100 s and remained elevated beyond 10 min. This approach is applicable to the detection of cariogenic bacteria in dental plaque. Thus, the allele-specific PCR products of Streptococcus mutans and Streptococcus sobrinus developed in this study were measured via the proposed bioluminescent assay. This protocol, which does not require expensive equipment, can be utilized to rapidly monitor cariogenic bacteria in dental plaque.

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

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

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

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

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

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

  7. [Effects of dopamine and adenosine on regulation of water-electrolyte exchange in Amoeba proteus].

    PubMed

    Bagrov, Ia Iu; Manusova, N B

    2014-01-01

    Dopamine and adenosine both regulate transport of sodium chloride in the renal tubules in mammals. We have studied the effect of dopamine and adenosine on spontaneous activity of contractile vacuole of Amoeba proteous. Both substances stimulated contractile vacuole. The effect of dopamine was suppressed by D2 receptor antagonist, haloperidol, but not by D1 antagonist, SCH 39166. Adenylate cyclase inhibitor, 2.5-dideoxyadenosine, suppressed the effect of dopamine, but not of adenosine. Inhibitor of protein kinase C, staurosporine, in contrast, blocked the effect of adenosine, but not dopamine. Notably, dopamine opposed effect of adenosine and vice versa. These results suggest that similar effects of dopamine and adenosine could be mediated by different intracellulare mechanisms.

  8. Crystal Structure of Schistosoma mansoni Adenosine Phosphorylase/5’-Methylthioadenosine Phosphorylase and Its Importance on Adenosine Salvage Pathway

    PubMed Central

    Torini, Juliana Roberta; Brandão-Neto, José; DeMarco, Ricardo; Pereira, Humberto D'Muniz

    2016-01-01

    Schistosoma mansoni do not have de novo purine pathways and rely on purine salvage for their purine supply. It has been demonstrated that, unlike humans, the S. mansoni is able to produce adenine directly from adenosine, although the enzyme responsible for this activity was unknown. In the present work we show that S. mansoni 5´-deoxy-5´-methylthioadenosine phosphorylase (MTAP, E.C. 2.4.2.28) is capable of use adenosine as a substrate to the production of adenine. Through kinetics assays, we show that the Schistosoma mansoni MTAP (SmMTAP), unlike the mammalian MTAP, uses adenosine substrate with the same efficiency as MTA phosphorolysis, which suggests that this enzyme is part of the purine pathway salvage in S. mansoni and could be a promising target for anti-schistosoma therapies. Here, we present 13 SmMTAP structures from the wild type (WT), including three single and one double mutant, and generate a solid structural framework for structure description. These crystal structures of SmMTAP reveal that the active site contains three substitutions within and near the active site when compared to it mammalian counterpart, thus opening up the possibility of developing specific inhibitors to the parasite MTAP. The structural and kinetic data for 5 substrates reveal the structural basis for this interaction, providing substract for inteligent design of new compounds for block this enzyme activity. PMID:27935959

  9. Dissecting striatal adenosine-cannabinoid receptor interactions. New clues from rats over-expressing adenosine A2A receptors.

    PubMed

    Ferré, Sergi; Sebastião, Ana Maria

    2016-03-01

    This Editorial highlights a study by Chiodi et al. () showing that the effects mediated by cannabinoid CB1 receptor (CB1R) activation in the striatum are significantly reduced in rats with neuronal over-expression of adenosine A2A receptors (A2AR). Two hypotheses are derived from that study. Hypothesis A: two subpopulations of pre-synaptic CB1R in corticostriatal glutamatergic terminals exist, one forming and another not forming heteromers with A2AR. Hypothesis B: CB1R are predominantly forming heteromers with A2AR. In the case of hypothesis A, the A2AR might be required for CB1R-A2AR heteromeric signaling, whereas non-heteromeric CB1R activity is inhibited by A2ARs. In the case of hypothesis B, up-regulation of A2ARs may perturb heteromeric stoichiometry, thus reducing CB1R functioning. In any case, pre-synaptic striatal A2AR-CB1R heteromers emerge as important targets of the effects of cannabinoids demonstrated at the neuronal and behavioral level. Read the highlighted article 'Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors' on page 907.

  10. Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

    PubMed

    Shah, Mrugeshkumar K; Kadowitz, Philip J

    2002-01-01

    The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.

  11. Picornaviral polymerase structure, function, and fidelity modulation.

    PubMed

    Peersen, Olve B

    2017-02-02

    Like all positive strand RNA viruses, the picornaviruses replicate their genomes using a virally encoded RNA-dependent RNA polymerase enzyme known as 3D(pol). Over the past decade we have made tremendous advances in our understanding of 3D(pol) structure and function, including the discovery of a novel mechanism for closing the active site that allows these viruses to easily fine tune replication fidelity and quasispecies distributions. This review summarizes current knowledge of picornaviral polymerase structure and how the enzyme interacts with RNA and other viral proteins to form stable and processive elongation complexes. The picornaviral RdRPs are among the smallest viral polymerases, but their fundamental molecular mechanism for catalysis appears to be generally applicable as a common feature of all positive strand RNA virus polymerases.

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

    PubMed Central

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

    1992-01-01

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

  13. Estimation of skeletal muscle interstitial adenosine during forearm dynamic exercise in humans

    NASA Technical Reports Server (NTRS)

    Costa, F.; Heusinkveld, J.; Ballog, R.; Davis, S.; Biaggioni, I.

    2000-01-01

    It has been proposed that adenosine is a metabolic signal that triggers activation of muscle afferents involved in the exercise pressor reflex. Furthermore, exogenous adenosine induces sympathetic activation that mimics the exercise pressor reflex, and blockade of adenosine receptors inhibits sympathetic activation induced by exercise. Thus, we hypothesize that adenosine is released locally by the muscle during exercise. We used microdialysis probes, placed in the flexor digitorium superficialis muscle, to estimate muscle interstitial adenosine levels in humans. We estimated resting in vivo muscle interstitial adenosine concentrations (0.292+/-0.058 micromol/L, n=4) by perfusing increasing concentrations of adenosine to determine the gradient produced in the dialysate. Muscle interstitial adenosine concentrations increased from 0.23+/-0.04 to 0.82+/-0.14 micromol/L (n=14, P<0.001) during intermittent dynamic exercise at 50% of maximal voluntary contraction. Lactate increased from 0.8+/-0.1 to 2.3+/-0.3 mmol/L (P<0.001). Lower intensity (15% maximal voluntary contraction) intermittent dynamic exercise increased adenosine concentrations from 0.104+/-0.02 to 0.42+/-0.16 micromol/L (n=7). The addition of ischemia to this low level of exercise produced a greater increase in adenosine (from 0.095+/-0.02 to 0.48+/-0.2 micromol/L) compared with nonischemic exercise (0. 095+/-0.02 to 0.25+/-0.12 micromol/L). These results indicate that microdialysis is useful in estimating adenosine concentrations and in reflecting changes in muscle interstitial adenosine during dynamic exercise in humans.

  14. Transcription termination by nuclear RNA polymerases

    PubMed Central

    Richard, Patricia; Manley, James L.

    2009-01-01

    Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human. PMID:19487567

  15. A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase.

    PubMed

    Piccirilli, J A; Moroney, S E; Benner, S A

    1991-10-22

    With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

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

    PubMed

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

    2015-02-01

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

  17. Adenosine induces apoptosis through TNFR1/RIPK1/P38 axis in colon cancer cells.

    PubMed

    Yu, Shunji; Hou, Daisen; Chen, Ping; Zhang, Qi; Lv, Bin; Ma, Yunfang; Liu, Fuchen; Liu, Hui; Song, Evelyn J; Yang, Dongqin; Liu, Jie

    2015-05-08

    Adenosine, a metabolite of ATP, ubiquitously exists in a wide range of organs and tissues. We previously reported that adenosine was implicated in apoptosis in many cancer cells by extrinsic and/or intrinsic pathways. Here, we found that adenosine suppresses the cell growth by induction of apoptosis of human colonic cancer cells through a novel mechanism. Adenosine suppresses the cell growth of human SW620 and SW480 colon cells in an adenosine transporter and adenosine kinase dependent manner. Moreover, the cell growth suppression is induced by apoptosis through activation of caspase-3 and PARP, and accumulation of ROS in cells. Importantly, we found that adenosine increases the expression of TNFR1 and RIPK1 and the phosphorylation of p38. Knockdown of TNFR1 or RIPK1 impairs the activation of p38, blocks the cleavage of PARP, and provides partially, yet significantly protection from cell death, including reducing the ROS generation in the colon cancer cells. These results indicate that a TNFR1/RIPK1/P38 axis is present in adenosine-induced apoptosis of colonic cancer cells. This axis triggers apoptosis and plays crucial roles in relay of the death signaling. Our study also provides additional experimental evidence for adenosine as a potent therapeutic drug in cancer therapy.

  18. Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

    PubMed Central

    Daddona, P E; Mitchell, B S; Meuwissen, H J; Davidson, B L; Wilson, J M; Koller, C A

    1983-01-01

    In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function. Images FIGURE 1 FIGURE 2 FIGURE 3 PMID:6603477

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    1991-01-01

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

  1. Intracoronary adenosine versus intravenous adenosine during primary PCI for ST-elevation myocardial infarction: which one offers better outcomes in terms of microvascular obstruction?

    PubMed

    Doolub, Gemina; Dall'armellina, Erica

    2013-01-01

    Aims. Previous studies have suggested that intravenous administration of adenosine improves myocardial reperfusion and reduces infarct size in ST-elevation myocardial infarction (STEMI) patients. Intracoronary administration of adenosine has shown conflicting results. Methods. In this retrospective, single-centre, blinded clinical study, we assessed whether selective intracoronary administration of adenosine distal to the occlusion site immediately before initial balloon inflation reduces microvascular obstruction (MVO) as assessed with cardiac magnetic resonance imaging (MRI). Using contrast-enhanced sequences, microvascular obstruction (MVO) was calculated. We found 81 patients presenting with STEMI within 12 h from symptom onset who were eligible for the study. In 80/81 (100%) patients receiving the study drug, MRI was performed on Day 1 after primary angioplasty. Results. The prevalence of MVO was reduced in the patients treated with intracoronary adenosine, (45%) compared to 85% of patients who were administered intravenous adenosine (P = 0.0043). We found that the size of MVO in patients receiving intracoronary adenosine was significantly reduced compared to 0.91 g in the intravenous-treated group (P = 0.027). There was no statistically significant difference in TIMI flow and clinical outcomes after primary PCI. Conclusion. We found significant evidence that selective high-dose intracoronary administration of adenosine distal to the occlusion site of the culprit lesion in STEMI patients results in a decrease in microvascular obstruction.

  2. Adenosine-to-inosine RNA editing meets cancer.

    PubMed

    Dominissini, Dan; Moshitch-Moshkovitz, Sharon; Amariglio, Ninette; Rechavi, Gideon

    2011-11-01

    The role of epigenetics in tumor onset and progression has been extensively addressed. Discoveries in the last decade completely changed our view on RNA. We now realize that its diversity lies at the base of biological complexity. Adenosine-to-inosine (A-to-I) RNA editing emerges a central generator of transcriptome diversity and regulation in higher eukaryotes. It is the posttranscriptional deamination of adenosine to inosine in double-stranded RNA catalyzed by enzymes of the adenosine deaminase acting on RNA (ADAR) family. Thought at first to be restricted to coding regions of only a few genes, recent bioinformatic analyses fueled by high-throughput sequencing revealed that it is a widespread modification affecting mostly non-coding repetitive elements in thousands of genes. The rise in scope is accompanied by discovery of a growing repertoire of functions based on differential decoding of inosine by the various cellular machineries: when recognized as guanosine, it can lead to protein recoding, alternative splicing or altered microRNA specificity; when recognized by inosine-binding proteins, it can result in nuclear retention of the transcript or its degradation. An imbalance in expression of ADAR enzymes with consequent editing dysregulation is a characteristic of human cancers. These alterations may be responsible for activating proto-oncogenes or inactivating tumor suppressors. While unlikely to be an early initiating 'hit', editing dysregulation seems to contribute to tumor progression and thus should be considered a 'driver mutation'. In this review, we examine the contribution of A-to-I RNA editing to carcinogenesis.

  3. Activity-Dependent Adenosine Release May Be Linked to Activation of Na+-K+ ATPase: An In Vitro Rat Study

    PubMed Central

    Sims, Robert Edward; Dale, Nicholas

    2014-01-01

    In the brain, extracellular adenosine increases as a result of neuronal activity. The mechanisms by which this occurs are only incompletely understood. Here we investigate the hypothesis that the Na+ influxes associated with neuronal signalling activate the Na+-K+ ATPase which, by consuming ATP, generates intracellular adenosine that is then released via transporters. By measuring adenosine release directly with microelectrode biosensors, we have demonstrated that AMPA-receptor evoked adenosine release in basal forebrain and cortex depends on extracellular Na+. We have simultaneously imaged intracellular Na+ and measured adenosine release. The accumulation of intracellular Na+ during AMPA receptor activation preceded adenosine release by some 90 s. By removing extracellular Ca2+, and thus preventing indiscriminate neuronal activation, we used ouabain to test the role of the Na+-K+ ATPase in the release of adenosine. Under conditions which caused a Na+ influx, brief applications of ouabain increased the accumulation of intracellular Na+ but conversely rapidly reduced extracellular adenosine levels. In addition, ouabain greatly reduced the amount of adenosine released during application of AMPA. Our data therefore suggest that activity of the Na+-K+ ATPase is directly linked to the efflux of adenosine and could provide a universal mechanism that couples adenosine release to neuronal activity. The Na+-K+ ATPase-dependent adenosine efflux is likely to provide adenosine-mediated activity-dependent negative feedback that will be important in many diverse functional contexts including the regulation of sleep. PMID:24489921

  4. Evidence for a substrate cycle between AMP and adenosine in isolated hepatocytes.

    PubMed Central

    Bontemps, F; Van den Berghe, G; Hers, H G

    1983-01-01

    The effect of adenosine on the metabolism of prelabeled adenine nucleotides was investigated in isolated hepatocytes. Adenosine caused an approximately equal to 2-fold increase in the ATP content of the cells. This effect was in part counteracted by an increased rate of adenine nucleotide catabolism that could be explained by a stimulation of both AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) and the cytoplasmic 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) because of the increased concentration of ATP. The unexpected finding that labeled adenosine was formed immediately after the addition of the unlabeled nucleoside could be explained by the trapping effect of adenosine. An accumulation of labeled adenosine was observed also in the presence of 5-iodotubercidin, a potent inhibitor of adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). Under these conditions, there was a decrease in the concentration of ATP in the cell and a 2- to 3-fold increase in the rate of formation of allantoin. This formation of adenosine was only slightly decreased by inhibition of the membranous 5'-nucleotidase; it led to the accumulation of S-adenosylhomocysteine in the presence of coformycin and an excess of L-homocysteine. It was concluded that, under basal conditions, the cytoplasmic 5'-nucleotidase present in the liver cell continuously produces adenosine, which is immediately reconverted into AMP by adenosine kinase, without giving rise to allantoin. This futile cycle between AMP and adenosine amounts to at least 20 nmol/min per g of liver and, thus, exceeds the basic rate of allantoin formation. PMID:6304684

  5. Effect of phentolamine on the hyperemic response to adenosine in patients with microvascular disease.

    PubMed

    Aarnoudse, Wilbert; Geven, Maartje; Barbato, Emanuele; Botman, Kees-joost; De Bruyne, Bernard; Pijls, Nico H J

    2005-12-15

    For accurate measurement of the fractional flow reserve (FFR) of the myocardium, the presence of maximum hyperemia is of paramount importance. It has been suggested that the hyperemic effect of the conventionally used hyperemic stimulus, adenosine, could be submaximal in patients who have microvascular dysfunction and that adding alpha-blocking agents could augment the hyperemic response in these patients. We studied the effect of the nonselective alpha-blocking agent phentolamine, which was administered in addition to adenosine after achieving hyperemia, in patients who had microvascular disease and those who did not. Thirty patients who were referred for percutaneous coronary intervention were selected. Of these 30 patients, 15 had strong indications for microvascular disease and 15 did not. FFR was measured using intracoronary adenosine, intravenous adenosine, and intracoronary papaverine before and after intracoronary administration of the nonselective alpha blocker phentolamine. In patients who did not have microvascular disease, no differences in hyperemic response to adenosine were noted, whether or not alpha blockade was given before adenosine administration; FFR levels before and after phentolamine were 0.76 and 0.75, respectively, using intracoronary adenosine (p = 0.10) and 0.75 and 0.74, respectively, using intravenous adenosine (p = 0.20). In contrast, in patients who had microvascular disease, some increase in hyperemic response was observed after administration of phentolamine; FFR levels decreased from 0.74 to 0.70 using intracoronary adenosine (p = 0.003) and from 0.75 to 0.72 using intravenous adenosine (p = 0.04). Although statistically significant, the observed further decrease in microvascular resistance after addition of phentolamine was small and did not affect clinical decision making in any patient. In conclusion, when measuring FFR, routinely adding an alpha-blocking agent to adenosine does not affect clinical decision making.

  6. Adenosine signaling contributes to ethanol-induced fatty liver in mice

    PubMed Central

    Peng, Zhongsheng; Borea, Pier Andrea; Wilder, Tuere; Yee, Herman; Chiriboga, Luis; Blackburn, Michael R.; Azzena, Gianfranco; Resta, Giuseppe; Cronstein, Bruce N.

    2009-01-01

    Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the biochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5′-nucleotidase, and adenosine production and adenosine receptor activation are known to play critical roles in the development of hepatic fibrosis. We therefore investigated whether adenosine and its receptors play a role in the development of alcohol-induced fatty liver. WT mice fed ethanol on the Lieber-DeCarli diet developed hepatic steatosis, including increased hepatic triglyceride content, while mice lacking ecto-5′-nucleotidase or adenosine A1 or A2B receptors were protected from developing fatty liver. Similar protection was also seen in WT mice treated with either an adenosine A1 or A2B receptor antagonist. Steatotic livers demonstrated increased expression of genes involved in fatty acid synthesis, which was prevented by blockade of adenosine A1 receptors, and decreased expression of genes involved in fatty acid metabolism, which was prevented by blockade of adenosine A2B receptors. In vitro studies supported roles for adenosine A1 receptors in promoting fatty acid synthesis and for A2B receptors in decreasing fatty acid metabolism. These results indicate that adenosine generated by ethanol metabolism plays an important role in ethanol-induced hepatic steatosis via both A1 and A2B receptors and suggest that targeting adenosine receptors may be effective in the prevention of alcohol-induced fatty liver. PMID:19221436

  7. Evidence for an A2/Ra adenosine receptor in the guinea-pig trachea

    PubMed Central

    Brown, C.M.; Collis, M.G.

    1982-01-01

    1 An attempt was made to determine whether the extracellular adenosine receptor that mediates relaxation in the guinea-pig trachea is of the A1/Ri or A2/Ra subtype. 2 Dose-response curves to adenosine and a number of 5′- and N6-substituted analogues were constructed for the isolated guinea-pig trachea, contracted with carbachol. 3 The 5′-substituted analogues of adenosine were the most potent compounds tested, the order of potency being 5′-N-cyclopropylcarboxamide adenosine (NCPCA) > 5′-N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine > L-N6-phenylisopropyladenosine (L-PIA) > adenosine > D-N6-phenylisopropyladenosine (D-PIA). 4 The difference in potency between the stereoisomers D- and L-PIA on the isolated trachea was at the most five fold. 5 Responses to low doses of adenosine and its analogues were attenuated after treatment with either theophylline or 8-phenyltheophylline. The responses to 2-chloroadenosine were affected to a lesser extent than were those to the other purines. 6 Adenosine transport inhibitors, dipyridamole and dilazep, potentiated responses to adenosine, did not affect those to NCPCA, NECA, L-PIA and D-PIA but significantly reduced the responses to high doses of 2-chloroadenosine. 7 Relaxations evoked by 9-β-D-xylofuranosyladenosine which can activate intracellular but not extracellular adenosine receptors, were attenuated by dipyridamole but unaffected by 8-phenyltheophylline. 8 The results support the existence of an extracellular A2/Ra subtype of adenosine receptor and an intracellular purine-sensitive site, both of which mediate relaxation. PMID:6286021

  8. Characterization of spontaneous, transient adenosine release in the caudate-putamen and prefrontal cortex.

    PubMed

    Nguyen, Michael D; Lee, Scott T; Ross, Ashley E; Ryals, Matthew; Choudhry, Vishesh I; Venton, B Jill

    2014-01-01

    Adenosine is a neuroprotective agent that inhibits neuronal activity and modulates neurotransmission. Previous research has shown adenosine gradually accumulates during pathologies such as stroke and regulates neurotransmission on the minute-to-hour time scale. Our lab developed a method using carbon-fiber microelectrodes to directly measure adenosine changes on a sub-second time scale with fast-scan cyclic voltammetry (FSCV). Recently, adenosine release lasting a couple of seconds has been found in murine spinal cord slices. In this study, we characterized spontaneous, transient adenosine release in vivo, in the caudate-putamen and prefrontal cortex of anesthetized rats. The average concentration of adenosine release was 0.17±0.01 µM in the caudate and 0.19±0.01 µM in the prefrontal cortex, although the range was large, from 0.04 to 3.2 µM. The average duration of spontaneous adenosine release was 2.9±0.1 seconds and 2.8±0.1 seconds in the caudate and prefrontal cortex, respectively. The concentration and number of transients detected do not change over a four hour period, suggesting spontaneous events are not caused by electrode implantation. The frequency of adenosine transients was higher in the prefrontal cortex than the caudate-putamen and was modulated by A1 receptors. The A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine, 6 mg/kg i.p.) increased the frequency of spontaneous adenosine release, while the A1 agonist CPA (N(6)-cyclopentyladenosine, 1 mg/kg i.p.) decreased the frequency. These findings are a paradigm shift for understanding the time course of adenosine signaling, demonstrating that there is a rapid mode of adenosine signaling that could cause transient, local neuromodulation.

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    1983-01-01

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

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

    PubMed Central

    2014-01-01

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

  12. Adenosine triphosphate (ATP) as a possible indicator of extraterrestrial biology

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.

    1974-01-01

    The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.

  13. Adenosine receptor expression and function in rat striatal cholinergic interneurons.

    PubMed

    Preston, Z; Lee, K; Widdowson, L; Freeman, T C; Dixon, A K; Richardson, P J

    2000-06-01

    Cholinergic neurons were identified in rat striatal slices by their size, membrane properties, sensitivity to the NK(1) receptor agonist (Sar(9), Met(O(2))(11)) Substance P, and expression of choline acetyltransferase mRNA. A(1) receptor mRNA was detected in 60% of the neurons analysed, and A(2A) receptor mRNA in 67% (n=15). The A(1) receptor agonist R-N(6)-(2-phenylisopropyl)adenosine (R-PIA) hyperpolarized cholinergic neurons in a concentration dependent manner sensitive to the A(1) antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 100 nM). In dual stimulus experiments, the A(2A) receptor antagonist 8-(3-chlorostyryl)caffeine (CSC, 500 nM) decreased release of [(3)H]-acetylcholine from striatal slices (S2/S1 0.78+/-0.07 versus 0.95+/-0.05 in control), as did adenosine deaminase (S2/S1 ratio 0.69+/-0.05), whereas the A(1) receptor antagonist DPCPX (100 nM) had no effect (S2/S1 1.05+/-0.14). In the presence of adenosine deaminase the adenosine A(2A) receptor agonist 2-p-((carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadeno sin e (CGS21680, 10 nM) increased release (S2/S1 ratio 1.03+/-0.05 versus 0.88+/-0.05 in control), an effect blocked by the antagonist CSC (500 nM, S2/S1 0.68+/-0.05, versus 0.73+/-0.08 with CSC alone). The combined superfusion of bicuculline (10 microM), saclofen (1 microM) and naloxone (10 microM) had no effect on the stimulation by CGS21680 (S2/S1 ratio 0.99+/-0.04). The A(1) receptor agonist R-PIA (100 nM) inhibited the release of [(3)H]-acetylcholine (S2/S1 ratio 0.70+/-0.03), an effect blocked by DPCPX (S2/S1 ratio 1.06+/-0.07). It is concluded that both A(1) and A(2A) receptors are expressed on striatal cholinergic neurons where they are functionally active.

  14. Adenosine conjugated lipidic nanoparticles for enhanced tumor targeting.

    PubMed

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

    2015-01-01

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

  15. Ribosome-inactivating lectins with polynucleotide:adenosine glycosidase activity.

    PubMed

    Battelli, M G; Barbieri, L; Bolognesi, A; Buonamici, L; Valbonesi, P; Polito, L; Van Damme, E J; Peumans, W J; Stirpe, F

    1997-05-26

    Lectins from Aegopodium podagraria (APA), Bryonia dioica (BDA), Galanthus nivalis (GNA), Iris hybrid (IRA) and Sambucus nigra (SNAI), and a new lectin-related protein from Sambucus nigra (SNLRP) were studied to ascertain whether they had the properties of ribosome-inactivating proteins (RIP). IRA and SNLRP inhibited protein synthesis by a cell-free system and, at much higher concentrations, by cells and had polynucleotide:adenosine glycosidase activity, thus behaving like non-toxic type 2 (two chain) RIP. APA and SNAI had much less activity, and BDA and GNA did not inhibit protein synthesis.

  16. The Probable Pathogenic Role of Adenosine in Malaria

    PubMed Central

    Onabanjo, A. O.; Maegraith, B. G.

    1970-01-01

    An increase in concentration of 0·14 mg./ml. of adenosine was demonstrated in the blood of Macaca mulatta infected with Plasmodium knowlesi at the advanced stages of the infection. The recorded increase may help to explain the physiological and pathological changes that occur in malaria, especially in relation to the dynamic and permeability functions of the smaller blood vessels. It is, however, suggested that the release of this agent into the circulation in greater concentration in the acute stages of malarial infection, could probably play a role in the pathogenesis of this disease. PMID:4992731

  17. Free RNA polymerase in Escherichia coli.

    PubMed

    Patrick, Michael; Dennis, Patrick P; Ehrenberg, Mans; Bremer, Hans

    2015-12-01

    The frequencies of transcription initiation of regulated and constitutive genes depend on the concentration of free RNA polymerase holoenzyme [Rf] near their promoters. Although RNA polymerase is largely confined to the nucleoid, it is difficult to determine absolute concentrations of [Rf] at particular locations within the nucleoid structure. However, relative concentrations of free RNA polymerase at different growth rates, [Rf]rel, can be estimated from the activities of constitutive promoters. Previous studies indicated that the rrnB P2 promoter is constitutive and that [Rf]rel in the vicinity of rrnB P2 increases with increasing growth rate. Recently it has become possible to directly visualize Rf in growing Escherichia coli cells. Here we examine some of the important issues relating to gene expression based on these new observations. We conclude that: (i) At a growth rate of 2 doublings/h, there are about 1000 free and 2350 non-specifically DNA-bound RNA polymerase molecules per average cell (12 and 28%, respectively, of 8400 total) which are in rapid equilibrium. (ii) The reversibility of the non-specific binding generates more than 1000 free RNA polymerase molecules every second in the immediate vicinity of the DNA. Of these, most rebind non-specifically to the DNA within a few ms; the frequency of non-specific binding is at least two orders of magnitude greater than specific binding and transcript initiation. (iii) At a given amount of RNA polymerase per cell, [Rf] and the density of non-specifically DNA-bound RNA polymerase molecules along the DNA both vary reciprocally with the amount of DNA in the cell. (iv) At 2 doublings/h an E. coli cell contains, on the average, about 1 non-specifically bound RNA polymerase per 9 kbp of DNA and 1 free RNA polymerase per 20 kbp of DNA. However some DNA regions (i.e. near active rRNA operons) may have significantly higher than average [Rf].

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

    PubMed Central

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

    2007-01-01

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

  19. Transient Delivery of Adenosine as a Novel Therapy to Prevent Epileptogenesis

    DTIC Science & Technology

    2014-08-01

    Using silk-based brain implants engineered to release adenosine we demonstrated that reversal of epigenetic changes prevents epileptogenesis. We...biodegradable silk-based brain implants ) to prevent epileptogenesis long term. These findings constitute a novel scientific advance with direct clinical...implications. Specifically, using bioengineered silk- based brain implants we demonstrated that transient delivery of a defined focal dose of adenosine

  20. Beneficial and detrimental role of adenosine signaling in diseases and therapy.

    PubMed

    Liu, Hong; Xia, Yang

    2015-11-15

    Adenosine is a major signaling nucleoside that orchestrates cellular and tissue adaptation under energy depletion and ischemic/hypoxic conditions by activation of four G protein-coupled receptors (GPCR). The regulation and generation of extracellular adenosine in response to stress are critical in tissue protection. Both mouse and human studies reported that extracellular adenosine signaling plays a beneficial role during acute states. However, prolonged excess extracellular adenosine is detrimental and contributes to the development and progression of various chronic diseases. In recent years, substantial progress has been made to understand the role of adenosine signaling in different conditions and to clarify its significance during the course of disease progression in various organs. These efforts have and will identify potential therapeutic possibilities for protection of tissue injury at acute stage by upregulation of adenosine signaling or attenuation of chronic disease progression by downregulation of adenosine signaling. This review is to summarize current progress and the importance of adenosine signaling in different disease stages and its potential therapeutic effects.

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

    PubMed Central

    2015-01-01

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

  2. Role of adenosine in postprandial and reactive hyperemia in canine jejunum.

    PubMed

    Sawmiller, D R; Chou, C C

    1992-10-01

    The role of adenosine in postprandial jejunal hyperemia was investigated by determining the effect of placement of predigested food into the jejunal lumen on blood flow and oxygen consumption before and during intra-arterial infusion of dipyridamole (1.5 microM arterial concn) or adenosine deaminase (9 U/ml arterial concn) in anesthetized dogs. Neither drug significantly altered resting jejunal blood flow and oxygen consumption. Before dipyridamole or deaminase, food placement increased blood flow by 30-36%, 26-42%, and 21-46%, and oxygen consumption by 13-22%, 21-22%, and 26-29%, during 0- to 3-, 4- to 7-, and 8- to 11-min placement periods, respectively. Adenosine deaminase abolished the entire 11-min hyperemia, whereas dipyridamole significantly enhanced the initial 7-min hyperemia (45-49%). Both drugs abolished the initial 7-min food-induced increase in oxygen consumption. Dipyridamole attenuated (14%), whereas deaminase did not alter (28%), the increased oxygen consumption that occurred at 8-11 min. Adenosine deaminase also prevented the food-induced increase in venoarterial adenosine concentration difference. In separate series of experiments, luminal placement of food significantly increased jejunal lymphatic adenosine concentration and release. Also, reactive hyperemia was accompanied by an increase in venous adenosine concentration and release. This study provides further evidence to support the thesis that adenosine plays a role in postprandial and reactive hyperemia in the canine jejunum.

  3. DNA polymerase activity of tomato fruit chromoplasts.

    PubMed

    Serra, E C; Carrillo, N

    1990-11-26

    DNA polymerase activity was measured in chromoplasts of ripening tomato fruits. Plastids isolated from young leaves or mature red fruits showed similar DNA polymerase activities. The same enzyme species was present in either chloroplasts or chromoplasts as judged by pH and temperature profiles, sensitivities towards different inhibitors and relative molecular mass (Mr 88 kDa). The activities analyzed showed the typical behaviour of plastid-type polymerases. The results presented here suggest that chromoplast maintain their DNA synthesis potential in fruit tissue at chloroplast levels. Consequently, the sharp decrease of the plastid chromosome transcription observed at the onset of fruit ripening could not be due to limitations in the availability of template molecules. Other mechanisms must be involved in the inhibition of chromoplast RNA synthesis.

  4. Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

    PubMed

    Chen, Y; Morera, S; Pasti, C; Angusti, A; Solaroli, N; Véron, M; Janin, J; Manfredini, S; Deville-Bonne, D

    2005-11-01

    NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

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

    PubMed

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

    2014-12-10

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

  6. Astrocyte-derived adenosine is central to the hypnogenic effect of glucose.

    PubMed

    Scharbarg, Emeric; Daenens, Marion; Lemaître, Frédéric; Geoffroy, Hélène; Guille-Collignon, Manon; Gallopin, Thierry; Rancillac, Armelle

    2016-01-12

    Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity.

  7. [Protective effect of adenosine receptor agonists in a model of spinal cord injury in rats].

    PubMed

    Sufianova, G Z; Usov, L A; Sufianov, A A; Perelomov, Iu P; Raevskaia, L Iu; Shapkin, A G

    2002-01-01

    Possibilities of the neuroprotector therapy using adenosine and cyclopentyladenosine (CPA), an adenosine receptor agonist, were studied on a model of spinal cord injury by compression in rats (most closely reproducing the analogous clinical pathological process in humans). The model was induced by slow, graded compression of the spinal cord at the thoracic level. Adenosine and CPA were introduced 60 min before injury by subcutaneous injections in a dose of 300 and 2.5 micrograms/kg, respectively. The protective effect was judged by comparing the neurological, electromyographic, and histopathological changes in animals with the model injury and in the control group (adenosine and CPA background). The A1-agonist CPA injections produced a pronounced, statistically significant neuroprotector effect on the given spinal cord injury model in rats. The neuroprotective effect of adenosine was significant but not as strong. It is concluded that it is expedient to use A-agonists in clinics.

  8. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis.

    PubMed

    Ng, Seng Kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2015-05-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR(+/-)) significantly protect ESMN from SOD1G93A(+) astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS.

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

    PubMed

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

    2014-09-01

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

  10. Suppression of adenosine 2a receptor (A2aR)-mediated adenosine signaling improves disease phenotypes in a mouse model of amyotrophic lateral sclerosis

    PubMed Central

    Ng, Seng kah; Higashimori, Haruki; Tolman, Michaela; Yang, Yongjie

    2017-01-01

    Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease in which the majority of upper and lower motor neurons are degenerated. Despite intensive efforts to identify drug targets and develop neuroprotective strategies, effective therapeutics for ALS remains unavailable. The identification and characterization of novel targets and pathways remain crucial in the development of ALS therapeutics. Adenosine is a major neuromodulator that actively regulates synaptic transmission. Interestingly, adenosine levels are significantly elevated in the cerebrospinal fluid (CSF) of progressing human ALS patients. In the current study, we showed that adenosine 2a receptor (A2aR), but not adenosine 1 receptor (A1R), is highly enriched in spinal (motor) neurons. A2aR expression is also selectively increased at the symptomatic onset in the spinal cords of SOD1G93A mice and end-stage human ALS spinal cords. Interestingly, we found that direct adenosine treatment is sufficient to induce embryonic stem cell-derived motor neuron (ESMN) cell death in cultures. Subsequent pharmacological inhibition and partial genetic ablation of A2aR (A2aR+/−) significantly protect ESMN from SOD1G93A+ astrocyte-induced cell death and delay disease progression of SOD1G93A mice. Taken together, our results provide compelling novel evidence that A2aR-mediated adenosine signaling contributes to the selective spinal motor neuron degeneration observed in the SOD1G93A mouse model of ALS. PMID:25779930

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

    PubMed Central

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

    2014-01-01

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

  12. The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates.

    PubMed

    Santner, Tobias; Siegmund, Vanessa; Marx, Andreas; Micura, Ronald

    2012-04-01

    Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.

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

    SciTech Connect

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

    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.

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

    PubMed

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

    2015-12-15

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

  15. Sawhorse waveform voltammetry for selective detection of adenosine, ATP, and hydrogen peroxide.

    PubMed

    Ross, Ashley E; Venton, B Jill

    2014-08-05

    Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters in vivo. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from -0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to -0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements in vivo; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes in vivo.

  16. Sawhorse Waveform Voltammetry for Selective Detection of Adenosine, ATP, and Hydrogen Peroxide

    PubMed Central

    2015-01-01

    Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters in vivo. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from −0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to −0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements in vivo; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes in vivo. PMID:25005825

  17. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

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

  18. Catalytic editing properties of DNA polymerases.

    PubMed Central

    Canard, B; Cardona, B; Sarfati, R S

    1995-01-01

    Enzymatic incorporation of 2',3'-dideoxynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479898

  19. The bacterial enhancer-dependent RNA polymerase

    PubMed Central

    Zhang, Nan; Darbari, Vidya C.; Glyde, Robert; Zhang, Xiaodong; Buck, Martin

    2016-01-01

    Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript. PMID:27789741

  20. Determining Annealing Temperatures for Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Porta, Angela R.; Enners, Edward

    2012-01-01

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

  1. Polymerase Chain Reaction for Educational Settings.

    ERIC Educational Resources Information Center

    Garrison, Stephen J.; dePamphillis, Claude

    1994-01-01

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

  2. Independent Structural Domains in Paramyxovirus Polymerase Protein*

    PubMed Central

    Dochow, Melanie; Krumm, Stefanie A.; Crowe, James E.; Moore, Martin L.; Plemper, Richard K.

    2012-01-01

    All enzymatic activities required for genomic replication and transcription of nonsegmented negative strand RNA viruses (or Mononegavirales) are believed to be concentrated in the viral polymerase (L) protein. However, our insight into the organization of these different enzymatic activities into a bioactive tertiary structure remains rudimentary. Fragments of Mononegavirales polymerases analyzed to date cannot restore bioactivity through trans-complementation, unlike the related L proteins of segmented NSVs. We investigated the domain organization of phylogenetically diverse Paramyxovirus L proteins derived from measles virus (MeV), Nipah virus (NiV), and respiratory syncytial virus (RSV). Through a comprehensive in silico and experimental analysis of domain intersections, we defined MeV L position 615 as an interdomain candidate in addition to the previously reported residue 1708. Only position 1708 of MeV and the homologous positions in NiV and RSV L also tolerated the insertion of epitope tags. Splitting of MeV L at residue 1708 created fragments that were unable to physically interact and trans-complement, but strikingly, these activities were reconstituted by the addition of dimerization tags to the fragments. Equivalently split fragments of NiV, RSV, and MeV L oligomerized with comparable efficiency in all homo- and heterotypic combinations, but only the homotypic pairs were able to trans-complement. These results demonstrate that synthesis as a single polypeptide is not required for the Mononegavirales polymerases to adopt a proper tertiary conformation. Paramyxovirus polymerases are composed of at least two truly independent folding domains that lack a traditional interface but require molecular compatibility for bioactivity. The functional probing of the L domain architecture through trans-complementation is anticipated to be applicable to all Mononegavirales polymerases. PMID:22215662

  3. The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re-uptake site.

    PubMed Central

    Nicholson, C. D.; Jackman, S. A.; Wilke, R.

    1989-01-01

    1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound. PMID:2474352

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

    SciTech Connect

    Andersson, Olov

    2014-02-01

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

  5. Adenosine regulation of the immune response initiated by ischemia reperfusion injury.

    PubMed

    Boros, D; Thompson, J; Larson, D F

    2016-03-01

    It is clinically established that adenosine has negative chronotropic, antiarrhythmic effects and reduces arterial blood pressure. Adenosine addition to cardioplegic solutions used in cardiac operations is clinically well tolerated and has been shown to improve myocardial protection in several studies. However, the mechanism of action remains unclear. Therefore, it is important to define the effect of adenosine on the inflammatory cascade as immune cell activation occurs early during ischemia reperfusion injury. Adenosine appears to mediate the initial steps of the inflammatory cascade via its four G-coupled protein receptors: A1, A2A, A2B, and A3, expressed on neutrophils, lymphocytes and macrophages. The adenosine receptor isotype dictates the immune response. More specifically, the A1 and A3 receptors stimulate a pro-inflammatory immune response whereas the A2A and A2B are immunosuppressive. As the adenosine receptors are important for cardiac pre-conditioning and post-conditioning, adenosine may regulate the inflammatory responses initiated during ischemia-mediated immune injury related to myocardial protection.

  6. The Role of Adenosine in Pulmonary Vein Isolation: A Critical Review

    PubMed Central

    Dallaglio, Paolo D.; Betts, Timothy R.; Ginks, Matthew; Bashir, Yaver; Anguera, Ignasi; Rajappan, Kim

    2016-01-01

    The cornerstone of atrial fibrillation (AF) ablation is pulmonary vein isolation (PVI), which can be achieved in more than 95% of patients at the end of the procedure. However, AF recurrence rates remain high and are related to recovery of PV conduction. Adenosine testing is used to unmask dormant pulmonary vein conduction (DC). The aim of this study is to review the available literature addressing the role of adenosine testing and determine the impact of ablation at sites of PV reconnection on freedom from AF. Adenosine infusion, by restoring the excitability threshold, unmasks reversible injury that could lead to recovery of PV conduction. The studies included in this review suggest that adenosine is useful to unmask nontransmural lesions at risk of reconnection and that further ablation at sites of DC is associated with improvement in freedom from AF. Nevertheless it has been demonstrated that adenosine is not able to predict all veins at risk of later reconnection, which means that veins without DC are not necessarily at low risk. The role of the waiting period in the setting of adenosine testing has also been analyzed, suggesting that in the acute phase adenosine use should be accompanied by enough waiting time. PMID:26981309

  7. Crystal structure of a model branchpoint-U2 snRNA duplex containing bulged adenosines.

    PubMed Central

    Berglund, J A; Rosbash, M; Schultz, S C

    2001-01-01

    Bulged nucleotides play a variety of important roles in RNA structure and function, frequently forming tertiary interactions and sometimes even participating in RNA catalysis. In pre-mRNA splicing, the U2 snRNA base pairs with the intron branchpoint sequence (BPS) to form a short RNA duplex that contains a bulged adenosine that ultimately serves as the nucleophile that attacks the 5' splice site. We have determined a 2.18-A resolution crystal structure of a self-complementary RNA designed to mimic the highly conserved yeast (Saccharomyces cerevisiae) branchpoint sequence (5'-UACUAACGUAGUA with the BPS italicized and the branchsite adenosine underlined) base paired with its complementary sequence from U2 snRNA. The structure shows a nearly ideal A-form helix from which two unpaired adenosines flip out. Although the adenosine adjacent to the branchsite adenosine is the one bulged out in the structure described here, either of these adenosines can serve as the nucleophile in mammalian but not in yeast pre-mRNA splicing. In addition, the packing of the bulged RNA helices within the crystal reveals a novel RNA tertiary interaction in which three RNA helices interact through bulged adenosines in the absence of any divalent metal ions. PMID:11350032

  8. Reaction of epichlorohydrin with adenosine, 2'-deoxyadenosine and calf thymus DNA: identification of adducts.

    PubMed

    Sund, Pernilla; Kronberg, Leif

    2006-06-01

    Epichlorohydrin (a probable human carcinogen) was allowed to react with adenosine and the adducts were characterized by NMR and UV spectroscopy, and mass spectrometry. The adduct initially formed was 1-(3-chloro-2-hydroxypropyl)-adenosine, which subsequently ring closures to 1,N(6)-(2-hydroxypropyl)-adenosine at neutral and basic conditions. At acid conditions, the N-1 adduct undergoes a slow deamination to yield 1-(3-chloro-2-hydroxypropyl)-inosine. Minor adducts identified were 7-(3-chloro-2-hydroxypropyl)-adenosine and 3-(3-chloro-2-hydroxypropyl)-adenosine which are easily deglycosylated, and an adduct where the epichlorohydrin residue was attached to the sugar moiety of adenosine. A diadduct, 1,N(6)-(2-hydroxypropyl)-N(6)-(3-chloro-2-hydroxypropyl)-adenosine was also identified. The reaction of epichlorohydrin with calf thymus DNA gave 1,N(6)-(2-hydroxypropyl)-deoxyadenosine and 3-(3-chloro-2-hydroxypropyl)-adenine (major adduct).

  9. GABAergic involvement in motor effects of an adenosine A(2A) receptor agonist in mice.

    PubMed

    Khisti, R T; Chopde, C T; Abraham, E

    2000-04-03

    Adenosine A(2A) agonists are known to induce catalepsy and inhibit dopamine mediated motor hyperactivity. An antagonistic interaction between adenosine A(2A) and dopamine D(2) receptors is known to regulate GABA-mediated neurotransmission in striatopallidal neurons. Stimulation of adenosine A(2A) and dopamine D(2) receptors has been shown to increase and inhibit GABA release respectively in pallidal GABAergic neurons. However, the role of GABAergic neurotransmission in the motor effects of adenosine A(2A) receptors is not yet known. Therefore in the present study the effect of GABAergic agents on adenosine A(2A) receptor agonist (NECA- or CGS 21680) induced catalepsy and inhibition of amphetamine elicited motor hyperactivity was examined. Pretreatment with GABA, the GABA(A) agonist muscimol or the GABA(B) agonist baclofen potentiated whereas the GABA(A) antagonist bicuculline attenuated NECA- or CGS 21680-induced catalepsy. However, the GABA(B) antagonists phaclophen and delta-aminovaleric acid had no effect. Administration of NECA or CGS 21680 not only reduced spontaneous locomotor activity but also antagonized amphetamine elicited motor hyperactivity. These effects of NECA and CGS 21680 were potentiated by GABA or muscimol and antagonized by bicuculline. These findings provide behavioral evidence for the role of GABA in the motor effects of adenosine A(2A) receptor agonists. Activation of adenosine A(2A) receptors increases GABA release which could reduce dopaminergic tone and induce catalepsy or inhibit amphetamine mediated motor hyperactivity.

  10. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells.

    PubMed

    Wilson, Jeffrey M; Ross, William G; Agbai, Oma N; Frazier, Renea; Figler, Robert A; Rieger, Jayson; Linden, Joel; Ernst, Peter B

    2009-04-15

    The endogenous purine nucleoside adenosine is an important antiinflammatory mediator that contributes to the control of CD4(+) T cell responses. While adenosine clearly has direct effects on CD4(+) T cells, it remains to be determined whether actions on APC such as dendritic cells (DC) are also important. In this report we characterize DC maturation and function in BMDC stimulated with LPS in the presence or absence of the nonselective adenosine receptor agonist NECA (5'-N-ethylcarboxamidoadenosine). We found that NECA inhibited TNF-alpha and IL-12 in a concentration-dependent manner, whereas IL-10 production was increased. NECA-treated BMDC also expressed reduced levels of MHC class II and CD86 and were less effective at stimulating CD4(+) T cell proliferation and IL-2 production compared with BMDC exposed to vehicle control. Based on real-time RT-PCR, the A(2A) adenosine receptor (A(2A)AR) and A(2B)AR were the predominant adenosine receptors expressed in BMDC. Using adenosine receptor subtype selective antagonists and BMDC derived from A(2A)AR(-/-) and A(2B)AR(-/-)mice, it was shown that NECA modulates TNF-alpha, IL-12, IL-10, and CD86 responses predominantly via A(2B)AR. These data indicate that engagement of A(2B)AR modifies murine BMDC maturation and suggest that adenosine regulates CD4(+) T cell responses by selecting for DC with impaired immunogencity.

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

    PubMed

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

    2014-09-01

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

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

    PubMed Central

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

    2009-01-01

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

  13. Adenosine-5'-phosphosulfate kinase is essential for Arabidopsis viability.

    PubMed

    Mugford, Sarah G; Matthewman, Colette A; Hill, Lionel; Kopriva, Stanislav

    2010-01-04

    In Arabidopsis thaliana, adenosine-5'-phosphosulfate kinase (APK) provides activated sulfate for sulfation of secondary metabolites, including the glucosinolates. We have successfully isolated three of the four possible triple homozygous mutant combinations of this family. The APK1 isoform alone was sufficient to maintain WT levels of growth and development. Analysis of apk1 apk2 apk3 and apk1 apk3 apk4 mutants suggests that APK3 and APK4 are functionally redundant, despite being located in cytosol and plastids, respectively. We were, however, unable to isolate apk1 apk3 apk4 mutants, most probably because the apk1 apk3 apk4 triple mutant combination is pollen lethal. Therefore, we conclude that APS kinase is essential for plant reproduction and viability.

  14. Late-onset adenosine deaminase deficiency presenting with Heck's disease.

    PubMed

    Artac, Hasibe; Göktürk, Bahar; Bozdemir, Sefika Elmas; Toy, Hatice; van der Burg, Mirjam; Santisteban, Ines; Hershfield, Michael; Reisli, Ismail

    2010-08-01

    Focal epithelial hyperplasia, also known as Heck's disease, is a rare but distinctive entity of viral etiology with characteristic clinical and histopathological features. It is a benign, asymptomatic disease of the oral mucosa caused by human papilloma viruses (HPV). Previous studies postulated an association between these lesions and immunodeficiency. Genetic deficiency of adenosine deaminase (ADA) results in varying degrees of immunodeficiency, including neonatal onset severe combined immunodeficiency (ADA-SCID), and milder, later onset immunodeficiency. We report a 12-year-old girl with the late onset-ADA deficiency presenting with Heck's disease. Our case report should draw attention to the possibility of immunodeficiency in patients with HPV-induced focal epithelial hyperplasia.

  15. Extraction and analysis of adenosine triphosphate from aquatic environments

    USGS Publications Warehouse

    Stephens, Doyle W.; Shultz, David J.

    1981-01-01

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

  16. Adenosine Monophosphate-Based Detection of Bacterial Spores

    NASA Technical Reports Server (NTRS)

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

    2009-01-01

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

  17. Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis.

    PubMed

    Young, Li-Sen; Harrison, Benjamin R; Narayana Murthy, U M; Moffatt, Barbara A; Gilroy, Simon; Masson, Patrick H

    2006-10-01

    Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-L-methionine pathway in the control of root gravitropism and cap morphogenesis.

  18. Cardiovascular selectivity of adenosine receptor agonists in anaesthetized dogs.

    PubMed Central

    Gerencer, R. Z.; Finegan, B. A.; Clanachan, A. S.

    1992-01-01

    1. In order to determine the relevance of adenosine (Ado) receptor classification obtained from in vitro methods to the cardiovascular actions of Ado agonists in vivo, the cardiovascular effects of adenosine 5'-monophosphate (AMP), N6-cyclohexyladenosine (CHA, 400 fold A1-selective), 5'-N-ethyl-carboxamidoadenosine (NECA, A1 approximately A2) and 2-phenylaminoadenosine (PAA, 5 fold A2-selective) were compared in open-chest, fentanyl-pentobarbitone anaesthetized dogs. 2. Graded doses of CHA (10 to 1000 micrograms kg-1), NECA (0.5 to 100 micrograms kg-1) or PAA (0.1 to 20 micrograms kg-1) were administered intravenously and changes in haemodynamics and myocardial contractility were assessed 10 min following each dose. The effects of graded infusions of AMP (200 to 1000 micrograms kg-1 min-1) were also evaluated. 3. AMP and each of the Ado analogues (NECA > PAA > CHA) increased the systemic vascular conductance index (SVCI) in a dose-dependent manner and reduced mean arterial pressure (MAP). At doses causing similar increases in SVCI, these agonists caused (i) similar reflex increases in heart rate (HR) and cardiac index (CI) and decreases in AV conduction interval (AVi) and (ii) similar increases in coronary vascular conductance (CVC). 4. After cardiac autonomic blockade with atropine (0.2 mg kg-1) and propranolol (1 mg kg-1), AMP, CHA and PAA still increased SVCI and CVC and decreased MAP. CHA and PAA had no marked effects on HR, CI or AVi. As in the absence of cardiac autonomic blockade, equieffective vasodilator doses of CHA and PAA had identical effects on CVC, CI and AVi.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1467827

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

    SciTech Connect

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

    2009-01-01

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

  20. Increased cerebral extracellular adenosine and decreased PGE2 during ethanol-induced inhibition of FBM.

    PubMed

    Watson, C S; White, S E; Homan, J H; Kimura, K A; Brien, J F; Fraher, L; Challis, J R; Bocking, A D

    1999-04-01

    Adenosine and PGE2 are neuromodulators, both of which inhibit fetal breathing movements (FBM). Although circulating PGE2 has been implicated as a mediator of ethanol-induced inhibition of FBM in the late-gestation ovine fetus, a role for adenosine has not been examined. The objective of this study was to determine the effect of maternal ethanol infusion on ovine fetal cerebral extracellular fluid adenosine and PGE2 concentrations by using in utero microdialysis and to relate any changes to ethanol-induced inhibition of FBM. Dialysate samples were obtained from the fetal parietal cortex over 70 h after surgery to determine steady-state extracellular fluid adenosine and PGE2 concentrations. On each of postoperative days 3 and 4, after a 2-h baseline period, ewes received a 1-h infusion of ethanol (1 g/kg maternal body wt) or an equivalent volume of saline, and the fetus was monitored for a further 11 h with 30-min dialysate samples collected throughout. Immediately after surgery, dialysate PGE2 and adenosine concentrations were 3.7 +/- 0.7 and 296 +/- 127 nM, respectively. PGE2 did not change over the 70 h, whereas adenosine decreased to 59 +/- 14 nM (P < 0.05) at 4 h and then remained unchanged. Ethanol decreased dialysate PGE2 concentration for 2 h (3.3 +/- 0.3 to 1.9 +/- 0.4 nM; P < 0.05) and increased adenosine concentration for 6 h (87 +/- 13 to a maximum of 252 +/- 59 nM, P < 0.05). Ethanol decreased FBM incidence from 47 +/- 7 to 16 +/- 5% (P < 0.01) for 8 h. Saline infusion did not change dialysate adenosine or PGE2 concentrations or FBM incidence. These data are consistent with the hypothesis that fetal cerebral adenosine, and not PGE2, is the primary mediator of ethanol-induced inhibition of FBM at 123 days of gestation in sheep.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    PubMed

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

    2016-01-18

    A panel of six luminescent iridium(III) complexes were synthesized and evaluated for their ability to act as G-quadruplex-selective probes. The novel iridium(III) complex 1 was found to be highly selective for G-quadruplex DNA, and was employed for the construction of a label-free G-quadruplex-based adenosine detection assay in aqueous solution. Two different detection strategies were investigated for adenosine detection, and the results showed that initial addition of adenosine to the adenosine aptamer gave superior results. The assay exhibited a linear response for adenosine in the concentration range of 5 to 120 μM (R(2) = 0.992), and the limit of detection for adenosine was 5 μM. Moreover, this assay was highly selective for adenosine over other nucleosides, and exhibited potential use for biological sample analysis.

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

    PubMed Central

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

    2016-01-01

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

  4. Deregulation of Adenosine Receptors in Psoriatic Epidermis: An Option for Therapeutic Treatment.

    PubMed

    Merighi, Stefania; Borea, Pier Andrea; Varani, Katia; Gessi, Stefania

    2017-01-01

    Purinergic signaling is involved in psoriasis, a chronic skin disease characterized by increased epidermis cell growth. In particular, Andrés et al. focus on the keratinocyte biology modulated by adenosine receptors providing evidence that the A2B subtype plays a prominent role in the reduction of keratinocyte proliferation whereas A2A and A2B agonists have antiinflammatory effects independent of adenosine receptors. The authors report that psoriatic epidermis presents a deregulated adenosine receptor expression profile with reduced A2B and increased A2A.

  5. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.

    PubMed

    Wigand, R

    1979-01-01

    The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.

  6. Benzodiazepine inhibition of adenosine uptake is not prevented by benzodiazepine antagonists.

    PubMed

    Morgan, P F; Lloyd, H G; Stone, T W

    1983-01-28

    Uptake of [3H]adenosine into rat cerebral cortex synaptosomes was studied. Hexobendine (10(-5) M) and the benzodiazepine agonists diazepam (10(-5) M) and flurazepam (10(-4) M) significantly inhibited this uptake, but only if the compounds were pre-incubated for 10 min in the case of the benzodiazepines. The benzodiazepine antagonists Ro15-1788 (10(-5) M) and CGS 8216 (10(-5) M) failed to reverse the action of benzodiazepine agonists or hexobendine on [3H]adenosine uptake. The results add weight to the view that inhibition of adenosine uptake processes by benzodiazepines do not contribute to their behavioural effects.

  7. Role of adenosine A2b receptor overexpression in tumor progression.

    PubMed

    Sepúlveda, Cesar; Palomo, Iván; Fuentes, Eduardo

    2016-12-01

    The adenosine A2b receptor is a G-protein coupled receptor. Its activation occurs with high extracellular adenosine concentration, for example in inflammation or hypoxia. These conditions are generated in the tumor environment. Studies show that A2b receptor is overexpressed in various tumor lines and biopsies from patients with different cancers. This suggests that A2b receptor can be used by tumor cells to promote progression. Thus A2b participates in different events, such as angiogenesis and metastasis, besides exerting immunomodulatory effects that protect tumor cells. Therefore, adenosine A2b receptor appears as an interesting therapeutic target for cancer treatment.

  8. Dopamine/adenosine interactions involved in effort-related aspects of food motivation.

    PubMed

    Salamone, John D; Correa, Merce

    2009-12-01

    Nucleus accumbens dopamine (DA) is involved in effort-related aspects of food motivation. Accumbens DA depletions reduce the tendency of rats to work for food, and alter effort-related choice, but leave other aspects of food motivation and appetite intact. DA and adenosine receptors interact to regulate effort-related processes. Adenosine A(2A) antagonists can reverse the effects of DA D(2) antagonists on effort-related choice, and intra-accumbens injections of a adenosine A(2A) agonist produce effects that are similar to those produced by accumbens DA depletion or antagonism. These studies have implications for understanding the neurochemical interactions that underlie activational aspects of motivation.

  9. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    DTIC Science & Technology

    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...via A2A receptors (A2AAR) is involved in retinal protection from diabetes -induced inflammation. Here we demonstrate that AKI-enhanced adenosine

  10. [Interaction of adenosin-3',5'-cyclosulfate with adenosine-3'5'-cyclophosphate dependent protein kinase and phosphodiesterase].

    PubMed

    Severin, E S; Tkachuk, V A; Guliaev, N N

    1976-02-01

    Interaction of adenosine-3',5'-cyclosulphate (cAMS) cAMP analogue, having sulphur atom instead of phosphorus in a six-term cyclic system with pig brain proteinkinase and rabbit skeletal muscle phosphodiesterase is studied. The affinity of proteinkinase to cAMS was found to be in 25000 times lower than the affinity of cAMP, the affinity of cAMS to the active site of phosphodiesterase being high enough. It is suggested that in the regulatory subunit of proteinkinase positive kationic group participates in nucleotide binding by interacting with negative oxygen atom of six-term cyclophosphate system. There is no such a group in the active site of phospodiesterase, because the absence of negative charge in case of cAMS only slightly affects the constant of cAMS binding by phosphodiesterase.

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

    PubMed

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

    2013-09-17

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

  12. Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

    SciTech Connect

    Delamere, N.A.; Socci, R.R.; King, K.L. )

    1990-10-01

    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.

  13. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

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

  14. Enzymatic initiation of DNA synthesis by yeast DNA polymerases.

    PubMed Central

    Plevani, P; Chang, L M

    1977-01-01

    Partially purified yeast RNA polymerases (RNA nucleotidyltransferases) initiate DNA synthesis by yeast DNA polymerase (DNA nucleotidyltransferase) I and to a lesser extent yeast DNA polymerase II in the replication of single-stranded DNA. The enzymatic initiation of DNA synthesis on phage fd DNA template occurs with dNTPs alone and is further stimulated by the presence of rNTPs in DNA polymerase I reactions. The presence of rNTPs has no effect on the RNA polymerase initiation of the DNA polymerase II reaction. RNA polymerases I and III are more efficient in initiation of DNA synthesis than RNA polymerase II. Analyses of the products of fd DNA replication show noncovalent linkage between the newly synthesized DNA and the template DNA, and covalent linkage between the newly synthesized RNA and DNA. PMID:325562

  15. The architecture of RNA polymerase fidelity.

    PubMed

    Kaplan, Craig D

    2010-06-22

    The basis for transcriptional fidelity by RNA polymerase is not understood, but the 'trigger loop', a conserved structural element that is rearranged in the presence of correct substrate nucleotides, is thought to be critical. A study just published in BMC Biology sheds new light on the ways in which the trigger loop may promote selection of correct nucleotide triphosphate substrates. See research article http://www.biomedcentral.com/1741-7007/8/54.

  16. DNA-polymerase inhibitors. Rifamycin derivatives.

    PubMed Central

    Frolova, L Y; Meldrays, Y A; Kochkina, L L; Giller, S A; Eremeyev, A V; Grayevskaya, N A; Kisselev, L L

    1977-01-01

    Ten new derivatives of the antibiotic rifamycin with variable side chains at position 3 were synthesized. The inhibitory activity of these derivatives against DNA-polymerases isolated from avian myeloblastosis virus, E. coli and calf thymus were studied at various conditions. 3-(2,4,6-trinitrophenylhydrazone-(methyl) rifamycin SV is a strong inhibitor for all the polymerases tested and belongs to the C class inhibitors of reverse transcriptase. 3-(monoallylhydrazone-(methyl) rifamycin SV possesses a selective action on polymerases: at 0.1 mg/ml concentration it almost completely inhibits the reverse transcriptase and less than half of the bacterial and eukaryotic enzymes. A drug is found which strongly inhibits the DNA-polymerases from E. coli and calf thymus and weakly the viral enzyme. The inhibitory effect on reverse transcriptase is independent of the choice of template-primer; it could be overcome by the addition of excess enzyme but not of excess template-primer; the inhibition could be completely reversed by dilution of the drug-enzyme mixture. From Lineweaver-Burk analysis, the inhibition is noncompetitive with respect to the template-primer and, thus the drugs bind to the site different from the active site for the template-primer. From protective action of the template-primer and other data it might be suggested that the rifamycin derivatives act at an early step(s) in DNA synthesis catalyzed by reverse transcriptase. The obtained data are in agreement with the results for other derivatives of rifamycin SV described in literature. PMID:68462

  17. DNA polymerase mu, a candidate hypermutase?

    PubMed Central

    Ruiz, J F; Domínguez, O; Laín de Lera, T; Garcia-Díaz, M; Bernad, A; Blanco, L

    2001-01-01

    A novel DNA polymerase (Pol mu) has been recently identified in human cells. The amino-acid sequence of Pol mu is 42% identical to that of terminal deoxynucleotidyl transferase (TdT), a DNA-independent DNA polymerase that contributes to antigen-receptor diversity. In this paper we review the evidence supporting the role of Pol mu in somatic hypermutation of immunoglobulin genes, a T-dependent process that selectively occurs at germinal centres: (i) preferential expression in secondary lymphoid organs; (ii) expression associated to developing germinal centres; and (iii) very low base discrimination during DNA-dependent DNA polymerization by Pol mu, a mutator phenotype enormously accentuated by the presence of activating Mn2+ ions. Moreover, its similarity to TdT, together with extrapolation to the crystal structure of DNA polymerase beta complexed (Pol beta) with DNA, allows us to discuss the structural basis for the unprecedented error proneness of Pol mu, and to predict that Pol mu is structurally well suited to participate also in DNA end-filling steps occurring both during V(D)J recombination and repair of DNA double-strand breaks that are processed by non-homologous end-joining. PMID:11205337

  18. Laboratory procedures manual for the firefly luciferase assay for adenosine triphosphate (ATP)

    NASA Technical Reports Server (NTRS)

    Chappelle, E. W.; Picciolo, G. L.; Curtis, C. A.; Knust, E. A.; Nibley, D. A.; Vance, R. B.

    1975-01-01

    A manual on the procedures and instruments developed for the adenosine triphosphate (ATP) luciferase assay is presented. Data cover, laboratory maintenance, maintenance of bacterial cultures, bacteria measurement, reagents, luciferase procedures, and determination of microbal susceptibility to antibiotics.

  19. Evidence that the positive inotropic effects of the alkylxanthines are not due to adenosine receptor blockade.

    PubMed Central

    Collis, M. G.; Keddie, J. R.; Torr, S. R.

    1984-01-01

    We investigated the possibility that the positive inotropic effects of the alkylxanthines are due to adenosine receptor blockade. The potency of 8-phenyltheophylline, theophylline and enprofylline as adenosine antagonists was assessed in vitro, using the guinea-pig isolated atrium, and in vivo, using the anaesthetized dog. The order of potency of the alkylxanthines as antagonists of the negative inotropic response to 2-chloroadenosine in vitro, and of the hypotensive response to adenosine in vivo was 8-phenyltheophylline greater than theophylline greater than enprofylline. The order of potency of the alkylxanthines as positive inotropic and chronotropic agents in the anaesthetized dog was enprofylline greater than theophylline greater than 8-phenyltheophylline. The results of this study indicate that the inotropic effects of the alkylxanthines in the anaesthetized dog are not due to adenosine receptor blockade. PMID:6322898

  20. Extracellular adenosine triphosphate affects the response of human macrophages infected with Mycobacterium tuberculosis.

    PubMed

    Dubois-Colas, Nicolas; Petit-Jentreau, Laetitia; Barreiro, Luis B; Durand, Sylvère; Soubigou, Guillaume; Lecointe, Cécile; Klibi, Jihène; Rezaï, Keyvan; Lokiec, François; Coppée, Jean-Yves; Gicquel, Brigitte; Tailleux, Ludovic

    2014-09-01

    Granulomas are the hallmark of Mycobacterium tuberculosis infection. As the host fails to control the bacteria, the center of the granuloma exhibits necrosis resulting from the dying of infected macrophages. The release of the intracellular pool of nucleotides into the surrounding medium may modulate the response of newly infected macrophages, although this has never been investigated. Here, we show that extracellular adenosine triphosphate (ATP) indirectly modulates the expression of 272 genes in human macrophages infected with M. tuberculosis and that it induces their alternative activation. ATP is rapidly hydrolyzed by the ecto-ATPase CD39 into adenosine monophosphate (AMP), and it is AMP that regulates the macrophage response through the adenosine A2A receptor. Our findings reveal a previously unrecognized role for the purinergic pathway in the host response to M. tuberculosis. Dampening inflammation through signaling via the adenosine A2A receptor may limit tissue damage but may also favor bacterial immune escape.

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

    PubMed Central

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

    2012-01-01

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

  2. RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits

    PubMed Central

    Carey, Lucas B

    2015-01-01

    Errors during transcription may play an important role in determining cellular phenotypes: the RNA polymerase error rate is >4 orders of magnitude higher than that of DNA polymerase and errors are amplified >1000-fold due to translation. However, current methods to measure RNA polymerase fidelity are low-throughout, technically challenging, and organism specific. Here I show that changes in RNA polymerase fidelity can be measured using standard RNA sequencing protocols. I find that RNA polymerase is error-prone, and these errors can result in splicing defects. Furthermore, I find that differential expression of RNA polymerase subunits causes changes in RNA polymerase fidelity, and that coding sequences may have evolved to minimize the effect of these errors. These results suggest that errors caused by RNA polymerase may be a major source of stochastic variability at the level of single cells. DOI: http://dx.doi.org/10.7554/eLife.09945.001 PMID:26652005

  3. Adenosine-activated potassium current in smooth muscle cells isolated from the pig coronary artery.

    PubMed Central

    Dart, C; Standen, N B

    1993-01-01

    1. The perforated patch technique with nystatin or amphotericin was used to record whole cell currents activated by adenosine in smooth muscle cells isolated enzymatically from pig coronary arteries. 2. Adenosine (5-40 microM) activated an outward current at a holding potential of 0 mV in 5 mM [K+]o and an inward current at -60 mV in 143 mM [K+]o. The dependence of the reversal potential for the adenosine-activated current on [K+]o suggests that it flows through K+ channels, while its current-voltage relation is consistent with the channels showing little voltage dependence. 3. The adenosine-activated current was inhibited by the sulphonylurea glibenclamide (5 microM) and by phencyclidine (5 microM). It was unaffected by charybdotoxin (50 nM) or apamin (100 nM), blockers of large and small conductance Ca(2+)-activated K+ channels respectively. 4. At -60 mV in 143 mM K+ solution, openings of single channels passing a current of just over -2 pA could sometimes be detected in the absence of adenosine. Openings became more frequent after the application of adenosine, with several levels then being detected. Openings of channels with a larger conductance were sometimes also seen in the presence of adenosine. Fluctuation analysis gave somewhat lower estimates of unitary current than did direct measurements. 5. The effect of adenosine could be mimicked by the A1 receptor agonist CCPA (2-chloro-N6-cyclopentyladenosine), while the A2 agonist CGS 21680 (2-p-(2-carboxethyl)phenethylamino-5'-N-ethylcarboxamido adenosine hydrochloride) was without effect. The response to adenosine was inhibited by the A1 antagonist DPCPX (8-cyclopentyl-1,3-dipropylxanthine), but was unaffected by the A2 antagonist CGS 15943A (5-amino-9-chloro-2-(2-furanyl)-1,2,4- triazolo[1,5-C]quinazoline monomethanesulphonate). 6. Our results suggest that adenosine acts at an A1 receptor to activate K+ channels. We consider it most likely that these are ATP-dependent K+ channels. We discuss the mechanism by

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

    PubMed Central

    2017-01-01

    Purpose Perioperative infusion of adenosine has been suggested to reduce the requirement for inhalation anesthetics, without causing serious adverse effects in humans. We conducted a meta-analysis of randomized controlled trials evaluating the effect of adenosine on postoperative analgesia. Methods We retrieved articles in computerized searches of Scopus, Web of Science, PubMed, EMBASE, and Cochrane Library databases, up to July 2016. We used adenosine, postoperative analgesia, and postoperative pain(s) as key words, with humans, RCT, and CCT as filters. Data of eligible studies were extracted, which included pain scores, cumulative opioid consumption, adverse reactions, and vital signs. Overall incidence rates, relative risk (RR), and 95% confidence intervals (CI) were calculated employing fixed-effects or random-effects models, depending on the heterogeneity of the included trials. Results In total, 757 patients from 9 studies were included. The overall effect of adenosine on postoperative VAS/VRS scores and postoperative opioid consumption was not significantly different from that of controls (P >0.1). The occurrence of PONV and pruritus was not statistically significantly different between an adenosine and nonremifentanil subgroup (P >0.1), but the rate of PONV occurrence was greater in the remifentanil subgroup (P <0.01). Time to first postoperative analgesic requirement in the adenosine group was not significantly difference from that of the saline group (SMD = 0.07, 95%CI: −0.28 to 0.41, P = 0.71); but this occurred significantly later than with remifentanil (SMD = 1.10, 95%CI: 2.48 to 4.06, P < 0.01). Time to hospital discharge was not significantly different between the control and adenosine groups (P = 0.78). The perioperative systolic blood pressure was significantly lower in the adenosine than in the control group in the mannitol subgroup (P < 0.01). The incidence of bradycardia, transient first- degree atrioventricular block, and tachycardia was not

  5. Adenosine induces a cholinergic tracheal reflex contraction in guinea pigs in vivo via an adenosine A1 receptor-dependent mechanism.

    PubMed

    Reynolds, Sandra M; Docherty, Reginald; Robbins, Jon; Spina, Domenico; Page, Clive P

    2008-07-01

    Adenosine induces dyspnea, cough, and airways obstruction in asthma, a phenomenon that also occurs in various sensitized animal models in which a neuronal involvement has been implicated. Although adenosine has been suggested to activate cholinergic nerves, the precise mechanism has not been established. In the present study, the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) induced a cholinergic reflex, causing tracheal smooth muscle contraction that was significantly inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 100 microg/kg) (P < 0.05) in anesthetized animals. Furthermore, the adenosine A(2) agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) induced a small reflex, whereas the A(3) selective agonist N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine (IB-MECA) was without effect. The tracheal reflex induced by CPA was also inhibited by recurrent nerve ligation or muscarinic receptor blockade (P < 0.001), indicating that a cholinergic neuronal mechanism of action accounted for this response. The cholinergic reflex in response to aerosolized CPA was significantly greater in passively sensitized compared with naive guinea pigs (P < 0.01). Chronic capsaicin treatment, which inhibited sensory nerve function, failed to inhibit CPA-induced reflex tracheal contractions in passively sensitized guinea pigs, although the local anesthetic lidocaine inhibited CPA-induced tracheal contractions. The effects of CPA on the reflex response was not dependent on the release of histamine from tissue mast cells or endogenous prostaglandins as shown by the lack of effect of the histamine H(1) receptor antagonist pyrilamine (1 mg/kg) or the cyclooxygenase inhibitor meclofenamic acid (3 mg/kg), respectively. In conclusion, activation of pulmonary adenosine A(1) receptors can stimulate cholinergic reflexes, and these reflexes are increased in allergic guinea pigs.

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

    PubMed Central

    Newby, A C

    1980-01-01

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

  7. Polarized fibronectin secretion induced by adenosine regulates bacterial–epithelial interaction in human intestinal epithelial cells

    PubMed Central

    2004-01-01

    Fibronectin (FN) is a multifunctional protein that plays important roles in many biological processes including cell adhesion and migration, wound healing and inflammation. Cellular FNs are produced by a wide variety of cell types including epithelial cells, which secrete them and often organize them into extensive extracellular matrices at their basal surface. However, regulation of FN synthesis and the polarity of FN secretion by intestinal epithelial cells have not been investigated. In the present study we investigated the role of adenosine, whose levels are up-regulated during inflammation, in modulating FN synthesis, the polarity of FN secretion and the downstream effects of the secreted FN. Polarized monolayers of T84 cells were used as an intestinal epithelial model. Adenosine added to either the apical or basolateral aspect of the cells led to a time- and dose-dependent accumulation of FN in the culture supernatants, polarized to the apical compartment and reached maximal levels 24 h after apical or basolateral addition of adenosine. Confocal microscopy confirmed that FN localized to the apical domain of model intestinal epithelial cells stimulated with apical or basolateral adenosine. The induction of FN was significantly down-regulated in response to the adenosine receptor antagonist alloxazine and was inhibited by cycloheximide. Moreover, adenosine increased FN promoter activity (3.5-fold compared with unstimulated controls) indicating that FN induction is, in part, transcriptionally regulated. Interestingly, we demonstrated that adenosine, as well as apical FN, significantly enhanced the adherence and invasion of Salmonella typhimurium into cultured epithelial cells. In summary, we have shown for the first time that FN, a classic extracellular matrix protein, is secreted into the apical compartment of epithelial cells in response to adenosine. FN may be a critical host factor that modulates adherence and invasion of bacteria, thus playing a key role

  8. Adenosine stimulates anabolic metabolism in developing castor bean (Ricinus communis L.) cotyledons.

    PubMed

    Flörchinger, Martin; Zimmermann, Marc; Traub, Michaela; Neuhaus, H Ekkehard; Möhlmann, Torsten

    2006-01-01

    In previous experiments it was shown that Castor-bean (Ricinus communis) endosperm releases carbohydrates, amino acids and nucleoside derivatives, which are subsequently imported into the developing cotyledons (Kombrink and Beevers in Plant Physiol 73:370-376, 1983). To investigate the importance of the most prominent nucleoside adenosine for the metabolism of growing Ricinus seedlings, we supplied adenosine to cotyledons of 5-days-old seedlings after removal of the endosperm. This treatment led to a 16% increase in freshweight of intact seedlings within 16 h, compared to controls. Using detached cotyledons, we followed uptake of radiolabelled adenosine and identified 40% of label in solubles (mostly ATP and ADP), 46% incorporation in RNA and 2.5% in DNA, indicating a highly active salvage pathway. About 7% of freshly imported adenosine entered the phloem, which indicates a major function of adenosine for cotyledon metabolism. Import and conversion of adenosine improved the energy content of cotyledons as revealed by a substantially increased ATP/ADP ratio. This effect was accompanied by slight increases in respiratory activity, decreased levels of hexose phosphates and increased levels of fructose-1,6-bisphosphate and triose phosphates. These alterations indicate a stimulation of glycolytic flux by activation of phosphofructokinase, and accordingly we determined a higher activity of this enzyme. Furthermore the rate of [(14)C]-sucrose driven starch biosynthesis in developing castor-bean is significantly increased by feeding of adenosine. In conclusion, our data indicate that adenosine imported from mobilizing endosperm into developing castor-bean cotyledons fulfils an important function as it promotes anabolic reactions in this rapidly developing tissue.

  9. Responsiveness of renal glomeruli to adenosine in streptozotocin-induced diabetic rats dependent on hyperglycaemia level.

    PubMed

    Szczepańska-Konkel, M; Jankowski, M; Stiepanow-Trzeciak, A; Rudzik, A; Pawełczyk, T; Angielski, S

    2003-03-01

    Glomerular filtration rate (GFR) in response to adenosine precursor, NAD, and glomeruli contractility in response to adenosine were evaluated in streptozotocin-induced diabetic rats with severe (blood glucose 27.8 +/- 1.2 mmol/L) and moderate hyperglycaemia (18.2 +/- 0.9 mmol/L) compared with nondiabetic (ND)-rats. In anaesthetised rats, basal GFR was greater in moderately diabetic rats compared with severely diabetic rats (p < 0.05) and ND-rats (p < 0.02). Intravenous infusion of 5 nmol x min(-1) x kg(-1) NAD reduced GFR and renal plasma flow (RPF) in diabetic rats but had no effect on these parameters in ND-rats. Moreover, NAD-induced reduction of GFR and RPF was greater in rats with severe diabetes (41% and 30%, respectively) than in with moderate diabetes (25% and 26%, respectively). Theophylline (0.2 micromol x min(-1) x kg(-1) ) abolished renal response to NAD. Isolated glomeruli contraction in response to adenosine, assessed by glomerular 3H-inulin space reduction, was lowered in moderately diabetic-group and enhanced in severely diabetic-group. compared with ND-group (p < 0.05). Adenosine A1-receptor antagonist DPCPX inhibited adenosine-induced glomeruli contraction. This differential response of diabetic renal glomeruli to adenosine suggests that impaired glomerular contractility in response to adenosine could be responsible for hyperfiltration in moderate diabets, whereas, the increased adenosine-dependent contractility of glomeruli in severe diabetes may increase the risk of acute renal failure in this condition.

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

    PubMed Central

    Costa, F; Biaggioni, I

    1994-01-01

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

  11. Opiate-induced Changes in Brain Adenosine Levels and Narcotic Drug Responses

    PubMed Central

    Wu, Manhong; Sahbaie, Peyman; Zheng, Ming; Lobato, Robert; Boison, Detlev; Clark, J. David; Peltz, Gary

    2012-01-01

    We have very little information about the metabolomic changes that mediate neurobehavioral responses, including addiction. It was possible that opioid-induced metabolomic changes in brain could mediate some of the pharmacodynamic effects of opioids. To investigate this, opiate-induced brain metabolomic responses were profiled using a semi-targeted method in C57BL/6 and 129Sv1 mice, which exhibit extreme differences in their tendency to become opiate dependent. Escalating morphine doses (10–40 mg/kg) administered over a 4-day period selectively induced a two-fold decrease (p<0.00005) in adenosine abundance in the brainstem of C57BL/6 mice, which exhibited symptoms of narcotic drug dependence; but did not decrease adenosine abundance in 129Sv1 mice, which do not exhibit symptoms of dependence. Based on this finding, the effect of adenosine on dependence was investigated in genetically engineered mice with alterations in adenosine tone in the brain and in pharmacologic experiments. Morphine withdrawal behaviors were significantly diminished (P<0.0004) in genetically engineered mice with reduced adenosine tone in the brainstem, and by treatment with an adenosine receptor1 (A1) agonist (2-chloro-N6-cyclopentyladenosine, 0.5 mg/kg) or an A2a receptor (A2a) antagonist (SCH 58261 1 mg/kg). These results indicate that adenosine homeostasis plays a crucial role in narcotic drug responses. Opiate-induced changes in brain adenosine levels may explain many important neurobehavioral features associated with opiate addiction and withdrawal. PMID:23098802

  12. Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices.

    PubMed

    Greene, R W; Haas, H L

    1985-09-01

    Intracellular recordings with a bridge amplifier of CA1 pyramidal neurones in vitro were employed to study the mechanisms of action of exogenously applied adenosine in the hippocampal slice preparation of the rat. Adenosine enhanced the calcium-dependent, long-duration after-hyperpolarization (a.h.p.) at least in part by a reduction in the rate of decay of the a.h.p. Both the reduced rate of decay and that of the control can be described with a single exponential. Antagonism of the calcium-dependent potassium current (and as a result, the a.h.p.) by bath application of CdCl2 or intracellular injection of EGTA (ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid) did not reduce the adenosine-evoked hyperpolarization or decrease in input resistance. Similarly, TEA (tetraethylammonium), which antagonizes both the voltage- and calcium-sensitive, delayed, outward rectification, had no effect on the adenosine-evoked changes in resting membrane properties. Adenosine did not affect the early, transient, outward rectification. During exposure to 4-aminopyridine (4-AP) in concentrations sufficient to antagonize this early rectification, the changes in resting membrane properties evoked by adenosine were unaffected. We conclude that the enhancement of the a.h.p. and accommodation by adenosine may be mediated by a change in the regulation of intracellular calcium. However, the mechanism responsible for the hyperpolarization and decrease in input resistance evoked by adenosine is both calcium and voltage insensitive. Thus, it appears distinct from that mediating the enhancement of the a.h.p. and accommodation.

  13. Adenosine actions on CA1 pyramidal neurones in rat hippocampal slices.

    PubMed Central

    Greene, R W; Haas, H L

    1985-01-01

    Intracellular recordings with a bridge amplifier of CA1 pyramidal neurones in vitro were employed to study the mechanisms of action of exogenously applied adenosine in the hippocampal slice preparation of the rat. Adenosine enhanced the calcium-dependent, long-duration after-hyperpolarization (a.h.p.) at least in part by a reduction in the rate of decay of the a.h.p. Both the reduced rate of decay and that of the control can be described with a single exponential. Antagonism of the calcium-dependent potassium current (and as a result, the a.h.p.) by bath application of CdCl2 or intracellular injection of EGTA (ethyleneglycolbis-(beta-aminoethyl ether)N,N'-tetraacetic acid) did not reduce the adenosine-evoked hyperpolarization or decrease in input resistance. Similarly, TEA (tetraethylammonium), which antagonizes both the voltage- and calcium-sensitive, delayed, outward rectification, had no effect on the adenosine-evoked changes in resting membrane properties. Adenosine did not affect the early, transient, outward rectification. During exposure to 4-aminopyridine (4-AP) in concentrations sufficient to antagonize this early rectification, the changes in resting membrane properties evoked by adenosine were unaffected. We conclude that the enhancement of the a.h.p. and accommodation by adenosine may be mediated by a change in the regulation of intracellular calcium. However, the mechanism responsible for the hyperpolarization and decrease in input resistance evoked by adenosine is both calcium and voltage insensitive. Thus, it appears distinct from that mediating the enhancement of the a.h.p. and accommodation. PMID:3932644

  14. Effect of chronic salt loading on adenosine metabolism and receptor expression in renal cortex and medulla in rats.

    PubMed

    Zou, A P; Wu, F; Li, P L; Cowley, A W

    1999-01-01

    Previous studies have shown that chronic salt loading increased renal interstitial adenosine concentrations and desensitized renal effects of adenosine, a phenomenon that could facilitate sodium excretion. However, the mechanisms responsible for the increased adenosine production and decreased adenosine response are poorly understood. This study examined the effects of the dietary high salt intake on adenosine metabolism and receptor expression in the renal cortex and medulla in Sprague Dawley rats. Fluorescent high-performance liquid chromatography analyses were performed to determine adenosine levels in snap-frozen kidney tissues. Comparing rats fed a normal (1% NaCl) versus high salt (4% NaCl) diet, renal adenosine concentrations in rats fed a high salt diet were significantly higher (cortex: 43+/-3 versus 85+/-4, P<0.05; medulla: 183+/-4 versus 302+/-8 nmol/g wet tissue, P<0.05). Increased adenosine concentrations were not associated with changes in the 5'-nucleotidase or adenosine deaminase activity, as determined by quantitative isoelectric focusing and gel electrophoresis. Western blot analyses showed that a high salt diet (4% NaCl for 3 weeks) downregulated A1 receptors (antinatriuretic type), did not alter A2A and A2B receptors (natriuretic type), and upregulated A3 receptors (function unknown) in both renal cortex and medulla. The data show that stimulation of adenosine production and downregulation of A1 receptors with salt loading may play an important role in adaptation in the kidney to promote sodium excretion.

  15. K+ depolarization evokes ATP, adenosine and glutamate release from glia in rat hippocampus: a microelectrode biosensor study

    PubMed Central

    Heinrich, A; Andó, RD; Túri, G; Rózsa, B; Sperlágh, B

    2012-01-01

    BACKGROUND AND PURPOSE This study was undertaken to characterize the ATP, adenosine and glutamate outflow evoked by depolarization with high K+ concentrations, in slices of rat hippocampus. EXPERIMENTAL APPROACH We utilized the microelectrode biosensor technique and extracellular electrophysiological recording for the real-time monitoring of the efflux of ATP, adenosine and glutamate. KEY RESULTS ATP, adenosine and glutamate sensors exhibited transient and reversible current during depolarization with 25 mM K+, with distinct kinetics. The ecto-ATPase inhibitor ARL67156 enhanced the extracellular level of ATP and inhibited the prolonged adenosine efflux, suggesting that generation of adenosine may derive from the extracellular breakdown of ATP. Stimulation-evoked ATP, adenosine and glutamate efflux was inhibited by tetrodotoxin, while exposure to Ca2+-free medium abolished ATP and adenosine efflux from hippocampal slices. Extracellular elevation of ATP and adenosine were decreased in the presence of NMDA receptor antagonists, D-AP-5 and ifenprodil, whereas non-NMDA receptor blockade by CNQX inhibited glutamate but not ATP and adenosine efflux. The gliotoxin fluoroacetate and P2X7 receptor antagonists inhibited the K+-evoked ATP, adenosine and glutamate efflux, while carbenoxolone in low concentration and probenecid decreased only the adenosine efflux. CONCLUSIONS AND IMPLICATIONS Our results demonstrated activity-dependent gliotransmitter release in the hippocampus in response to ongoing neuronal activity. ATP and glutamate were released by P2X7 receptor activation into extracellular space. Although the increased extracellular levels of adenosine did derive from released ATP, adenosine might also be released directly via pannexin hemichannels. LINKED ARTICLE This article is commented on by Sershen, pp. 1000–1002 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.02072.x PMID:22394324

  16. Expression of adenosine receptors in monocytes from patients with bronchial asthma

    PubMed Central

    Yuryeva, Ksenia; Saltykova, Irina; Ogorodova, Ludmila; Kirillova, Natalya; Kulikov, Evgeny; Korotkaya, Elena; Iakovleva, Yulia; Feoktistov, Igor; Sazonov, Alexey; Ryzhov, Sergey

    2015-01-01

    Adenosine is generated from adenosine triphosphate, which is released by stressed and damaged cells. Adenosine levels are significantly increased in patients with bronchial asthma (BA) and mediate mast cell degranulation and bronchoconstriction. Over the last decade, increasing evidence has shown that adenosine can modulate the innate immune response during monocytes differentiation towards mature myeloid cells. These adenosine-differentiated myeloid cells, characterized by co-expression of monocytes/macrophages and dendritic cell markers such as CD14 and CD209, produce high levels of pro-inflammatory cytokines, thus contributing to the pathogenesis of BA and chronic obstructive pulmonary disease. We found that expression of ADORA2A and ADORA2B are increased in monocytes obtained from patients with BA, and are associated with the generation of CD14posCD209pos pro-inflammatory cells. A positive correlation between expression of ADORA2B and IL-6 was identified in human monocytes and may explain the increased expression of IL-6 mRNA in asthmatics. Taken together, our results suggest that monocyte-specific expression of A2 adenosine receptors plays an important role in pro-inflammatory activation of human monocytes, thus contributing to the progression of asthma. PMID:26232643

  17. Unraveling the role of adenosine in remote ischemic preconditioning-induced cardioprotection.

    PubMed

    Randhawa, Puneet Kaur; Jaggi, Amteshwar Singh

    2016-06-15

    Remote ischemic preconditioning (RIPC) induced by alternate cycles of preconditioning ischemia and reperfusion protects the heart against sustained ischemia-reperfusion-induced injury. This technique has been translated to clinical levels in patients undergoing various surgical interventions including coronary artery bypass graft surgery, abdominal aortic aneurysm repair, percutaneous coronary intervention and heart valve surgery. Adenosine is a master regulator of energy metabolism and reduces myocardial ischemia-reperfusion-induced injury. Furthermore, adenosine is a critical trigger as well as a mediator in RIPC-induced cardioprotection and scientists have demonstrated the role of adenosine by showing an increase in its levels in the systemic circulation during RIPC delivery. Furthermore, the blockade of cardioprotective effects of RIPC in the presence of specific adenosine receptor blockers and transgenic animals with targeted ablation of A1 receptors has also demonstrated its critical role in RIPC. The studies have shown that adenosine may elicit cardioprotection via activation of neurogenic pathway. The present review describes the possible role and mechanism of adenosine in mediating RIPC-induced cardioprotection.

  18. Effect of coadministration of caffeine and either adenosine agonists or cyclic nucleotides on ketorolac analgesia.

    PubMed

    Aguirre-Bañuelos, P; Castañeda-Hernández, G; López-Muñoz, F J; Granados-Soto, V

    1999-07-21

    Caffeine potentiation of ketorolac-induced antinociception in the pain-induced functional impairment model in rats was assessed. Caffeine alone was ineffective, but increased the effect of ketorolac without affecting its pharmacokinetics. Intra-articular administration of adenosine and N6-cyclohexyladenosine (CHA, an adenosine A1 receptor agonist), but not 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS-21680, an adenosine A2A receptor agonist), significantly increased ketorolac antinociception. This effect was not local, as contralateral administration was also effective. Ipsilateral and contralateral administration of adenosine and CHA also increased antinociception by ketorolac-caffeine. Intra-articular 8-Bromo-adenosine cyclic 3',5'-hydrogen phosphate sodium or 8-Bromo-guanosine-3',5'-cyclophosphate sodium (cGMP) given ipsilaterally or contralaterally did not affect ketorolac-induced antinociception. Nevertheless, ipsilateral, but not contralateral, administration of 8-Br-cGMP significantly increased antinociception by ketorolac-caffeine, suggesting a local effect. The results suggest that caffeine potentiation of ketorolac antinociception is mediated, at least partially, by a local increase in cGMP and rule out the participation of adenosine receptor blockade.

  19. Kinetics of N6-(Δ2-Isopentenyl)Adenosine Degradation in Tobacco Cells

    PubMed Central

    Terrine, Claude; Laloue, Michel

    1980-01-01

    Uptake and degradation of the cytokinin, N6-(Δ2-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N6(Δ2-isopentenyl)[8-14C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin. Consequently, only rates of N6-(Δ2-isopentenyl)adenosine degradation measured during the first 3 hours of incubation with [8-14C]-N-6(Δ2-isopentenyl)adenosine are representative of the intrinsic in vivo cytokinin degradative activity of tobacco cells. Within these limits, it appears that cytokinin degradative activity is high in cytokinin-autonomous tobacco cells, as indicated by the half life of the supplied N6(Δ2 isopentenyl adenosine (about 3 hours) when it is supplied at the physiological concentration of 0.2 micromolar. This cytokinin degradative activity appears to be under the control of cytokinins themselves because N6-(Δ2-isopentenyl)adenosine degradative activity is increased several-fold following a 3- to 4-hour delay after these cells have been exposed to a cytokinin. PMID:16661337

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1990-11-01

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

  2. CD73-Dependent Generation of Adenosine and Endothelial Adora2b Signaling Attenuate Diabetic Nephropathy

    PubMed Central

    Tak, Eunyoung; Ridyard, Douglas; Kim, Jae-Hwan; Zimmerman, Michael; Werner, Tilmann; Wang, Xiaoxin X.; Shabeka, Uladzimir; Seo, Seong-Wook; Christians, Uwe; Klawitter, Jost; Moldovan, Radu; Garcia, Gabriela; Levi, Moshe; Haase, Volker; Ravid, Katya; Eltzschig, Holger K.

    2014-01-01

    Nucleotide phosphohydrolysis by the ecto-5′-nucleotidase (CD73) is the main source for extracellular generation of adenosine. Extracellular adenosine subsequently signals through four distinct adenosine A receptors (Adora1, Adora2a, Adora2b, or Adora3). Here, we hypothesized a functional role for CD73-dependent generation and concomitant signaling of extracellular adenosine during diabetic nephropathy. CD73 transcript and protein levels were elevated in the kidneys of diabetic mice. Genetic deletion of CD73 was associated with more severe diabetic nephropathy, whereas treatment with soluble nucleotidase was therapeutic. Transcript levels of renal adenosine receptors showed a selective induction of Adora2b during diabetic nephropathy. In a transgenic reporter mouse, Adora2b expression localized to the vasculature and increased after treatment with streptozotocin. Adora2b−/− mice experienced more severe diabetic nephropathy, and studies in mice with tissue-specific deletion of Adora2b in tubular epithelia or vascular endothelia implicated endothelial Adora2b signaling in protection from diabetic nephropathy. Finally, treatment with a selective Adora2b agonist (BAY 60–6583) conveyed potent protection from diabetes-associated kidney disease. Taken together, these findings implicate CD73-dependent production of extracellular adenosine and endothelial Adora2b signaling in kidney protection during diabetic nephropathy. PMID:24262796

  3. Localization of Adenosine Triphosphatase Activity on the Chloroplast Envelope in Tendrils of Pisum sativum1

    PubMed Central

    Sabnis, Dinkar D.; Gordon, Mildred; Galston, Arthur W.

    1970-01-01

    When samples of pea tendril tissue were incubated in the Wachstein-Meisel medium for the demonstration of adenosine triphosphatases, deposits of lead reaction product were localized between the membranes of the chloroplast envelope. The presence of Mg2+ was necessary for adenosine triphosphatase activity, and Ca2+ could not substitute for this requirement. Varying the pH of incubation to 5.5 or 9.4 inhibited enzyme activity, as did the addition of p-chloromercuribenzoic acid or N-ethylmaleimide. The adenosine triphosphatase was apparently inactivated or degraded when the plants were grown in the dark for 24 hours prior to incubation. The enzyme was substrate-specific for adenosine triphosphate; no reaction was obtained with adenosine diphosphate, uridine triphosphate, inosine triphosphate, p-nitrophenyl phosphate, and sodium β-glycerophosphate. Sites of nonspecific depositions of lead are described. The adenosine triphosphatase on the chloroplast envelope may be involved in the light-induced contraction of this organelle. Images PMID:4245003

  4. Evidence that sigma factors are components of chloroplast RNA polymerase.

    PubMed Central

    Troxler, R F; Zhang, F; Hu, J; Bogorad, L

    1994-01-01

    Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae. PMID:8159791

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

    PubMed

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

    2005-11-23

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

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

    SciTech Connect

    Culliton, B.J.

    1990-08-31

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

  7. Adenosine deaminase in cell transformation. Biophysical manifestation of membrane dynamics.

    PubMed

    Porat, N; Gill, D; Parola, A H

    1988-10-15

    Cell transformation is associated with a dramatic collapse of a graphic fingerprint characteristic of normal cells, as measured by phase fluorimetry. This is demonstrated on adenosine deaminase (ADA, EC 3.5.4.4), an established malignancy marker. ADA activity is known to decrease markedly in chick embryo fibroblasts (CEF) transformed by Rous sarcoma virus. The high affinity between the catalytic small subunit ADA (SS-ADA) and its membranal complexing protein (ADCP) (which abounds on the plasma membrane of CEF) allowed the hybridization of fluorescent labeled SS-ADA with native ADCP on CEF. Multifrequency differential phase fluorimetry responded remarkably to the state of this hybrid membrane protein. The transformation process is shown to have led to increased membrane fluidity and rotational mobility of ADCP as well as to its reduced availability to SS-ADA binding. The hypothesis of protein vertical sinking into the lipid core of the membrane is now given support by our spectroscopic data. Additional models are considered. A regulatory role is thus suggested for the complexing protein, which may also account for (a) reduced ADA activity in transformed cells and (b) detachment, exclusive to normal cells, upon addition of SS-ADA in excess.

  8. Intracellular Adenosine Triphosphate Deprivation through Lanthanide-Doped Nanoparticles.

    PubMed

    Tian, Jing; Zeng, Xiao; Xie, Xiaoji; Han, Sanyang; Liew, Oi-Wah; Chen, Yei-Tsung; Wang, Lianhui; Liu, Xiaogang

    2015-05-27

    Growing interest in lanthanide-doped nanoparticles for biological and medical uses has brought particular attention to their safety concerns. However, the intrinsic toxicity of this new class of optical nanomaterials in biological systems has not been fully evaluated. In this work, we systematically evaluate the long-term cytotoxicity of lanthanide-doped nanoparticles (NaGdF4 and NaYF4) to HeLa cells by monitoring cell viability (mitochondrial activity), adenosine triphosphate (ATP) level, and cell membrane integrity (lactate dehydrogenase release), respectively. Importantly, we find that ligand-free lanthanide-doped nanoparticles induce intracellular ATP deprivation of HeLa cells, resulting in a significant decrease in cell viability after exposure for 7 days. We attribute the particle-induced cell death to two distinct cell death pathways, autophagy and apoptosis, which are primarily mediated via the interaction between the nanoparticle and the phosphate group of cellular ATP. The understanding gained from the investigation of cytotoxicity associated with lanthanide-doped nanoparticles provides keen insights into the safe use of these nanoparticles in biological systems.

  9. Effects of DDT on eggshell quality and calcium adenosine triphosphatase.

    PubMed

    Kolaja, G J; Hinton, D E

    1977-11-01

    Adult mallard ducks were fed a diet containing 50 ppm DDT for 6 months. Eggs laid during this period were collected and eggshell weight, thickness, and calcium were determined. Chronic ingestion of DDT resulted in production of eggshells that were significantly thinner and lighter than those of controls. Total calcium of thinned eggshells was also reduced; however, calcium per gram of eggshell was not altered, indicating that other eggshell constituents were not incorporated as well. Calcium adenosine triphosphatase activity in the microsomal fraction of eggshell gland epithelium was assayed in control and DDT-fed ducks. Enzyme activity in DDT-fed ducks was reduced to 65% of control values. Since Ca-ATPase has been shown to be associated with calcium transport, enzyme inhibition may be responsible for decreased eggshell weight and thickness. Electron microscopic evaluation of microsomal fractions showed elements of the plasma membrane, including cilia and microvilli, as well as rough and smooth endoplasmic reticulum. Inhibition of calcium transport at the plasma membrane of mucosal epithelium is proposed as a possible mechanism of DDT-induced eggshell thinning.

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

    NASA Astrophysics Data System (ADS)

    Garai, Ashok; Mani, Jesrael; Chowdhury, Debashish

    2012-04-01

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

  11. Adenosine Deaminase Deficiency – More Than Just an Immunodeficiency

    PubMed Central

    Whitmore, Kathryn V.; Gaspar, Hubert B.

    2016-01-01

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

  12. Altered adenosine-to-inosine RNA editing in human cancer.

    PubMed

    Paz, Nurit; Levanon, Erez Y; Amariglio, Ninette; Heimberger, Amy B; Ram, Zvi; Constantini, Shlomi; Barbash, Zohar S; Adamsky, Konstantin; Safran, Michal; Hirschberg, Avi; Krupsky, Meir; Ben-Dov, Issachar; Cazacu, Simona; Mikkelsen, Tom; Brodie, Chaya; Eisenberg, Eli; Rechavi, Gideon

    2007-11-01

    Adenosine-to-inosine (A-to-I) RNA editing was recently shown to be abundant in the human transcriptome, affecting thousands of genes. Employing a bioinformatic approach, we identified significant global hypoediting of Alu repetitive elements in brain, prostate, lung, kidney, and testis tumors. Experimental validation confirmed this finding, showing significantly reduced editing in Alu sequences within MED13 transcripts in brain tissues. Looking at editing of specific recoding and noncoding sites, including in cancer-related genes, a more complex picture emerged, with a gene-specific editing pattern in tumors vs. normal tissues. Additionally, we found reduced RNA levels of all three editing mediating enzymes, ADAR, ADARB1, and ADARB2, in brain tumors. The reduction of ADARB2 correlated with the grade of malignancy of glioblastoma multiforme, the most aggressive of brain tumors, displaying a 99% decrease in ADARB2 RNA levels. Consistently, overexpression of ADAR and ADARB1 in the U87 glioblastoma multiforme cell line resulted in decreased proliferation rate, suggesting that reduced A-to-I editing in brain tumors is involved in the pathogenesis of cancer. Altered epigenetic control was recently shown to play a central role in oncogenesis. We suggest that A-to-I RNA editing may serve as an additional epigenetic mechanism relevant to cancer development and progression.

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

    PubMed

    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.

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

    PubMed

    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.

  15. Conditions for Using DNA Polymerase I as an RNA-Dependent DNA Polymerase

    PubMed Central

    Gulati, S. C.; Kacian, D. L.; Spiegelman, S.

    1974-01-01

    Conditions are described for using Escherichia coli DNA polymerase I for synthesizing complementary DNA copies of natural RNA molecules, which are suitable for use in hybridization experiments. The molar ratio of enzyme to template is critical; below a certain level, synthesis is not observed. Hybrids formed with the complementary DNA are of comparable specificity and stability to those formed with complementary DNAs synthesized by viral RNA-directed DNA polymerase. Synthesis of dA-dT polymers, a common occurrence with this enzyme, can be eliminated by including distamycin in the reaction mixture. PMID:4133845

  16. Interplay between polymerase II- and polymerase III-assisted expression of overlapping genes.

    PubMed

    Lukoszek, Radoslaw; Mueller-Roeber, Bernd; Ignatova, Zoya

    2013-11-15

    Up to 15% of the genes in different genomes overlap. This architecture, although beneficial for the genome size, represents an obstacle for simultaneous transcription of both genes. Here we analyze the interference between RNA-polymerase II (Pol II) and RNA-polymerase III (Pol III) when transcribing their target genes encoded on opposing strands within the same DNA fragment in Arabidopsis thaliana. The expression of a Pol II-dependent protein-coding gene negatively correlated with the transcription of a Pol III-dependent, tRNA-coding gene set. We suggest that the architecture of the overlapping genes introduces an additional layer of control of gene expression.

  17. Generation and characterization of influenza A viruses with altered polymerase fidelity

    PubMed Central

    Cheung, Peter PH; Watson, Simon J.; Choy, Ka-Tim; Sia, Sin Fun; Wong, Diana DY; Poon, Leo LM; Kellam, Paul; Guan, Yi; Peiris, JS Malik; Yen, Hui-Ling

    2014-01-01

    Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analog that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post-inoculation is associated with a 10-fold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis. PMID:25183443

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

    PubMed Central

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

    2011-01-01

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

  19. Adenosine promotes burst activity in guinea-pig geniculocortical neurones through two different ionic mechanisms.

    PubMed Central

    Pape, H C

    1992-01-01

    1. The mechanisms of action of adenosine were examined in relay neurones of the dorsal lateral geniculate nucleus (LGND) using in vitro intracellular recording techniques in guinea-pig thalamic slices. 2. Adenosine hyperpolarized LGND relay neurones due to an increase in membrane potassium conductance. The K+ currents generated by near maximal stimulation of adenosine and GABAB receptors were non-additive. 3. Blockage of membrane K+ conductances by barium unmasked a second response to adenosine; an outward shift of the current versus voltage relationship negative to -65 mV associated with an increase in membrane input resistance. The beta-adrenoceptor agonist isoprenaline elicited an inward current in the same voltage range, which was inhibited and replaced by an outward current during activation of adenosine receptors. The effects of adenosine were due to a decrease in amplitude and rate of rise of the hyperpolarization-activated cation current, Ih. Maximal reduction by 66% of Ih amplitude occurred near the range of half-activation. 4. Both responses to adenosine were mimicked by the selective A1 receptor agonists N6-cyclopentyladenosine or N6-cyclohexyladenosine, and reversibly blocked by the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 5. The decrease in Ih by adenosine may be mediated by an inhibition of adenylyl cyclase activity and hence a decrease in the intracellular level of cyclic AMP, since local application of the adenylyl cyclase inhibitor 2',3'-dideoxyadenosine imitated the decrease in Ih. Local application of the adenylyl cyclase stimulant forskolin or 8-bromo-cyclic AMP resulted in an enhancement in Ih, and forskolin inhibited the action on Ih evoked by N6-cyclopentyladenosine. 6. The adenosine-induced effects interacted with the intrinsic electrophysiological properties of LGND neurones in that (i) the hyperpolarization due to an increase in K+ conductance inhibited single spike firing and promoted calcium

  20. Adenosine effects on inhibitory synaptic transmission and excitation–inhibition balance in the rat neocortex

    PubMed Central

    Zhang, Pei; Bannon, Nicholas M; Ilin, Vladimir; Volgushev, Maxim; Chistiakova, Marina

    2015-01-01

    Abstract Adenosine might be the most widespread neuromodulator in the brain: as a metabolite of ATP it is present in every neuron and glial cell. However, how adenosine affects operation of neurons and networks in the neocortex is poorly understood, mostly because modulation of inhibitory transmission by adenosine has been so little studied. To clarify adenosine's role at inhibitory synapses, and in excitation–inhibition balance in pyramidal neurons, we recorded pharmacologically isolated inhibitory responses, compound excitatory–inhibitory responses and spontaneous events in layer 2/3 pyramidal neurons in slices from rat visual cortex. We show that adenosine (1–150 μm) suppresses inhibitory transmission to these neurons in a concentration-dependent and reversible manner. The suppression was mediated by presynaptic A1 receptors (A1Rs) because it was blocked by a selective A1 antagonist, DPCPX, and associated with changes of release indices: paired-pulse ratio, inverse coefficient of variation and frequency of miniature events. At some synapses (12 out of 24) we found evidence for A2ARs: their blockade led to a small but significant increase of the magnitude of adenosine-mediated suppression. This effect of A2AR blockade was not observed when A1Rs were blocked, suggesting that A2ARs do not have their own effect on transmission, but can modulate the A1R-mediated suppression. At both excitatory and inhibitory synapses, the magnitude of A1R-mediated suppression and A2AR–A1R interaction expressed high variability, suggesting high heterogeneity of synapses in the sensitivity to adenosine. Adenosine could change the balance between excitation and inhibition at a set of inputs to a neuron bidirectionally, towards excitation or towards inhibition. On average, however, these bidirectional changes cancelled each other, and the overall balance of excitation and inhibition was maintained during application of adenosine. These results suggest that changes of adenosine

  1. An A-T linker adapter polymerase chain reaction method for chromosome walking without restriction site cloning bias.

    PubMed

    Trinh, Quoclinh; Xu, Wentao; Shi, Hui; Luo, Yunbo; Huang, Kunlun

    2012-06-01

    A-T linker adapter polymerase chain reaction (PCR) was modified and employed for the isolation of genomic fragments adjacent to a known DNA sequence. The improvements in the method focus on two points. The first is the modification of the PO(4) and NH(2) groups in the adapter to inhibit the self-ligation of the adapter or the generation of nonspecific products. The second improvement is the use of the capacity of rTaq DNA polymerase to add an adenosine overhang at the 3' ends of digested DNA to suppress self-ligation in the digested DNA and simultaneously resolve restriction site clone bias. The combination of modifications in the adapter and in the digested DNA leads to T/A-specific ligation, which enhances the flexibility of this method and makes it feasible to use many different restriction enzymes with a single adapter. This novel A-T linker adapter PCR overcomes the inherent limitations of the original ligation-mediated PCR method such as low specificity and a lack of restriction enzyme choice. Moreover, this method also offers higher amplification efficiency, greater flexibility, and easier manipulation compared with other PCR methods for chromosome walking. Experimental results from 143 Arabidopsis mutants illustrate that this method is reliable and efficient in high-throughput experiments.

  2. Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors

    PubMed Central

    Tahirov, Tahir H; Makarova, Kira S; Rogozin, Igor B; Pavlov, Youri I; Koonin, Eugene V

    2009-01-01

    Background Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved. Results We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol ε evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol ε parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol ε, and the other one to the common ancestor of Pol α, Pol δ, and Pol ζ. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polε shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases. Conclusion Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal

  3. Fluorescence detection of adenosine triphosphate in an aqueous solution using a combination of copper(II) complexes.

    PubMed

    Kataev, Evgeny; Arnold, René; Rüffer, Tobias; Lang, Heinrich

    2012-08-06

    Fluorescent ligands have been designed to form ternary complexes with a Cu(II) cation and phosphates in a buffer solution at physiological pH 7.4. It has been shown that a combination of two different ligands and CuCl(2) allows one to achieve high adenosine triphosphate/adenosine diphosphate, adenosine 5'-monophosphate selectivity, and ratiometric fluorescence sensing, while separately each ligand complex does not have such properties.

  4. Polymerase chain reaction assay for avian polyomavirus.

    PubMed

    Phalen, D N; Wilson, V G; Graham, D L

    1991-05-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV.

  5. Polymerase chain reaction assay for avian polyomavirus.

    PubMed Central

    Phalen, D N; Wilson, V G; Graham, D L

    1991-01-01

    A polymerase chain reaction assay was developed for detection of budgerigar fledgling disease virus (BFDV). The assay used a single set of primers complementary to sequences located in the putative coding region for the BFDV VP1 gene. The observed amplification product had the expected size of 550 bp and was confirmed to derive from BFDV DNA by its restriction digestion pattern. This assay was specific for BFDV and highly sensitive, being able to detect as few as 20 copies of the virus. By using the polymerase chain reaction, BFDV was detected in adult, nestling, and embryo budgerigar (Melopsitticus undulatus) tissue DNAs and in sera from adult and nestling budgerigars. These results suggest the possibility of persistent infections in adult birds and lend further support to previously described evidence of possible in ovo transmission. BFDV was also detected in chicken embryo fibroblast cell cultures and chicken eggs inoculated with the virus. A 550-bp product with identical restriction enzyme sites was amplified from a suspected polyomavirus isolated from a peach-faced lovebird (Agapornis pesonata) and from tissue DNA from a Hahn's macaw (Ara nobilis) and a sun conure (Aratinga solstitialis) with histological lesions suggestive of polyomavirus infection. These fragments also hybridized with a BFDV-derived probe, proving that they were derived from a polyomavirus very similar, if not identical, to BFDV. Images PMID:1647403

  6. Solving the RNA polymerase I structural puzzle

    SciTech Connect

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  7. Pharmacological and biochemical characterization of adenosine receptors in the human malignant melanoma A375 cell line

    PubMed Central

    Merighi, Stefania; Varani, Katia; Gessi, Stefania; Cattabriga, Elena; Iannotta, Valeria; Ulouglu, Canan; Leung, Edward; Borea, Pier Andrea

    2001-01-01

    The present work characterizes, from a pharmacological and biochemical point of view, adenosine receptors in the human malignant melanoma A375 cell line. Adenosine receptors were detected by RT – PCR experiments. A1 receptors were characterized using [3H]-DPCPX binding with a KD of 1.9±0.2 nM and Bmax of 23±7 fmol mg−1 of protein. A2A receptors were studied with [3H]-SCH 58261 binding and revealed a KD of 5.1±0.2 nM and a Bmax of 220±7 fmol mg−1 of protein. A3 receptors were studied with the new A3 adenosine receptor antagonist [3H]-MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 3.3±0.7 nM and Bmax of 291±50 fmol mg−1 of protein. The pharmacological profile of radioligand binding on A375 cells was established using typical adenosine ligands which displayed a rank order of potency typical of the different adenosine receptor subtype. Thermodynamic data indicated that radioligand binding to adenosine receptor subtypes in A375 cells was entropy- and enthalpy-driven. In functional assays the high affinity A2A agonists HE-NECA, CGS 21680 and A2A – A2B agonist NECA were able to increase cyclic AMP accumulation in A375 cells whereas A3 agonists Cl-IB-MECA, IB-MECA and NECA were able to stimulate Ca2+ mobilization. In conclusion, all these data indicate, for the first time, that adenosine receptors with a pharmacological and biochemical profile typical of the A1, A2A, A2B and A3 receptor subtype are present on A375 melanoma cell line. PMID:11704641

  8. Dual Effect of Adenosine A1 Receptor Activation on Renal O2 Consumption.

    PubMed

    Babich, Victor; Vadnagara, Komal; Di Sole, Francesca

    2015-12-01

    The high requirement of O2 in the renal proximal tubule stems from a high rate of Na(+) transport. Adenosine A1 receptor (A1R) activation regulates Na(+) transport in this nephron segment. Thus, the effect of the acute activation and the mechanisms of A1R on the rate of O2 consumption were evaluated. The A1R-antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX) and adenosine deaminase (ADA), which metabolize endogenous adenosine, reduced O2 consumption (40-50%). Replacing Na(+) in the buffer reversed the ADA- or CPX-mediated reduction of O2 consumption. Blocking the Na/H-exchanger activity, which decreases O2 usage per se, did not enhance the ADA- or CPX-induced inhibition of O2 consumption. These data indicate that endogenous adenosine increases O2 usage via the activation of Na(+) transport. In the presence of endogenous adenosine, A1R was further activated by the A1R-agonist N(6)-cyclopentyladenosine (CPA); CPA inhibited O2 usage (30%) and this effect also depended on Na(+) transport. Moreover, a low concentration of CPA activated O2 usage in tissue pretreated with ADA, whereas a high concentration of CPA inhibited O2 usage; both effects depended on Na(+). Protein kinase C signaling mediated the inhibitory effect of A1R, while adenylyl cyclase mediated its stimulatory effect on O2 consumption. In summary, increasing the local concentrations of adenosine can either activate or inhibit O2 consumption via A1R, and this mechanism depends on Na(+) transport. The inhibition of O2 usage by A1R activation might restore the compromised balance between energy supply and demand under pathophysiological conditions, such as renal ischemia, which results in high adenosine production.

  9. Structural basis of the substrate specificity of Bacillus cereus adenosine phosphorylase

    SciTech Connect

    Dessanti, Paola; Zhang, Yang; Allegrini, Simone; Tozzi, Maria Grazia; Sgarrella, Francesco; Ealick, Steven E.

    2012-03-01

    Adenosine phosphorylase from B. cereus shows a strong preference for adenosine over other 6-oxopurine nucleosides. Mutation of Asp204 to asparagine reduces the efficiency of adenosine cleavage but does not affect inosine cleavage, effectively reversing the substrate specificity. The structures of D204N complexes explain these observations. Purine nucleoside phosphorylases catalyze the phosphorolytic cleavage of the glycosidic bond of purine (2′-deoxy)nucleosides, generating the corresponding free base and (2′-deoxy)ribose 1-phosphate. Two classes of PNPs have been identified: homotrimers specific for 6-oxopurines and homohexamers that accept both 6-oxopurines and 6-aminopurines. Bacillus cereus adenosine phosphorylase (AdoP) is a hexameric PNP; however, it is highly specific for 6-aminopurines. To investigate the structural basis for the unique substrate specificity of AdoP, the active-site mutant D204N was prepared and kinetically characterized and the structures of the wild-type protein and the D204N mutant complexed with adenosine and sulfate or with inosine and sulfate were determined at high resolution (1.2–1.4 Å). AdoP interacts directly with the preferred substrate through a hydrogen-bond donation from the catalytically important residue Asp204 to N7 of the purine base. Comparison with Escherichia coli PNP revealed a more optimal orientation of Asp204 towards N7 of adenosine and a more closed active site. When inosine is bound, two water molecules are interposed between Asp204 and the N7 and O6 atoms of the nucleoside, thus allowing the enzyme to find alternative but less efficient ways to stabilize the transition state. The mutation of Asp204 to asparagine led to a significant decrease in catalytic efficiency for adenosine without affecting the efficiency of inosine cleavage.

  10. Caffeine and propranolol block the increase in rat pineal melatonin production produced by stimulation of adenosine receptors.

    PubMed

    Babey, A M; Palmour, R M; Young, S N

    1994-07-18

    The adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) injected i.p. during the light period increased rat pineal melatonin levels and this increase was blocked by simultaneous administration of the non-selective adenosine receptor antagonist caffeine. A single dose of the adenosine A1 agonist cyclopentyladenosine had no effect on nocturnal melatonin production. The NECA-stimulated increase was also blocked by the beta-adrenergic receptor antagonist propranolol. Given alone, neither caffeine nor propranolol had any effect on melatonin levels. The results point to an intermediate role for beta-adrenergic receptors in the adenosine-stimulated increase of melatonin production.

  11. Ameliorative effect of adenosine on hypoxia-reoxygenation injury in LLC-PK1, a porcine kidney cell line.

    PubMed

    Yonehana, T; Gemba, M

    1999-06-01

    We studied the effects of adenosine on injury caused by hypoxia and reoxygenation in LLC-PK1 cells. Lactate dehydrogenase and gamma-glutamyltranspeptidase were released from cells exposed to hypoxia for 6 hr and then reoxygenation for 1 hr. The addition of adenosine at 100 microM to the medium before hypoxia began significantly decreased enzyme leakage into medium during both hypoxia and reoxygenation. The adenosine A1-receptor agonist, R(-)-N6-(2-phenylisopropyl)adenosine (R-PIA), at the concentration of 100 microM, did not affect enzyme release, but the adenosine A2-receptor agonist 2-p-[2-car-boxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosi ne hydrochloride (CGS 21680) at the concentration of 100 nM, suppressed the injury caused by hypoxia and reoxygenation. There were decreases in cAMP contents and ATP levels in LLC-PK1 cells injured by hypoxia and reoxygenation. Adenosine (100 microM) restored ATP levels in the cells during reoxygenation. With adenosine, the intracellular cAMP level was increased prominently during reoxygenation. These results suggest that adenosine protects LLC-PK1 cells from injury caused by hypoxia and reoxygenation by increasing the intracellular cAMP level via adenosine A2 receptor.

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

    PubMed Central

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

    2014-01-01

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

  13. The A2B adenosine receptor promotes Th17 differentiation via stimulation of dendritic cell IL-6.

    PubMed

    Wilson, Jeffrey M; Kurtz, Courtney C; Black, Steven G; Ross, William G; Alam, Mohammed S; Linden, Joel; Ernst, Peter B

    2011-06-15

    Adenosine is an endogenous metabolite produced during hypoxia or inflammation. Previously implicated as an anti-inflammatory mediator in CD4(+) T cell regulation, we report that adenosine acts via dendritic cell (DC) A(2B) adenosine receptor (A(2B)AR) to promote the development of Th17 cells. Mouse naive CD4(+) T cells cocultured with DCs in the presence of adenosine or the stable adenosine mimetic 5'-(N-ethylcarboximado) adenosine resulted in the differentiation of IL-17- and IL-22-secreting cells and elevation of mRNA that encode signature Th17-associated molecules, such as IL-23R and RORγt. The observed response was similar when DCs were generated from bone marrow or isolated from small intestine lamina propria. Experiments using adenosine receptor antagonists and cells from A(2B)AR(-/-) or A(2A)AR(-/-)/A(2B)AR(-/-) mice indicated that the DC A(2B)AR promoted the effect. IL-6, stimulated in a cAMP-independent manner, is an important mediator in this pathway. Hence, in addition to previously noted direct effects of adenosine receptors on regulatory T cell development and function, these data indicated that adenosine also acts indirectly to modulate CD4(+) T cell differentiation and suggested a mechanism for putative proinflammatory effects of A(2B)AR.

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

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

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

  15. The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity

    PubMed Central

    Randi, Lorenzo; Perrone, Alessandro; Maturi, Mirko; Dal Piaz, Fabrizio; Camerani, Michela; Hochkoeppler, Alejandro

    2016-01-01

    We report in the present study on the catalytic properties of the Deinococcus radiodurans DNA polymerase III α subunit (αDr). The αDr enzyme was overexpressed in Escherichia coli, both in soluble form and as inclusion bodies. When purified from soluble protein extracts, αDr was found to be tightly associated with E. coli RNA polymerase, from which αDr could not be dissociated. On the contrary, when refolded from inclusion bodies, αDr was devoid of E. coli RNA polymerase and was purified to homogeneity. When assayed with different DNA substrates, αDr featured slower DNA extension rates when compared with the corresponding enzyme from E. coli (E. coli DNA Pol III, αEc), unless under high ionic strength conditions or in the presence of manganese. Further assays were performed using a ssDNA and a dsDNA, whose recombination yields a DNA substrate. Surprisingly, αDr was found to be incapable of recombination-dependent DNA polymerase activity, whereas αEc was competent in this action. However, in the presence of the RecA recombinase, αDr was able to efficiently extend the DNA substrate produced by recombination. Upon comparing the rates of RecA-dependent and RecA-independent DNA polymerase activities, we detected a significant activation of αDr by the recombinase. Conversely, the activity of αEc was found maximal under non-recombination conditions. Overall, our observations indicate a sharp contrast between the catalytic actions of αDr and αEc, with αDr more performing under recombination conditions, and αEc preferring DNA substrates whose extension does not require recombination events. PMID:27789781

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

    PubMed

    Nescerecka, Alina; Juhna, Talis; Hammes, Frederik

    2016-09-15

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

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

    PubMed

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

    2016-03-01

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

  18. Autoimmune dysregulation and purine metabolism in adenosine deaminase deficiency.

    PubMed

    Sauer, Aisha Vanessa; Brigida, Immacolata; Carriglio, Nicola; Aiuti, Alessandro

    2012-01-01

    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.

  19. The influence of lactic acid on adenosine release from skeletal muscle in anaesthetized dogs.

    PubMed Central

    Ballard, H J

    1991-01-01

    1. In anaesthetized and artificially ventilated dogs, a gracilis muscle was vascularly isolated and perfused at a constant flow rate of 11.9 +/- 2.2 ml min-1 100 g-1 (mean +/- S.E.M., n = 16; equivalent to 170.2 +/- 21.3% of its resting free flow). 2. Stimulation (3 Hz) of the obturator nerve produced twitch contractions of the gracilis muscle, reduced venous pH from 7.366 +/- 0.027 to 7.250 +/- 0.031 (n = 5), increased oxygen consumption from 0.62 +/- 0.24 to 2.76 +/- 0.46 ml min-1 100 g-1 (n = 5) and increased adenosine release from -0.40 +/- 0.14 (net uptake) to 1.36 +/- 0.50 nmol min-1 100 g-1 (n = 8). 3. Infusion of lactic acid (4.2 mM) into the artery reduced venous pH to 7.281 +/- 0.026 (n = 5) and increased adenosine release to 0.96 +/- 0.40 nmol min-1 100 g-1 (n = 8), but did not significantly alter oxygen consumption (0.80 +/- 0.19 ml min-1 100 g-1; n = 5). Stimulation (3 Hz) in the presence of lactic acid infusion produced no further significant changes in venous pH or adenosine release, but increased oxygen consumption to 2.53 +/- 0.37 ml min-1 100 g-1 (n = 5). 4. Infusion of a range of lactic acid concentrations (> or = 1.83 mM) produced dose-dependent increases in adenosine release. The maximum lactic acid concentration tested (5.95 mM) reduced venous pH to 7.249 +/- 0.023 (n = 5) and increased adenosine release to 2.64 +/- 1.26 nmol min-1 100 g-1 (n = 6). 5. A strong correlation existed between the adenosine release and the venous pH (r = -0.92); points obtained during muscle stimulation and/or lactic acid infusion fell on a single correlation line. 6. The vasoactivity of adenosine administered by close-arterial injection was unaltered by infusion of either lactic acid (7.2 mM) or saline. 7. These results suggest that the release of adenosine from skeletal muscle can be induced by a decrease in pH (probably at an intracellular site), and that this mechanism may contribute to the release of adenosine during muscle contractions. PMID:1841964

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

    PubMed

    Lebon, Guillaume; Edwards, Patricia C; Leslie, Andrew G W; Tate, Christopher G

    2015-06-01

    The adenosine A2A receptor (A(2A)R) plays a key role in transmembrane signaling mediated by the endogenous agonist adenosine. Here, we describe the crystal structure of human A2AR thermostabilized in an active-like conformation bound to the selective agonist 2-[p-(2-carboxyethyl)phenylethyl-amino]-5'-N-ethylcarboxamido adenosine (CGS21680) at a resolution of 2.6 Å. Comparison of A(2A)R structures bound to either CGS21680, 5'-N-ethylcarboxamido adenosine (NECA), UK432097 [6-(2,2-diphenylethylamino)-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-tetrahydrofuran-2-yl]-N-[2-[[1-(2-pyridyl)-4-piperidyl]carbamoylamino]ethyl]purine-2-carboxamide], or adenosine shows that the adenosine moiety of the ligands binds to the receptor in an identical fashion. However, an extension in CGS21680 compared with adenosine, the (2-carboxyethyl)phenylethylamino group, binds in an extended vestibule formed from transmembrane regions 2 and 7 (TM2 and TM7) and extracellular loops 2 and 3 (EL2 and EL3). The (2-carboxyethyl)phenylethylamino group makes van der Waals contacts with side chains of amino acid residues Glu169(EL2), His264(EL3), Leu267(7.32), and Ile274(7.39), and the amine group forms a hydrogen bond with the side chain of Ser67(2.65). Of these residues, only Ile274(7.39) is absolutely conserved across the human adenosine receptor subfamily. The major difference between the structures of A(2A)R bound to either adenosine or CGS21680 is that the binding pocket narrows at the extracellular surface when CGS21680 is bound, due to an inward tilt of TM2 in that region. This conformation is stabilized by hydrogen bonds formed by the side chain of Ser67(2.65) to CGS21680, either directly or via an ordered water molecule. Mutation of amino acid residues Ser67(2.65), Glu169(EL2), and His264(EL3), and analysis of receptor activation either in the presence or absence of ligands implicates this region in modulating the level of basal activity of A(2A)R.

  1. Label-free electrochemical aptasensor for adenosine detection based on cascade signal amplification strategy.

    PubMed

    Shen, Jing; Wang, Hongyang; Li, Chunxiang; Zhao, Yanyan; Yu, Xijuan; Luo, Xiliang

    2017-04-15

    In this work, a simple and highly sensitive label-free electrochemical aptasensor for adenosine detection was developed based on target-aptamer binding triggered nicking endonuclease-assisted strand-replacement DNA polymerization and rolling circle amplification (RCA) strategy. The magnetic beads (MB) probe, which was attached the aptamer of adenosine and mDNA, was firstly fabricated. In the presence of adenosine, mDNA was released from MB upon recognition of the aptamer to target adenosine. The released mDNA as the primer activated autonomous DNA polymerization/nicking process and accompanied by the continuous release of replicated DNA fragments. Subsequently, numerous released DNA fragments were captured on the working electrode, and then as initiators to trigger the downstream RCA process leading to the formation of a long ssDNA concatemer for loading large amounts of Ru(NH3)6(3+). Therefore, a conspicuously amplified electrochemical signal through the developed dual-amplification strategy could be achieved. This method exhibited a high sensitivity toward adenosine with a detection limit of 0.032nM. Also, it exhibited high selectivity to different nucleoside families and good reproducibility. This design opens new horizons for integrating different disciplines, presenting a versatile tool for ultrasensitive detecting organic small molecules in medical research and clinical diagnosis.

  2. Squalenoyl adenosine nanoparticles provide neuroprotection after stroke and spinal cord injury

    NASA Astrophysics Data System (ADS)

    Gaudin, Alice; Yemisci, Müge; Eroglu, Hakan; Lepetre-Mouelhi, Sinda; Turkoglu, Omer Faruk; Dönmez-Demir, Buket; Caban, Seçil; Sargon, Mustafa Fevzi; Garcia-Argote, Sébastien; Pieters, Grégory; Loreau, Olivier; Rousseau, Bernard; Tagit, Oya; Hildebrandt, Niko; Le Dantec, Yannick; Mougin, Julie; Valetti, Sabrina; Chacun, Hélène; Nicolas, Valérie; Desmaële, Didier; Andrieux, Karine; Capan, Yilmaz; Dalkara, Turgay; Couvreur, Patrick

    2014-12-01

    There is an urgent need to develop new therapeutic approaches for the treatment of severe neurological trauma, such as stroke and spinal cord injuries. However, many drugs with potential neuropharmacological activity, such as adenosine, are inefficient upon systemic administration because of their fast metabolization and rapid clearance from the bloodstream. Here, we show that conjugation of adenosine to the lipid squalene and the subsequent formation of nanoassemblies allows prolonged circulation of this nucleoside, providing neuroprotection in mouse stroke and rat spinal cord injury models. The animals receiving systemic administration of squalenoyl adenosine nanoassemblies showed a significant improvement of their neurologic deficit score in the case of cerebral ischaemia, and an early motor recovery of the hindlimbs in the case of spinal cord injury. Moreover, in vitro and in vivo studies demonstrated that the nanoassemblies were able to extend adenosine circulation and its interaction with the neurovascular unit. This Article shows, for the first time, that a hydrophilic and rapidly metabolized molecule such as adenosine may become pharmacologically efficient owing to a single conjugation with the lipid squalene.

  3. The Effect of Endogenous Adenosine on Neuronal Activity in Rats: An FDG PET Study

    PubMed Central

    Paul, Soumen; Zhang, Dali; Mzengeza, Shadreck; Ko, Ji Hyun

    2016-01-01

    ABSTRACT 2–18F‐fluorodeoxy‐D‐glucose (FDG) is a glucose analog that is taken up by cells and phosphorylated. The amount of FDG accumulated by cells is a measure of the rate of glycolysis, which reflects cellular activity. As the levels and actions of the neuromodulator adenosine are dynamically regulated by neuronal activity, this study was designed to test whether endogenous adenosine affects tissue accumulation of FDG as assessed by positron emission tomography (PET) or by postmortem analysis of tissue radioactivity. Rats were given an intraperitoneal injection of the adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropyl‐xanthine (DPCPX, 3 mg/kg), the adenosine kinase inhibitor ABT‐702 (3 mg/kg), or vehicle 10 minutes prior to an intravenous injection of FDG (15.4 ± 0.7 MBq per rat). Rats were then subjected to a 15 minute static PET scan. Reconstructed images were normalized to FDG PET template for rats and standard uptake values (SUVs) were calculated. To examine the regional effect of active treatment compared to vehicle, statistical parametric mapping analysis was performed. Whole‐brain FDG uptake was not affected by drug treatment. Significant regional hypometabolism was detected, particularly in cerebellum, of DPCPX‐ and ABT‐702 treated rats, relative to vehicle‐treated rats. Thus, endogenous adenosine can affect FDG accumulation although this effect is modest in quiescent rats. PMID:27082948

  4. Adenosine uptake is the major effector of extracellular ATP toxicity in human cervical cancer cells

    PubMed Central

    Mello, Paola de Andrade; Filippi-Chiela, Eduardo Cremonese; Nascimento, Jéssica; Beckenkamp, Aline; Santana, Danielle Bertodo; Kipper, Franciele; Casali, Emerson André; Nejar Bruno, Alessandra; Paccez, Juliano Domiraci; Zerbini, Luiz Fernando; Wink, Marcia Rosângela; Lenz, Guido; Buffon, Andréia

    2014-01-01

    In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells. PMID:25103241

  5. The impact of adenosine and A(2B) receptors on glucose homoeostasis.

    PubMed

    Rüsing, D; Müller, C E; Verspohl, E J

    2006-12-01

    Adenosine and adenosine receptor antagonists are involved in glucose homoeostasis. The participating receptors are not known, mainly due to a lack of specific agonists and antagonists, but are reasonable targets for anti-diabetic therapy. The stable, albeit nonselective, adenosine analogue NECA (5'-N-ethylcarboxamidoadenosine) (10 microM) reduced glucose-stimulated insulin release from INS-1 cells. This was mimicked by A(1)-(CHA), A(2A)-(CGS-21680) and A(3)-receptor agonists (Cl-IB-MECA). Two newly synthesized A(2B)-receptor antagonists, PSB-53 and PSB-1115, counteracted the inhibitory effect of NECA. These in-vitro effects were mirrored by in-vivo data with respect to CHA, CGS and Cl-IB-MECA. Distinct concentrations of either PSB-53 or PSB-1115 reversed the decrease in plasma insulin induced by NECA. This was not mimicked by a corresponding change in blood glucose. The effect of PSB-1115 was also obvious in diabetic GotoKakizaki rats: plasma insulin was increased whereas blood glucose was unchanged. During most experiments the effects on blood glucose were not impressive probably because of the physiologically necessary homoeostasis. The adenosine levels were not different in normal Wistar rats and in diabetic GotoKakzaki rats. Altogether the A(2B)-receptor antagonists showed an anti-diabetic potential mainly by increasing plasma insulin levels under conditions when the adenosine tonus was elevated in-vivo and increased insulin release in-vitro.

  6. Phosphorylation potential and adenosine release during norepinephrine infusion in guinea pig heart

    SciTech Connect

    He, Miao-Xiang; Wangler, R.D.; Dillon, P.F.; Romig, G.D.; Sparks, H.V. )

    1987-11-01

    This study tested the hypothesis that adenosine released from isolated guinea pig hearts in response to norepinephrine is related to the cellular phosphorylation potential (PP;(ATP)/(ADP)(P{sub i})), where P{sub i} is inorganic phosphate. {sup 31}P-nuclear magnetic resonance (NMR) was used to measure the relative concentrations of P{sub i}, phosphocreatine (PCr), and ATP. After a control period, norepinephrine was infused for 20 min during which {sup 31}P-NMR spectra and samples of venous effluent were collected every minute. With norepinephrine infusion, PCr decreased rapidly to 72% of control by 8 min and then recovered to 80% of control for the remaining 12 min. ATP fell slowly to 70% of control over 20 min. P{sub i} increased to a peak at 2 min, then declined slowly to a steady state from 8 to 20 min. Adenosine release increased at 7 min and then slowly fell to a steady state from 10 to 20 min. There is hyperbolic relationship between adenosine release and PP; when the PP declines, a level is reached below which there is a rapid increase in adenosine release. These data support the hypothesis that adenosine release is regulated by the cellular PP as a closely related variable.

  7. Endogenous adenosine release is involved in the control of heart rate in rats.

    PubMed

    Jammes, Yves; Joulia, Fabrice; Steinberg, Jean Guillaume; Ravailhe, Sylvie; Delpierre, Stéphane; Condo, Jocelyne; Guieu, Regis; Delliaux, Stéphane

    2015-08-01

    Intravenous (i.v.) injections of adenosine exert marked effects on heart rate (HR) and arterial blood pressure (BP), but the role of an endogenous adenosine release by vagal stimulation has not been evaluated. In anaesthetized rats, we examined HR and BP changes induced by 1 min electrical vagal stimulation in the control condition, and then after i.v. injections of (i) atropine, (ii) propranolol, (iii) caffeine, (iv) 8 cyclopentyl-1,3-dipropylxanthine (DPCPX), or (v) dipyridamole to increase the plasma concentration of adenosine (APC). APC was measured by chromatography in the arterial blood before and at the end of vagal stimulation. The decrease in HR in the controls during vagal stimulation was markedly attenuated, but persisted after i.v. injections of atropine and propranolol. When first administered, DPCPX modestly but significantly reduced the HR response to vagal stimulation, but this disappeared after i.v. caffeine administration. Both the HR and BP responses were significantly accentuated after i.v. injection of dipyridamole. Vagal stimulation induced a significant increase in APC, proportional to the magnitude of HR decrease. Our data suggest that the inhibitory effects of electrical vagal stimulations on HR and BP were partly mediated through the activation of A1 and A2 receptors by an endogenous adenosine release. Our experimental data could help to understand the effects of ischemic preconditioning, which are partially mediated by adenosine.

  8. Adenosine augments IL-10-induced STAT3 signaling in M2c macrophages.

    PubMed

    Koscsó, Balázs; Csóka, Balázs; Kókai, Endre; Németh, Zoltán H; Pacher, Pál; Virág, László; Leibovich, S Joseph; Haskó, György

    2013-12-01

    The alternatively activated macrophage phenotype induced by IL-10 is called M2c. Adenosine is an endogenous purine nucleoside that accumulates in the extracellular space in response to metabolic disturbances, hypoxia, inflammation, physical damage, or apoptosis. As adenosine is known to regulate classically activated M1 and IL4- and IL-13-activated M2a macrophages, the goal of the present study was to explore its effects on M2c macrophages. We found that adenosine augmented the IL-10-induced expression of TIMP-1 and arginase-1 by the mouse macrophage cell line RAW 264.7 and by mouse BMDMs. The effects of AR stimulation on IL-10-induced TIMP-1 or arginase-1 expression were lacking in A2BAR KO macrophages. The role of A2BAR on TIMP-1 production of RAW 264.7 cells was confirmed with specific agonist BAY606583 and antagonist PSB0788. AR stimulation augmented IL-10-induced STAT3 phosphorylation in macrophages, and pharmacological inhibition or silencing of STAT3 using siRNA reduced the stimulatory effect of AR stimulation on TIMP-1 production. In contrast to its stimulatory effect on IL-10-induced STAT3 activation, adenosine inhibited IL-6-induced STAT3 phosphorylation and SAA3 expression. In conclusion, adenosine enhances IL-10-induced STAT3 signaling and M2c macrophage activation.

  9. Comparative transcriptome analysis of Bacillus subtilis responding to dissolved oxygen in adenosine fermentation.

    PubMed

    Yu, Wen-Bang; Gao, Shu-Hong; Yin, Chun-Yun; Zhou, Ying; Ye, Bang-Ce

    2011-01-01

    Dissolved oxygen (DO) is an important factor for adenosine fermentation. Our previous experiments have shown that low oxygen supply in the growth period was optimal for high adenosine yield. Herein, to better understand the link between oxygen supply and adenosine productivity in B. subtilis (ATCC21616), we sought to systematically explore the effect of DO on genetic regulation and metabolism through transcriptome analysis. The microarrays representing 4,106 genes were used to study temporal transcript profiles of B. subtilis fermentation in response to high oxygen supply (agitation 700 r/min) and low oxygen supply (agitation 450 r/min). The transcriptome data analysis revealed that low oxygen supply has three major effects on metabolism: enhance carbon metabolism (glucose metabolism, pyruvate metabolism and carbon overflow), inhibit degradation of nitrogen sources (glutamate family amino acids and xanthine) and purine synthesis. Inhibition of xanthine degradation was the reason that low oxygen supply enhanced adenosine production. These provide us with potential targets, which can be modified to achieve higher adenosine yield. Expression of genes involved in energy, cell type differentiation, protein synthesis was also influenced by oxygen supply. These results provided new insights into the relationship between oxygen supply and metabolism.

  10. [Adenosine-dependent death of Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16 (author's transl)].

    PubMed

    Kaltwasser, H; Glaeser, H

    1976-01-01

    Heterotrophic growth with fructose and autotrophic growth with hydrogen and carbon dioxide was entirely inhibited by adenosine at a concentration of 0.6 mg/ml in Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16. Growth inhibition was not accompanied by a detectable uptake of the nucleoside. Adenosine caused a rapid inhibition of protein synthesis, followed by a decrease in nucleic acid formation. Enzyme synthesis was also impared, whilst cell respiration remained uneffected. Adenosin also caused a drastic but temporary decrease in viable cell counts. Cells incubated in presence of adenosine and fructose for several days, however, were no longer susceptable to this nucleoside. Adenosine-dependent growth inhibition turned out to be contingent upon the nature of the organic substrate. Cells growing with succinate, glutamate or peptone were less effected than cells, growing autotrophically or with fructose. No inhibition was observed in fructose-growing cells, if amino acids were also present in the medium. Several other nucleosides tested, did not show such growth inhibition, except desoxyadenosine, which, however, did not effect viable cell counts.

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

    PubMed Central

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

    2014-01-01

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

  12. Sulfur-Containing 1,3-Dialkylxanthine Derivatives as Selective Antagonists at A1-Adenosine Receptors

    PubMed Central

    Kiriasis, Leonidas; Barone, Suzanne; Bradbury, Barton J.; Kammula, Udai; Campagne, Jean Michel; Secunda, Sherrie; Daly, John W.; Neumeyer, John L.; Pfleiderer, Wolfgang

    2012-01-01

    Sulfur-containing analogues of 8-substituted xanthines were prepared in an effort to increase selectivity or potency as antagonists at adenosine receptors. Either cyclopentyl or various aryl substituents were utilized at the 8-position, because of the association of these groups with high potency at A1-adenosine receptors. Sulfur was incorporated on the purine ring at positions 2 and/or 6, in the 8-position substituent in the form of 2- or 3-thienyl groups, or via thienyl groups separated from an 8-aryl substituent through an amide-containing chain. The feasibility of using the thienyl group as a prosthetic group for selective iodination via its Hg2+ derivative was explored. Receptor selectivity was determined in binding assays using membrane homogenates from rat cortex [[3H]-N6-(phenylisopropyl) adenosine as radioligand] or striatum [[3H]-5′-(N-ethylcarbamoyl)adenosine as radioligand] for A1- and A2-adenosine receptors, respectively. Generally, 2-thio-8-cycloalkylxanthines were at least as A1 selective as the corresponding oxygen analogue. 2-Thio-8-aryl derivatives tended to be more potent at A2 receptors than the oxygen analogue. 8-[4-[(Carboxymethyl)oxy]phenyl]-1,3-dipropyl-2-thioxanthine ethyl ester was >740-fold A1 selective. PMID:2754711

  13. Astrocyte-derived adenosine is central to the hypnogenic effect of glucose

    PubMed Central

    Scharbarg, Emeric; Daenens, Marion; Lemaître, Frédéric; Geoffroy, Hélène; Guille-Collignon, Manon; Gallopin, Thierry; Rancillac, Armelle

    2016-01-01

    Sleep has been hypothesised to maintain a close relationship with metabolism. Here we focus on the brain structure that triggers slow-wave sleep, the ventrolateral preoptic nucleus (VLPO), to explore the cellular and molecular signalling pathways recruited by an increase in glucose concentration. We used infrared videomicroscopy on ex vivo brain slices to establish that glucose induces vasodilations specifically in the VLPO via the astrocytic release of adenosine. Real-time detection by in situ purine biosensors further revealed that the adenosine level doubles in response to glucose, and triples during the wakefulness period. Finally, patch-clamp recordings uncovered the depolarizing effect of adenosine and its A2A receptor agonist, CGS-21680, on sleep-promoting VLPO neurons. Altogether, our results provide new insights into the metabolically driven release of adenosine. We hypothesise that adenosine adjusts the local energy supply to local neuronal activity in response to glucose. This pathway could contribute to sleep-wake transition and sleep intensity. PMID:26755200

  14. Circadian variations of adenosine level in blood and liver and its possible physiological significance.

    PubMed

    Chagoya de Sánchez, V; Hernández-Muñoz, R; Díaz-Muñoz, M; Villalobos, R; Glender, W; Vidrio, S; Suárez, J; Yañez, L

    1983-09-12

    The role of adenosine as a possible physiological modulator was explored by measuring its concentration in different tissues during a 24-hour period. Initially the circadian variations of adenosine and other purine compounds such as inosine, hypoxanthine, uric acid and adenine nucleotides were studied in the rat blood. A daily cyclic response was observed, with low levels of adenosine from 08.00 - 20.00 h, followed by an increase from this time on. Inosine and hypoxanthine levels were elevated during the day and low at night. The uric acid changes observed indicate that the decrease in purine catabolism coincides with a decrease in inosine and hypoxanthine levels and an increase in adenosine. The blood adenine nucleotides, energy charge and phosphorylation potential remained constant during the day and showed oscillatory changes during the night. Similar studies were made in the liver, a primary source of circulating purines. Liver adenosine was high during the night while inosine and hypoxanthine remained low along the 24 hours. The results suggest that liver purine metabolism might participate in the maintenance and renewal of the blood purine pool and in the energy state of erythrocytes in vivo.

  15. The A2a adenosine receptor modulates the reinforcement efficacy and neurotoxicity of MDMA.

    PubMed

    Ruiz-Medina, Jessica; Ledent, Catherine; Carretón, Olga; Valverde, Olga

    2011-04-01

    Adenosine is an endogenous purine nucleoside that plays a neuromodulatory role in the central nervous system. A2a adenosine receptors have been involved in reward-related processes, inflammatory phenomena and neurotoxicity reactions. In the present study, we investigated the role of A2a adenosine receptors on the acute pharmacological effects, reinforcement and neuroinflammation induced by MDMA administration. First, the acute effects of MDMA on body temperature, locomotor activity and anxiety-like responses were measured in A2a knockout mice and wild-type littermates. Second, MDMA reinforcing properties were evaluated using the intravenous self-administration paradigm. Finally, we assessed striatal astrogliosis and microgliosis as markers of MDMA neurotoxicity. Our results showed that acute MDMA produced a biphasic effect on body temperature and increased locomotor activity and anxiogenic-like responses in both genotypes. However, MDMA reinforcing properties were dramatically affected by the lack of A2a adenosine receptors. Thus, wild-type mice maintained MDMA self-administration under a fixed ratio 1 reinforcement schedule, whereas the operant response appeared completely abolished in A2a knockout mice. In addition, the MDMA neurotoxic regime produced an enhanced inflammatory response in striatum of wild-type mice, revealed by a significant increase in glial expression, whereas such activation was attenuated in mutant mice. This is the first report indicating that A2a adenosine receptors play a key role in reinforcement and neuroinflammation induced by the widely used psychostimulant.

  16. Nucleotide binding interactions modulate dNTP selectivity and facilitate 8-oxo-dGTP incorporation by DNA polymerase lambda

    PubMed Central

    Burak, Matthew J.; Guja, Kip E.; Garcia-Diaz, Miguel

    2015-01-01

    8-Oxo-7,8,-dihydro-2′-deoxyguanosine triphosphate (8-oxo-dGTP) is a major product of oxidative damage in the nucleotide pool. It is capable of mispairing with adenosine (dA), resulting in futile, mutagenic cycles of base excision repair. Therefore, it is critical that DNA polymerases discriminate against 8-oxo-dGTP at the insertion step. Because of its roles in oxidative DNA damage repair and non-homologous end joining, DNA polymerase lambda (Pol λ) may frequently encounter 8-oxo-dGTP. Here, we have studied the mechanisms of 8-oxo-dGMP incorporation and discrimination by Pol λ. We have solved high resolution crystal structures showing how Pol λ accommodates 8-oxo-dGTP in its active site. The structures indicate that when mispaired with dA, the oxidized nucleotide assumes the mutagenic syn-conformation, and is stabilized by multiple interactions. Steady-state kinetics reveal that two residues lining the dNTP binding pocket, Ala510 and Asn513, play differential roles in dNTP selectivity. Specifically, Ala510 and Asn513 facilitate incorporation of 8-oxo-dGMP opposite dA and dC, respectively. These residues also modulate the balance between purine and pyrimidine incorporation. Our results shed light on the mechanisms controlling 8-oxo-dGMP incorporation in Pol λ and on the importance of interactions with the incoming dNTP to determine selectivity in family X DNA polymerases. PMID:26220180

  17. A 30-year-old female Behçet’s disease patient with recurrent pleural and pericardial effusion and elevated adenosine deaminase levels: case report

    PubMed Central

    Choi, Joon Young; Kim, Sung-Hwan; Kwok, Seung-Ki; Jung, Jung Im; Lee, Kyo-Young; Kim, Tae-Jung

    2016-01-01

    Behçet’s disease is a systemic disease which may involve various organs. We describe a case of a patient diagnosed as pleuropericardial involvement of Behçet’s disease. A 30-year-old woman visited our clinic presented with left pleuritic chest pain for s days. She had been diagnosed as Behçet’s disease and admitted to our clinic due to pericardial and pleural effusion repeatedly in past two years. In the previous studies, effusion analysis revealed to be lympho-dominant exudate with high adenosine deaminase level. Acid-fast bacilli (AFB) culture and polymerase chain reaction (PCR) for mycobacterial tuberculosis (M.TB) were negative in the pericardial tissue, and pathologic finding showed mild endothelitis with micro-thrombi formation in the lumen. The patient had been treated with antituberculous medication for a year. In the current admission, chest computed tomography (CT) again showed left pleural effusion without other significant lesion. Pleural fluid analysis was similar with the previous study. Video-assisted thoracoscopic pleural biopsy was performed to obtain the definite diagnosis. Pathology confirmed the diagnosis as pleuropericardial involvement of Behçet’s disease, and we treated the patient with oral steroid in the out-patient department. Pleuropericardial involvement of Behçet’s disease may mimic TB pleurisy or pericarditis due to high adenosine deaminase (ADA) level in effusion analysis. Clinicians should keep in mind that Behçet’s disease may manifest as pleural or pericardial effusion, and pathologic confirmation could be helpful for the definite diagnosis. PMID:27499994

  18. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats.

    PubMed

    Iglesias, Inmaculada; Albasanz, Jose Luis; Martín, Mairena

    2014-12-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine.

  19. Effect of Caffeine Chronically Consumed During Pregnancy on Adenosine A1 and A2A Receptors Signaling in Both Maternal and Fetal Heart from Wistar Rats

    PubMed Central

    Iglesias, Inmaculada; Albasanz, Jose Luis

    2014-01-01

    Background: Caffeine is the most widely consumed psychoactive substance in the world, even during pregnancy. Its stimulatory effects are mainly due to antagonism of adenosine actions by blocking adenosine A1 and A2A receptors. Previous studies have shown that caffeine can cross the placenta and therefore modulate these receptors not only in the fetal brain but also in the heart. Methods: In the present work, the effect of caffeine chronically consumed during pregnancy on A1 and A2A receptors in Wistar rat heart, from both mothers and their fetuses, were studied using radioligand binding, Western-blotting, and adenylyl cyclase activity assays, as well as reverse transcription polymerase chain reaction. Results: Caffeine did not significantly alter A1R neither at protein nor at gene expression level in both the maternal and fetal heart. On the contrary, A2AR significantly decreased in the maternal heart, although mRNA was not affected. Gi and Gs proteins were also preserved. Finally, A1R-mediated inhibition of adenylyl cyclase activity did not change in the maternal heart, but A2AR mediated stimulation of this enzymatic activity significantly decreased according to the detected loss of this receptor. Conclusions: Opposite to the downregulation and desensitization of the A1R/AC pathway previously reported in the brain, these results show that this pathway is not affected in rat heart after caffeine exposure during pregnancy. In addition, A2AR is downregulated and desensitized in the maternal heart, suggesting a differential modulation of these receptor-mediated pathways by caffeine. PMID:25538864

  20. Conformational flexibility of bacterial RNA polymerase

    PubMed Central

    Darst, Seth A.; Opalka, Natacha; Chacon, Pablo; Polyakov, Andrey; Richter, Catherine; Zhang, Gongyi; Wriggers, Willy

    2002-01-01

    The structure of Escherichia coli core RNA polymerase (RNAP) was determined by cryo-electron microscopy and image processing of helical crystals to a nominal resolution of 15 Å. Because of the high sequence conservation between the core RNAP subunits, we were able to interpret the E. coli structure in relation to the high-resolution x-ray structure of Thermus aquaticus core RNAP. A very large conformational change of the T. aquaticus RNAP x-ray structure, corresponding to opening of the main DNA/RNA channel by nearly 25 Å, was required to fit the E. coli map. This finding reveals, at least partially, the range of conformational flexibility of the RNAP, which is likely to have functional implications for the initiation of transcription, where the DNA template must be loaded into the channel. PMID:11904365

  1. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  2. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  3. A Cross-chiral RNA Polymerase Ribozyme

    PubMed Central

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer.1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes prior to the origin of RNA-based life.3,4 Once an RNA enzyme arose that could catalyze the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D-and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness. Such a cross-chiral RNA polymerase has now been developed using in vitro evolution. The D-RNA enzyme, consisting of 83 nucleotides, catalyzes the joining of L-mono- or oligonucleotide substrates on a complementary L-RNA template, and similarly for the L-enzyme with D-substrates and a D-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme's activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides. PMID:25363769

  4. RNA binding and replication by the poliovirus RNA polymerase

    SciTech Connect

    Oberste, M.S.

    1988-01-01

    RNA binding and RNA synthesis by the poliovirus RNA-dependent RNA polymerase were studied in vitro using purified polymerase. Templates for binding and RNA synthesis studies were natural RNAs, homopolymeric RNAs, or subgenomic poliovirus-specific RNAs synthesized in vitro from cDNA clones using SP6 or T7 RNA polymerases. The binding of the purified polymerase to poliovirion and other RNAs was studied using a protein-RNA nitrocellulose filter binding assay. A cellular poly(A)-binding protein was found in the viral polymerase preparations, but was easily separated from the polymerase by chromatography on poly(A) Sepharose. The binding of purified polymerase to {sup 32}P-labeled ribohomopolymeric RNAs was examined, and the order of binding observed was poly(G) >>> poly(U) > poly(C) > poly(A). The K{sub a} for polymerase binding to poliovirion RNA and to a full-length negative strand transcript was about 1 {times} 10{sup 9} M{sup {minus}1}. The polymerase binds to a subgenomic RNAs which contain the 3{prime} end of the genome with a K{sub a} similar to that for virion RNA, but binds less well to 18S rRNA, globin mRNA, and subgenomic RNAs which lack portions of the 3{prime} noncoding region.

  5. Functional oligomerization of poliovirus RNA-dependent RNA polymerase.

    PubMed Central

    Pata, J D; Schultz, S C; Kirkegaard, K

    1995-01-01

    Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis. Images FIGURE 1 FIGURE 2 FIGURE 5 FIGURE 6 FIGURE 8 PMID:7489508

  6. Basic mechanism of transcription by RNA polymerase II

    PubMed Central

    Svetlov, Vladimir; Nudler, Evgeny

    2012-01-01

    RNA polymerase II-like enzymes carry out transcription of genomes in Eukaryota, Archaea, and some viruses. They also exhibit fundamental similarity to RNA polymerases from bacteria, chloroplasts, and mitochondria. In this review we take an inventory of recent studiesilluminating different steps of basic transcription mechanism, likely common for most multi-subunit RNA polymerases. Through the amalgamation of structural and computational chemistry data we attempt to highlight the most feasible reaction pathway for the two-metal nucleotidyl transfer mechanism, and to evaluate the way catalysis can be linked to translocation in the mechano-chemical cycle catalyzed by RNA polymerase II. PMID:22982365

  7. High-Throughput Polymerase Fidelity Evolution in Microfluidic Droplets

    NASA Astrophysics Data System (ADS)

    Collins, Jesse; de Paz, Alexandra; Cybulski, Ted; Bhan, Namita; Zhang, Huidan; Weitz, Dave; Tyo, Keith; Kording, Konrad

    Polymerases are technologically important as a tool in molecular biology, and are scientifically important for their role in DNA replication and inheritance. We study large numbers (at least millions) of polymerase mutants by compartmentalizing each gene in a droplet in a microfluidic device. Also in each droplet are in vitro transcription and translation proteins, such that mutant polymerases can be generated to extend their own gene along a known DNA template. Reading the resulting sequence tells us both the mutant gene sequence and the number of and particular errors that the resulting mutant polymerase made during extension. This work is supported by the NIH.

  8. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

    PubMed Central

    Bernad, A; Zaballos, A; Salas, M; Blanco, L

    1987-01-01

    The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases. Images Fig. 1. Fig. 2. Fig. 4. PMID:3127204

  9. Elongation of primed DNA templates by eukaryotic DNA polymerases.

    PubMed Central

    Ikeda, J E; Longiaru, M; Horwitz, M S; Hurwitz, J

    1980-01-01

    The combined action of DNA polymerase alpha and DNA polymerase beta leads to the synthesis of full-length linear DNA strands with phi X174 DNA templates containing an RNA primer. The reaction can be carried out in two stages. In the first stage, DNA polymerase alpha catalyzes the synthesis of a chain that averaged 230 deoxynucleotides long and was covalently linked to the RNA primer. In the second stage, DNA polymerase beta elongates the DNA strand covalently attached to the RNA primer to full length. With DNA primers, DNA polymerase alpha catalyzes only limited deoxynucleotide addition whereas DNA polymerase beta alone elongates DNA primed templates to full length. DNA polymerase beta can also stimulate the synthesis of adenovirus DNA in vitro in the presence of a cytosol extract from adenovirus-infected cells. In all of these systems, dNMP incorporation catalyzed by DNA polymerase beta was sensitive to N-ethylmaleimide; however, this polymerase activity was resistant to N-ethylmaleimide with poly(rA) x (dT) as the primer template. Images PMID:6160581

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

    PubMed

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

    2014-08-07

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

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

    PubMed

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

    2014-04-01

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

  12. Schisandrin B inhibits cell growth and induces cellular apoptosis and autophagy in mouse hepatocytes and macrophages: implications for its hepatotoxicity

    PubMed Central

    Zhang, Yi; Zhou, Zhi-Wei; Jin, Hua; Hu, Chengbin; He, Zhi-Xu; Yu, Zhi-Ling; Ko, Kam-Ming; Yang, Tianxin; Zhang, Xueji; Pan, Si-Yuan; Zhou, Shu-Feng

    2015-01-01

    A number of drugs and herbal compounds have been documented to cause hepatoxicity. Schisandrin B (Sch B) is an active dibenzocyclooctadiene isolated from Schisandrae fructus, with a wide array of pharmacological activities. However, the potential hepatotoxicity of Sch B is a major safety concern, and the underlying mechanism for Sch B-induced liver toxic effects is not fully elucidated. In the present study, we aimed to investigate the liver toxic effects and the molecular mechanisms of Sch B in mouse liver and macrophage cells. The results have shown that Sch B exhibits potent grow inhibitory, proapoptotic, and proautophagic effects in AML-12 and RAW 264.7 cells. Sch B markedly arrested cells in G1 phase in both cell lines, accompanied by the down-regulation of cyclin dependent kinase 2 (CDK2) and cyclin D1 and up-regulation of p27 Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl) B-cell lymphoma 2 (Bcl-2), but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax). Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP) in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and

  13. Effect of nanostructured TiO2 crystal phase on photoinduced apoptosis of breast cancer epithelial cells

    PubMed Central

    Lagopati, Nefeli; Tsilibary, Effie-Photini; Falaras, Polycarpos; Papazafiri, Panagiota; Pavlatou, Evangelia A; Kotsopoulou, Eleni; Kitsiou, Paraskevi

    2014-01-01

    Purpose The use of nanoparticles has seen exponential growth in the area of health care, due to the unique physicochemical properties of nanomaterials that make them desirable for medical applications. The aim of this study was to examine the effects of crystal phase-nanostructured titanium dioxide particles on bioactivity/cytotoxicity in breast cancer epithelial cells. Materials and methods Cultured Michigan Cancer Foundation (MCF)-7 and human breast adenocarcinoma (MDA-MB-468) breast cancer epithelial cells were exposed to ultraviolet A light (wavelength 350 nm) for 20 minutes in the presence of aqueous dispersions of two different nanostructured titanium dioxide (TiO2) crystal phases: anatase and an anatase–rutile mixture. Detailed characterization of each titanium dispersion was performed by dynamic light scattering. A 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) colorimetric assay was employed to estimate the percentage of viable cells after each treatment. Western blot analysis of protein expression and characterization, as well as a deoxyribonucleic acid (DNA)-laddering assay, were used to detect cell apoptosis. Results Our results documented that 100% anatase TiO2 nanoparticles (110–130 nm) exhibited significantly higher cytotoxicity in the highly malignant MDA-MB-468 cancer cells than anatase– rutile mixtures (75%/25%) with the same size. On the contrary, MCF-7 cells (characterized by low invasive properties) were not considerably affected. Exposure of MDA-MB-468 cells to pure anatase nanoparticles or anatase–rutile mixtures for 48 hours resulted in increased proapoptotic Bax expression, caspase-mediated poly(adenosine diphosphate ribose) polymerase (PARP) cleavage, DNA fragmentation, and programmed cell death/apoptosis. Conclusion The obtained results indicated that pure anatase TiO2 nanoparticles exhibit superior cytotoxic effects compared to anatase–rutile mixtures of the same size. The molecular mechanism of TiO2

  14. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β signaling pathway in H9c2 cardiomyocytes

    SciTech Connect

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang; Yi, Kyu Yang; Chung, Hun-Jong; Park, Jong Seok; Kim, Bokyung; Feng, Zhong-Ping; Shin, Hwa-Sup

    2013-04-01

    Poly(adenosine 5′-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3β (GSK-3β), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3β activation. In addition, inhibiting the Akt/GSK-3β pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3β pathway. - Highlights: ► 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ► 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ► 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ► 5-AIQ activated Akt and GSK-3

  15. CD73-adenosine: a next-generation target in immuno-oncology.

    PubMed

    Allard, David; Allard, Bertrand; Gaudreau, Pierre-Olivier; Chrobak, Pavel; Stagg, John

    2016-02-01

    Cancer immunotherapy has entered in a new era with the development of first-generation immune checkpoint inhibitors targeting the PD1/PD-L1 and CTLA-4 pathways. In this context, considerable research effort is being deployed to find the next generation of cancer immunotherapeutics. The CD73-adenosine axis constitutes one of the most promising pathways in immuno-oncology. We and others have demonstrated the immunosuppressive role of CD73-adenosine in cancer and established proof-of-concept that the targeted blockade of CD73 or adenosine receptors could effectively promote anti-tumor immunity and enhance the activity of first-generation immune checkpoint blockers. With Phase I clinical trials now underway evaluating anti-CD73 or anti-A2A therapies in cancer patients, we here discuss the fundamental, preclinical and clinical findings related to the role of the CD73-adenosinergic pathway in tumor immunity.

  16. SERS detection of low-concentration adenosine by silver nanoparticles on silicon nanoporous pyramid arrays structure

    NASA Astrophysics Data System (ADS)

    Zhang, C.; Man, B. Y.; Jiang, S. Z.; Yang, C.; Liu, M.; Chen, C. S.; Xu, S. C.; Qiu, H. W.; Li, Z.

    2015-08-01

    A novel surface-enhanced Raman spectroscopy (SERS) substrate based on uniform silver nanoparticles/silicon nanoporous pyramid arrays (Ag/PS) is prepared and SERS behaviors to adenosine are discussed and compared. With a low concentration of 10-7 M, the characteristic Raman bands of adenosine demonstrate the significantly high SERS sensitivity of the prepared Ag/PS substrate. A reasonable linear correlation is obtained between the intensity of SERS signal and the adenosine concentration from 10-2 to 10-7M in log scale. These results imply that the Ag/PS with regular pyramids array might be an effective substrate for performing label-free sensitive SERS detections of biomolecule.

  17. Neuromodulation and metamodulation by adenosine: Impact and subtleties upon synaptic plasticity regulation.

    PubMed

    Sebastião, Ana M; Ribeiro, Joaquim A

    2015-09-24

    Synaptic plasticity mechanisms, i.e. the sequence of events that underlies persistent changes in synaptic strength as a consequence of transient alteration in neuronal firing, are greatly influenced by the 'chemical atmosphere' of the synapses, that is to say by the presence of molecules at the synaptic cleft able to fine-tune the activity of other molecules more directly related to plasticity. One of those fine tuners is adenosine, known for a long time as an ubiquitous neuromodulator and metamodulator and recognized early as influencing synaptic plasticity. In this review we will refer to the mechanisms that adenosine can use to affect plasticity, emphasizing aspects of the neurobiology of adenosine relevant to its ability to control synaptic functioning. This article is part of a Special Issue entitled Brain and Memory.

  18. 8-(2-Furyl)adenine derivatives as A₂A adenosine receptor ligands.

    PubMed

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Thomas, Ajiroghene; Klotz, Karl-Norbert; Federico, Stephanie; Cacciari, Barbara; Spalluto, Giampiero; Volpini, Rosaria

    2013-01-01

    Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2-furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A₂A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N(6)-position takes to different effects on the A₂A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A₂A receptor crystal structures provides an interpretation of the obtained biological results.

  19. Role of adenosine in the antiepileptic effects of deep brain stimulation

    PubMed Central

    Miranda, Maisa F.; Hamani, Clement; de Almeida, Antônio-Carlos G.; Amorim, Beatriz O.; Macedo, Carlos E.; Fernandes, Maria José S.; Nobrega, José N.; Aarão, Mayra C.; Madureira, Ana Paula; Rodrigues, Antônio M.; Andersen, Monica L.; Tufik, Sergio; Mello, Luiz E.; Covolan, Luciene

    2014-01-01

    Despite the effectiveness of anterior thalamic nucleus (AN) deep brain stimulation (DBS) for the treatment of epilepsy, mechanisms responsible for the antiepileptic effects of this therapy remain elusive. As adenosine modulates neuronal excitability and seizure activity in animal models, we hypothesized that this nucleoside could be one of the substrates involved in the effects of AN DBS. We applied 5 days of stimulation to rats rendered chronically epileptic by pilocarpine injections and recorded epileptiform activity in hippocampal slices. We found that slices from animals given DBS had reduced hippocampal excitability and were less susceptible to develop ictal activity. In live animals, AN DBS significantly increased adenosine levels in the hippocampus as measured by microdialysis. The reduced excitability of DBS in vitro was completely abolished in animals pre-treated with A1 receptor antagonists and was strongly potentiated by A1 receptor agonists. We conclude that some of the antiepileptic effects of DBS may be mediated by adenosine. PMID:25324724

  20. Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex

    SciTech Connect

    Florio, C.; Rosati, A.M.; Traversa, U.; Vertua, R. )

    1991-01-01

    In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6-cyclohexyl-({sup 3}H)adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks. Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system.

  1. The breakdown of adenosine triphosphate in the contraction cycle of the frog sartorius muscle

    PubMed Central

    Mommaerts, W. F. H. M.; Wallner, A.

    1967-01-01

    1. It is confirmed that a fluorodinitrobenzene (FDNB)-treated frog sartorius muscle does not split phosphorylcreatine in the course of its contraction cycle, but does use adenosine triphosphate (ATP). 2. Good stoicheiometric relations between the diminution of ATP and the formation of adenosine diphosphate (ADP), adenosine monophosphate (AMP) and phosphate are obtained, and in a 0·2 sec tetanus at 0° C the net break-down of ATP amounts to 0·27, the total equivalent break-down to 0·34 μmoles/g. 3. There is no difference in this quantity between muscles interrupted at the height of contraction and those that have also relaxed, and, in experiments specifically designed to determine relaxation metabolism separately, no such metabolism is found. Thus, all the ATP-break-down occurs in the contraction phase. PMID:6065882

  2. Autoradiographic localization of adenosine receptors in rat brain using (/sup 3/H)cyclohexyladenosine

    SciTech Connect

    Goodman, R.R.; Synder, S.H.

    1982-09-01

    Adenosine (A1) receptor binding sites have been localized in rat brain by an in vitro light microscopic autoradiographic method. The binding of (/sup 3/H)N6-cyclohexyladenosine to slide-mounted rat brain tissue sections has the characteristics of A1 receptors. It is saturable with high affinity and has appropriate pharmacology and stereospecificity. The highest densities of adenosine receptors occur in the molecular layer of the cerebellum, the molecular and polymorphic layers of the hippocampus and dentate gyrus, the medial geniculate body, certain thalamic nuclei, and the lateral septum. High densities also are observed in certain layers of the cerebral cortex, the piriform cortex, the caudate-putamen, the nucleus accumbens, and the granule cell layer of the cerebellum. Most white matter areas, as well as certain gray matter areas, such as the hypothalamus, have negligible receptor concentrations. These localizations suggest possible central nervous system sites of action of adenosine.

  3. Cocaine exposure modulates dopamine and adenosine signaling in the fetal brain

    PubMed Central

    Kubrusly, Regina C. C.; Bhide, Pradeep G.

    2009-01-01

    Exposure to cocaine during the fetal period can produce significant lasting changes in the structure and function of the brain. Cocaine exerts its effects on the developing brain by blocking monoamine transporters and impairing monoamine receptor signaling. Dopamine is a major central target of cocaine. In a mouse model, we show that cocaine exposure from embryonic day 8 (E8) to E14 produces significant reduction in dopamine transporter activity, attenuation of dopamine D1-receptor function and upregulation of dopamine D2-receptor function. Cocaine’s effects on the D1-receptor are at the level of protein expression as well as activity. The cocaine exposure also produces significant increases in basal cAMP levels in the striatum and cerebral cortex. The increase in the basal cAMP levels was independent of dopamine receptor activity. In contrast, blocking the adenosine A2a receptor downregulated of the basal cAMP levels in the cocaine-exposed brain to physiological levels, suggesting the involvement of adenosine receptors in mediating cocaine’s effects on the embryonic brain. In support of this suggestion, we found that the cocaine exposure downregulated adenosine transporter function. We also found that dopamine D2- and adenosine A2a-receptors antagonize each other’s function in the embryonic brain in a manner consistent with their interactions in the mature brain. Thus, our data show that prenatal cocaine exposure produces direct effects on both the dopamine and adenosine systems. Furthermore, the dopamine D2 and adenosine A2a receptor interactions in the embryonic brain discovered in this study unveil a novel substrate for cocaine’s effects on the developing brain. PMID:19765599

  4. Effect of adenosine system in the action of oseltamivir on behavior in mice.

    PubMed

    Uchiyama, Hidemori; Hiromura, Makoto; Shiratani, Tomonori; Kuroki, Hiroaki; Honda, Sinichiro; Kosako, Kazuhiro; Soeda, Shinji; Inoue, Kazuhide; Toda, Akihisa

    2015-07-10

    Abnormal behaviors and death associated with the use of oseltamivir (Tamiflu(®)) have emerged as a major issue in influenza patients. We have previously reported that the mechanisms underlying the effects of caffeine, a non-selective adenosine A1/A2 receptor antagonist, combined with oseltamivir. Oseltamivir is rapidly hydrolyzed to its active form (oseltamivir carboxylate, OCB). In this study, we investigated the effects of an adenosine system and OCB on the action of oseltamivir on mice behavior. Oseltamivir for 1 day (150 mg/kg, intraperitoneally (i.p.)) alone did not affect ambulation at 2 h post-injection. However, caffeine (10 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, caffeine (30 mg/kg, i.p.) in combination with oseltamivir for 3 days increased ambulation, but caffeine (10 mg/kg, i.p.) in combination with oseltamivir for 3 days did not increase. These enhancements were inhibited by an adenosine A2 receptor agonist, CGS21680 (0.2 mg/kg, subcutaneously (s.c.)). Furthermore, an adenosine A2 receptor antagonist, SCH58261 (1 and 3 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, SCH58261 (3 mg/kg, i.p.) in combination with oseltamivir for 3 days increased ambulation, but SCH58261 (1 mg/kg, i.p.) in combination with oseltamivir for 3 days did not. Conversely, in phenobarbital (PB)-treated mice, caffeine (3 mg/kg, i.p.) in combination with oseltamivir for 1 day increased ambulation. Moreover, OCB for 1 day (0.3 μg/mouse intracerebroventricular (i.c.v.)) alone increased ambulation. These findings suggest that the actions of oseltamivir may involve the adenosine systems and its metabolism. Our findings suggest an interaction between the central blockade of adenosine A2 receptors by caffeine and OCB-induced behavioral changes.

  5. Extracellular adenosine concentrations during in vitro ischaemia in rat hippocampal slices

    PubMed Central

    Latini, Serena; Bordoni, Francesca; Pedata, Felicita; Corradetti, Renato

    1999-01-01

    The application of an ischaemic insult in hippocampal slices results in the depression of synaptic transmission, mainly attributed to the activation of A1 adenosine receptors by adenosine released in the extracellular space. To estimate the concentration of endogenous adenosine acting at the receptor level during an ischaemic episode, we recorded field e.p.s.ps (fe.p.s.ps) from hippocampal slices, and evaluated the ability of the selective A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), to reverse the fe.p.s.p. depression induced by in vitro ischaemia. A relationship between the IC50 of an antagonist and the endogenous concentration of a neurotransmitter has been used for pharmacological analysis. The complete and reversible depression of fe.p.s.p. in the CA1 region induced by 5 min ischaemia was decreased in the presence of DPCPX (50–500 nM). 8-Phenyltheophylline (10 μM) abolished the depression of fe.p.s.ps during the ischaemic period, while a small (peak effect 12±4%) decrease in fe.p.s.ps was observed during the initial phase of reperfusion. In the time-interval of maximal depression of fe.p.s.ps., IC50 and adenosine concentration changed as function of time with a good degree of correlation. The maximal value of adenosine concentration was 30 μM. Our data provide an estimation of the adenosine concentration reached at the receptor level during an ischaemic episode, with a higher time discrimination (15 s) than that achieved with any biochemical approach. This estimation may be useful in order to establish appropriate concentrations of purinergic compounds to be tested for their pharmacological effects during an ischaemic episode. PMID:10401564

  6. Synthesis of adenosine-imprinted microspheres for the recognition of ADP-ribosylated proteins.

    PubMed

    Gong, Xia; Tang, Biao; Liu, Jing Jing; You, Xiang Yu; Gu, Jing; Deng, Jiao Yu; Xie, Wei-Hong

    2017-01-15

    Core-shell structural adenosine-imprinted microspheres were prepared via a two-step procedure. Polystyrene core particles (CP) were firstly prepared via a reversible addition-fragmentation chain transfer (RAFT) polymerization leaving the iniferter on the surface of the cores, then a molecularly imprinted polymer (MIP) shell was synthesized on the surface of the cores by using acrylamide (AAm) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The formation and growth of the MIP layer were seen dependent on the initiator (AIBN), AAm and the polymerization time used within the polymerization. SEM/TEM images showed that the dimensions of the cores and shells were 2μM and 44nm, respectively. The MIP microspheres exhibited a fast rebinding rate within 2h and a maximum adsorption capacity of 177μg per gram for adenosine. The adsorption fitted a Langmuir-Freundlich (LF) isotherm model with a KLF value of 41mL/μg and a qm value of 177μg/g for the MIP microspheres. The values were larger than those for a non-molecularly imprinted polymer (NIP) particles (5mL/μg and 88μg/g) indicating a better adsorption ability towards adenosine. The MIP microspheres showed a good selectivity for adenosine with a higher adsorption (683nmol/g) for adenosine than that (91nmol/g, 24nmol/g and 54nmol/g) for guanosine, cytidine and uridine respectively. Further experiment proved that the adenosine-imprinted polymer microspheres also had a good selectivity for ADP-ribosylated proteins that the MIP could extract the ADP-ribosylated proteins from the cell extract samples.

  7. Adenosine transport systems on dissociated brain cells from mouse, guinea-pig, and rat

    SciTech Connect

    Johnston, M.E.; Geiger, J.D. )

    1990-09-01

    The kinetics and sodium dependence of adenosine transport were determined using an inhibitor-stop method on dissociated cell body preparations obtained from mouse, guinea-pig and rat brain. Transport affinity (KT) values for the high affinity adenosine transport systems KT(H) were significantly different between these three species; mean +/- SEM values were 0.34 +/- 0.1 in mouse, 0.9 +/- 0.2 in rat, and 1.5 +/- 0.5 microM in guinea-pig. The KT values for the low affinity transport system KT(L) were not different between the three species. Brain cells from rat displayed a significantly greater maximal capacity to accumulate (3H)adenosine (Vmax) than did mouse or guinea-pig for the high affinity system, or than did mouse for the low affinity system. When sodium chloride was replaced in the transport medium with choline chloride, the KT(H) values for guinea-pig and rat were both increased by approximately 100%; only in rat did the change reach statistical significance. The sodium-dependence of adenosine transport in mouse brain was clearly absent. The differences between KT(H) values in mouse and those in guinea-pig or rat were accentuated in the absence of sodium. The differences in kinetic values, ionic requirements, and pharmacological characteristics between adenosine transporters in CNS tissues of mouse, guinea-pig and rat may help account for some of the variability noted among species in terms of their physiological responses to adenosine.

  8. Adenosine potentiates the therapeutic effects of neural stem cells expressing cytosine deaminase against metastatic brain tumors.

    PubMed

    Kang, Wonyoung; Seol, Ho Jun; Seong, Dong-Ho; Kim, Jandi; Kim, Yonghyun; Kim, Seung U; Nam, Do-Hyun; Joo, Kyeung Min

    2013-09-01

    Tumor-tropic properties of neural stem cells (NSCs) provide a novel approach with which to deliver targeting therapeutic genes to brain tumors. Previously, we developed a therapeutic strategy against metastatic brain tumors using a human NSC line (F3) expressing cytosine deaminase (F3.CD). F3.CD converts systemically administered 5-fluorocytosine (5-FC), a blood-brain barrier permeable nontoxic prodrug, into the anticancer agent 5-fluorouracil (5-FU). In this study, we potentiated a therapeutic strategy of treatment with nucleosides in order to chemically facilitate the endogenous conversion of 5-FU to its toxic metabolite 5-FU ribonucleoside (5-FUR). In vitro, 5-FUR showed superior cytotoxic activity against MDA-MB-435 cancer cells when compared to 5-FU. Although adenosine had little cytotoxic activity, the addition of adenosine significantly potentiated the in vitro cytotoxicity of 5-FU. When MDA-MB‑435 cells were co-cultured with F3.CD cells, F3.CD cells and 5-FC inhibited the growth of MDA-MB-435 cells more significantly in the presence of adenosine. Facilitated 5-FUR production by F3.CD was confirmed by an HPLC analysis of the conditioned media derived from F3.CD cells treated with 5-FC and adenosine. In vivo systemic adenosine treatment also significantly potentiated the therapeutic effects of F3.CD cells and 5-FC in an MDA-MB-435 metastatic brain tumor model. Simple adenosine addition improved the antitumor activity of the NSCs carrying the therapeutic gene. Our results demonstrated an increased therapeutic potential, and thereby, clinical applicability of NSC-based gene therapy.

  9. Adenosine A1 and A3 receptors protect astrocytes from hypoxic damage.

    PubMed

    Björklund, Olga; Shang, Mingmei; Tonazzini, Ilaria; Daré, Elisabetta; Fredholm, Bertil B

    2008-10-31

    Brain levels of adenosine are elevated during hypoxia. Through effects on adenosine receptors (A(1), A(2A), A(2B) and A(3)) on astrocytes, adenosine can influence functions such as glutamate uptake, reactive gliosis, swelling, as well as release of neurotrophic and neurotoxic factors having an impact on the outcome of metabolic stress. We have studied the roles of these receptors in astrocytes by evaluating their susceptibility to damage induced by oxygen deprivation or exposure to the hypoxia mimic cobalt chloride (CoCl(2)). Hypoxia caused ATP breakdown and purine release, whereas CoCl(2) (0.8 mM) mainly reduced ATP by causing cell death in human D384 astrocytoma cells. Further experiments were conducted in primary astrocytes prepared from specific adenosine receptor knock-out (KO) and wild type (WT) mice. In WT cells purine release following CoCl(2) exposure was mainly due to nucleotide release, whereas hypoxia-induced intracellular ATP breakdown followed by nucleoside efflux. N-ethylcarboxamidoadenosine (NECA), an unselective adenosine receptor agonist, protected from cell death following hypoxia. Cytotoxicity was more pronounced in A(1)R KO astrocytes and tended to be higher in WT cells in the presence of the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Genetic deletion of A(2A) receptor resulted in less prominent effects. A(3)R KO glial cells were more affected by hypoxia than WT cells. Accordingly, the A(3) receptor agonist 2-chloro-N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) reduced ATP depletion caused by hypoxic conditions. It also reduced apoptosis in human astroglioma D384 cells after oxygen deprivation. In conclusion, the data point to a cytoprotective role of adenosine mediated by both A(1) and A(3) receptors in primary mouse astrocytes.

  10. Adenosine receptor antagonists alter the stability of human epileptic GABAA receptors

    PubMed Central

    Roseti, Cristina; Martinello, Katiuscia; Fucile, Sergio; Piccari, Vanessa; Mascia, Addolorata; Di Gennaro, Giancarlo; Quarato, Pier Paolo; Manfredi, Mario; Esposito, Vincenzo; Cantore, Gianpaolo; Arcella, Antonella; Simonato, Michele; Fredholm, Bertil B.; Limatola, Cristina; Miledi, Ricardo; Eusebi, Fabrizio

    2008-01-01

    We examined how the endogenous anticonvulsant adenosine might influence γ-aminobutyric acid type A (GABAA) receptor stability and which adenosine receptors (ARs) were involved. Upon repetitive activation (GABA 500 μM), GABAA receptors, microtransplanted into Xenopus oocytes from neurosurgically resected epileptic human nervous tissues, exhibited an obvious GABAA-current (IGABA) run-down, which was consistently and significantly reduced by treatment with the nonselective adenosine receptor antagonist CGS15943 (100 nM) or with adenosine deaminase (ADA) (1 units/ml), that inactivates adenosine. It was also found that selective antagonists of A2B (MRS1706, 10 nM) or A3 (MRS1334, 30 nM) receptors reduced IGABA run-down, whereas treatment with the specific A1 receptor antagonist DPCPX (10 nM) was ineffective. The selective A2A receptor antagonist SCH58261 (10 nM) reduced or potentiated IGABA run-down in ≈40% and ≈20% of tested oocytes, respectively. The ADA-resistant, AR agonist 2-chloroadenosine (2-CA) (10 μM) potentiated IGABA run-down but only in ≈20% of tested oocytes. CGS15943 administration again decreased IGABA run-down in patch-clamped neurons from either human or rat neocortex slices. IGABA run-down in pyramidal neurons was equivalent in A1 receptor-deficient and wt neurons but much larger in neurons from A2A receptor-deficient mice, indicating that, in mouse cortex, GABAA-receptor stability is tonically influenced by A2A but not by A1 receptors. IGABA run-down from wt mice was not affected by 2-CA, suggesting maximal ARs activity by endogenous adenosine. Our findings strongly suggest that cortical A2–A3 receptors alter the stability of GABAA receptors, which could offer therapeutic opportunities. PMID:18809912

  11. Bench-to-bedside review: Adenosine receptors – promising targets in acute lung injury?

    PubMed Central

    Schepp, Carsten P; Reutershan, Jörg

    2008-01-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are life-threatening disorders that have substantial adverse effects on outcomes in critically ill patients. ALI/ARDS develops in response to pulmonary or extrapulmonary injury and is characterized by increased leakage from the pulmonary microvasculature and excessive infiltration of polymorphonuclear cells into the lung. Currently, no therapeutic strategies are available to control these fundamental pathophysiological processes in human ALI/ARDS. In a variety of animal models and experimental settings, the purine nucleoside adenosine has been demonstrated to regulate both endothelial barrier integrity and polymorphonuclear cell trafficking in the lung. Adenosine exerts its effects through four G-protein-coupled receptors (A1, A2A, A2B, and A3) that are expressed on leukocytes and nonhematopoietic cells, including endothelial and epithelial cells. Each type of adenosine receptor (AR) is characterized by a unique pharmacological and physiological profile. The development of selective AR agonists and antagonists, as well as the generation of gene-deficient mice, has contributed to a growing understanding of the cellular and molecular processes that are critically involved in the development of ALI/ARDS. Adenosine-dependent pathways are involved in both protective and proinflammatory effects, highlighting the need for a detailed characterization of the distinct pathways. This review summarizes current experimental observations on the role of adenosine signaling in the development of acute lung injury and illustrates that adenosine and ARs are promising targets that may be exploited in the development of innovative therapeutic strategies. PMID:18828873

  12. [Conformation of adenosine deaminase in complexes with inhibitors: application of selective quenching of fluorescence emission].

    PubMed

    Vermishian, I G; Sharoian, S G; Antonian, A A; Grigorian, N A; Mardanian, S S; Khoetsian, A V; Markarian, Sh A

    2008-01-01

    The effect of inhibitors, 1-deazaadenosine (1-dAdo) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), on the conformation of adenosine deaminase was studied using the method of selective quenching of fluorescence emission by acrylamide, I- and Cs+. Both in free adenosine deaminase and in its complexes with the inhibitors, the wavelength maxima and half-width of the emission characterize the environment of fluorescing tryptophan residues in adenosine deaminase as weak polar with limited access to solvent. The formation of complexes with the ground state inhibitors used did not quench or change the main emission characteristics of tryptophan fluorescence in adenosine deaminase. Small blue shifts of emission maxima were observed upon quenching in all three samples. The Stern-Volmer parameters of tryptophan fluorescence quenching by acrylamide were not essentially influenced by complex formation of the enzyme with the inhibitors: in general, the folding of the enzyme molecule in the complexes is not perturbed. On the contrary, the emission quenching by charged heavy ions, I- and Cs+, in the complexes was hindered in comparison with free adenosine deaminase. In the complex with 1-deazaadenosine, the parameters for quenching by both ions evidence the essential worsening of their interaction with tryptophans. In the complex with erythro-9-(2-hydroxy-3-nonyl)adenine, along with the worse quenching by I-, complete prohibition of quenching by Cs+ was observed. These data indicate that the local environments of fluorescing tryptophan residues is substantially distorted compared with free adenosine deaminase, which leads to their screening from charged heavy ions.

  13. Abnormal regulation of DNA replication and increased lethality in ataxia telangiectasia cells exposed to carcinogenic agents

    SciTech Connect

    Jaspers, N.G.; de Wit, J.; Regulski, M.R.; Bootsma, D.

    1982-01-01

    The effect of different carcinogenic agents on the rate of semiconservative DNA replication in normal and ataxia telangiectasis (AT) cells was investigated. The rate of DNA synthesis in all AT cell strains tested was depressed to a significantly lesser extent than in normal cells after exposure to X-rays under oxia or hypoxia or to bleomycin, agents to which AT cells are hypersensitive. In contrast, inhibition of DNA replication in normal human and AT cells was similar after treatment with some DNA-methylating agents or mitomycin C. Colony-forming ability of AT cells treated with these agents was not different from normal cells. Treatment with 4-nitroquinoline 1-oxide elicited a variable response in both AT and normal cell strains. In some strains, including those shown to be hypersensitive to the drug by other workers, the inhibition of DNA synthesis was more pronounced than in other cell strains, but no significant difference between AT and normal cells could be detected. The rejoining of DNA strand breaks induced by X-rays, measured by DNA elution techniques, occurred within l2 hr after treatment and could not be correlated with the difference in DNA synthesis inhibition in AT and normal cells. After low doses of X-rays, AT cells rejoined single-strand breaks slightly more slowly than did normal cells. The rate of DNA replication in X-irradiation AT and normal cells was not affected by nicotinamide, an inhibitor of poly(adenosine diphosphate ribose) synthesis. These data indicate that the diminished inhibition of DNA replication in carcinogen-treated AT cells (a) is a general characteristic of all AT cell strains, (b) correlates with AT cellular hypersensitivity, (c) is not directly caused by the bulk of the DNA strand breaks produced by carcinogenic agents, and (d) is not based on differences in the induction of poly(adenosine diphosphate ribose) synthesis between X-irradiated AT and normal cells.

  14. Platelet-derived growth factor receptor-α-positive cells and not smooth muscle cells mediate purinergic hyperpolarization in murine colonic muscles.

    PubMed

    Kurahashi, Masaaki; Mutafova-Yambolieva, Violeta; Koh, Sang Don; Sanders, Kenton M

    2014-09-15

    Enteric inhibitory neurotransmission is an important feature of the neural regulation of gastrointestinal motility. Purinergic neurotransmission, via P2Y1 receptors, mediates one phase of inhibitory neural control. For decades, ATP has been assumed to be the purinergic neurotransmitter and smooth muscle cells (SMCs) have been considered the primary targets for inhibitory neurotransmission. Recent experiments have cast doubt on both of these assumptions and suggested that another cell type, platelet-derived growth factor receptor-α-positive (PDGFRα(+)) cells, is the target for purinergic neurotransmission. We compared responses of PDGFRα(+) cells and SMCs to several purine compounds to determine if these cells responded in a manner consistent with enteric inhibitory neurotransmission. ATP hyperpolarized PDGFRα(+) cells but depolarized SMCs. Only part of the ATP response in PDGFRα(+) cells was blocked by MRS 2500, a P2Y1 antagonist. ADP, MRS 2365, β-NAD, and adenosine 5-diphosphate-ribose, P2Y1 agonists, hyperpolarized PDGFRα(+) cells, and these responses were blocked by MRS 2500. Adenosine 5-diphosphate-ribose was more potent in eliciting hyperpolarization responses than β-NAD. P2Y1 agonists failed to elicit responses in SMCs. Small hyperpolarization responses were elicited in SMCs by a small-conductance Ca(2+)-activated K(+) channel agonist, cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine, consistent with the low expression and current density of small-conductance Ca(2+)-activated K(+) channels in these cells. Large-amplitude hyperpolarization responses, elicited in PDGFRα(+) cells, but not SMCs, by P2Y1 agonists are consistent with the generation of inhibitory junction potentials in intact muscles in response to purinergic neurotransmission. The responses of PDGFRα(+) cells and SMCs to purines suggest that SMCs are unlikely targets for purinergic neurotransmission in colonic muscles.

  15. A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

    PubMed

    Riewe, David; Grosman, Lukasz; Fernie, Alisdair R; Zauber, Henrik; Wucke, Cornelia; Geigenberger, Peter

    2008-10-01

    Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

  16. Binary Drugs: Conjugates of Purines and a Peptide That Bind to Both Adenosine and Substance P Receptors

    PubMed Central

    Jacobson, Kenneth A.; Lipkowski, Andrzej W.; Moody, Terry W.; Padgett, William; Pijl, Evelyn; Kirk, Kenneth L.; Daly, John W.

    2012-01-01

    A “functionalized congener” approach to adenosine receptor antagonists has provided a means to synthesize highly potent peptide conjugates of 1,3-dialkylxanthines. The antagonist XAC, such a functionalized xanthine amine congener, has been attached to a segment derived from the neurotransmitter peptide substance P (SP) to form a binary drug that binds to both receptors with Ki values of 35 nM (central A1-adenosine) and 300 nM (striatal SP). Coupling of the functionalized adenosine agonist N6-[p-(carboxymethyl)phenyl]adenosine to an SP C-terminal peptide also resulted in a binary drug that binds to both receptors. The demonstration that the biochemical properties of two unrelated drugs, both of which act through binding at extracellular receptors, may be combined in the same molecule suggests a novel strategy for drug design. In principle, a combined effect of the two different substances that produce the same final effect (e.g., hypotension by adenosine agonists and by SP analogues) might occur in vivo. Adenosine analogues have analgesic properties, and the binary drug derived from substance P and adenosine agonists or antagonists might provide useful tools for probing interrelationships of SP pathways and sites for the antinociceptive action of adenosine. PMID:2441057

  17. Adenosine Generated in the Bone Marrow Niche Through a CD38-Mediated Pathway Correlates With Progression of Human Myeloma

    PubMed Central

    Horenstein, Alberto L; Quarona, Valeria; Toscani, Denise; Costa, Federica; Chillemi, Antonella; Pistoia, Vito; Giuliani, Nicola; Malavasi, Fabio

    2016-01-01

    Human myeloma cells express CD38 at high levels and grow in hypoxic niches inside the bone marrow. Myeloma cells respond to hypoxia with metabolic changes leading to aerobic glycolysis, thus reducing adenosine triphosphate (ATP) and increasing NAD+. Our hypothesis is that these conditions favor the enzymatic pathways involved in the production of adenosine in the niche. Within the niche, NAD+ is able to activate a discontinuous adenosinergic pathway that relies upon CD38, CD203a and CD73 or TRACP, according to the environmental pH. The observed variability in adenosine concentrations in bone marrow aspirates is a result of the interactions taking place among myeloma and other cells in the bone marrow niche. A pilot study showed that adenosine profiles differ during disease progression. Adenosine levels were significantly higher in the bone marrow plasma of patients with symptomatic myeloma and correlated with ISS staging, suggesting that adenosine is produced in the myeloma niche at micromolar levels by an ectoenzymatic network centered on CD38. Adenosine levels increase with disease aggressiveness, a finding that supports adenosine as a potential marker of myeloma progression. PMID:27761584

  18. Role of renal medullary adenosine in the control of blood flow and sodium excretion.

    PubMed

    Zou, A P; Nithipatikom, K; Li, P L; Cowley, A W

    1999-03-01

    This study determined the levels of adenosine in the renal medullary interstitium using microdialysis and fluorescence HPLC techniques and examined the role of endogenous adenosine in the control of medullary blood flow and sodium excretion by infusing the specific adenosine receptor antagonists or agonists into the renal medulla of anesthetized Sprague-Dawley rats. Renal cortical and medullary blood flows were measured using laser-Doppler flowmetry. Analysis of microdialyzed samples showed that the adenosine concentration in the renal medullary interstitial dialysate averaged 212 +/- 5.2 nM, which was significantly higher than 55.6 +/- 5.3 nM in the renal cortex (n = 9). Renal medullary interstitial infusion of a selective A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 300 pmol. kg-1. min-1, n = 8), did not alter renal blood flows, but increased urine flow by 37% and sodium excretion by 42%. In contrast, renal medullary infusion of the selective A2 receptor blocker 3, 7-dimethyl-1-propargylxanthine (DMPX; 150 pmol. kg-1. min-1, n = 9) decreased outer medullary blood flow (OMBF) by 28%, inner medullary blood flows (IMBF) by 21%, and sodium excretion by 35%. Renal medullary interstitial infusion of adenosine produced a dose-dependent increase in OMBF, IMBF, urine flow, and sodium excretion at doses from 3 to 300 pmol. kg-1. min-1 (n = 7). These effects of adenosine were markedly attenuated by the pretreatment of DMPX, but unaltered by DPCPX. Infusion of a selective A3 receptor agonist, N6-benzyl-5'-(N-ethylcarbonxamido)adenosine (300 pmol. kg-1. min-1, n = 6) into the renal medulla had no effect on medullary blood flows or renal function. Glomerular filtration rate and arterial pressure were not changed by medullary infusion of any drugs. Our results indicate that endogenous medullary adenosine at physiological concentrations serves to dilate medullary vessels via A2 receptors, resulting in a natriuretic response that overrides the tubular A1 receptor

  19. New QSAR combined strategy for the design of A1 adenosine receptor agonists.

    PubMed

    González, Maykel Pérez; Besada, Pedro; González Moa, Maria José; Teijeira, Marta; Terán, Carmen

    2008-02-15

    Combined discriminant and regression analysis was carried out on a series of 167 A1 adenosine receptor agonists to identify the best linear and nonlinear models for the design of new compounds with a better biological profile. On the basis of the best linear discriminant analysis and both linear and nonlinear Multi Layer Perceptron neural networks regression, we have designed and synthesized 14 carbonucleoside analogues of adenosine. Their biological activities were predicted and experimentally measured to demonstrate the capability of our model to avoid the prediction of false positives. A good agreement was found between the calculated and observed biological activity.

  20. Exploiting Protein Conformational Change to Optimize Adenosine-Derived Inhibitors of HSP70

    PubMed Central

    2016-01-01

    HSP70 is a molecular chaperone and a key component of the heat-shock response. Because of its proposed importance in oncology, this protein has become a popular target for drug discovery, efforts which have as yet brought little success. This study demonstrates that adenosine-derived HSP70 inhibitors potentially bind to the protein with a novel mechanism of action, the stabilization by desolvation of an intramolecular salt-bridge which induces a conformational change in the protein, leading to high affinity ligands. We also demonstrate that through the application of this mechanism, adenosine-derived HSP70 inhibitors can be optimized in a rational manner. PMID:27119979

  1. Regulation of photoreceptor gap junction phosphorylation by adenosine in zebrafish retina

    PubMed Central

    Li, Hongyan; Chuang, Alice Z.; O’Brien, John

    2014-01-01

    Electrical coupling of photoreceptors through gap junctions suppresses voltage noise, routes rod signals into cone pathways, expands the dynamic range of rod photoreceptors in high scotopic and mesopic illumination, and improves detection of contrast and small stimuli. In essentially all vertebrates, connexin 35/36 (gene homologues Cx36 in mammals, Cx35 in other vertebrates) is the major gap junction protein observed in photoreceptors, mediating rod-cone, cone-cone, and possibly rod-rod communication. Photoreceptor coupling is dynamically controlled by the day/night cycle and light/dark adaptation, and is directly correlated with phosphorylation of Cx35/36 at two sites, serine110 and serine 276/293 (homologous sites in teleost fish and mammals respectively). Activity of protein kinase A (PKA) plays a key role during this process. Previous studies have shown that activation of dopamine D4 receptors on photoreceptors inhibits adenylyl cyclase, down-regulates cAMP and PKA activity, and leads to photoreceptor uncoupling, imposing the daytime/light condition. In this study we explored the role of adenosine, a nighttime signal with a high extracellular concentration at night and a low concentration in the day, in regulating photoreceptor coupling by examining photoreceptor Cx35 phosphorylation in zebrafish retina. Adenosine enhanced photoreceptor Cx35 phosphorylation in daytime, but with a complex dose-response curve. Selective pharmacological manipulations revealed that adenosine A2a receptors provide a potent positive drive to phosphorylate photoreceptor Cx35 under the influence of endogenous adenosine at night. A2a receptors can be activated in the daytime as well by micromolar exogenous adenosine. However, the higher affinity adenosine A1 receptors are also present and have an antagonistic though less potent effect. Thus the nighttime/darkness signal adenosine provides a net positive drive on Cx35 phosphorylation at night, working in opposition to dopamine to

  2. Adenosine Receptor Regulation of Coronary Blood Flow in Ossabaw Miniature SwineS⃞

    PubMed Central

    Long, Xin; Mokelke, Eric A.; Neeb, Zachary P.; Alloosh, Mouhamad; Edwards, Jason M.

    2010-01-01

    Adenosine clearly regulates coronary blood flow (CBF); however, contributions of specific adenosine receptor (AR) subtypes (A1, A2A, A2B, A3) to CBF in swine have not been determined. ARs generally decrease (A1, A3) or increase (A2A, A2B) cyclic adenosine monophosphate, a major mediator of vasodilation. We hypothesized that A1 antagonism potentiates coronary vasodilation and coronary stent deployment in dyslipidemic Ossabaw swine elicits impaired vasodilation to adenosine that is associated with increased A1/A2A expression. The left main coronary artery was accessed with a guiding catheter allowing intracoronary infusions. After placement of a flow wire into the left circumflex coronary artery the responses to bolus infusions of adenosine were obtained. Steady-state infusion of AR-specific agents was achieved by using a small catheter fed over the flow wire in control pigs. CBF was increased by the A2-nonselective agonist 2-phenylaminoadenosine (CV1808) in a dose-dependent manner. Baseline CBF was increased by the highly A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), but not changed by other AR-specific agents. The nonselective A2 antagonist 3,7-dimethyl-1-propargylxanthine and A2A-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385) abolished adenosine-induced CBF, whereas A2B and A3 antagonism had no effect. Dyslipidemia and stenting decreased adenosine-induced CBF ∼70%, whereas A1, A2A, and A2B mRNA were up-regulated in dyslipidemic versus control >5-fold and there was no change in the ratio of A1/A2A protein in microvessels distal to the stent. In control Ossabaw swine A1 antagonism by DPCPX positively regulated basal CBF. Impaired adenosine-induced CBF after stenting in dyslipidemia is most likely caused by the altered balance between A1 and A2A signaling, not receptor expression. PMID:20855445

  3. Termination of idiopathic sustained monomorphic ventricular tachycardia by intravenous adenosine in a pregnant woman.

    PubMed

    Hasdemir, Can; Musayev, Oktay; Alkan, Mustafa B; Can, Levent H; Kultursay, Hakan

    2009-11-01

    A 34-year-old pregnant woman presented to the emergency department with the complaints of palpitations at 32 weeks gestation. The diagnosis of right ventricular outflow tract ventricular tachycardia (VT) was made. Intravenous 5 mg of metoprolol and 25 mg of diltiazem did not terminate the VT. Ten milligrams of adenosine were administered. Within 10 s of adenosine administration, sustained VT converted to repetitive monomorphic VT and within 30 s to normal sinus rhythm. The mother and the foetus tolerated the medications well. Non-stress test for the assessment of the foetal well-being was normal.

  4. Adenosine(5') oligophospho-(5') guanosines and guanosine(5') oligophospho-(5') guanosines in human platelets.

    PubMed Central

    Schlüter, H; Grobeta, I; Bachmann, J; Kaufmann, R; van der Giet, M; Tepel, M; Nofer, J R; Assmann, G; Karas, M; Jankowski, J; Zidek, W

    1998-01-01

    We isolated and identified nucleoside(5') oligophospho-(5') nucleosides containing adenosine and guanosine (ApnG; n = 3-6) as well as diguanosine polyphosphates (GpnG; n = 3-6) in human platelets. For identification, UV spectrometry, matrix-assisted laser desorption/ionization, postsource decay matrix-assisted laser desorption/ionization mass spectrometry, and enzymatic cleavage experiments were used. The adenosine(5') oligophospho-(5') guanosines act as vasoconstrictors and growth factors. The diguanosine polyphosphates are potent modulators of growth in vascular smooth muscle cells, but do not affect vascular tone. PMID:9449703

  5. Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats?

    PubMed

    Camara, Henrique; Rodrigues, Juliano Quintella Dantas; Alves, Gabriel Andrade; da Silva Junior, Edilson Dantas; Caricati-Neto, Afonso; Garcia, Antônio G; Jurkiewicz, Aron

    2015-12-05

    Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration-effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000μM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1-300µM and cardiac arrest at 1000μM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels.

  6. Repetitive systemic morphine alters activity-dependent plasticity of Schaffer-collateral-CA1 pyramidal cell synapses: involvement of adenosine A1 receptors and adenosine deaminase.

    PubMed

    Sadegh, Mehdi; Fathollahi, Yaghoub

    2014-10-01

    The effectiveness of O-pulse stimulation (TPS) for the reversal of O-pattern primed bursts (PB)-induced long-term potentiation (LTP) were examined at the Schaffer-collateral-CA1 pyramidal cell synapses of hippocampal slices derived from rats chronically treated with morphine (M-T). The results showed that slices derived from both control and M-T rats had normal field excitatory postsynaptic potential (fEPSP)-LTP, whereas PS-LTP in slices from M-T rats was significantly greater than that from control slices. When morphine was applied in vitro to slices derived from rats chronically treated with morphine, the augmentation of PS-LTP was not seen. TPS given 30 min after LTP induction failed to reverse the fEPSP- or PS-LTP in both groups of slices. However, TPS delivered in the presence of long-term in vitro morphine caused the PS-LTP reversal. This effect was blocked by the adenosine A1 receptor (A1R) antagonist CPX (200 nM) and furthermore was enhanced by the adenosine deaminase (ADA) inhibitor EHNA (10 μM). Interestingly, TPS given 30 min after LTP induction in the presence of EHNA (10 μM) can reverse LTP in morphine-exposed control slices in vitro. These results suggest adaptive changes in the hippocampus area CA1 in particular in adenosine system following repetitive systemic morphine. Chronic in vivo morphine increases A1R and reduces ADA activity in the hippocampus. Consequently, adenosine can accumulate because of a stimulus train-induced activity pattern in CA1 area and takes the opportunity to work as an inhibitory neuromodulator and also to enable CA1 to cope with chronic morphine. In addition, adaptive mechanisms are differentially working in the dendrite layer rather than the somatic layer of hippocampal CA1.

  7. A general strategy for expanding polymerase function by droplet microfluidics

    PubMed Central

    Larsen, Andrew C.; Dunn, Matthew R.; Hatch, Andrew; Sau, Sujay P.; Youngbull, Cody; Chaput, John C.

    2016-01-01

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson–Crick base pairing. PMID:27044725

  8. A general strategy for expanding polymerase function by droplet microfluidics.

    PubMed

    Larsen, Andrew C; Dunn, Matthew R; Hatch, Andrew; Sau, Sujay P; Youngbull, Cody; Chaput, John C

    2016-04-05

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson-Crick base pairing.

  9. RNA polymerase backtracking in gene regulation and genome instability.

    PubMed

    Nudler, Evgeny

    2012-06-22

    RNA polymerase is a ratchet machine that oscillates between productive and backtracked states at numerous DNA positions. Since its first description 15 years ago, backtracking--the reversible sliding of RNA polymerase along DNA and RNA--has been implicated in many critical processes in bacteria and eukaryotes, including the control of transcription elongation, pausing, termination, fidelity, and genome instability.

  10. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  11. Problem-Solving Test: Real-Time Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: polymerase chain reaction, DNA amplification, electrophoresis, breast cancer, "HER2" gene, genomic DNA, "in vitro" DNA synthesis, template, primer, Taq polymerase, 5[prime][right arrow]3[prime] elongation activity, 5[prime][right arrow]3[prime] exonuclease activity, deoxyribonucleoside…

  12. A Practical Polymerase Chain Reaction Laboratory for Introductory Biology Classes.

    ERIC Educational Resources Information Center

    Bowlus, R. David; Grether, Susan C.

    1996-01-01

    Presents a polymerase chain reaction (PCR) laboratory exercise that can be performed by introductory biology students in 1 45- to 55-minute class period. Includes a general description of the polymerase chain reaction, materials needed, procedure, and details of interest to teachers. (JRH)

  13. Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.

    PubMed

    Serebryannyy, Leonid A; Parilla, Megan; Annibale, Paolo; Cruz, Christina M; Laster, Kyle; Gratton, Enrico; Kudryashov, Dmitri; Kosak, Steven T; Gottardi, Cara J; de Lanerolle, Primal

    2016-09-15

    Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. However, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin-RNA-polymerase-II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.

  14. DNA polymerase as a molecular motor and pump.

    PubMed

    Sengupta, Samudra; Spiering, Michelle M; Dey, Krishna K; Duan, Wentao; Patra, Debabrata; Butler, Peter J; Astumian, R Dean; Benkovic, Stephen J; Sen, Ayusman

    2014-03-25

    DNA polymerase is responsible for synthesizing DNA, a key component in the running of biological machinery. Using fluorescence correlation spectroscopy, we demonstrate that the diffusive movement of a molecular complex of DNA template and DNA polymerase enhances during nucleotide incorporation into the growing DNA template. The diffusion coefficient of the complex also shows a strong dependence on its inorganic cofactor, Mg2+ ions. When exposed to gradients of either nucleotide or cofactor concentrations, an ensemble of DNA polymerase complex molecules shows collective movement toward regions of higher concentrations. By immobilizing the molecular complex on a patterned gold surface, we demonstrate the fabrication of DNA polymerase-powered fluid pumps. These miniature pumps are capable of transporting fluid and tracer particles in a directional manner with the pumping speed increasing in the presence of the cofactor. The role of DNA polymerase as a micropump opens up avenues for designing miniature fluid pumps using enzymes as engines.

  15. Role of polymerase η in mitochondrial mutagenesis of Saccharomyces cerevisiae

    SciTech Connect

    Chatterjee, Nimrat; Pabla, Ritu; Siede, Wolfram

    2013-02-08

    Highlights: ► DNA polymerase η is detectable in mitochondria of budding yeast. ► Pol η reduces UV-induced mitochondrial base pair substitutions and frameshifts. ► For UV-induced base pair substitutions, Pol η and Pol ζ interact epistatically. -- Abstract: DNA polymerase η mostly catalyzes an error-free bypass of the most frequent UV lesions, pyrimidine dimers of the cyclobutane-type. In addition to its nuclear localization, we show here for the first time its mitochondrial localization in budding yeast. In mitochondria, this polymerase improves bypass replication fidelity opposite UV damage as shown in base pair substitution and frameshift assays. For base pair substitutions, polymerase η appears to be related in function and epistatic to DNA polymerase ζ which, however, plays the opposite role in the nucleus.

  16. The polymerase slips and PIPO exists

    PubMed Central

    White, K Andrew

    2015-01-01

    Plant viruses that contain plus-sensed single-stranded RNA genomes are highly abundant in nature. As the equivalents of large mRNAs, these viral genomes utilize a wide variety of gene expression strategies for the production of their encoded proteins. The potyviruses, which are among the most agriculturally important members in this category, contain a single large open reading frame (ORF) coding for a polyprotein that is processed into functional units. For many years, the products derived from the full-length polyprotein were thought to be the only functional viral proteins. However, this notion was dispelled when an additional essential viral ORF, PIPO, was discovered encoded in an alternative reading frame. Since then, the PIPO protein—P3N-PIPO, which mediates virus movement in plants—has been intensively studied, but its mode of expression remained elusive until now. Two articles, one in this issue of EMBO Reports, now report that slippage of the viral polymerase during viral genome replication is responsible for shifting PIPO into a translated reading frame, thereby allowing for production of P3N-PIPO 1,2. This mechanism of gene expression represents a novel strategy for plant viruses. PMID:26160653

  17. Conserved Endonuclease Function of Hantavirus L Polymerase

    PubMed Central

    Rothenberger, Sylvia; Torriani, Giulia; Johansson, Maria U.; Kunz, Stefan; Engler, Olivier

    2016-01-01

    Hantaviruses are important emerging pathogens belonging to the Bunyaviridae family. Like other segmented negative strand RNA viruses, the RNA-dependent RNA polymerase (RdRp) also known as L protein of hantaviruses lacks an intrinsic “capping activity”. Hantaviruses therefore employ a “cap snatching” strategy acquiring short 5′ RNA sequences bearing 5′cap structures by endonucleolytic cleavage from host cell transcripts. The viral endonuclease activity implicated in cap snatching of hantaviruses has been mapped to the N-terminal domain of the L protein. Using a combination of molecular modeling and structure–function analysis we confirm and extend these findings providing evidence for high conservation of the L endonuclease between Old and New World hantaviruses. Recombinant hantavirus L endonuclease showed catalytic activity and a defined cation preference shared by other viral endonucleases. Based on the previously reported remarkably high activity of hantavirus L endonuclease, we established a cell-based assay for the hantavirus endonuclase function. The robustness of the assay and its high-throughput compatible format makes it suitable for small molecule drug screens to identify novel inhibitors of hantavirus endonuclease. Based on the high degree of similarity to RdRp endonucleases, some candidate inhibitors may be broadly active against hantaviruses and other emerging human pathogenic Bunyaviruses. PMID:27144576

  18. Transcriptional proofreading in dense RNA polymerase traffic

    NASA Astrophysics Data System (ADS)

    Sahoo, Mamata; Klumpp, Stefan

    2011-12-01

    The correction of errors during transcription involves the diffusive backward translocation (backtracking) of RNA polymerases (RNAPs) on the DNA. A trailing RNAP on the same template can interfere with backtracking as it progressively restricts the space that is available for backward translocation and thereby ratchets the backtracked RNAP forward. We analyze the resulting negative impact on proofreading theoretically using a driven lattice gas model of transcription under conditions of dense RNAP traffic. The fraction of errors that are corrected is calculated exactly for the case of a single RNAP; for multi-RNAP transcription, we use simulations and an analytical approximation and find a decrease with increasing traffic density. Moreover, we ask how the parameters of the system have to be set to keep down the impact of the interference of a trailing RNAP. Our analysis uncovers a surprisingly simple picture of the design of the error correction system: its efficiency is essentially determined by the rate for the initial backtracking step, while the value of the cleavage rate ensures that the correction mechanism remains efficient at high transcription rates. Finally, we argue that our analysis can also be applied to cases with transcription-translation coupling where the leading ribosome on the transcript assumes the role of the trailing RNAP.

  19. Solving the RNA polymerase I structural puzzle

    PubMed Central

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-01-01

    Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution. PMID:25286842

  20. Integrated polymerase chain reaction/electrophoresis instrument

    DOEpatents

    Andresen, Brian D.

    2000-01-01

    A new approach and instrument for field identification of micro-organisms and DNA fragments using a small and disposable device containing integrated polymerase chain reaction (PCR) enzymatic reaction wells, attached capillary electrophoresis (CE) channels, detectors, and read-out all on/in a small hand-held package. The analysis instrument may be made inexpensively, for example, of plastic, and thus is disposable, which minimizes cross contamination and the potential for false positive identification between samples. In addition, it is designed for multiple users with individual applications. The integrated PCR/CE is manufactured by the PCR well and CE channels are "stamped" into plastic depressions where conductive coatings are made in the wells and ends of the CE microchannels to carry voltage and current to heat the PCR reaction mixtures and simultaneously draw DNA bands up the CE channels. Light is transmitted through the instrument at appropriate points and detects PCR bands and identifies DNA fragments by size (retention time) and quantifies each by the amount of light generated as each phototransistor positioned below each CE channel detects a passing band. The instrument is so compact that at least 100 PCR/CE reactions/analyses can be performed easily on one detection device.

  1. Actinobaculum suis Detection Using Polymerase Chain Reaction

    PubMed Central

    Amigo, Cristina Román; de Gobbi, Debora Dirani Sena; Gomes, Vasco Túlio de Moura; Perina, Danilo do Prado; Nogueira de Lima Filsner, Pedro Henrique; Costa, Barbara Letícia Pereira; Spindola, Maria Garcia; Ferreira, Thais Sebastiana Porfida; Brandão, Paulo Eduardo; Moreno, Andrea Micke

    2012-01-01

    Actinobaculum suis is an important agent related to urinary infection in swine females. Due to its fastidious growth characteristics, the isolation of this anaerobic bacterium is difficult, thus impairing the estimation of its prevalence. The purpose of this study was to develop and test a polymerase chain reaction (PCR) for the detection and identification of A. suis and then compare these results with traditional isolation methods. Bacterial isolation and PCR were performed on one hundred and ninety-two urine samples from sows and forty-five preputial swabs from boars. The results indicate that this PCR was specific for A. suis, presenting a detection limit between 1.0 × 101 CFU/mL and 1.0 × 102 CFU/mL. A. suis frequencies, as measured by PCR, were 8.9% (17/192) in sow urine samples and 82.2% (37/45) in preputial swabs. Assessed using conventional culturing techniques, none of the urine samples were positive for A. suis; however, A. suis was detected in 31.1% (14/45) of the swabs. This PCR technique was shown to be an efficient method for the detection of A. suis in urine and preputial swabs. PMID:23346017

  2. Adenovirus DNA polymerase is a phosphoprotein.

    PubMed

    Ramachandra, M; Nakano, R; Mohan, P M; Rawitch, A B; Padmanabhan, R

    1993-01-05

    Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

  3. Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

    PubMed

    Podestá, Dolores; García-Herreros, María I; Cannata, Joaquín J B; Stoppani, Andrés O M; Fernández Villamil, Silvia H

    2004-06-01

    Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

  4. Structural Determinant for Switching between the Polymerase and Exonuclease Modes in the PCNA-Replicative DNA Polymerase Complex

    NASA Astrophysics Data System (ADS)

    Nishida, Hirokazu; Mayanagi, Kouta; Ishino, Yoshizumi; Morikawa, Kosuke

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with a cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction. Electron microscopy analyses confirmed that this complex structure exists among the multiple functional configurations in solution. Together with data from mutational analyses, this structural study indicated that the novel interaction between a stretched loop of PCNA and the PfuPol Thumb domain is quite important, in addition to the authentic PCNA-polymerase recognition site (PIP box). A comparison of the present structures with the previously reported structures of polymerases complexed with DNA suggested that the second interaction site plays a crucial role in switching between the polymerase and exonuclease modes, by stabilizing only the polymerase mode. This proposed mechanism of fidelity control of replicative DNA polymerases was supported by experiments, in which a mutation within the second interaction site caused an enhancement in the exonuclease activity in the presence of PCNA.

  5. Structural determinant for switching between the polymerase and exonuclease modes in the PCNA-replicative DNA polymerase complex

    PubMed Central

    Nishida, Hirokazu; Mayanagi, Kouta; Kiyonari, Shinichi; Sato, Yuichi; Oyama, Takuji; Ishino, Yoshizumi; Morikawa, Kosuke

    2009-01-01

    Proliferating cell nuclear antigen (PCNA) is responsible for the processivity of DNA polymerase. We determined the crystal structure of Pyrococcus furiosus DNA polymerase (PfuPol) complexed with the cognate monomeric PCNA, which allowed us to construct a convincing model of the polymerase-PCNA ring interaction, with unprecedented configurations of the two molecules. Electron microscopic analyses indicated that this complex structure exists in solution. Our structural study revealed that an interaction occurs between a stretched loop of PCNA and the PfuPol Thumb domain, in addition to the authentic PCNA-polymerase recognition site (PIP box). Comparisons of the present structure with the previously reported structures of polymerases complexed with DNA, suggested that the second interaction plays a crucial role in switching between the polymerase and exonuclease modes, by inducing a PCNA-polymerase complex configuration that favors synthesis over editing. This putative mechanism for fidelity control of replicative DNA polymerases is supported by experiments, in which mutations at the second interaction site caused enhancements in the exonuclease activity in the presence of PCNA. PMID:19934045

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

    PubMed Central

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

    2017-01-01

    Adenosine monophosphate-activated protein kinase (AMPK) is an important energy sensor which is activated by increases in adenosine monophosphate (AMP)/adenosine triphosphate (ATP) ratio and/or adenosine diphosphate (ADP)/ATP ratio, and increases different metabolic pathways such as fatty acid oxidation, glucose transport and mitochondrial biogenesis. In this sense, AMPK maintains cellular energy homeostasis by induction of catabolism and inhibition of ATP-consuming biosynthetic pathways to preserve ATP levels. Several studies indicate a reduction of AMPK sensitiv