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Sample records for adenosine diphosphate ribose

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

    Science.gov (United States)

    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. Roles and mechanisms of the CD38/cyclic adenosine diphosphate ribose/Ca2+ signaling pathway

    Institute of Scientific and Technical Information of China (English)

    Wenjie; Wei; Richard; Graeff; Jianbo; Yue

    2014-01-01

    Mobilization of intracellular Ca2+ stores is involved inmany diverse cell functions, including: cell proliferation;differentiation; fertilization; muscle contraction; secre-tion of neurotransmitters, hormones and enzymes;and lymphocyte activation and proliferation. Cyclic ad-enosine diphosphate ribose(cADPR) is an endogenousCa2+ mobilizing nucleotide present in many cell typesand species, from plants to animals. cADPR is formedby ADP-ribosyl cyclases from nicotinamide adenine di-nucleotide. The main ADP-ribosyl cyclase in mammalsis CD38, a multi-functional enzyme and a type Ⅱ mem-brane protein. It has been shown that many extracel-lular stimuli can induce cADPR production that leadsto calcium release or influx, establishing cADPR as asecond messenger. cADPR has been linked to a widevariety of cellular processes, but the molecular mecha-nisms regarding cADPR signaling remain elusive. Theaim of this review is to summarize the CD38/cADPR/Ca2+ signaling pathway, focusing on the recent advanc-es involving the mechanism and physiological functionsof cADPR-mediated Ca2+ mobilization.

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

    Institute of Scientific and Technical Information of China (English)

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

    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.

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

    Science.gov (United States)

    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

  5. Novel treatment strategies in triple-negative breast cancer: specific role of poly(adenosine diphosphate-ribose polymerase inhibition

    Directory of Open Access Journals (Sweden)

    Audeh MW

    2014-10-01

    Full Text Available M William Audeh Division of Medical Oncology, Samuel Oschin Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA Abstract: Inhibitors of the poly(adenosine triphosphate-ribose polymerase (PARP-1 enzyme induce synthetic lethality in cancers with ineffective DNA (DNA repair or homologous repair deficiency, and have shown promising clinical activity in cancers deficient in DNA repair due to germ-line mutation in BRCA1 and BRCA2. The majority of breast cancers arising in carriers of BRCA1 germ-line mutations, as well as half of those in BRCA2 carriers, are classified as triple-negative breast cancer (TNBC. TNBC is a biologically heterogeneous group of breast cancers characterized by the lack of immunohistochemical expression of the ER, PR, or HER2 proteins, and for which the current standard of care in systemic therapy is cytotoxic chemotherapy. Many “sporadic” cases of TNBC appear to have indicators of DNA repair dysfunction similar to those in BRCA-mutation carriers, suggesting the possible utility of PARP inhibitors in a subset of TNBC. Significant genetic heterogeneity has been observed within the TNBC cohort, creating challenges for interpretation of prior clinical trial data, and for the design of future clinical trials. Several PARP inhibitors are currently in clinical development in BRCA-mutated breast cancer. The use of PARP inhibitors in TNBC without BRCA mutation will require biomarkers that identify cancers with homologous repair deficiency in order to select patients likely to respond. Beyond mutations in the BRCA genes, dysfunction in other genes that interact with the homologous repair pathway may offer opportunities to induce synthetic lethality when combined with PARP inhibition. Keywords: PARP, triple negative breast cancer, PARP inhibitors

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

    Energy Technology Data Exchange (ETDEWEB)

    Azad, Arun, E-mail: arun.azad@bccancer.bc.ca [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Pathology, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Bukczynska, Patricia; Jackson, Susan [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Haput, Ygal; Cullinane, Carleen [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia); McArthur, Grant A.; Solomon, Benjamin [Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Division of Cancer Medicine, Peter MacCallum Cancer Centre, East Melbourne, Victoria (Australia); Department of Medicine, St. Vincent' s Hospital, University of Melbourne, Parkville, Victoria (Australia); Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria (Australia)

    2014-02-01

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

  7. Extracellular Adenosine Diphosphate Ribose Mobilizes Intracellular Ca2+ via Purinergic-Dependent Ca2+ Pathways in Rat Pulmonary Artery Smooth Muscle Cells

    Directory of Open Access Journals (Sweden)

    Chun Huang

    2015-11-01

    Full Text Available Background/Aims: Adenosine diphosphate ribose (ADPR, a product of β-NAD+ metabolism generated by the multifunctional enzyme CD38, is recognized as a novel signaling molecule. The catalytic site of CD38 orients extracellularly or intracellularly, capable of generating ADPR outside and inside the cells. CD38-dependent pathways have been characterized in pulmonary artery smooth muscle cells (PASMCs; however the physiological function of extracellular ADPR is unclear. Methods: Ca2+ mobilizing and proliferative effects of extracellular ADPR were characterized and compared with the ATP-induced responses in rat PASMCs; and the expression of purinergic receptor (P2X and P2Y subtypes were examined in pulmonary arteries. Results: ADPR elicited concentration-dependent increase in [Ca2+]i with a fast transient and a sustained phase in PASMCs. The sustained phase was abolished by Ca2+ removal and inhibited by the non-selective cation channel blocker SKF-96365, but was unaffected by TRPM2 antagonists or nifedipine. The purinergic receptor (P2X antagonist pyridoxal-phosphate-6-azophenyl-2', 4'-disulfonate inhibited partially the transient and the sustained Ca2+ response, while the P2(XY inhibitor suramin and the phospholipase C inhibitor U73122 abolished the sustained Ca2+ influx. The P2Y1 antagonist MRS2179 had no effect on the response. By contrast, ATP and ADP activated Ca2+ response exhibited a high and a low affinity component, and the pharmacological profile of ATP-induced Ca2+ response was distinctive from that of ADPR. BrdU incorporation assay showed that ADPR caused significant inhibition whereas ATP caused slight stimulation of PASMC proliferation. RT-PCR analysis found that almost all P2X and P2Y subtypes are expressed in PAs. Conclusion: ADPR and ATP activate Ca2+ responses through different combinations of multiple purinergic receptor subtypes; and extracellular ADPR may exert an autocrine/paracrine action via purinergic receptors on PASMCs.

  8. Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

    Science.gov (United States)

    Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

    2001-01-01

    An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

  9. Radioimmunoassay for guanosin-5'-diphosphate-3'-diphosphate and adenosine-5'-triphosphate-3'-diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Hamagishi, Y.; Oki, T.; Tone, H.; Inui, T. (Sanraku-Ocean Co. Ltd., Fujisawa, Kanagawa (Japan). Central Research Lab.)

    1980-12-01

    A radioimmunoassay for guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and adenosine-5'-triphosphate-3'-diphosphate (pppApp) has been developed. The assay method is based on competition of an unlabeled highly phosphorylated nucleotide with /sup 3/H-labeled highly phosphorylated nucleotide for binding sites on a specific antibody. Antibodies to ppGpp and pppApp were obtained by immunizing rabbits with the antigen prepared by conjugating ppGpp with human serum albumin using 1-ethyl-3-(3-dimethylaminoprophyl)carbodiimide, and with the antigen prepared by conjugating 8-(6-aminohexyl)amino-adenosine-5'-triphosphate-3'-diphosphate with human serum albumin using glutaraldehyde, respectively. Antibody-bound /sup 3/H-labeled highly phosphorylated nucleotides were separated from the free /sup 3/H-labeled highly phosphorylated nucleotides by selective adsorption on dextran-coated charcoal. Displacement plots were linear over a concentration range of 5 - 1,000 pmol/assay tube in a log-probit percentage plot. Application of this method to biological systems offers improved accuracy and convenience compared with the previous /sup 32/PO/sub 4/-labeling technique.

  10. The effect of ticlopidine administration to humans on the binding of adenosine diphosphate to blood platelets

    NARCIS (Netherlands)

    Lips, J.P.M.; Sixma, J.J.; Schiphorst, M.E.

    1980-01-01

    Administration of Ticlopidine to human volunteers resulted in a prolonged bleeding time and decreased or absent aggregation of platelets with collagen and epinephrine. Adenosine diphosphate (ADP) induced platelet aggregation was initiated by a normal shape change, but the rate of the first wave of a

  11. Early Cessation of Adenosine Diphosphate Receptor Inhibitors Among Acute Myocardial Infarction Patients Treated With Percutaneous Coronary Intervention

    DEFF Research Database (Denmark)

    Fosbøl, Emil L; Ju, Christine; Anstrom, Kevin J

    2016-01-01

    BACKGROUND: Guidelines recommend the use of adenosine diphosphate receptor inhibitor (ADPri) therapy for 1 year postacute myocardial infarction; yet, early cessation of therapy occurs frequently in clinical practice. METHODS AND RESULTS: We examined 11 858 acute myocardial infarction patients tre...... adverse cardiovascular event risk. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01088503....

  12. Adenosine-diphosphate (ADP) receptor antagonists for the prevention of cardiovascular disease in type 2 diabetes mellitus (Review)

    NARCIS (Netherlands)

    Valentine, N.; Laar, F.A. van de; Driel, M.L. van

    2012-01-01

    BACKGROUND: Cardiovascular disease (CVD) is the most prevalent complication of type 2 diabetes with an estimated 65% of people with type 2 diabetes dying from a cause related to atherosclerosis. Adenosine-diphosphate (ADP) receptor antagonists like clopidogrel, ticlopidine, prasugrel and ticagrelor

  13.   Adenosine-diphosphate (ADP) reduces infarct size and improves porcine heart function after myocardial infarction

    DEFF Research Database (Denmark)

    Bune, Laurids Touborg; Larsen, Jens Kjærgaard Rolighed; Thaning, Pia;

    2013-01-01

    (UTP) are both released during myocardial ischemia, influencing hemodynamics. Both mediate the release of tissue plasminogen activator (t-PA), which can reduce infarct size (IS). The objective of this study was to investigate whether exogenous ADP and UTP administration during reperfusion could reduce......Acute myocardial infarction continues to be a major cause of morbidity and mortality. Timely reperfusion can substantially improve outcomes and the administration of cardioprotective substances during reperfusion is therefore highly attractive. Adenosine diphosphate (ADP) and uridine-5-triphoshate...... myocardial IS and whether this correlated to t-PA release or improvements in hemodynamic responses. Hemodynamic variables and t-PA were measured in 22 pigs before, during, and after 45 min of left anterior coronary artery occlusion. During reperfusion, the pigs were randomized to 240 min of intracoronary...

  14. P2X receptors regulate adenosine diphosphate release from hepatic cells.

    Science.gov (United States)

    Chatterjee, Cynthia; Sparks, Daniel L

    2014-12-01

    Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2-4 h. Nucleotide release from hepatic cells is stimulated by the Ca(2+) ionophore, ionomycin, and by the P2 receptor agonist, 2'3'-O-(4-benzoyl-benzoyl)-adenosine 5'-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10-100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca(2+) levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.

  15. Antibody to poly(adenosine diphosphate-ribose) polymerase and its use in chromatin analysis.

    OpenAIRE

    1982-01-01

    To facilitate investigations on the organization of poly (ADP-Rib) polymerase in chromatin, and to elucidate its biological function, polymerase purified from HeLa nuclei was used to elicit antibodies in mice. The anti-polymerase sera was found to be specific by multiple criteria. The association of polymerase with oligonucleosomes of differing chain size was determined by the specific binding of polymerase antibody (and as control, anti-histone H3) to nitrocellulose transfers of native elect...

  16. Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

    Directory of Open Access Journals (Sweden)

    Brans Alain

    2007-08-01

    Full Text Available Abstract Background We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP, in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. Results Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5–7.0 and a high Km for ThDP (5 mM, suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65. Conclusion This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells.

  17. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose-5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-d-ribosyl a-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  18. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    The three conserved aspartic acid residues of the 5-phospho-D-ribosyl α-1-diphosphate binding site (213-GRDCVLVDDMIDTGGT-228) of Escherichia coli phosphoribosyl diphosphate synthetase were studied by analysis of the mutant enzymes D220E, D220F, D221A, D224A, and D224S. The mutant enzymes showed...... an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...

  19. Online cleanup of accelerated solvent extractions for determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly using high-performance liquid chromatography.

    Science.gov (United States)

    Xue, Xiaofeng; Wang, Feng; Zhou, Jinhui; Chen, Fang; Li, Yi; Zhao, Jing

    2009-06-10

    Determination of the levels of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly is important for the study of its pharmacological activities, health benefits, and adenosine phosphate degradation. In this study was developed a novel method to determine ATP, ADP, and AMP levels in royal jelly using accelerated solvent extraction (ASE) followed by online cleanup and high-performance liquid chromatography (HPLC) with diode array detection (DAD). The optimum extraction conditions were obtained using an 11 mL ASE cell, ethanol/water (5:5 v/v) as the extraction solvent, 1500 psi, 80 degrees C, a 5 min static time, and a 60% flush volume. Optimum separation of the three compounds was achieved in AMP levels in 15 samples of royal jelly of different origins was performed. Sample results indicated that the AMP concentration was 24.2-2214.4 mg kg(-1), whereas ATP and ADP were not detectable or present only at low levels.

  20. Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

    OpenAIRE

    Brans Alain; Makarchikov Alexander F; Bettendorff Lucien

    2007-01-01

    Abstract Background We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. Results Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried o...

  1. Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate.

    Science.gov (United States)

    Bonello, Laurent; Tantry, Udaya S; Marcucci, Rossella; Blindt, Ruediger; Angiolillo, Dominick J; Becker, Richard; Bhatt, Deepak L; Cattaneo, Marco; Collet, Jean Philippe; Cuisset, Thomas; Gachet, Christian; Montalescot, Gilles; Jennings, Lisa K; Kereiakes, Dean; Sibbing, Dirk; Trenk, Dietmar; Van Werkum, Jochem W; Paganelli, Franck; Price, Matthew J; Waksman, Ron; Gurbel, Paul A

    2010-09-14

    The addition of clopidogrel to aspirin treatment reduces ischemic events in a wide range of patients with cardiovascular disease. However, recurrent ischemic event occurrence during dual antiplatelet therapy, including stent thrombosis, remains a major concern. Platelet function measurements during clopidogrel treatment demonstrated a variable and overall modest level of P2Y(12) inhibition. High on-treatment platelet reactivity to adenosine diphosphate (ADP) was observed in selected patients. Multiple studies have now demonstrated a clear association between high on-treatment platelet reactivity to ADP measured by multiple methods and adverse clinical event occurrence. However, the routine measurement of platelet reactivity has not been widely implemented and recommended in the guidelines. Reasons for the latter include: 1) a lack of consensus on the optimal method to quantify high on-treatment platelet reactivity and the cutoff value associated with clinical risk; and 2) limited data to support that alteration of therapy based on platelet function measurements actually improves outcomes. This review provides a consensus opinion on the definition of high on-treatment platelet reactivity to ADP based on various methods reported in the literature and proposes how this measurement may be used in the future care of patients. Copyright © 2010 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  2. Effects of cumene hydroperoxide on adenosine diphosphate ribosyl transferase in mononuclear leukocytes of patients with adenomatous polyps in the colon.

    Science.gov (United States)

    Markowitz, M M; Johnson, D B; Pero, R W; Winawer, S J; Miller, D G

    1988-03-01

    We have studied the effects of plasma and of cumene hydroperoxide (CUM) on adenosine diphosphate ribosyl transferase (ADPRT) from mononuclear leukocytes (HML) of patients with colonic adenomatous polyps (n = 22), with colonic hyperplastic polyps (n = 5) and with neither type of polyp (controls) (n = 6). ADPRT was measured after incubation of HML with plasma alone (termed the plasma value), and with plasma plus CUM (50 microM) (the activated value); the difference elicited by CUM was termed the induced value. There was no significant difference in values between the control and hyperplastic polyp groups: these were combined for further analysis. The plasma (P = 0.038), activated (P = 0.009) and induced (P = 0.0024) values of the combined group all differed significantly from those of the adenoma group. At low exposures, CUM stimulated both ADPRT and unscheduled DNA synthesis and, at higher exposures, inactivated both. Pretreatment of HML with vitamin E protected against these effects of CUM, while pretreatment with diamide (which depletes GSH) accentuated the effects. This study demonstrates a differential reaction of ADPRT in patients harboring colonic adenomas and suggests that the origin of this difference may lie in cellular responses to oxidative stress.

  3. Consensus and update on the definition of on-treatment platelet reactivity to adenosine diphosphate associated with ischemia and bleeding.

    Science.gov (United States)

    Tantry, Udaya S; Bonello, Laurent; Aradi, Daniel; Price, Matthew J; Jeong, Young-Hoon; Angiolillo, Dominick J; Stone, Gregg W; Curzen, Nick; Geisler, Tobias; Ten Berg, Jurrien; Kirtane, Ajay; Siller-Matula, Jolanta; Mahla, Elisabeth; Becker, Richard C; Bhatt, Deepak L; Waksman, Ron; Rao, Sunil V; Alexopoulos, Dimitrios; Marcucci, Rossella; Reny, Jean-Luc; Trenk, Dietmar; Sibbing, Dirk; Gurbel, Paul A

    2013-12-17

    Dual antiplatelet therapy with aspirin and a P2Y12 receptor blocker is a key strategy to reduce platelet reactivity and to prevent thrombotic events in patients treated with percutaneous coronary intervention. In an earlier consensus document, we proposed cutoff values for high on-treatment platelet reactivity to adenosine diphosphate (ADP) associated with post-percutaneous coronary intervention ischemic events for various platelet function tests (PFTs). Updated American and European practice guidelines have issued a Class IIb recommendation for PFT to facilitate the choice of P2Y12 receptor inhibitor in selected high-risk patients treated with percutaneous coronary intervention, although routine testing is not recommended (Class III). Accumulated data from large studies underscore the importance of high on-treatment platelet reactivity to ADP as a prognostic risk factor. Recent prospective randomized trials of PFT did not demonstrate clinical benefit, thus questioning whether treatment modification based on the results of current PFT platforms can actually influence outcomes. However, there are major limitations associated with these randomized trials. In addition, recent data suggest that low on-treatment platelet reactivity to ADP is associated with a higher risk of bleeding. Therefore, a therapeutic window concept has been proposed for P2Y12 inhibitor therapy. In this updated consensus document, we review the available evidence addressing the relation of platelet reactivity to thrombotic and bleeding events. In addition, we propose cutoff values for high and low on-treatment platelet reactivity to ADP that might be used in future investigations of personalized antiplatelet therapy.

  4. Comparison of cationic propyl gallate and adenosine diphosphate for the measurement of aspirin effectivity with optical aggregometry.

    Science.gov (United States)

    Motovska, Zuzana; Sujanova, Zdenka; Wimmerova, Sona; Ardo, Jan; Skrakova, Marcela; Widimsky, Petr

    2007-10-01

    To compare the newer inductor of platelet aggregation cationic propyl gallate (CPG) with adenosine diphosphate (ADP) for the examination of aspirin (ASA) effectivity with optical aggregometry. In total,116 patients were prospectively enrolled with a stable cardiovascular disease, taking ASA 100 mg/day for >or=1 month. The control group consisted of 62 healthy volunteers. A platelet aggregation was investigated by optical aggregometry (aggregometer LASER 4x; BIO ART, Sint-Katelijne-Waver, Belgium). CPG and ADP were added as aggregating agents. The measured parameters were CPG-slope (%/min) and ADP max (%). Using the CPG-slope values from the control group, the CPG-slope cut-off value was determined to define a laboratory ASA-noneffectively treated patient. The values from control group followed a normal distribution (Shapiro-Wilk test). We calculated the cut-off value using the 1-tailed 95% confidence interval. The CPG-slope cut-off value was 79 %/min for an ASA-effectively treated patient. We marked the patients as laboratory ASA-noneffective treated when the CPG-slope was >79%/min. In the same way we defined the cut-off value for ADP-max. We identified the aspirin treatment as ineffective when the value of ADP-max was >62%. The values of CPG-slope and ADP-max were in close correlation in the group of patients treated with aspirin with a highly significant correlation index (r=0.671, Pinductors, the proportion of ASA-noneffectively treated patients was 25%. Using both tests, 72.4% of patients were equally divided. ASA-noneffectively treated patients were commonly more obese (46.2%), had hypertension (94.9%) and hypercholesterolemia (73.7%), and were less commonly treated with statins (30.8%) than the aspirin effectively treated patients (42%, 88.2%, 59.2%, and 42.1%, respectively). The detected differences were not statistically significant. Cationic propyl gallate is an optimal inductor for optical aggregometry to monitor laboratory effectiveness of aspirin

  5. Use of tailored loading-dose clopidogrel in patients undergoing selected percutaneous coronary intervention based on adenosine diphosphate-mediated platelet aggregation

    Institute of Scientific and Technical Information of China (English)

    MENG Kang; L(U) Shu-zheng; ZHU Hua-gang; CHEN Xin; GE Chang-jiang; SONG Xian-tao

    2010-01-01

    Background Adenosine phosphate-mediated platelet aggregation is a prognostic factor for major adverse cardiac events in patients who have undergone selective percutaneous coronary interventions. This study aimed to assess whether an adjusted loading dose of clopidogrel could more effectively inhibit platelet aggregation in patients undergoing selected percutaneous coronary intervention.Methods A total of 205 patients undergoing selected percutaneous coronary intervention were enrolled in this multicenter, prospective, randomized study. Patients receiving domestic clopidogrel (n=104) served as the Talcom (Taijia)group; others (n=101) received Plavix, the Plavix group, Patients received up to 3 additional 300-mg loading doses of clopidogrel to decrease the adenosine phosphate-mediated platelet aggregation index by more than 50% (the primary endpoint) compared with the baseline. The secondary endpoint was major adverse cardiovascular events at 12 months.Results Compared with the rational loading dosage, the tailored loading dosage better inhibited platelet aggregation based on a >50% decrease in adenosine phosphate-mediated platelet aggregation (rational loading dosage vs. tailored loading dosage, 48% vs. 73%, P=0.028). There was no significant difference in the eligible index between the Talcom and Plavix groups (47% vs. 49% at 300 mg; 62% vs. 59% at 600 mg; 74% vs. 72% at 900 mg; P >0.05) based on a standard adenosine diphosphate-mediated platelet aggregation decrease of >50%. After 12 months of follow-up, there were no significant differences in major adverse cardiac events (2.5% vs. 2.9%, P=5.43). No acute or subacute stent thrombosis events occurred.Conclusion An adjusted loading dose of clopidogrel could have significant effects on antiplatelet aggregation compared with a rational dose, decreasing 1-year major adverse cardiac events in patients undergoing percutaneous coronary interventions based on adenosine phosphate-mediated platelet aggregation with no

  6. Safety, tolerability, and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, compared with clopidogrel, in patients with non-ST-segment elevation acute coronary syndrome: primary results of the DISPERSE-2 trial

    DEFF Research Database (Denmark)

    Cannon, Christopher P; Husted, Steen; Harrington, Robert A;

    2007-01-01

    OBJECTIVES: Our goal was to compare the safety and initial efficacy of AZD6140, the first reversible oral adenosine diphosphate receptor antagonist, with clopidogrel in patients with non-ST-segment elevation acute coronary syndromes (NSTE-ACS). BACKGROUND: AZD6140 achieves higher mean levels of p...

  7. Effects of mutagenesis of aspartic acid residues in the putative phosphoribosyl diphosphate binding site of Escherichia coli phosphoribosyl diphosphate synthetase on metal ion specificity and ribose 5-phosphate binding

    DEFF Research Database (Denmark)

    Willemoës, Martin; Nilsson, Dan; Hove-Jensen, Bjarne

    1996-01-01

    an increase in KM for ribose 5-phosphate in the presence of at least one of the divalent metal ions Mg2+, Mn2+, Co2+, or Cd2+, with the most dramatic changes revealed by the D220E and D220F enzymes in the presence of Co2+ and the D221A enzyme in the presence of Mn2+ or Co2+. The D220F and D221A enzymes both...... showed large decreases in Vapp in the presence of the various divalent metal ions, except for the D221A enzyme in the presence of Co2+. Vapp of the D220E enzyme was similar to that of the wild-type enzyme in the presence of Mg2+, Mn2+, or Cd2+, whereas the Vapp was increased in the presence of Co2+. Vapp...... enzymes were dependent on the metal ion present, suggesting a function of the investigated aspartic acid residues both in the binding of ribose 5-phosphate, possibly via a divalent metal ion, and in the interaction with a divalent metal ion during catalysis...

  8. Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. I. Measurement of concentration and size of single platelets and aggregates.

    Science.gov (United States)

    Bell, D N; Spain, S; Goldsmith, H L

    1989-11-01

    A double infusion flow system and particle sizing technique were developed to study the effect of time and shear rate on adenosine diphosphate-induced platelet aggregation in Poiseuille flow. Citrated platelet-rich plasma, PRP, and 2 microM ADP were simultaneously infused into a 40-microliters cylindrical mixing chamber at a fixed flow ratio, PRP/ADP = 9:1. After rapid mixing by a rotating magnetic stirbar, the platelet suspension flowed through 1.19 or 0.76 mm i.d. polyethylene tubing for mean transit times, t, from 0.1 to 86 s, over a range of mean tube shear rate, G, from 41.9 to 1,000 s-1. Known volumes of suspension were collected into 0.5% buffered glutaraldehyde, and all particles in the volume range 1-10(5) microns 3 were counted and sized using a model ZM particle counter (Coulter Electronics Inc., Hialeah, FL) and a logarithmic amplifier. The decrease in the single platelet concentration served as an overall index of aggregation. The decrease in the total particle concentration was used to calculate the collision capture efficiency during the early stages of aggregation, and aggregate growth was followed by changes in the volume fraction of particles of successively increasing size. Preliminary results demonstrate that both collision efficiency and particle volume fraction reveal important aspects of the aggregation process not indicated by changes in the single platelet concentration alone.

  9. Adenosine Diphosphate Ribosylation Factor-GTPaseActivating Protein Stimulates the Transport of AUX1Endosome, Which Relies on Actin Cytoskeletal Organization in Rice Root DevelopmentF

    Institute of Scientific and Technical Information of China (English)

    Cheng Du; Yunyuan XU; Yingdian Wang; Kang Chong

    2011-01-01

    Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

  10. Binding of Divalent Magnesium by Escherichia coli Phosphoribosyl Diphosphate Synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates MgATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-d-ribosyl a-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, a,ß-methylene ATP and (+)-1-a,2-a,3...... of substrates and products indicated a role of Mg2+ in preparing the active site of phosphoribosyl diphosphate synthetase for binding of the highly phosphorylated ligands MgATP and phosphoribosyl diphosphate, as evaluated by analysis of the effects of the inhibitors adenosine and ribose 1,5-bisphosphate....... Calcium ions, which inhibit the enzyme even in the presence of high concentrations of Mg2+, appeared to compete with free Mg2+ for binding to its activator site on the enzyme. Analysis of the inhibition of Mg2+ binding by MgADP indicated that MgADP binding to the allosteric site may occur in competition...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-09-17

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

  12. A novel dynamic layer-by-layer assembled nano-scale biointerface: functionality tests with platelet adhesion and aggregate morphology influenced by adenosine diphosphate.

    Science.gov (United States)

    Watson, Melanie G; Lopez, Juan M; Paun, Mihaela; Jones, Steven A

    2013-11-01

    An improved biointerface was developed, dynamic layer-by-layer self-assembly surface (d-LbL), and utilized as a biologically-active substrate for platelet adhesion and aggregation. Possible clinical applications for this research include improved anti-coagulation surfaces. This work demonstrated the functionality of d-LbL biointerfaces in the presence of platelet-rich-plasma (PRP) with the addition of 20 μM adenosine diphosphate (ADP), a thrombus activator. The surface morphology of the experimental control, plain PRP, was compared to PRP containing additional ADP (PRP + ADP) and resulted in an expected increase of platelet adhesions along the fibrinogen d-LbL substrate. The d-LbL process was used to coat glass slides with fibrinogen, Poly (sodium 4-styrene-sulfonate), and Poly (diallydimethlyammonium chloride). Slides were exposed to PRP under flow and static conditions with and without 20 μM of ADP. Fluorescence microscopy (FM), phase contrast microscopy (PCM), atomic force microscopy (AFM), and field emission-scanning electron microscopy (FE-SEM) were used to evaluate platelet adhesions under the influence of varied shear conditions. PCM images illustrated differences between the standard LbL and d-LbL substrates. FM images provided percent surface coverage values. For high-shear conditions, percent surface coverage values increased when using ADP whereas plain PRP exposure displayed no significant increase. AFM scans also displayed higher mean peak height values and unique surface characteristics for PRP + ADP as opposed to plain PRP. FE-SEM images revealed platelet adhesions along the biointerface and unique characteristics of the d-LbL surface. In conclusion, PRP + ADP was more effective at increasing platelet aggregation, especially under high shear conditions, providing further validation of the improved biointerface.

  13. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots.

    Science.gov (United States)

    Chung, Tze-Wen; Lin, Pei-Yi; Wang, Shoei-Shen; Chen, Yen-Fung

    2014-01-01

    Chitosan nanoparticles (NPs) decorated with adenosine diphosphate (ADP) (ANPs) or fibrinogen (FNPs) were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP) or FNP (clot/FNP) were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1 ± 14.0, 251.0 ± 9.8, and 326.5 ± 14.5 nm and zeta potentials of 24.1 ± 0.5, 20.6 ± 1.9, and 15.3 ± 1.5 mV (n=4), respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR) spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM) micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP), clot/ANP, and clot/FNP (at 0.05 wt%) were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc) of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM), were significantly (P<0.05) (n=4) shorter than that of a clot induced by a phosphate-buffered solution (PBS) (clot/PBS) (63.6% ± 3.1%, 48.3% ± 6.2%, and 63.2% ± 4.7%, respectively). The ΔF2 values in the spectra of frequency shifts associated with the propagation of fibrin networks in the clot/ANPs and clot/FNPs were significantly lower than those of clot/PBS. Interestingly, texture profile analysis of the compressional properties showed significantly lower hardness and compressibility in clot/NPs and clot/ANPs (P<0.05 or better) (n=4) compared with

  14. Adenosine diphosphate-decorated chitosan nanoparticles shorten blood clotting times, influencing the structures and varying the mechanical properties of the clots

    Directory of Open Access Journals (Sweden)

    Chung TW

    2014-03-01

    Full Text Available Tze-Wen Chung,1,3 Pei-Yi Lin,2 Shoei-Shen Wang,2 Yen-Fung Chen31Department of Biomedical Engineering, National Yang-Ming University, 2Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, Republic of China; 3Department of Chemical and Materials Engineering, National Yunlin University of Science and Technology, Yunlin, Taiwan, Republic of ChinaAbstract: Chitosan nanoparticles (NPs decorated with adenosine diphosphate (ADP (ANPs or fibrinogen (FNPs were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP or FNP (clot/FNP were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1±14.0, 251.0±9.8, and 326.5±14.5 nm and zeta potentials of 24.1±0.5, 20.6±1.9, and 15.3±1.5 mV (n=4, respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP, clot/ANP, and clot/FNP (at 0.05 wt% were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM, were significantly (P<0.05 (n=4 shorter than that of a clot induced by a phosphate-buffered solution (PBS (clot/PBS (63.6%±3.1%, 48.3%±6.2%, and 63.2%±4.7%, respectively. The ∆F2

  15. Binding of divalent magnesium by Escherichia coli phosphoribosyl diphosphate synthetase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne

    1997-01-01

    The mechanism of binding of the substrates Mg x ATP and ribose 5-phosphate as well as Mg2+ to the enzyme 5-phospho-D-ribosyl (alpha-1-diphosphate synthetase from Escherichia coli has been analyzed. By use of the competive inhibitors of ATP and ribose 5-phosphate binding, alpha,beta-methylene ATP ...

  16. Macro Domain from Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Is an Efficient ADP-ribose Binding Module: CRYSTAL STRUCTURE AND BIOCHEMICAL STUDIES.

    Science.gov (United States)

    Cho, Chao-Cheng; Lin, Meng-Hsuan; Chuang, Chien-Ying; Hsu, Chun-Hua

    2016-03-01

    The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) encodes the conserved macro domain within non-structural protein 3. However, the precise biochemical function and structure of the macro domain is unclear. Using differential scanning fluorimetry and isothermal titration calorimetry, we characterized the MERS-CoV macro domain as a more efficient adenosine diphosphate (ADP)-ribose binding module than macro domains from other CoVs. Furthermore, the crystal structure of the MERS-CoV macro domain was determined at 1.43-Å resolution in complex with ADP-ribose. Comparison of macro domains from MERS-CoV and other human CoVs revealed structural differences in the α1 helix alters how the conserved Asp-20 interacts with ADP-ribose and may explain the efficient binding of the MERS-CoV macro domain to ADP-ribose. This study provides structural and biophysical bases to further evaluate the role of the MERS-CoV macro domain in the host response via ADP-ribose binding but also as a potential target for drug design.

  17. Adenosine diphosphate ribosylation of dinitrogenase reductase and adenylylation of glutamine synthetase control ammonia excretion in ethylenediamine-resistant mutants of Azospirillum brasilense Sp7.

    Science.gov (United States)

    Srivastava, A; Tripathi, A K

    2006-10-01

    Azospirillum brasilense is a nitrogen-fixing, root-colonizing bacterium that brings about plant-growth-promoting effects mainly because of its ability to produce phytohormones. Ethylenediamine (EDA)-resistant mutants of A. brasilense were isolated and screened for their higher ability to decrease acetylene and release ammonia in the medium. One of the mutants showed considerably higher levels of acetylene decrease and ammonia excretion. Nitrogenase activity of this mutant was relatively resistant to inhibition by NH(4)Cl. Adenosine triphosphate ribosylation of dinitrogenase reductase in the mutant did not increase even in presence of 10 mM NH(4)Cl. Although the mutant showed decreased glutamine synthetase (GS) activity, neither the levels of GS synthesized by the mutant nor the NH (4) (+) -binding site in the GS differed from those of the parent. The main reason for the release of ammonia by the mutant seems to be the fixation of higher levels of nitrogen than its GS can assimilate, as well as higher levels of adenylylation of GS, which may decrease ammonia assimilation.

  18. Host cell poly(ADP-ribose glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

    Directory of Open Access Journals (Sweden)

    Salomé C Vilchez Larrea

    Full Text Available Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose glycohydrolase in a trypanosomatid (TcPARG. In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl pyrrolidinediol or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

  19. Structures of the human poly (ADP-ribose glycohydrolase catalytic domain confirm catalytic mechanism and explain inhibition by ADP-HPD derivatives.

    Directory of Open Access Journals (Sweden)

    Julie A Tucker

    Full Text Available Poly(ADP-ribose glycohydrolase (PARG is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG. Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR, adenosine 5'-diphosphate (hydroxymethylpyrrolidinediol (ADP-HPD and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

  20. Combining Higher-Energy Collision Dissociation and Electron-Transfer/Higher-Energy Collision Dissociation Fragmentation in a Product-Dependent Manner Confidently Assigns Proteomewide ADP-Ribose Acceptor Sites.

    Science.gov (United States)

    Bilan, Vera; Leutert, Mario; Nanni, Paolo; Panse, Christian; Hottiger, Michael O

    2017-02-07

    Protein adenosine diphosphate (ADP)-ribosylation is a physiologically and pathologically important post-translational modification. Recent technological advances have improved analysis of this complex modification and have led to the discovery of hundreds of ADP-ribosylated proteins in both cultured cells and mouse tissues. Nevertheless, accurate assignment of the ADP-ribose acceptor site(s) within the modified proteins identified has remained a challenging task. This is mainly due to poor fragmentation of modified peptides. Here, using an Orbitrap Fusion Tribrid mass spectrometer, we present an optimized methodology that not only drastically improves the overall localization scores for ADP-ribosylation acceptor sites but also boosts ADP-ribosylated peptide identifications. First, we systematically compared the efficacy of higher-energy collision dissociation (HCD), electron-transfer dissociation with supplemental collisional activation (ETcaD), and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation methods when determining ADP-ribose acceptor sites within complex cellular samples. We then tested the combination of HCD and EThcD fragmentation, which were employed in a product-dependent manner, and the unique fragmentation properties of the ADP-ribose moiety were used to trigger targeted fragmentation of only the modified peptides. The best results were obtained with a workflow that included initial fast, high-energy HCD (Orbitrap, FT) scans, which produced intense ADP-ribose fragmentation ions. These potentially ADP-ribosylated precursors were then selected and analyzed via subsequent high-resolution HCD and EThcD fragmentation. Using these resulting high-quality spectra, we identified a xxxxxxKSxxxxx modification motif where lysine can serve as an ADP-ribose acceptor site. Due to the appearance of serine within this motif and its close presence to the lysine, further analysis revealed that serine serves as a new ADP-ribose acceptor site

  1. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Andersen, Rune W.; Lo Leggio, Leila; Hove-Jensen, Bjarne

    2015-01-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg2+-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP...... PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate...

  2. Dermal benefits of topical D-ribose

    Directory of Open Access Journals (Sweden)

    Linda M Shecterle

    2009-09-01

    Full Text Available Linda M Shecterle, John A St. CyrJacqmar, Inc., Minneapolis, MN, USAAbstract: Our aging skin undergoes changes with reductions in collagenous and elastic fibers, fibroblasts, mast cells, and macrophages with free radical production, which can result in reduced skin tone and wrinkle formation. Fibroblasts are important for dermal integrity and function with a decrease in function producing less skin tone, thinning, and wrinkle formation. Dermal levels of adenosine triphosphate (ATP decline with aging, potentially altering dermal function. Supplemental D-ribose, a natural occurring carbohydrate, enhances ATP regeneration. D-ribosebased studies demonstrated benefits in both cell culture fibroblastic activities and a subsequent clinical study in women with decreased skin tone with wrinkles. Supplemental D-ribose may offer this needed cellular benefit.Keywords: dermal, fibroblast, ATP, aging, wrinkles

  3. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.B.; Wildung, M.R.; Burke, C.C.; Gershenzon, J.

    1999-03-02

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate. 5 figs.

  4. Geranyl diphosphate synthase from mint

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, Rodney Bruce (Pullman, WA); Wildung, Mark Raymond (Colfax, WA); Burke, Charles Cullen (Moscow, ID); Gershenzon, Jonathan (Jena, DE)

    1999-01-01

    A cDNA encoding geranyl diphosphate synthase from peppermint has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID No:1) is provided which codes for the expression of geranyl diphosphate synthase (SEQ ID No:2) from peppermint (Mentha piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for geranyl diphosphate synthase or for a base sequence sufficiently complementary to at least a portion of the geranyl diphosphate synthase DNA or RNA to enable hybridization therewith (e.g., antisense geranyl diphosphate synthase RNA or fragments of complementary geranyl diphosphate synthase DNA which are useful as polymerase chain reaction primers or as probes for geranyl diphosphate synthase or related genes). In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding geranyl diphosphate synthase. Thus, systems and methods are provided for the recombinant expression of geranyl diphosphate synthase that may be used to facilitate the production, isolation and purification of significant quantities of recombinant geranyl diphosphate synthase for subsequent use, to obtain expression or enhanced expression of geranyl diphosphate synthase in plants in order to enhance the production of monoterpenoids, to produce geranyl diphosphate in cancerous cells as a precursor to monoterpenoids having anti-cancer properties or may be otherwise employed for the regulation or expression of geranyl diphosphate synthase or the production of geranyl diphosphate.

  5. Effect of PD 128763, a new potent inhibitor of poly(ADP-ribose) polymerase, on X-ray-induced cellular recovery processes in Chinese hamster V79 cells

    Energy Technology Data Exchange (ETDEWEB)

    Arundel-Suto, C.M.; Scavone, S.V.; Turner, W.R.; Suto, M.J.; Sebolt-Leopold, J.S. (Warner-Lambert Company, Ann Arbor, MI (USA))

    1991-06-01

    The modifying effects of PD 128763 (3,4-dihydro-5-methyl-1(2H)-isoquinolinone), a potent inhibitor of poly(adenosine-diphosphate (ADP)-ribose) polymerase, on radiation-induced cell killing were examined in Chinese hamster V79 cells. This compound has an IC50 value against the purified enzyme approximately 50X lower than 3-aminobenzamide (3-AB), a widely used specific inhibitor of the enzyme. Exposure of exponentially growing cells to a noncytotoxic concentration (0.5 mM) of PD 128763 for 2 h immediately following X irradiation increased their radiation sensitivity, modifying both the shoulder and the slope of the survival curve. When recovery from sublethal damage and potentially lethal damage was examined in exponential and plateau-phase cells, respectively, postirradiation incubation with 0.5 mM PD 128763 was found not only to inhibit both these processes fully, but also to enhance further the level of radiation-induced cell killing. This is in contrast to the slight effect seen with the less potent inhibitor, 3-AB. The results presented suggest that the mechanism of radiosensitization by PD 128763 is related to the potent inhibition of poly(ADP-ribose) polymerase by this compound.

  6. Manganese dipyridoxyl diphosphate:

    DEFF Research Database (Denmark)

    H, Brurok; Ardenkjær-Larsen, Jan Henrik; G, Hansson

    1999-01-01

    Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent for magnetic resonance imaging (MRI) of the liver. Aims of the study were to examine if MnDPDP possesses superoxide dismutase (SOD) mimetic activity in vitro, and if antioxidant protection can be demonstrated in an ex vivo rat heart...

  7. Poly(ADP-ribose) polymerase-1 protects from oxidative stress induced endothelial dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Gebhard, Catherine; Staehli, Barbara E. [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zurich, Raemistrasse 100, 8091 Zurich (Switzerland); Shi, Yi; Camici, Giovanni G.; Akhmedov, Alexander; Hoegger, Lisa; Lohmann, Christine [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Matter, Christian M. [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zurich, Raemistrasse 100, 8091 Zurich (Switzerland); Hassa, Paul O.; Hottiger, Michael O. [Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Malinski, Tadeusz [Department of Chemistry and Biochemistry, Ohio University, Athens, OH (United States); Luescher, Thomas F. [Cardiovascular Research, Physiology Institute, University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland); Cardiology, Cardiovascular Center, University Hospital Zurich, Raemistrasse 100, 8091 Zurich (Switzerland); and others

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer The nuclear enzyme PARP-1 is a downstream effector of oxidative stress. Black-Right-Pointing-Pointer PARP-1 protects from oxidative stress induced endothelial dysfunction. Black-Right-Pointing-Pointer This effect is mediated through inhibition of vasoconstrictor prostanoid production. Black-Right-Pointing-Pointer Thus, PARP-1 may play a protective role as antioxidant defense mechanism. -- Abstract: Background: Generation of reactive oxygen species (ROS) is a key feature of vascular disease. Activation of the nuclear enzyme poly (adenosine diphosphate [ADP]-ribose) polymerase-1 (PARP-1) is a downstream effector of oxidative stress. Methods: PARP-1(-/-) and PARP-1(+/+) mice were injected with paraquat (PQ; 10 mg/kg i.p.) to induce intracellular oxidative stress. Aortic rings were suspended in organ chambers for isometric tension recording to analyze vascular function. Results: PQ treatment markedly impaired endothelium-dependent relaxations to acetylcholine in PARP-1(-/-), but not PARP-1(+/+) mice (p < 0.0001). Maximal relaxation was 45% in PQ treated PARP-1(-/-) mice compared to 79% in PARP-1(+/+) mice. In contrast, endothelium-independent relaxations to sodium nitroprusside (SNP) were not altered. After PQ treatment, L-NAME enhanced contractions to norepinephrine by 2.0-fold in PARP-1(-/-) mice, and those to acetylcholine by 3.3-fold, respectively, as compared to PARP-1(+/+) mice. PEG-superoxide dismutase (SOD) and PEG-catalase prevented the effect of PQ on endothelium-dependent relaxations to acetylcholine in PARP-1(-/-) mice (p < 0.001 vs. PQ treated PARP-1(+/+) mice. Indomethacin restored endothelium-dependent relaxations to acetylcholine in PQ treated PARP-1(-/-) mice (p < 0.05 vs. PQ treated PARP-1(+/+). Conclusion: PARP-1 protects from acute intracellular oxidative stress induced endothelial dysfunction by inhibiting ROS induced production of vasoconstrictor prostanoids.

  8. Bis(4-methoxybenzylammonium dihydrogen diphosphate

    Directory of Open Access Journals (Sweden)

    Adel Elboulali

    2013-02-01

    Full Text Available In the title compound, 2C8H12NO+·H2P2O72−, the linked PO4 groups of the diphosphate anion are almost eclipsed and the P—O—P angle is 134.45 (7°. In the crystal, infinite ribbons of H2P2O72− anions propagate in [100], being linked by strong O—H...O hydrogen bonds. The 4-methoxybenzylammonium cations bond to the diphosphate chains by N—H...O and C—H...O links, and are themselves linked by C—H...π interactions.

  9. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens;

    2017-01-01

    is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP......Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds...... synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number...

  10. Reactions of fac-[Re(CO)3(H2O)3]+ with nucleoside diphosphates and thiamine diphosphate in aqueous solution investigated by multinuclear NMR spectroscopy.

    Science.gov (United States)

    Adams, Kristie M; Marzilli, Patricia A; Marzilli, Luigi G

    2007-10-29

    Products formed between monoester diphosphates (MDPs) and fac-[Re(CO)3(H2O)3]OTf at pH 3.6 were examined. Such adducts of the fac-[Re(CO)3]+ moiety have an uncommon combination of properties for an "inert" metal center in that sharp NMR signals can be observed, yet the products are equilibrating at rates allowing NMR EXSY cross-peaks to be observed. Thiamine diphosphate (TDP) and uridine 5'-diphosphate (5'-UDP) form 1:1 bidentate {Palpha,Pbeta} chelates, in which the MDP binds Re(I) via Palpha and Pbeta phosphate groups. Asymmetric centers are created at Re(I) (RRe/SRe) and Palpha (Delta/Lambda), leading to four diastereomers. The two mirror pairs of diastereomers (RReDelta/SReLambda) and (RReLambda/SReDelta) for TDP (no ribose) and for all four diastereomers (RReDelta, RReLambda, SReDelta, SReLambda) for 5'-UDP (asymmetric ribose) gave two and four sets of NMR signals for the bound MDP, respectively. 31Palpha-31Palpha EXSY cross-peaks indicate that the fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- isomers interchange slowly on the NMR time scale, with an average k approximately equal to 0.8 s(-1) at 32 degrees C; the EXSY cross-peaks could arise from chirality changes at only Re(I) or at only Palpha. Guanosine 5'-diphosphate (5'-GDP), with a ribose moiety and a Re(I)-binding base, formed both possible diastereomers (RRe and SRe) of the fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- macrochelate, with one slightly more abundant diastereomer suggested to be RRe by Mn2+ ion 1H NMR signal line-broadening combined with distances from molecular models. Interchange of the diastereomers requires that the coordination site of either N7 or Pbeta move to the H2O site. 31Palpha-31Palpha EXSY cross-peaks indicate a k approximately equal to 0.5 s(-1) at 32 degrees C for RRe-to-SRe interchange. The similarity of the rate constants for interchange of fac-[Re(CO)3(H2O)({Palpha,Pbeta}MDP)]- and fac-[Re(CO)3(H2O)({N7,Pbeta}GDP)]- adducts suggest strongly that interchange of Pbeta and H2O coordination

  11. Structure activity relationship of uridine 5′-diphosphate analogues at the human P2Y6 receptor

    Science.gov (United States)

    Besada, Pedro; Shin, Dae Hong; Costanzi, Stefano; Ko, Hyojin; Mathé, Christophe; Gagneron, Julien; Gosselin, Gilles; Maddileti, Savitri; Harden, T. Kendall; Jacobsona, Kenneth A.

    2012-01-01

    The structure activity relationships and molecular modeling of the uracil nucleotide-activated P2Y6 receptor have been studied. A series of UDP analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group was synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4-position, with the synthesis of 4-substituted-thiouridine-5′-diphosphate analogues, as well as at positions 3 and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3′,5′-diphosphate analogue, a 3′-diphosphate analogue and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 0.15 μM) was equipotent to UDP, while substitutions of the 2′-hydroxyl (amino, azido) greatly reduce potency. 2- and 4-Thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the β-phosphate of 5′-UDP and analogs is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 μM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S) conformation (P= 126°, 1′-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligands PMID:16942026

  12. Adenosine and adenosine receptors: Newer therapeutic perspective

    Directory of Open Access Journals (Sweden)

    Manjunath S

    2009-01-01

    Full Text Available Adenosine, a purine nucleoside has been described as a ′retaliatory metabolite′ by virtue of its ability to function in an autocrine manner and to modify the activity of a range of cell types, following its extracellular accumulation during cell stress or injury. These effects are largely protective and are triggered by binding of adenosine to any of the four adenosine receptor subtypes namely A1, A2a, A2b, A3, which have been cloned in humans, and are expressed in most of the organs. Each is encoded by a separate gene and has different functions, although overlapping. For instance, both A1 and A2a receptors play a role in regulating myocardial oxygen consumption and coronary blood flow. It is a proven fact that adenosine plays pivotal role in different physiological functions, such as induction of sleep, neuroprotection and protection against oxidative stress. Until now adenosine was used for certain conditions like paroxysmal supraventricular tachycardia (PSVT and Wolff Parkinson White (WPW syndrome. Now there is a growing evidence that adenosine receptors could be promising therapeutic targets in a wide range of conditions including cardiac, pulmonary, immunological and inflammatory disorders. After more than three decades of research in medicinal chemistry, a number of selective agonists and antagonists of adenosine receptors have been discovered and some have been clinically evaluated, although none has yet received regulatory approval. So this review focuses mainly on the newer potential role of adenosine and its receptors in different clinical conditions.

  13. D-ribose benefits restless legs syndrome.

    Science.gov (United States)

    Shecterle, Linda; Kasubick, Robert; St Cyr, John

    2008-11-01

    Restless legs syndrome is a neurological disorder characterized by unpleasant sensations and pain, predominantly in the lower extremities while at rest, accompanied by an uncontrollable urge for movement for relief. We report on two affected male individuals, a father and son, ages 71 and 47, from a family in which three generations carry the diagnosis. To evaluate any potential benefit of D-ribose in this condition, each individual orally consumed 5-g doses of D-ribose daily at different trial stages. Each stage lasted 3 weeks with a 2-week washout period between stages. The initial stage involved a single 5 gm dose of D-ribose consumed at breakfast. Throughout the second stage, D-ribose was taken at breakfast and lunch. In the third stage, D-ribose was taken at all meals, breakfast, lunch, and dinner. Diaries by the subjects pertaining to their documentation and severity of restless legs syndrome symptoms was compiled. During the initial stage both men reported a general feeling of more energy and less fatigue, most notably after exercise, without any significant changes in their symptoms. With the increase in the daily dose of D-ribose, in the second stage, their leg twitching and the feeling to move during the day was reduced for 1 subject, and rarely present in the other. Both still experienced the unpleasant sensations during the night. However, during the final stage, a further increase in the daily dose of D-ribose eliminated their daily symptoms and the symptoms at night were of a lesser degree and had a later occurrence. Both men reported that D-ribose did not totally eliminate their discomfort, but the severity and onset of symptoms affecting their quality of life was substantially improved with D-ribose without any adverse reactions.

  14. Activation of thiamin diphosphate in enzymes.

    Science.gov (United States)

    Hübner, G; Tittmann, K; Killenberg-Jabs, M; Schäffner, J; Spinka, M; Neef, H; Kern, D; Kern, G; Schneider, G; Wikner, C; Ghisla, S

    1998-06-29

    Activation of the coenzyme ThDP was studied by measuring the kinetics of deprotonation at the C2 carbon of thiamin diphosphate in the enzymes pyruvate decarboxylase, transketolase, pyruvate dehydrogenase complex, pyruvate oxidase, in site-specific mutant enzymes and in enzyme complexes containing coenzyme analogues by proton/deuterium exchange detected by 1H-NMR spectroscopy. The respective deprotonation rate constant is above the catalytic constant in all enzymes investigated. The fast deprotonation requires the presence of an activator in pyruvate decarboxylase from yeast, showing the allosteric regulation of this enzyme to be accomplished by an increase in the C2-H dissociation rate of the enzyme-bound thiamin diphosphate. The data of the thiamin diphosphate analogues and of the mutant enzymes show the N1' atom and the 4'-NH2 group to be essential for the activation of the coenzyme and a conserved glutamate involved in the proton abstraction mechanism of the enzyme-bound thiamin diphosphate.

  15. Recycling of 5'-methylthioadenosine-ribose carbon atoms into methionine in tomato tissue in relation to ethylene production.

    Science.gov (United States)

    Wang, S Y; Adams, D O; Lieberman, M

    1982-07-01

    The ribose moiety of 5'-methylthioadenosine (MTA) is metabolized to form the four-carbon unit (2-aminobutyrate) of methionine in tomato tissue (Lycopersicon esculentum Mill., cv. Pik Red). When [U-(14)C-adenosine] MTA was administered to tomato tissue slices, label was recovered in 5-methylthioribose (MTR), methionine, 1-aminocyclopropane-1-carboxylic acid (ACC), C(2)H(4) and other unidentified compounds. However, when [U-(14)C-ribose]MTR was administered, radioactivities were recovered in methionine, ACC and C(2)H(4), but not MTA. This suggests that C(2)H(4) formed in tomato pericarp tissue may be derived from the ribose portion of MTA via MTR, methionine and ACC. The conversion of MTR to methionine is not inhibited by aminoethoxyvinylglycine (AVG), but is O(2) dependent. These data present a new salvage pathway for methionine biosynthesis which may be important in relation to polyamine and ethylene biosynthesis in tomato tissue.

  16. Neer Award 2016: reduced muscle degeneration and decreased fatty infiltration after rotator cuff tear in a poly(ADP-ribose) polymerase 1 (PARP-1) knock-out mouse model.

    Science.gov (United States)

    Kuenzler, Michael B; Nuss, Katja; Karol, Agnieszka; Schär, Michael O; Hottiger, Michael; Raniga, Sumit; Kenkel, David; von Rechenberg, Brigitte; Zumstein, Matthias A

    2017-05-01

    Disturbed muscular architecture, atrophy, and fatty infiltration remain irreversible in chronic rotator cuff tears even after repair. Poly (adenosine 5'-diphosphate-ribose) polymerase 1 (PARP-1) is a key regulator of inflammation, apoptosis, muscle atrophy, muscle regeneration, and adipocyte development. We hypothesized that the absence of PARP-1 would lead to a reduction in damage to the muscle subsequent to combined tenotomy and neurectomy in a PARP-1 knockout (KO) mouse model. PARP-1 KO and wild-type C57BL/6 (WT group) mice were analyzed at 1, 6, and 12 weeks (total n = 84). In all mice, the supraspinatus and infraspinatus muscles of the left shoulder were detached and denervated. Macroscopic analysis, magnetic resonance imaging, gene expression analysis, immunohistochemistry, and histology were used to assess the differences in PARP-1 KO and WT mice. The muscles in the PARP-1 KO group had significantly less retraction, atrophy, and fatty infiltration after 12 weeks than in the WT group. Gene expression of inflammatory, apoptotic, adipogenic, and muscular atrophy genes was significantly decreased in PARP-1 KO mice in the first 6 weeks. Absence of PARP-1 leads to a reduction in muscular architectural damage, early inflammation, apoptosis, atrophy, and fatty infiltration after combined tenotomy and neurectomy of the rotator cuff muscle. Although the macroscopic reaction to injury is similar in the first 6 weeks, the ability of the muscles to regenerate was much greater in the PARP-1 KO group, leading to a near-normalization of the muscle after 12 weeks. Copyright © 2017 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.

  17. Ammonium ytterbium(III diphosphate(V

    Directory of Open Access Journals (Sweden)

    Michal Dušek

    2008-12-01

    Full Text Available The title compound, NH4YbP2O7, crystallizes in the KAlP2O7 structure type and consists of distorted YbO6 octahedra and bent P2O74− diphosphate units forming together a three-dimensional network. There are channels in the structure running along the c axis, where the NH4+ cations are located. They are connected via N—H...O hydrogen bonds to the terminal O atoms of the diphosphate anions.

  18. Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance.

    Science.gov (United States)

    Hove-Jensen, Bjarne; Andersen, Kasper R; Kilstrup, Mogens; Martinussen, Jan; Switzer, Robert L; Willemoës, Martin

    2017-03-01

    Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species.

  19. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...

  20. Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine.

    Science.gov (United States)

    Ross, Ashley E; Venton, B Jill

    2012-07-07

    Adenosine is a neuromodulator that regulates neurotransmission. Adenosine can be monitored using fast-scan cyclic voltammetry at carbon-fiber microelectrodes and ATP is a possible interferent in vivo because the electroactive moiety, adenine, is the same for both molecules. In this study, we investigated carbon-fiber microelectrodes coated with Nafion and carbon nanotubes (CNTs) to enhance the sensitivity of adenosine and decrease interference by ATP. Electrodes coated in 0.05 mg mL(-1) CNTs in Nafion had a 4.2 ± 0.2 fold increase in current for adenosine, twice as large as for Nafion alone. Nafion-CNT electrodes were 6 times more sensitive to adenosine than ATP. The Nafion-CNT coating did not slow the temporal response of the electrode. Comparing different purine bases shows that the presence of an amine group enhances sensitivity and that purines with carbonyl groups, such as guanine and hypoxanthine, do not have as great an enhancement after Nafion-CNT coating. The ribose group provides additional sensitivity enhancement for adenosine over adenine. The Nafion-CNT modified electrodes exhibited significantly more current for adenosine than ATP in brain slices. Therefore, Nafion-CNT modified electrodes are useful for sensitive, selective detection of adenosine in biological samples.

  1. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP.

    Science.gov (United States)

    Andersen, Rune W; Leggio, Leila Lo; Hove-Jensen, Bjarne; Kadziola, Anders

    2015-03-01

    The enzyme 5-phosphoribosyl-1-α-diphosphate (PRPP) synthase (EC 2.7.6.1) catalyses the Mg(2+)-dependent transfer of a diphosphoryl group from ATP to the C1 hydroxyl group of ribose 5-phosphate resulting in the production of PRPP and AMP. A nucleotide sequence specifying Sulfolobus solfataricus PRPP synthase was synthesised in vitro with optimised codon usage for expression in Escherichia coli. Following expression of the gene in E. coli PRPP synthase was purified by heat treatment and ammonium sulphate precipitation and the structure of S. solfataricus PRPP synthase was determined at 2.8 Å resolution. A bent dimer oligomerisation was revealed, which seems to be an abundant feature among PRPP synthases for defining the adenine specificity of the substrate ATP. Molecular replacement was used to determine the S. solfataricus PRPP synthase structure with a monomer subunit of Methanocaldococcus jannaschii PRPP synthase as a search model. The two amino acid sequences share 35 % identity. The resulting asymmetric unit consists of three separated dimers. The protein was co-crystallised in the presence of AMP and ribose 5-phosphate, but in the electron density map of the active site only AMP and a sulphate ion were observed. Sulphate ion, reminiscent of the ammonium sulphate precipitation step of the purification, seems to bind tightly and, therefore, presumably occupies and blocks the ribose 5-phosphate binding site. The activity of S. solfataricus PRPP synthase is independent of phosphate ion.

  2. Motesanib diphosphate in progressive differentiated thyroid cancer

    DEFF Research Database (Denmark)

    Sherman, Steven I; Wirth, Lori J; Droz, Jean-Pierre

    2008-01-01

    BACKGROUND: The expression of vascular endothelial growth factor (VEGF) is characteristic of differentiated thyroid cancer and is associated with aggressive tumor behavior and a poor clinical outcome. Motesanib diphosphate (AMG 706) is a novel oral inhibitor of VEGF receptors, platelet......-derived growth-factor receptor, and KIT. METHODS: In an open-label, single-group, phase 2 study, we treated 93 patients who had progressive, locally advanced or metastatic, radioiodine-resistant differentiated thyroid cancer with 125 mg of motesanib diphosphate, administered orally once daily. The primary end...... point was an objective response as assessed by an independent radiographic review. Additional end points included the duration of the response, progression-free survival, safety, and changes in serum thyroglobulin concentration. RESULTS: Of the 93 patients, 57 (61%) had papillary thyroid carcinoma...

  3. How thiamine diphosphate is activated in enzymes.

    Science.gov (United States)

    Kern, D; Kern, G; Neef, H; Tittmann, K; Killenberg-Jabs, M; Wikner, C; Schneider, G; Hübner, G

    1997-01-03

    The controversial question of how thiamine diphosphate, the biologically active form of vitamin B1, is activated in different enzymes has been addressed. Activation of the coenzyme was studied by measuring thermodynamics and kinetics of deprotonation at the carbon in the 2-position (C2) of thiamine diphosphate in the enzymes pyruvate decarboxylase and transketolase by use of nuclear magnetic resonance spectroscopy, proton/deuterium exchange, coenzyme analogs, and site-specific mutant enzymes. Interaction of a glutamate with the nitrogen in the 1'-position in the pyrimidine ring activated the 4'-amino group to act as an efficient proton acceptor for the C2 proton. The protein component accelerated the deprotonation of the C2 atom by several orders of magnitude, beyond the rate of the overall enzyme reaction. Therefore, the earlier proposed concerted mechanism or stabilization of a C2 carbanion can be excluded.

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

    Science.gov (United States)

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

    2002-03-01

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

  5. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  6. Adenosine receptor neurobiology: overview.

    Science.gov (United States)

    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.

  7. Synthesis of phosphonate and phostone analogues of ribose-1-phosphates

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    Pitak Nasomjai

    2009-07-01

    Full Text Available The synthesis of phosphonate analogues of ribose-1-phosphate and 5-fluoro-5-deoxyribose-1-phosphate is described. Preparations of both the α- and β-phosphonate anomers are reported for the ribose and 5-fluoro-5-deoxyribose series and a synthesis of the corresponding cyclic phostones of each α-ribose is also reported. These compounds have been prepared as tools to probe the details of fluorometabolism in S. cattleya.

  8. Synthesis of phosphonate and phostone analogues of ribose-1-phosphates.

    Science.gov (United States)

    Nasomjai, Pitak; O'Hagan, David; Slawin, Alexandra M Z

    2009-07-27

    The synthesis of phosphonate analogues of ribose-1-phosphate and 5-fluoro-5-deoxyribose-1-phosphate is described. Preparations of both the alpha- and beta-phosphonate anomers are reported for the ribose and 5-fluoro-5-deoxyribose series and a synthesis of the corresponding cyclic phostones of each alpha-ribose is also reported. These compounds have been prepared as tools to probe the details of fluorometabolism in S. cattleya.

  9. A Versatile Photoactivatable Probe Designed to Label the Diphosphate Binding Site of Farnesyl Diphosphate Utilizing Enzymes

    Science.gov (United States)

    Henry, Olivier; Lopez-Gallego, Fernando; Agger, Sean A.; Schmidt-Dannert, Claudia; Sen, Stephanie; Shintani, David; Cornish, Katrina; Distefano, Mark D.

    2009-01-01

    Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit S. cerevisiae protein farnesyltransferase (ScPFTase) with IC50 values of 5.8 (meta isomer) and 3.0 µM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a KI of 0.46 µM. Radiolabeled forms of both analogues selectively labelled the β-subunit of ScPFTase. The para isomer was also shown to label E. coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes. PMID:19447628

  10. Adenosine and sleep

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Inhibition of aortic vessel adenosine diphosphate degradation by cadmium and mercury.

    Science.gov (United States)

    Togna, G; Dolci, N; Caprino, L

    1984-01-01

    The effects of cadmium and mercury on ADP breakdown by vessel walls were investigated. These metals reduce the ADP clearance promoted by arterial tissue. This effect could be attributed to the inhibition of vessel wall ADP-ase enzyme, which plays an important role in the genesis of thrombotic phenomena.

  12. The inhibitory activity of ginsenoside Rp4 in adenosine diphosphate-induced platelet aggregation

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    Young-Min Son

    2017-01-01

    Conclusion: G-Rp4 significantly inhibited ADP-induced platelet aggregation and this is mediated via modulating the intracellular signaling molecules. These results indicate that G-Rp4 could be a potential candidate as a therapeutic agent against platelet-related cardiovascular diseases.

  13. Adenosine diphosphate glucose pyrophosphorylase genes in wheat: differential expression and gene mapping.

    Science.gov (United States)

    Ainsworth, C; Hosein, F; Tarvis, M; Weir, F; Burrell, M; Devos, K M; Gale, M D

    1995-01-01

    A full-length cDNA clone representing the large (shrunken-2) subunit of ADP-glucose pyrophosphorylase (AGP; EC 2.7.7.27) has been isolated from a cDNA library prepared from developing grain of hexaploid wheat (Triticum aestivum L., cv. Chinese Spring). The 2084-bp cDNA insert contains an open reading frame of 1566 nucleotides and primer-extension analysis indicated that the 5' end is 10 nucleotides shorter than the mRNA. The deduced protein contains 522 amino acids (57.8 kDa) and includes a putative transit peptide of 62 amino acids (6.5 kDa). The similarity of the deduced protein to the small subunit of AGP and to other AGP genes from plants and microorganisms is discussed. Northern hybridisation shows that the Agp1 genes (encoding the small subunit in the wheat endosperm) and the Agp2 genes (encoding the large subunit in the wheat endosperm) are differentially expressed in the wheat grain. Transcripts from both gene sets accumulate to high levels in the endosperm during grain development with the majority of the expression in the endopsperm rather than the embryo and pericarp layers. Although enzyme activity is detected in developing grains prior to 10 d post anthesis, only the Agp1 genes are active at this time (the Agp2 genes are not expressed until 10 d post anthesis). The possibility that the enzyme expressed during early grain development is a homotetramer of small subunits is discussed. The Agp1 and Agp2 genes are arranged as triplicate sets of single-copy homoeoloci in wheat. The Agp2 genes are located on the long arms of chromosomes 1A, 1B and 1D, about 80 cM from the centromere. The Agp1 genes have been mapped to a position just distal to the centromere on the long arms of chromosomes 7A, 7B and 7D.

  14. RESIDUAL PLATELET REACTIVITY DURING THERAPY WITH INHIBITORS OF CYCLOOXIGENASE OR ADENOSINE DIPHOSPHATE RECEPTORS

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    A. A. Lomonosova

    2012-01-01

    Full Text Available Aim. To compare effects of acetylsalicylic acid (ASA and two clopidogrel drugs on residual platelet aggregative reactivity (RPAR. Material and methods. Patients (n=40 with ischemic heart disease aged under 70 years were involved into the crossover study. Clinical examination included questionnaire survey , blood pressure (BP measurement, ECG registration, 24-hour ECG and BP monitoring, determination of blood levels of total cholesterol, high density lipoproteins, triglycerides, transaminases, and creatinine, complete blood cell count, including platelets number and hemoglobin level. Besides evaluation of the platelet aggregation by optical aggregometry was performed initially , after one week ASA treatment and after every next 3 week clopidogrel treatment period.  Results. RPAR during ASA monotherapy was 56.4±0.3%. There were no significant differences in effects of original and generic clopidogrel on RPAR. Сlopidogrel therapy reduced RPAR more significantly (42.2±0.2% than ASA monotherapy did (p=0.0003. Authors proposed definition for high level of RPAR during therapy - it is platelet aggregation more than 46%. Data analysis taking into account this criterion showed that a number of patients with high RPAR was 70 and 30% among patients treated with enterosoluble ASA and clopidogrel, respectively. Conclusion. Study results show that a significant number of patients receiving antiplatelet monotherapy does not achieve the target level of RPAR(<46%. These results may be a rationale for combined therapy in patients of this type.

  15. Localization of Adenosine Triphosphatase Activity on the Chloroplast Envelope in Tendrils of Pisum sativum1

    Science.gov (United States)

    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

  16. Rv0989c encodes a novel (E)-geranyl diphosphate synthase facilitating decaprenyl diphosphate biosynthesis in Mycobacterium tuberculosis.

    Science.gov (United States)

    Mann, Francis M; Thomas, Jill A; Peters, Reuben J

    2011-02-04

    Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the "missing" GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from Mtb, resolving previous discrepancies between biochemical and genetic studies of cell wall biosynthesis.

  17. Studies on thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Leeper, F J; Hawksley, D; Mann, S; Perez Melero, C; Wood, M D H

    2005-08-01

    The 3-deaza analogue of TPP (thiamine diphosphate), a close mimic of the ylid intermediate, has been synthesized and is an extremely potent inhibitor of a variety of TPP-dependent enzymes, binding much more tightly than TPP itself. Results using deazaTPP complexed with the E1 subunit of PDH (pyruvate dehydrogenase) have led to a novel proposal about the mechanism of this enzyme. The 2-substituted forms of deazaTPP, which mimic other intermediates in the catalytic mechanism, can also be synthesized and 2-(1-hydroxyethyl)deazaTPP is also an extremely potent inhibitor of PDC (pyruvate decarboxylase). Attachment of such 2-substituents is expected to be a way to introduce selectivity in the inhibition of various TPP-dependent enzymes.

  18. Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase.

    Science.gov (United States)

    Bergstrom, J D; Bostedor, R G; Masarachia, P J; Reszka, A A; Rodan, G

    2000-01-01

    Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.

  19. Daily supplementation of D-ribose shows no therapeutic benefits in the MHC-I transgenic mouse model of inflammatory myositis.

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    William Coley

    Full Text Available BACKGROUND: Current treatments for idiopathic inflammatory myopathies (collectively called myositis focus on the suppression of an autoimmune inflammatory response within the skeletal muscle. However, it has been observed that there is a poor correlation between the successful suppression of muscle inflammation and an improvement in muscle function. Some evidence in the literature suggests that metabolic abnormalities in the skeletal muscle underlie the weakness that continues despite successful immunosuppression. We have previously shown that decreased expression of a purine nucleotide cycle enzyme, adenosine monophosphate deaminase (AMPD1, leads to muscle weakness in a mouse model of myositis and may provide a mechanistic basis for muscle weakness. One of the downstream metabolites of this pathway, D-ribose, has been reported to alleviate symptoms of myalgia in patients with a congenital loss of AMPD1. Therefore, we hypothesized that supplementing exogenous D-ribose would improve muscle function in the mouse model of myositis. We treated normal and myositis mice with daily doses of D-ribose (4 mg/kg over a 6-week time period and assessed its effects using a battery of behavioral, functional, histological and molecular measures. RESULTS: Treatment with D-ribose was found to have no statistically significant effects on body weight, grip strength, open field behavioral activity, maximal and specific forces of EDL, soleus muscles, or histological features. Histological and gene expression analysis indicated that muscle tissues remained inflamed despite treatment. Gene expression analysis also suggested that low levels of the ribokinase enzyme in the skeletal muscle might prevent skeletal muscle tissue from effectively utilizing D-ribose. CONCLUSIONS: Treatment with daily oral doses of D-ribose showed no significant effect on either disease progression or muscle function in the mouse model of myositis.

  20. Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate.

    Science.gov (United States)

    Schilmiller, Anthony L; Schauvinhold, Ines; Larson, Matthew; Xu, Richard; Charbonneau, Amanda L; Schmidt, Adam; Wilkerson, Curtis; Last, Robert L; Pichersky, Eran

    2009-06-30

    We identified a cis-prenyltransferase gene, neryl diphosphate synthase 1 (NDPS1), that is expressed in cultivated tomato (Solanum lycopersicum) cultivar M82 type VI glandular trichomes and encodes an enzyme that catalyzes the formation of neryl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. mRNA for a terpene synthase gene, phellandrene synthase 1 (PHS1), was also identified in these glands. It encodes an enzyme that uses neryl diphosphate to produce beta-phellandrene as the major product as well as a variety of other monoterpenes. The profile of monoterpenes produced by PHS1 is identical with the monoterpenes found in type VI glands. PHS1 and NDPS1 map to chromosome 8, and the presence of a segment of chromosome 8 derived from Solanum pennellii LA0716 causes conversion from the M82 gland monoterpene pattern to that characteristic of LA0716 plants. The data indicate that, contrary to the textbook view of geranyl diphosphate as the "universal" substrate of monoterpene synthases, in tomato glands neryl diphosphate serves as a precursor for the synthesis of monoterpenes.

  1. Adenosine improves cardiomyocyte respiratory efficiency.

    Science.gov (United States)

    Babsky, A M; Doliba, M M; Doliba, N M; Osbakken, M D

    1998-01-01

    The role of adenosine on the regulation of mitochondrial function has been studied. In order to evaluate this the following experiments were done in isolated rat cardiomyocites and mitochondria using polarographic techniques. Cardiomyocyte oxygen consumption (MVO2) and mitochondrial respiratory function (State 3 and State 4, respiratory control index, and ADP/O ratio) were evaluated after exposure to adenosine. Cardiomyocyte MVO2 was significantly lower in cells previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) than in cells not exposed to adenosine (control). Addition of dipyridamole (10 microM) or 8-(p-Sulfophenyl) theophylline (50 microM) to cardiomyocytes before adenosine incubation prevented the adenosine-induced changes in MVO2. Mitochondria obtained from isolated perfused beating heart previously perfused with adenosine (10 microM, 30 min heart perfusion) also resulted in significant increases in ADP/O and respiratory control index compared to matching control. Mitochondria isolated from cardiomyocytes previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) resulted in a significant increase in mitochondrial ADP/O ratio compared to control. Adenosine-induced decrease in cardiomyocyte MVO2 may be related to an increase in efficiency of mitochondrial oxidative phosphorylation, and more economical use of oxygen, which is necessary for survival under ischemic stress.

  2. Effect of guanosine 5'-diphosphate 3'-diphosphate and related nucleoside polyphosphates on induction of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli.

    Science.gov (United States)

    Yoshimoto, A; Oki, T; Inui, T

    1978-10-04

    Exogenous addition of guanosine and adenosine 5'-(mono, di and tri) phosphate 3'-diphosphates (pppGpp, ppGpp, pGpp, pppApp, ppApp and pApp) stimulated the synthesis of tryptophanase and beta-galactosidase in permeabilized cells of Escherichia coli. From the results obtained with ppGpp and pppApp, this effect appeared to be at a transcriptional level and depended greatly on the growth condition; the largest effect was observed in cells under shiftdown or grown on poor enrgy source. ppGpp and pppApp, unlike cyclic AMP, did not act to overcome the inhibition of enzyme induction by glucose, but in combination with cyclic AMP caused a synergistic stimulation effect. In the shiftdown cells, ppGpp and pppApp gave 30% or more stimulation effect on tryptophanase induction while cyclic AMP did not stimulate induction. There was therefore a pronounced difference between cyclic AMP and ppGpp or pppApp in stimulatory function.

  3. Trapping Poly(ADP-Ribose) Polymerase.

    Science.gov (United States)

    Shen, Yuqiao; Aoyagi-Scharber, Mika; Wang, Bing

    2015-06-01

    Recent findings indicate that a major mechanism by which poly(ADP-ribose) polymerase (PARP) inhibitors kill cancer cells is by trapping PARP1 and PARP2 to the sites of DNA damage. The PARP enzyme-inhibitor complex "locks" onto damaged DNA and prevents DNA repair, replication, and transcription, leading to cell death. Several clinical-stage PARP inhibitors, including veliparib, rucaparib, olaparib, niraparib, and talazoparib, have been evaluated for their PARP-trapping activity. Although they display similar capacity to inhibit PARP catalytic activity, their relative abilities to trap PARP differ by several orders of magnitude, with the ability to trap PARP closely correlating with each drug's ability to kill cancer cells. In this article, we review the available data on molecular interactions between these clinical-stage PARP inhibitors and PARP proteins, and discuss how their biologic differences might be explained by the trapping mechanism. We also discuss how to use the PARP-trapping mechanism to guide the development of PARP inhibitors as a new class of cancer therapy, both for single-agent and combination treatments. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  4. A STUDY OF THE NUCLEOSIDE TRI- AND DIPHOSPHATE ACTIVITIES OF RAT LIVER MICROSOMES

    Science.gov (United States)

    Ernster, Lars; Jones, Lois C.

    1962-01-01

    Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment

  5. ADP-ribose in glycation and glycoxidation reactions.

    Science.gov (United States)

    Jacobson, E L; Cervantes-Laurean, D; Jacobson, M K

    1997-01-01

    Glycation is initiated by reaction of a reducing sugar with a protein amino group to generate a Schiff base adduct. Following an Amadori rearrangement to form a ketoamine adduct, a complex chemistry involving oxidation often leads to protein glycoxidation products referred to as advanced glycosylation end products (AGE). The AGE include protein carboxymethyllysine (CML) residues and a heterogeneous group of complex modifications characterized by high fluorescence and protein-protein cross links. The sugar sources for the glycoxidation of intracellular proteins are not well defined but pentoses have been implicated because they are efficient precursors for the formation of the fluorescent AGE, pentosidine. ADP-ribose, generated from NAD by ADP-ribose transfer reactions, is a likely intracellular source of a reducing pentose moiety. Incubation of ADP-ribose with histones results in the formation of ketoamine glycation conjugates and also leads to the rapid formation of protein CML residues, histone H1 dimers, and highly fluorescent products with properties similar to the AGE. ADP-ribose is much more efficient than other possible pentose donors for glycation and glycoxidation of protein amino groups. Recently developed methods that differentiate nonenzymic modifications of proteins by ADP-ribose from enzymic modifications now allow investigations to establish whether some protein modifications by monomers of ADP-ribose in vivo represent glycation and glycoxidation.

  6. Effects of adenosine on lymphangiogenesis.

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    Bénédicte Lenoir

    Full Text Available BACKGROUND: The lymphatic system controls tissue homeostasis by draining protein-rich lymph to the vascular system. Lymphangiogenesis, the formation of lymphatic vessels, is a normal event in childhood but promotes tumor spread and metastasis during adulthood. Blocking lymphangiogenesis may therefore be of therapeutic interest. Production of adenosine is enhanced in the tumor environment and contributes to tumor progression through stimulation of angiogenesis. In this study, we determined whether adenosine affects lymphangiogenesis. METHODS: Lymphatic endothelial cells (HMVEC-dLy were cultured in presence of adenosine and their proliferation, migration and tube formation was assessed. Gelatin sponges embedded with the stable analogue of adenosine 2-chloro adenosine were implanted in mice ear and lymphangiogenesis was quantified. Mice were intravenously injected with adenoviruses containing expression vector for 5'-endonucleotidase, which plays a major role in the formation of adenosine. RESULTS: In vitro, we observed that adenosine decreased the proliferation of lymphatic endothelial cells, their migration and tube formation. However, in vivo, gelatin sponges containing 2-chloro adenosine and implanted in mice ear displayed an elevated level of lymphangiogenesis (2.5-fold, p<0.001. Adenovirus-mediated over-expression of cytosolic 5'-nucleotidase IA stimulated lymphangiogenesis and the recruitment of macrophages in mouse liver. Proliferation of lymphatic endothelial cells was enhanced (2-fold, p<0.001 when incubated in the presence of conditioned medium from murine macrophages. CONCLUSION: We have shown that adenosine stimulates lymphangiogenesis in vivo, presumably through a macrophage-mediated mechanism. This observation suggests that blockade of adenosine receptors may help in anti-cancer therapies.

  7. Effect of 5-Aminoisoquinolinone on the Adhesion of Colon Carcinoma

    Institute of Scientific and Technical Information of China (English)

    WANG Ya-lan; HAO Lan-xiang

    2007-01-01

    Objective: 5-Aminoisoquinolinone, a water-soluble, potent inhibitor of the activity of poly (adenosine 5'-diphosphate ribose) polymerase, plays an important role in the tissue injury associated with ischaemia-reperfusion injury and inflammation by inhibiting the activity of poly (adenosine 5'-diphosphate ribose) polymerase and the expression of cell adhesion molecules such as ICAM-1, P-selectin et al. But how about it in the tumor is not clear. The aim of the present study was to study the effects of 5-Aminoisoquinolinon on the adhesion of colon carcinoma line HT-29 cells to human umbilical vein endothelial cells; and the effects of 5-Aminoisoquinolinon on the expression of ICAM-1, P-selectin and the activity of poly (adenosine 5'-diphosphate ribose) polymerase in colon carcinoma HT-29 cells. Methods: The adhesion of HT-29 cells to human umbilical vein endothelial cells was detected by adhesive experiment. Immunocytochemically Streptavidin-Peroxidase method was used to investigate the expression of ICAM-1, P-selectin and Poly (adenosine 5'-diphosphate ribose)( the product of poly (adenosine 5'-diphosphate ribose) polymerase activation). Results: the results of the adhesion assay of HT-29 cells to HUVEC showed that the OD570 value in each 5-AIQ-treated group was significant lower than that in the control group (5-AIQ-untreated) in a dose-dependent manner. The expression of ICAM-1, P-selectin and Poly (adenosine 5'-diphosphate ribose) was significant lower in 5-Aminoisoquinolinone-treated HT-29 cell group than that in 5-Aminoisoquinolinone- untreated groups. Conclusion: The data suggest that 5-Aminoisoquinolinone can inhibit the adhesion of HT-29 cells to human umbilical vein endothelial cells. 5-Aminoisoquinolinone also can inhibit poly (adenosine 5'-diphosphate ribose) polymerase activation and the expressions of ICAM-1 and P-selectin in HT-29 cells. 5-Aminoisoquinolinone probably contributes to the prevention of tumor cell metastasis. Further study is needed.

  8. L-Ribose isomerase and mannose-6-phosphate isomerase: properties and applications for L-ribose production.

    Science.gov (United States)

    Xu, Zheng; Sha, Yuanyuan; Liu, Chao; Li, Sha; Liang, Jinfeng; Zhou, Jiahai; Xu, Hong

    2016-11-01

    L-Ribose is a synthetic L-form monosaccharide. It is a building block of many novel nucleotide analog anti-viral drugs. Bio-production of L-ribose relies on a two-step reaction: (i) conversion of L-arabinose to L-ribulose by the catalytic action of L-arabinose isomerase (L-AI) and (ii) conversion of L-ribulose to L-ribose by the catalytic action of L-ribose isomerase (L-RI, EC 5.3.1.B3) or mannose-6-phosphate isomerase (MPI, EC 5.3.1.8, alternately named as phosphomannose isomerase). Between the two enzymes, L-RI is a rare enzyme that was discovered in 1996 by Professor Izumori's group, whereas MPI is an essential enzyme in metabolic pathways in humans and microorganisms. Recent studies have focused on their potentials for industrial production of L-ribose. This review summarizes the applications of L-RI and MPI for L-ribose production.

  9. Imaging Adenosine Triphosphate (ATP).

    Science.gov (United States)

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

    2016-08-01

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

  10. A new motif for inhibitors of geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Foust, Benjamin J; Allen, Cheryl; Holstein, Sarah A; Wiemer, David F

    2016-08-15

    The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure-function relationship which is dependent on the nature of the alkyl group at the α-carbon.

  11. Some neural effects of adenosin.

    Science.gov (United States)

    Haulică, I; Brănişteanu, D D; Petrescu, G H

    1978-01-01

    The possible neural effects of adenosine were investigated by using electrophysiological techniques at the level of some central and peripheral synapses. The evoked potentials in the somatosensorial cerebral cortex are influenced according to both the type of administration and the level of the electrical stimulation. While the local application does not induce significant alterations, the intrathalamic injections and the perfusion of the IIIrd cerebral ventricle do change the distribution of activated units at the level of different cortical layers especially during the peripheral stimulation. The frequency of spontaneous miniature discharges intracellularly recorded in the neuromuscular junction (mepp) is significantly depressed by adenosine. This effect is calcium- and dose-dependent. The end plate potentials (EPP) were also depressed. The statistical binomial analysis of the phenomenon indicated that adenosine induces a decrease if the presynaptic pool of the available transmitter. The data obtained demonstrate a presynaptic inhibitory action of adenosine beside its known vascular and metaholic effects.

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

    Science.gov (United States)

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

    2017-01-29

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

  13. D-ribose inhibits DNA repair synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zunica, G.; Marini, M.; Brunelli, M.A.; Chiricolo, M.; Franceschi, C.

    1986-07-31

    D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

  14. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity

    NARCIS (Netherlands)

    Yang, T.; Gao, L.; Hu, H.; Stoopen, G.M.; Wang, C.; Jongsma, M.A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP).

  15. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemolsynthase activity

    NARCIS (Netherlands)

    Yang, T.; Gao, L.; Hu, H.; Stoopen, G.M.; Wang, C.; Jongsma, M.A.

    2014-01-01

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme inthe biosynthesis of pyrethrins, the most widely used plant-derivedpesticide.CDScatalyzes c1’-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP).

  16. Niacin, poly(ADP-ribose) polymerase-1 and genomic stability

    NARCIS (Netherlands)

    Hageman, G.J.; Stierum, R.H.

    2001-01-01

    Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD+ (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD+ is the sole s

  17. Ribose 5-Phosphate Isomerase Investigations for the Undergraduate Biochemistry Laboratory

    Science.gov (United States)

    Jewett, Kathy; Sandwick, Roger K.

    2011-01-01

    The enzyme ribose 5-phosphate isomerase (RpiA) has many features that make it attractive as a focal point of a semester-long, advanced biochemistry laboratory for undergraduate students. The protein can easily and inexpensively be isolated from spinach using traditional purification techniques. Characterization of RpiA enzyme activity can be…

  18. Glycosidation of Methanol with Ribose: An Interdisciplinary Undergraduate Laboratory Experiment

    Science.gov (United States)

    Simon, Erin; Cook, Katie; Pritchard, Meredith R.; Stripe, Wayne; Bruch, Martha; Bendinskas, Kestutis

    2010-01-01

    This exercise provides students hands-on experience with the topics of glycosidation, hemiacetal and acetal formation, proton nuclear magnetic resonance ([superscript 1]H NMR) spectroscopy, and kinetic and thermodynamic product formation. In this laboratory experiment, the methyl acetal of ribose is synthesized, and the kinetic and thermodynamic…

  19. Niacin, poly(ADP-ribose) polymerase-1 and genomic stability

    NARCIS (Netherlands)

    Hageman, G.J.; Stierum, R.H.

    2001-01-01

    Nicotinic acid (NA) and nicotinamide (NAM), commonly called niacin, are the dietary precursors for NAD+ (nicotinamide adenine dinucleotide), which is required for DNA synthesis, as well as for the activity of the enzyme poly(ADP-ribose) polymerase-1 (PARP-1; EC 2.4.2.30) for which NAD+ is the sole

  20. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors.

    Science.gov (United States)

    Bettendorff, Lucien; Wins, Pierre

    2009-06-01

    Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thiamin is phosphorylated to higher phosphate derivatives. Thiamin diphosphate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysiological role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some proteins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A soluble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli, but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP.

  1. The Metabolism of 2-Caroboxy-4-Ketopentitol Diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Moses, V.; Calvin, M.

    1958-07-15

    2-Carboxy-4-ketopentitol is converted enzymatically by a cell-free preparation from spinach leaves into a substance undergoing acid-lactone interconversion. This substance has no phosphate or letone group and is probably a dicarboxylic, six-carbon sugar acid or the saccharic or saccharinic acid type. The significance of these findings with regard to the metabolic role of 2-carboxy-4-ketopentitol diphosphate is discussed.

  2. Electrophysiologic effects of adenosine triphosphate on rabbit sinoatrial node pace maker cells via P1 receptors

    Institute of Scientific and Technical Information of China (English)

    RENLei-Ming; LIJun-Xia; SHIChen-Xia; ZHAODing

    2003-01-01

    AIM: To study the electrophysiologic effects of adenosine triphosphate (ATP) on rabbit sinoatrial node pacemakercells and the receptors related with the action of ATP. METHODS: Intracellular microelectrode method was usedto record the parameters of action potential (AP) in the rabbit sinoatrial nodes. RESULTS: ATP (0.1-3 mmol/L)decreased the rate of pacemaker firing (RPF) by 16 %-43 % and velocity of diastolic depolarization (VDD) by 33 %-67 %, increased the amplitude of AP (APA) by 6 %-9 % and maximal rate of depolarization (Vmax) by 30 %-76 %,shortened APD50 by 7 %-12 % and APD90 by 6.3 %-9 %, concentration-dependently. The effects of ATP, adenos-ine (Ado), and adenosine diphosphate at the same concentration on AP were not different from each other significantly.Neither uridine triphosphate nor, α,β-methylene ATP had significant electrophysiologic effects on the sinoatrialnode of rabbits. Both the electrophysiologic effects of ATP and Ado on pacemaker cells were inhibited by P1receptor antagonist aminophylline 0.1 mmol/L (P0.05). CONCLUSION: There are nofunctional P2X1 and P2Y2 receptors on pacemaker cells of the rabbit sinoatrial nodes, and the electrophysiologiceffects of ATP in the rabbit sinoatrial node pacemaker cells are mediated via P1 receptors by Ado degraded fromATP.

  3. Regulation of adenosine levels during cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Stephanie CHU; Wei XIONG; Dali ZHANG; Hanifi SOYLU; Chao SUN; Benedict C ALBENSI; Fiona E PARKINSON

    2013-01-01

    Adenosine is a neuromodulator with its level increasing up to 100-fold during ischemic events,and attenuates the excitotoxic neuronal injury.Adenosine is produced both intracellularly and extracellularly,and nucleoside transport proteins transfer adenosine across plasma membranes.Adenosine levels and receptor-mediated effects of adenosine are regulated by intracellular ATP consumption,cellular release of ATP,metabolism of extracellular ATP (and other adenine nucleotides),adenosine influx,adenosine efflux and adenosine metabolism.Recent studies have used genetically modified mice to investigate the relative contributions of intra-and extracellular pathways for adenosine formation.The importance of cortical or hippocampal neurons as a source or a sink of adenosine under basal and hypoxic/ischemic conditions was addressed through the use of transgenic mice expressing human equilibrative nucleoside transporter 1 (hENT1) under the control of a promoter for neuron-specific enolase.From these studies,we conclude that ATP consumption within neurons is the primary source of adenosine in neuronal cultures,but not in hippocampal slices or in vivo mice exposed to ischemic conditions.

  4. Reprogramming cellular events by poly(ADP-ribose)-binding proteins

    Science.gov (United States)

    Pic, Émilie; Ethier, Chantal; Dawson, Ted M.; Dawson, Valina L.; Masson, Jean-Yves; Poirier, Guy G.; Gagné, Jean-Philippe

    2013-01-01

    Poly(ADP-ribosyl)ation is a posttranslational modification catalyzed by the poly(ADP-ribose) polymerases (PARPs). These enzymes covalently modify glutamic, aspartic and lysine amino acid side chains of acceptor proteins by the sequential addition of ADP-ribose (ADPr) units. The poly(ADP-ribose) (pADPr) polymers formed alter the physico-chemical characteristics of the substrate with functional consequences on its biological activities. Recently, non-covalent binding to pADPr has emerged as a key mechanism to modulate and coordinate several intracellular pathways including the DNA damage response, protein stability and cell death. In this review, we describe the basis of non-covalent binding to pADPr that has led to the emerging concept of pADPr-responsive signaling pathways. This review emphasizes the structural elements and the modular strategies developed by pADPr-binding proteins to exert a fine-tuned control of a variety of pathways. Poly(ADP-ribosyl)ation reactions are highly regulated processes, both spatially and temporally, for which at least four specialized pADPr-binding modules accommodate different pADPr structures and reprogram protein functions. In this review, we highlight the role of well-characterized and newly discovered pADPr-binding modules in a diverse set of physiological functions. PMID:23268355

  5. Activation of thiamin diphosphate and FAD in the phosphatedependent pyruvate oxidase from Lactobacillus plantarum

    OpenAIRE

    Tittmann, Kai; Proske, Daniela; Spinka, Michael; Ghisla, Sandro; Rudolph, Rainer; Hübner, Gerhard; Kern, Gunther

    1998-01-01

    The phosphate- and oxygen-dependent pyruvate oxidase from Lactobacillus plantarum is a homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per subunit. A kinetic analysis of the partial reactions in the overall oxidative conversion of pyruvate to acetyl phosphate and CO2 shows an indirect activation of the thiamine diphosphate by FAD that is mediated by the protein moiety. The rate constant of the initial step, the deprotonation of C2-H of thiamine diphosphate, increases 10-fold...

  6. Global transcriptome response in Lactobacillus sakei during growth on ribose

    Directory of Open Access Journals (Sweden)

    Naterstad Kristine

    2011-06-01

    Full Text Available Abstract Background Lactobacillus sakei is valuable in the fermentation of meat products and exhibits properties that allow for better preservation of meat and fish. On these substrates, glucose and ribose are the main carbon sources available for growth. We used a whole-genome microarray based on the genome sequence of L. sakei strain 23K to investigate the global transcriptome response of three L. sakei strains when grown on ribose compared with glucose. Results The function of the common regulated genes was mostly related to carbohydrate metabolism and transport. Decreased transcription of genes encoding enzymes involved in glucose metabolism and the L-lactate dehydrogenase was observed, but most of the genes showing differential expression were up-regulated. Especially transcription of genes directly involved in ribose catabolism, the phosphoketolase pathway, and in alternative fates of pyruvate increased. Interestingly, the methylglyoxal synthase gene, which encodes an enzyme unique for L. sakei among lactobacilli, was up-regulated. Ribose catabolism seems closely linked with catabolism of nucleosides. The deoxyribonucleoside synthesis operon transcriptional regulator gene was strongly up-regulated, as well as two gene clusters involved in nucleoside catabolism. One of the clusters included a ribokinase gene. Moreover, hprK encoding the HPr kinase/phosphatase, which plays a major role in the regulation of carbon metabolism and sugar transport, was up-regulated, as were genes encoding the general PTS enzyme I and the mannose-specific enzyme II complex (EIIman. Putative catabolite-responsive element (cre sites were found in proximity to the promoter of several genes and operons affected by the change of carbon source. This could indicate regulation by a catabolite control protein A (CcpA-mediated carbon catabolite repression (CCR mechanism, possibly with the EIIman being indirectly involved. Conclusions Our data shows that the ribose uptake

  7. Novel aspects of extracellular adenosine dynamics revealed by adenosine sensor cells

    Directory of Open Access Journals (Sweden)

    Kunihiko Yamashiro

    2017-01-01

    Full Text Available Adenosine modulates diverse physiological and pathological processes in the brain, including neuronal activities, blood flow, and inflammation. However, the mechanisms underlying the dynamics of extracellular adenosine are not fully understood. We have recently developed a novel biosensor, called an adenosine sensor cell, and we have characterized the neuronal and astrocytic pathways for elevating extracellular adenosine. In this review, the physiological implications and therapeutic potential of the pathways revealed by the adenosine sensor cells are discussed. We propose that the multiple pathways regulating extracellular adenosine allow for the diverse functions of this neuromodulator, and their malfunctions cause various neurological and psychiatric disorders.

  8. Proteomic investigation of phosphorylation sites in poly(ADP-ribose) polymerase-1 and poly(ADP-ribose) glycohydrolase.

    Science.gov (United States)

    Gagné, Jean-Philippe; Moreel, Xavier; Gagné, Pierre; Labelle, Yves; Droit, Arnaud; Chevalier-Paré, Mélissa; Bourassa, Sylvie; McDonald, Darin; Hendzel, Michael J; Prigent, Claude; Poirier, Guy G

    2009-02-01

    Phosphorylation is a very common post-translational modification event known to modulate a wide range of biological responses. Beyond the regulation of protein activity, the interrelation of phosphorylation with other post-translational mechanisms is responsible for the control of diverse signaling pathways. Several observations suggest that phosphorylation of poly(ADP-ribose) polymerase-1 (PARP-1) regulates its activity. There is also accumulating evidence to suggest the establishment of phosphorylation-dependent assembly of PARP-1-associated multiprotein complexes. Although it is relatively straightforward to demonstrate phosphorylation of a defined target, identification of the actual amino acids involved still represents a technical challenge for many laboratories. With the use of a combination of bioinformatics-based predictions tools for generic and kinase-specific phosphorylation sites, in vitro phosphorylation assays and mass spectrometry analysis, we investigated the phosphorylation profile of PARP-1 and poly(ADP-ribose) glycohydrolase (PARG), two major enzymes responsible for poly(ADP-ribose) turnover. Mass spectrometry analysis revealed the phosphorylation of several serine/threonine residues within important regulatory domains and motifs of both enzymes. With the use of in vivo microirradiation-induced DNA damage, we show that altered phosphorylation at specific sites can modify the dynamics of assembly and disassembly of PARP-1 at sites of DNA damage. By documenting and annotating a collection of known and newly identified phosphorylation sites, this targeted proteomics study significantly advances our understanding of the roles of phosphorylation in the regulation of PARP-1 and PARG.

  9. Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate.

    Science.gov (United States)

    Graue, C; Klingenberg, M

    1979-06-05

    The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.

  10. Resistance to aspirin is increased by ST-elevation myocardial infarction and correlates with adenosine diphosphate levels

    Directory of Open Access Journals (Sweden)

    Öhlin Hans

    2005-07-01

    Full Text Available Abstract Background To be fully activated platelets are dependent on two positive feedback loops; the formation of thromboxane A2 by cyclooxygenase in the platelets and the release of ADP. We wanted to evaluate the effect of aspirin on platelet function in patients with acute coronary syndromes and we hypothesized that increased levels of ADP in patients with acute coronary syndromes could contribute to aspirin resistance. Methods Platelet activity in 135 patients admitted for chest pain was assessed with PFA-100. An epinephrine-collagen cartridge (EPI-COLL was used for the detection of aspirin resistance together with an ADP-collagen cartridge (ADP-COLL. ADP was measured with hplc from antecubital vein samples. Three subgroups were compared: chest pain with no sign of cardiac disease (NCD, NonST-elevation myocardial infarction (NSTEMI and STEMI. Results Platelet activation was increased for the STEMI group compared NCD. Aspirin resistance defined as Conclusion Platelets are activated and aspirin resistance is more frequent in STEMI, probably due to a general activation of platelets. ADP levels are increased in STEMI and correlates with platelet activation. Increased levels of ADP could be one reason for increased platelet activity and aspirin resistance.

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

    Science.gov (United States)

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

    2017-03-08

    Numerous compounds such as protein and double-stranded DNA have been shown to efficiently inhibit intrinsic peroxidase-mimic activity in Fe3O4 nanoparticles (NP) and other related nanomaterials. However, only a few studies have focused on finding new compounds for enhancing the catalytic activity of Fe3O4 NP-related nanomaterials. Herein, phosphate containing adenosine analogs are reported to enhance the oxidation reaction of hydrogen peroxide (H2O2) and amplex ultrared (AU) for improving the peroxidase-like activity in Fe3O4 NPs. This enhancement is suggested to be a result of the binding of adenosine analogs to Fe(2+)/Fe(3+) sites on the NP surface and from adenosine 5'-monophosphate (AMP) acting as the distal histidine residue of horseradish peroxidase for activating H2O2. Phosphate containing adenosine analogs revealed the following trend for the enhanced activity of Fe3O4 NPs: AMP > adenosine 5'-diphosphate > adenosine 5'-triphosphate. The peroxidase-like activity in the Fe3O4 NPs progressively increased with increasing AMP concentration and polyadenosine length. The Michaelis constant for AMP attached Fe3O4 NPs is 5.3-fold lower and the maximum velocity is 2.7-fold higher than those of the bare Fe3O4 NPs. Furthermore, on the basis of AMP promoted peroxidase mimicking activity in the Fe3O4 NPs and the adsorption of protein on the NP surface, a selective fluorescent turn-off system for the detection of urinary protein is developed.

  12. Pathologic overproduction: the bad side of adenosine.

    Science.gov (United States)

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

    2017-03-02

    Adenosine is an endogenous ubiquitous purine nucleoside, increased by hypoxia, ischemia and tissue damage that mediates a number of physiopathological effects by interacting with four G-protein-coupled receptors, identified as A1 , A2A , A2B , and A3 . Physiological and acutely-increased adenosine is associated with beneficial effects mostly including vasodilation and decrease of 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 pathologic overproduction of adenosine analysing when, where and how adenosine exerts its detrimental effects through the body.

  13. Nuclear magnetic resonance-based quantification of organic diphosphates.

    Science.gov (United States)

    Lenevich, Stepan; Distefano, Mark D

    2011-01-15

    Phosphorylated compounds are ubiquitous in life. Given their central role, many such substrates and analogs have been prepared for subsequent evaluation. Prior to biological experiments, it is typically necessary to determine the concentration of the target molecule in solution. Here we describe a method where concentrations of stock solutions of organic diphosphates and bisphosphonates are quantified using (31)P nuclear magnetic resonance (NMR) spectroscopy with standard instrumentation using a capillary tube with a secondary standard. The method is specific and is applicable down to a concentration of 200 μM. The capillary tube provides the reference peak for quantification and deuterated solvent for locking.

  14. Domain relationships in thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Duggleby, Ronald G

    2006-08-01

    Three-dimensional structures have been determined for 13 different enzymes that use thiamine diphosphate (ThDP) as a cofactor. These enzymes fall into five families, where members within a family have similar structures. In different families, there are similarities between some domains that clearly point to a common ancestor for all of these enzymes. Where the enzyme structures differ, evolutionary relationships between families can be discerned. Here, I present an analysis of these families and propose an evolutionary pathway to explain the diversity of structures that are now known.

  15. D-Ribose Induces Cellular Protein Glycation and Impairs Mouse Spatial Cognition

    OpenAIRE

    Chanshuai Han; Yang Lu; Yan Wei; Ying Liu; Rongqiao He

    2011-01-01

    BACKGROUND: D-ribose, an important reducing monosaccharide, is highly active in the glycation of proteins, and results in the rapid production of advanced glycation end products (AGEs) in vitro. However, whether D-ribose participates in glycation and leads to production of AGEs in vivo still requires investigation. METHODOLOGY/PRINCIPAL FINDINGS: Here we treated cultured cells and mice with D-ribose and D-glucose to compare ribosylation and glucosylation for production of AGEs. Treatment with...

  16. STUDIES OF DISSOLVED OXYGEN IN PRODUCTION OF D-RIBOSE BY FERMENTATION

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    D-ribose as a component unit of ribonucleic acidoccurs in all living matted Ribitol occurs as acomponent unit of vitamin BZ and teichoic acid whichis a building block of the cell wall. Thus, D-ribose isa physiologically important substance.More recently, D-ribose has been much utilizedas a starting material for synthetic production ofnucleotide condiments. The studies on D-ribosehave been paid much attention recently, possiblybecause this pentose can be used as synthesizeantiviral and anticancer drugs. Ther...

  17. Rates of Decomposition of Ribose and other Sugars: Implications for Chemical Evolution

    Science.gov (United States)

    Larralde, Rosa; Robertson, Michael P.; Miller, Stanley L.

    1995-01-01

    The existence of the RNA world, in which RNA acted as a catalyst as well as an informational macromolecule, assumes a large prebiotic source of ribose or the existence of pre-RNA molecules with backbones different from ribose-phosphate. The generally accepted prebiotic synthesis of ribose, the formose reaction, yields numerous sugars without any selectivity. Even if there were a selective synthesis of ribose, there is still the problem of stability. Sugars are known to be unstable in strong acid or base, but there are few data for neutral solutions. Therefore, we have measured the rate of decomposition of ribose between pH 4 and pH 8 from 40 C to 120 C. The ribose half-lives are very short (73 min at pH 7.0 and 100 C and 44 years at pH 7.0 and 0 C). The other aldopentoses and aldohexoses have half-lives within an order of magnitude of these values, as do 2-deoxyribose, ribose 5-phosphate, and ribose 2,4bisphosphate. These results suggest that the backbone of the first genetic material could not have contained ribose or other sugars because of their instability.

  18. A procedure for the preparation and isolation of nucleoside-5’-diphosphates

    Directory of Open Access Journals (Sweden)

    Heidi J. Korhonen

    2015-04-01

    Full Text Available Tris[bis(triphenylphosphoranylideneammonium] pyrophosphate (PPN pyrophosphate was used in the SN2 displacements of the tosylate ion from 5’-tosylnucleosides to afford nucleoside-5’-diphosphates. Selective precipitation permitted the direct isolation of nucleoside-5’-diphosphates from crude reaction mixtures.

  19. Poly(glycidyl methacrylate-co-N-methylolacrylamide-co-ethylene dimethacrylate) monolith coupled to high-performance liquid chromatography for the determination of adenosine phosphates in royal jelly.

    Science.gov (United States)

    Liu, Dan; Zhang, Tianbin; Cheng, Yechun; Jia, Qiong

    2014-07-01

    A polymer monolith microextraction method coupled with high-performance liquid chromatography was developed for the determination of adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate. The monolithic column was synthesized inside fused-silica capillaries using thermal initiation free-radical polymerization with glycidyl methacrylate as the monomer, ethylene dimethacrylate as the cross-linker, cyclohexanol, and 1-dodecanol as the porogen. N-Methylolacrylamide, an important hydrophilic monomer, was incorporated into the polymerization mixture to enhance the hydrophilicity of the poly(glycidyl methacrylate-co-ethylene dimethacrylate) column. The obtained poly(glycidyl methacrylate-co-N-methylolacrylamide-co-ethylene dimethacrylate) monolith was characterized by scanning electron microscopy, Fourier-transform infrared spectra, and X-ray photoelectron spectroscopy. Optimum conditions for the preconcentration and separation of the target adenosines were also investigated. Under the optimum conditions, we obtained acceptable linearities, low limits of detection, and good relative standard deviations. The developed polymer monolith microextraction with high-performance liquid chromatography method exhibited a good performance with recovery values in the range of 76.9-104.7% when applied to the determination of the adenosines in five royal jelly samples.

  20. Adenosine, Energy Metabolism, and Sleep

    Directory of Open Access Journals (Sweden)

    Tarja Porkka-Heiskanen

    2003-01-01

    Full Text Available While the exact function of sleep remains unknown, it is evident that sleep was developed early in phylogenesis and represents an ancient and vital strategy for survival. Several pieces of evidence suggest that the function of sleep is associated with energy metabolism, saving of energy, and replenishment of energy stores. Prolonged wakefulness induces signs of energy depletion in the brain, while experimentally induced, local energy depletion induces increase in sleep, similarly as would a period of prolonged wakefulness. The key molecule in the induction of sleep appears to be adenosine, which induces sleep locally in the basal forebrain.

  1. [Interaction of pyruvate dehydrogenase complex from the heart muscle with thiamine diphosphate and its derivatives].

    Science.gov (United States)

    Strumilo, S A; Kiselevskiĭ, Iu V; Taranda, N I; Zabrodskaia, S V; Oparin, D A

    1989-01-01

    Inhibitory effects of 23 thiamin derivatives on the bovine heart pyruvate dehydrogenase complex (PDC) were studied. Oxythiamin diphosphate and tetrahydroxythiamin diphosphate exhibited the most pronounced effect on the PDC activity, affecting the complex by a competitive type of inhibition for thiamin diphosphate (TDP). The apparent affinity of TDP and the anticoenzyme derivatives for apo PDC depended on presence of phosphate and divalent metal ions. Phosphate considerably increased the Km values for TDP (up to 0.17 microM) and the Ki values for oxythiamin diphosphate (0.40 microM) as well as for tetrahydroxythiamin diphosphate (0.23 microM). In presence of Mn2+, Km value for TDP was 3.5-fold lower as compared with Mg2+ containing medium.

  2. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution.

    OpenAIRE

    Lindqvist, Y; Schneider, G; Ermler, U.; Sundström, M

    1992-01-01

    The crystal structure of Saccharomyces cerevisiae transketolase, a thiamine diphosphate dependent enzyme, has been determined to 2.5 A resolution. The enzyme is a dimer with the active sites located at the interface between the two identical subunits. The cofactor, vitamin B1 derived thiamine diphosphate, is bound at the interface between the two subunits. The enzyme subunit is built up of three domains of the alpha/beta type. The diphosphate moiety of thiamine diphosphate is bound to the enz...

  3. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    Science.gov (United States)

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  4. Noncovalent protein interaction with poly(ADP-ribose).

    Science.gov (United States)

    Malanga, Maria; Althaus, Felix R

    2011-01-01

    Compared to most common posttranslational modifications of proteins, a peculiarity of poly(ADP-ribosyl)ation is the molecular heterogeneity and complexity of the reaction product, poly(ADP-ribose) (PAR). In fact, protein-bound PAR consists of variously sized (2-200 ADP-ribose residues) linear or branched molecules, negatively charged at physiological pH. It is now clear that PAR not only affects the function of the polypeptide to which it is covalently bound, but it can also influence the activity of other proteins by engaging specific noncovalent interactions. In the last 10 years, the family of PAR-binding proteins has been rapidly growing and functional studies have expanded the regulatory potential of noncovalent -protein targeting by PAR far beyond initial assumptions.In this chapter, methods are described for: (1) PAR synthesis and analysis; (2) detecting PAR-binding proteins in protein mixtures; (3) defining affinity and specificity of PAR binding to individual proteins or protein fragments; and (4) identifying PAR molecules selectively involved in the interaction.

  5. Recognition of the iso-ADP-ribose moiety in poly(ADP-ribose) by WWE domains suggests a general mechanism for poly(ADP-ribosyl)ation-dependent ubiquitination.

    Science.gov (United States)

    Wang, Zhizhi; Michaud, Gregory A; Cheng, Zhihong; Zhang, Yue; Hinds, Thomas R; Fan, Erkang; Cong, Feng; Xu, Wenqing

    2012-02-01

    Protein poly(ADP-ribosyl)ation and ubiquitination are two key post-translational modifications regulating many biological processes. Through crystallographic and biochemical analysis, we show that the RNF146 WWE domain recognizes poly(ADP-ribose) (PAR) by interacting with iso-ADP-ribose (iso-ADPR), the smallest internal PAR structural unit containing the characteristic ribose-ribose glycosidic bond formed during poly(ADP-ribosyl)ation. The key iso-ADPR-binding residues we identified are highly conserved among WWE domains. Binding assays further demonstrate that PAR binding is a common function for the WWE domain family. Since many WWE domain-containing proteins are known E3 ubiquitin ligases, our results suggest that protein poly(ADP-ribosyl)ation may be a general mechanism to target proteins for ubiquitination.

  6. Interactions Between Metal Ions and Carbohydrates: Coordination Behavior of D-Ribose to Lanthanide Ions

    Institute of Scientific and Technical Information of China (English)

    苏允兰; 杨丽敏; 翁诗甫; 吴瑾光

    2002-01-01

    Lanthanum chloride α-D-ribopyranose pentahydrate complex was prepared and speculated its structure from the similar IR spectra of corresponding praseodymium and neodymium-D-ribose complexes, which reveal the coordination behavior of D-ribose to lanthanide ions and give us a model of the interactions between metal ions and carbohydrates.

  7. Effect of heat shock on poly(ADP-ribose) synthetase and DNA repair in Drosophila cells

    Energy Technology Data Exchange (ETDEWEB)

    Nolan, N.L.; Kidwell, W.R.

    1982-04-01

    Poly(ADP-ribose) synthetase, a chromatin-bound enzyme which attaches polyanionic chains of ADP-ribose to nuclear proteins, was found to be temperature sensitive in intact Drosophila melanogaster cells. The synthetase was completely inactivated by heat-shocking the cells at 37/sup 0/C for 5 min, a condition which had no appreciable effect on the subsequent growth of Drosophila cells at their physiological temperature. The heat-shock effect on synthetase was reversible; enzyme activity began to reappear about 2 hr post heat shock. During the 2-hr interval when poly(ADP-ribose) synthetase was absent, the cells were competent in repair of ..gamma..-ray-induced DNA strand breaks as shown by DNA sedimentation studies on alkaline sucrose gradients. It is thus concluded that poly(ADP-ribose) synthesis is unnecessary for repair of DNA strand breaks introduced by irradiation. The same conclusion was reached from the fact that two inhibitors of poly(ADP-ribose) synthetase 3-aminobenzamide and 5-methylnicotinamide, failed to block repair of ..gamma..-ray-induced DNA chain breaks even though both inhibitors reduced the amount of poly(ADP-ribose) synthesized in cells by 50-75%. Although it was found that the repair of DNA strand breaks is independent of poly(ADP-ribose) synthesis, irradiation does activate the synthetase in control cells, as shown by radioimmunoassay of poly(ADP-ribose) levels.

  8. Profiling of Ribose Methylations in RNA by High-Throughput Sequencing

    DEFF Research Database (Denmark)

    Birkedal, Ulf; Christensen-Dalsgaard, Mikkel; Krogh, Nicolai;

    2015-01-01

    Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing-based method...

  9. Perspectives of biotechnological production of L-ribose and its purification.

    Science.gov (United States)

    Hu, Chao; Li, Liangzhi; Zheng, Yayue; Rui, Lilian; Hu, Cuiying

    2011-11-01

    L-ribose is a non-natural and expensive sugar that can be used as an important intermediate for the synthesis of L-nucleoside analogues, which are used as antiviral drugs. In contrast to chemical production, biotechnological methods can produce L-ribose from biomass under environmentally friendly conditions. In this mini-review, various strategies for synthesizing L-ribose by applying microorganisms and their enzymes are discussed, including microbial biotransformation and biocatalysis by engineering bacteria. Furthermore, subsequent isolation-and-purification techniques, as an integral step in the whole process, are accordingly described, containing the special introduction of a promising strategy of L-ribose separation. Particularly, further researches and outlook for the improvement of L-ribose preparation was solely stressed. Compared with each method, this mini-review provides a panorama of respective advantages and disadvantages existing in them.

  10. Ribose and related sugars from ultraviolet irradiation of interstellar ice analogs

    Science.gov (United States)

    Meinert, Cornelia; Myrgorodska, Iuliia; de Marcellus, Pierre; Buhse, Thomas; Nahon, Laurent; Hoffmann, Søren V.; d'Hendecourt, Louis Le Sergeant; Meierhenrich, Uwe J.

    2016-04-01

    Ribose is the central molecular subunit in RNA, but the prebiotic origin of ribose remains unknown. We observed the formation of substantial quantities of ribose and a diversity of structurally related sugar molecules such as arabinose, xylose, and lyxose in the room-temperature organic residues of photo-processed interstellar ice analogs initially composed of H2O, CH3OH, and NH3. Our results suggest that the generation of numerous sugar molecules, including the aldopentose ribose, may be possible from photochemical and thermal treatment of cosmic ices in the late stages of the solar nebula. Our detection of ribose provides plausible insights into the chemical processes that could lead to formation of biologically relevant molecules in suitable planetary environments.

  11. Bacillus caldolyticus prs gene encoding phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Krath, Britta N.; Hove-Jensen, Bjarne

    1996-01-01

    The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-1-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

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

    Science.gov (United States)

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

    2016-07-01

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

  13. Impairment of skeletal muscle adenosine triphosphate–sensitive K+ channels in patients with hypokalemic periodic paralysis

    Science.gov (United States)

    Tricarico, Domenico; Servidei, Serenella; Tonali, Pietro; Jurkat-Rott, Karin; Camerino, Diana Conte

    1999-01-01

    The adenosine triphosphate (ATP)–sensitive K+ (KATP) channel is the most abundant K+ channel active in the skeletal muscle fibers of humans and animals. In the present work, we demonstrate the involvement of the muscular KATP channel in a skeletal muscle disorder known as hypokalemic periodic paralysis (HOPP), which is caused by mutations of the dihydropyridine receptor of the Ca2+ channel. Muscle biopsies excised from three patients with HOPP carrying the R528H mutation of the dihydropyridine receptor showed a reduced sarcolemma KATP current that was not stimulated by magnesium adenosine diphosphate (MgADP; 50–100 μM) and was partially restored by cromakalim. In contrast, large KATP currents stimulated by MgADP were recorded in the healthy subjects. At channel level, an abnormal KATP channel showing several subconductance states was detected in the patients with HOPP. None of these were surveyed in the healthy subjects. Transitions of the KATP channel between subconductance states were also observed after in vitro incubation of the rat muscle with low-K+ solution. The lack of the sarcolemma KATP current observed in these patients explains the symptoms of the disease, i.e., hypokalemia, depolarization of the fibers, and possibly the paralysis following insulin administration. PMID:10074484

  14. Structure and Mechanism of the Farnesyl Diphosphate Synthase from Trypanosoma cruzi: Implications for Drug Design

    Energy Technology Data Exchange (ETDEWEB)

    Gabelli,S.; McLellan, J.; Montalvetti, A.; Oldfield, E.; Docampo, R.; Amzel, L.

    2006-01-01

    Typanosoma cruzi, the causative agent of Chagas disease, has recently been shown to be sensitive to the action of the bisphosphonates currently used in bone resorption therapy. These compounds target the mevalonate pathway by inhibiting farnesyl diphosphate synthase (farnesyl pyrophosphate synthase, FPPS), the enzyme that condenses the diphosphates of C{sub 5} alcohols (isopentenyl and dimethylallyl) to form C{sub 10} and C{sub 15} diphosphates (geranyl and farnesyl). The structures of the T. cruzi FPPS (TcFPPS) alone and in two complexes with substrates and inhibitors reveal that following binding of the two substrates and three Mg2+ ions, the enzyme undergoes a conformational change consisting of a hinge-like closure of the binding site. In this conformation, it would be possible for the enzyme to bind a bisphosphonate inhibitor that spans the sites usually occupied by dimethylallyl diphosphate (DMAPP) and the homoallyl moiety of isopentenyl diphosphate. This observation may lead to the design of new, more potent anti-trypanosomal bisphosphonates, because existing FPPS inhibitors occupy only the DMAPP site. In addition, the structures provide an important mechanistic insight: after its formation, geranyl diphosphate can swing without leaving the enzyme, from the product site to the substrate site to participate in the synthesis of farnesyl diphosphate.

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

    Science.gov (United States)

    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.

  16. Surface exposed amino acid differences between mesophilic and thermophilic phosphoribosyl diphosphate synthase

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; McGuire, James N

    2004-01-01

    The amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the thermophile Bacillus caldolyticus is 81% identical to the amino acid sequence of 5-phospho-alpha-D-ribosyl 1-diphosphate synthase from the mesophile Bacillus subtilis. Nevertheless the enzyme from the two organisms...... competitive with respect to ATP. A predicted structure of the B. caldolyticus enzyme based on homology modelling with the structure of B. subtilis 5-phospho-alpha-D-ribosyl 1-diphosphate synthase shows 92% of the amino acid differences to be on solvent exposed surfaces in the hexameric structure....

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

    Science.gov (United States)

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

    2015-01-20

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

  18. Fast determination of adenosine 5'-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography.

    Science.gov (United States)

    Zhou, Ling; Xue, XiaoFeng; Zhou, JinHui; Li, Yi; Zhao, Jing; Wu, LiMing

    2012-09-12

    To obtain insight into the metabolic regulation of adenosine 5'-triphosphate (ATP) in royal jelly and to determine whether ATP and its catabolites can be used as objective parameters to evaluate the freshness and quality of royal jelly (RJ), a rapid ultraperformance liquid chromatography (UPLC) method has been developed for feasible separation and quantitation of ATP and its catabolites in RJ, namely, adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx). The analytes in the sample were extracted using 5% precooled perchloric acid. Chromatographic separation was performed on a Waters Acquity UPLC system with a Waters BEH Shield RP18 column and gradient elution based on a mixture of two solvents: solvent A, 50 mM phosphate buffer (pH 6.5); and solvent B, acetonitrile. The recoveries were in the range of 86.0-102.3% with RSD of no more than 3.6%. The correlation coefficients of six analytes were high (r(2) ≥ 0.9988) and within the test ranges. The limits of detection and quantification for the investigated compounds were lower, at 0.36-0.68 and 1.22-2.30 mg/kg, respectively. The overall intra- and interday RSDs were no more than 1.8%. The developed method was successfully applied to the analysis of the analytes in samples. The results showed that ATP in RJ sequentially degrades to ADP, AMP, IMP, HxR, and Hx during storage.

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

    Science.gov (United States)

    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.

  20. Mast cell adenosine receptors function: a focus on the A3 adenosine receptor and inflammation

    Directory of Open Access Journals (Sweden)

    Noam eRudich

    2012-06-01

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

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

    Science.gov (United States)

    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.

  2. Adenosine Deaminase Activities in Hyperlipidaemic Patients ...

    African Journals Online (AJOL)

    Journal of Health and Visual Sciences ... Abstract. Adenosine Deaminase Activities, markers of cellular-mediated immunity ... were statistically significantly higher (P<0.001) in the test groups than in the control groups (10.7+3iu/1) respectively.

  3. Changing ribulose diphosphate carboxylase/oxygenase activity in ripening tomato fruit.

    Science.gov (United States)

    Bravdo, B A; Palgi, A; Lurie, S

    1977-08-01

    Tomato fruit (Lycopersicum esculentum Mill) from green, pink, and red stages were assayed for changes in the activity of ribulose diphosphate carboxylase and oxygenase, phosphoenolpyruvate carboxylase, changes in the levels of glycolate and respiratory gas exchange. The ribulose diphosphate carboxylase activity decreased as the fruit ripened. By comparison, the ribulose diphosphate oxygenase activity increased during the transition from the green to the pink stage, and declined afterward. The changes in the endogenous glycolate levels and the respiratory gas exchange, as observed at different stages of ripening, resembled the changes in the ribulose diphosphate oxygenase activity. The utilization of glycolate in further metabolic activity may result in the formation of peroxidases required for the onset of ripening.

  4. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    OpenAIRE

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-01-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported.

  5. Structural insight into the interaction of ADP-ribose with the PARP WWE domains.

    Science.gov (United States)

    He, Fahu; Tsuda, Kengo; Takahashi, Mari; Kuwasako, Kanako; Terada, Takaho; Shirouzu, Mikako; Watanabe, Satoru; Kigawa, Takanori; Kobayashi, Naohiro; Güntert, Peter; Yokoyama, Shigeyuki; Muto, Yutaka

    2012-11-02

    The WWE domain is often identified in proteins associated with ubiquitination or poly-ADP-ribosylation. Structural information about WWE domains has been obtained for the ubiquitination-related proteins, such as Deltex and RNF146, but not yet for the poly-ADP-ribose polymerases (PARPs). Here we determined the solution structures of the WWE domains from PARP11 and PARP14, and compared them with that of the RNF146 WWE domain. NMR perturbation experiments revealed the specific differences in their ADP-ribose recognition modes that correlated with their individual biological activities. The present structural information sheds light on the ADP-ribose recognition modes by the PARP WWE domains.

  6. A standard numbering scheme for thiamine diphosphate-dependent decarboxylases

    Directory of Open Access Journals (Sweden)

    Vogel Constantin

    2012-11-01

    Full Text Available Abstract Background Standard numbering schemes for families of homologous proteins allow for the unambiguous identification of functionally and structurally relevant residues, to communicate results on mutations, and to systematically analyse sequence-function relationships in protein families. Standard numbering schemes have been successfully implemented for several protein families, including lactamases and antibodies, whereas a numbering scheme for the structural family of thiamine-diphosphate (ThDP -dependent decarboxylases, a large subfamily of the class of ThDP-dependent enzymes encompassing pyruvate-, benzoylformate-, 2-oxo acid-, indolpyruvate- and phenylpyruvate decarboxylases, benzaldehyde lyase, acetohydroxyacid synthases and 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD is still missing. Despite a high structural similarity between the members of the ThDP-dependent decarboxylases, their sequences are diverse and make a pairwise sequence comparison of protein family members difficult. Results We developed and validated a standard numbering scheme for the family of ThDP-dependent decarboxylases. A profile hidden Markov model (HMM was created using a set of representative sequences from the family of ThDP-dependent decarboxylases. The pyruvate decarboxylase from S. cerevisiae (PDB: 2VK8 was chosen as a reference because it is a well characterized enzyme. The crystal structure with the PDB identifier 2VK8 encompasses the structure of the ScPDC mutant E477Q, the cofactors ThDP and Mg2+ as well as the substrate analogue (2S-2-hydroxypropanoic acid. The absolute numbering of this reference sequence was transferred to all members of the ThDP-dependent decarboxylase protein family. Subsequently, the numbering scheme was integrated into the already established Thiamine-diphosphate dependent Enzyme Engineering Database (TEED and was used to systematically analyze functionally and structurally relevant

  7. Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase

    Energy Technology Data Exchange (ETDEWEB)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W. (UIUC); (Iowa State); (Penn)

    2011-09-20

    The structure of ent-copalyl diphosphate synthase reveals three {alpha}-helical domains ({alpha}, {beta} and {gamma}), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the {beta}{gamma} domains in ent-copalyl diphosphate synthase but exclusively in the {alpha} domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions.

  8. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation.

    Science.gov (United States)

    Rose, Nicholas D; Regan, John M

    2015-12-01

    Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD(+), respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP(+), respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190 mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  9. Changes in phosphorylation of adenosine phosphate and redox state of nicotinamide-adenine dinucleotide (phosphate) in Geobacter sulfurreducens in response to electron acceptor and anode potential variation

    KAUST Repository

    Rose, Nicholas D.

    2015-12-01

    © 2015 Elsevier B.V. Geobacter sulfurreducens is one of the dominant bacterial species found in biofilms growing on anodes in bioelectrochemical systems. The intracellular concentrations of reduced and oxidized forms of nicotinamide-adenine dinucleotide (NADH and NAD+, respectively) and nicotinamide-adenine dinucleotide phosphate (NADPH and NADP+, respectively) as well as adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) were measured in G. sulfurreducens using fumarate, Fe(III)-citrate, or anodes poised at different potentials (110, 10, -90, and -190mV (vs. SHE)) as the electron acceptor. The ratios of CNADH/CNAD+ (0.088±0.022) and CNADPH/CNADP+ (0.268±0.098) were similar under all anode potentials tested and with Fe(III)-citrate (reduced extracellularly). Both ratios significantly increased with fumarate as the electron acceptor (0.331±0.094 for NAD and 1.96±0.37 for NADP). The adenylate energy charge (the fraction of phosphorylation in intracellular adenosine phosphates) was maintained near 0.47 under almost all conditions. Anode-growing biofilms demonstrated a significantly higher molar ratio of ATP/ADP relative to suspended cultures grown on fumarate or Fe(III)-citrate. These results provide evidence that the cellular location of reduction and not the redox potential of the electron acceptor controls the intracellular redox potential in G. sulfurreducens and that biofilm growth alters adenylate phosphorylation.

  10. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer

    DEFF Research Database (Denmark)

    Mirza, Mansoor R; Monk, Bradley J; Herrstedt, Jørn

    2016-01-01

    Background Niraparib is an oral poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) 1/2 inhibitor that has shown clinical activity in patients with ovarian cancer. We sought to evaluate the efficacy of niraparib versus placebo as maintenance treatment for patients with platinum-sensitive, ...

  11. Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Kasparyan, Elena; Richter, Michael; Dresen, Carola; Walter, Lydia S; Fuchs, Georg; Leeper, Finian J; Wacker, Tobias; Andrade, Susana L A; Kolter, Geraldine; Pohl, Martina; Müller, Michael

    2014-12-01

    The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon-carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

  12. Ribulose diphosphate carboxylase of the cyanobacterium Spirulina platensis

    Energy Technology Data Exchange (ETDEWEB)

    Terekhova, I.V.; Chernyad' ev, I.I.; Doman, N.G.

    1986-11-20

    The ribulose diphosphate (RDP) carboxylase activity of the cyanobacterium Spirulina platensis is represented by two peaks when a cell homogenate is centrifuged in a sucrose density gradient. In the case of differential centrifugation (40,000 g, 1 h), the activity of the enzyme was distributed between the supernatant liquid (soluble form) and the precipitate (carboxysomal form). From the soluble fraction, in which 80-95% of the total activity of the enzyme is concentrated, electrophoretically homogeneous RDP carboxylase was isolated by precipitation with ammonium sulfate and centrifugation in a sucrose density gradient. The purified enzyme possessed greater electrophoretic mobility in comparison with the RDP carboxylase of beans Vicia faba. The molecular weight of the enzyme, determined by gel filtration, was 450,000. The enzyme consists of monotypic subunits with a molecular weight of 53,000. The small subunits were not detected in electrophoresis in polyacrylamide gel in the presence of SDS after fixation and staining of the gels by various methods.

  13. Crystal structures of phosphoketolase: thiamine diphosphate-dependent dehydration mechanism.

    Science.gov (United States)

    Suzuki, Ryuichiro; Katayama, Takane; Kim, Byung-Jun; Wakagi, Takayoshi; Shoun, Hirofumi; Ashida, Hisashi; Yamamoto, Kenji; Fushinobu, Shinya

    2010-10-29

    Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H(2)O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.

  14. Propioin synthesis using thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Mikolajek, Renaud J; Spiess, Antje C; Pohl, Martina; Büchs, Jochen

    2009-01-01

    Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluorescens and benzoylformate decarboxylase (BFD, EC 4.1.1.7) from Pseudomonas putida are thiamine diphosphate-dependent enzymes. These enzymes share a common tetrameric structure and catalyze various C--C-bond forming and breaking reactions. Here we describe a detailed study of the asymmetric synthesis of propioin from propanal catalyzed by BAL or BFD in aqueous solution in a batch reactor. Both enzymes are deactivated in the presence of high concentration of propanal. Compared to BAL, BFD is more stable under reaction conditions as well as during storage. The kinetic studies showed a typical Michaelis-Menten kinetic for BAL with a maximal specific reaction rate of 26.2 U/mg and an unusually high K(M) of 415 mM, whereas the v/[S]-plot for BFD is almost linear in the concentration range (100-1500 mM) investigated. Both enzymes produce propioin with opposite enantiomeric excess: BAL produced the (S)-propioin (ee of 35%), whereas BFD yielded the (R)-enantiomer (ee of 67%).

  15. Maillard Reactions of Ribose 5‐Phosphate and Amino Acids

    National Research Council Canada - National Science Library

    SANDWICK, ROGER; JOHANSON, MATTHEW; BREUER, ELIZABETH

    2005-01-01

    A bstract : An important metabolite in nucleotide synthesis, ribose 5‐phosphate (R5P) undergoes Maillard reactions at a rate significantly faster than most common sugars and sugar phosphates of its type...

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

    Science.gov (United States)

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

    2003-12-01

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

  17. Metabolic roles of poly(ADP-ribose) polymerases.

    Science.gov (United States)

    Vida, András; Márton, Judit; Mikó, Edit; Bai, Péter

    2017-03-01

    Poly(ADP-ribosyl)ation (PARylation) is an evolutionarily conserved reaction that had been associated with numerous cellular processes such as DNA repair, protein turnover, inflammatory regulation, aging or metabolic regulation. The metabolic regulatory tasks of poly(ADP-ribose) polymerases (PARPs) are complex, it is based on the regulation of metabolic transcription factors (e.g. SIRT1, nuclear receptors, SREBPs) and certain cellular energy sensors. PARP over-activation can cause damage to mitochondrial terminal oxidation, while the inhibition of PARP-1 or PARP-2 can induce mitochondrial oxidation by enhancing the mitotropic tone of gene transcription and signal transduction. These PARP-mediated processes impact on higher order metabolic regulation that modulates lipid metabolism, circadian oscillations and insulin secretion and signaling. PARP-1, PARP-2 and PARP-7 are related to metabolic diseases such as diabetes, alcoholic and non-alcoholic fatty liver disease (AFLD, NAFLD), or on a broader perspective to Warburg metabolism in cancer or the metabolic diseases accompanying aging.

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

    Science.gov (United States)

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

    2013-11-01

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

  19. Adenosine stress protocols for myocardial perfusion imaging

    Directory of Open Access Journals (Sweden)

    Baškot Branislav

    2008-01-01

    Full Text Available Background/Aim. Treadmill test combined with myocardial perfusion scintigraphy (MPS is a commonly used technique in the assessment of coronary artery disease. There are many patients, however, who may not be able to undergo treadmill test. Such patients would benefit from pharmacological stress procedures combined with MPS. The most commonly used pharmacological agents for cardiac stress are coronary vasodilatators (adenosine, dipyridamol and catecholamines. Concomitant low-level treadmill exercise with adenosine pharmacologic stress (AdenoEX during MPS has become commonly used in recent years. A number of studies have demonstrated a beneficial impact of AdenoEX protocol. The aim of the study was, besides introducing into practice the two types of protocols of pharmatological stress test with adenosine, as a preparation for MPS, to compare and monitor the frequency of their side effects to quality, acquisition, as well as to standardize the onset time of acquisition (diagnostic imaging for both protocols. Methods. A total of 130 patients underwent pharmacological stress test with adenosine (vasodilatator. In 108 of the patients we performed concomitant exercise (AdenoEX of low level (50W by a bicycle ergometar. In 28 of the patients we performed Adenosine abbreviated protocol (AdenoSCAN. Side effects of adenosine were followed and compared between the two kinds of protocols AdenoEX and AdenoSCAN. Also compared were image quality and suggested time of acquisition after the stress test. Results. Numerous side effects were found, but being short-lived they did not require any active interventions. The benefit of AdenoEX versus AdenoSCAN included decreased side effects (62% vs 87%, improved safety and patients tolerance, improved target-to-background ratios because of less subdiaphragmatic activity, earlier acquisition, and improved sensitivity. Conclusion. The safety and efficacy of adenosine pharmacological stress is even better with concomitant

  20. Assessment of Hematological and Biochemical parameters with extended D-Ribose ingestion

    Directory of Open Access Journals (Sweden)

    Frelich Angela

    2008-09-01

    Full Text Available Abstract D-ribose, a naturally occurring pentose carbohydrate, has been shown to replenish high- energy phosphates following myocardial ischemia and high intensity, repetitive exercise. Human studies have mainly involved short-term assessment, including potential toxicity. Reports describing adverse effects of D-ribose with prolonged ingestion have been lacking. Therefore, this study assessed the toxicity of extended consumption of D-ribose in healthy adults. Nineteen subjects ingested 20 grams/Day (10 grams, twice a Day of ribose with serial measurements of biochemical and hematological parameters at Days 0, 7, and 14. No significant toxic changes over the 14-day assessment period occurred in complete blood count, albumin, alkaline phosphatase, gamma glutamyltransferase, alanine amiotransferase, and aspartate aminotransferase. However, D-ribose did produce an asymptomatic, mild hypoglycemia of short duration. Uric acid levels increased at Day 7, but decreased to baseline values by Day 14. D-ribose consumption for 14 days appears not to produce significant toxic changes in both hematological and biochemical parameters in healthy human volunteers.

  1. Ribose mediated crosslinking of collagen-hydroxyapatite hybrid scaffolds for bone tissue regeneration using biomimetic strategies.

    Science.gov (United States)

    Krishnakumar, Gopal Shankar; Gostynska, Natalia; Campodoni, Elisabetta; Dapporto, Massimiliano; Montesi, Monica; Panseri, Silvia; Tampieri, Anna; Kon, Elizaveta; Marcacci, Maurilio; Sprio, Simone; Sandri, Monica

    2017-08-01

    This study explores for the first time the application of ribose as a highly biocompatible agent for the crosslinking of hybrid mineralized constructs, obtained by bio-inspired mineralization of self-assembling Type I collagen matrix with magnesium-doped-hydroxyapatite nanophase, towards a biomimetic mineralized 3D scaffolds (MgHA/Coll) with excellent compositional and structural mimicry of bone tissue. To this aim, two different crosslinking mechanisms in terms of pre-ribose glycation (before freeze drying) and post-ribose glycation (after freeze drying) were investigated. The obtained results explicate that with controlled freeze-drying, highly anisotropic porous structures with opportune macro-micro porosity are obtained. The physical-chemical features of the scaffolds characterized by XRD, FTIR, ICP and TGA demonstrated structural mimicry analogous to the native bone. The influence of ribose greatly assisted in decreasing solubility and increased enzymatic resistivity of the scaffolds. In addition, enhanced mechanical behaviour in response to compressive forces was achieved. Preliminary cell culture experiments reported good cytocompatibility with extensive cell adhesion, proliferation and colonization. Overall, scaffolds developed by pre-ribose glycation process are preferred, as the related crosslinking technique is more facile and robust to obtain functional scaffolds. As a proof of concept, we have demonstrated that ribose crosslinking is cost-effective, safe and functionally effective. This study also offers new insights and opportunities in developing promising scaffolds for bone tissue engineering. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Ribose 5-phosphate isomerase B knockdown compromises Trypanosoma brucei bloodstream form infectivity.

    Science.gov (United States)

    Loureiro, Inês; Faria, Joana; Clayton, Christine; Macedo-Ribeiro, Sandra; Santarém, Nuno; Roy, Nilanjan; Cordeiro-da-Siva, Anabela; Tavares, Joana

    2015-01-01

    Ribose 5-phosphate isomerase is an enzyme involved in the non-oxidative branch of the pentose phosphate pathway, and catalyzes the inter-conversion of D-ribose 5-phosphate and D-ribulose 5-phosphate. Trypanosomatids, including the agent of African sleeping sickness namely Trypanosoma brucei, have a type B ribose-5-phosphate isomerase. This enzyme is absent from humans, which have a structurally unrelated ribose 5-phosphate isomerase type A, and therefore has been proposed as an attractive drug target waiting further characterization. In this study, Trypanosoma brucei ribose 5-phosphate isomerase B showed in vitro isomerase activity. RNAi against this enzyme reduced parasites' in vitro growth, and more importantly, bloodstream forms infectivity. Mice infected with induced RNAi clones exhibited lower parasitaemia and a prolonged survival compared to control mice. Phenotypic reversion was achieved by complementing induced RNAi clones with an ectopic copy of Trypanosoma cruzi gene. Our results present the first functional characterization of Trypanosoma brucei ribose 5-phosphate isomerase B, and show the relevance of an enzyme belonging to the non-oxidative branch of the pentose phosphate pathway in the context of Trypanosoma brucei infection.

  3. Two Eucommia farnesyl diphosphate synthases exhibit distinct enzymatic properties leading to end product preferences.

    Science.gov (United States)

    Kajiura, Hiroyuki; Suzuki, Nobuaki; Tokumoto, Yuji; Yoshizawa, Takuya; Takeno, Shinya; Fujiyama, Kazuhito; Kaneko, Yoshinobu; Matsumura, Hiroyoshi; Nakazawa, Yoshihisa

    2017-08-01

    Farnesyl diphosphate synthase (FPS) is an essential enzyme in the biosynthesis of prenyl precursors for the production of primary and secondary metabolites, including sterols, dolichols, carotenoids and ubiquinones, and for the modification of proteins. Here we identified and characterized two FPSs (EuFPS1 and EuFPS2) from the plant Eucommia ulmoides. The EuFPSs had seven highly conserved prenyltransferase-specific domains that are critical for activity. Complementation and biochemical analyses using bacterially produced recombinant EuFPS isoforms showed that the EuFPSs had FPP synthesis activities both in vivo and in vitro. In addition to the typical reaction mechanisms of FPS, EuFPSs utilized farnesyl diphosphate (FPP) as an allylic substrate and participated in further elongation of the isoprenyl chain, resulting in the synthesis of geranylgeranyl diphosphate. However, despite the high amino acid similarities between the two EuFPS isozymes, their specific activities, substrate preferences, and final reaction products were different. The use of dimethylallyl diphosphate (DMAPP) as an allylic substrate highlighted the differences between the two enzymes: depending on the pH, the metal ion cofactor, and the cofactor concentration, EuFPS2 accumulated geranyl diphosphate as an intermediate product at a constant rate, whereas EuFPS1 synthesized little geranyl diphosphate. The reaction kinetics of the EuFPSs demonstrated that isopentenyl diphosphate and DMAPP were used both as substrates and as inhibitors of EuFPS activity. Taken together, the results indicate that the biosynthesis of FPP is highly regulated by various factors indispensable for EuFPS reactions in plants. Copyright © 2017. Published by Elsevier B.V.

  4. Metal ions control product specificity of isoprenyl diphosphate synthases in the insect terpenoid pathway

    Science.gov (United States)

    Frick, Sindy; Nagel, Raimund; Schmidt, Axel; Bodemann, René R.; Rahfeld, Peter; Pauls, Gerhard; Brandt, Wolfgang; Gershenzon, Jonathan; Boland, Wilhelm; Burse, Antje

    2013-01-01

    Isoprenyl diphosphate synthases (IDSs) produce the ubiquitous branched-chain diphosphates of different lengths that are precursors of all major classes of terpenes. Typically, individual short-chain IDSs (scIDSs) make the C10, C15, and C20 isoprenyl diphosphates separately. Here, we report that the product length synthesized by a single scIDS shifts depending on the divalent metal cofactor present. This previously undescribed mechanism of carbon chain-length determination was discovered for a scIDS from juvenile horseradish leaf beetles, Phaedon cochleariae. The recombinant enzyme P. cochleariae isoprenyl diphosphate synthase 1 (PcIDS1) yields 96% C10-geranyl diphosphate (GDP) and only 4% C15-farnesyl diphosphate (FDP) in the presence of Co2+ or Mn2+ as a cofactor, whereas it yields only 18% C10 GDP but 82% C15 FDP in the presence of Mg2+. In reaction with Co2+, PcIDS1 has a Km of 11.6 μM for dimethylallyl diphosphate as a cosubstrate and 24.3 μM for GDP. However, with Mg2+, PcIDS1 has a Km of 1.18 μM for GDP, suggesting that this substrate is favored by the enzyme under such conditions. RNAi targeting PcIDS1 revealed the participation of this enzyme in the de novo synthesis of defensive monoterpenoids in the beetle larvae. As an FDP synthase, PcIDS1 could be associated with the formation of sesquiterpenes, such as juvenile hormones. Detection of Co2+, Mn2+, or Mg2+ in the beetle larvae suggests flux control into C10 vs. C15 isoprenoids could be accomplished by these ions in vivo. The dependence of product chain length of scIDSs on metal cofactor identity introduces an additional regulation for these branch point enzymes of terpene metabolism. PMID:23440195

  5. Adenosine receptor targeting in health and disease.

    Science.gov (United States)

    Gessi, Stefania; Merighi, Stefania; Fazzi, Debora; Stefanelli, Angela; Varani, Katia; Borea, Pier Andrea

    2011-12-01

    The adenosine receptors A(1), A(2A), A(2B) and A(3) are important and ubiquitous mediators of cellular signaling that play vital roles in protecting tissues and organs from damage. In particular, adenosine triggers tissue protection and repair by different receptor-mediated mechanisms, including increasing the oxygen supply:demand ratio, pre-conditioning, anti-inflammatory effects and the stimulation of angiogenesis. The state of the art of the role of adenosine receptors which have been proposed as targets for drug design and discovery, in health and disease, and an overview of the ligands for these receptors in clinical development. Selective ligands of A(1), A(2A), A(2B) and A(3) adenosine receptors are likely to find applications in the treatment of pain, ischemic conditions, glaucoma, asthma, arthritis, cancer and other disorders in which inflammation is a feature. The aim of this review is to provide an overview of the present knowledge regarding the role of these adenosine receptors in health and disease.

  6. Modulation and metamodulation of synapses by adenosine.

    Science.gov (United States)

    Ribeiro, J A; Sebastião, A M

    2010-06-01

    The presence of adenosine in all nervous system cells (neurones and glia) together with its intensive release following insults makes adenosine as a sort of 'regulator' of synaptic communication, leading to the homeostatic coordination of brain function. Besides the direct actions of adenosine on the neurosecretory mechanisms, to tune neurotransmitter release, adenosine receptors interact with other receptors as well as with transporters as part of its attempt to fine-tune synaptic transmission. This review will focus on examples of the different ways adenosine can use to modulate or metamodulate synapses, in other words, to trigger or brake the action of some neurotransmitters and neuromodulators, to cross-talk with other G protein-coupled receptors, with ionotropic receptors and with receptor kinases as well as with transporters. Most of these interactions occur through A2A receptors, which in spite of their low density in some brain areas, such as the hippocampus, may function as amplifiers of the signalling of other mediators at synapses.

  7. ADP-ribosylhydrolase 3 (ARH3), Not Poly(ADP-ribose) Glycohydrolase (PARG) Isoforms, Is Responsible for Degradation of Mitochondrial Matrix-associated Poly(ADP-ribose)*

    Science.gov (United States)

    Niere, Marc; Mashimo, Masato; Agledal, Line; Dölle, Christian; Kasamatsu, Atsushi; Kato, Jiro; Moss, Joel; Ziegler, Mathias

    2012-01-01

    Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3−/− mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism. PMID:22433848

  8. ADP-ribosylhydrolase 3 (ARH3), not poly(ADP-ribose) glycohydrolase (PARG) isoforms, is responsible for degradation of mitochondrial matrix-associated poly(ADP-ribose).

    Science.gov (United States)

    Niere, Marc; Mashimo, Masato; Agledal, Line; Dölle, Christian; Kasamatsu, Atsushi; Kato, Jiro; Moss, Joel; Ziegler, Mathias

    2012-05-11

    Important cellular processes are regulated by poly(ADP-ribosyl)ation. This protein modification is catalyzed mainly by nuclear poly(ADP-ribose) polymerase (PARP) 1 in response to DNA damage. Cytosolic PARP isoforms have been described, whereas the presence of poly(ADP-ribose) (PAR) metabolism in mitochondria is controversial. PAR is degraded by poly(ADP-ribose) glycohydrolase (PARG). Recently, ADP-ribosylhydrolase 3 (ARH3) was also shown to catalyze PAR-degradation in vitro. PARG is encoded by a single, essential gene. One nuclear and three cytosolic isoforms result from alternative splicing. The presence and origin of a mitochondrial PARG is still unresolved. We establish here the genetic background of a human mitochondrial PARG isoform and investigate the molecular basis for mitochondrial poly(ADP-ribose) degradation. In common with a cytosolic 60-kDa human PARG isoform, the mitochondrial protein did not catalyze PAR degradation because of the absence of exon 5-encoded residues. In mice, we identified a transcript encoding an inactive cytosolic 52-kDa PARG lacking the mitochondrial targeting sequence and a substantial portion of exon 5. Thus, mammalian PARG genes encode isoforms that do not catalyze PAR degradation. On the other hand, embryonic fibroblasts from ARH3(-/-) mice lack most of the mitochondrial PAR degrading activity detected in wild-type cells, demonstrating a potential involvement of ARH3 in PAR metabolism.

  9. Hyperthermal (1-100 eV) nitrogen ion scattering damage to D-ribose and 2-deoxy-D-ribose films.

    Science.gov (United States)

    Deng, Zongwu; Bald, Ilko; Illenberger, Eugen; Huels, Michael A

    2007-10-14

    Highly charged heavy ion traversal of a biological medium can produce energetic secondary fragment ions. These fragment ions can in turn cause collisional and reactive scattering damage to DNA. Here we report hyperthermal (1-100 eV) scattering of one such fragment ion (N(+)) from biologically relevant sugar molecules D-ribose and 2-deoxy-D-ribose condensed on polycrystalline Pt substrate. The results indicate that N(+) ion scattering at kinetic energies down to 10 eV induces effective decomposition of both sugar molecules and leads to the desorption of abundant cation and anion fragments. Use of isotope-labeled molecules (5-(13)C D-ribose and 1-D D-ribose) partly reveals some site specificity of the fragment origin. Several scattering reactions are also observed. Both ionic and neutral nitrogen atoms abstract carbon from the molecules to form CN(-) anion at energies down to approximately 5 eV. N(+) ions also abstract hydrogen from hydroxyl groups of the molecules to form NH(-) and NH(2) (-) anions. A fraction of OO(-) fragments abstract hydrogen to form OH(-). The formation of H(3)O(+) ions also involves hydrogen abstraction as well as intramolecular proton transfer. These findings suggest a variety of severe damaging pathways to DNA molecules which occur on the picosecond time scale following heavy ion irradiation of a cell, and prior to the late diffusion-limited homogeneous chemical processes.

  10. Adenosine monophosphate forms ordered arrays in multilamellar lipid matrices: insights into assembly of nucleic acid for primitive life.

    Directory of Open Access Journals (Sweden)

    Laura Toppozini

    Full Text Available A fundamental question of biology is how nucleic acids first assembled and then were incorporated into the earliest forms of cellular life 4 billion years ago. The polymerization of nucleotides is a condensation reaction in which phosphodiester bonds are formed. This reaction cannot occur in aqueous solutions, but guided polymerization in an anhydrous lipid environment could promote a non-enzymatic condensation reaction in which oligomers of single stranded nucleic acids are synthesized. We used X-ray scattering to investigate 5'-adenosine monophosphate (AMP molecules captured in a multilamellar phospholipid matrix composed of dimyristoylphosphatidylcholine. Bragg peaks corresponding to the lateral organization of the confined AMP molecules were observed. Instead of forming a random array, the AMP molecules are highly entangled, with the phosphate and ribose groups in close proximity. This structure may facilitate polymerization of the nucleotides into RNA-like polymers.

  11. Changes in NAD/ADP-ribose metabolism in rectal cancer

    Directory of Open Access Journals (Sweden)

    L. Yalcintepe

    2005-03-01

    Full Text Available The extent of ADP-ribosylation in rectal cancer was compared to that of the corresponding normal rectal tissue. Twenty rectal tissue fragments were collected during surgery from patients diagnosed as having rectal cancer on the basis of pathology results. The levels of ADP-ribosylation in rectum cancer tissue samples (95.9 ± 22.1 nmol/ml was significantly higher than in normal tissues (11.4 ± 4 nmol/ml. The level of NAD+ glycohydrolase and ADP-ribosyl cyclase activities in rectal cancer and normal tissue samples were measured. Cancer tissues had significantly higher NAD+ glycohydrolase and ADP-ribosyl cyclase activities than the control tissues (43.3 ± 9.1 vs 29.2 ± 5.2 and 6.2 ± 1.6 vs 1.6 ± 0.4 nmol mg-1 min-1. Approximately 75% of the NAD+ concentration was consumed as substrate in rectal cancer, with changes in NAD+/ADP-ribose metabolism being observed. When [14C]-ADP-ribosylated tissue samples were subjected to SDS-PAGE, autoradiographic analysis revealed that several proteins were ADP-ribosylated in rectum tissue. Notably, the radiolabeling of a 113-kDa protein was remarkably greater than that in control tissues. Poly(ADP-ribosylation of the 113-kDa protein in rectum cancer tissues might be enhanced with its proliferative activity, and poly(ADP-ribosylation of the same protein in rectum cancer patients might be an indicator of tumor diagnosis.

  12. The role of adenosine receptors and endogenous adenosine in citalopram-induced cardiovascular toxicity

    Directory of Open Access Journals (Sweden)

    Kubilay Oransay

    2014-01-01

    Full Text Available Aim: We investigated the role of adenosine in citalopram-induced cardiotoxicity. Materials and Methods: Protocol 1: Rats were randomized into four groups. Sodium cromoglycate was administered to rats. Citalopram was infused after the 5% dextrose, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX; A 1 receptor antagonist, 8-(-3-chlorostyryl-caffeine (CSC; A 2a receptor antagonist, or dimethyl sulfoxide (DMSO administrations. Protocol 2: First group received 5% dextrose intraperitoneally 1 hour prior to citalopram. Other rats were pretreated with erythro-9-(2-hydroxy-3-nonyl adenine (EHNA; inhibitor of adenosine deaminase and S-(4-Nitrobenzyl-6-thioinosine (NBTI; inhibitor of facilitated adenosine transport. After pretreatment, group 2 received 5% dextrose and group 3 received citalopram. Adenosine concentrations, mean arterial pressure (MAP, heart rate (HR,  QRS duration and QT interval were evaluated. Results: In the dextrose group, citalopram infusion caused a significant decrease in MAP and HR and caused a significant prolongation in QRS and QT. DPCPX infusion significantly prevented the prolongation of the QT interval when compared to control. In the second protocol, citalopram infusion did not cause a significant change in plasma adenosine concentrations, but a significant increase observed in EHNA/NBTI groups. In EHNA/NBTI groups, citalopram-induced MAP and HR reductions, QRS and QT prolongations were more significant than the dextrose group. Conclusions: Citalopram may lead to QT prolongation by stimulating adenosine A 1 receptors without affecting the release of adenosine.

  13. An enzyme-coupled continuous fluorescence assay for farnesyl diphosphate synthases.

    Science.gov (United States)

    Dozier, Jonathan K; Distefano, Mark D

    2012-02-01

    Farnesyl diphosphate synthase (FDPS) catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to farnesyl diphosphate, a crucial metabolic intermediate in the synthesis of cholesterol, ubiquinone, and prenylated proteins; consequently, much effort has gone into developing inhibitors that target FDPS. Currently most FDPS assays either use radiolabeled substrates and are discontinuous or monitor pyrophosphate release and not farnesyl diphosphate (FPP) creation. Here we report the development of a continuous coupled enzyme assay for FDPS activity that involves the subsequent incorporation of the FPP product of that reaction into a peptide via the action of protein farnesyltransferase (PFTase). By using a dansylated peptide whose fluorescence quantum yield increases upon farnesylation, the rate of FDPS-catalyzed FPP production can be measured. We show that this assay is more sensitive than existing coupled assays, that it can be used to conveniently monitor FDPS activity in a 96-well plate format, and that it can reproduce IC(50) values for several previously reported FDPS inhibitors. This new method offers a simple, safe, and continuous method to assay FDPS activity that should greatly facilitate the screening of inhibitors of this important target.

  14. Examination of the thiamin diphosphate binding site in yeast transketolase by site-directed mutagenesis.

    Science.gov (United States)

    Meshalkina, L; Nilsson, U; Wikner, C; Kostikowa, T; Schneider, G

    1997-03-01

    The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site-directed mutagenesis. Replacement of E162, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild-type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25-80-fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.

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

    Science.gov (United States)

    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.

  16. Bioconversion of lactose/whey to fructose diphosphate with recombinant Saccharomyces cerevisiae cells

    Energy Technology Data Exchange (ETDEWEB)

    Compagno, C.; Tura, A.; Ranzi, B.M.; Martegani, E. (Univ. di Milano (Italy))

    1993-07-01

    Genetically engineered Saccharomyces cerevisiae strains that express Escherichia coli [beta]-galactosidase gene are able to bioconvert lactose or whey into fructose-1,6-diphosphate (FDP). High FDP yields from whey were obtained with an appropriate ratio between cell concentration and inorganic phosphate. The biomass of transformed cells can be obtained from different carbon sources, according to the expression vector bearing the lacZ gene. The authors showed that whey can be used as the carbon source for S. cerevisiae growth and as the substrate for bioconversion to fructose diphosphate.

  17. Fruit color mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

    Science.gov (United States)

    Isoprenoids are a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). In plants, IPP is synthesized in the cytoplasm from mevalonic acid via the “MVA pathway” a...

  18. Novel iron complexes bearing N6-substituted adenosine derivatives: synthesis, magnetic, 57Fe Mössbauer, DFT, and in vitro cytotoxicity studies.

    Science.gov (United States)

    Trávnícek, Zdenek; Mikulík, Jirí; Cajan, Michal; Zboril, Radek; Popa, Igor

    2008-09-15

    Iron complexes (1-7) involving N6-benzyladenosine derivatives of the predominant composition [Fe(L(n))Cl(3)].H(2)O {where L(1)=N6-(2-fluorobenzyl)adenosine (1), L(2)=N6-(4-fluorobenzyl)adenosine (2), L(3)=N6-(2-trifluoromethylbenzyl)adenosine (3), L(4)=N6-(3-trifluoromethylbenzyl)adenosine (4), L(5)=N6-(4-trifluoromethylbenzyl)adenosine (5), L(6)=N6-(4-trifluoromethoxybenzyl)adenosine (6), and L(7)=N6-(4-chlorobenzyl)adenosine (7)} have been synthesized. The compounds have been characterized by elemental analysis, variable-temperature and in-field 57Fe Mössbauer, ES+ MS, FTIR, 1H and 13C NMR spectroscopies, magnetochemical and conductivity measurements, thermal (TGA/DSC/DTA) analyses, and DFT calculations. It has been found that the organic molecule is coordinated to iron via N7 atom of the appropriate adenosine derivative and the products are represented by mixtures of complexes with various iron oxidation (Fe(III)/Fe(II)) and spin states (S=5/2, 4/2, 3/2, 2/2) and geometries (tetrahedral or trigonal bipyramidal). It is caused by the fact that partial redox processes proceed during the reactions due to the presence of a ribose moiety, which is oxidized to the corresponding 5'-ribotic acid, and simultaneously, a portion of Fe(III) cations is reduced to Fe(II) ones. Moreover, a significant effect of crystal water molecules on stereochemistry, and hence, on magnetic and spectral properties of the prepared complexes has been found. The compounds have been tested for their in vitro cytotoxicity against the following human cancer cell lines: malignant melanoma (G-361), osteogenic sarcoma (HOS), chronic myelogenous leukemia (K-562), and breast adenocarcinoma (MCF-7). The most important results have been obtained for complex 2 with IC(50) values 8-16 microM against HOS, K-562, and MCF-7 cell lines, and for complex 6 with IC(50) value 4 microM against MCF-7 cell line.

  19. AMP is an adenosine A1 receptor agonist.

    Science.gov (United States)

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

    2012-02-17

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

  20. Augmentation of poly(ADP-ribose) polymerase-dependent neuronal cell death by acidosis.

    Science.gov (United States)

    Zhang, Jian; Li, Xiaoling; Kwansa, Herman; Kim, Yun Tai; Yi, Liye; Hong, Gina; Andrabi, Shaida A; Dawson, Valina L; Dawson, Ted M; Koehler, Raymond C; Yang, Zeng-Jin

    2017-06-01

    Tissue acidosis is a key component of cerebral ischemic injury, but its influence on cell death signaling pathways is not well defined. One such pathway is parthanatos, in which oxidative damage to DNA results in activation of poly(ADP-ribose) polymerase and generation of poly(ADP-ribose) polymers that trigger release of mitochondrial apoptosis-inducing factor. In primary neuronal cultures, we first investigated whether acidosis per sé is capable of augmenting parthanatos signaling initiated pharmacologically with the DNA alkylating agent, N-methyl- N'-nitro- N-nitrosoguanidine. Exposure of neurons to medium at pH 6.2 for 4 h after N-methyl- N'-nitro- N-nitrosoguanidine washout increased intracellular calcium and augmented the N-methyl- N'-nitro- N-nitrosoguanidine-evoked increase in poly(ADP-ribose) polymers, nuclear apoptosis-inducing factor , and cell death. The augmented nuclear apoptosis-inducing factor and cell death were blocked by the acid-sensitive ion channel-1a inhibitor, psalmotoxin. In vivo, acute hyperglycemia during transient focal cerebral ischemia augmented tissue acidosis, poly(ADP-ribose) polymers formation, and nuclear apoptosis-inducing factor , which was attenuated by a poly(ADP-ribose) polymerase inhibitor. Infarct volume from hyperglycemic ischemia was decreased in poly(ADP-ribose) polymerase 1-null mice. Collectively, these results demonstrate that acidosis can directly amplify neuronal parthanatos in the absence of ischemia through acid-sensitive ion channel-1a . The results further support parthanatos as one of the mechanisms by which ischemia-associated tissue acidosis augments cell death.

  1. Adenosine: An immune modulator of inflammatory bowel diseases

    Institute of Scientific and Technical Information of China (English)

    Jeff Huaqing Ye; Vazhaikkurichi M Rajendran

    2009-01-01

    Inflammatory bowel disease (IBD) is a common and lifelong disabling gastrointestinal disease. Emerging treatments are being developed to target inflammatory cytokines which initiate and perpetuate the immune response. Adenosine is an important modulator of inflammation and its anti-inflammatory effects have been well established in humans as well as in animal models. High extracellular adenosine suppresses and resolves chronic inflammation in IBD models. High extracellular adenosine levels could be achieved by enhanced adenosine absorption and increased de novo synthesis. Increased adenosine concentration leads to activation of the A2a receptor on the cell surface of immune and epithelial cells that would be a potential therapeutic target for chronic intestinal inflammation. Adenosine is transported via concentrative nucleoside transporter and equilibrative nucleoside transporter transporters that are localized in apical and basolateral membranes of intestinal epithelial cells, respectively. Increased extracellular adenosine levels activate the A2a receptor, which would reduce cytokines responsible for chronic inflammation.

  2. The alterations in adenosine nucleotides and lactic acid levels in striated muscles following death with cervical dislocation or electric shock.

    Science.gov (United States)

    Pençe, Halime Hanim; Pençe, Sadrettin; Kurtul, Naciye; Bakan, Ebubekir; Kök, Ahmet Nezih; Kocoglu, Hasan

    2003-01-01

    In this study, changes in adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and lactic acid levels in masseter, triceps, and quadriceps muscles obtained from right and left sides of Spraque-Dawley rats following two different types of death were investigated. The samples were taken immediately and 120 minutes after death occurred either by cervical dislocation or electric shock. ATP concentrations in the muscles of masseter, triceps, and quadriceps were lower in samples obtained 120 minutes after death than that of samples obtained immediately after death. ADP, AMP, and lactic acid concentrations in these muscles were higher in samples obtained 120 minutes after death than those obtained immediately after death. A positive linear correlation was determined between ATP and ADP concentrations in quadriceps muscles of the rats killed with cervical dislocation and in masseter muscles of the rats killed with electric shock. When the rats killed with cervical dislocation and with electric shock were compared, ADP, AMP, and lactic acid concentrations were lower in the former than in the latter for both times (immediately and 120 minutes after death occurred). In the case of electric shock, ATP is consumed faster because of immediate contractions during death, resulting in a faster rigor mortis. This finding was confirmed with higher lactic acid levels in muscles of the rats killed with electric shock than the other group. In the cervical dislocation and electric shock group rats, ATP decreased in different levels in the three different muscle types mentioned above, being much decline in masseter in cervical dislocation and in quadriceps in electric shock group. This may be caused by low mass and less glycogen storage of masseter and by near localisation of electrode to quadriceps. One can conclude that the occurrence of rigor mortis is closely related to the mode of death.

  3. Higher cytoplasmic and nuclear poly(ADP-ribose) polymerase expression in familial than in sporadic breast cancer

    NARCIS (Netherlands)

    Klauke, M.L.; Hoogerbrugge-van der Linden, N.; Budczies, J.; Bult, P.; Prinzler, J.; Radke, C.; Krieken, J.H. van; Dietel, M.; Denkert, C.; Muller, B.M.

    2012-01-01

    Poly(ADP-ribose) polymerase 1 (PARP) is a key element of the single-base excision pathway for repair of DNA single-strand breaks. To compare the cytoplasmic and nuclear poly(ADP-ribose) expression between familial (BRCA1, BRCA2, or non BRCA1/2) and sporadic breast cancer, we investigated 39 sporadic

  4. Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

    Science.gov (United States)

    Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

    2013-07-01

    Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

  5. Phosphorylation of nm23/nucleoside diphosphate kinase by casein kinase 2 in vitro

    DEFF Research Database (Denmark)

    Engel, M; Issinger, O G; Lascu, I;

    1994-01-01

    We have investigated phosphorylation of human nucleoside diphosphate kinase (NDPK) and of homologous NDPK from different species by human casein kinase 2 (CK-2). The human NDPK isotypes A and B were phosphorylated by CK-2 in vitro both when the purified proteins and total lysate of HL-60 leukemia...

  6. ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

    NARCIS (Netherlands)

    Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

  7. Intersubunit ionic interactions stabilize the nucleoside diphosphate kinase of Mycobacterium tuberculosis

    DEFF Research Database (Denmark)

    Georgescauld, Florian; Moynie, Lucile; Habersetzer, Johann;

    2013-01-01

    Most nucleoside diphosphate kinases (NDPKs) are hexamers. The C-terminal tail interacting with the neighboring subunits is crucial for hexamer stability. In the NDPK from Mycobacterium tuberculosis (Mt) this tail is missing. The quaternary structure of Mt-NDPK is essential for full enzymatic acti...

  8. Purification and properties of phosphoribosyl-diphosphate synthetase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Arnvig, Kirsten; Hove-Jensen, Bjarne; Switzer, Robert L.

    1990-01-01

    Phosphoribosyl-diphosphate (PPRibP) synthetase from Bacillus subtiliis has been purified to near homogeneity from an Escherichia coli Δprs strain bearing the cloned B. subtilis prs gene, encoding PPRibP synthentase, on a plasmid. The Mr of the subunit (34,000) and its amino-terminal amino acid...

  9. Chrysanthemyl diphosphate synthase operates in planta as a bifunctional enzyme with chrysanthemol synthase activity.

    Science.gov (United States)

    Yang, Ting; Gao, Liping; Hu, Hao; Stoopen, Geert; Wang, Caiyun; Jongsma, Maarten A

    2014-12-26

    Chrysanthemyl diphosphate synthase (CDS) is the first pathway-specific enzyme in the biosynthesis of pyrethrins, the most widely used plant-derived pesticide. CDS catalyzes c1'-2-3 cyclopropanation reactions of two molecules of dimethylallyl diphosphate (DMAPP) to yield chrysanthemyl diphosphate (CPP). Three proteins are known to catalyze this cyclopropanation reaction of terpene precursors. Two of them, phytoene and squalene synthase, are bifunctional enzymes with both prenyltransferase and terpene synthase activity. CDS, the other member, has been reported to perform only the prenyltransferase step. Here we show that the NDXXD catalytic motif of CDS, under the lower substrate conditions prevalent in plants, also catalyzes the next step, converting CPP into chrysanthemol by hydrolyzing the diphosphate moiety. The enzymatic hydrolysis reaction followed conventional Michaelis-Menten kinetics, with a Km value for CPP of 196 μm. For the chrysanthemol synthase activity, DMAPP competed with CPP as substrate. The DMAPP concentration required for half-maximal activity to produce chrysanthemol was ∼100 μm, and significant substrate inhibition was observed at elevated DMAPP concentrations. The N-terminal peptide of CDS was identified as a plastid-targeting peptide. Transgenic tobacco plants overexpressing CDS emitted chrysanthemol at a rate of 0.12-0.16 μg h(-1) g(-1) fresh weight. We propose that CDS should be renamed a chrysanthemol synthase utilizing DMAPP as substrate.

  10. Two solanesyl diphosphate synthases with different subcellular localizations and their respective physiological roles in Oryza sativa.

    Science.gov (United States)

    Ohara, Kazuaki; Sasaki, Kanako; Yazaki, Kazufumi

    2010-06-01

    Long chain prenyl diphosphates are crucial biosynthetic precursors of ubiquinone (UQ) in many organisms, ranging from bacteria to humans, as well as precursors of plastoquinone in photosynthetic organisms. The cloning and characterization of two solanesyl diphosphate synthase genes, OsSPS1 and OsSPS2, in Oryza sativa is reported here. OsSPS1 was highly expressed in root tissue whereas OsSPS2 was found to be high in both leaves and roots. Enzymatic characterization using recombinant proteins showed that both OsSPS1 and OsSPS2 could produce solanesyl diphosphates as their final product, while OsSPS1 showed stronger activity than OsSPS2. However, an important biological difference was observed between the two genes: OsSPS1 complemented the yeast coq1 disruptant, which does not form UQ, whereas OsSPS2 only very weakly complemented the growth defect of the coq1 mutant. HPLC analyses showed that both OsSPS1 and OsSPS2 yeast transformants produced UQ9 instead of UQ6, which is the native yeast UQ. According to the complementation study, the UQ9 levels in OsSPS2 transformants were much lower than that of OsSPS1. Green fluorescent protein fusion analyses showed that OsSPS1 localized to mitochondria, while OsSPS2 localized to plastids. This suggests that OsSPS1 is involved in the supply of solanesyl diphosphate for ubiquinone-9 biosynthesis in mitochondria, whereas OsSPS2 is involved in providing solanesyl diphosphate for plastoquinone-9 formation. These findings indicate that O. sativa has a different mechanism for the supply of isoprenoid precursors in UQ biosynthesis from Arabidopsis thaliana, in which SPS1 provides a prenyl moiety for UQ9 at the endoplasmic reticulum.

  11. METOVITAN PREVENTS THE ACCUMULATION OF THIAMINE DIPHOSPHATE OXIDIZED FORM IN RAT TISSUES UNDER IRRADIATION

    Directory of Open Access Journals (Sweden)

    Parkhomenko Yu. M.

    2015-08-01

    Full Text Available The aim of the research was to test the ability of the drug "Metovitan" to prevent the redox balance disturbance in the tissues and thiamine diphosphate irreversible oxidation upon exposure of ionizing radiation on the body. The rats were subjected to a single exposure of the X-ray therapeutic instrument RUM-17 to create a dose of 0.5, 1.0 and 5.0 Gray. Preparation "Metovitan" was administered at a dose of 25 mg per 1 kg body weight for 22–24 h before irradiation. Contents of thiamine diphosphate, reduced SH-groups and reactive oxygen species in blood and brain were determined using previously described methods. It has been shown that the active form of the thiamine diphosphate content in the blood was decreased depending on the doses (from 0.5 to 5.0 Gray. At the same time the content of thiamine diphosphate oxidized form was increased. Furthermore the critical changes occurred in metabolic processes redox state parameters, namely, the level of free SH-groups was reduced and the level of reactive oxygen species was increased. Similar changes were observed in the brain tissue. The Metovitan single administration to the animals the day before irradiation, at 25 mg per 1 kg of body weight dose, promotes the protection of the intracellular thiamine diphosphate and redox status in animal tissues (blood, brain tissue from the negative influence of irradiation (at 0.5; 1.0 Gray doses. These results give reason to recommend the drug for treating of the staff that is involved in the elimination of radioactive contamination. To provide the protection from the higher doses, other scheme of the treatment should be probably recommended.

  12. Characterization of an Isopentenyl Diphosphate Isomerase involved in the Juvenile Hormone pathway in Aedes aegypti

    Science.gov (United States)

    Diaz, Miguel; Mayoral, Jaime G.; Priestap, Horacio; Nouzova, Marcela; Rivera-Perez, Crisalejandra; Noriega, Fernando G.

    2012-01-01

    Isopentenyl diphosphate isomerase (IPPI) is an enzyme involved in the synthesis of juvenile hormone (JH) in the corpora allata (CA) of insects. IPPI catalyzes the conversion of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP); afterwards IPP and DMAPP condense in a head-to-tail manner to produce geranyl diphosphate (GPP), this head-to-tail condensation can be repeated, by the further reaction of GPP with IPP, yielding the JH precursor farnesyl diphosphate. An IPPI expressed sequence tag (EST) was obtained from an Aedes aegypti corpora-allata + corpora cardiaca library. Its full-length cDNA encodes a 244-aa protein that shows a high degree of similarity with type I IPPIs from other organisms, particularly for those residues that have important roles in catalysis, metal coordination and interaction with the diphosphate moiety of the IPP. Heterologous expression produced a recombinant protein that metabolized IPP into DMAPP; treatment of DMAPP with phosphoric acid produced isoprene, a volatile compound that was measured with an assay based on a solid-phase micro extraction protocol and direct analysis by gas chromatography. A. aegypti IPPI (AaIPPI) required Mg2+ or Mn2+ but not Zn2+ for full activity and it was entirely inhibited by iodoacetamide. Real time PCR experiments showed that AaIPPI is highly expressed in the CA. Changes in AaIPPI mRNA levels in the CA in the pupal and adult female mosquito corresponded well with changes in JH synthesis (Li et al., 2003). This is the first molecular and functional characterization of an isopentenyl diphosphate isomerase involved in the production of juvenile hormone in the CA of an insect. PMID:22782071

  13. Vault-poly-ADP-ribose polymerase in the Octopus vulgaris brain: a regulatory factor of actin polymerization dynamic.

    Science.gov (United States)

    De Maio, Anna; Natale, Emiliana; Rotondo, Sergio; Di Cosmo, Anna; Faraone-Mennella, Maria Rosaria

    2013-09-01

    Our previous behavioural, biochemical and immunohistochemical analyses conducted in selected regions (supra/sub oesophageal masses) of the Octopus vulgaris brain detected a cytoplasmic poly-ADP-ribose polymerase (more than 90% of total enzyme activity). The protein was identified as the vault-free form of vault-poly-ADP-ribose polymerase. The present research extends and integrates the biochemical characterization of poly-ADP-ribosylation system, namely, reaction product, i.e., poly-ADP-ribose, and acceptor proteins, in the O. vulgaris brain. Immunochemical analyses evidenced that the sole poly-ADP-ribose acceptor was the octopus cytoskeleton 50-kDa actin. It was present in both free, endogenously poly-ADP-ribosylated form (70kDa) and in complex with V-poly-ADP-ribose polymerase and poly-ADP-ribose (260kDa). The components of this complex, alkali and high salt sensitive, were purified and characterized. The kind and the length of poly-ADP-ribose corresponded to linear chains of 30-35 ADP-ribose units, in accordance with the features of the polymer synthesized by the known vault-poly-ADP-ribose polymerase. In vitro experiments showed that V-poly-ADP-ribose polymerase activity of brain cytoplasmic fraction containing endogenous actin increased upon the addition of commercial actin and was highly reduced by ATP. Anti-actin immunoblot of the mixture in the presence and absence of ATP showed that the poly-ADP-ribosylation of octopus actin is a dynamic process balanced by the ATP-dependent polymerization of the cytoskeleton protein, a fundamental mechanism for synaptic plasticity.

  14. Aminopyrimidine derivatives as adenosine antagonists / Janke Kleynhans

    OpenAIRE

    Kleynhans, Janke

    2013-01-01

    Aims of this project - The aim of this study was to design and synthesise novel 2-aminopyrimidine derivatives as potential adenosine A1 and A2A receptor antagonists. Background and rationale - Parkinson’s disease is the second most common neurodegenerative disorder (after Alzheimer’s disease) and is characterised by the selective death of the dopaminergic neurons of the nigro-striatal pathway. Distinctive motor symptoms include bradykinesia, muscle rigidity and tremor, while non-m...

  15. Frontalin pheromone biosynthesis in the mountain pine beetle, Dendroctonus ponderosae, and the role of isoprenyl diphosphate synthases.

    Science.gov (United States)

    Keeling, Christopher I; Chiu, Christine C; Aw, Tidiane; Li, Maria; Henderson, Hannah; Tittiger, Claus; Weng, Hong-Biao; Blomquist, Gary J; Bohlmann, Joerg

    2013-11-19

    The mountain pine beetle (Dendroctonus ponderosae Hopkins) is the most destructive pest of western North American pine forests. Adult males produce frontalin, an eight-carbon antiaggregation pheromone, via the mevalonate pathway, as part of several pheromones that initiate and modulate the mass attack of host trees. Frontalin acts as a pheromone, attractant, or kairomone in most Dendroctonus species, other insects, and even elephants. 6-Methylhept-6-en-2-one, a frontalin precursor, is hypothesized to originate from 10-carbon geranyl diphosphate (GPP), 15-carbon farnesyl diphosphate (FPP), or 20-carbon geranylgeranyl diphosphate (GGPP) via a dioxygenase- or cytochrome P450-mediated carbon-carbon bond cleavage. To investigate the role of isoprenyl diphosphate synthases in pheromone biosynthesis, we characterized a bifunctional GPP/FPP synthase and a GGPP synthase in the mountain pine beetle. The ratio of GPP to FPP produced by the GPP/FPP synthase was highly dependent on the ratio of the substrates isopentenyl diphosphate and dimethylallyl diphosphate used in the assay. Transcript levels in various tissues and life stages suggested that GGPP rather than GPP or FPP is used as a precursor to frontalin. Reduction of transcript levels by RNA interference of the isoprenyl diphosphate synthases identified GGPP synthase as having the largest effect on frontalin production, suggesting that frontalin is derived from a 20-carbon isoprenoid precursor rather than from the 10- or 15-carbon precursors.

  16. The Effects of Ribose on Mechanical and Physicochemical Properties of Cold Water Fish Gelatin Films

    Directory of Open Access Journals (Sweden)

    Neda Javadian

    2014-06-01

    Full Text Available Native fish gelatin has some disadvantages such as high hydrophilic, and solubility in cold water. Mixing with other biopolymers and crosslinking by sugars may improve functional properties of fish gelatin. So in this research, the effects of ribose were investigated on moisture sorption isotherm, solubility in water, and mechanical properties of cold water fish gelatin (CWFG films. Ribose sugar was incorporated into CWFG solutions at different concentrations (e.g. 0, 2, 4, and 6% w/w dried gelatin. Physicochemical properties such as water solubility, moisture sorption isotherm and mechanical properties of the films were measured according to ASTM standards. Results showed that incorporation of ribose sugar significantly improved functional properties of CWFG films. Solubility, moisture content and monolayer water content of the matrixes were decreased by increasing the ribose contents. Mechanical properties of biocomposites were improved more than 20% and moisture sorption isotherm curve significantly shifted to lower moisture contents. The results of this study could be explored for commercial use, depending on industrial needs for either production of edible films or for packaging purposes.

  17. The Sound of Silence: RNAi in Poly (ADP-Ribose Research

    Directory of Open Access Journals (Sweden)

    Felix R. Althaus

    2012-12-01

    Full Text Available Poly(ADP-ribosyl-ation is a nonprotein posttranslational modification of proteins and plays an integral part in cell physiology and pathology. The metabolism of poly(ADP-ribose (PAR is regulated by its synthesis by poly(ADP-ribose polymerases (PARPs and on the catabolic side by poly(ADP-ribose glycohydrolase (PARG. PARPs convert NAD+ molecules into PAR chains that interact covalently or noncovalently with target proteins and thereby modify their structure and functions. PAR synthesis is activated when PARP1 and PARP2 bind to DNA breaks and these two enzymes account for almost all PAR formation after genotoxic stress. PARG cleaves PAR molecules into free PAR and finally ADP-ribose (ADPR moieties, both acting as messengers in cellular stress signaling. In this review, we discuss the potential of RNAi to manipulate the levels of PARPs and PARG, and consequently those of PAR and ADPR, and compare the results with those obtained after genetic or chemical disruption.

  18. Role of poly(ADP-ribose) synthetase in inflammation and ischaemia-reperfusion.

    Science.gov (United States)

    Szabó, C; Dawson, V L

    1998-07-01

    Oxidative and nitrosative stress can trigger DNA strand breakage, which then activates the nuclear enzyme poly(ADP-ribose) synthetase (PARS). This enzyme has also been termed poly(ADP-ribose) polymerase (PARP) or poly(ADP-ribose) transferase (pADPRT). Rapid activation of the enzyme depletes the intracellular concentration of its substrate, nicotinamide adenine dinucleotide, thus slowing the rate of glycolysis, electron transport and subsequently ATP formation. This process can result in cell dysfunction and cell death. In this article, Csaba Szabó and Valina Dawson overview the impact of pharmacological inhibition or genetic inactivation of PARS on the course of oxidant-induced cell death in vitro, and in inflammation and reperfusion injury in vivo. A major trigger for DNA damage in pathophysiological conditions is peroxynitrite, a cytotoxic oxidant formed by the reaction between the free radicals nitric oxide and superoxide. The pharmacological inhibition of poly(ADP-ribose) synthetase is a novel approach for the experimental therapy of various forms of inflammation and shock, stroke, myocardial and intestinal ischaemia-reperfusion, and diabetes mellitus.

  19. Synthesis of Gabosine A and N from Ribose by the Use of Ring-Closing Metathesis

    DEFF Research Database (Denmark)

    Monrad, Rune Nygaard; Fanefjord, Mette; Hansen, Flemming Gundorph;

    2009-01-01

    -methylallyl bromide. The functionalized octa-1,7-diene, thus obtained, is converted into the six-membered gabosine skeleton by ring-closing olefin metathesis. Subsequent protective group manipulations and oxidation gives rise to gabosine N in a total of 8 steps from ribose while the synthesis of gabosine...

  20. Escherichia coli rpiA> gene encoding ribose phosphate isomerase A

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Maigaard, Marianne

    1993-01-01

    The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment was seque...

  1. VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

    Directory of Open Access Journals (Sweden)

    Laura Lafon-Hughes

    2014-10-01

    Full Text Available Poly-ADP-ribose (PAR is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs and degraded by poly-ADP-ribose-glycohydrolase (PARG. Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair. Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt. In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO. PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

  2. Escherichia coli rpiA gene encoding ribose phosphate isomerase A

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Maigaard, Marianne

    1993-01-01

    The rpiA gene encoding ribose phosphate isomerase A was cloned from phage 1A2(471) of the Kohara gene library. Subcloning, restriction, and complementation analyses revealed an 1,800-bp SspI-generated DNA fragment that contained the entire control and coding sequences. This DNA fragment...

  3. Selective deletion of the A1 adenosine receptor abolishes heart-rate slowing effects of intravascular adenosine in vivo.

    Directory of Open Access Journals (Sweden)

    Michael Koeppen

    Full Text Available OBJECTIVE: Intravenous adenosine induces temporary bradycardia. This is due to the activation of extracellular adenosine receptors (ARs. While adenosine can signal through any of four ARs (A1AR, A2AAR, A2BAR, A3AR, previous ex vivo studies implicated the A1AR in the heart-rate slowing effects. Here, we used comparative genetic in vivo studies to address the contribution of individual ARs to the heart-rate slowing effects of intravascular adenosine. METHODS AND RESULTS: We studied gene-targeted mice for individual ARs to define their in vivo contribution to the heart-rate slowing effects of adenosine. Anesthetized mice were treated with a bolus of intravascular adenosine, followed by measurements of heart-rate and blood pressure via a carotid artery catheter. These studies demonstrated dose-dependent slowing of the heart rate with adenosine treatment in wild-type, A2AAR(-/-, A2BAR(-/-, or A3AR(-/- mice. In contrast, adenosine-dependent slowing of the heart-rate was completely abolished in A1AR(-/- mice. Moreover, pre-treatment with a specific A1AR antagonist (DPCPX attenuated the heart-rate slowing effects of adenosine in wild-type, A2AAR(-/-, or A2BAR(-/- mice, but did not alter hemodynamic responses of A1AR(-/- mice. CONCLUSIONS: The present studies combine pharmacological and genetic in vivo evidence for a selective role of the A1AR in slowing the heart rate during adenosine bolus injection.

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

    Science.gov (United States)

    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.

  5. Protective effect of D-ribose against inhibition of rats testes function at excessive exercise

    Directory of Open Access Journals (Sweden)

    Chigrinskiy E.A.

    2011-09-01

    Full Text Available An increasing number of research studies point to participation in endurance exercise training as having significant detrimental effects upon reproductive hormonal profiles in men. The means used for prevention and correction of fatigue are ineffective for sexual function recovery and have contraindications and numerous side effects. The search for substances effectively restoring body functions after overtraining and at the same time sparing the reproductive function, which have no contraindications precluding their long and frequent use, is an important trend of studies. One of the candidate substances is ribose used for correction of fatigue in athletes engaged in some sports.We studied the role of ribose deficit in metabolism of the testes under conditions of excessive exercise and the potentialities of ribose use for restoration of the endocrine function of these organs.45 male Wistar rats weighing 240±20 g were used in this study. Animals were divided into 3 groups (n=15: control; excessive exercise; excessive exercise and received ribose treatment. Plasma concentrations of lactic, β-hydroxybutyric, uric acids, luteinizing hormone, total and free testosterone were measured by biochemical and ELISA methods. The superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase activities and uric acids, malondialdehyde, glutathione, ascorbic acids, testosterone levels were estimated in the testes sample.Acute disorders of purine metabolism develop in rat testes under conditions of excessive exercise. These disorders are characterized by enhanced catabolism and reduced reutilization of purine mononucleotides and activation of oxidative stress against the background of reduced activities of the pentose phosphate pathway and antioxidant system. Administration of D-ribose to rats subjected to excessive exercise improves purine reutilization, stimulates the pentose phosphate pathway work

  6. Effects of adenosine agonist R-phenylisopropyl-adenosine on halothane anesthesia and antinociception in rats

    Institute of Scientific and Technical Information of China (English)

    Hai-chun MA; Yan-fen WANG; Chun-sheng FENG; Hua ZHAO; Shuji DOHI

    2005-01-01

    Aim: To investigate the antinociceptive effect of adenosine agonist Rphenylisopropyl-adenosine (R-PIA) given to conscious rats by intracerebroventricular (ICV) and intrathecal (IT), and identify the effect of R-PIA on minimum alveolar concentration (MAC) of halothane with pretreatment of A1 receptor an tagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) or K+ channel blocker 4-aminopyridine (4-AP). Methods: Sprague-Dawley rats were implanted with 24 gauge stainless steel guide cannula using stereotaxic apparatus and ICV method, and an IT catheter (PE-10, 8.5 cm) was inserted into the lumbar subarachnoid space, while the rats were under pentobarbital anesthesia. After one week of recovery from surgery, rats were randomly assigned to one of the following protocols: MAC of halothane, or tail-flick latency. All measurements were performed after R-PIA (0.8-2.0 μg) microinjection into ICV and IT with or without pretreatment of DPCPX or 4-AP. Results: Microinjection of adenosine agonist R PIA in doses of 0.8-2.0 μg into ICV and IT produced a significant dose- and time dependent antinociceptive action as reflected by increasing latency times and ICV administration of adenosine agonist R-PIA (0.8 μg) reducing halothane anes thetic requirements (by 29%). The antinociception and reducing halothane requirements effected by adenosine agonist R-PIA was abolished by DPCPX and 4-AP. Conclusion: ICV and IT administration of adenosine agonist R-PIA produced an antinociceptive effect in a dose-dependent manner and decreased hal othane MAC with painful stimulation through activation of A1 receptor subtype, and the underlying mechanism involves K+ channel activation.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

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

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

    Directory of Open Access Journals (Sweden)

    Nathan T. Fried

    2017-03-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2011-06-01

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

  11. Pretreatment with adenosine and adenosine A1 receptor agonist protects against intestinal ischemia-reperfusion injury in rat

    Institute of Scientific and Technical Information of China (English)

    V Haktan Ozacmak; Hale Sayan

    2007-01-01

    AIM: To examine the effects of adenosine and A1 receptor activation on reperfusion-induced small intestinal injury.METHODS: Rats were randomized into groups with sham operation, ischemia and reperfusion, and systemic treatments with either adenosine or 2-chloro-N6-cyclopentyladenosine, A1 receptor agonist or 8-cyclopentyl-1,3-dipropylxanthine, A1 receptor antagonist, plus adenosine before ischemia. Following reperfusion, contractions of ileum segments in response to KCl, carbachol and substance P were recorded. Tissue myeloperoxidase,malondialdehyde, and reduced glutathione levels were measured.RESULTS: Ischemia significantly decreased both contraction and reduced glutathione level which were ameliorated by adenosine and agonist administration. Treatment also decreased neutrophil infiltration and membrane lipid peroxidation. Beneficial effects of adenosine were abolished by pretreatment with A1 receptor antagonist.CONCLUSION: The data suggest that adenosine and A1 receptor stimulation attenuate ischemic intestinal injury via decreasing oxidative stress, lowering neutrophil infiltration, and increasing reduced glutathione content.

  12. Thermal Behavior of d-Ribose Adsorbed on Silica: Effect of Inorganic Salt Coadsorption and Significance for Prebiotic Chemistry.

    Science.gov (United States)

    Akouche, Mariame; Jaber, Maguy; Zins, Emilie-Laure; Maurel, Marie-Christine; Lambert, Jean-Francois; Georgelin, Thomas

    2016-10-24

    Understanding ribose reactivity is a crucial step in the "RNA world" scenario because this molecule is a component of all extant nucleotides that make up RNA. In solution, ribose is unstable and susceptible to thermal destruction. We examined how ribose behaves upon thermal activation when adsorbed on silica, either alone or with the coadsorption of inorganic salts (MgCl2 , CaCl2 , SrCl2 , CuCl2 , FeCl2 , FeCl3 , ZnCl2 ). A combination of (13) C NMR, in situ IR, and TGA analyses revealed a variety of phenomena. When adsorbed alone, ribose remains stable up to 150 °C, at which point ring opening is observed, together with minor oxidation to a lactone. All the metal salts studied showed specific interactions with ribose after dehydration, resulting in the formation of polydentate metal ion complexes. Anomeric equilibria were affected, generally favoring ribofuranoses. Zn(2+) stabilized ribose up to higher temperatures than bare silica (180 to 200 °C). Most other cations had an adverse effect on ribose stability, with ring opening already upon drying at 70 °C. In addition, alkaline earth cations catalyzed the dehydration of ribose to furfural and, to variable degrees, its further decarbonylation to furan. Transition-metal ions with open d-shells took part in redox reactions with ribose, either as reagents or as catalysts. These results allow the likelihood of prebiotic chemistry scenarios to be evaluated, and may also be of interest for the valorization of biomass-derived carbohydrates by heterogeneous catalysis.

  13. Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase

    DEFF Research Database (Denmark)

    Willemoës, Martin; Hove-Jensen, Bjarne; Larsen, Sine

    2000-01-01

    saturation with ribose 5-phosphate leads to the binding of Mg2+ and substrates via a slow pathway where Pi binds to the enzyme last. The random mechanism for Pi binding was further supported by studies with competitive inhibitors of Mg2+, MgATP, and ribose 5-phosphate that all appeared noncompetitive when...... varying Pi at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing Pi concentrations. Results from ADP inhibition of Pi activation suggest that these effectors compete for binding to a common regulatory site....

  14. Mechanism of protection of adenosine from sulphate radical anion and repair of adenosine radicals by caffeic acid in aqueous solution

    Indian Academy of Sciences (India)

    M Sudha Swaraga; L Charitha; M Adinarayana

    2005-07-01

    The photooxidation of adenosine in presence of peroxydisulphate (PDS) has been studied by spectrophotometrically measuring the absorbance of adenosine at 260 nm. The rates of oxidation of adenosine by sulphate radical anion have been determined in the presence of different concentrations of caffeic acid. Increase in [caffeic acid] is found to decrease the rate of oxidation of adenosine suggesting that caffeic acid acts as an efficient scavenger of $SO_{4}^{\\bullet-}$ and protects adenosine from it. Sulphate radical anion competes for adenosine as well as for caffeic acid. The quantum yields of photooxidation of adenosine have been calculated from the rates of oxidation of adenosine and the light intensity absorbed by PDS at 254 nm, the wavelength at which PDS is activated to sulphate radical anion. From the results of experimentally determined quantum yields (exptl) and the quantum yields calculated (cal) assuming caffeic acid acting only as a scavenger of $SO_{4}^{\\bullet-}$ show that exptl values are lower than cal values. The ' values, which are experimentally found quantum yield values at each caffeic acid concentration and corrected for $SO_{4}^{\\bullet-}$ scavenging by caffeic acid, are also found to be greater than exptl values. These observations suggest that the transient adenosine radicals are repaired by caffeic acid in addition to scavenging of sulphate radical anions.

  15. Electroacupuncture improves neuropathic pain Adenosine,adenosine 5'-triphosphate disodium and their receptors perhaps change simultaneously

    Institute of Scientific and Technical Information of China (English)

    Wen Ren; Wenzhan Tu; Songhe Jiang; Ruidong Cheng; Yaping Du

    2012-01-01

    Applying a stimulating current to acupoints through acupuncture needles-known as electroacupuncture-has the potential to produce analgesic effects in human subjects and experimental animals.When acupuncture was applied in a rat model,adenosine 5'-triphosphate disodium in the extracellular space was broken down into adenosine,which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process.Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture.The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves.In neuropathic pain,there is upregulation of P2X purinoceptor 3(P2X3)receptor expression in dorsal root ganglion neurons.Conversely,the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated.The pathways upon which electroacupuncture appear to act are interwoven with pain pathways,and electroacupuncture stimuli converge with impulses originating from painful areas.Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.

  16. A New Method of Crystallization of Octahydro Trisodium Salt of Fructose-1,6-diphosphate

    Institute of Scientific and Technical Information of China (English)

    应汉杰; 欧阳平凯

    2002-01-01

    In order to overcome the elementary heterogeneous nucleation while octahydro trisodium salt of fructose-1,6-diphosphate(FDPNaa.8 H2O) is crystallized with ethanol precipitation at low temperature, a new crystallizationmethod with alcohol precipitation combined with salt precipitation has been presented. The ethanol-sodium ac-etate system for crystallization of salt of fructose-1,6-diphosphate is based on the mechanism of crystallization ofFDPNa3.8 H2O in the ethanol-low temperature system. It is found that crystal size may be controlled by regulatingtemperature or pH value of solution in the crystallization process, and the crystal yield increases to 95% from 78%which obtained in the ethanol-low temperature system.

  17. Molecular mechanism of allosteric substrate activation in a thiamine diphosphate-dependent decarboxylase.

    Science.gov (United States)

    Versées, Wim; Spaepen, Stijn; Wood, Martin D H; Leeper, Finian J; Vanderleyden, Jos; Steyaert, Jan

    2007-11-30

    Thiamine diphosphate-dependent enzymes are involved in a wide variety of metabolic pathways. The molecular mechanism behind active site communication and substrate activation, observed in some of these enzymes, has since long been an area of debate. Here, we report the crystal structures of a phenylpyruvate decarboxylase in complex with its substrates and a covalent reaction intermediate analogue. These structures reveal the regulatory site and unveil the mechanism of allosteric substrate activation. This signal transduction relies on quaternary structure reorganizations, domain rotations, and a pathway of local conformational changes that are relayed from the regulatory site to the active site. The current findings thus uncover the molecular mechanism by which the binding of a substrate in the regulatory site is linked to the mounting of the catalytic machinery in the active site in this thiamine diphosphate-dependent enzyme.

  18. New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E. coli.

    Science.gov (United States)

    Beigi, Maryam; Waltzer, Simon; Zarei, Mostafa; Müller, Michael

    2014-12-10

    The intermolecular asymmetric Stetter reaction is a rarely found biocatalysts transformation. MenD, the second enzyme of the menaquinone biosynthetic pathway, catalyzes as a physiological reaction a Stetter-like addition of α-ketoglutarate to isochorismate. The substrate range of MenD for similar 1,4-additions is highly restricted. All other thiamine diphosphate dependent enzymes known to act as stetterases are members of the PigD enzyme subfamily, which accept aliphatic and aromatic α,β-unsaturated ketones and thioesters as Michael acceptor substrates. Here, we describe the unexpected activity of MenD with short-chain α,β-unsaturated acids and derivatives as substrates in Stetter reactions. MenD possesses a characteristic substrate range with respect to Michael acceptor substrates which is distinctly different from the classical stetterases. This provides biocatalytic access to new types of products which are not related to the products currently accessible by thiamine diphosphate dependent enzyme catalysis.

  19. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Directory of Open Access Journals (Sweden)

    Juhi Sikarwar

    2013-01-01

    Full Text Available Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3. The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA chromatography.

  20. Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    Science.gov (United States)

    Sikarwar, Juhi; Kaushik, Sanket; Sinha, Mau; Kaur, Punit; Sharma, Sujata; Singh, Tej P.

    2013-01-01

    Acinetobacter baumannii is a multidrug resistant pathogenic bacteria associated with hospital acquired infections. This bacterium possesses a variety of resistance mechanisms which makes it more difficult to control the bacterium with conventional drugs, and, so far no effective drug treatment is available against it. Nucleoside diphosphate kinase is an important enzyme, which maintains the total nucleotide triphosphate pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs. The role of nucleoside diphosphate kinase (Ndk) has also been observed in pathogenesis in other organisms. However, intensive studies are needed to decipher its other putative roles in Acinetobacter baumannii. In the present study, we have successfully cloned the gene encoding Ndk and achieved overexpression in bacterial host BL-21 (DE3). The overexpressed protein is further purified by nickel-nitrilotriacetic acid (Ni-NTA) chromatography. PMID:23662205

  1. Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate

    Energy Technology Data Exchange (ETDEWEB)

    Salowe, S.P.; Ator, M.A.; Stubbe, J.A.

    1987-06-16

    Ribonucleoside diphosphate reductase (RDPR) from Escherichia coli was completely inactivated by 1 equiv of the mechanism-based inhibitor 2'-azido-2'-azido-2'-deoxyuridine 5'-diphosphate (N/sub 3/UDP). Incubation of RDPR with (3'-/sup 3/H)N/sub 3/UDP resulted in 0.2 mol of /sup 3/H released to solvent per mole of enzyme inactivated, indicating that cleavage of the 3' carbon-hydrogen bond occurred in the reaction. Incubation of RDPR with (..beta..-/sup 32/P)N/sub 3/UDP resulted in stoichiometric production of inorganic pyrophosphate. One equivalent of uracil was eliminated from N/sub 3/UDP, but no azide release was detected. Analysis of the reaction of RDPR with (/sup 15/N/sub 3/)N/sub 3/UDP by mass spectrometry revealed that the azide moiety was converted to 0.9 mol of nitrogen gas per mole of enzyme inactivated. The tyrosyl radical of the B2 subunit was destroyed during the inactivation by N/sub 3/UDP as reported previously, while the specific activity of the B1 subunit was reduced by half. Incubation of (5'-/sup 3/H)N/sub 3/UDP with RDPR resulted in stoichiometric covalent radiolabeling of the enzyme. Separation of the enzyme's subunits by chromatofocusing revealed that the modification was specific for the B1 subunit.

  2. Serum adenosine deaminase activity in cutaneous anthrax.

    Science.gov (United States)

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

    2014-07-06

    Adenosine deaminase (ADA) activity has been discovered in several inflammatory conditions; however, there are no data associated with cutaneous anthrax. The aim of this study was to investigate serum ADA activity in patients with cutaneous anthrax. Sixteen patients with cutaneous anthrax and 17 healthy controls were enrolled. We measured ADA activity; peripheral blood leukocyte, lymphocyte, neutrophil, and monocyte counts; erythrocyte sedimentation rate; and C reactive protein levels. Serum ADA activity was significantly higher in patients with cutaneous anthrax than in the controls (panthrax.

  3. Expression pattern of the coparyl diphosphate synthase gene in developing rice anthers.

    Science.gov (United States)

    Fukuda, Ari; Nemoto, Keisuke; Chono, Makiko; Yamaguchi, Shinjiro; Nakajima, Masatoshi; Yamagishi, Junko; Maekawa, Masahiko; Yamaguchi, Isomaro

    2004-08-01

    Rice anthers contain high concentrations of gibberellins A(4) and A(7). To understand their physiological roles, we examined the site of their biosynthesis by analyzing the expression pattern of a gene (OsCPS) encoding coparyl diphosphate synthase in developing rice flowers. Expression was apparent in the anthers 1-2 days before flowering, and CPS mRNA accumulated in the maturing pollen.

  4. Inclusion of thiamine diphosphate and S-adenosylmethionine at their chemically active sites.

    Science.gov (United States)

    Schrader, Thomas; Fokkens, Michael; Klärner, Frank-Gerrit; Polkowska, Jolanta; Bastkowski, Frank

    2005-12-09

    [structure: see text] Molecular clips functionalized by phosphonate or phosphate groups bind thiamine diphosphate (TPP) and S-adenosylmethionine (SAM) with high affinity in water; both sulfur-based cofactors transfer organic groups to biomolecules. For TPP, various analytical tools point toward a simultaneous insertion of both heterocyclic rings into the electron-rich clip cavity. Similarly, SAM is also embedded with its sulfonium moiety inside the receptor cavity. This paves the way for enzyme models and direct interference with enzymatic processes.

  5. A novel chloroplastic isopentenyl diphosphate isomerase gene from Jatropha curcas: Cloning, characterization and subcellular localization

    OpenAIRE

    Wei, Lei; Yin, Li; Hu,Xiaole; Xu, Ying; Chen,Fang

    2014-01-01

    Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene ...

  6. Role of Arginine-304 in the Diphosphate-Triggered Active Site Closure Mechanism of Trichodiene Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Vedula,L.; Cane, D.; Christianson, D.

    2005-01-01

    The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PPi) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PPi bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg{sup 2+}{sub 3-}PP{sub i}, in which R304 donates hydrogen bonds to PP{sub i} and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP{sub i} in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP{sub i} and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg{sup 2+}{sub 3-}PP{sub i} binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP{sub i} leaving group.

  7. Structure and Mechanism of the Diterpene Cyclase ent-Copalyl Diphosphate Synthase

    Science.gov (United States)

    Köksal, Mustafa; Hu, Huayou; Coates, Robert M.; Peters, Reuben J.; Christianson, David W.

    2011-01-01

    The structure of ent-copalyl diphosphate synthase (CPS) reveals three α-helical domains (α, β, γ), as also observed in the related diterpene cyclase taxadiene synthase. However, active sites are located at the interface of the βγ domains in CPS but exclusively in the α domain of taxadiene synthase. Modular domain architecture in plant diterpene cyclases enables the evolution of alternative active sites and chemical strategies for catalyzing isoprenoid cyclization reactions. PMID:21602811

  8. Efficient diterpene production in yeast by engineering Erg20p into a geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Ignea, Codruta; Trikka, Fotini A; Nikolaidis, Alexandros K; Georgantea, Panagiota; Ioannou, Efstathia; Loupassaki, Sofia; Kefalas, Panagiotis; Kanellis, Angelos K; Roussis, Vassilios; Makris, Antonios M; Kampranis, Sotirios C

    2015-01-01

    Terpenes have numerous applications, ranging from pharmaceuticals to fragrances and biofuels. With increasing interest in producing terpenes sustainably and economically, there has been significant progress in recent years in developing methods for their production in microorganisms. In Saccharomyces cerevisiae, production of the 20-carbon diterpenes has so far proven to be significantly less efficient than production of their 15-carbon sesquiterpene counterparts. In this report, we identify the modular structure of geranylgeranyl diphosphate synthesis in yeast to be a major limitation in diterpene yields, and we engineer the yeast farnesyl diphosphate synthase Erg20p to produce geranylgeranyl diphosphate. Using a combination of protein and genetic engineering, we achieve significant improvements in the production of sclareol and several other isoprenoids, including cis-abienol, abietadiene and β-carotene. We also report the development of yeast strains carrying the engineered Erg20p, which support efficient isoprenoid production and can be used as a dedicated chassis for diterpene production or biosynthetic pathway elucidation. The design developed here can be applied to the production of any GGPP-derived isoprenoid and is compatible with other yeast terpene production platforms.

  9. Cloning and Characterization of Farnesyl Diphosphate Synthase Gene Involved in Triterpenoids Biosynthesis from Poria cocos

    Directory of Open Access Journals (Sweden)

    Jianrong Wang

    2014-12-01

    Full Text Available Poria cocos (P. cocos has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%. The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP from geranyl diphosphate (GPP and isopentenyl diphosphate (IPP. Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  10. A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases

    Science.gov (United States)

    DeGraw, Amanda J.; Zhao, Zongbao; Strickland, Corey L.; Taban, A. Huma; Hsieh, John; Michael, Jefferies; Xie, Wenshuang; Shintani, David; McMahan, Colleen; Cornish, Katrina; Distefano, Mark D.

    2008-01-01

    A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and prenyltransferases. While these initial analogues proved to be excellent structural analogues with good cross linking capability, they lack the stability needed when the goals include isolation of cross-linked species, tryptic digestion, and subsequent peptide sequencing. Here, the synthesis of a benzophenone-based farnesyl diphosphate analogue containing a stable phosphonophosphate group is described. Inhibition kinetics, photolabeling experiments, as well as x-ray crystallographic analysis with a protein prenyltransferase are described, verifying this compound as a good isoprenoid mimetic. In addition, the utility of this new analogue was explored by using it to photoaffinity label crude protein extracts obtained from Hevea brasiliensis latex. Those experiments suggest that a small protein, Rubber Elongation Factor, interacts directly with farnesyl diphosphate during rubber biosynthesis. These results indicate that this benzophenone-based isoprenoid analogue will be useful for identifying enzymes that utilize farnesyl diphosphate as a substrate. PMID:17477573

  11. Adaptive evolution of the chrysanthemyl diphosphate synthase gene involved in irregular monoterpene metabolism

    Directory of Open Access Journals (Sweden)

    Liu Ping-Li

    2012-11-01

    Full Text Available Abstract Background Chrysanthemyl diphosphate synthase (CDS is a key enzyme in biosynthetic pathways producing pyrethrins and irregular monoterpenes. These compounds are confined to plants of the tribe Anthemideae of the Asteraceae, and play an important role in defending the plants against herbivorous insects. It has been proposed that the CDS genes arose from duplication of the farnesyl diphosphate synthase (FDS gene and have different function from FDSs. However, the duplication time toward the origin of CDS and the evolutionary force behind the functional divergence of the CDS gene are still unknown. Results Two duplication events were detected in the evolutionary history of the FDS gene family in the Asteraceae, and the second duplication led to the origin of CDS. CDS occurred after the divergence of the tribe Mutisieae from other tribes of Asteraceae but before the birth of the Anthemideae tribe. After its origin, CDS accumulated four mutations in sites homologous to the substrate-binding and catalysis sites of FDS. Of these, two sites were involved in the binding of the nucleophilic substrate isopentenyl diphosphate in FDS. Maximum likelihood analyses showed that some sites in CDS were under positive selection and were scattered throughout primary sequences, whereas in the three-dimensional structure model they clustered in the large central cavity. Conclusion Positive selection associated with gene duplication played a major role in the evolution of CDS.

  12. Cloning and characterization of farnesyl diphosphate synthase gene involved in triterpenoids biosynthesis from Poria cocos.

    Science.gov (United States)

    Wang, Jianrong; Li, Yangyuan; Liu, Danni

    2014-12-02

    Poria cocos (P. cocos) has long been used as traditional Chinese medicine and triterpenoids are the most important pharmacologically active constituents of this fungus. Farnesyl pyrophosphate synthase (FPS) is a key enzyme of triterpenoids biosynthesis. The gene encoding FPS was cloned from P. cocos by degenerate PCR, inverse PCR and cassette PCR. The open reading frame of the gene is 1086 bp in length, corresponding to a predicted polypeptide of 361 amino acid residues with a molecular weight of 41.2 kDa. Comparison of the P. cocos FPS deduced amino acid sequence with other species showed the highest identity with Ganoderma lucidum (74%). The predicted P. cocos FPS shares at least four conserved regions involved in the enzymatic activity with the FPSs of varied species. The recombinant protein was expressed in Pichia pastoris and purified. Gas chromatography analysis showed that the recombinant FPS could catalyze the formation of farnesyl diphosphate (FPP) from geranyl diphosphate (GPP) and isopentenyl diphosphate (IPP). Furthermore, the expression profile of the FPS gene and content of total triterpenoids under different stages of development and methyl jasmonate treatments were determined. The results indicated that there is a positive correlation between the activity of FPS and the amount of total triterpenoids produced in P. cocos.

  13. The ribose and glycine Maillard reaction in the interstellar medium (ISM): A theoretical study

    Indian Academy of Sciences (India)

    Abraham F Jalbout; Md Abul Haider Shipar

    2008-05-01

    Possibility of the Maillard reaction to take place in the gaseous phase in the interstellar medium was investigated by using Density Functional Theory (DFT) computations. Cyclic ribose (c-Rib)/open-chain ribose (c-Rib) and glycine were taken as the model. Mechanisms have been proposed, and possibility of the formation of different compounds have been evaluated through calculating the Gibb’s free energy changes for different steps of the reaction by following the total mass balance. The result reveals that both c-Rib and Rib can participate in the reaction, and c-Rib is more efficient than Rib. The reactions under basic and neutral conditions are supposed to be the first and second most favourable. Acidic conditions and the isoelectric point of glycine were unfeasible for the reaction. The kinetics of the mechanics are briefly addressed in this work.

  14. 13C-labeled D-ribose: chemi-enzymic synthesis of various isotopomers.

    Science.gov (United States)

    Serianni, A S; Bondo, P B

    1994-04-01

    Current interest in the use of heteronuclear multidimensional NMR methods to assess the structures, conformations and/or dynamics of oligonucleotides in solution has created an immediate need for nucleosides and their derivatives labeled in various ways with stable isotopes (13C, 2H, 15N and/or 17,18O). This short review focuses exclusively on chemienzymic methods to introduce one or more 13C labels into D-ribose, a precursor to ribo- and 2'-deoxyribonucleosides. It will be demonstrated that five convenient reactions, applied in specific sequences, provide access to 26 of the 32 13C-labeled isotopomers of D-ribose in acceptable yields. While not explicitly discussed herein, these same reactions, appropriately modified, can also be used to insert one or more 2H and/or 17,18O isotopes into this aldopentose.

  15. Metabolic flexibility of d-ribose producer strain of Bacillus pumilus under environmental perturbations

    DEFF Research Database (Denmark)

    Srivastava, Rajesh K.; Maiti, Soumen K.; Das, Debasish;

    2012-01-01

    The metabolic reaction rate vector is a bridge that links gene and protein expression alterations to the phenotypic endpoint. We present a simple approach for the estimation of flux distribution at key branch points in the metabolic network by using substrate uptake, metabolite secretion rate......, and biomass growth rate for transketolase (tkt) deficient Bacillus pumilus ATCC 21951. We find that the glucose-6-phosphate (G6P) and pseudo catabolic/anabolic branch points are flexible in the d-ribose-producing tkt deficient strain of B. pumilus. The normalized flux through the pentose phosphate pathway...... (PPP) varied from 1.5 to 86 % under different growth conditions, thereby enabling substantial extracellular accumulation of d-ribose under certain conditions. Interestingly, the flux through PPP was affected by the extracellular phosphate concentration and dissolved oxygen concentration. This metabolic...

  16. Structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC118.

    Science.gov (United States)

    Lobley, Carina M C; Aller, Pierre; Douangamath, Alice; Reddivari, Yamini; Bumann, Mario; Bird, Louise E; Nettleship, Joanne E; Brandao-Neto, Jose; Owens, Raymond J; O'Toole, Paul W; Walsh, Martin A

    2012-12-01

    The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Å resolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and β D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.

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

    Science.gov (United States)

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

    2015-02-24

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

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

    Science.gov (United States)

    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

  19. Endogenous adenosine curtails lipopolysaccharide-stimulated tumour necrosis factor synthesis

    NARCIS (Netherlands)

    Eigler, A; Greten, T F; Sinha, B; Haslberger, C; Sullivan, G W; Endres, S

    1997-01-01

    Recent studies have demonstrated the inhibitory effect of exogenous adenosine on TNF production. During inflammation endogenous adenosine levels are elevated and may be one of several anti-inflammatory mediators that reduce TNF synthesis. In the present study the authors investigated this role of ad

  20. Adenosine receptor modulation: potential implications in veterinary medicine.

    Science.gov (United States)

    Dip, Ramiro G

    2009-01-01

    Adenosine is a purine nucleoside whose concentration increases during inflammation and hypoxia and the many roles of this molecule are becoming better understood. Increased reactivity to adenosine of the airways of asthmatic but not of normal subjects underlines the role of adenosine in airway inflammation. The identification and pharmacological characterisation of different adenosine receptors have stimulated the search for subtype-specific ligands able to modulate the effects of this molecule in a directed way. Several compounds of different chemical classes have been identified as having potential drawbacks, including side effects resulting from the broad distribution of the receptors across the organism, have prevented clinical application. In this article, the effects of adenosine's different receptors and the intracellular signalling pathways are reviewed. The potential of adenosine receptor modulation as a therapeutic target for chronic airway inflammation is considered, taking equine recurrent airway disease and feline asthma as examples of naturally occurring airway obstructive diseases. Other potential applications for adenosine receptor modulation are also discussed. As the intrinsic molecular events of adenosine's mechanism of action become uncovered, new concrete therapeutic approaches will become available for the treatment of various conditions in veterinary medicine.

  1. Extending the Clinical Phenotype of Adenosine Deaminase 2 Deficiency.

    Science.gov (United States)

    Ben-Ami, Tal; Revel-Vilk, Shoshana; Brooks, Rebecca; Shaag, Avraham; Hershfield, Michael S; Kelly, Susan J; Ganson, Nancy J; Kfir-Erenfeld, Shlomit; Weintraub, Michael; Elpeleg, Orly; Berkun, Yackov; Stepensky, Polina

    2016-10-01

    Adenosine deaminase 2 deficiency is an autoinflammatory disease, characterized by various forms of vasculitis. We describe 5 patients with adenosine deaminase 2 deficiency with various hematologic manifestations, including pure red cell aplasia, with no evidence for vasculitis. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Adenosine Receptors: Expression, Function and Regulation

    Directory of Open Access Journals (Sweden)

    Sandeep Sheth

    2014-01-01

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

  3. Poly(ADP-ribosylation) at successive stages of rooster spermatogenesis. Levels of polymeric ADP-ribose in vivo and poly(ADP-ribose) polymerase activity and turnover of ADP-ribosyl residues in vitro.

    Science.gov (United States)

    Corominas, M; Mezquita, C

    1985-12-25

    Rooster testis cells were separated by sedimentation at unit gravity and the in vivo levels of polymeric ADP-ribose were determined both in intact cells and isolated nuclei by fluorescence methods. Poly(ADP-ribose) polymerase activity was assayed after cell permeabilization or after isolation of nuclei. The turnover of ADP-ribosyl residues was determined in isolated nuclei using benzamide. The content of poly(ADP-ribose), the poly(ADP-ribose) polymerase activity, and the turnover of ADP-ribosyl residues, decreased during the differentiation of the germinal cell line, especially at the end of spermiogenesis. Treatment of cells with 1 mM dimethyl sulfate for 1 h resulted in a marked stimulation of poly(ADP-ribose) polymerase activity in meiotic and premeiotic cells and also in round and late spermatids. The enzymatic activity was not detected and could not be induced in mature spermatozoa. These cells, however, still contained polymeric ADP-ribose with a 2% of branched form.

  4. Cloning and expression of cDNA for human poly(ADP-ribose)polymerase

    Energy Technology Data Exchange (ETDEWEB)

    Alkhatib, H.M.; Chen, D.; Cherney, B.; Bhatia, K.; Notario, V.; Giri, C.; Stein, G.; Slattery, E.; Roeder, R.G.; Smulson, M.E.

    1987-03-01

    cDNAs encoding poly(ADP-ribose) polymerase from a human hepatoma lambdagt11 cDNA library were isolated by immunological screening. One insert of 1.3 kilobases (kb) consistently hybridized on RNA gel blots to an mRNA species of 3.6-3.7 kb, which is consistent with the size of RNA necessary to code for the polymerase protein (116 kDa). This insert was subsequently used in both in vitro hybrid selection and hybrid-arrested translation studies. An mRNA species from HeLa cells of 3.6-3.7 kb was selected that was translated into a 116-kDa protein, which was selectively immunoprecipitated with anti-poly(ADP-ribose) polymerase. To confirm that the 1.3-kb insert from lambdagt11 encodes for poly(ADP-ribose) polymerase, the insert was used to screen a 3- to 4-kb subset of a transformed human fibroblast cDNA library in the Okayama-Berg vector. One of these vectors was tested in transient transfection experiments in COS cells. This cDNA insert contained the complete coding sequence for polymerase. Using pcD-p(ADPR)P as probe, it was observed that the level of poly(ADP-ribose) polymerase mRNA was elevated at 5 and 7 hr of S phase of the HeLa cell cycle, but was unaltered when artificial DNA strand breaks are introduced in HeLa cells by alkylating agents.

  5. A New Farnesyl Diphosphate Synthase Gene from Taxus media Rehder: Cloning, Characterization and Functional Complementation

    Institute of Scientific and Technical Information of China (English)

    Zhi-Hua Liao; Min Chen; Yi-Fu Gong; Zhu-Gang Li; Kai-Jing Zuo; Peng Wang; Feng Tan; Xiao-Fen Sun; Ke-Xuan Tang

    2006-01-01

    Farnesyl diphosphate synthase (FPS; EC 2.5.1.10) catalyzes the production of 15-carbon farnesyl diphosphate which is a branch-point intermediate for many terpenoids. This reaction is considered to be a ratelimiting step in terpenoid biosynthesis. Here we report for the first time the cloning of a new full-length cDNA encoding farnesyl diphosphate synthase from a gymnosperm plant species, Taxus media Rehder,designated as TmFPS1. The full-length cDNA of TmFPS1 (GenBank accession number: AY461811) was 1 464bp with a 1 056-bp open reading frame encoding a 351-amino acid polypeptide with a calculated molecular weight of 40.3 kDa and a theoretical pl of 5.07. Bioinformatic analysis revealed that TmFPS1 contained all five conserved domains of prenyltransferases, and showed homology to other FPSs of plant origin. Phylogenetic analysis showed that farnesyl diphosphate synthases can be divided into two groups: one of prokaryotic origin and the other of eukaryotic origin. TmFPS1 was grouped with FPSs of plant origin. Homologybased structural modeling showed that TmFPS1 had the typical spatial structure of FPS, whose most prominent structural feature is the arrangement of 13 core helices around a large central cavity in which the catalytic reaction takes place. Our bioinformatic analysis strongly suggests that TmFPS1 is a functional gene. Southern blot analysis revealed that TmFPS1 belongs to a small FPS gene family in T. media. Northern blot analysis indicated that TmFPS1 is expressed in all tested tissues, including the needles, stems and roots of T. media. Subsequently, functional complementation with TmFPS1 in a FPS-deficient mutant yeast demonstrated that TmFPS1 did encode farnesyl diphosphate synthase, which rescued the yeast mutant.This study will be helpful in future investigations aiming at understanding the detailed role of FPS in terpenoid biosynthesis flux control at the molecular genetic level.

  6. ADP-ribose polymer - a novel and general biomarker of human cancers of head & neck, breast, and cervix

    OpenAIRE

    Sharan Rajeshwar N; Kataki Amal C; Lakadong Rennie O

    2010-01-01

    Abstract Background Poly-ADP-ribosylation, a reversible post-translational modification of primarily chromosomal proteins, is involved in various cellular and molecular processes including carcinogenesis. ADP-ribose polymer or poly-ADP-ribose adducts are enzymatically added onto or stripped off the target chromosomal proteins during this metabolic process. Due to this, the chromatin superstructure is reversibly altered, which significantly influences the pattern of gene expression. We hypothe...

  7. Does inhibition of poly(ADP-ribose) polymerase prevent energy overconsumption under microgravity?

    Science.gov (United States)

    Dobrota, C.; Piso, M. I.; Keul, A.

    When plants are exposed to a stress signal they expend a lot of energy and exhibit enhanced respiration rates This is partially due to a breakdown in the NAD pool caused by the enhanced activity PARP which uses NAD as a substrate to synthesize polymers of ADP-ribose Stress-induced depletion of NAD results in a similar depletion of energy since ATP molecules are required to resynthesize the depleted NAD It seems that plants with lowered poly ADP ribosyl ation activity appear tolerant to multiple stresses Inhibiting PARP activity prevents energy overconsumption under stress allowing normal mitochondrial respiration We intend to study if the microgravity is perceived by plants as a stress factor and if experimental inhibition of poly ADP-ribose polymerase may improve the energetic level of the cells References DeBlock M Verduyn C De Brouwer D and Cornelissen M 2005 Poly ADP-ribose polymerase in plants affects energy homeostasis cell death and stress tolerance The Plant Journal 41 95--106 Huang S Greenway H Colmerm T D and Millar A H 2005 Protein synthesis by rice coleoptiles during prolonged anoxia Implications for glycolysis growth and energy utilization Annals of Botany 96 703--715 Mittler R Vanderauwera S Gollery M and Van Breusegem F 2005 Reactive oxygen gene network of plants Trends in Plant Science 9 10 490-498

  8. Crystal structures and enzyme mechanisms of a dual fucose mutarotase/ribose pyranase.

    Science.gov (United States)

    Lee, Kwang-Hoon; Ryu, Kyoung-Seok; Kim, Min-Sung; Suh, Hye-Young; Ku, Bonsu; Song, Young-Lan; Ko, Sunggeon; Lee, Weontae; Oh, Byung-Ha

    2009-08-07

    Escherichia coli FucU (Fucose Unknown) is a dual fucose mutarotase and ribose pyranase, which shares 44% sequence identity with its human counterpart. Herein, we report the structures of E. coli FucU and mouse FucU bound to L-fucose and delineate the catalytic mechanisms underlying the interconversion between stereoisomers of fucose and ribose. E. coli FucU forms a decameric toroid with each active site formed by two adjacent subunits. While one subunit provides most of the fucose-interacting residues including a catalytic tyrosine residue, the other subunit provides a catalytic His-Asp dyad. This active-site feature is critical not only for the mutarotase activity toward L-fucose but also for the pyranase activity toward D-ribose. Structural and biochemical analyses pointed that mouse FucU assembles into four different oligomeric forms, among which the smallest homodimeric form is most abundant and would be the predominant species under physiological conditions. This homodimer has two fucose-binding sites that are devoid of the His-Asp dyad and catalytically inactive, indicating that the mutarotase and the pyranase activities appear dispensable in vertebrates. The defective assembly of the mouse FucU homodimer into the decameric form is due to an insertion of two residues at the N-terminal extreme, which is a common aspect of all the known vertebrate FucU proteins. Therefore, vertebrate FucU appears to serve for as yet unknown function through the quaternary structural alteration.

  9. Poly (ADP-ribose polymerase 1 is required for protein localization to Cajal body.

    Directory of Open Access Journals (Sweden)

    Elena Kotova

    2009-02-01

    Full Text Available Recently, the nuclear protein known as Poly (ADP-ribose Polymerase1 (PARP1 was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose, PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.

  10. The role of poly(ADP-ribose) in the DNA damage signaling network.

    Science.gov (United States)

    Malanga, Maria; Althaus, Felix R

    2005-06-01

    DNA damage signaling is crucial for the maintenance of genome integrity. In higher eukaryotes a NAD+-dependent signal transduction mechanism has evolved to protect cells against the genome destabilizing effects of DNA strand breaks. The mechanism involves 2 nuclear enzymes that sense DNA strand breaks, poly(ADP-ribose) polymerase-1 and -2 (PARP-1 and PARP-2). When activated by DNA breaks, these PARPs use NAD+ to catalyze their automodification with negatively charged, long and branched ADP-ribose polymers. Through recruitment of specific proteins at the site of damage and regulation of their activities, these polymers may either directly participate in the repair process or coordinate repair through chromatin unfolding, cell cycle progression, and cell survival-cell death pathways. A number of proteins, including histones, DNA topoisomerases, DNA methyltransferase-1 as well as DNA damage repair and checkpoint proteins (p23, p21, DNA-PK, NF-kB, XRCC1, and others) can be targeted in this manner; the interaction involves a specific poly(ADP-ribose)-binding sequence motif of 20-26 amino acids in the target domains.

  11. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    Energy Technology Data Exchange (ETDEWEB)

    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.

  12. 2'-O-ribose methylation of cap2 in human: function and evolution in a horizontally mobile family.

    Science.gov (United States)

    Werner, Maria; Purta, Elzbieta; Kaminska, Katarzyna H; Cymerman, Iwona A; Campbell, David A; Mittra, Bidyottam; Zamudio, Jesse R; Sturm, Nancy R; Jaworski, Jacek; Bujnicki, Janusz M

    2011-06-01

    The 5' cap of human messenger RNA consists of an inverted 7-methylguanosine linked to the first transcribed nucleotide by a unique 5'-5' triphosphate bond followed by 2'-O-ribose methylation of the first and often the second transcribed nucleotides, likely serving to modify efficiency of transcript processing, translation and stability. We report the validation of a human enzyme that methylates the ribose of the second transcribed nucleotide encoded by FTSJD1, henceforth renamed HMTR2 to reflect function. Purified recombinant hMTr2 protein transfers a methyl group from S-adenosylmethionine to the 2'-O-ribose of the second nucleotide of messenger RNA and small nuclear RNA. Neither N(7) methylation of the guanosine cap nor 2'-O-ribose methylation of the first transcribed nucleotide are required for hMTr2, but the presence of cap1 methylation increases hMTr2 activity. The hMTr2 protein is distributed throughout the nucleus and cytosol, in contrast to the nuclear hMTr1. The details of how and why specific transcripts undergo modification with these ribose methylations remains to be elucidated. The 2'-O-ribose RNA cap methyltransferases are present in varying combinations in most eukaryotic and many viral genomes. With the capping enzymes in hand their biological purpose can be ascertained.

  13. Occurrence and stability of lone pair–π stacking interactions between ribose and nucleobases in functional RNAs

    KAUST Repository

    Chawla, Mohit

    2017-08-18

    The specific folding pattern and function of RNA molecules lies in various weak interactions, in addition to the strong base-base pairing and stacking. One of these relatively weak interactions, characterized by the stacking of the O4′ atom of a ribose on top of the heterocycle ring of a nucleobase, has been known to occur but has largely been ignored in the description of RNA structures. We identified 2015 ribose–base stacking interactions in a high-resolution set of non-redundant RNA crystal structures. They are widespread in structured RNA molecules and are located in structural motifs other than regular stems. Over 50% of them involve an adenine, as we found ribose-adenine contacts to be recurring elements in A-minor motifs. Fewer than 50% of the interactions involve a ribose and a base of neighboring residues, while approximately 30% of them involve a ribose and a nucleobase at least four residues apart. Some of them establish inter-domain or inter-molecular contacts and often implicate functionally relevant nucleotides. In vacuo ribose-nucleobase stacking interaction energies were calculated by quantum mechanics methods. Finally, we found that lone pair–π stacking interactions also occur between ribose and aromatic amino acids in RNA–protein complexes.

  14. Chrysanthemyl diphosphate synthase: Isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium

    OpenAIRE

    Rivera, Susan B.; Swedlund, Bradley D.; King, Gretchen J.; Bell, Russell N.; Hussey, Charles E.; Shattuck-Eidens, Donna M.; Wrobel, Wislawa M.; Peiser, Galen D.; Poulter, C. Dale

    2001-01-01

    Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1′-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPa...

  15. Fine-tuning of Smad protein function by poly(ADP-ribose polymerases and poly(ADP-ribose glycohydrolase during transforming growth factor β signaling.

    Directory of Open Access Journals (Sweden)

    Markus Dahl

    Full Text Available BACKGROUND: Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose polymerase 1 (PARP-1 negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose glycohydrolase (PARG can remove poly(ADP-ribose chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling. METHODS: A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference. RESULTS: TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7 and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1. CONCLUSION

  16. Structure of trihydrated rare-earth acid diphosphates LnHP 2O 7·3H 2O ( Ln=La, Er)

    Science.gov (United States)

    Ben Moussa, S.; Ventemillas, S.; Cabeza, A.; Gutierrez-Puebla, E.; Sanz, J.

    2004-06-01

    In trihydrated lanthanum acid-diphosphates LnHP 2O 7·3H 2O, prepared from acid LnCl 3 and Na 4P 2O 7 solutions (pH=1), two crystal forms were obtained. Layered structures of two representative members of this family have been determined by single-crystal X-ray diffraction (XRD) technique. In the case of orthorhombic LaHP 2O 7·3H 2O (type I), lanthanum cations are ninefold coordinated and diphosphate groups adopt a staggered (alternated) configuration. In the case of triclinic ErHP 2O 7·3H 2O (type II), erbium cations are eightfold coordinated and diphosphate groups adopt an eclipsed configuration. In agreement with Infrared (IR) spectroscopic data, a bended configuration for diphosphate groups has been deduced. In both structures, one-dimensional chains of edge-sharing rare-earth polyhedra are linked together by diphosphate groups to form the phosphate layers. In both diphosphates, PO 4 and HPO 4 environments have been identified by 31P MAS-NMR technique. In the two compounds, OH groups of HPO 4 tetrahedra point out of diphosphate planes interacting with adjacent layers. In La-diphosphate, the interaction between HPO 4 groups and water molecules of adjacent layers is favored; however, in Er-diphosphate, the interaction between phosphate acid groups of contiguous layers is produced. Based on structural information deduced, differences detected in IR and NMR spectra of two disphosphates are discussed.

  17. Specific partial reduction of geranylgeranyl diphosphate by an enzyme from the thermoacidophilic archaeon Sulfolobus acidocaldarius yields a reactive prenyl donor, not a dead-end product.

    Science.gov (United States)

    Sato, Sho; Murakami, Motomichi; Yoshimura, Tohru; Hemmi, Hisashi

    2008-06-01

    Geranylgeranyl reductase from Sulfolobus acidocaldarius was shown to catalyze the reduction of geranylgeranyl groups in the precursors of archaeal membrane lipids, generally reducing all four double bonds. However, when geranylgeranyl diphosphate was subjected to the reductase reaction, only three of the four double bonds were reduced. Mass spectrometry and acid hydrolysis indicated that the allylic double bond was preserved in the partially reduced product derived from geranylgeranyl diphosphate. Thus, the reaction product was shown to be phytyl diphosphate, which is a substrate for archaeal prenyltransferases, unlike the completely reduced compound phytanyl diphosphate.

  18. C2-alpha-lactylthiamin diphosphate is an intermediate on the pathway of thiamin diphosphate-dependent pyruvate decarboxylation. Evidence on enzymes and models.

    Science.gov (United States)

    Zhang, Sheng; Liu, Min; Yan, Yan; Zhang, Zhen; Jordan, Frank

    2004-12-24

    Thiamin diphosphate (ThDP)-dependent decarboxylations are usually assumed to proceed by a series of covalent intermediates, the first one being the C2-trimethylthiazolium adduct with pyruvate, C2-alpha-lactylthiamin diphosphate (LThDP). Herein is addressed whether such an intermediate is kinetically competent with the enzymatic turnover numbers. In model studies it is shown that the first-order rate constant for decarboxylation can indeed exceed 50 s(-1) in tetrahydrofuran as solvent, approximately 10(3) times faster than achieved in previous model systems. When racemic LThDP was exposed to the E91D yeast pyruvate decarboxylase variant, or to the E1 subunit of the pyruvate dehydrogenase complex (PDHc-E1) from Escherichia coli, it was partitioned between reversion to pyruvate and decarboxylation. Under steady-state conditions, the rate of these reactions is severely limited by the release of ThDP from the enzyme. Under pre-steady-state conditions, the rate constant for decarboxylation on exposure of LThDP to the E1 subunit of the pyruvate dehydrogenase complex was 0.4 s(-1), still more than a 100-fold slower than the turnover number. Because these experiments include binding, decarboxylation, and oxidation (for detection purposes), this is a lower limit on the rate constant for decarboxylation. The reasons for this slow reaction most likely include a slow conformational change of the free LThDP to the V conformation enforced by the enzyme. Between the results from model studies and those from the two enzymes, it is proposed that LThDP is indeed on the decarboxylation pathway of the two enzymes studied, and once LThDP is bound the protein needs to provide little assistance other than a low polarity environment.

  19. Vasoconstrictor and vasodilator effects of adenosine in the kidney

    DEFF Research Database (Denmark)

    Hansen, Pernille B; Schnermann, Jurgen

    2003-01-01

    Adenosine is an ATP breakdown product that in most vessels causes vasodilatation and that contributes to the metabolic control of organ perfusion, i.e., to the match between oxygen demand and oxygen delivery. In the renal vasculature, in contrast, adenosine can produce vasoconstriction, a respons...... activation from changes in vascular adenosine concentration, a characteristic that is ideally suited for the role of renal adenosine as a paracrine factor in the control of glomerular function.......Adenosine is an ATP breakdown product that in most vessels causes vasodilatation and that contributes to the metabolic control of organ perfusion, i.e., to the match between oxygen demand and oxygen delivery. In the renal vasculature, in contrast, adenosine can produce vasoconstriction, a response...... that has been suggested to be an organ-specific version of metabolic control designed to restrict organ perfusion when transport work increases. However, the vasoconstriction elicited by an intravenous infusion of adenosine is only short lasting, being replaced within 1-2 min by vasodilatation. It appears...

  20. Temporal variations of adenosine metabolism in human blood.

    Science.gov (United States)

    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.

  1. Increased Cortical Extracellular Adenosine Correlates with Seizure Termination

    Science.gov (United States)

    Van Gompel, Jamie J.; Bower, Mark R.; Worrell, Gregory A.; Stead, Matt; Chang, Su-Youne; Goerss, Stephan J.; Kim, Inyong; Bennet, Kevin E.; Meyer, Fredric B.; Marsh, W. Richard; Blaha, Charles D.; Lee, Kendall H.

    2014-01-01

    Objective Seizures are currently defined by their electrographic features. However, neuronal networks are intrinsically dependent upon neurotransmitters of which little is known regarding their peri-ictal dynamics. Evidence supports adenosine as having a prominent role in seizure termination, as its administration can terminate and reduce seizures in animal models. Further, microdialysis studies in humans suggest adenosine is elevated peri-ictally, but the relationship to the seizure is obscured by its temporal measurement limitations. Because electrochemical techniques can provide vastly superior temporal resolution, we test the hypothesis that extracellular adenosine concentrations rise during seizure termination in an animal model and humans using electrochemistry. Methods White farm swine (n=45) were used in an acute cortical model of epilepsy and 10 human epilepsy patients were studied during intraoperative electrocorticography (Ecog). Wireless Instantaneous Neurotransmitter Concentration Sensor (WINCS) based fast scan cyclic voltametry (FSCV) and fixed potential amperometry were obtained utilizing an adenosine specific triangular waveform or biosensors respectively. Results Simultaneous Ecog and electrochemistry demonstrated an average adenosine rise of 260% compared to baseline at 7.5 ± 16.9 seconds with amperometry (n=75 events) and 2.6 ± 11.2 seconds with FSCV (n=15 events) prior to electrographic seizure termination. In agreement with these animal data, adenosine elevation prior to seizure termination in a human patient utilizing FSCV was also seen. Significance Simultaneous Ecog and electrochemical recording supports the hypothesis that adenosine rises prior to seizure termination, suggesting that adenosine itself may be responsible for seizure termination. Future work using intraoperative WINCS based FSCV recording may help to elucidate the precise relationship between adenosine and seizure termination. PMID:24483230

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

    Science.gov (United States)

    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.

  3. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference

    Directory of Open Access Journals (Sweden)

    Xianan Zhang

    2015-11-01

    Full Text Available Isopentenyl diphosphate isomerase (IPI catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP and dimethylallyl diphosphate (DMAPP during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1 was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  4. Functional Analysis of the Isopentenyl Diphosphate Isomerase of Salvia miltiorrhiza via Color Complementation and RNA Interference.

    Science.gov (United States)

    Zhang, Xianan; Guan, Hongyu; Dai, Zhubo; Guo, Juan; Shen, Ye; Cui, Guanghong; Gao, Wei; Huang, Luqi

    2015-11-10

    Isopentenyl diphosphate isomerase (IPI) catalyzes the isomerization between the common terpene precursor substances isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) during the terpenoid biosynthesis process. In this study, tissue expression analysis revealed that the expression level of the Salvia miltiorrhiza IPI1 gene (SmIPI1) was higher in the leaves than in the roots and stems. Furthermore, color complementation and RNA interference methods were used to verify the function of the SmIPI1 gene from two aspects. A recombinant SmIPI1 plasmid was successfully constructed and transferred into engineered E. coli for validating the function of SmIPI1 through the color difference in comparison to the control group; the observed color difference indicated that SmIPI1 served in promoting the accumulation of lycopene. Transformant hairy root lines with RNA interference of SmIPI1 were successfully constructed mediated by Agrobacterium rhizogenes ACCC 10060. RNA interference hairy roots had a severe phenotype characterized by withering, deformity or even death. The mRNA expression level of SmIPI1 in the RSi3 root line was only 8.4% of that of the wild type. Furthermore the tanshinone content was too low to be detected in the RNA interference lines. These results suggest that SmIPI1 plays a critical role in terpenoid metabolic pathways. Addition of an exogenous SmIPI1 gene promoted metabolic flow toward the biosynthesis of carotenoids in E. coli, and SmIPI1 interference in S. miltiorrhiza hairy roots may cause interruption of the 2-C-methyl-D-erythritol-4-phosphate metabolic pathway.

  5. Purification, crystallization and preliminary structural analysis of nucleoside diphosphate kinase from Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Misra, Gauri [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Aggarwal, Anita [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Mittal, Sonia [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India); Singh, Yogendra [Institute of Genomics and Integrative Biology, Mall Road, Delhi 110 007 (India); Ramachandran, Ravishankar, E-mail: r-ravishankar@cdri.res.in [Molecular and Structural Biology Division, Central Drug Research Institute, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226 001 (India)

    2007-12-01

    Nucleoside diphosphate kinase from B. anthracis has been crystallized. Preliminary crystallographic analysis shows that there is one monomer in the asymmetric unit of the crystal. Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 µl 10 mg ml{sup −1} protein in 50 mM Tris–HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 µl reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å. The crystals are hexagonal in shape and belong to space group P6{sub 3}22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V{sub M}) of 2.1 Å{sup 3} Da{sup −1} and a solvent content of about 36.9%.

  6. Structural Basis for Nucleotide Binding and Reaction Catalysis in Mevalonate Diphosphate Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; McWhorter, William J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2012-09-17

    Mevalonate diphosphate decarboxylase (MDD) catalyzes the final step of the mevalonate pathway, the Mg{sup 2+}-ATP dependent decarboxylation of mevalonate 5-diphosphate (MVAPP), producing isopentenyl diphosphate (IPP). Synthesis of IPP, an isoprenoid precursor molecule that is a critical intermediate in peptidoglycan and polyisoprenoid biosynthesis, is essential in Gram-positive bacteria (e.g., Staphylococcus, Streptococcus, and Enterococcus spp.), and thus the enzymes of the mevalonate pathway are ideal antimicrobial targets. MDD belongs to the GHMP superfamily of metabolite kinases that have been extensively studied for the past 50 years, yet the crystallization of GHMP kinase ternary complexes has proven to be difficult. To further our understanding of the catalytic mechanism of GHMP kinases with the purpose of developing broad spectrum antimicrobial agents that target the substrate and nucleotide binding sites, we report the crystal structures of wild-type and mutant (S192A and D283A) ternary complexes of Staphylococcus epidermidis MDD. Comparison of apo, MVAPP-bound, and ternary complex wild-type MDD provides structural information about the mode of substrate binding and the catalytic mechanism. Structural characterization of ternary complexes of catalytically deficient MDD S192A and D283A (k{sub cat} decreased 10{sup 3}- and 10{sup 5}-fold, respectively) provides insight into MDD function. The carboxylate side chain of invariant Asp{sup 283} functions as a catalytic base and is essential for the proper orientation of the MVAPP C3-hydroxyl group within the active site funnel. Several MDD amino acids within the conserved phosphate binding loop ('P-loop') provide key interactions, stabilizing the nucleotide triphosphoryl moiety. The crystal structures presented here provide a useful foundation for structure-based drug design.

  7. Sugar-metal ion interactions: the complicated coordination structures of cesium ion with D-ribose and myo-inositol.

    Science.gov (United States)

    Hu, Haijian; Xue, Junhui; Wen, Xiaodong; Li, Weihong; Zhang, Chao; Yang, Limin; Xu, Yizhuang; Zhao, Guozhong; Bu, Xiaoxia; Liu, Kexin; Chen, Jia'er; Wu, Jinguang

    2013-11-18

    The novel cesium chloride-D-ribose complex (CsCl·C5H10O5; Cs-R) and cesium chloride-myo-inositol complex (CsCl·C6H12O6; Cs-I) have been synthesized and characterized using X-ray diffraction and FTIR, FIR, THz, and Raman spectroscopy. Cs(+) is eight-coordinated to three chloride ions, O1 and O2 from one D-ribose molecule, O1 from another D-ribose molecule, and O4 and O5 from the third D-ribose molecule in Cs-R. For one D-ribose molecule, the oxygen atom O1 in the ring is coordinated to two cesium ions as an oxygen bridge, O2 is cocoordinated with O1 to one of the two cesium ions, and O4 and O5 are coordinated to the third cesium ion, respectively. O3 does not coordinate to metal ions and only takes part in forming hydrogen bonds. One chloride ion is connected to three cesium ions. Thus, a complicated structure of Cs-D-ribose forms. For Cs-I, Cs(+) is 10-coordinated to three chloride ions, O1 and O2 from one myo-inositol molecule, O3 and O4 from another myo-inositol molecule, O5 and O6 from the third myo-inositol molecule, and O6 from the fourth myo-inositol molecule. One metal ion is connected to four ligands, and one myo-inositol is coordinated to four Cs(+) ions, which is also a complicated coordination structure. Crystal structure results, FTIR, FIR, THz, and Raman spectra provide detailed information on the structure and coordination of hydroxyl groups to metal ions in the cesium chloride-D-ribose and cesium chloride-myo-inositol complexes.

  8. Enzymatic production of fructose 1,6-diphosphate using crude cell extract of Bacillus stearothermophilus.

    Science.gov (United States)

    Widjaja, A; Yasuda, M; Ogino, H; Nakajima, H; Ishikawa, H

    1999-01-01

    The enzymatic production of fructose 1,6-diphosphate (FDP) from glucose was performed in a batch reactor and a semibatch reactor using the crude cell extract of Bacillus stearothermophilus which contains all four enzymes required for the synthesis. The experimental results of the yield and the time courses of FDP production obtained using various enzyme concentrations were in good agreement with the theoretical predictions calculated based on the differential equations including the rate equations of the four enzymes, which were determined using the purified enzymes of B. stearothermophilus.

  9. METOVITAN PREVENTS ACCUMULATION OF THIAMIN DIPHOSPHATE OXYGENIZED FORM IN RAT TISSUES UNDER IRRADIATION

    OpenAIRE

    Yu. M. Parkhomenko; G. V. Donchenko; L. I. Chehovskaya; S. P. Stepanenko; O. A. Mejenskaya; E. N. Gorban

    2015-01-01

    The aim of the research was to test the ability of the drug “Metovitan” to prevent the redox balance disturbance in the tissues and thiamine diphosphate irreversible oxidation upon exposure of ionizing radiation on the body. The rats were undergo to a single exposure of the X-ray therapeutic instrument RUM-17 to create a dose of 0.5, 1.0 and 5.0 Gy. Preparation “Metovitan” were administered at a dose of 25 mg per 1 kg body weight for 22–24 h before irradiation. Contents of thiamin...

  10. Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase.

    Science.gov (United States)

    Ospanov, R V; Kochetov, G A; Kurganov, B I

    2007-01-01

    The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP TK...ThDP TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.

  11. Chemoenzymatic Synthesis of 4-Fluoro-N-Acetylhexosamine Uridine Diphosphate Donors: Chain Terminators in Glycosaminoglycan Synthesis.

    Science.gov (United States)

    Schultz, Victor L; Zhang, Xing; Linkens, Kathryn; Rimel, Jenna; Green, Dixy E; DeAngelis, Paul L; Linhardt, Robert J

    2017-02-17

    Unnatural uridine diphosphate (UDP)-sugar donors, UDP-4-deoxy-4-fluoro-N-acetylglucosamine (4FGlcNAc) and UDP-4-deoxy-4-fluoro-N-acetylgalactosamine (4FGalNAc), were prepared using both chemical and chemoenzymatic syntheses relying on N-acetylglucosamine-1-phosphate uridylyltransferase (GlmU). The resulting unnatural UDP-sugar donors were then tested as substrates in glycosaminoglycan synthesis catalyzed by various synthases. UDP-4FGlcNAc was transferred onto an acceptor by Pastuerella multocida heparosan synthase 1 and subsequently served as a chain terminator.

  12. K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells.

    Science.gov (United States)

    Beech, D J; Zhang, H; Nakao, K; Bolton, T B

    1993-10-01

    1. Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein. 2. In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mM guanosine 5'-diphosphate (GDP) induced a slowly developing outward K-current at -37 mV that reached a maximum on average of 72 +/- 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mM GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mM, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mM but had almost no effect at 10 mM. 3. In 14% of inside-out patches exposed to 1 mM GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mM:130 mM K-gradient and the unitary conductance was 24 pS. 4. Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nM and the inhibition was fully reversible on wash-out. 5. IK(GDP) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward IK(GDP). 6. Inclusion of 1 mM ATP in the recording pipette solution reduced IK(GDP) and also attenuated its decline during long (25 min) recordings. 7. When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-D-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed. 8. These K channels appear similar to

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

    Science.gov (United States)

    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.

  14. Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

    Directory of Open Access Journals (Sweden)

    Leibly David J

    2011-10-01

    Full Text Available Abstract Background Ribose-5-phosphate isomerase is an enzyme that catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate. This family of enzymes naturally occurs in two distinct classes, RpiA and RpiB, which play an important role in the pentose phosphate pathway and nucleotide and co-factor biogenesis. Results Although RpiB occurs predominantly in bacteria, here we report crystal structures of a putative RpiB from the pathogenic fungus Coccidioides immitis. A 1.9 Å resolution apo structure was solved by combined molecular replacement and single wavelength anomalous dispersion (SAD phasing using a crystal soaked briefly in a solution containing a high concentration of iodide ions. RpiB from C. immitis contains modest sequence and high structural homology to other known RpiB structures. A 1.8 Å resolution phosphate-bound structure demonstrates phosphate recognition and charge stabilization by a single positively charged residue whereas other members of this family use up to five positively charged residues to contact the phosphate of ribose-5-phosphate. A 1.7 Å resolution structure was obtained in which the catalytic base of C. immitis RpiB, Cys76, appears to form a weakly covalent bond with the central carbon of malonic acid with a bond distance of 2.2 Å. This interaction may mimic that formed by the suicide inhibitor iodoacetic acid with RpiB. Conclusion The C. immitis RpiB contains the same fold and similar features as other members of this class of enzymes such as a highly reactive active site cysteine residue, but utilizes a divergent phosphate recognition strategy and may recognize a different substrate altogether.

  15. Amino acids allosterically regulate the thiamine diphosphate-dependent alpha-keto acid decarboxylase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Werther, Tobias; Spinka, Michael; Tittmann, Kai; Schütz, Anja; Golbik, Ralph; Mrestani-Klaus, Carmen; Hübner, Gerhard; König, Stephan

    2008-02-29

    The gene rv0853c from Mycobacterium tuberculosis strain H37Rv codes for a thiamine diphosphate-dependent alpha-keto acid decarboxylase (MtKDC), an enzyme involved in the amino acid degradation via the Ehrlich pathway. Steady state kinetic experiments were performed to determine the substrate specificity of MtKDC. The mycobacterial enzyme was found to convert a broad spectrum of branched-chain and aromatic alpha-keto acids. Stopped-flow kinetics showed that MtKDC is allosterically activated by alpha-keto acids. Even more, we demonstrate that also amino acids are potent activators of this thiamine diphosphate-dependent enzyme. Thus, metabolic flow through the Ehrlich pathway can be directly regulated at the decarboxylation step. The influence of amino acids on MtKDC catalysis was investigated, and implications for other thiamine diphosphate-dependent enzymes are discussed.

  16. The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis.

    Science.gov (United States)

    Thabet, Insaf; Guirimand, Grégory; Courdavault, Vincent; Papon, Nicolas; Godet, Stéphanie; Dutilleul, Christelle; Bouzid, Sadok; Giglioli-Guivarc'h, Nathalie; Clastre, Marc; Simkin, Andrew J

    2011-11-15

    Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

  17. Rosuvastatin increases extracellular adenosine formation in humans in vivo: a new perspective on cardiovascular protection.

    OpenAIRE

    Meijer, P; Oyen, W.J.G.; Dekker, D.; Broek, P.H.H. van den; Wouters, C.W.; Boerman, O.C.; Scheffer, G. J.; Smits, P; Rongen, G.A.P.J.M.

    2009-01-01

    OBJECTIVE: Statins may increase extracellular adenosine formation from adenosine monophosphate by enhancing ecto-5'-nucleotidase activity. This theory was tested in humans using dipyridamole-induced vasodilation as a read-out for local adenosine formation. Dipyridamole inhibits the transport of extracellular adenosine into the cytosol resulting in increased extracellular adenosine and subsequent vasodilation. In addition, we studied the effect of statin therapy in a forearm model of ischemia-...

  18. Adenosine Deaminase Activity in Diabetic and Obese Patients ...

    African Journals Online (AJOL)

    Journal of Health and Visual Sciences ... Abstract. Adenosine deaminase (ADA) commonly associated with severe combined ... The results (mean±) show that the mean values in the test groups were significantly higher than the controls ...

  19. Inhibition of uptake of adenosine into human blood platelets

    NARCIS (Netherlands)

    Lips, J.P.M.; Sixma, J.J.; Trieschnigg, A.C.

    1980-01-01

    Adenosine transport into human blood platelets is mediated by two independent systems with different affinities. Both systems transport only purine nucleosides and no pyrimidine nucleosides. In experiments with differently substituted purine nucleosides, purines and analogues, differences in carrier

  20. Mw Spectroscopy Coupled with Ultrafast UV Laser Vaporization: {RIBOSE} Found in the Gas Phase

    Science.gov (United States)

    Cocinero, Emilio J.; Ecija, Patricia; Basterretxea, Francisco J.; Fernandez, Jose A.; Castano, Fernando; Lesarri, Alberto; Grabow, Jens-Uwe

    2012-06-01

    Sugars are aldoses or ketoses with multiple hydroxy groups which have been elusive to spectroscopic studies. Here we report a rotational study of the aldopentose ribose. According to any standard textbook aldopentoses can exhibit either linear forms, cyclic five-membered (furanose) structures or six-membered (pyranose) rings, occurring either as α- or β- anomers depending on the orientation of the hydroxy group at C-1 (anomeric carbon). β-Furanose is predominant in ribonucleosides, RNA, ATP and other biochemically relevant derivatives, but is β-furanose the native form also of free ribose? Recent condensed-phase X-ray and older NMR studies delivered conflicting results. In order to solve this question we conducted a microwave study on D-ribose that, owing to ultrafast UV laser vaporization, has become the first C-5 sugar observed with rotational resolution. The spectrum revealed six conformations of free ribose, preferentially adopting β-pyranose chairs as well as higher-energy α-pyranose forms. The method also allowed for unambiguous distinction between different orientations of the hydroxy groups, which stabilize the structures by cooperative hydrogen-bond networks. No evidence was observed of the α-/β-furanoses or linear forms found in the biochemical derivatives. i) D. Šišak, L. B. McCusker, G. Zandomeneghi, B. H. Meier, D. Bläser, R. Boese, W. B. Schweizer, R. Gylmour and J. D. Dunitz Angew. Chem. Int. Ed. 49, 4503, 2010. ii) W. Saenger Angew. Chem. Int. Ed. 49, 6487, 2010. i) M. Rudrum, and D. F. Shaw, J. Chem. Soc. 52, 1965. ii) R. U. Lemieux and J. D. Stevens Can. J. Chem. 44, 249, 1966. iii) E. Breitmaier and U. Hollstein Org. Magn. Reson. 8, 573, 1976. E. J. Cocinero, A. Lesarri, P. Écija, F. J. Basterretxea, J. U. Grabow, J. A. Fernández and F. Castaño Angew. Chem. Int. Ed. in press: DOI: 10.1002/anie.201107973, 2012.

  1. Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle.

    Science.gov (United States)

    Chang, Y C; Soman, G; Graves, D J

    1986-09-30

    An enzymatic activity present in high-speed supernatant fluids of rat skeletal muscle was found that catalyzes the release of ADP-ribose from ADP-ribosylated-modified lysozyme. The nature of the product was proved by chromatographic studies and proton nuclear magnetic resonance spectroscopy. The enzyme activity is stimulated by Mg2+, dithioerythritol, and flouride. These results and those published earlier (Soman, G., Mickelson, J.R., Louis, C.F., and Graves, D.J. (1984) Biochem. Biophys. Res. Commun. 120, 973-980) show that ADP-ribosylation is a reversible process in skeletal muscle.

  2. Poly (ADP-ribose) polymerase inhibitor:an evolving paradigm in the treatment of prostate cancer

    Institute of Scientific and Technical Information of China (English)

    Jingsong Zhang

    2014-01-01

    Recent phase I studies have reported single-agent activities of poly (ADP-ribose) polymerase (PARP) inhibitor in sporadic and in BRCA-mutant prostate cancers. Two of the most common genetic alterations in prostate cancer, ETS gene rearrangement and loss of PTEN, have been linked to increased sensitivity to PARP inhibitor in preclinical models. Emerging evidence also suggests that PARP1 plays an important role in mediating the transcriptional activities of androgen receptor (AR) and ETS gene rearrangement. In this article, the preclinical work and early-phase clinical trials in developing PARP inhibitor-based therapy as a new treatment paradigm for metastatic prostate cancer are reviewed.

  3. Poly (ADP-ribose polymerase inhibitor: an evolving paradigm in the treatment of prostate cancer

    Directory of Open Access Journals (Sweden)

    Jingsong Zhang

    2014-06-01

    Full Text Available Recent phase I studies have reported single-agent activities of poly (ADP-ribose polymerase (PARP inhibitor in sporadic and in BRCA-mutant prostate cancers. Two of the most common genetic alterations in prostate cancer, ETS gene rearrangement and loss of PTEN, have been linked to increased sensitivity to PARP inhibitor in preclinical models. Emerging evidence also suggests that PARP1 plays an important role in mediating the transcriptional activities of androgen receptor (AR and ETS gene rearrangement. In this article, the preclinical work and early-phase clinical trials in developing PARP inhibitor-based therapy as a new treatment paradigm for metastatic prostate cancer are reviewed.

  4. An Ancient Fingerprint Indicates the Common Ancestry of Rossmann-Fold Enzymes Utilizing Different Ribose-Based Cofactors.

    Science.gov (United States)

    Laurino, Paola; Tóth-Petróczy, Ágnes; Meana-Pañeda, Rubén; Lin, Wei; Truhlar, Donald G; Tawfik, Dan S

    2016-03-01

    Nucleoside-based cofactors are presumed to have preceded proteins. The Rossmann fold is one of the most ancient and functionally diverse protein folds, and most Rossmann enzymes utilize nucleoside-based cofactors. We analyzed an omnipresent Rossmann ribose-binding interaction: a carboxylate side chain at the tip of the second β-strand (β2-Asp/Glu). We identified a canonical motif, defined by the β2-topology and unique geometry. The latter relates to the interaction being bidentate (both ribose hydroxyls interacting with the carboxylate oxygens), to the angle between the carboxylate and the ribose, and to the ribose's ring configuration. We found that this canonical motif exhibits hallmarks of divergence rather than convergence. It is uniquely found in Rossmann enzymes that use different cofactors, primarily SAM (S-adenosyl methionine), NAD (nicotinamide adenine dinucleotide), and FAD (flavin adenine dinucleotide). Ribose-carboxylate bidentate interactions in other folds are not only rare but also have a different topology and geometry. We further show that the canonical geometry is not dictated by a physical constraint--geometries found in noncanonical interactions have similar calculated bond energies. Overall, these data indicate the divergence of several major Rossmann-fold enzyme classes, with different cofactors and catalytic chemistries, from a common pre-LUCA (last universal common ancestor) ancestor that possessed the β2-Asp/Glu motif.

  5. ADP-ribose polymer - a novel and general biomarker of human cancers of head & neck, breast, and cervix

    Directory of Open Access Journals (Sweden)

    Sharan Rajeshwar N

    2010-10-01

    Full Text Available Abstract Background Poly-ADP-ribosylation, a reversible post-translational modification of primarily chromosomal proteins, is involved in various cellular and molecular processes including carcinogenesis. ADP-ribose polymer or poly-ADP-ribose adducts are enzymatically added onto or stripped off the target chromosomal proteins during this metabolic process. Due to this, the chromatin superstructure is reversibly altered, which significantly influences the pattern of gene expression. We hypothesize that a decrease in the concentration of total poly-ADP-ribose adducts of peripheral blood lymphocyte (PBL proteins strongly correlates with the incidence of human cancer. Results Using a novel immunoprobe assay, we show a statistically significant (P ≤ 0.001 reduction (~ 42 to 49% in the level of poly-ADP-ribose adducts of PBL proteins of patients with advanced cancers of head & neck (H & N region (comprising fourteen distinct cancers at different sites, breast and cervix in comparison to healthy controls. Conclusions These findings imply potential utility of the poly-ADP-ribose adducts of PBL proteins as a novel and general biomarker of human cancers with potentials of significant clinical and epidemiological applications.

  6. Adenosine Deaminase Deficiency – More Than Just an Immunodeficiency

    OpenAIRE

    Kathryn Victoria Whitmore; Hubert Bobby Gaspar

    2016-01-01

    Adenosine deaminase (ADA) deficiency is best known as a form of severe combined immunodeficiency (SCID) which results from mutations in the gene encoding adenosine deaminase. Affected patients present with clinical and immunological manifestations typical of a severe combined immunodeficiency. Therapies are currently available that can that target these immunological disturbances and treated patients show varying degrees of clinical improvement. However, there is now a growing body of evidenc...

  7. Low-dose adenosine stress echocardiography: Detection of myocardial viability

    Science.gov (United States)

    Djordjevic-Dikic, Ana; Ostojic, Miodrag; Beleslin, Branko; Nedeljkovic, Ivana; Stepanovic, Jelena; Stojkovic, Sinisa; Petrasinovic, Zorica; Nedeljkovic, Milan; Saponjski, Jovica; Giga, Vojislav

    2003-01-01

    Objective The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Background Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Methods Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals) echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of ≥ 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 ± 2 months) were available in 24 revascularized patients. Results Wall motion score index improved from rest 1.55 ± 0.30 to 1.33 ± 0.26 at low-dose adenosine (p < 0.001). Of the 257 segments with baseline dyssynergy, adenosine echocardiography identified 122 segments as positive for viability, and 135 as necrotic since no improvement of systolic thickening was observed. Follow-up wall motion score index was 1.31 ± 0.30 (p < 0.001 vs. rest). The sensitivity of adenosine echo test for identification of viable segments was 87%, while specificity was 95%, and diagnostic accuracy 90%. Positive and negative predictive values were 97% and 80%, respectively. Conclusion Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability. PMID:12812523

  8. The A3 adenosine receptor: history and perspectives.

    Science.gov (United States)

    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.

  9. Low-dose adenosine stress echocardiography: Detection of myocardial viability

    Directory of Open Access Journals (Sweden)

    Nedeljkovic Milan

    2003-06-01

    Full Text Available Abstract Objective The aim of this study was to evaluate the diagnostic potential of low-dose adenosine stress echocardiography in detection of myocardial viability. Background Vasodilation through low dose dipyridamole infusion may recruit contractile reserve by increasing coronary flow or by increasing levels of endogenous adenosine. Methods Forty-three patients with resting dyssynergy, due to previous myocardial infarction, underwent low-dose adenosine (80, 100, 110 mcg/kg/min in 3 minutes intervals echocardiography test. Gold standard for myocardial viability was improvement in systolic thickening of dyssinergic segments of ≥ 1 grade at follow-up. Coronary angiography was done in 41 pts. Twenty-seven patients were revascularized and 16 were medically treated. Echocardiographic follow up data (12 ± 2 months were available in 24 revascularized patients. Results Wall motion score index improved from rest 1.55 ± 0.30 to 1.33 ± 0.26 at low-dose adenosine (p Conclusion Low-dose adenosine stress echocardiography test has high diagnostic potential for detection of myocardial viability in the group of patients with left ventricle dysfunction due to previous myocardial infarction. Low dose adenosine stress echocardiography may be adequate alternative to low-dose dobutamine test for evaluation of myocardial viability.

  10. Cloning, expression and characterization of a nucleoside diphosphate kinase (NDPK) gene from tobacco

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Nucleoside diphosphate kinase (NDPK) is a housekeeping enzyme that maintains the intracellular levels of all (d)NTPs used in biosynthesis except ATP. Here we report that a full-length cDNA encoding nucleoside diphosphate kinase (NDPK) was cloned using yeast two-hybrid approach. A tobacco NDPK gene was obtained and designated as NtNDPK1 . NtNDPK1 is 704 bp in length and encodes a putative 16.2 kD protein of 148 amino acids. Phylogenic analysis showed that NtNDPK1 is highly homologous to other plant NDPK genes and identified as type Ⅰ (NDPK1). RNA-gel blot analysis showed that there was no significant difference of NtNDPK1 expression in roots, stems, leaves and buds. And expression of NtNDPK1 was induced by ABA and PEG and repressed by NaCl, but not significantly affected by Paraquat, wounding and low temperature (4℃) treatments, indicating that NtNDPK1 may play a certain role in response to abiotic stress. In vitro phosphorylation assay demonstrated that NtNDPK1 had autophosphorylation activity.

  11. Isolation and characterization of a copalyl diphosphate synthase gene promoter from Salvia miltiorrhiza

    Directory of Open Access Journals (Sweden)

    Piotr Szymczyk

    2016-09-01

    Full Text Available The promoter, 5' UTR, and 34-nt 5' fragments of protein encoding region of the Salvia miltiorrhiza copalyl diphosphate synthase gene were cloned and characterized. No tandem repeats, miRNA binding sites, or CpNpG islands were observed in the promoter, 5' UTR, or protein encoding fragments. The entire isolated promoter and 5' UTR is 2235 bp long and contains repetitions of many cis-active elements, recognized by homologous transcription factors, found in Arabidopsis thaliana and other plant species. A pyrimidine-rich fragment with only 6 non-pyrimidine bases was localized in the 33-nt stretch from nt 2185 to 2217 in the 5' UTR. The observed cis-active sequences are potential binding sites for trans-factors that could regulate spatio-temporal CPS gene expression in response to biotic and abiotic stress conditions. Obtained results are initially verified by in silico and co-expression studies based on A. thaliana microarray data. The quantitative RT-PCR analysis confirmed that the entire 2269-bp copalyl diphosphate synthase gene fragment has the promoter activity. Quantitative RT-PCR analysis was used to study changes in CPS promoter activity occurring in response to the application of four selected biotic and abiotic regulatory factors; auxin, gibberellin, salicylic acid, and high-salt concentration.

  12. A cesium copper vanadyl-diphosphate: Synthesis, crystal structure and physical properties

    Science.gov (United States)

    Shvanskaya, Larisa; Yakubovich, Olga; Bychkov, Andrey; Shcherbakov, Vasiliy; Golovanov, Alexey; Zvereva, Elena; Volkova, Olga; Vasiliev, Alexander

    2015-02-01

    A non-centrosymmetric orthorhombic diphosphate, Cs2Cu1+x(VO)2-x(P2O7)2 (x=0.1) with a=13.7364(2) Å, b=9.2666(2) Å, c=11.5678(2) Å, Z=4, has been isolated. Its 3D framework is built from Cu atoms in square pyramidal and square planar coordination, VO5 tetragonal pyramids and P2O7 diphosphate groups, sharing vertices. Large channels are fulfilled by cesium atoms. The ESR study reveals a similarity in behaviour of two paramagnetic (Cu and V) subsystems. The temperature dependences of the ESR linewidth and static magnetic susceptibility data present evidences for a cluster type magnetic ordering in the title compound at T*=22 K. The weakness of the relevant anomalies reflects presumably obvious Cu2+ ions and (VO)2+ units disorder in the system. It is supposed that the charge and geometry of the framework are controlled by the Cu2+/(VO)2+ ratio; its variation may lead to a design of new materials.

  13. Studies on the mammary tumor-inhibiting effects of diethylstilbestrol and its mono- and diphosphate.

    Science.gov (United States)

    Schneider, M R; von Angerer, E; Prekajac, J; Brade, W P

    1986-01-01

    Diethylstilbestrol (DES), diethylstilbestrol monophosphate (DES-MP) and diethylstilbestrol diphosphate (DES-DP) were tested for their estrogen receptor affinity, estrogenic potency and mammary tumor-inhibiting activity in vitro and in vivo. DES had a much higher receptor binding affinity than its mono- or diphosphate. All three compounds inhibited the growth of the hormone-dependent MCF-7 and hormone-independent MDA-MB 231 breast cancer line only at relatively high concentrations. The estrogenic potency in the immature mouse uterine weight test decreased in the order DES greater than DES-MP much greater than DES-DP. The hormone-dependent MXT mammary tumor of the mouse was inhibited by all three compounds at a dosage of 1.0 mg/kg per week. At a dose of 0.01 mg/kg, DES, DES-MP, and DES-DP stimulated the tumor growth. Thus, for the first time, a biphasic effect on tumor growth was demonstrated in intact mature animals. As the effects of all three compounds were similar in this assay, a cleavage of the phosphate groups is likely. A decrease in estrogenic potency concomitant with a retained antitumor effect of DES-MP and DES-DP compared to DES was not demonstrable in the mature mouse using the MXT assay, only in the uterotrophic test in the immature mouse.

  14. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase

    Energy Technology Data Exchange (ETDEWEB)

    Jeudy, Sandra [Information Génomique et Structurale, CNRS UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Coutard, Bruno [Architecture et Fonction des Macromolecules Biologiques, CNRS UMR 6098, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Lebrun, Régine [IBSM, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France); Abergel, Chantal, E-mail: chantal.abergel@igs.cnrs-mrs.fr [Information Génomique et Structurale, CNRS UPR 2589, 31 Chemin Joseph Aiguier, 13402 Marseille CEDEX 20 (France)

    2005-06-01

    A. polyphaga mimivirus, the largest known double-stranded DNA virus, is the first virus to exhibit a nucleoside diphosphate kinase gene. The expression and crystallization of the viral NDK are reported. The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004 ▶), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005 ▶), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P2{sub 1}3, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.

  15. The genes and enzymes involved in the biosynthesis of thiamin and thiamin diphosphate in yeasts.

    Science.gov (United States)

    Kowalska, Ewa; Kozik, Andrzej

    2008-01-01

    Thiamin (vitamin B1) is an essential molecule for all living organisms. Its major biologically active derivative is thiamin diphosphate, which serves as a cofactor for several enzymes involved in carbohydrate and amino acid metabolism. Important new functions for thiamin and its phosphate esters have recently been suggested, e.g. in gene expression regulation by influencing mRNA structure, in DNA repair after UV illumination, and in the protection of some organelles against reactive oxygen species. Unlike higher animals, which rely on nutritional thiamin intake, yeasts can synthesize thiamin de novo. The biosynthesis pathways include the separate synthesis of two precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate and 5-(2-hydroxyethyl)-4-methylthiazole phosphate, which are then condensed into thiamin monophosphate. Additionally, yeasts evolved salvage mechanisms to utilize thiamin and its dephosphorylated late precursors, 4-amino-5-hydroxymethyl-2-methylpyrimidine and 5-(2-hydroxyethyl)-4-methylthiazole, from the environment. The current state of knowledge on the discrete steps of thiamin biosynthesis in yeasts is far from satisfactory; many intermediates are postulated only by analogy to the much better understood biosynthesis process in bacteria. On the other hand, the genetic mechanisms regulating thiamin biosynthesis in yeasts are currently under extensive exploration. Only recently, the structures of some of the yeast enzymes involved in thiamin biosynthesis, such as thiamin diphosphokinase and thiazole synthase, were determined at the atomic resolution, and mechanistic proposals for the catalysis of particular biosynthetic steps started to emerge.

  16. Reversed-phase ion-pair high-performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues.

    Science.gov (United States)

    Kozlov, Vyacheslav V; Danilov, Leonid L

    2016-02-01

    A reversed-phase ion-pair high-performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion-pair reagent, and the dependence of the separation quality on pH of ion-pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. 1-(beta-D-Erythrofuranosyl)adenosine.

    Science.gov (United States)

    Kline, Paul C; Zhao, Hongqiu; Noll, Bruce C; Oliver, Allen G; Serianni, Anthony S

    2010-04-01

    The title compound, also known as beta-erythroadenosine, C(9)H(11)N(5)O(3), (I), a derivative of beta-adenosine, (II), that lacks the C5' exocyclic hydroxymethyl (-CH(2)OH) substituent, crystallizes from hot ethanol with two independent molecules having different conformations, denoted (IA) and (IB). In (IA), the furanose conformation is (O)T(1)-E(1) (C1'-exo, east), with pseudorotational parameters P and tau(m) of 114.4 and 42 degrees, respectively. In contrast, the P and tau(m) values are 170.1 and 46 degrees, respectively, in (IB), consistent with a (2)E-(2)T(3) (C2'-endo, south) conformation. The N-glycoside conformation is syn (+sc) in (IA) and anti (-ac) in (IB). The crystal structure, determined to a resolution of 2.0 A, of a cocrystal of (I) bound to the enzyme 5'-fluorodeoxyadenosine synthase from Streptomyces cattleya shows the furanose ring in a near-ideal (O)E (east) conformation (P = 90 degrees and tau(m) = 42 degrees) and the base in an anti (-ac) conformation.

  18. Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation.

    Science.gov (United States)

    Dölle, Christian; Niere, Marc; Lohndal, Emilia; Ziegler, Mathias

    2010-02-01

    Poly-ADP-ribose polymerases (PARPs) use NAD(+) as substrate to generate polymers of ADP-ribose. We targeted the catalytic domain of human PARP1 as molecular NAD(+) detector into cellular organelles. Immunochemical detection of polymers demonstrated distinct subcellular NAD(+) pools in mitochondria, peroxisomes and, surprisingly, in the endoplasmic reticulum and the Golgi complex. Polymers did not accumulate within the mitochondrial intermembrane space or the cytosol. We demonstrate the suitability of this compartment-specific NAD(+) and poly-ADP-ribose turnover to establish intra-organellar protein localization. For overexpressed proteins, genetically endowed with PARP activity, detection of polymers indicates segregation from the cytosol and consequently intra-organellar residence. In mitochondria, polymer build-up reveals matrix localization of the PARP fusion protein. Compared to presently used fusion tags for subcellular protein localization, these are substantial improvements in resolution. We thus established a novel molecular tool applicable for studies of subcellular NAD metabolism and protein localization.

  19. Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: insight into the receptor activation switch.

    Science.gov (United States)

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Kachler, Sonja; Falgner, Nico; Marucci, Gabriella; Thomas, Ajiroghene; Cristalli, Gloria; Volpini, Rosaria; Klotz, Karl-Norbert

    2014-01-15

    A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.

  20. Pcal_1127, a highly stable and efficient ribose-5-phosphate pyrophosphokinase from Pyrobaculum calidifontis.

    Science.gov (United States)

    Bibi, Tahira; Perveen, Sumera; Aziz, Iram; Bashir, Qamar; Rashid, Naeem; Imanaka, Tadayuki; Akhtar, Muhammad

    2016-11-01

    Analysis of the genome sequence of Pyrobaculum calidifontis revealed the presence of an open reading frame Pcal_1127 annotated as ribose-5-phosphate pyrophosphokinase. To examine the properties of Pcal_1127 the coding gene was cloned, expressed in Escherichia coli, and the purified gene product was characterized. Pcal_1127 exhibited higher activity when ATP was replaced by dATP as pyrophosphate donor. Phosphate and EDTA activated the enzyme activity and equivalent amount of activity was detected with ATP and dATP in their presence. Recombinant Pcal_1127 could utilize all the four nucleotides as pyrophosphate donors with a marked preference for ATP. Optimum temperature and pH for the enzyme activity were 55 °C and 10.5, respectively. A unique feature of Pcal_1127 was its stability against temperature as well as denaturants. Pcal_1127 exhibited more than 95 % residual activity after heating for 4 h at 90 °C and a half-life of 15 min in the boiling water. The enzyme activity was not affected by the presence of 8 M urea or 4 M guanidinium chloride. Pcal_1127 was a highly efficient enzyme with a catalytic efficiency of 5183 mM(-1) s(-1). These features make Pcal_1127, a novel and unique ribose-5-phosphate pyrophosphokinase.

  1.  Poly(ADP-ribose polymerase (PARP inhibitors in BRCA1/2 cancer therapy

    Directory of Open Access Journals (Sweden)

    Katarzyna Kluzek

    2012-06-01

    Full Text Available  A majority of currently used anticancer drugs belong to a group of chemical agents that damage DNA. The efficiency of the treatment is limited by effective DNA repair systems functioning in cancer cells. Many chemotherapeutic compounds cause strong systemic toxicity. Therefore, there is still a need for new anticancer agents which are less toxic for nontransformed cells and selectively kill cancer cells. One of the most promising molecular targets in cancer therapy is poly(ADP-ribose polymerases (PARP. PARP play an essential role in repairing DNA strand breaks. Small molecule inhibitors of these enzymes have been developed and have proved to be extremely toxic for cancer cells that lack the functional BRCA1 and BRCA2 proteins that are involved in homologous recombination, a complex repair mechanism of DNA double strand breaks. Mutations in BRCA1/2 genes are associated with genetically inherited breast and ovarian cancers. Therefore PARP inhibitors may prove to be very effective and selective in the treatment of these cancer types. This review is focused on the function of BRCA1/2 proteins and poly(ADP-ribose polymerases in DNA repair systems, especially in the homologous recombination process. A short history of the studies that led to synthesis of high specificity small molecule PARP inhibitors is also presented, as well as the results of clinical trials concerning the most effective PARP inhibitors in view of their potential application in oncological treatment, particularly breast cancers.

  2. FRET Response of a Modified Ribose Receptor Expressed in the Diatom Thalassiosira pseudonana

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Hanna

    2011-08-26

    The ability to insert complex proteins into silica has many applications including biosensing. Previous research has demonstrated how to direct proteins to the biosilica of diatoms [1]. Here, we show that a complex fusion protein that includes an enzyme, a bacterial ribose periplasmic binding protein, flanked by fluorescent proteins constituting a FRET pair can remain functional in the frustules of living diatoms. A Sil3 tag is attached to the N-terminal end to localize the fusion protein to frustules of the diatom Thalassiosira pseudonana. When ribose was applied, a larger decrease in FRET response was seen in transformed cells than in untransformed cells. Multiple forms of the expression vector were tested to find the optimal system; specifically, a one-vector system was compared to a two-vector system and the gDNA version of the Sil3 localization tag was compared to the cDNA version. The optimal system was found to be a one-vector system with the genomic version of the Sil3 tag to direct the protein to the frustules. Localization of the enzyme to the frustules was further confirmed through cell fluorescence imaging.

  3. Characterization of hMTr1, a human Cap1 2'-O-ribose methyltransferase.

    Science.gov (United States)

    Bélanger, François; Stepinski, Janusz; Darzynkiewicz, Edward; Pelletier, Jerry

    2010-10-22

    Cellular eukaryotic mRNAs are capped at their 5' ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2'-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2'-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo.

  4. Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death.

    Science.gov (United States)

    Blenn, Christian; Althaus, Felix R; Malanga, Maria

    2006-06-15

    PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.

  5. Poly(ADP-ribose)--a unique natural polymer structural features, biological role and approaches to the chemical synthesis.

    Science.gov (United States)

    Drenichev, Mikhail S; Mikhailov, Sergey N

    2015-01-01

    Poly(ADP-ribose) (PAR) is a natural polymer, taking part in numerous important cellular processes. Several enzymes are involved in biosynthesis and degradation of PAR. One of them, poly(ADP-ribose)polymerase-1 (PARP-1) is considered to be a perspective target for the design of new drugs, affecting PAR metabolism. The structure of PAR was established by enzymatic hydrolysis and further analysis of the products, but total chemical synthesis of PAR hasn't been described yet. Several approaches have been developed on the way to chemical synthesis of this unique biopolymer.

  6. Role of A3 adenosine receptor in diabetic neuropathy.

    Science.gov (United States)

    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.

  7. Differential and Concordant Roles for Poly(ADP-Ribose) Polymerase 1 and Poly(ADP-Ribose) in Regulating WRN and RECQL5 Activities.

    Science.gov (United States)

    Khadka, Prabhat; Hsu, Joseph K; Veith, Sebastian; Tadokoro, Takashi; Shamanna, Raghavendra A; Mangerich, Aswin; Croteau, Deborah L; Bohr, Vilhelm A

    2015-12-01

    Poly(ADP-ribose) (PAR) polymerase 1 (PARP1) catalyzes the poly(ADP-ribosyl)ation (PARylation) of proteins, a posttranslational modification which forms the nucleic acid-like polymer PAR. PARP1 and PAR are integral players in the early DNA damage response, since PARylation orchestrates the recruitment of repair proteins to sites of damage. Human RecQ helicases are DNA unwinding proteins that are critical responders to DNA damage, but how their recruitment and activities are regulated by PARPs and PAR is poorly understood. Here we report that all human RecQ helicases interact with PAR noncovalently. Furthermore, we define the effects that PARP1, PARylated PARP1, and PAR have on RECQL5 and WRN, using both in vitro and in vivo assays. We show that PARylation is involved in the recruitment of RECQL5 and WRN to laser-induced DNA damage and that RECQL5 and WRN have differential responses to PARylated PARP1 and PAR. Furthermore, we show that the loss of RECQL5 or WRN resulted in increased sensitivity to PARP inhibition. In conclusion, our results demonstrate that PARP1 and PAR actively, and in some instances differentially, regulate the activities and cellular localization of RECQL5 and WRN, suggesting that PARylation acts as a fine-tuning mechanism to coordinate their functions in time and space during the genotoxic stress response.

  8. A functional (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase exhibits diurnal regulation of expression in Stevia rebaudiana (Bertoni).

    Science.gov (United States)

    Kumar, Hitesh; Kumar, Sanjay

    2013-09-15

    The leaves of stevia [Stevia rebaudiana (Bertoni)] are a rich source of steviol glycosides that are used as non-calorific sweetener in many countries around the world. Steviol moiety of steviol glycosides is synthesized via plastidial 2C-methyl-D-erythritol 4-phosphate pathway, where (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) is the key enzyme. HDR catalyzes the simultaneous conversion of (E)-4-hydroxy-3-methylbut-2-enyl diphosphate into five carbon isoprenoid units, isopentenyl diphosphate and dimethylallyl diphosphate. Stevia HDR (SrHDR) successfully rescued HDR lethal mutant strain MG1655 araispH upon genetic complementation, suggesting SrHDR to encode a functional protein. The gene exhibited diurnal variation in expression. To identify the possible regulatory elements, upstream region of the gene was cloned and putative cis-acting elements were detected by in silico analysis. Electrophoretic mobility shift assay, using a putative light responsive element GATA showed the binding of nuclear proteins (NP) isolated from leaves during light period of the day, but not with the NP from leaves during the dark period. Data suggested the involvement of GATA box in light mediated gene regulation of SrHDR in stevia.

  9. Intracoronary adenosine improves myocardial perfusion in late reperfused myocardial infarction

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Background Myocardial perfusion associates with clinical syndromes and prognosis.Adenosine could improve myocardial perfusion of acute myocardial infarction within 6 hours,but few data are available on late perfusion of myocardial infarction (MI).This study aimed at quantitatively evaluating the value of intracoronary adenosine improving myocardial perfusion in late reperfused MI with myocardial contrast echocardiography(MCE).Methods Twenty-six patients with anterior wall infarcts were divided randomly into 2 groups:adenosine group(n=12) and normal saline group(n=14).Their history of myocardial infarction was about 3-12 weeks.Adenosine or normalsaline was given when the guiding wire crossed the lesion through percutaneous coronary intervention(PCI),then the balloon was dilated and stent(Cypher/Cypher select)was implanted at the lesion.Contrast pulse sequencing MCE with Sonovue contrast via the coronary route was done before PCI and 30 minutes after PCI.Video densitometry and contrast filled-blank area were calculated with the CUSQ off-line software.Heart function and cardiac events were followed up within 30 days.Results Perfusion in the segments of the criminal occlusive coronary artery in the adenosine group was better than that in the saline group(5.71±0.29 vs 4.95±1.22,P<0.05).Ischemic myocardial segment was deminished significantly afterPCI,but the meliorated area was bigger in the adenosine group than in the saline group((1.56±0.60)cm2 vs(1.02±0.56) cm2,P<0.05).The video densitometry in critical segments was also improved significantly in the adenosine group (5.53±0.36 vs 5.26±0.35,P<0.05).Left ventricular ejection fraction(LVEF)was improved in all patients after PCI,but EF was not significant between the two groups((67±6)% vs(62±7)%,P>0.05).There was no in-hospital or 30-day major adverse cardiac event(MACE)in the adenosine group but 3 MACE in the saline group in 30 days after PCI.Conclusions Adenosine could improve myocardial microvascular

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

    Science.gov (United States)

    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. 8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist

    Science.gov (United States)

    Kirchberger, Tanja; Moreau, Christelle; Wagner, Gerd K.; Fliegert, Ralf; Siebrands, Cornelia C.; Nebel, Merle; Schmid, Frederike; Harneit, Angelika; Odoardi, Francesca; Flügel, Alexander; Potter, Barry V. L.; Guse, Andreas H.

    2009-01-01

    cADPR (cyclic ADP-ribose) is a universal Ca2+ mobilizing second messenger. In T-cells cADPR is involved in sustained Ca2+ release and also in Ca2+ entry. Potential mechanisms for the latter include either capacitative Ca2+ entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N1-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N1-cIDPR evoked Ca2+ signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca2+ signalling induced by 8-Br-N1-cIDPR consisted of Ca2+ release and Ca2+ entry. Whereas Ca2+ release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca2+ entry was inhibited by the Ca2+ entry blockers Gd3+ (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca2+ entry evoked by 8-Br-N1-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H2O2 was enhanced dramatically in those cells, Ca2+ signalling induced by 8-Br-N1-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N1-cIDPR. In summary, the sensitivity to the Ca2+ entry blockers Gd3+ and SKF-96365 is in favour of the concept of capacitative Ca2+ entry, secondary to store depletion by 8-Br-N1-cIDPR. Taken together, 8-Br-N1-cIDPR appears to be the first cADPR agonist affecting Ca2+ release and secondary Ca2+ entry, but without effect on TRPM2. PMID:19492987

  12. Comparison of the acid-base properties of ribose and 2'-deoxyribose nucleotides.

    Science.gov (United States)

    Mucha, Ariel; Knobloch, Bernd; Jezowska-Bojczuk, Małgorzata; Kozłowski, Henryk; Sigel, Roland K O

    2008-01-01

    The extent to which the replacement of a ribose unit by a 2'-deoxyribose unit influences the acid-base properties of nucleotides has not hitherto been determined in detail. In this study, by potentiometric pH titrations in aqueous solution, we have measured the acidity constants of the 5'-di- and 5'-triphosphates of 2'-deoxyguanosine [i.e., of H(2)(dGDP)(-) and H(2)(dGTP)(2-)] as well as of the 5'-mono-, 5'-di-, and 5'-triphosphates of 2'-deoxyadenosine [i.e., of H(2)(dAMP)(+/-), H(2)(dADP)(-), and H(2)(dATP)(2-)]. These 12 acidity constants (of the 56 that are listed) are compared with those of the corresponding ribose derivatives (published data) measured under the same experimental conditions. The results show that all protonation sites in the 2'-deoxynucleotides are more basic than those in their ribose counterparts. The influence of the 2'-OH group is dependent on the number of 5'-phosphate groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is most significantly enhanced (by about 0.2 pK units), while the effect on the phosphate groups and the N1H or N1H(+) sites is less pronounced but clearly present. In addition, (1)H NMR chemical shift change studies in dependence on pD in D(2)O have been carried out for the dAMP, dADP, and dATP systems, which confirmed the results from the potentiometric pH titrations and showed the nucleotides to be in their anti conformations. Overall, our results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact basis for the calculation, determination, and understanding of perturbed pK(a) values in DNAzymes and ribozymes, as needed for the delineation of acid-base mechanisms in catalysis.

  13. Structure of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Entamoeba histolytica.

    Science.gov (United States)

    Edwards, Thomas E; Gardberg, Anna S; Phan, Isabelle Q H; Zhang, Yang; Staker, Bart L; Myler, Peter J; Lorimer, Donald D

    2015-05-01

    Uridine diphosphate N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the final step in the synthesis of UDP-GlcNAc, which is involved in cell-wall biogenesis in plants and fungi and in protein glycosylation. Small-molecule inhibitors have been developed against UAP from Trypanosoma brucei that target an allosteric pocket to provide selectivity over the human enzyme. A 1.8 Å resolution crystal structure was determined of UAP from Entamoeba histolytica, an anaerobic parasitic protozoan that causes amoebic dysentery. Although E. histolytica UAP exhibits the same three-domain global architecture as other UAPs, it appears to lack three α-helices at the N-terminus and contains two amino acids in the allosteric pocket that make it appear more like the enzyme from the human host than that from the other parasite T. brucei. Thus, allosteric inhibitors of T. brucei UAP are unlikely to target Entamoeba UAPs.

  14. Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

    Science.gov (United States)

    Dyda, F; Furey, W; Swaminathan, S; Sax, M; Farrenkopf, B; Jordan, F

    1990-10-15

    Single crystals of the thiamin diphosphate (the vitamin B1 coenzyme)-dependent enzyme pyruvate decarboxylase (EC 4.1.1.1) from brewers' yeast have been grown using polyethylene glycol as a precipitating agent. Crystals of the homotetrameric version alpha 4 of the holoenzyme are triclinic, space group P1, with cell constants a = 81.0, b = 82.4, c = 116.6 A, alpha = 69.5 beta = 72.6, gamma = 62.4 degrees. The crystals are reasonably stable in a rotating anode x-ray beam and diffract to at least 2.5 A resolution. The Vm value of 2.55 A/dalton is consistent with a unit cell containing four subunits with mass of approximately 60 kDa each. Rotation function results with native data indicate strong non-crystallographic 222 symmetry relating the four identical subunits, thus density averaging methods are likely to play a role in the structure determination.

  15. Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

    Science.gov (United States)

    Liu, Xiaoqing; Bisswanger, Hans

    2005-01-01

    Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.

  16. Acanthamoeba polyphaga mimivirus NDK: preliminary crystallographic analysis of the first viral nucleoside diphosphate kinase.

    Science.gov (United States)

    Jeudy, Sandra; Coutard, Bruno; Lebrun, Régine; Abergel, Chantal

    2005-06-01

    The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004), Science, 306, 1344-1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005), Acta Cryst. F61, 212-215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P2(1)3, with unit-cell parameter 99.425 A. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.

  17. Additional diterpenes from Physcomitrella patens synthesized by copalyl diphosphate/kaurene synthase (PpCPS/KS).

    Science.gov (United States)

    Zhan, Xin; Bach, Søren Spanner; Hansen, Nikolaj Lervad; Lunde, Christina; Simonsen, Henrik Toft

    2015-11-01

    The bifunctional diterpene synthase, copalyl diphosphate/kaurene synthase from the moss Physcomitrella patens (PpCPS/KS), catalyses the formation of at least four diterpenes, including ent-beyerene, ent-sandaracopimaradiene, ent-kaur-16-ene, and 16-hydroxy-ent-kaurene. The enzymatic activity has been confirmed through generation of a targeted PpCPS/KS knock-out mutant in P. patens via homologous recombination, through transient expression of PpCPS/KS in Nicotiana benthamiana, and expression of PpCPS/KS in E. coli. GC-MS analysis of the knock-out mutant shows that it lacks the diterpenoids, supporting that all are products of PpCPS/KS as observed in N. benthamiana and E. coli. These results provide additional knowledge of the mechanism of this bifunctional diterpene synthase, and are in line with proposed reaction mechanisms in kaurene biosynthesis.

  18. Crystal Structures of Staphylococcus epidermidis Mevalonate Diphosphate Decarboxylase Bound to Inhibitory Analogs Reveal New Insight into Substrate Binding and Catalysis

    Energy Technology Data Exchange (ETDEWEB)

    Barta, Michael L.; Skaff, D. Andrew; McWhorter, William J.; Herdendorf, Timothy J.; Miziorko, Henry M.; Geisbrecht, Brian V. (UMKC)

    2011-10-28

    The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 {angstrom} resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 {angstrom} resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 {angstrom} resolution). Comparison of these structures provides a physical basis for the significant differences in K{sub i} values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser{sup 192} as making potential contributions to catalysis. Significantly, Ser {yields} Ala substitution of this side chain decreases k{sub cat} by {approx}10{sup 3}-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 {angstrom} cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.

  19. Stathmin slows down guanosine diphosphate dissociation from tubulin in a phosphorylation-controlled fashion.

    Science.gov (United States)

    Amayed, P; Carlier, M F; Pantaloni, D

    2000-10-10

    Stathmin is an important protein that interacts with tubulin and regulates microtubule dynamics in a phosphorylation-controlled fashion. Here we show that the dissociation of guanosine 5'-diphosphate (GDP) from beta-tubulin is slowed 20-fold in the (tubulin)(2)-stathmin ternary complex (T(2)S). The kinetics of GDP or guanosine 5'-triphosphate (GTP) dissociation from tubulin have been monitored by the change in tryptophan fluorescence of tubulin upon exchanging 2-amino-6-mercapto-9-beta-ribofuranosylpurine 5'-diphosphate (S6-GDP) for tubulin-bound guanine nucleotide. At molar ratios of stathmin to tubulin lower than 0.5, biphasic kinetics were observed, indicating that the dynamics of the complex is extremely slow, consistent with its high stability. The method was used to characterize the effects of phosphorylation of stathmin on its interaction with tubulin. The serine-to-glutamate substitution of all four phosphorylatable serines of stathmin (4E-stathmin) weakens the stability of the T(2)S complex by about 2 orders of magnitude. The phosphorylation of serines 16 and 63 in stathmin has a more severe effect and weakens the stability of T(2)S 10(4)-fold. The rate of GDP dissociation is lowered only 7-fold and 4-fold in the complexes of tubulin with 4E-stathmin and diphosphostathmin, respectively. Sedimentation velocity studies support the conclusions of nucleotide exchange data and show that the T(2)S complexes formed between tubulin and 4E-stathmin or diphosphostathmin are less compact than the highly stable T(2)S complex. The correlation between the effect of phosphorylation of stathmin on the stability of T(2)S complex measured in vitro and on the function of stathmin in vivo is discussed.

  20. An intersubunit disulfide bridge stabilizes the tetrameric nucleoside diphosphate kinase of Aquifex aeolicus.

    Science.gov (United States)

    Boissier, Fanny; Georgescauld, Florian; Moynié, Lucile; Dupuy, Jean-William; Sarger, Claude; Podar, Mircea; Lascu, Ioan; Giraud, Marie-France; Dautant, Alain

    2012-06-01

    The nucleoside diphosphate kinase (Ndk) catalyzes the reversible transfer of the γ-phosphate from nucleoside triphosphate to nucleoside diphosphate. Ndks form hexamers or two types of tetramers made of the same building block, namely, the common dimer. The secondary interfaces of the Type I tetramer found in Myxococcus xanthus Ndk and of the Type II found in Escherichia coli Ndk involve the opposite sides of subunits. Up to now, the few available structures of Ndk from thermophiles were hexameric. Here, we determined the X-ray structures of four crystal forms of the Ndk from the hyperthermophilic bacterium Aquifex aeolicus (Aa-Ndk). Aa-Ndk displays numerous features of thermostable proteins and is made of the common dimer but it is a tetramer of Type I. Indeed, the insertion of three residues in a surface-exposed spiral loop, named the Kpn-loop, leads to the formation of a two-turn α-helix that prevents both hexamer and Type II tetramer assembly. Moreover, the side chain of the cysteine at position 133, which is not present in other Ndk sequences, adopts two alternate conformations. Through the secondary interface, each one forms a disulfide bridge with the equivalent Cys133 from the neighboring subunit. This disulfide bridge was progressively broken during X-ray data collection by radiation damage. Such crosslinks counterbalance the weakness of the common-dimer interface. A 40% decrease of the kinase activity at 60°C after reduction and alkylation of the protein corroborates the structural relevance of the disulfide bridge on the tetramer assembly and enzymatic function.

  1. Improving monoterpene geraniol production through geranyl diphosphate synthesis regulation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Jianzhi; Bao, Xiaoming; Li, Chen; Shen, Yu; Hou, Jin

    2016-05-01

    Monoterpenes have wide applications in the food, cosmetics, and medicine industries and have recently received increased attention as advanced biofuels. However, compared with sesquiterpenes, monoterpene production is still lagging in Saccharomyces cerevisiae. In this study, geraniol, a valuable acyclic monoterpene alcohol, was synthesized in S. cerevisiae. We evaluated three geraniol synthases in S. cerevisiae, and the geraniol synthase Valeriana officinalis (tVoGES), which lacked a plastid-targeting peptide, yielded the highest geraniol production. To improve geraniol production, synthesis of the precursor geranyl diphosphate (GPP) was regulated by comparing three specific GPP synthase genes derived from different plants and the endogenous farnesyl diphosphate synthase gene variants ERG20 (G) (ERG20 (K197G) ) and ERG20 (WW) (ERG20 (F96W-N127W) ), and controlling endogenous ERG20 expression, coupled with increasing the expression of the mevalonate pathway by co-overexpressing IDI1, tHMG1, and UPC2-1. The results showed that overexpressing ERG20 (WW) and strengthening the mevalonate pathway significantly improved geraniol production, while expressing heterologous GPP synthase genes or down-regulating endogenous ERG20 expression did not show positive effect. In addition, we constructed an Erg20p(F96W-N127W)-tVoGES fusion protein, and geraniol production reached 66.2 mg/L after optimizing the amino acid linker and the order of the proteins. The best strain yielded 293 mg/L geraniol in a fed-batch cultivation, a sevenfold improvement over the highest titer previously reported in an engineered S. cerevisiae strain. Finally, we showed that the toxicity of geraniol limited its production. The platform developed here can be readily used to synthesize other monoterpenes.

  2. Mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound intermediates and kinetic isotope effects.

    Science.gov (United States)

    Chakraborty, Sumit; Nemeria, Natalia; Yep, Alejandra; McLeish, Michael J; Kenyon, George L; Jordan, Frank

    2008-03-25

    Direct spectroscopic observation of thiamin diphosphate-bound intermediates was achieved on the enzyme benzaldehyde lyase, which carries out reversible and highly enantiospecific conversion of ( R)-benzoin to benzaldehyde. The key enamine intermediate could be observed at lambda max 393 nm in the benzoin breakdown direction and in the decarboxylase reaction starting with benzoylformate. With benzaldehyde as substrate, no intermediates could be detected, only formation of benzoin at 314 nm. To probe the rate-limiting step in the direction of ( R)-benzoin synthesis, the (1)H/ (2)H kinetic isotope effect was determined for benzaldehyde labeled at the aldehyde position and found to be small (1.14 +/- 0.03), indicating that ionization of the C2alphaH from C2alpha-hydroxybenzylthiamin diphosphate is not rate limiting. Use of the alternate substrates benzoylformic and phenylpyruvic acids (motivated by the observation that while a carboligase, benzaldehyde lyase could also catalyze the slow decarboxylation of 2-oxo acids) enabled the observation of the substrate-thiamin covalent intermediate via the 1',4'-iminopyrimidine tautomer, characteristic of all intermediates with a tetrahedral C2 substituent on ThDP. The reaction of benzaldehyde lyase with the chromophoric substrate analogue ( E)-2-oxo-4(pyridin-3-yl)-3-butenoic acid and its decarboxylated product ( E)-3-(pyridine-3-yl)acrylaldehyde enabled the detection of covalent adducts with both. Neither adduct underwent further reaction. An important finding of the studies is that all thiamin-related intermediates are in a chiral environment on benzaldehyde lyase as reflected by their circular dichroism signatures.

  3. Repeated febrile convulsions impair hippocampal neurons and cause synaptic damage in immature rats:neuroprotective effect of fructose-1,6-diphosphate

    Institute of Scientific and Technical Information of China (English)

    Jianping Zhou; Fan Wang; Jun Zhang; Hui Gao; Yufeng Yang; Rongguo Fu

    2014-01-01

    Fructose-1,6-diphosphate is a metabolic intermediate that promotes cell metabolism. We hy-pothesize that fructose-1,6-diphosphate can protect against neuronal damage induced by febrile convulsions. Hot-water bathing was used to establish a repetitive febrile convulsion model in rats aged 21 days, equivalent to 3-5 years in humans. Ninety minutes before each seizure induc-tion, rats received an intraperitoneal injection of low- or high-dose fructose-1,6-diphosphate (500 or 1,000 mg/kg, respectively). Low- and high-dose fructose-1,6-diphosphate prolonged the latency and shortened the duration of seizures. Furthermore, high-dose fructose-1,6-di-phosphate effectively reduced seizure severity. Transmission electron microscopy revealed that 24 hours after the last seizure, high-dose fructose-1,6-diphosphate reduced mitochondrial swelling, rough endoplasmic reticulum degranulation, Golgi dilation and synaptic cleft size, and increased synaptic active zone length, postsynaptic density thickness, and synaptic interface cur-vature in the hippocampal CA1 area. The present findings suggest that fructose-1,6-diphosphate is a neuroprotectant against hippocampal neuron and synapse damage induced by repeated fe-brile convulsion in immature rats.

  4. Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels

    NARCIS (Netherlands)

    Perraud, AL; Takanishi, CL; Shen, B; Kang, S; Smith, MK; Schmitz, C; Knowles, HM; Ferraris, D; Li, WX; Zhang, J; Stoddard, BL; Scharenberg, AM

    2005-01-01

    TRPM2 is a member of the transient receptor potential melastatin-related (TRPM) family of cation channels, which possesses both ion channel and ADP-ribose hydrolase functions. TRPM2 has been shown to gate in response to oxidative and nitrosative stresses, but the mechanism through which TRPM2 gating

  5. Inhibition of poly(ADP-ribose polymerase interferes with Trypanosoma cruzi infection and proliferation of the parasite.

    Directory of Open Access Journals (Sweden)

    Salomé C Vilchez Larrea

    Full Text Available Poly(ADP-ribosylation is a post-translational covalent modification of proteins catalyzed by a family of enzymes termed poly(ADP-ribose polymerases (PARPs. In the human genome, 17 different genes have been identified that encode members of the PARP superfamily. Poly (ADP-ribose metabolism plays a role in a wide range of biological processes. In Trypanosoma cruzi, PARP enzyme appears to play a role in DNA repair mechanisms and may also be involved in controlling the different phases of cell growth. Here we describe the identification of potent inhibitors for T. cruzi PARP with a fluorescence-based activity assay. The inhibitors were also tested on T. cruzi epimastigotes, showing that they reduced ADP-ribose polymer formation in vivo. Notably, the identified inhibitors are able to reduce the growth rate of T. cruzi epimastigotes. The best inhibitor, Olaparib, is effective at nanomolar concentrations, making it an efficient chemical tool for chacterization of ADP-ribose metabolism in T. cruzi. PARP inhibition also decreases drastically the amount of amastigotes but interestingly has no effect on the amount of trypomastigotes in the cell culture. Knocking down human PARP-1 decreases both the amount of amastigotes and trypomastigotes in cell culture, indicating that the effect would be mainly due to inhibition of human PARP-1. The result suggests that the inhibition of PARP could be a potential way to interfere with T. cruzi infection.

  6. A glucose transporter can mediate ribose uptake: definition of residues that confer substrate specificity in a sugar transporter.

    Science.gov (United States)

    Naula, Christina M; Logan, Flora J; Logan, Flora M; Wong, Pui Ee; Barrett, Michael P; Burchmore, Richard J

    2010-09-24

    Sugars, the major energy source for many organisms, must be transported across biological membranes. Glucose is the most abundant sugar in human plasma and in many other biological systems and has been the primary focus of sugar transporter studies in eukaryotes. We have previously cloned and characterized a family of glucose transporter genes from the protozoan parasite Leishmania. These transporters, called LmGT1, LmGT2, and LmGT3, are homologous to the well characterized glucose transporter (GLUT) family of mammalian glucose transporters. We have demonstrated that LmGT proteins are important for parasite viability. Here we show that one of these transporters, LmGT2, is a more effective carrier of the pentose sugar d-ribose than LmGT3, which has a 6-fold lower relative specificity (V(max)/K(m)) for ribose. A pair of threonine residues, located in the putative extracellular loops joining transmembrane helices 3 to 4 and 7 to 8, define a filter that limits ribose approaching the exofacial substrate binding pocket in LmGT3. When these threonines are substituted by alanine residues, as found in LmGT2, the LmGT3 permease acquires ribose permease activity that is similar to that of LmGT2. The location of these residues in hydrophilic loops supports recent suggestions that substrate recognition is separated from substrate binding and translocation in this important group of transporters.

  7. Poly (ADP-ribose) polymerase-1 gene polymorphism in various Chinese nationalities

    Institute of Scientific and Technical Information of China (English)

    Hairong Liang; Junli Shao; Yuting Gao; Linhua Liu; Juanxiu Dai; Yun He; Huanwen Tang

    2011-01-01

    Poly (ADP-ribose) polymerase-1 (PARP-1) can exacerbate ischemic brain injury and lessen ischemic neuronal death, which may be associated with PARP-1 polymorphisms. The present study investigated human PARP-1 gene polymorphisms in various Chinese nationalities, the results of which could potentially help in the treatment and prevention of neurologic diseases. Genetic polymorphisms of seven exons in the PARP-1 gene, in 898 Chinese Han, Buyi, Shui, Miao, and Zhuang subjects, were investigated by PCR-single-strand conformation polymorphism. A single-strand conformation polymorphism variant in exons 12, 13, 16, and 17 of the PARP-1 gene was identified in 148 people, with two stationary bands showing three degenerative single strands.Results showed that the PARP-1 gene polymorphisms exist in various nationalities, and may act as a biomarker for susceptibility to disease.

  8. The Role of Poly(ADP-ribose Polymerase-1 in Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Samuel García

    2015-01-01

    Full Text Available Poly(ADP-ribose polymerase-1 (PARP-1 is a nuclear enzyme with a crucial role in the maintenance of genomic stability. In addition to the role of PARP-1 in DNA repair, multiple studies have also demonstrated its involvement in several inflammatory diseases, such as septic shock, asthma, atherosclerosis, and stroke, as well as in cancer. In these diseases, the pharmacological inhibition of PARP-1 has shown a beneficial effect, suggesting that PARP-1 regulates their inflammatory processes. In recent years, we have studied the role of PARP-1 in rheumatoid arthritis, as have other researchers, and the results have shown that PARP-1 has an important function in the development of this disease. This review summarizes current knowledge on the effects of PARP-1 in rheumatoid arthritis.

  9. Synthesis of Thienamycin methyl ester from 2-deoxy-D-ribose via Kinugasa reaction.

    Science.gov (United States)

    Soluch, Magdalena; Grzeszczyk, Barbara; Staszewska-Krajewska, Olga; Chmielewski, Marek; Furman, Bartłomiej

    2016-03-01

    A novel synthesis of thienamycin is described. The crucial step of the synthesis is based on Cu(I)-mediated Kinugasa cycloaddition/rearrangement cascade reaction between terminal acetylene derived from D-lactic acid and suitable, partially protected, five-membered cyclic nitrone obtained from 2-deoxy-D-ribose. The reaction was performed in the presence of tetramethylguanidine as a base to provide 5,6-trans substituted carbapenam as the main product. Thus obtained carbapenam 11 with (5R,6S) configuration at the azetidinone ring was subsequently subjected to oxidation/deprotection/oxidation reaction sequence to afford the β-keto ester 20, which was directly transformed into N,O-protected methyl ester of thienamycin.

  10. Poly(ADP-ribose) polymerase-13 and RNA regulation in immunity and cancer.

    Science.gov (United States)

    Todorova, Tanya; Bock, Florian J; Chang, Paul

    2015-06-01

    Post-transcriptional regulation of RNA is an important mechanism for activating and resolving cellular stress responses. Poly(ADP-ribose) polymerase-13 (PARP13), also known as ZC3HAV1 and zinc-finger antiviral protein (ZAP), is an RNA-binding protein that regulates the stability and translation of specific mRNAs, and modulates the miRNA silencing pathway to globally affect miRNA targets. These functions of PARP13 are important components of the cellular response to stress. In addition, the ability of PARP13 to restrict oncogenic viruses and to repress the prosurvival cytokine receptor tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 4 (TRAILR4) suggests that it can be protective against malignant transformation and cancer development. The relevance of PARP13 to human health and disease make it a promising therapeutic target.

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

    Science.gov (United States)

    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.

  12. Conformational influence of the ribose 2'-hydroxyl group: crystal structures of DNA-RNA chimeric duplexes

    Science.gov (United States)

    Egli, M.; Usman, N.; Rich, A.

    1993-01-01

    We have crystallized three double-helical DNA-RNA chimeric duplexes and determined their structures by X-ray crystallography at resolutions between 2 and 2.25 A. The two self-complementary duplexes [r(G)d(CGTATACGC)]2 and [d(GCGT)r(A)d(TACGC)]2, as well as the Okazaki fragment d(GGGTATACGC).r(GCG)d(TATACCC), were found to adopt A-type conformations. The crystal structures are non-isomorphous, and the crystallographic environments for the three chimeras are different. A number of intramolecular interactions of the ribose 2'-hydroxyl groups contribute to the stabilization of the A-conformation. Hydrogen bonds between 2'-hydroxyls and 5'-oxygens or phosphate oxygens, in addition to the previously observed hydrogen bonds to 1'-oxygens of adjacent riboses and deoxyriboses, are observed in the DNA-RNA chimeric duplexes. The crystalline chimeric duplexes do not show a transition between the DNA A- and B-conformations. CD spectra suggest that the Okazaki fragment assumes an A-conformation in solution as well. In this molecule the three RNA residues may therefore lock the complete decamer in the A-conformation. Crystals of an all-DNA strand with the same sequence as the self-complementary chimeras show a morphology which is different from those of the chimera crystals. Moreover, the oligonucleotide does not match any of the sequence characteristics of DNAs usually adopting the A-conformation in the crystalline state (e.g., octamers with short alternating stretches of purines and pyrimidines). In DNA-RNA chimeric duplexes, it is therefore possible that a single RNA residue can drive the conformational equilibrium toward the A-conformation.

  13. Evaluation of the sorption of Eu(III) in titanium diphosphate; Evaluacion de la sorcion de Eu(III) en difosfato de titanio

    Energy Technology Data Exchange (ETDEWEB)

    Ortiz O, H.B.; Ordonez R, E.; Fernandez V, S.M. [ININ, Carretera Mexico-Toluca Km 36.5, Salazar, Estado de Mexico (Mexico)]. e-mail: hortiz@nuclear.inin.mx

    2007-07-01

    In this work its are presented: the synthesis, physicochemical characterization and the surface parameters estimation that can be related with the retention properties of the titanium diphosphate for the actinides of valence III (Pu, Am, Cm among others), using the Eu{sup 3+} like a chemical analog. The surface area, hydration time, zero charge point, density of active sites and the surface species distribution in the titanium diphosphate are reported. This information was used to explain the retention of the Eu(lll) in the surface of the titanium diphosphate. (Author)

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

    Energy Technology Data Exchange (ETDEWEB)

    Hung, Szu-Ying; Shih, Ya-Chen [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); Tseng, Wei-Lung, E-mail: tsengwl@mail.nsysu.edu.tw [Department of Chemistry, National Sun Yat-sen University, Taiwan (China); School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Taiwan (China); Center for Nanoscience and Nanotechnology, National Sun Yat-sen University, Taiwan (China); Center for Stem Cell Research, Kaohsiung Medical University, Taiwan (China)

    2015-02-01

    Graphical abstract: A simple, enzyme-free, label-free, sensitive and selective system was developed for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles as an efficient quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate and as a recognition element for adenosine. - Highlights: • The proposed method can detect adenosine with more than 1000-fold selectivity. • The analysis of adenosine is rapid (∼6 min) using the proposed method. • This method provided better sensitivity for adenosine as compared to aptamer-based sensors. • This method can be applied for the determination of adenosine in urine. - Abstract: This study describes the development of a simple, enzyme-free, label-free, sensitive, and selective system for detecting adenosine based on the use of Tween 20-stabilized gold nanoparticles (Tween 20-AuNPs) as an efficient fluorescence quencher for boron dipyrromethene-conjugated adenosine 5′-triphosphate (BODIPY-ATP) and as a recognition element for adenosine. BODIPY-ATP can interact with Tween 20-AuNPs through the coordination between the adenine group of BODIPY-ATP and Au atoms on the NP surface, thereby causing the fluorescence quenching of BODIPY-ATP through the nanometal surface energy transfer (NSET) effect. When adenosine attaches to the NP surface, the attached adenosine exhibits additional electrostatic attraction to BODIPY-ATP. As a result, the presence of adenosine enhances the efficiency of AuNPs in fluorescence quenching of BODIPY-ATP. The AuNP-induced fluorescence quenching of BODIPY-ATP progressively increased with an increase in the concentration of adenosine; the detection limit at a signal-to-noise ratio of 3 for adenosine was determined to be 60 nM. The selectivity of the proposed system was more than 1000-fold for adenosine over any adenosine analogs and other nucleotides. The proposed system combined with a phenylboronic acid-containing column was successfully applied to the

  15. Adenosine thiamine triphosphate accumulates in Escherichia coli cells in response to specific conditions of metabolic stress

    Directory of Open Access Journals (Sweden)

    Zorzi Willy

    2010-05-01

    Full Text Available Abstract Background E. coli cells are rich in thiamine, most of it in the form of the cofactor thiamine diphosphate (ThDP. Free ThDP is the precursor for two triphosphorylated derivatives, thiamine triphosphate (ThTP and the newly discovered adenosine thiamine triphosphate (AThTP. While, ThTP accumulation requires oxidation of a carbon source, AThTP slowly accumulates in response to carbon starvation, reaching ~15% of total thiamine. Here, we address the question whether AThTP accumulation in E. coli is triggered by the absence of a carbon source in the medium, the resulting drop in energy charge or other forms of metabolic stress. Results In minimal M9 medium, E. coli cells produce AThTP not only when energy substrates are lacking but also when their metabolization is inhibited. Thus AThTP accumulates in the presence of glucose, when glycolysis is blocked by iodoacetate, or in the presence lactate, when respiration is blocked by cyanide or anoxia. In both cases, ATP synthesis is impaired, but AThTP accumulation does not appear to be a direct consequence of reduced ATP levels. Indeed, in the CV2 E. coli strain (containing a thermolabile adenylate kinase, the ATP content is very low at 37°C, even in the presence of metabolizable substrates (glucose or lactate and under these conditions, the cells produce ThTP but not AThTP. Furthermore, we show that ThTP inhibits AThTP accumulation. Therefore, we conclude that a low energy charge is not sufficient to trigger AThTP accumulation and the latter can only accumulate under conditions where no ThTP is synthesized. We further show that AThTP production can also be induced by the uncoupler CCCP but, unexpectedly, this requires the presence of pyruvate or a substrate yielding pyruvate (such a D-glucose or L-lactate. Under the conditions described, AThTP production is not different when RelA or SpoT mutants are used. Conclusions In E. coli, AThTP accumulates in response to two different conditions of

  16. The Use of Adenosine Agonists to Treat Nerve Agent-Induced Seizure and Neuropathology

    Science.gov (United States)

    2016-09-01

    kainate, adenosine and neuropeptide Y receptors. Neurochemical Research. 28: 1501-1515. 23. Bjorness, T. E. & R. W. Greene. 2009. Adenosine and sleep ...al. 2004. Adenosine and sleep -wake regulation. Progress in Neurobiology. 73: 379-396. 31. Schubert, P., et al. 1997. Protective mechanisms of...effects of adenosine by caffeine or 8-(p-sulfophenyl)theophylline. The Journal of Pharmacology and Experimental Therapeutics. 240: 428-432. 44

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

    OpenAIRE

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

    2016-01-01

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

  18. Development of coronary vasospasm during adenosine-stress myocardial perfusion CT imaging

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jeong Gu; Choi, Seong Hoon; Kang, Byeong Seong; Bang, Min Aeo; Kwon, Woon Jeong [Dept. of Radiology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan (Korea, Republic of)

    2015-06-15

    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.

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

    Science.gov (United States)

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

    2017-07-01

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

  20. Adenosine A(3) receptor-induced CCL2 synthesis in cultured mouse astrocytes

    NARCIS (Netherlands)

    Wittendorp, MC; Boddeke, HWGM; Biber, K

    2004-01-01

    During neuropathological conditions, high concentrations of adenosine are released, stimulating adenosine receptors in neurons and glial cells. It has recently been shown that stimulation of adenosine receptors in glial cells induces the release of neuroprotective substances such as NGF, S-100beta,

  1. DMPD: Shaping of monocyte and macrophage function by adenosine receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17056121 Shaping of monocyte and macrophage function by adenosine receptors. Hasko ...tml) (.csml) Show Shaping of monocyte and macrophage function by adenosine receptors. PubmedID 17056121 Titl...e Shaping of monocyte and macrophage function by adenosine receptors. Authors Has

  2. The role of glial adenosine receptors in neural resilience and the neurobiology of mood disorders

    NARCIS (Netherlands)

    Calker, D; Biber, K

    2005-01-01

    Adenosine receptors were classified into A(1)- and A(2)-receptors in the laboratory of Bernd Hamprecht more than 25 years ago. Adenosine receptors are instrumental to the neurotrophic effects of glia cells. Both microglia and astrocytes release after stimulation via adenosine receptors factors that

  3. Contributory role of adenosine deaminase in metabolic syndrome

    African Journals Online (AJOL)

    olayemitoyin

    Summary: Adenosine deaminase (ADA) is an enzyme of purine metabolism ... as obesity, insulin resistance, fasting hyperglycaemia, lipid abnormalities and ... Body mass index (BMI), fasting blood glucose (FBG), Glycated ... regulation of intracellular and extra cellular ... studies have indicated that defective signalling from.

  4. No role of interstitial adenosine in insulin-mediated vasodilation

    DEFF Research Database (Denmark)

    Dela, F; Stallknecht, B

    1999-01-01

    The mechanisms behind the vasodilatory effect of insulin are not fully understood, but nitric oxide plays an important role. We have investigated the possibility that insulin mediates vasodilatation in the human skeletal muscle via an increase in extracellular adenosine concentrations. In eight h...

  5. Adenosine receptor blockade reduces splanchnic hyperemia in cirrhotic rats.

    Science.gov (United States)

    Lee, S S; Chilton, E L; Pak, J M

    1992-06-01

    To explore a possible role for adenosine in the pathogenesis of the splanchnic hyperemia of cirrhosis, we administered 8-phenyltheophylline, a specific adenosine receptor antagonist, to rats with biliary cirrhosis caused by bile duct ligation and to control sham-operated rats. Micro-Doppler flow studies showed that a 10-mumol/kg dose of 8-phenyltheophylline completely abolished the superior mesenteric hyperemic response to infusions of exogenous adenosine in both cirrhotic and control rats. Analysis of regional blood flows by radioactive microspheres demonstrated that this dose of 8-phenyltheophylline in cirrhotic rats significantly increased portal tributary vascular resistance by 60% and decreased portal tributary blood flow by 26%. This decrease was entirely the result of a 42% reduction in the intestinal blood flow. 8-phenyltheophylline did not affect cardiac output, arterial pressure or any other extrasplanchnic hemodynamic variables in cirrhotic rats. No detectable effect of 8-phenyltheophylline was seen in sham-operated rats. These results suggest that adenosine may be involved in the genesis of splanchnic hyperemia in cirrhotic rats.

  6. Adenosine receptor modulation of seizure susceptibility in rats

    Energy Technology Data Exchange (ETDEWEB)

    Szot, P.

    1987-01-01

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

  7. Searching Inhibitors of Adenosine Kinase by Simulation Methods

    Institute of Scientific and Technical Information of China (English)

    ZHU Rui-Xin; ZHANG Xing-Long; DONG Xi-Cheng; CHEN Min-Bo

    2006-01-01

    Searching new inhibitors of adenosine kinase (AK) is still drawing attention of experimental scientists. A better and solid model is here proposed by means of simulation methods from different ways, the direct analysis of receptor itself, the conventional 3D-QSAR methods and the integration of docking method and the conventional QSAR analysis.

  8. 21 CFR 864.7040 - Adenosine triphosphate release assay.

    Science.gov (United States)

    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...

  9. CD39/adenosine pathway is involved in AIDS progression.

    Directory of Open Access Journals (Sweden)

    Maria Nikolova

    2011-07-01

    Full Text Available HIV-1 infection is characterized by a chronic activation of the immune system and suppressed function of T lymphocytes. Regulatory CD4+ CD25(high FoxP3+CD127(low T cells (Treg play a key role in both conditions. Here, we show that HIV-1 positive patients have a significant increase of Treg-associated expression of CD39/ENTPD1, an ectoenzyme which in concert with CD73 generates adenosine. We show in vitro that the CD39/adenosine axis is involved in Treg suppression in HIV infection. Treg inhibitory effects are relieved by CD39 down modulation and are reproduced by an adenosine-agonist in accordance with a higher expression of the adenosine A2A receptor on patients' T cells. Notably, the expansion of the Treg CD39+ correlates with the level of immune activation and lower CD4+ counts in HIV-1 infected patients. Finally, in a genetic association study performed in three different cohorts, we identified a CD39 gene polymorphism that was associated with down-modulated CD39 expression and a slower progression to AIDS.

  10. Adenosine Receptor Heteromers and their Integrative Role in Striatal Function

    Directory of Open Access Journals (Sweden)

    Sergi Ferré

    2007-01-01

    Full Text Available By analyzing the functional role of adenosine receptor heteromers, we review a series of new concepts that should modify our classical views of neurotransmission in the central nervous system (CNS. Neurotransmitter receptors cannot be considered as single functional units anymore. Heteromerization of neurotransmitter receptors confers functional entities that possess different biochemical characteristics with respect to the individual components of the heteromer. Some of these characteristics can be used as a “biochemical fingerprint” to identify neurotransmitter receptor heteromers in the CNS. This is exemplified by changes in binding characteristics that are dependent on coactivation of the receptor units of different adenosine receptor heteromers. Neurotransmitter receptor heteromers can act as “processors” of computations that modulate cell signaling, sometimes critically involved in the control of pre- and postsynaptic neurotransmission. For instance, the adenosine A1-A2A receptor heteromer acts as a concentration-dependent switch that controls striatal glutamatergic neurotransmission. Neurotransmitter receptor heteromers play a particularly important integrative role in the “local module” (the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit, where they act as processors mediating computations that convey information from diverse volume-transmitted signals. For instance, the adenosine A2A-dopamine D2 receptor heteromers work as integrators of two different neurotransmitters in the striatal spine module.

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

    Science.gov (United States)

    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

  12. Biosynthesis of the diterpenoid lycosantalonol via nerylneryl diphosphate in Solanum lycopersicum.

    Science.gov (United States)

    Matsuba, Yuki; Zi, Jiachen; Jones, A Daniel; Peters, Reuben J; Pichersky, Eran

    2015-01-01

    We recently reported that three genes involved in the biosynthesis of monoterpenes in trichomes, a cis-prenyltransferase named neryl diphosphate synthase 1 (NDPS1) and two terpene synthases (TPS19 and TPS20), are present in close proximity to each other at the tip of chromosome 8 in the genome of the cultivated tomato (Solanum lycopersicum). This terpene gene "cluster" also contains a second cis-prenyltransferase gene (CPT2), three other TPS genes, including TPS21, and the cytochrome P450-oxidoreductase gene CYP71BN1. CPT2 encodes a neryneryl diphosphate synthase. Co-expression in E. coli of CPT2 and TPS21 led to the formation of the diterpene lycosantalene, and co-expression in E. coli of CPT2, TPS21 and CYP71BN1 led to the formation of lycosantalonol, an oxidation product of lycosantalene. Here we show that maximal expression of all three genes occurs in the petiolule part of the leaf, but little expression of these genes occurs in the trichomes present on the petiolules. While lycosantalene or lycosantalonol cannot be detected in the petiolules of wild-type plants (or anywhere else in the plant), lycosantalene and lycosantalonol are detected in petiolules of transgenic tomato plants expressing CPT2 under the control of the 35S CaMV promoter. These results suggest that lycosantalene and lycosantalonol are produced in the petiolules and perhaps in other tissues of wild-type plants, but that low rate of synthesis, controlled by the rate-limiting enzyme CPT2, results in product levels that are too low for detection under our current methodology. It is also possible that these compounds are further modified in the plant. The involvement of CPT2, TPS21 and CYP71BN1 in a diterpenoid biosynthetic pathway outside the trichomes, together with the involvement of other genes in the cluster in the synthesis of monoterpenes in trichomes, indicates that this cluster is further evolving into "sub-clusters" with unique biochemical, and likely physiological, roles.

  13. Organization of monoterpene biosynthesis in Mentha. Immunocytochemical localizations of geranyl diphosphate synthase, limonene-6-hydroxylase, isopiperitenol dehydrogenase, and pulegone reductase.

    Science.gov (United States)

    Turner, Glenn W; Croteau, Rodney

    2004-12-01

    We present immunocytochemical localizations of four enzymes involved in p-menthane monoterpene biosynthesis in mint: the large and small subunits of peppermint (Mentha x piperita) geranyl diphosphate synthase, spearmint (Mentha spicata) (-)-(4S)-limonene-6-hydroxylase, peppermint (-)-trans-isopiperitenol dehydrogenase, and peppermint (+)-pulegone reductase. All were localized to the secretory cells of peltate glandular trichomes with abundant labeling corresponding to the secretory phase of gland development. Immunogold labeling of geranyl diphosphate synthase occurred within secretory cell leucoplasts, (-)-4S-limonene-6-hydroxylase labeling was associated with gland cell endoplasmic reticulum, (-)-trans-isopiperitenol dehydrogenase labeling was restricted to secretory cell mitochondria, while (+)-pulegone reductase labeling occurred only in secretory cell cytoplasm. We discuss this pathway compartmentalization in relation to possible mechanisms for the intracellular movement of monoterpene metabolites, and for monoterpene secretion into the extracellular essential oil storage cavity.

  14. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  15. Peculiarities of the inhibition of the pyruvate dehydrogenase complex by thiamine thiazolone diphosphate in vitro and in intact mitochondria

    Energy Technology Data Exchange (ETDEWEB)

    Yakovleva, G.M.; Strumilo, S.A.; Gorenshtein, B.I.; Ostrovskii, Yu.M.

    1986-07-10

    Thiamine thiazolone diphosphate (TTPP) possesses the ability to penetrate through the mitochondrial membrane and inhibit the pyruvate dehydrogenase complex in intact mitochondria, TTPP inhibits the activity of the complex of animal origin according to a mixed type (K/sub i/ 5 x 10/sup -8/ M) and yeast pyruvate decarboxylase according to a competitive type (K/sub i/ 5 x 10/sup -6/ M) with respect to thiamine diphosphate (TPP). Decarboxylation of pyruvate in intact and lysed rat liver and brain mitochondria is inhibited in the presence of TTPP significantly more weakly than the total activity of the pyruvate dehydrogenase complex, determined according to the formation of acetyl-CoA. It is suggested that TTPP, as an analog of the transition state, acts only in dehydrogenase reactions but not at the stage of simple decarboxylation of pyruvate.

  16. Saccharomyces pastorianus as cell factory to improve production of fructose 1,6-diphosphate using novel fermentation strategies

    Directory of Open Access Journals (Sweden)

    Chiara Schiraldi

    2015-08-01

    Full Text Available Enzymatic phosphorylation of glucose with inorganic phosphate, mediated by permeabilized yeast cells, is one of the methods commonly used to manufacture fructose 1,6-diphosphate, a compound of pharmaceutical interest. This process requires high concentrations of yeast active biomass, that is the catalyst of bioconversion of glucose and inorganic phosphate into fructose 1,6-diphosphate. In this study we firstly describe the high cell density production of a brewer's Saccharomyces strain (Saccharomyces pastorianus DSM 6581, focusing on the optimization of medium composition and exploiting fed-batch strategies and novel technologies based on membrane bioreactors. In fed-batch fermentation an appropriate exponential feed profile was set up to maintain the glucose concentration in the bioreactor below 0.9 g·L-1, thus yielding reproducibly 58 g dry weight biomass per liter in 80 h fermentation, improving eight-fold batch processes output. In addition a higher final biomass density was reached when implementing a microfiltration strategy (70 g dry weight biomass, that led to a productivity of 2.1 gcdw·L-1·h-1, 2.4-fold the fed-batch one. Successively, this biomass was opportunely permeabilized and proved capable of catalyzing the bioconversion of glucose into fructose 1,6-diphosphate. Acting on critical parameters of the bioconversion (substrates molar ratio, catalyst concentration and permeabilization agent, fructose 1,6-diphosphate was produced, after 3 h of process, at 56.3 ± 1 g·L-1 with a yield of 80% of the theoretical value.

  17. Feasibility of gas/solid carboligation: conversion of benzaldehyde to benzoin using thiamine diphosphate-dependent enzymes.

    Science.gov (United States)

    Mikolajek, R; Spiess, A C; Büchs, J

    2007-05-10

    A carboligation was investigated for the first time as an enzymatic gas phase reaction, where benzaldehyde was converted to benzoin using thiamine diphosphate (ThDP)-dependent enzymes, namely benzaldehyde lyase (BAL) and benzoylformate decarboxylase (BFD). The biocatalyst was immobilized per deposition on non-porous support. Some limitations of the gas/solid biocatalysis are discussed based on this carboligation and it is also demonstrated that the solid/gas system is an interesting tool for more volatile products.

  18. A tailor-made chimeric thiamine diphosphate dependent enzyme for the direct asymmetric synthesis of (S)-benzoins.

    Science.gov (United States)

    Westphal, Robert; Vogel, Constantin; Schmitz, Carlo; Pleiss, Jürgen; Müller, Michael; Pohl, Martina; Rother, Dörte

    2014-08-25

    Thiamine diphosphate dependent enzymes are well known for catalyzing the asymmetric synthesis of chiral α-hydroxy ketones from simple prochiral substrates. The steric and chemical properties of the enzyme active site define the product spectrum. Enzymes catalyzing the carboligation of aromatic aldehydes to (S)-benzoins have not so far been identified. We were able to close this gap by constructing a chimeric enzyme, which catalyzes the synthesis of various (S)-benzoins with excellent enantiomeric excess (>99%) and very good conversion.

  19. Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

    OpenAIRE

    Singleton Charles K; Song Qilin

    2002-01-01

    Abstract Background Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. Results Here we examine Th...

  20. Intracellular signalling pathways in the vasoconstrictor response of mouse afferent arterioles to adenosine

    DEFF Research Database (Denmark)

    Hansen, Pernille B. Lærkegaard; Friis, Ulla Glenert; Uhrenholt, Torben Rene

    2007-01-01

    AIMS: Adenosine causes vasoconstriction of afferent arterioles of the mouse kidney through activation of adenosine A(1) receptors and Gi-mediated stimulation of phospholipase C. In the present study, we further explored the signalling pathways by which adenosine causes arteriolar vasoconstriction....... METHODS AND RESULTS: Adenosine (10(-7) M) significantly increased the intracellular calcium concentration in mouse isolated afferent arterioles measured by fura-2 fluorescence. Pre-treatment with thapsigargin (2 microM) blocked the vasoconstrictor action of adenosine (10(-7) M) indicating that release...

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

    Directory of Open Access Journals (Sweden)

    Cátia Vieira

    2014-01-01

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

  2. p53-Independent induction of Fas and apoptosis in leukemic cells by an adenosine derivative, Cl-IB-MECA

    Science.gov (United States)

    Kim, Seong Gon; Ravi, Gnana; Hoffmann, Carsten; Jung, Yun-Jin; Kim, Min; Chen, Aishe; Jacobson, Kenneth A.

    2016-01-01

    A3 adenosine receptor (A3AR) agonists have been reported to influence cell death and survival. The effects of an A3AR agonist, 1-[2-chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-β-D-ribofuranonamide (Cl-IB-MECA), on apoptosis in two human leukemia cell lines, HL-60 and MOLT-4, were investigated. Cl-IB-MECA (≥30 μM) increased the apoptotic fractions, as determined using fluorescence-activated cell sorting (FACS) analysis, and activated caspase 3 and poly-ADP-ribose-polymerase. Known messengers coupled to A3AR (phospholipase C and intracellular calcium) did not seem to play a role in the induction of apoptosis. Neither dantrolene nor BAPTA-AM affected the Cl-IB-MECA-induced apoptosis. Cl-IB-MECA failed to activate phospholipase C in HL-60 cells, while UTP activated it through endogenous P2Y2 receptors. Induction of apoptosis during a 48 hr exposure to Cl-IB-MECA was not prevented by the A3AR antagonists [5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate] (MRS 1220) or N-[9-chloro-2-(2-furanyl)[l,2,4]triazolo[l,5-c]quinazolin-5-yl]benzeneacetamide (MRS 1523). Furthermore, higher concentrations of MRS 1220, which would also antagonize A1 and A2A receptors, were ineffective in preventing the apoptosis. Although Cl-IB-MECA has been shown in other systems to cause apoptosis through an A3AR-mediated mechanism, in these cells it appeared to be an adenosine receptor-independent effect, which required prolonged incubation. In both HL-60 and MOLT-4 cells, Cl-IB-MECA induced the expression of Fas, a death receptor. This induction of Fas was not dependent upon p53, because p53 is not expressed in an active form in either HL-60 or MOLT-4 cells. Cl-IB-MECA-induced apoptosis in HL-60 cells was augmented by an agonistic Fas antibody, CH-11, and this increase was suppressed by the antagonistic anti-Fas antibody ZB-4. Therefore, Cl-IB-MECA induced apoptosis via a novel, p53-independent up-regulation of Fas. Published by

  3. Small-Animal PET Study of Adenosine A(1) Receptors in Rat Brain : Blocking Receptors and Raising Extracellular Adenosine

    NARCIS (Netherlands)

    Paul, Soumen; Khanapur, Shivashankar; Rybczynska, Anna A.; Kwizera, Chantal; Sijbesma, Jurgen W. A.; Ishiwata, Kiichi; Willemsen, Antoon T. M.; Elsinga, Philip H.; Dierckx, Rudi A. J. O.; van Waarde, Aren

    2011-01-01

    Activation of adenosine A(1) receptors (A(1)R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A(1)R can be visualized using 8-dicyclopropylmethyl-1-C-11-methyl-3-propyl-xanthine (C-11-MPDX) and PET. This study aims to test whether C-11-MPDX

  4. Small-Animal PET Study of Adenosine A(1) Receptors in Rat Brain : Blocking Receptors and Raising Extracellular Adenosine

    NARCIS (Netherlands)

    Paul, Soumen; Khanapur, Shivashankar; Rybczynska, Anna A.; Kwizera, Chantal; Sijbesma, Jurgen W. A.; Ishiwata, Kiichi; Willemsen, Antoon T. M.; Elsinga, Philip H.; Dierckx, Rudi A. J. O.; van Waarde, Aren

    2011-01-01

    Activation of adenosine A(1) receptors (A(1)R) in the brain causes sedation, reduces anxiety, inhibits seizures, and promotes neuroprotection. Cerebral A(1)R can be visualized using 8-dicyclopropylmethyl-1-C-11-methyl-3-propyl-xanthine (C-11-MPDX) and PET. This study aims to test whether C-11-MPDX c

  5. Biosynthesis of anthecotuloide, an irregular sesquiterpene lactone from Anthemis cotula L. (Asteraceae) via a non-farnesyl diphosphate route.

    Science.gov (United States)

    van Klink, John; Becker, Hans; Andersson, Susannah; Boland, Wilhelm

    2003-05-07

    Retrobiosynthetic analysis of the allergenic sesquiterpene lactone, anthecotuloide, suggested that this natural product could be formed either by head to head condensation of geranyl diphosphate with dimethylallyl diphosphate, or from farnesyl diphosphate (FPP), the accepted regular sesquiterpene precursor via the rearrangement of a germacranolide precursor. Isotopic labelling of anthecotuloide has now been achieved by feeding [1-(13)C]-glucose, [U-13C6]-glucose and [6,6-(2)H2]-glucose to aseptically grown plantlets of Anthemis cotula(family Asteraceae). Analysis of labelling patterns and absolute 13C abundances using quantitative 13C NMR spectroscopy showed that the isoprene building blocks of this sesquiterpene are formed exclusively via the MEP terpene biosynthetic pathway. This was supported by results from an experiment using [U-13C6]-glucose. A deuterium labelling experiment using [6,6-(2)H2]-glucose supported the original proposal and showed that anthecotuloide is formed from a non FPP precursor. Isotope ratio mass spectrometry suggested that there were two pathways for sesquiterpene biosynthesis in A. cotula.

  6. Clinical Application of Poly(ADP-Ribose) Polymerase Inhibitors in High-Grade Serous Ovarian Cancer.

    Science.gov (United States)

    Parkes, Eileen E; Kennedy, Richard D

    2016-05-01

    : High-grade serous ovarian cancer is characterized by genomic instability, with one half of all tumors displaying defects in the important DNA repair pathway of homologous recombination. Given the action of poly(ADP-ribose) polymerase (PARP) inhibitors in targeting tumors with deficiencies in this repair pathway by loss of BRCA1/2, ovarian tumors could be an attractive population for clinical application of this therapy. PARP inhibitors have moved into clinical practice in the past few years, with approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA) within the past 2 years. The U.S. FDA approval of olaparib applies to fourth line treatment in germline BRCA-mutant ovarian cancer, and European EMA approval to olaparib maintenance in both germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. In order to widen the ovarian cancer patient population that would benefit from PARP inhibitors, predictive biomarkers based on a clear understanding of the mechanism of action are required. Additionally, a better understanding of the toxicity profile is needed if PARP inhibitors are to be used in the curative, rather than the palliative, setting. We reviewed the development of PARP inhibitors in phase I-III clinical trials, including combination trials of PARP inhibitors and chemotherapy/antiangiogenics, the approval for these agents, the mechanisms of resistance, and the outstanding issues, including the development of biomarkers and the rate of long-term hematologic toxicities with these agents. The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib has recently received approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA), with a second agent (rucaparib) likely to be approved in the near future. However, the patient population with potential benefit from PARP inhibitors is likely wider than that of germline BRCA mutation-associated disease, and biomarkers are in development to enable the

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  9. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Science.gov (United States)

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  10. Evaluation of serum nucleoside diphosphate kinase A for the detection of colorectal cancer

    Science.gov (United States)

    Otero-Estévez, Olalla; De Chiara, Loretta; Barcia-Castro, Leticia; Páez de la Cadena, María; Rodríguez-Berrocal, Francisco Javier; Cubiella, Joaquín; Hernández, Vicent; Martínez-Zorzano, Vicenta Soledad

    2016-01-01

    We previously described the over-expression of nucleoside diphosphate kinase A (NDKA) in tumours and serum from colorectal cancer (CRC) patients, suggesting its use as biomarker. In this study we evaluated the diagnostic accuracy of serum NDKA to detect advanced neoplasia (CRC or advanced adenomas). Furthermore, the performance of NDKA was compared with the faecal immunochemical test (FIT). The study population included a case-control cohort and a screening cohort (511 asymptomatic first-degree relatives of CRC patients that underwent a colonoscopy and a FIT). Serum NDKA was elevated in CRC patients in the case-control cohort (p = 0.002). In the screening cohort, NDKA levels were higher for advanced adenomas (p = 0.010) and advanced neoplasia (p = 0.006) compared to no neoplasia. Moreover, elevated NDKA was associated with severe characteristics of adenomas (≥3 lesions, size ≥ 1 cm or villous component). Setting specificity to 85%, NDKA showed a sensitivity of 30.19% and 29.82% for advanced adenomas and advanced neoplasia, respectively. NDKA combined with FIT (100 ng/mL cut-off) detected advanced adenomas and advanced neoplasia with 45.28% and 49.12% sensitivity, with specificity close to 90%. The combination of serum NDKA and FIT can improve the detection of advanced neoplasia, mainly for lesions located on the proximal colon, in asymptomatic individuals with CRC family-risk. PMID:27222072

  11. Thiamine diphosphate binds to intermediates in the assembly of adenovirus fiber knob trimers in Escherichia coli.

    Science.gov (United States)

    Schulz, Ryan; Zhang, Yian-Biao; Liu, Chang-Jun; Freimuth, Paul

    2007-12-01

    Assembly of the adenovirus (Ad) homotrimeric fiber protein is nucleated by its C-terminal knob domain, which itself can trimerize when expressed as a recombinant protein fragment. The non-interlocked, globular structure of subunits in the knob trimer implies that trimers assemble from prefolded monomers through a dimer intermediate, but these intermediates have not been observed and the mechanism of assembly therefore remains uncharacterized. Here we report that expression of the Ad serotype 2 (Ad2) knob was toxic for thi- strains of Escherichia coli, which are defective in de novo synthesis of thiamine (vitamin B1). Ad2 knob trimers isolated from a thi+ strain copurified through multiple chromatography steps with a small molecule of mass equivalent to that of thiamine diphosphate (ThDP). Mutant analysis did not implicate any specific site for ThDP binding. Our results suggest that ThDP may associate with assembly intermediates and become trapped in assembled trimers, possibly within one of several large cavities that are partially solvent-accessible or buried completely within the trimer interior.

  12. New function of the amino group of thiamine diphosphate in thiamine catalysis.

    Science.gov (United States)

    Meshalkina, L E; Kochetov, G A; Hübner, G; Tittmann, K; Golbik, R

    2009-03-01

    In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4'-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase--4'-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4'-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH2-methylated ThDP exhibits dihydroxyethyl-4'-methylamino-ThDP-synthase activity. Thus, the 4'-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.

  13. Isolation and functional analysis of two Cistus creticus cDNAs encoding geranylgeranyl diphosphate synthase.

    Science.gov (United States)

    Pateraki, Irene; Kanellis, Angelos K

    2008-05-01

    Cistus creticus ssp. creticus is an indigenous shrub of the Mediterranean area. The glandular trichomes covering its leaf surfaces secrete a resin called "ladanum", which among others contains a number of specific labdane-type diterpenes that exhibit antibacterial and antifungal action as well as in vitro and in vivo cytotoxic and cytostatic activity against human cancer cell lines. In view of the properties and possible future exploitation of these metabolites, it was deemed necessary to study the geranylgeranyl diphosphate synthase enzyme (GGDPS, EC 2.5.1.30), a short chain prenyltransferase responsible for the synthesis of the precursor molecule of all diterpenes. In this work, we present the cloning, functional characterisation and expression profile at the gene and protein levels of two differentially expressed C. creticus full-length cDNAs, CcGGDPS1 and CcGGDPS2. Heterologous yeast cell expression system showed that these cDNAs exhibited GGDPS enzyme activity. Gene and protein expression analyses suggest that this enzyme is developmentally and tissue-regulated showing maximum expression in trichomes and smallest leaves (0.5-1.0cm). This work is the first attempt to study the terpenoid biosynthesis at the molecular level in C. creticus ssp. creticus.

  14. Geosmin biosynthesis. Streptomyces coelicolor germacradienol/germacrene D synthase converts farnesyl diphosphate to geosmin.

    Science.gov (United States)

    Jiang, Jiaoyang; He, Xiaofei; Cane, David E

    2006-06-28

    Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.

  15. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 degrees C

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, Christopher R.; Felmy, Andrew R.; Clark, Sue B.

    2010-11-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO2)(3)(PO4)(2)center dot 4H(2)O) and Na autunite (Na[UO2PO4]center dot xH(2)O) at 23 and 50 degrees C in NaClO4-HClO4 solutions at pC(H+) = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 in solutions were equilibrated at 23 and 50 degrees C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K-sp, for TDT was determined to be -49.7 and -51.3 at 23 and 50 degrees C respectively. log K for Na autunite was determined to be -24.4 (23 degrees C) and -24.1 +/- 0.2 (50 degrees C).

  16. Solubility of triuranyl diphosphate tetrahydrate (TDT) and Na autunite at 23 and 50 C

    Energy Technology Data Exchange (ETDEWEB)

    Armstrong, C.R.; Clark, S.B. [Washington State Univ., Pullman, WA (United States); Felmy, A.R. [Pacific Northwest National Lab., Richland, WA (United States)

    2010-07-01

    In this report we present experimental solubility data for well-characterized triuranyl diphosphate tetrahydrate (TDT: (UO{sub 2}){sub 3}(PO{sub 4}){sub 2} . 4H{sub 2}O) and Na autunite (Na[UO{sub 2}PO{sub 4}] . xH{sub 2}O) at 23 and 50 C in NaClO{sub 4}-HClO{sub 4} solutions at pC{sub H1} = 2. Duplicate samples of TDT in 0.1, 0.5, 1.0, 2.0 and 5.0 m solutions were equilibrated at 23 and 50 C. TDT solid was synthesized and characterized with ICP-OES, ATR-IR and powder XRD before and after solubility experiments. The pH of the suspensions were monitored throughout the experiments. Equilibrium was achieved from undersaturation with respect to TDT and oversaturation for Na autunite. Steady-state conditions were achieved in all cases within 82 d. TDT was unstable at ionic strengths above 0.1 m, where its complete conversion to Na autunite was observed. The ion-interaction model was used to interpret the experimental solubility data. The solubility product, log K{sub sp}, for TDT was determined to be -49.7 and -51.3 at 23 and 50 C respectively. log K{sub sp} for Na autunite was determined to be -24.4 (23 C) and -24.1 {+-} 0.2 (50 C). (orig.)

  17. The uridine diphosphate glucuronosyltransferases: quantitative structure-activity relationships for hydroxyl polychlorinated biphenyl substrates

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Degao [Dalian University of Technology, Department of Environmental Science and Technology, Dalian (China)

    2005-10-01

    Quantitative structure-activity relationships (QSARs), which relate the glucuronidation of hydroxyl polychlorinated biphenyls (OH-PCBs) - catalyzed by the uridine diphosphate glucuronosyltransferases (UGTs) - to their physicochemical properties and molecular structural parameters, can be used to predict the rate constants and interpret the mechanism of glucuronidation. In this study, QSARs have been developed that use 23 semi-empirical calculated quantum chemical descriptors to predict the logarithms of the constants 1/K{sub m} and V{sub max}, related to enzyme kinetics. A partial least squares regression method was used to select the optimal set of descriptors to minimize the multicollinearity between the descriptors, as well as to maximize the cross-validated coefficient (Q{sup 2} {sub cum}) values. The key descriptors affecting log(1/K{sub m}) were E{sub lumo}- E{sub homo} (the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital) and q{sub C}{sup -} (the largest negative net atomic charge on a carbon atom), while the key descriptors affecting log V{sub max} were the polarizability {alpha}, the Connolly solvent-excluded volume (CSEV), and logP (the logarithm of the partition coefficient for octanol/water). From the results obtained it can be concluded that hydrophobic and electronic aspects of OH-PCBs are important in the glucuronidation of OH-PCBs. (orig.)

  18. Metabolism of farnesyl diphosphate in tobacco BY-2 cells treated with squalestatin.

    Science.gov (United States)

    Hartmann, M A; Wentzinger, L; Hemmerlin, A; Bach, T J

    2000-12-01

    Plant isoprenoids represent a large group of compounds with a wide range of physiological functions. In the cytosol, isoprenoids are synthesized via the classical acetate/mevalonate pathway. In this pathway, farnesyl diphosphate (FPP) occupies a central position, from which isoprene units are dispatched to the different classes of isoprenoids, with sterols as the major end products. The present work deals with effects of squalestatin (SQ) on the metabolism of FPP in proliferating and synchronized cultured tobacco cv. Bright Yellow-2 cells. SQ is a potent inhibitor of squalene synthase (SQS), the first committed enzyme in the sterol pathway. At nanomolar concentrations, SQ severely impaired cell growth and sterol biosynthesis, as attested by the rapid decrease in SQS activity. At the same time, it triggered a several-fold increase in both the enzymic activity and mRNA levels of 3-hydroxy-3-methylglutaryl CoA reductase. When SQ was added to cells synchronized by aphidicolin treatment, it was found to block the cell cycle at the end of G(1) phase, but no cell death was induced. Tobacco cells were also fed exogenous tritiated trans-trans farnesol, the allylic alcohol derived from FPP, in the presence and absence of SQ. Evidence is presented that this compound was incorporated into sterols and ubiquinone Q(10). In the presence of SQ, the sterol pathway was inhibited, but no increase in the radioactivity of ubiquinone was observed, suggesting that this metabolic channel was already saturated under normal conditions.

  19. In vitro synthesis and characterization of guanosine 3',5'-bis(diphosphate).

    Science.gov (United States)

    Hardiman, Timo; Windeisen, Volker; Ewald, Jennifer C; Zibek, Susanne; Schlack, Petra; Rebell, Jochen; Reuss, Matthias; Siemann-Herzberg, Martin

    2008-12-15

    The intracellular alarmone guanosine 3',5'-bis(diphosphate) (ppGpp) has been thoroughly investigated over the past 40 years and has become one of the best-known effectors in bacterial physiology. ppGpp is also of great importance for biotechnological applications. Systems biology research, involving experimental and mathematical approaches, has contributed a great deal to uncovering the alarmone's complex regulatory effects. HPLC analysis and UV detection are used to quantify intracellular ppGpp. The samples analyzed also contain other phosphorylated guanine nucleotides and, therefore, are spiked with a standard ppGpp solution. A rapidly growing number of laboratories are turning to synthesizing the nucleotide in vitro involving time-consuming protocols and yielding only low amounts of ppGpp. The current article provides a protocol for the preparation of large quantities of a ribosome extract that contains high ppGpp synthesis activity. The demonstrated upscaling from shaking flask to bioreactor cultivation involves the continuous and refrigerated harvest of the biomass. (13)C NMR analysis enabled the structural characterization of the synthesis product and was complemented by mass spectrometry and methods that are commonly used to identify ppGpp.

  20. Boric acid as a mobile phase additive for high performance liquid chromatography separation of ribose, arabinose and ribulose.

    Science.gov (United States)

    De Muynck, Cassandra; Beauprez, Joeri; Soetaert, Wim; Vandamme, Erick J

    2006-01-01

    A new high performance liquid chromatographic (HPLC) method is described for the analysis of ribose, arabinose and ribulose mixtures obtained from (bio)chemical isomerization processes. These processes gain importance since the molecules can be used for the synthesis of antiviral therapeutics. The HPLC method uses boric acid as a mobile phase additive to enhance the separation on an Aminex HPX-87K column. By complexing with boric acid, the carbohydrates become negatively charged, thus elute faster from the column by means of ion exlusion and are separated because the complexation capacity with boric acid differs from one carbohydrate to another. Excellent separation between ribose, ribulose and arabinose was achieved with concentrations between 0.1 and 10 gL(-1) of discrete sugar.

  1. Ab initio treatment of ion-induced charge transfer dynamics of isolated 2-deoxy-D-ribose.

    Science.gov (United States)

    Bacchus-Montabonel, Marie-Christine

    2014-08-21

    Modeling-induced radiation damage in biological systems, in particular, in DNA building blocks, is of major concern in cancer therapy studies. Ion-induced charge-transfer dynamics may indeed be involved in proton and hadrontherapy treatments. We have thus performed a theoretical approach of the charge-transfer dynamics in collision of C(4+) ions and protons with isolated 2-deoxy-D-ribose in a wide collision energy range by means of ab initio quantum chemistry molecular methods. The comparison of both projectile ions has been performed with regard to previous theoretical and experimental results. The charge transfer appears markedly less efficient with the 2-deoxy-D-ribose target than that with pyrimidine nucleobases, which would induce an enhancement of the fragmentation process in agreement with experimental measurements. The mechanism has been analyzed with regard to inner orbital excitations, and qualitative tendencies have been pointed out for studies on DNA buiding block damage.

  2. A single and two step isomerization process for d-tagatose and l-ribose bioproduction using l-arabinose isomerase and d-lyxose isomerase.

    Science.gov (United States)

    Patel, Manisha J; Akhani, Rekha C; Patel, Arti T; Dedania, Samir R; Patel, Darshan H

    2017-02-01

    l-ribose and d-tagatose are biochemically synthesized using sugar isomerases. The l-arabinose isomerase gene from Shigella flexneri (Sf-AI) was cloned and expressed in Escherichia coli BL-21. Sf-AI was applied for the bioproduction of d-tagatose from d-galactose. l-ribose synthesis was performed by two step isomerization using Sf-AI and d-lyxose/ribose isomerase from Cohnella laevoribosii. The overall 22.3% and 25% conversion rate were observed for d-tagatose and l-ribose production from d-galactose and l-arabinose respectively. In the present manuscript, synthesis of rare sugars from naturally available sugars is discussed along with the biochemical characterization of Sf-AI and its efficiency. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  4. PARP2 Is the Predominant Poly(ADP-Ribose Polymerase in Arabidopsis DNA Damage and Immune Responses.

    Directory of Open Access Journals (Sweden)

    Junqi Song

    2015-05-01

    Full Text Available Poly (ADP-ribose polymerases (PARPs catalyze the transfer of multiple poly(ADP-ribose units onto target proteins. Poly(ADP-ribosylation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390, rather than PARP1 (At2g31320, makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose glycohydrolase (PARG enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosylation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosylation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation.

  5. Activation of Poly(ADP-Ribose) Polymerase by Myocardial Ischemia and Coronary Reperfusion in Human Circulating Leukocytes

    OpenAIRE

    Tóth-Zsámboki, Emese; Horváth, Eszter; Vargova, Katarina; Pankotai, Eszter; Murthy, Kanneganti; Zsengellér, Zsuzsanna; Bárány, Tamás; Pék, Tamás; Fekete, Katalin; Kiss, Róbert Gábor; Préda, István; Lacza, Zsombor; Gerö, Domokos; SzabÓ, Csaba

    2006-01-01

    Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 2...

  6. Genetic alteration of poly(ADP-ribose) polymerase-1 in human germ cell tumors.

    Science.gov (United States)

    Shiokawa, Motoko; Masutani, Mitsuko; Fujihara, Hisako; Ueki, Keisuke; Nishikawa, Ryo; Sugimura, Takashi; Kubo, Harumi; Nakagama, Hitoshi

    2005-02-01

    Accumulated evidence suggests that poly(ADP-ribose) polymerase-1 (PARP-1) is involved in DNA repair, cell-death induction, differentiation and tumorigenesis. Parp-1 deficiency also induces trophoblast differentiation from mouse embryonic stem cells during teratocarcinoma-like tumor formation. To understand the relationship of PARP-1 dysfunction and development of germ cell tumors, we conducted a genetic analysis of the PARP-1 gene in human germ cell tumors. Sixteen surgical specimens of germ cell tumors that developed in the brain and testes were used. Two known single nucleotide polymorphisms (SNPs) (Val762Ala and Lys940Arg), which are listed in the SNP database of the NCBI (National Center for Biotechnology Information), were detected. In both cases, cSNPs encoded amino acids located within peptide stretches in the catalytic domain, which are highly conserved among various animal species. Furthermore, another novel sequence alteration, a base change of ATG to ACG, was identified in a tumor specimen, which would result in the amino acid substitution, Met129Thr. This base change was observed in one allele of both tumor and normal tissues, suggesting that it is either a rare SNP or a germline mutation of the PARP-1 gene. Notably, the amino acid Met129 is located within the second zinc finger domain, which is essential for DNA binding and is conserved among animal species. One SNP in intron 2 and one in the upstream 5'-UTR (untranslated region) were also detected.

  7. Sumoylation of poly(ADP-ribose) polymerase 1 inhibits its acetylation and restrains transcriptional coactivator function.

    Science.gov (United States)

    Messner, Simon; Schuermann, David; Altmeyer, Matthias; Kassner, Ingrid; Schmidt, Darja; Schär, Primo; Müller, Stefan; Hottiger, Michael O

    2009-11-01

    Poly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated nuclear protein and functions as a molecular stress sensor. At the cellular level, PARP1 has been implicated in a wide range of processes, such as maintenance of genome stability, cell death, and transcription. PARP1 functions as a transcriptional coactivator of nuclear factor kappaB (NF-kappaB) and hypoxia inducible factor 1 (HIF1). In proteomic studies, PARP1 was found to be modified by small ubiquitin-like modifiers (SUMOs). Here, we characterize PARP1 as a substrate for modification by SUMO1 and SUMO3, both in vitro and in vivo. PARP1 is sumoylated at the single lysine residue K486 within its automodification domain. Interestingly, modification of PARP1 with SUMO does not affect its ADP-ribosylation activity but completely abrogates p300-mediated acetylation of PARP1, revealing an intriguing crosstalk of sumoylation and acetylation on PARP1. Genetic complementation of PARP1-depleted cells with wild-type and sumoylation-deficient PARP1 revealed that SUMO modification of PARP1 restrains its transcriptional coactivator function and subsequently reduces gene expression of distinct PARP1-regulated target genes.

  8. A Novel and Selective Poly (ADP-Ribose) Polymerase Inhibitor Ameliorates Chemotherapy-Induced Painful Neuropathy

    Science.gov (United States)

    Ta, Lauren E.; Schmelzer, James D.; Bieber, Allan J.; Loprinzi, Charles L.; Sieck, Gary C.; Brederson, Jill D.; Low, Philip A.; Windebank, Anthony J.

    2013-01-01

    Background Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose) polymerase (PARP) inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888) would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice. Results An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p.) injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment. Conclusion Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy. PMID:23326593

  9. A novel and selective poly (ADP-ribose polymerase inhibitor ameliorates chemotherapy-induced painful neuropathy.

    Directory of Open Access Journals (Sweden)

    Lauren E Ta

    Full Text Available BACKGROUND: Chemotherapy-induced neuropathy is the principle dose limiting factor requiring discontinuation of many chemotherapeutic agents, including cisplatin and oxaliplatin. About 30 to 40% of patients receiving chemotherapy develop pain and sensory changes. Given that poly (ADP-ribose polymerase (PARP inhibition has been shown to provide neuroprotection, the current study was developed to test whether the novel PARP inhibitor compound 4a (analog of ABT-888 would attenuate pain in cisplatin and oxaliplatin-induced neuropathy in mice. RESULTS: An established chemotherapy-induced painful neuropathy model of two weekly cycles of 10 intraperitoneal (i.p. injections separated by 5 days rest was used to examine the therapeutic potential of the PARP inhibitor compound 4a. Behavioral testing using von Frey, paw radiant heat, cold plate, and exploratory behaviors were taken at baseline, and followed by testing at 3, 6, and 8 weeks from the beginning of drug treatment. CONCLUSION: Cisplatin-treated mice developed heat hyperalgesia and mechanical allodynia while oxaliplatin-treated mice exhibited cold hyperalgesia and mechanical allodynia. Co-administration of 50 mg/kg or 25 mg/kg compound 4a with platinum regimen, attenuated cisplatin-induced heat hyperalgesia and mechanical allodynia in a dose dependent manner. Similarly, co-administration of 50 mg/kg compound 4a attenuated oxaliplatin-induced cold hyperalgesia and mechanical allodynia. These data indicate that administration of a novel PARP inhibitor may have important applications as a therapeutic agent for human chemotherapy-induced painful neuropathy.

  10. Poly (ADP) ribose polymerase inhibition: A potential treatment of malignant peripheral nerve sheath tumor.

    Science.gov (United States)

    Kivlin, Christine M; Watson, Kelsey L; Al Sannaa, Ghadah A; Belousov, Roman; Ingram, Davis R; Huang, Kai-Lieh; May, Caitlin D; Bolshakov, Svetlana; Landers, Sharon M; Kalam, Azad Abul; Slopis, John M; McCutcheon, Ian E; Pollock, Raphael E; Lev, Dina; Lazar, Alexander J; Torres, Keila E

    2016-01-01

    Poly (ADP) ribose polymerase (PARP) inhibitors, first evaluated nearly a decade ago, are primarily used in malignancies with known defects in DNA repair genes, such as alterations in breast cancer, early onset 1/2 (BRCA1/2). While no specific mutations in BRCA1/2 have been reported in malignant peripheral nerve sheath tumors (MPNSTs), MPNST cells could be effectively targeted with a PARP inhibitor to drive cells to synthetic lethality due to their complex karyotype and high level of inherent genomic instability. In this study, we assessed the expression levels of PARP1 and PARP2 in MPNST patient tumor samples and correlated these findings with overall survival. We also determined the level of PARP activity in MPNST cell lines. In addition, we evaluated the efficacy of the PARP inhibitor AZD2281 (Olaparib) in MPNST cell lines. We observed decreased MPNST cell proliferation and enhanced apoptosis in vitro at doses similar to, or less than, the doses used in cell lines with established defective DNA repair genes. Furthermore, AZD2281 significantly reduced local growth of MPNST xenografts, decreased the development of macroscopic lung metastases, and increased survival of mice with metastatic disease. Our results suggest that AZD2281 could be an effective therapeutic option in MPNST and should be further investigated for its potential clinical use in this malignancy.

  11. Antioxidant Effects of Potassium Ascorbate with Ribose Therapy in a Case with Prader Willi Syndrome

    Directory of Open Access Journals (Sweden)

    C. Anichini

    2012-01-01

    Full Text Available Oxidative stress (OS is involved in several human diseases, including obesity, diabetes, atherosclerosis, carcinogenesis, as well as genetic diseases. We previously found that OS occurs in Down Syndrome as well as in Beckwith-Wiedemann Syndrome (BWS. Here we describe the clinical case of a female patient with Prader Willi Syndrome (PWS, a genomic imprinting disorder, characterized by obesity, atherosclerosis and diabetes mellitus type 2, pathologies in which a continuous and important production of free radicals takes place. We verified the presence of OS by measuring a redox biomarkers profile including total hydroperoxides (TH, non protein-bound iron (NPBI, thiols (SH, advanced oxidation protein products (AOPP and isoprostanes (IPs. Thus we introduced in therapy an antioxidant agent, namely potassium ascorbate with ribose (PAR, in addition to GH therapy and we monitored the redox biomarkers profile for four years. A progressive decrease in OS biomarkers occurred until their normalization. In the meantime a weight loss was observed together with a steady growth in standards for age and sex.

  12. Poly(ADP-ribose) polymerase-dependent energy depletion occurs through inhibition of glycolysis

    Science.gov (United States)

    Andrabi, Shaida A.; Umanah, George K. E.; Chang, Calvin; Stevens, Daniel A.; Karuppagounder, Senthilkumar S.; Gagné, Jean-Philippe; Poirier, Guy G.; Dawson, Valina L.; Dawson, Ted M.

    2014-01-01

    Excessive poly(ADP-ribose) (PAR) polymerase-1 (PARP-1) activation kills cells via a cell-death process designated “parthanatos” in which PAR induces the mitochondrial release and nuclear translocation of apoptosis-inducing factor to initiate chromatinolysis and cell death. Accompanying the formation of PAR are the reduction of cellular NAD+ and energetic collapse, which have been thought to be caused by the consumption of cellular NAD+ by PARP-1. Here we show that the bioenergetic collapse following PARP-1 activation is not dependent on NAD+ depletion. Instead PARP-1 activation initiates glycolytic defects via PAR-dependent inhibition of hexokinase, which precedes the NAD+ depletion in N-methyl-N-nitroso-N-nitroguanidine (MNNG)-treated cortical neurons. Mitochondrial defects are observed shortly after PARP-1 activation and are mediated largely through defective glycolysis, because supplementation of the mitochondrial substrates pyruvate and glutamine reverse the PARP-1–mediated mitochondrial dysfunction. Depleting neurons of NAD+ with FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, does not alter glycolysis or mitochondrial function. Hexokinase, the first regulatory enzyme to initiate glycolysis by converting glucose to glucose-6-phosphate, contains a strong PAR-binding motif. PAR binds to hexokinase and inhibits hexokinase activity in MNNG-treated cortical neurons. Preventing PAR formation with PAR glycohydrolase prevents the PAR-dependent inhibition of hexokinase. These results indicate that bioenergetic collapse induced by overactivation of PARP-1 is caused by PAR-dependent inhibition of glycolysis through inhibition of hexokinase. PMID:24987120

  13. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium.

    Science.gov (United States)

    Cooper, Karen L; Dashner, Erica J; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye; Hudson, Laurie G

    2016-01-15

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations.

  14. Inhibition of poly(ADP-ribose)polymerase-1 and DNA repair by uranium

    Science.gov (United States)

    Cooper, Karen L.; Dashner, Erica J.; Tsosie, Ranalda; Cho, Young Mi; Lewis, Johnnye

    2015-01-01

    Uranium has radiological and non-radiological effects within biological systems and there is increasing evidence for genotoxic and carcinogenic properties attributable to uranium through its heavy metal properties. In this study, we report that low concentrations of uranium (as uranyl acetate; <10 μM) is not cytotoxic to human embryonic kidney cells or normal human keratinocytes; however, uranium exacerbates DNA damage and cytotoxicity induced by hydrogen peroxide, suggesting that uranium may inhibit DNA repair processes. Concentrations of uranyl acetate in the low micromolar range inhibited the zinc finger DNA repair protein poly(ADP-ribose) polymerase (PARP)-1 and caused zinc loss from PARP-1 protein. Uranyl acetate exposure also led to zinc loss from the zinc finger DNA repair proteins Xeroderma Pigmentosum, Complementation Group A (XPA) and aprataxin (APTX). In keeping with the observed inhibition of zinc finger function of DNA repair proteins, exposure to uranyl acetate enhanced retention of induced DNA damage. Co-incubation of uranyl acetate with zinc largely overcame the impact of uranium on PARP-1 activity and DNA damage. These findings present evidence that low concentrations of uranium can inhibit DNA repair through disruption of zinc finger domains of specific target DNA repair proteins. This may provide a mechanistic basis to account for the published observations that uranium exposure is associated with DNA repair deficiency in exposed human populations. PMID:26627003

  15. Photo Decoloration of Methylene Blue with Ribose under Optimum Conditions by Visible Radiation

    Institute of Scientific and Technical Information of China (English)

    AZMAT, Rafia; UDDIN, Fahim

    2009-01-01

    Photo decoloration of the methylene blue (MB) with reducing sugar, ribose (RH), was investigated on an especially designed optical processor using monochromatic radiation of 661 nm through a red filter. The dye molecule gets ex- cited into triplet transient species (MBT) during flushing with lifetime of 10.1 μs into acetate buffered aqueous alcoholic medium, which later on reduces to protonated leuco dye (MBH). Pbotolysis of the aqueous aicholic medium bose into respective acid while hydrogen abstraction and 2e- reduced the dye (MB) into MBH by following reaction MBr++RCHO→MBH+RCOOH. Influences of different operational parameters suggest the imperative role of acidity, reductant and temperature where positive relations with quantum yield (φ) were established under the opti- mum conditions while elevated concentration of dye resulted in the decrease in the absorption of number of photon consequently, and no change in the φ was observed. A highly solvated transient complex was reported in the reduc- tion of MB and RH through thermodynamics activation parameters. A mechanism of MBT formation and its reduc- tion into MBH has been discussed.

  16. Differential Role of Poly(ADP-ribose polymerase in D. discoideum growth and development

    Directory of Open Access Journals (Sweden)

    Begum Rasheedunnisa

    2011-03-01

    Full Text Available Abstract Background Poly(ADP-ribose polymerase is evolutionarily conserved as a responder to various forms of stress. Though PARP's role in cell death is well addressed, its role in development and multicellularity is still an enigma. We have previously reported the role of PARP in oxidative stress induced delayed development of D. discoideum. Results In the current study we highlight the involvement of PARP during D. discoideum development. Oxidative stress affects expression of aca and cAR1 thus affecting aggregation. Although parp expression is not affected during oxidative stress but it is involved during normal development as confirmed by our PARP down-regulation studies. Constitutive PARP down-regulation resulted in blocked development while no effect was observed on D. discoideum growth. Interestingly, stage specific PARP down-regulation arrested development at the slug stage. Conclusion These results emphasize that PARP is essential for complex differentiation and its function may be linked to multicellularity. This is the first report where the involvement of PARP during normal multicellular development in D. discoideum, an ancient eukaryote, is established which could be of evolutionary significance. Thus our study adds one more role to the multitasking function of PARP.

  17. Functional localization of two poly(ADP-ribose)-degrading enzymes to the mitochondrial matrix.

    Science.gov (United States)

    Niere, Marc; Kernstock, Stefan; Koch-Nolte, Friedrich; Ziegler, Mathias

    2008-01-01

    Recent discoveries of NAD-mediated regulatory processes in mitochondria have documented important roles of this compartmentalized nucleotide pool in addition to energy transduction. Moreover, mitochondria respond to excessive nuclear NAD consumption arising from DNA damage-induced poly-ADP-ribosylation because poly(ADP-ribose) (PAR) can trigger the release of apoptosis-inducing factor from the organelles. To functionally assess mitochondrial NAD metabolism, we overexpressed the catalytic domain of nuclear PAR polymerase 1 (PARP1) and targeted it to the matrix, which resulted in the constitutive presence of PAR within the organelles. As a result, stably transfected HEK293 cells exhibited a decrease in NAD content and typical features of respiratory deficiency. Remarkably, inhibiting PARP activity revealed PAR degradation within mitochondria. Two enzymes, PAR glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3), are known to cleave PAR. Both full-length ARH3 and a PARG isoform, which arises from alternative splicing, localized to the mitochondrial matrix. This conclusion was based on the direct demonstration of their PAR-degrading activity within mitochondria of living cells. The visualization of catalytic activity establishes a new approach to identify submitochondrial localization of proteins involved in the metabolism of NAD derivatives. In addition, targeted PARP expression may serve as a compartment-specific "knock-down" of the NAD content which is readily detectable by PAR formation.

  18. Mechanical unfolding of ribose binding protein and its comparison with other periplasmic binding proteins.

    Science.gov (United States)

    Kotamarthi, Hema Chandra; Narayan, Satya; Ainavarapu, Sri Rama Koti

    2014-10-01

    Folding and unfolding studies on large, multidomain proteins are still rare despite their high abundance in genomes of prokaryotes and eukaryotes. Here, we investigate the unfolding properties of a 271 residue, two-domain ribose binding protein (RBP) from the bacterial periplasm using single-molecule force spectroscopy. We observe that RBP predominately unfolds via a two-state pathway with an unfolding force of ∼80 pN and an unfolding contour length of ∼95 nm. Only a small population (∼15%) of RBP follows three-state pathways. The ligand binding neither increases the mechanical stability nor influences the unfolding flux of RBP through different pathways. The kinetic partitioning between two-state and three-state pathways, which has been reported earlier for other periplasmic proteins, is also observed in RBP, albeit to a lesser extent. These results provide important insights into the mechanical stability and unfolding processes of large two-domain proteins and highlight the contrasting features upon ligand binding. Protein structural topology diagrams are used to explain the differences in the mechanical unfolding behavior of RBP with other periplasmic binding proteins.

  19. Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

    Science.gov (United States)

    Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

    2017-02-23

    Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

  20. Cyclic ADP-ribose as a universal calcium signal molecule in the nervous system.

    Science.gov (United States)

    Higashida, Haruhiro; Salmina, Alla B; Olovyannikova, Raissa Ya; Hashii, Minako; Yokoyama, Shigeru; Koizumi, Keita; Jin, Duo; Liu, Hong-Xiang; Lopatina, Olga; Amina, Sarwat; Islam, Mohammad Saharul; Huang, Jian-Jun; Noda, Mami

    2007-01-01

    beta-NAD(+) is as abundant as ATP in neuronal cells. beta-NAD(+) functions not only as a coenzyme but also as a substrate. beta-NAD(+)-utilizing enzymes are involved in signal transduction. We focus on ADP-ribosyl cyclase/CD38 which synthesizes cyclic ADP-ribose (cADPR), a universal Ca(2+) mobilizer from intracellular stores, from beta-NAD(+). cADPR acts through activation/modulation of ryanodine receptor Ca(2+) releasing Ca(2+) channels. cADPR synthesis in neuronal cells is stimulated or modulated via different pathways and various factors. Subtype-specific coupling of various neurotransmitter receptors with ADP-ribosyl cyclase confirms the involvement of the enzyme in signal transduction in neurons and glial cells. Moreover, cADPR/CD38 is critical in oxytocin release from the hypothalamic cell dendrites and nerve terminals in the posterior pituitary. Therefore, it is possible that pharmacological manipulation of intracellular cADPR levels through ADP-ribosyl cyclase activity or synthetic cADPR analogues may provide new therapeutic opportunities for treatment of neurodevelopmental disorders.

  1. [Healing of osseous defects by guided bone regeneration using ribose cross linked collagen membranes].

    Science.gov (United States)

    Tal, H

    2004-07-01

    The ultimate goal of periodontal therapy has long been the complete regeneration of the periodontal attachment apparatus. Guided Tissue Regeneration (GTR) and Guided Bone Regeneration (GBR) are two regenerative procedures which converted this goal from a dream to reality. In search of a biocompatible resorbable tissue barrier, collagen, being a natural protein and a weak antigen, has attracted much interest and became the focus of much intention during the 80's and the 90's. The understanding that cross linking of collagen with aldehyde sugars, especially ribose, produces collagen which is highly resistant to resorption in vivo led to the development of a "natural" Crossed-Linked Collagen Barrier (CB-SX). Animal and Human studies have shown that the newly developed membrane is biocompatible, remains intact in the tissues 6 months and more, and results in impressive guided tissue/bone regeneration. Spontaneous early exposure of the membrane is common but the healing potential of the resulted tissue dehiscence is favorable with no tendency for bacterial infection. The commercial version of the CB-SX is especially suitable for GBR procedures; it is highly recommended that the gingival flaps involved will properly be released, will lack tension, and be thoroughly sutured.

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

    Science.gov (United States)

    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

  3. Ribose Supplementation Alone or with Elevated Creatine Does Not Preserve High Energy Nucleotides or Cardiac Function in the Failing Mouse Heart.

    Directory of Open Access Journals (Sweden)

    Kiterie M E Faller

    Full Text Available Reduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE.FOUR GROUPS WERE STUDIED: sham; myocardial infarction (MI; MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN pool was decreased to a similar amount (8-14% in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV dysfunction (3-fold reduction in ejection fraction and LV hypertrophy (32-47% increased mass. Ejection fraction closely correlated with infarct size independently of treatment (r(2 = 0.63, p<0.0001, but did not correlate with myocardial creatine or TAN levels.Elevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated.

  4. Hypoglycemic neuronal death and cognitive impairment are prevented by poly(ADP-ribose) polymerase inhibitors administered after hypoglycemia.

    Science.gov (United States)

    Suh, Sang Won; Aoyama, Koji; Chen, Yongmei; Garnier, Philippe; Matsumori, Yasuhiko; Gum, Elizabeth; Liu, Jialing; Swanson, Raymond A

    2003-11-19

    Severe hypoglycemia causes neuronal death and cognitive impairment. Evidence suggests that hypoglycemic neuronal death involves excitotoxicity and DNA damage. Poly(ADP-ribose) polymerase-1 (PARP-1) normally functions in DNA repair, but promotes cell death when extensively activated by DNA damage. Cortical neuron cultures were subjected to glucose deprivation to assess the role of PARP-1 in hypoglycemic neuronal death. PARP-1-/- neurons and wild-type, PARP-1+/+ neurons treated with the PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone both showed increased resistance to glucose deprivation. A rat model of insulin-induced hypoglycemia was used to assess the therapeutic potential of PARP inhibitors after hypoglycemia. Rats subjected to severe hypoglycemia (30 min EEG isoelectricity) accumulated both nitrotyrosine and the PARP-1 product, poly(ADP-ribose), in vulnerable neurons. Treatment with PARP inhibitors immediately after hypoglycemia blocked production of poly(ADP-ribose) and reduced neuronal death by >80% in most brain regions examined. Increased neuronal survival was also achieved when PARP inhibitors were administered up to 2 hr after blood glucose correction. Behavioral and histological assessments performed 6 weeks after hypoglycemia confirmed a sustained salutary effect of PARP inhibition. These results suggest that PARP-1 activation is a major factor mediating hypoglycemic neuronal death and that PARP-1 inhibitors can rescue neurons that would otherwise die after severe hypoglycemia.

  5. Adenosine gates synaptic plasticity at hippocampal mossy fiber synapses

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  9. Adenosine receptors and stress : Studies using methylmercury, caffeine and hypoxia

    OpenAIRE

    Björklund, Olga

    2008-01-01

    Brain development is a precisely organized process that can be disturbed by various stress factors present in the diet (e.g. exposure to xenobiotics) as well as insults such as decreased oxygen supply. The consequent adverse changes in nervous system function may not necessarily be apparent until a critical age when neurodevelopmental defects may be unmasked by a subsequent challenge. Adenosine and its receptors (AR) (A1, A2A, A2B and A3) which participate in the brain stres...

  10. Synthesis of novel chromene scaffolds for adenosine receptors.

    Science.gov (United States)

    Costa, Marta; Areias, Filipe; Castro, Marian; Brea, Jose; Loza, María I; Proença, Fernanda

    2011-06-07

    A one-pot procedure was developed for the synthesis of novel 3-[amino(methoxy)methylene]-2-oxo-3,4-dihydro-2H-chromen-4-yl)-3-cyanoacetamides and chromeno[3,4-c]pyridine-1-carbonitriles from the reaction of 2-oxo-2H-chromene-3-carbonitriles and cyanoacetamides. These chromene derivatives were identified as new scaffolds for adenosine receptors and the hits 3a, 3c, 5a, and 5b were found.

  11. Severe combined immunodeficiency due to adenosine deaminase deficiency.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. Adenosine receptors in post-mortem human brain.

    Science.gov (United States)

    James, S; Xuereb, J H; Askalan, R; Richardson, P J

    1992-01-01

    1. Adenosine A2-like binding sites were characterized in post-mortem human brain membranes by examining several compounds for their ability to displace [3H]-CGS 21680 (2[p-(2 carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) binding. 2. Two A2-like binding sites were identified in the striatum. 3. The more abundant striatal site was similar to the A2a receptor previously described in rat striatum, both in its pharmacological profile and striatal localization. 4. The less abundant striatal site had a pharmacological profile similar to that of the binding site characterized in the other brain regions examined. This was intermediate in character between A1 and A2 and may represent another adenosine receptor subtype. 5. The co-purification of [3H]-CGS 21680 binding during immunoisolation of human striatal cholinergic membranes was used to assess the possible cholinergic localization of A2-like binding sites in the human striatum. Only the more abundant striatal site co-purified with cholinergic membranes. This suggests that this A2a-like site is present on cholinergic neurones in the human striatum.

  14. Identification of widespread adenosine nucleotide binding in Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

    Directory of Open Access Journals (Sweden)

    Jim Sun

    Full Text Available Defining the mechanisms of Mycobacterium tuberculosis (Mtb persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2 dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk and obtained a stable mutant (Mtb Ndk-AS that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk

  16. Thiamin diphosphate-dependent enzymes: from enzymology to metabolic regulation, drug design and disease models.

    Science.gov (United States)

    Bunik, Victoria I; Tylicki, Adam; Lukashev, Nikolay V

    2013-12-01

    Bringing a knowledge of enzymology into research in vivo and in situ is of great importance in understanding systems biology and metabolic regulation. The central metabolic significance of thiamin (vitamin B1 ) and its diphosphorylated derivative (thiamin diphosphate; ThDP), and the fundamental differences in the ThDP-dependent enzymes of metabolic networks in mammals versus plants, fungi and bacteria, or in health versus disease, suggest that these enzymes are promising targets for biotechnological and medical applications. Here, the in vivo action of known regulators of ThDP-dependent enzymes, such as synthetic structural analogs of the enzyme substrates and thiamin, is analyzed in light of the enzymological data accumulated during half a century of research. Mimicking the enzyme-specific catalytic intermediates, the phosphonate analogs of 2-oxo acids selectively inhibit particular ThDP-dependent enzymes. Because of their selectivity, use of these compounds in cellular and animal models of ThDP-dependent enzyme malfunctions improves the validity of the model and its predictive power when compared with the nonselective and enzymatically less characterized oxythiamin and pyrithiamin. In vitro studies of the interaction of thiamin analogs and their biological derivatives with potential in vivo targets are necessary to identify and attenuate the analog selectivity. For both the substrate and thiamin synthetic analogs, in vitro reactivities with potential targets are highly relevant in vivo. However, effective concentrations in vivo are often higher than in vitro studies would suggest. The significance of specific inihibition of the ThDP-dependent enzymes for the development of herbicides, antibiotics, anticancer and neuroprotective strategies is discussed.

  17. Flagellar Radial Spokes Contain a Ca2+-stimulated Nucleoside Diphosphate Kinase

    Science.gov (United States)

    Patel-King, Ramila S.; Gorbatyuk, Oksana; Takebe, Sachiko; King, Stephen M.

    2004-01-01

    The radial spokes are required for Ca2+-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca2+-independent manner, whereas IQ2 and IQ3 show Ca2+-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca2+. This Ca2+-responsive enzyme, which accounts for ∼45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca2+-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes. PMID:15194815

  18. Bacterial and plant HAD enzymes catalyse a missing phosphatase step in thiamin diphosphate biosynthesis.

    Science.gov (United States)

    Hasnain, Ghulam; Roje, Sanja; Sa, Na; Zallot, Rémi; Ziemak, Michael J; de Crécy-Lagard, Valérie; Gregory, Jesse F; Hanson, Andrew D

    2016-01-15

    The penultimate step of thiamin diphosphate (ThDP) synthesis in plants and many bacteria is dephosphorylation of thiamin monophosphate (ThMP). Non-specific phosphatases have been thought to mediate this step and no genes encoding specific ThMP phosphatases (ThMPases) are known. Comparative genomic analysis uncovered bacterial haloacid dehalogenase (HAD) phosphatase family genes (from subfamilies IA and IB) that cluster on the chromosome with, or are fused to, thiamin synthesis genes and are thus candidates for the missing phosphatase (ThMPase). Three typical candidates (from Anaerotruncus colihominis, Dorea longicatena and Syntrophomonas wolfei) were shown to have efficient in vivo ThMPase activity by expressing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro assays confirmed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arabidopsis thaliana ThMPase homologue (At4g29530) of unknown function whose expression pattern and compartmentation fit with a role in ThDP synthesis was shown to have in vivo ThMPase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional inactivation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, indicating that Arabidopsis has at least one other ThMPase gene. The Zea mays orthologue of At4g29530 (GRMZM2G035134) was also shown to have ThMPase activity. These data identify HAD genes specifying the elusive ThMPase activity, indicate that ThMPases are substrate-specific rather than general phosphatases and suggest that different evolutionary lineages have recruited ThMPases independently from different branches of the HAD family.

  19. Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

    Science.gov (United States)

    Vaccaro, J A; Crane, E J; Harris, T K; Washabaugh, M W

    1995-10-01

    Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 < or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Heterologous expression of active human uridine diphosphate glucuronosyltransferase 1A3 in Chinese hamster lung cells

    Institute of Scientific and Technical Information of China (English)

    Ya-Kun Chen; Xin Li; Shu-Qing Chen; Su Zeng

    2005-01-01

    AIM: To obtain the active human recombinant uridine diphosphate glucuronosyltransferase 1A3 (UGT1A3) enzyme from Chinese hamster lung (CHL) cells.METHODS: The full-length UGT1A3 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR)using total RNA from human liver as template. The correct fragment confirmed by sequencing was subcloned into the mammalian expression vector pcDNA3.1 (+), and the recombinant vector was transfected into CHL cells using a calcium phosphate method. Expressed UGT1A3 protein was prepared from CHL cells resistant to neomycin (G418). Then the protein was added into a reaction mixture for glucuronidation of quercetin. The glucuronidation activity of UGT1A3 was determined by reverse phase-high performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD). The quercetin glucuronide was confirmed by hydrolysis with β-glucuronidase. Control experiments were performed in parallel. The transcriptions of recombinants were also determined by RT-PCR.RESULTS: The gene was confirmed to be an allele (UGT1A3-3) of UGT1A3 by DNA sequencing. The fragment was introduced into pcDNA3.1 (+) successfully. Several colonies were obtained under the selection pressure of G418.The result of RT-PCR showed transcription of recombinants in mRNA level. Glucuronidation assay and HPLC analysis indicated UGT1A3 expressed heterologously in CHL cells was in an active form, and one of the gulcuronides corresponding to quercetin was also detected.CONCLUSION: Correct sequence of UGT1A3 gene can be obtained, and active UGT1A3 enzyme is expressed heterologously in CHL cells.

  1. Mycobacterium tuberculosis nucleoside diphosphate kinase inactivates small GTPases leading to evasion of innate immunity.

    Directory of Open Access Journals (Sweden)

    Jim Sun

    Full Text Available Defining the mechanisms of Mycobacterium tuberculosis (Mtb persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2 dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk and obtained a stable mutant (Mtb Ndk-AS that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67(phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67(phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67(phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk

  2. Observation of an Acryloyl–Thiamin Diphosphate Adduct in the First Step of Clavulanic Acid Biosynthesis

    Science.gov (United States)

    Merski, Matthew

    2011-01-01

    The first committed biosynthetic step toward clavulanic acid, the clinically-important β-lactamase inhibitor, is catalyzed by the thiamin diphosphate (ThDP)-dependent enzyme N2-(2-carboxyethyl)arginine synthase (CEAS). This protein carries out a unique reaction among ThDP-dependent processes in which a C–N bond is formed, and an electrophilic acryloyl–thiazolium intermediate of ThDP is proposed to be involved, unlike the nucleophilic enamine species typically generated by this class of enzymes. Here we present evidence for the existence of the putative acryloyl adduct, and report the unexpected observation of a long-wavelength chromophore (λ = 433 nm), which we attribute to this enzyme bound species. Chemical models were synthesized that both confirm its expected absorption (λ = 310–320 nm), and exclude self-condensation and intramolecular imine formation with the cofactor as its cause. Circular dichroism experiments and others discount charge transfer as a likely explanation for the ~120 nm red shift of the chromophore (~25 kcal). Examples are well-known of charged molecules that exhibit significantly red-shifted UV-visible spectra compared to their neutral forms as, for example, polyene cations and dyes such as indigo and the cyanines. Rhodopsin is the classic biochemical example where the protein (opsin)-bound protonated Schiff base of retinal displays a remarkable range of red-shifted absorptions modulated by the protein environment. Similar tuning of the chromophoric behavior of the enzyme-bound CEAS acryloyl•ThDP species may be occurring. PMID:18052280

  3. Molecular cloning and characterization of three isoprenyl diphosphate synthase genes from alfalfa.

    Science.gov (United States)

    Sun, Yan; Long, Ruicai; Kang, Junmei; Zhang, Tiejun; Zhang, Ze; Zhou, He; Yang, Qingchuan

    2013-02-01

    Isoprenoid is the precursor for the biosynthesis of saponins, abscisic acid, gibberellins, chlorophylls and many other products in plants. Saponins are an important group of bioactive plant natural products. The alfalfa (Medicago sativa L.) saponins are glycosides of different triterpene aglycones and possess many biological activities. We isolated three genes (MsFPPS, MsGPPS and MsGGPPS) encoding isoprenyl diphosphate synthases (IDS) from alfalfa via a homology-based PCR approach. The enzyme activity assay of purified recombined MsFPPS and MsGGPPS expressed in Escherichia coli indicated that they all had IDS activity. Expression analysis of the three genes in different alfalfa tissues using real time PCR displayed that they were expressed in all tissues although they had a different expression patterns. MsFPPS and MsGPS displayed a significant increase in transcript level in response to methyl jasmonate, but the transcript level of MsGGPPS decreased obviously. To elucidate the functions of the three IDSs, their overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in tobacco plants was applied and analyzed. The T(0) transgenic plants of MsFPPS showed high levels of squalene content when compared with control. However, no differences were detected in T(0) transgenic plants of MsGPPS and MsGGPPS. In addition, the overexpression of MsFPPS induced senescence response in transgenic plant leaves. This result may indicate that MsFPPS performs a role not only in phytosterol and triterpene biosynthesis, but also in growth regulation.

  4. The antihyperalgesic effect of cytidine-5'-diphosphate-choline in neuropathic and inflammatory pain models.

    Science.gov (United States)

    Bagdas, Deniz; Sonat, Fusun Ak; Hamurtekin, Emre; Sonal, Songul; Gurun, Mine Sibel

    2011-09-01

    This study was designed to test the effects of intracerebroventricularly (i.c.v.) administered CDP-choline (cytidine-5'-diphosphate-choline; citicoline) and its metabolites in rat models of inflammatory and neuropathic pain. The i.c.v. administration of CDP-choline (0.5, 1.0 and 2.0 µmol) produced a dose and time-dependent reversal of mechanical hyperalgesia in both carrageenan-induced inflammatory and chronic constriction injury-induced neuropathic pain models in rats. The antihyperalgesic effect of CDP-choline was similar to that observed with an equimolar dose of choline (1 µmol). The CDP-choline-induced antihyperalgesic effect was prevented by central administration of the neuronal high-affinity choline uptake inhibitor hemicholinium-3 (1 µg), the nonselective nicotinic receptor antagonist mecamylamine (50 µg), the α7-selective nicotinic ACh receptor antagonist, α-bungarotoxin (2 µg) and the γ-aminobutyric acid B receptor antagonist CGP-35348 (20 µg). In contrast, i.c.v. pretreatment with the nonselective opioid receptor antagonist naloxone (10 µg) only prevented the CDP-choline-induced antihyperalgesic effect in the neuropathic pain model while the nonselective muscarinic receptor antagonist atropine (10 µg) did not alter the antihyperalgesic effect in the two models. These results indicate that CDP-choline-elicited antihyperalgesic effect in different models of pain occurs through mechanisms that seem to involve an interaction with supraspinal α7-selective nicotinic ACh receptors, and γ-aminobutyric acid B receptors, whereas central opioid receptors have a role only in the neuropathic pain model.

  5. Poly (ADP-ribose) polymerase inhibitor efficacy in head and neck cancer.

    Science.gov (United States)

    Heitmann, Jana; Geeleher, Paul; Zuo, Zhixiang; Weichselbaum, Ralph R; Vokes, Everett E; Fetscher, Sebastian; Seiwert, Tanguy Y

    2014-09-01

    Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown single agent activity against tumors with deficiencies in the DNA repair mechanism homologous recombination including, but not limited to those harboring BRCA mutations. We hypothesized that, in the context of homologous recombination deficiency (HRD), PARPi could have an effect in head and neck cancer (HNC). We evaluated TCGA data for evidence of HRD using a copy number data signature established for breast cancer. The comparative potency of three PARPi was evaluated using cell viability assays in a panel of HNC cell lines and response was compared to BRCA-deficient breast cancer cell lines. The change in foci formation of γH2AX and RAD51 was assessed with immunofluorescent staining after exposure to a PARPi. Baseline gene expression was analyzed using microarray data. We found a subgroup in the TCGA HNC cohort harboring genomic aberrations consistent with HRD in breast cancer. Rucaparib activity was superior to olaparib and veliparib and showed single agent activity in a subset of HNC cell lines that was comparable to BRCA-deficient breast cancer cell lines. Rucaparib-sensitive and rucaparib-resistant groups showed significant differences in γH2AX and RAD51 foci formation after rucaparib exposure. Expression of genes involved in chromosome structure was strongly associated with rucaparib resistance. We demonstrate that PARPi are effective in a subset of HNC cell lines and propose that HRD may be present in HNC in vivo suggesting that these compounds could play a role in the treatment of HNC. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Clinical Trials of Poly(ADP-Ribose) Polymerase Inhibitors for Cancer Therapy: A Review.

    Science.gov (United States)

    Buege, Michael; Mahajan, Pramod B

    2015-01-01

    Poly(ADP-Ribose) Polymerase (PARP) is a family of enzymes involved in DNA repair, genome stability, cellular energy metabolism and cell division. Inhibition of PARP-1, the well characterized member of this family, has been explored as a strategy for enhancing anti-cancer activity of existing drugs and for developing new drugs. Recently unique enzymatic properties and biological functions of PARP-2 and PARP-3 have been discovered, further expanding the utility of PARP as a target for cancer pharmacotherapy. We compare and contrast the structural and enzymatic properties of these three members of the PARP family. Interactions of these enzymes with proteins specific to different DNA repair pathways are summarized. Further, we evaluate progress on development of PARP inhibitors as anticancer agents. Results of Phase I and Phase II clinical trials of seven PARP inhibitors, used alone or in combination with known anticancer agents are reviewed highlighting common observations regarding the maximum tolerable dose, adverse reactions profile, PARP inhibition and anticancer effects. While further clinical studies are warranted, based on current data, Olaparib (Ola), Veliparib (Veli) and Rucaparib (Ruca) offer considerable potential. Prolonged exposure to Ola and Veli leads to resistant cancer cells, primarily through restoration of the HR pathway, overexpression of the P-glycoprotein efflux pump or modulation of PARP expression. Some resistant cancer cells continue to respond to platinum based drugs, encouraging further development of PARP inhibitors for cancer treatment. Future course of this research, specifically focusing on use of PARP inhibition as a strategy for personalized cancer therapy, is discussed.

  7. Poly (ADP-ribose) polymerase inhibition enhances trastuzumab antitumour activity in HER2 overexpressing breast cancer.

    Science.gov (United States)

    García-Parra, Jetzabel; Dalmases, Alba; Morancho, Beatriz; Arpí, Oriol; Menendez, Silvia; Sabbaghi, MohammadA; Zazo, Sandra; Chamizo, Cristina; Madoz, Juan; Eroles, Pilar; Servitja, Sonia; Tusquets, Ignasi; Yelamos, Jose; Lluch, Ana; Arribas, Joaquin; Rojo, Federico; Rovira, Ana; Albanell, Joan

    2014-10-01

    Poly (ADP-ribose) polymerase (PARP) inhibitors have shown promising results in Breast Cancer (BRCA) deficient breast cancer, but not in molecularly unselected patient populations. Two lines of research in this field are needed: the identification of novel subsets of patients that could potentially benefit from PARP inhibitors and the discovery of suitable targeted therapies for combination strategies. We tested PARP inhibition, alone or combined with the anti-HER2 antibody trastuzumab on HER2+ breast cancer. We used two PARP inhibitors in clinical development, olaparib and rucaparib, as well as genetic downmodulation of PARP-1 for in vitro studies. DNA damage was studied by the formation of γH2AX foci and comet assay. Finally, the in vivo anti-tumour effect of olaparib and trastuzumab was examined in nude mice subcutaneously implanted with BT474 cells. In a panel of four HER2 overexpressing breast cancer cell lines, both olaparib and rucaparib significantly decreased cell growth and enhanced anti-tumour effects of trastuzumab. Cells exposed to olaparib and trastuzumab had greater DNA damage than cells exposed to each agent alone. Mechanistic exploratory assays showed that trastuzumab downmodulated the homologous recombination protein proliferating cell nuclear antigen (PCNA). Combination treatment in the BT474 xenograft model resulted in enhanced growth inhibition, reduced tumour cell proliferation, and increased DNA damage and apoptosis. Taken together, our results show that PARP inhibition has antitumour effects and increases trastuzumab activity in HER2 overexpressing breast cancer. These findings make this novel combination a promising strategy for clinical development. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex.

    Science.gov (United States)

    Sümegi, B; Alkonyi, I

    1983-11-02

    In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2016-12-01

    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.

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

    Science.gov (United States)

    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.

  12. Acute hyperammonemia and systemic inflammation is associated with increased extracellular brain adenosine in rats

    DEFF Research Database (Denmark)

    Bjerring, Peter Nissen; Dale, Nicholas; Larsen, Fin Stolze

    2015-01-01

    Acute liver failure (ALF) can lead to brain edema, cerebral hyperperfusion and intracranial hypertension. These complications are thought to be mediated by hyperammonemia and inflammation leading to altered brain metabolism. As increased levels of adenosine degradation products have been found...... in brain tissue of patients with ALF we investigated whether hyperammonemia could induce adenosine release in brain tissue. Since adenosine is a potent vasodilator and modulator of cerebral metabolism we furthermore studied the effect of adenosine receptor ligands on intracranial pressure (ICP......) and cerebral blood flow (CBF). We measured the adenosine concentration with biosensors in rat brain slices exposed to ammonia and in a rat model with hyperammonemia and systemic inflammation. Exposure to ammonia in concentrations from 0.15-10 mM led to increases in the cortical adenosine concentration up to 18...

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  14. Adenosine contributes to blood flow regulation in the exercising human leg by increasing prostaglandin and nitric oxide formation

    DEFF Research Database (Denmark)

    Mortensen, Stefan; Nyberg, Michael; Thaning, Pia

    2009-01-01

    /min); (2) whether adenosine-induced vasodilation is mediated via formation of prostaglandins and/or NO; and (3) the femoral arterial and venous plasma adenosine concentrations during leg exercise with the microdialysis technique in a total of 24 healthy, male subjects. Inhibition of adenosine receptors......+/-8%, and 66+/-8%, respectively (Pplasma adenosine concentrations were similar at rest and during exercise. These results suggest that adenosine contributes to the regulation of skeletal muscle blood flow by stimulating prostaglandin and NO synthesis.......Adenosine can induce vasodilation in skeletal muscle, but to what extent adenosine exerts its effect via formation of other vasodilators and whether there is redundancy between adenosine and other vasodilators remain unclear. We tested the hypothesis that adenosine, prostaglandins, and NO act...

  15. Structural and thermodynamic basis of the inhibition of Leishmania major farnesyl diphosphate synthase by nitrogen-containing bisphosphonates

    Energy Technology Data Exchange (ETDEWEB)

    Aripirala, Srinivas [Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States); Gonzalez-Pacanowska, Dolores [López-Neyra Institute of Parasitology and Biomedicine, 18001 Granada (Spain); Oldfield, Eric [University of Illinois at Urbana-Champaign, Urbana, IL 61801 (United States); Kaiser, Marcel [University of Basel, Petersplatz 1, CH-4003 Basel (Switzerland); Amzel, L. Mario, E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Gabelli, Sandra B., E-mail: mamzel@jhmi.edu [Johns Hopkins University School of Medicine, 725 N. Wolfe Street WBSB 604, Baltimore, MD 21205 (United States); Johns Hopkins University School of Medicine, Baltimore, MD 21205 (United States); Johns Hopkins University, 725 North Wolfe Street WBSB 605, Baltimore, MD 21210 (United States)

    2014-03-01

    Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneous leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.

  16. Involvement of an ent-copalyl diphosphate synthase in tissue-specific accumulation of specialized diterpenes in Andrographis paniculata.

    Science.gov (United States)

    Misra, Rajesh Chandra; Garg, Anchal; Roy, Sudeep; Chanotiya, Chandan Singh; Vasudev, Prema G; Ghosh, Sumit

    2015-11-01

    Ent-labdane-related diterpene (ent-LRD) specialized (i.e. secondary) metabolites of the medicinal plant kalmegh (Andrographis paniculata) have long been known for several pharmacological activities. However, our understanding of the ent-LRD biosynthetic pathway has remained largely incomplete. Since ent-LRDs accumulate in leaves, we carried out a comparative transcriptional analysis using leaf and root tissues, and identified 389 differentially expressed transcripts, including 223 transcripts that were preferentially expressed in leaf tissue. Analysis of the transcripts revealed various specialized metabolic pathways, including transcripts of the ent-LRD biosynthetic pathway. Two class II diterpene synthases (ApCPS1 and ApCPS2) along with one (ApCPS1') and two (ApCPS2' and ApCPS2″) transcriptional variants that were the outcomes of alternative splicing of the precursor mRNA and alternative transcriptional termination, respectively, were identified. ApCPS1 and ApCPS2 encode for 832- and 817-amino acids proteins, respectively, and are phylogenetically related to the dicotyledons ent-copalyl diphosphate synthases (ent-CPSs). The spatio-temporal patterns of ent-LRD metabolites accumulation and gene expression suggested a likely role for ApCPS1 in general (i.e. primary) metabolism, perhaps by providing precursor for the biosynthesis of phytohormone gibberellin (GA). However, ApCPS2 is potentially involved in tissue-specific accumulation of ent-LRD specialized metabolites. Bacterially expressed recombinant ApCPS2 catalyzed the conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP), the general precursor of diterpenes to ent-copalyl diphosphate (ent-CPP), the precursor of ent-LRDs. Taken together, these results advance our understanding of the tissue-specific accumulation of specialized ent-LRDs of medicinal importance.

  17. Poly(ADP-ribose) binds to the splicing factor ASF/SF2 and regulates its phosphorylation by DNA topoisomerase I.

    Science.gov (United States)

    Malanga, Maria; Czubaty, Alicja; Girstun, Agnieszka; Staron, Krzysztof; Althaus, Felix R

    2008-07-18

    Human DNA topoisomerase I plays a dual role in transcription, by controlling DNA supercoiling and by acting as a specific kinase for the SR-protein family of splicing factors. The two activities are mutually exclusive, but the identity of the molecular switch is unknown. Here we identify poly(ADP-ribose) as a physiological regulator of the two topoisomerase I functions. We found that, in the presence of both DNA and the alternative splicing factor/splicing factor 2 (ASF/SF2, a prototypical SR-protein), poly(ADP-ribose) affected topoisomerase I substrate selection and gradually shifted enzyme activity from protein phosphorylation to DNA cleavage. A likely mechanistic explanation was offered by the discovery that poly(ADP-ribose) forms a high affinity complex with ASF/SF2 thereby leaving topoisomerase I available for directing its action onto DNA. We identified two functionally important domains, RRM1 and RS, as specific poly(ADP-ribose) binding targets. Two independent lines of evidence emphasize the potential biological relevance of our findings: (i) in HeLa nuclear extracts, ASF/SF2, but not histone, phosphorylation was inhibited by poly(ADP-ribose); (ii) an in silico study based on gene expression profiling data revealed an increased incidence of alternative splicing within a subset of inflammatory response genes that are dysregulated in cells lacking a functional poly(ADP-ribose) polymerase-1. We propose that poly(ADP-ribose) targeting of topoisomerase I and ASF/SF2 functions may participate in the regulation of gene expression.

  18. Endogenous adenosine and hemorrhagic shock: effects of caffeine administration or caffeine withdrawal.

    OpenAIRE

    Conlay, L A; Evoniuk, G; Wurtman, R J

    1988-01-01

    Plasma adenosine concentrations doubled when rats were subjected to 90 min of profound hemorrhagic shock. Administration of caffeine (20 mg per kg of body weight), an adenosine-receptor antagonist, attenuated the hemorrhage-induced decrease in blood pressure. In contrast, chronic caffeine consumption (0.1% in drinking water), followed by a brief period of caffeine withdrawal, amplified the hypotensive response to hemorrhage. These data suggest that endogenous adenosine participates in the hyp...

  19. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia.

    OpenAIRE

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

    2014-01-01

    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by ...

  20. Interaction between Intrathecal Gabapentin and Adenosine in the Formalin Test of Rats

    OpenAIRE

    Yoon, Myung Ha; Choi, Jeong Il; Park, Heon Chang; Bae, Hong Beom

    2004-01-01

    Spinal gabapentin and adenosine have been known to display an antinociceptive effect. We evaluated the nature of the interaction between gabapentin and adenosine in formalin-induced nociception at the spinal level. Male Sprague-Dawley rats were prepared for intrathecal catheterization. Pain was evoked by injection of formalin solution (5%, 50 µL) into the hindpaw. After examination of the effects of gabapentin and adenosine, the resulting interaction was investigated with isobolographic and f...

  1. Endogenous adenosine and hemorrhagic shock: effects of caffeine administration or caffeine withdrawal.

    OpenAIRE

    Conlay, L A; Evoniuk, G; Wurtman, R.J.

    1988-01-01

    Plasma adenosine concentrations doubled when rats were subjected to 90 min of profound hemorrhagic shock. Administration of caffeine (20 mg per kg of body weight), an adenosine-receptor antagonist, attenuated the hemorrhage-induced decrease in blood pressure. In contrast, chronic caffeine consumption (0.1% in drinking water), followed by a brief period of caffeine withdrawal, amplified the hypotensive response to hemorrhage. These data suggest that endogenous adenosine participates in the hyp...

  2. Regeneration of hemopoietic and lymphoid tissues following total-body irradiation and therapeutic administration of thiamin diphosphate. [Mice, gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Vavrova, J.; Nouza, K.; Petjrek, P.

    1977-01-01

    Analysis was made of the mechanism of therapeutic application of thiamine diphosphate (TDP) in radiation sickness in mice. This agent increased the number of endogenous colonies in the spleen and incorporation of /sup 59/Fe in spleen and bone marrow with sublethal doses of radiation (500 and 600 R) and has no effect with a lethal dose (750 R). After administration of TDP to mice exposed to 500 R radiation, there is faster DNA synthesis in the spleen, thymus and bone marrow, as well as reliable increase in number of nuclear cells in femoral marrow.

  3. Topical adenosine increases the proportion of thick hair in Caucasian men with androgenetic alopecia.

    Science.gov (United States)

    Iwabuchi, Tokuro; Ideta, Ritsuro; Ehama, Ritsuko; Yamanishi, Haruyo; Iino, Masato; Nakazawa, Yosuke; Kobayashi, Takashi; Ohyama, Manabu; Kishimoto, Jiro

    2016-05-01

    Adenosine is an effective treatment for androgenetic alopecia (AGA) in Japanese men and women. Adenosine exerts its effects by significantly increasing the proportion of thick hair. In this study, we assessed the clinical outcome of adenosine treatment for 6 months in 38 Caucasian men. The change in proportion of thick hair (≥60 μm) compared with baseline in the adenosine group was significantly higher than that in the placebo group (P thick hair in Caucasian men with AGA as well as in Japanese men and women.

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

    Science.gov (United States)

    Cheon, Yong-Pil

    2016-06-01

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

  5. Higher number of pentosidine cross-links induced by ribose does not alter tissue stiffness of cancellous bone

    Energy Technology Data Exchange (ETDEWEB)

    Willems, Nop M.B.K., E-mail: n.willems@acta.nl [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Langenbach, Geerling E.J. [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Stoop, Reinout [Dept. of Metabolic Health Research, TNO, P.O. Box 2215, 2301 CE Leiden (Netherlands); Toonder, Jaap M.J. den [Dept. of Mechanical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Mulder, Lars [Dept. of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven (Netherlands); Zentner, Andrej [Dept. of Orthodontics, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands); Everts, Vincent [Dept. of Oral Cell Biology and Functional Anatomy, MOVE Research Institute, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University, Gustav Mahlerlaan 3004, 1081 LA Amsterdam (Netherlands)

    2014-09-01

    The role of mature collagen cross-links, pentosidine (Pen) cross-links in particular, in the micromechanical properties of cancellous bone is unknown. The aim of this study was to examine nonenzymatic glycation effects on tissue stiffness of demineralized and non-demineralized cancellous bone. A total of 60 bone samples were derived from mandibular condyles of six pigs, and assigned to either control or experimental groups. Experimental handling included incubation in phosphate buffered saline alone or with 0.2 M ribose at 37 °C for 15 days and, in some of the samples, subsequent complete demineralization of the sample surface using 8% EDTA. Before and after experimental handling, bone microarchitecture and tissue mineral density were examined by means of microcomputed tomography. After experimental handling, the collagen content and the number of Pen, hydroxylysylpyridinoline (HP), and lysylpyridinoline (LP) cross-links were estimated using HPLC, and tissue stiffness was assessed by means of nanoindentation. Ribose treatment caused an up to 300-fold increase in the number of Pen cross-links compared to nonribose-incubated controls, but did not affect the number of HP and LP cross-links. This increase in the number of Pen cross-links had no influence on tissue stiffness of both demineralized and nondemineralized bone samples. These findings suggest that Pen cross-links do not play a significant role in bone tissue stiffness. - Highlights: • The assessment of effects of glycation in bone using HPLC, microCT, and nanoindentation • Ribose incubation: 300‐fold increase in the number of pentosidine cross-links • 300‐fold increase in the number of pentosidine cross-links: no changes in bone tissue stiffness.

  6. Occurrence of Tourmaline in Metasedimentary Rocks of the Isua Supracrustal Belt, Greenland: Implications for Ribose Stabilization in Hadean Marine Sediments

    Science.gov (United States)

    Mishima, Shinpei; Ohtomo, Yoko; Kakegawa, Takeshi

    2016-06-01

    Abiotic formation of RNA was important for the emergence of terrestrial life, but the acknowledged difficulties of generating and stabilizing ribose have often raised questions regarding how the first RNA might have formed. Previous researchers have proposed that borate could have stabilized ribose; however, the availability of borate on the early Earth has been the subject of intense debate. In order to examine whether borate was available on the early Earth, this study examined metasedimentary rocks from the Isua Supracrustal Belt. Garnet, biotite, and quartz comprise the major constituents of the examined rocks. Field relationships and the chemical compositions of the examined rocks suggest sedimentary origin. The present study found that garnet crystals contain a number of inclusions of tourmaline (a type of borosilicate mineral). All tourmaline crystals are Fe-rich and categorized as schorl. Both garnet and tourmaline often contain graphite inclusions and this close association of tourmaline with garnet and graphite has not been recognized previously. Garnet-biotite and graphite geothermometers suggest that the tourmaline in garnet experienced peak metamorphic conditions (~500 °C and 5 kbar). The mineralogical characteristics of the tourmaline and the whole rock composition indicate that the tourmaline formed authigenically in the sediment during diagenesis and/or early metamorphism. Clay minerals in modern sediments have the capability to adsorb and concentrate borate, which could lead to boron enrichment during diagenesis, followed by tourmaline formation under metamorphic conditions. Clay minerals, deposited on the early Archean seafloor, were the precursors of the garnet and biotite in the examined samples. The studied tourmaline crystals were most likely formed in the same way as modern tourmaline in marine sediments. Therefore, boron enrichment by clays must have been possible even during the early Archean. Thus, similar enrichment could have been

  7. Poly(ADP-ribose polymerase (PARP-1 is not involved in DNA double-strand break recovery

    Directory of Open Access Journals (Sweden)

    Fernet Marie

    2003-07-01

    Full Text Available Abstract Background The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H2O2 and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose polymerase (PARP-1 in DNA double-strand break repair. Results PARP-1-/- were considerably more sensitive than PARP-1+/+ 3T3s to induced cell kill by γ-rays and H2O2. However, the two cell lines did not show any significant difference in the susceptibility to neocarzinostatin below 1.5 nM drug. Restoration of PARP-1 expression in PARP-1-/- 3T3s by retroviral transfection of the full PARP-1 cDNA did not induce any change in neocarzinostatin response. Moreover the incidence and the rejoining kinetics of neocarzinostatin-induced DNA double-strand breaks were identical in PARP-1+/+ and PARP-1-/- 3T3s. Poly(ADP-ribose synthesis following γ-rays and H2O2 was observed in PARP-1-proficient cells only. In contrast neocarzinostatin, even at supra-lethal concentration, was unable to initiate PARP-1 activation yet it induced H2AX histone phosphorylation in both PARP1+/+ and PARP-1-/- 3T3s as efficiently as γ-rays and H2O2. Conclusions The results show that PARP-1 is not a major determinant of DNA double-strand break recovery with either strand break rejoining or cell survival as an endpoint. Even though both PARP-1 and ATM activation are major determinants of the cell response to γ-rays and H2O2, data suggest that PARP-1-dependent poly(ADP-ribose synthesis and ATM-dependent H2AX phosphorylation, are not inter-related in the repair pathway of neocarzinostatin-induced DNA double-strand breaks.

  8. Occurrence of Tourmaline in Metasedimentary Rocks of the Isua Supracrustal Belt, Greenland: Implications for Ribose Stabilization in Hadean Marine Sediments.

    Science.gov (United States)

    Mishima, Shinpei; Ohtomo, Yoko; Kakegawa, Takeshi

    2016-06-01

    Abiotic formation of RNA was important for the emergence of terrestrial life, but the acknowledged difficulties of generating and stabilizing ribose have often raised questions regarding how the first RNA might have formed. Previous researchers have proposed that borate could have stabilized ribose; however, the availability of borate on the early Earth has been the subject of intense debate. In order to examine whether borate was available on the early Earth, this study examined metasedimentary rocks from the Isua Supracrustal Belt. Garnet, biotite, and quartz comprise the major constituents of the examined rocks. Field relationships and the chemical compositions of the examined rocks suggest sedimentary origin. The present study found that garnet crystals contain a number of inclusions of tourmaline (a type of borosilicate mineral). All tourmaline crystals are Fe-rich and categorized as schorl. Both garnet and tourmaline often contain graphite inclusions and this close association of tourmaline with garnet and graphite has not been recognized previously. Garnet-biotite and graphite geothermometers suggest that the tourmaline in garnet experienced peak metamorphic conditions (~500 °C and 5 kbar). The mineralogical characteristics of the tourmaline and the whole rock composition indicate that the tourmaline formed authigenically in the sediment during diagenesis and/or early metamorphism. Clay minerals in modern sediments have the capability to adsorb and concentrate borate, which could lead to boron enrichment during diagenesis, followed by tourmaline formation under metamorphic conditions. Clay minerals, deposited on the early Archean seafloor, were the precursors of the garnet and biotite in the examined samples. The studied tourmaline crystals were most likely formed in the same way as modern tourmaline in marine sediments. Therefore, boron enrichment by clays must have been possible even during the early Archean. Thus, similar enrichment could have been

  9. 葡萄糖发酵液D-核糖含量的高效液相色谱分析%High Performance Liquid Chromatography Analysis of D-Ribose Content in Glucose Zymotic Fluid

    Institute of Scientific and Technical Information of China (English)

    张津枫; 邓国才; 杨四海; 陈荣悌; 王健刚

    2001-01-01

    The method has been put forward to analyze the content of D-ribose obtained from glucose zymotic fluid.D-ribose was determined by HPLC,on a Sugar Pak Ⅰ column,by using a refractive index detector and a mobile phase of pure water.We have also studied the separation conditions of sugars.It has many advantages,such as rapid analysis,high sensitivity and high precision and good reproducibility.The recovery of D-ribose were 95.5%-104%,RSD were less than 1.5%,the detection limit of D-Ribose was 50 ng.

  10. Update on Poly-ADP-ribose polymerase inhibition for ovarian cancer treatment.

    Science.gov (United States)

    Papa, Anselmo; Caruso, Davide; Strudel, Martina; Tomao, Silverio; Tomao, Federica

    2016-09-15

    Despite standard treatment for epithelial ovarian cancer (EOC), that involves cytoreductive surgery followed by platinum-based chemotherapy, and initial high response rates to these, up to 80 % of patients experience relapses with a median progression-free survival of 12-18 months. There remains an urgent need for novel targeted therapies to improve clinical outcomes in ovarian cancer. Of the many targeted therapies currently under evaluation, the most promising strategies developed thus far are antiangiogenic agents and Poly(ADP-ribose) polymerase (PARP) inhibitors. Particularly, PARP inhibitors are active in cells that have impaired repair of DNA by the homologous recombination (HR) pathway. Cells with mutated breast related cancer antigens (BRCA) function have HR deficiency, which is also present in a significant proportion of non-BRCA-mutated ovarian cancer ("BRCAness" ovarian cancer). The prevalence of germline BRCA mutations in EOC has historically been estimated to be around 10-15 %. However, recent reports suggest that this may be a gross underestimate, especially in women with high-grade serous ovarian cancer (HGSOC). The emergence of the DNA repair pathway as a rational target in various cancers led to the development of the PARP inhibitors. The concept of tumor-selective synthetic lethality heralded the beginning of an eventful decade, culminating in the approval by regulatory authorities both in Europe as a maintenance therapy and in the United States treatment for advanced recurrent disease of the first oral PARP inhibitor, olaparib, for the treatment of BRCA-mutated ovarian cancer patients. Other PARP inhibitors are clearly effective in this disease and, within the next years, the results of ongoing randomized trials will clarify their respective roles. This review will discuss the different PARP inhibitors in development and the potential use of this class of agents in the future. Moreover, combination strategies involving PARP inhibitors are

  11. Binding of nitrogen-containing bisphosphonates (N-BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chuan-Hsiang; Gabelli, Sandra B.; Oldfield, Eric; Amzel, L. Mario (UIUC); (JHU-MED)

    2010-11-15

    Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy-related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen-containing bisphosphonates (N-BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti-trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N-BP inhibitors show that the C-1 hydroxyl and the nitrogen-containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N-BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N-BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS.

  12. Active site binding modes of inhibitors of Staphylococcus aureus mevalonate diphosphate decarboxylase from docking and molecular dynamics simulations.

    Science.gov (United States)

    Addo, James K; Skaff, D Andrew; Miziorko, Henry M

    2016-01-01

    Bacterial mevalonate diphosphate decarboxylase (MDD) is an attractive therapeutic target for antibacterial drug development. In this work, we discuss a combined docking and molecular dynamics strategy toward inhibitor binding to bacterial MDD. The docking parameters utilized in this study were first validated with observations for the inhibitors 6-fluoromevalonate diphosphate (FMVAPP) and diphosphoglycolylproline (DPGP) using existing structures for the Staphylococcus epidermidis enzyme. The validated docking protocol was then used to predict structures of the inhibitors bound to Staphylococcus aureus MDD using the unliganded crystal structure of Staphylococcus aureus MDD. We also investigated a possible interactions improvement by combining this docking method with molecular dynamics simulations. Thus, the predicted docking structures were analyzed in a molecular dynamics trajectory to generate dynamic models and reinforce the predicted binding modes. FMVAPP is predicted to make more extensive contacts with S. aureus MDD, forming stable hydrogen bonds with Arg144, Arg193, Lys21, Ser107, and Tyr18, as well as making stable hydrophobic interactions with Tyr18, Trp19, and Met196. The differences in predicted binding are supported by experimentally determined Ki values of 0.23 ± 0.02 and 34 ± 8 μM, for FMVAPP and DPGP, respectively. The structural information coupled with the kinetic characterization obtained from this study should be useful in defining the requirements for inhibition as well as in guiding the selection of active compounds for inhibitor optimization.

  13. The Thiamine diphosphate dependent Enzyme Engineering Database: A tool for the systematic analysis of sequence and structure relations

    Directory of Open Access Journals (Sweden)

    Radloff Robert

    2010-02-01

    Full Text Available Abstract Background Thiamine diphosphate (ThDP-dependent enzymes form a vast and diverse class of proteins, catalyzing a wide variety of enzymatic reactions including the formation or cleavage of carbon-sulfur, carbon-oxygen, carbon-nitrogen, and especially carbon-carbon bonds. Although very diverse in sequence and domain organisation, they share two common protein domains, the pyrophosphate (PP and the pyrimidine (PYR domain. For the comprehensive and systematic comparison of protein sequences and structures the Thiamine diphosphate (ThDP-dependent Enzyme Engineering Database (TEED was established. Description The TEED http://www.teed.uni-stuttgart.de contains 12048 sequence entries which were assigned to 9443 different proteins and 379 structure entries. Proteins were assigned to 8 different superfamilies and 63 homologous protein families. For each family, the TEED offers multisequence alignments, phylogenetic trees, and family-specific HMM profiles. The conserved pyrophosphate (PP and pyrimidine (PYR domains have been annotated, which allows the analysis of sequence similarities for a broad variety of proteins. Human ThDP-dependent enzymes are known to be involved in many diseases. 20 different proteins and over 40 single nucleotide polymorphisms (SNPs of human ThDP-dependent enzymes were identified in the TEED. Conclusions The online accessible version of the TEED has been designed to serve as a navigation and analysis tool for the large and diverse family of ThDP-dependent enzymes.

  14. Molecular cloning and catalytic activity of a membrane-bound prenyl diphosphate phosphatase from Croton stellatopilosus Ohba.

    Science.gov (United States)

    Nualkaew, Natsajee; Guennewich, Nils; Springob, Karin; Klamrak, Anuwatchakit; De-Eknamkul, Wanchai; Kutchan, Toni M

    2013-07-01

    Geranylgeraniol (GGOH), a bioactive acyclic diterpene with apoptotic induction activity, is the immediate precursor of the commercial anti-peptic, plaunotol (18-hydroxy geranylgeraniol), which is found in Croton stellatopilosus (Ohba). From this plant, a cDNA encoding a prenyl diphosphate phosphatase (CsPDP), which catalyses the dephosphorylation of geranylgeranyl diphosphate (GGPP) to GGOH, was isolated using a PCR approach. The full-length cDNA contained 888bp and encoded a 33.6 kDa protein (295 amino acids) that was phylogenetically grouped into the phosphatidic acid phosphatase (PAP) enzyme family. The deduced amino acid sequence showed 6 hydrophobic transmembrane regions with 57-85% homology to the sequences of other plant PAPs. The recombinant CsPDP and its 4 truncated constructs exhibited decreasing dephosphorylation activities relative to the lengths of the N-terminal deletions. While the full-length CsPDP successfully performed the two sequential monodephosphorylation steps on GGPP to form GGOH, the larger N-terminal deletion in the truncated enzymes appeared to specifically decrease the catalytic efficiency of the second monodephosphorylation step. The information presented here on the CsPDP cDNA and factors affecting the dephosphorylation activity of its recombinant protein may eventually lead to the discovery of the specific GGPP phosphatase gene and enzyme that are involved in the formation of GGOH in the biosynthetic pathway of plaunotol in C. stellatopilosus.

  15. Molecular cloning and functional expression analysis of a new gene encoding geranylgeranyl diphosphate synthase from hazel (Corylus avellana L. Gasaway).

    Science.gov (United States)

    Wang, Yechun; Miao, Zhiqi; Tang, Kexuan

    2010-10-01

    Geranylgeranyl diphosphate synthase (GGPPS) [EC 2.5.1.29] catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes such as taxol. Herein, a full-length cDNA encoding GGPPS (designated as CgGGPPS) was cloned and characterized from hazel (Corylus avellana L. Gasaway), a taxol-producing angiosperms. The full-length cDNA of CgGGPPS was 1515 bp with a 1122 bp open reading frame (ORF) encoding a 373 amino acid polypeptide. The CgGGPPS genomic DNA sequence was also obtained, revealing CgGGPPS gene was not interrupted by an intron. Southern blot analysis indicated that CgGGPPS belonged to a small gene family. Tissue expression pattern analysis indicated that CgGGPPS expressed the highest in leaves. RT-PCR analysis indicated that CgGGPPS expression could be induced by exogenous methyl jasmonate acid. Furthermore, carotenoid accumulation was observed in Escherichia coli carrying pACCAR25ΔcrtE plasmid carrying CgGGPPS. The result revealed that cDNA encoded a functional GGPP synthase.

  16. Adenosine conjugated lipidic nanoparticles for enhanced tumor targeting.

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Chun eYang

    2013-06-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  3. Lewis acid catalyzed cyclization of glycals/2-deoxy-D-ribose with arylamines: additional findings on product structure and reaction diastereoselectivity.

    Science.gov (United States)

    Du, Chengtang; Li, Fulong; Zhang, Xuefeng; Hu, Wenxiang; Yao, Qizheng; Zhang, Ao

    2011-11-01

    The cyclization reactions of arylamines with 2-deoxy-D-ribose or glycals were reinvestigated in the current report. In the montmorillonite KSF- or InCl(3)-initiated reactions of 2-deoxy-D-ribose with arylamines, a pair of diastereomeric tetrahydro-2H-pyran-fused tetrahydroquinolines was obtained in a nearly 1:1 ratio where the structure of one diastereomer was incorrectly assigned in the literature. Meanwhile, the diastereoselectivity in InBr(3)-catalyzed cyclization of glycals with arylamines was also incorrectly reported previously. It was found that high diastereomeric selectivity was achieved only when a C5-substituted glycal was used; otherwise, a pair of diastereomers was obtained in moderate yield with 1:1 diastereomeric ratio. Furthermore, tetrahydrofuran-fused tetrahydroquinolines 5b and 5b' were also prepared successfully by using TBDPS-protected ribose as the glycal precursor and montmorillonite KSF as the activator.

  4. Complex coordinated extracellular metabolism: Acid phosphatases activate diluted human leukocyte proteins to generate energy flow as NADPH from purine nucleotide ribose.

    Science.gov (United States)

    Hibbs, John B; Vavrin, Zdenek; Cox, James E

    2016-08-01

    Complex metabolism is thought to occur exclusively in the crowded intracellular environment. Here we report that diluted enzymes from lysed human leukocytes produce extracellular energy. Our findings involve two pathways: the purine nucleotide catabolic pathway and the pentose phosphate pathway, which function together to generate energy as NADPH. Glucose6P fuel for NADPH production is generated from structural ribose of purine ribonucleoside monophosphates, ADP, and ADP-ribose. NADPH drives glutathione reductase to reduce an oxidized glutathione disulfide-glutathione redox couple. Acid phosphatases initiate ribose5P salvage from purine ribonucleoside monophosphates, and transaldolase controls the direction of carbon chain flow through the nonoxidative branch of the pentose phosphate pathway. These metabolic control points are regulated by pH. Biologically, this energy conserving metabolism could function in perturbed extracellular spaces.

  5. Silencing of poly(ADP-ribose) glycohydrolase sensitizes lung cancer cells to radiation through the abrogation of DNA damage checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Nakadate, Yusuke [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Kodera, Yasuo; Kitamura, Yuka [Shien-Lab, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Tachibana, Taro [Department of Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585 (Japan); Tamura, Tomohide [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Koizumi, Fumiaki, E-mail: fkoizumi@ncc.go.jp [Division of Thoracic Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    2013-11-29

    Highlights: •Radiosensitization by PARG silencing was observed in multiple lung cancer cells. •PAR accumulation was enhanced by PARG silencing after DNA damage. •Radiation-induced G2/M arrest and checkpoint activation were impaired by PARG siRNA. -- Abstract: Poly(ADP-ribose) glycohydrolase (PARG) is a major enzyme that plays a role in the degradation of poly(ADP-ribose) (PAR). PARG deficiency reportedly sensitizes cells to the effects of radiation. In lung cancer, however, it has not been fully elucidated. Here, we investigated whether PARG siRNA contributes to an increased radiosensitivity using 8 lung cancer cell lines. Among them, the silencing of PARG induced a radiosensitizing effect in 5 cell lines. Radiation-induced G2/M arrest was largely suppressed by PARG siRNA in PC-14 and A427 cells, which exhibited significantly enhanced radiosensitivity in response to PARG knockdown. On the other hand, a similar effect was not observed in H520 cells, which did not exhibit a radiosensitizing effect. Consistent with a cell cycle analysis, radiation-induced checkpoint signals were not well activated in the PC-14 and A427 cells when treated with PARG siRNA. These results suggest that the increased sensitivity to radiation induced by PARG knockdown occurs through the abrogation of radiation-induced G2/M arrest and checkpoint activation in lung cancer cells. Our findings indicate that PARG could be a potential target for lung cancer treatments when used in combination with radiotherapy.

  6. Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks.

    Science.gov (United States)

    Desnoyers, S; Kirkland, J B; Poirier, G G

    1996-06-21

    Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD+ to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase+RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.

  7. ExpandplusCrystal Structures of Poly(ADP-ribose) Polymerase-1 (PARP-1) Zinc Fingers Bound to DNA

    Energy Technology Data Exchange (ETDEWEB)

    M Langelier; J Planck; S Roy; J Pascal

    2011-12-31

    Poly(ADP-ribose) polymerase-1 (PARP-1) has two homologous zinc finger domains, Zn1 and Zn2, that bind to a variety of DNA structures to stimulate poly(ADP-ribose) synthesis activity and to mediate PARP-1 interaction with chromatin. The structural basis for interaction with DNA is unknown, which limits our understanding of PARP-1 regulation and involvement in DNA repair and transcription. Here, we have determined crystal structures for the individual Zn1 and Zn2 domains in complex with a DNA double strand break, providing the first views of PARP-1 zinc fingers bound to DNA. The Zn1-DNA and Zn2-DNA structures establish a novel, bipartite mode of sequence-independent DNA interaction that engages a continuous region of the phosphodiester backbone and the hydrophobic faces of exposed nucleotide bases. Biochemical and cell biological analysis indicate that the Zn1 and Zn2 domains perform distinct functions. The Zn2 domain exhibits high binding affinity to DNA compared with the Zn1 domain. However, the Zn1 domain is essential for DNA-dependent PARP-1 activity in vitro and in vivo, whereas the Zn2 domain is not strictly required. Structural differences between the Zn1-DNA and Zn2-DNA complexes, combined with mutational and structural analysis, indicate that a specialized region of the Zn1 domain is re-configured through the hydrophobic interaction with exposed nucleotide bases to initiate PARP-1 activation.

  8. Different Modulating Effects of Adenosine on Neonatal and Adult Polymorphonuclear Leukocytes

    Directory of Open Access Journals (Sweden)

    Pei-Chen Hou

    2012-01-01

    Full Text Available Polymorphonuclear leukocytes (PMNs are the major leukocytes in the circulation and play an important role in host defense. Intact PMN functions include adhesion, migration, phagocytosis, and reactive oxygen species (ROS release. It has been known for a long time that adenosine can function as a modulator of adult PMN functions. Neonatal plasma has a higher adenosine level than that of adults; however, little is known about the modulating effects of adenosine on neonatal PMNs. The aim of this study was to investigate the effects of adenosine on neonatal PMN functions. We found that neonatal PMNs had impaired adhesion, chemotaxis, and ROS production abilities, but not phagocytosis compared to adult PMNs. As with adult PMNs, adenosine could suppress the CD11b expressions of neonatal PMNs, but had no significant suppressive effect on phagocytosis. In contrast to adult PMNs, adenosine did not significantly suppress chemotaxis and ROS production of neonatal PMNs. This may be due to impaired phagocyte reactions and a poor neonatal PMN response to adenosine. Adenosine may not be a good strategy for the treatment of neonatal sepsis because of impaired phagocyte reactions and poor response.

  9. The effect of circulating adenosine on cerebral haemodynamics and headache generation in healthy subjects

    DEFF Research Database (Denmark)

    Birk, S; Petersen, K.A.; Kruuse, Christina Rostrup

    2005-01-01

    been investigated in man and reports regarding the effect of intravenous adenosine on cerebral blood flow are conflicting. Twelve healthy participants received adenosine 80, 120 microg kg(-1) min(-1) and placebo intravenously for 20 min, in a double-blind, three-way, crossover, randomized design...

  10. Genetically Controlled Upregulation of Adenosine A(1) Receptor Expression Enhances the Survival of Primary Cortical Neurons

    NARCIS (Netherlands)

    Serchov, Tsvetan; Atas, Hasan-Cem; Normann, Claus; van Calker, Dietrich; Biber, Knut

    2012-01-01

    Adenosine has a key endogenous neuroprotective role in the brain, predominantly mediated by the adenosine A(1) receptor (A(1)R). This has been mainly explored using pharmacological tools and/or receptor knockout mice strains. It has long been suggested that the neuroprotective effects of A(1)R are i

  11. Extracellular ATP and adenosine : The Yin and Yang in immune responses?

    NARCIS (Netherlands)

    Faas, M. M.; Saez, T.; de Vos, P.

    Extracellular adenosine 50-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under

  12. Treatment of paroxysmal supraventricular tachycardia with intravenous injection of adenosine triphosphate.

    OpenAIRE

    Saito, D.; Ueeda, M; Abe, Y.; Tani, H; Nakatsu, T.; Yoshida, H.; Haraoka, S; Nagashima, H

    1986-01-01

    Intravenous adenosine triphosphate rapidly terminated all 11 episodes of paroxysmal supraventricular tachycardia in 10 patients. Eight patients reported side effects but these resolved within 20 seconds and did not require treatment. Adenosine triphosphate is a suitable agent for the rapid termination of paroxysmal supraventricular tachycardia.

  13. Photomodulation of G protein-coupled adenosine receptors by a novel light-switchable ligand.

    Science.gov (United States)

    Bahamonde, María Isabel; Taura, Jaume; Paoletta, Silvia; Gakh, Andrei A; Chakraborty, Saibal; Hernando, Jordi; Fernández-Dueñas, Víctor; Jacobson, Kenneth A; Gorostiza, Pau; Ciruela, Francisco

    2014-10-15

    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 N(6) 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.

  14. Adenosine testing after cryoballoon pulmonary vein isolation improves long-term clinical outcome

    NARCIS (Netherlands)

    Y. van Belle (Yves); P. Janse (Petter); N. de Groot (Natasja); W. Anné (Wim); D.A.M.J. Theuns (Dominic); L.J.L.M. Jordaens (Luc)

    2012-01-01

    textabstractBackground Adenosine infusion after pulmonary vein isolation (PVI) with radiofrequency energy reveals dormant muscular sleeves and predicts atrial fibrillation (AF) recurrence. The aim of our study was to determine whether adenosine could reveal dormant PV sleeves after cryoballoon isola

  15. The ischemic preconditioning effect of adenosine in patients with ischemic heart disease

    Directory of Open Access Journals (Sweden)

    Berglund Margareta

    2009-11-01

    Full Text Available Abstract Introduction In vivo and in vitro evidence suggests that adenosine and its agonists play key roles in the process of ischemic preconditioning. The effects of low-dose adenosine infusion on ischemic preconditioning have not been thoroughly studied in humans. Aims We hypothesised that a low-dose adenosine infusion could reduce the ischemic burden evoked by physical exercise and improve the regional left ventricular (LV systolic function. Materials and methods We studied nine severely symptomatic male patients with severe coronary artery disease. Myocardial ischemia was induced by exercise on two separate occasions and quantified by Tissue Doppler Echocardiography. Prior to the exercise test, intravenous low-dose adenosine or placebo was infused over ten minutes according to a randomized, double blind, cross-over protocol. The LV walls were defined as ischemic if a reduction, no increment, or an increment of Results PSV increased from baseline to maximal exercise in non-ischemic walls both during placebo (P = 0.0001 and low-dose adenosine infusion (P = 0.0009. However, in the ischemic walls, PSV increased only during low-dose adenosine infusion (P = 0.001, while no changes in PSV occurred during placebo infusion (P = NS. Conclusion Low-dose adenosine infusion reduced the ischemic burden and improved LV regional systolic function in the ischemic walls of patients with exercise-induced myocardial ischemia, confirming that adenosine is a potential preconditioning agent in humans.

  16. Adenosine Amine Congener as a Cochlear Rescue Agent

    Directory of Open Access Journals (Sweden)

    Srdjan M. Vlajkovic

    2014-01-01

    Full Text Available We have previously shown that adenosine amine congener (ADAC, a selective A1 adenosine receptor agonist, can ameliorate noise- and cisplatin-induced cochlear injury. Here we demonstrate the dose-dependent rescue effects of ADAC on noise-induced cochlear injury in a rat model and establish the time window for treatment. Methods. ADAC (25–300 μg/kg was administered intraperitoneally to Wistar rats (8–10 weeks old at intervals (6–72 hours after exposure to traumatic noise (8–16 kHz, 110 dB sound pressure level, 2 hours. Hearing sensitivity was assessed using auditory brainstem responses (ABR before and 12 days after noise exposure. Pharmacokinetic studies investigated ADAC concentrations in plasma after systemic (intravenous administration. Results. ADAC was most effective in the first 24 hours after noise exposure at doses >50 μg/kg, providing up to 21 dB protection (averaged across 8–28 kHz. Pharmacokinetic studies demonstrated a short (5 min half-life of ADAC in plasma after intravenous administration without detection of degradation products. Conclusion. Our data show that ADAC mitigates noise-induced hearing loss in a dose- and time-dependent manner, but further studies are required to establish its translation as a clinical otological treatment.

  17. Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation.

    Directory of Open Access Journals (Sweden)

    Peter A Keyel

    Full Text Available Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA, which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

  18. Adenosine signaling and the energetic costs of induced immunity.

    Directory of Open Access Journals (Sweden)

    Brian P Lazzaro

    2015-04-01

    Full Text Available Life history theory predicts that trait evolution should be constrained by competing physiological demands on an organism. Immune defense provides a classic example in which immune responses are presumed to be costly and therefore come at the expense of other traits related to fitness. One strategy for mitigating the costs of expensive traits is to render them inducible, such that the cost is paid only when the trait is utilized. In the current issue of PLOS Biology, Bajgar and colleagues elegantly demonstrate the energetic and life history cost of the immune response that Drosophila melanogaster larvae induce after infection by the parasitoid wasp Leptopilina boulardi. These authors show that infection-induced proliferation of defensive blood cells commands a diversion of dietary carbon away from somatic growth and development, with simple sugars instead being shunted to the hematopoetic organ for rapid conversion into the raw energy required for cell proliferation. This metabolic shift results in a 15% delay in the development of the infected larva and is mediated by adenosine signaling between the hematopoietic organ and the central metabolic control organ of the host fly. The adenosine signal thus allows D. melanogaster to rapidly marshal the energy needed for effective defense and to pay the cost of immunity only when infected.

  19. Adenosine Deaminase Deficiency - More Than Just an Immunodeficiency

    Directory of Open Access Journals (Sweden)

    Kathryn Victoria Whitmore

    2016-08-01

    Full Text Available Adenosine deaminase (ADA deficiency is best known as a form of severe combined immunodeficiency (SCID which results from mutations in the gene encoding adenosine deaminase. Affected patients present with clinical and immunological manifestations typical of a severe combined immunodeficiency. Therapies are currently available that can that 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.

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

    Science.gov (United States)

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

    2008-01-01

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

  1. Study of the thorium phosphate-diphosphate (TPD) dissolution: kinetic aspect - thermodynamic aspect: analysis of the neo-formed phases; Etude de la dissolution du phosphate diphosphate de thorium: - aspect cinetique - aspect thermodynamique: analyse des phases neoformees

    Energy Technology Data Exchange (ETDEWEB)

    Thomas, A.Ch

    2000-10-06

    The aim of this work is to study the aqueous corrosion of the thorium phosphate-diphosphate (TPD), of the formula Th{sub 4}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, in the framework of the actinides immobilization. In order to complete the anterior studies concerning solid solutions where thorium is substituted by a tetravalent ion (uranium (IV) or plutonium (IV)) in the TPD structure, compounds of thorium and neptunium phosphate-diphosphate, of formula Th{sub 4-x}Np{sub x}(PO{sub 4}){sub 4}P{sub 2}O{sub 7}, have been prepared. Furthermore, a new chemical way of synthesis has been investigated in order to sinter solids solution of thorium and uranium phosphate-diphosphate (TUPD) in good conditions. The TPD dissolution study showed two principals steps. The first one corresponds to the control of element concentration by the material dissolution whereas the second corresponds to the formation of secondary precipitates for which thermodynamic equilibrium controls the concentration of the species in solution. Leaching tests have been performed varying several independent parameters in order to determine the TPD dissolution rate. The partial orders related to the protons or to the hydroxide ions have been found between 0.35 and 0.45 whereas the apparent dissolution rate constants are in the range 1.10{sup -5} for 9.10{sup -5} g.m{sup -2}.j{sup -1} for acidic and basic media. The neo-formed phases have been characterized after the dissolution of TPD and TUPD. We found that the TPD leaching in acidic medium leads to the formation of the crystallized thorium phosphate-hydrogen-phosphate (TPHP), of formula Th{sub 2}(PO{sub 4}){sub 2}(HPO{sub 4}), x H{sub 2}O, whereas the TUPD dissolution leads to the TPHP and an other compound, of formula (UO{sub 2}){sub 3}(PO{sub 4}){sub 2}, 5 H{sub 2}O. We calculated its solubility product which is in good agreement with those found in the literature. The phases formed during the leaching of solids containing plutonium; americium or curium (Th

  2. Effect of mild temperature shift on poly(ADP-ribose) and γH2AX levels in cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Yamashita, Sachiko [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Tanaka, Masakazu [Department of Microbiology, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka 573-1010 (Japan); Sato, Teruaki [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Ida, Chieri [Department of Applied Life Studies, College of Nagoya Women’s University, 3-40 Shioji-cho, Mizuho-ku, Nagoya-shi, Aichi 467-8610 (Japan); Ohta, Narumi; Hamada, Takashi; Uetsuki, Taichi; Nishi, Yoshisuke [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan); Moss, Joel [Cardiovascular and Pulmonary Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1590 (United States); Miwa, Masanao, E-mail: m_miwa@nagahama-i-bio.ac.jp [Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura, Nagahama, Shiga 526-0829 (Japan)

    2016-08-05

    Poly (ADP-ribose) (PAR) is rapidly synthesized by PAR polymerases (PARPs) upon activation by DNA single- and double-strand breaks. In this study, we examined the quantitative amount of PAR in HeLa cells cultured within the physiological temperatures below 41 °C for verification of the effect of shifting-up or -down the temperature from 37.0 °C on the DNA breaks, whether the temperature-shift caused breaks that could be monitored by the level of PAR. While PAR level did not change significantly when HeLa cells were cultured at 33.5 °C or 37.0 °C, it was significantly increased 2- and 3-fold when cells were cultured for 12 h and 24 h, respectively, at 40.5 °C as compared to 37.0 °C. Similar to the results with HeLa cells, PAR level was increased 2-fold in CHO-K1 cells cultured at 40.5 °C for 24 h as compared to 37.0 °C. As the cellular levels of PAR polymerase1 (PARP1) and PAR glycohydrolase (PARG), a major degradation enzyme for PAR, did not seem to change significantly, this increase could be caused by activation of PARP1 by DNA strand breaks. In fact, γH2AX, claimed to be a marker of DNA double-strand breaks, was found in cell extracts of HeLa cells and CHO-K1 cells at elevated temperature vs. 37.0 °C, and these γH2AX signals were intensified in the presence of 3-aminobenzamide, a PARP inhibitor. The γH2AX immunohistochemistry results in HeLa cells were consistent with Western blot analyses. In HeLa cells, proliferation was significantly suppressed at 40.5 °C in 72 h-continuous cultures and decreased viabilities were also observed after 24–72 h at 40.5 °C. Flow cytometric analyses showed that the HeLa cells were arrested at G2/M after temperature shift-up to 40.5 °C. These physiological changes were potentiated in the presence of 3-aminobenzamide. Decrease in growth rates, increased cytotoxicity and G2/M arrest, were associated with the temperature-shift to 40.5 °C and are indirect evidence of DNA breaks. In addition to γH2AX

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    femoral artery infusion of adenosine (1 mg * min(-1) * litre thigh volume(-1)), which has previously been shown to induce maximal whole thigh blood flow of ~8 L/min. This response was compared to the blood flow induced by moderate-high intensity one-leg dynamic knee extension exercise. Adenosine increased...... muscle. Additionally, it remains to be determined what proportion of adenosine-induced flow elevation is specifically directed to muscle only. In the present study we measured thigh muscle capillary nutritive blood flow in nine healthy young men using positron emission tomography at rest and during...... muscle blood flow on average to 40 +/- 7 ml. min(-1) per 100g(-1) of muscle and an aggregate value of 2.3 +/- 0.6 L * min(-1) for the whole thigh musculature. Adenosine also induced a substantial change in blood flow distribution within individuals. Muscle blood flow during adenosine infusion...

  4. Interstitial and plasma adenosine stimulate nitric oxide and prostacyclin formation in human skeletal muscle

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Mortensen, Stefan Peter; Thaning, Pia;

    2010-01-01

    One major unresolved issue in muscle blood flow regulation is that of the role of circulating versus interstitial vasodilatory compounds. The present study determined adenosine-induced formation of NO and prostacyclin in the human muscle interstitium versus in femoral venous plasma to elucidate....... In young healthy humans, microdialysate was collected at rest, during arterial infusion of adenosine, and during interstitial infusion of adenosine through microdialysis probes inserted into musculus vastus lateralis. Muscle interstitial NO and prostacyclin increased with arterial and interstitial infusion...... levels. These findings provide novel insight into the role of adenosine in skeletal muscle blood flow regulation and vascular function by revealing that both interstitial and plasma adenosine have a stimulatory effect on NO and prostacyclin formation. In addition, both skeletal muscle and microvascular...

  5. New chromene scaffolds for adenosine A(2A) receptors: synthesis, pharmacology and structure-activity relationships.

    Science.gov (United States)

    Areias, Filipe; Costa, Marta; Castro, Marián; Brea, José; Gregori-Puigjané, Elisabet; Proença, M Fernanda; Mestres, Jordi; Loza, María I

    2012-08-01

    In silico screening of a collection of 1584 academic compounds identified a small molecule hit for the human adenosine A(2A) receptor (pK(i) = 6.2) containing a novel chromene scaffold (3a). To explore the structure-activity relationships of this new chemical series for adenosine receptors, a focused library of 43 2H-chromene-3-carboxamide derivatives was synthesized and tested in radioligand binding assays at human adenosine A(1), A(2A), A(2B) and A(3) receptors. The series was found to be enriched with bioactive compounds for adenosine receptors, with 14 molecules showing submicromolar affinity (pK(i) ≥ 6.0) for at least one adenosine receptor subtype. These results provide evidence that the chromene scaffold, a core structure present in natural products from a wide variety of plants, vegetables, and fruits, constitutes a valuable source for novel therapeutic agents. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A...... of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate...... that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies....

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

    Institute of Scientific and Technical Information of China (English)

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

    2012-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2014-01-01

    After hypoxia, ischemia, or inlfammatory 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, lfow 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 ifrst 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.

  9. 2-(1-Hexyn-1-yl)adenosine-induced intraocular hypertension is mediated via K+ channel opening through adenosine A2A receptor in rabbits.

    Science.gov (United States)

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

    2005-08-22

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

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

    Science.gov (United States)

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

    2005-04-01

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

  11. Inhibition by adenosine of histamine and leukotriene release from human basophils.

    Science.gov (United States)

    Peachell, P T; Lichtenstein, L M; Schleimer, R P

    1989-06-01

    Adenosine inhibited the release of histamine and leukotriene C4 (LTC4) from immunologically-activated basophils in a dose-dependent manner. Structural congeners of adenosine also attenuated the elaboration of these two mediators from stimulated basophils and a rank order of potency for the inhibition was observed following the sequence 2-chloroadenosine greater than or equal to N-ethylcarboxamidoadenosine (NECA) greater than adenosine greater than or equal to R-phenylisopropyladenosine (R-PIA) greater than or equal to S-PIA. These same nucleosides modulated the generation of LTC4 more potently than the release of histamine. A number of methylxanthines, which are antagonists of cell surface adenosine receptors, reversed the inhibition by adenosine and its congeners of the release of both histamine and LTC4 to varying extents. Dipyridamole and nitrobenzylthioinosine (NBTI), agents that block the intracellular uptake of adenosine, antagonized the inhibition of histamine release by adenosine (and 2-chloroadenosine) but failed to reverse the attenuation of LTC4 generation by the nucleoside. These same uptake blockers were unable to antagonize the inhibitory effects of NECA on either histamine or LTC4 release. In purified basophils, NECA and R-PIA, and in that order of decreasing reactivity, increased total cell cyclic adenosine monophosphate (cAMP) levels and inhibited the stimulated release of mediators. In total, these results suggest that the basophil possesses a cell surface adenosine receptor which, on the basis of both pharmacological and biochemical criteria, most closely conforms to an A2/Ra-like receptor. However, in addition to an interaction at the cell surface, studies with agents that block the intracellular uptake of adenosine suggest that the nucleoside may also exert intracellular effects when countering the release of histamine (but not LTC4).

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

    Science.gov (United States)

    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.

  13. The Regulation of Skeletal Muscle Active Hyperemia: The Differential Role of Adenosine in Muscles of Varied Fiber Types

    Science.gov (United States)

    1986-04-21

    response. Proctor (1984) found theophylline, a competitiv~ antagonist of adenosine, attenuated the response in the low-oxidative hamster cremaster ...exogenously applied adenosine in hamster cremaster muscle but did not affect the vascular response to muscle stimulation. Differences in the exact drugs...found that the addition of adenosine deaminase to the suffusion solution adjacent to the arterioles of the transilluminated hamster cremaster muscle

  14. 75 FR 8981 - Prospective Grant of Exclusive License: Treatment of Glaucoma by Administration of Adenosine A3...

    Science.gov (United States)

    2010-02-26

    ... Glaucoma by Administration of Adenosine A3 Antagonists AGENCY: National Institutes of Health, Public Health.../092,292, entitled ``A3 Adenosine Receptor Antagonists,'' filed July 10, 1998 , PCT Application PCT/US99/ 15562, entitled''A3 Adenosine Receptor Antagonists,'' filed July 2, 1999 , U.S. Patent...

  15. Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver

    Directory of Open Access Journals (Sweden)

    Chen Ren

    2012-10-01

    Full Text Available Abstract Background Natural rubber produced by plants, known as polyisoprene, is the most widely used isoprenoid polymer. Plant polyisoprenes can be classified into two types; cis-polyisoprene and trans-polyisoprene, depending on the type of polymerization of the isoprene unit. More than 2000 species of higher plants produce latex consisting of cis-polyisoprene. Hevea brasiliensis (rubber tree produces cis-polyisoprene, and is the key source of commercial rubber. In contrast, relatively few plant species produce trans-polyisoprene. Currently, trans-polyisoprene is mainly produced synthetically, and no plant species is used for its commercial production. Results To develop a plant-based system suitable for large-scale production of trans-polyisoprene, we selected a trans-polyisoprene-producing plant, Eucommia ulmoides Oliver, as the target for genetic transformation. A full-length cDNA (designated as EuIPI, Accession No. AB041629 encoding isopentenyl diphosphate isomerase (IPI was isolated from E. ulmoides. EuIPI consisted of 1028 bp with a 675-bp open reading frame encoding a protein with 224 amino acid residues. EuIPI shared high identity with other plant IPIs, and the recombinant protein expressed in Escherichia coli showed IPI enzymatic activity in vitro. EuIPI was introduced into E. ulmoides via Agrobacterium-mediated transformation. Transgenic lines of E. ulmoides overexpressing EuIPI showed increased EuIPI expression (up to 19-fold that of the wild-type and a 3- to 4-fold increase in the total content of trans-polyisoprenes, compared with the wild-type (non-transgenic root line control. Conclusions Increasing the expression level of EuIPI by overexpression increased accumulation of trans-polyisoprenes in transgenic E. ulmoides. IPI catalyzes the conversion of isopentenyl diphosphate to its highly electrophilic isomer, dimethylallyl diphosphate, which is the first step in the biosynthesis of all isoprenoids, including polyisoprene. Our

  16. A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

    Science.gov (United States)

    Liu, Yan; Luo, Shi-Hong; Schmidt, Axel; Wang, Guo-Dong; Sun, Gui-Ling; Grant, Marcus; Kuang, Ce; Yang, Min-Jie; Jing, Shu-Xi; Li, Chun-Huan; Schneider, Bernd; Gershenzon, Jonathan; Li, Sheng-Hong

    2016-03-01

    Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms.

  17. Chronic alcoholism in rats induces a compensatory response, preserving brain thiamine diphosphate, but the brain 2-oxo acid dehydrogenases are inactivated despite unchanged coenzyme levels.

    Science.gov (United States)

    Parkhomenko, Yulia M; Kudryavtsev, Pavel A; Pylypchuk, Svetlana Yu; Chekhivska, Lilia I; Stepanenko, Svetlana P; Sergiichuk, Andrej A; Bunik, Victoria I

    2011-06-01

    Thiamine-dependent changes in alcoholic brain were studied using a rat model. Brain thiamine and its mono- and diphosphates were not reduced after 20 weeks of alcohol exposure. However, alcoholism increased both synaptosomal thiamine uptake and thiamine diphosphate synthesis in brain, pointing to mechanisms preserving thiamine diphosphate in the alcoholic brain. In spite of the unchanged level of the coenzyme thiamine diphosphate, activities of the mitochondrial 2-oxoglutarate and pyruvate dehydrogenase complexes decreased in alcoholic brain. The inactivation of pyruvate dehydrogenase complex was caused by its increased phosphorylation. The inactivation of 2-oxoglutarate dehydrogenase complex (OGDHC) correlated with a decrease in free thiols resulting from an elevation of reactive oxygen species. Abstinence from alcohol following exposure to alcohol reactivated OGDHC along with restoration of the free thiol content. However, restoration of enzyme activity occurred before normalization of reactive oxygen species levels. Hence, the redox status of cellular thiols mediates the action of oxidative stress on OGDHC in alcoholic brain. As a result, upon chronic alcohol consumption, physiological mechanisms to counteract the thiamine deficiency and silence pyruvate dehydrogenase are activated in rat brain, whereas OGDHC is inactivated due to impaired antioxidant ability.

  18. New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

    Science.gov (United States)

    Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

    2009-04-03

    Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

  19. Implications of secondary structure prediction and amino acid sequence comparison of class I and class II phosphoribosyl diphosphate synthases on catalysis, regulation, and quaternary structure

    DEFF Research Database (Denmark)

    Krath, B N; Hove-Jensen, B

    2001-01-01

    Spinach 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP) synthase isozyme 4 was synthesized in Escherichia coli and purified to near homogeneity. The activity of the enzyme is independent of P(i); it is inhibited by ADP in a competitive manner, indicating a lack of an allosteric site; and it accepts...

  20. Ion-Pairing Liquid Chromatography Coupled with Mass Spectrometry for the Simultaneous Determination of Nucleosides and Nucleotides%离子对液相色谱-质谱应用于核苷和核苷酸的同时测定

    Institute of Scientific and Technical Information of China (English)

    蔡宗苇; 钱天秀; 杨斯敏

    2004-01-01

    Adenosine and its corresponding nucleotides adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate ( ADP ) and adenosine 5'-triphos-phate (ATP) are important biomolecules that provide energy and substrates for various cellular bio-chemical processes. There have been strong