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Sample records for b-nicotinamide adenine dinucleotide

  1. Methemoglobinemia and eccentrocytosis in equine erythrocyte flavin adenine dinucleotide deficiency.

    PubMed

    Harvey, J W; Stockham, S L; Scott, M A; Johnson, P J; Donald, J J; Chandler, C J

    2003-11-01

    This report describes erythrocyte biochemical findings in an adult Spanish mustang mare that exhibited persistent methemoglobinemia, eccentrocytosis, and pyknocytosis that were not related to the consumption or administration of an exogenous oxidant. The methemoglobinemia was attributed to a deficiency in cytochrome-b5 reductase (Cb5R) activity, and the eccentrocytes and pyknocytes were attributed to a marked deficiency in reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase (GR) activity that resulted in decreased reduced glutathione concentration within erythrocytes. The GR activity increased to a near-normal value after addition of flavin adenine dinucleotide (FAD) to the enzyme assay, indicating a deficiency of FAD in erythrocytes. The methemoglobinemia, eccentrocytosis, and pyknocytosis were attributed to deficiency of FAD in erythrocytes because the GR and Cb5R enzymes use FAD as a cofactor. This deficiency in FAD results from a defect in erythrocyte riboflavin metabolism, which has not been documented previously in animals. PMID:14608016

  2. Orthophosphite-Nicotinamide Adenine Dinucleotide Oxidoreductase from Pseudomonas fluorescens

    PubMed Central

    Malacinski, George M.; Konetzka, W. A.

    1967-01-01

    Information was obtained on the general properties and specificity of orthophosphite-nicotinamide adenine dinucleotide oxidoreductase. The enzyme was extracted from Pseudomonas fluorescens 195 grown in medium containing orthophosphite as the sole source of phosphorus. An enzyme preparation suitable for characterization was obtained from crude extracts by use of high-speed centrifugation, protamine sulfate precipitation, ammonium sulfate fractionation, and Sephadex gel filtration. The enzyme exhibited maximal activity at pH 7.0, and was inactivated within 6 min at 37 C. Arsenite, hypophosphite, nitrite, selenite, and tellurite were not oxidized by the enzyme. Sulfite inhibited the enzymatic oxidation of orthophosphite in an apparent competitive manner. PMID:4381632

  3. Selective inhibition of nicotinamide adenine dinucleotide kinases by dinucleoside disulfide mimics of nicotinamide adenine dinucleotide analogues.

    PubMed

    Petrelli, Riccardo; Sham, Yuk Yin; Chen, Liqiang; Felczak, Krzysztof; Bennett, Eric; Wilson, Daniel; Aldrich, Courtney; Yu, Jose S; Cappellacci, Loredana; Franchetti, Palmarisa; Grifantini, Mario; Mazzola, Francesca; Di Stefano, Michele; Magni, Giulio; Pankiewicz, Krzysztof W

    2009-08-01

    Diadenosine disulfide (5) was reported to inhibit NAD kinase from Listeria monocytogenes and the crystal structure of the enzyme-inhibitor complex has been solved. We have synthesized tiazofurin adenosine disulfide (4) and the disulfide 5, and found that these compounds were moderate inhibitors of human NAD kinase (IC(50)=110 microM and IC(50)=87 microM, respectively) and Mycobacterium tuberculosis NAD kinase (IC(50)=80 microM and IC(50)=45 microM, respectively). We also found that NAD mimics with a short disulfide (-S-S-) moiety were able to bind in the folded (compact) conformation but not in the common extended conformation, which requires the presence of a longer pyrophosphate (-O-P-O-P-O-) linkage. Since majority of NAD-dependent enzymes bind NAD in the extended conformation, selective inhibition of NAD kinases by disulfide analogues has been observed. Introduction of bromine at the C8 of the adenine ring restricted the adenosine moiety of diadenosine disulfides to the syn conformation making it even more compact. The 8-bromoadenosine adenosine disulfide (14) and its di(8-bromoadenosine) analogue (15) were found to be the most potent inhibitors of human (IC(50)=6 microM) and mycobacterium NAD kinase (IC(50)=14-19 microM reported so far. None of the disulfide analogues showed inhibition of lactate-, and inosine monophosphate-dehydrogenase (IMPDH), enzymes that bind NAD in the extended conformation. PMID:19596199

  4. Intermolecular interactions of reduced nicotinamide adenine dinucleotide (NADH) in solution

    NASA Astrophysics Data System (ADS)

    Jasensky, Joshua; Junaid Farooqi, M.; Urayama, Paul

    2008-10-01

    Nicotinamide adenine dinucleotide (NAD^+/NADH) is a coenzyme involved in cellular respiration as an electron transporter. In aqueous solution, the molecule exhibits a folding transition characterized by the stacking of its aromatic moieties. A transition to an unfolded conformation is possible using chemical denaturants like methanol. Because the reduced NADH form is fluorescent, the folding transition can be monitored using fluorescence spectroscopy, e.g., via a blue-shift in the UV-excited emission peak upon methanol unfolding. Here we present evidence of interactions between NADH molecules in solution. We measure the excited-state emission from NADH at various concentrations (1-100 μM in MOPS buffer, pH 7.5; 337-nm wavelength excitation). Unlike for the folded form, the emission peak wavelength of the unfolded form is concentration dependent, exhibiting a red-shift with higher NADH concentration, suggesting the presence of intermolecular interactions. An understanding of NADH spectra in solution would assist in interpreting intercellular NADH measurements used for the in vivo monitoring cellular energy metabolism.

  5. Nicotinic acid adenine dinucleotide phosphate (NAADP) and Ca2+ mobilization.

    PubMed

    Mándi, Miklós; Bak, Judit

    2008-01-01

    Many physiological processes are controlled by a great diversity of Ca2+ signals that depend on Ca2+ entry into the cell and/or Ca2+ release from internal Ca2+ stores. Ca2+ mobilization from intracellular stores is gated by a family of messengers including inositol-1,4,5-trisphosphate (InsP3), cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). There is increasing evidence for a novel intracellular Ca2+ release channel that may be targeted by NAADP and that displays properties distinctly different from the well-characterized InsP3 and ryanodine receptors. These channels appear to localize on a wider range of intracellular organelles, including the acidic Ca2+ stores. Activation of the NAADP-sensitive Ca2+ channels evokes complex changes in cytoplasmic Ca2+ levels by means of channel chatter with other intracellular Ca2+ channels. The recent demonstration of changes in intracellular NAADP levels in response to physiologically relevant extracellular stimuli highlights the significance of NAADP as an important regulator of intracellular Ca2+ signaling. PMID:18569524

  6. Ototoxic Model of Oxaliplatin and Protection from Nicotinamide Adenine Dinucleotide

    PubMed Central

    Dalian, Ding; Haiyan, Jiang; Yong, Fu; Yongqi, Li; Salvi, Richard

    2014-01-01

    Oxaliplatin, an anticancer drug commonly used to treat colorectal cancer and other tumors, has a number of serious side effects, most notably neuropathy and ototoxicity. To gain insights into its ototoxic profile, oxaliplatin was applied to rat cochlear organ cultures. Consistent with it neurotoxic propensity, oxaliplatin selectively damaged nerve fibers at a very low dose 1 μM. In contrast, the dose required to damage hair cells and spiral ganglion neurons was 50 fold higher (50 μM). Oxailiplatin-induced cochlear lesions initially increased with dose, but unexpectedly decreased at very high doses. This non-linear dose response could be related to depressed oxaliplatin uptake via active transport mechanisms. Previous studies have demonstrated that axonal degeneration involves biologically active processes which can be greatly attenuated by nicotinamide adenine dinucleotide (NAD+). To determine if NAD+ would protect spiral ganglion axons and the hair cells from oxaliplatin damage, cochlear cultures were treated with oxaliplatin alone at doses of 10 μM or 50 μM respectively as controls or combined with 20 mM NAD+. Treatment with 10 μM oxaliplatin for 48 hours resulted in minor damage to auditory nerve fibers, but spared cochlear hair cells. However, when cochlear cultures were treated with 10 μM oxaliplatin plus 20 mM NAD+, most auditory nerve fibers were intact. 50 μM oxaliplatin destroyed most of spiral ganglion neurons and cochlear hair cells with apoptotic characteristics of cell fragmentations. However, 50 μM oxaliplatin plus 20 mM NAD+ treatment greatly reduced neuronal degenerations and hair cell missing. The results suggested that NAD+ provides significant protection against oxaliplatin-induced neurotoxicity and ototoxicity, which may be due to its actions of antioxidant, antiapoptosis, and energy supply. PMID:25419212

  7. Glutamate Synthase: Properties of the Reduced Nicotinamide Adenine Dinucleotide-Dependent Enzyme from Saccharomyces cerevisiae

    PubMed Central

    Roon, Robert J.; Even, Harvey L.; Larimore, Fred

    1974-01-01

    A reduced nicotinamide adenine dinucleotide (NADH)-dependent glutamate synthase has been detected and partially purified from crude extracts of Saccharomyces cerevisiae. The enzyme is specific for NADH, glutamine, and α-ketoglutarate (Km values of 2.6 μM, 1.0 mM, and 140 μM, respectively) and has a pH optimum between 7.1 and 7.7. The stoichiometry of the reaction has been determined as 2 mol of glutamate synthesized per mol of glutamine consumed. Glutamate synthase can be distinguished from either of the glutamate dehydrogenases of yeast on the basis of its substrate requirements and behavior during agarose gel and ion exchange chromatography. Variations in the specific activity of glutamate synthase, which occur in response to changes in the growth medium, are similar in character to those observed with the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase. PMID:4362465

  8. Affinity chromatography of nicotinamide–adenine dinucleotide-linked dehydrogenases on immobilized derivatives of the dinucleotide

    PubMed Central

    Barry, Standish; O'Carra, Pádraig

    1973-01-01

    1. Three established methods for immobilization of ligands through primary amino groups promoted little or no attachment of NAD+ through the 6-amino group of the adenine residue. Two of these methods (coupling to CNBr-activated agarose and to carbodi-imide-activated carboxylated agarose derivatives) resulted instead in attachment predominantly through the ribosyl residues. Other immobilized derivatives were prepared by azolinkage of NAD+ (probably through the 8 position of the adenine residue) to a number of different spacer-arm–agarose derivatives. 2. The effectiveness of these derivatives in the affinity chromatography of a variety of NAD-linked dehydrogenases was investigated, applying rigorous criteria to distinguish general or non-specific adsorption effects from truly NAD-specific affinity (bio-affinity). The ribosyl-attached NAD+ derivatives displayed negligible bio-affinity for any of the NAD-linked dehydrogenases tested. The most effective azo-linked derivative displayed strong bio-affinity for glycer-aldehyde 3-phosphate dehydrogenase, weaker bio-affinity for lactate dehydrogenase and none at all for malate dehydrogenase, although these three enzymes have very similar affinities for soluble NAD+. Alcohol dehydrogenase and xanthine dehydrogenase were subject to such strong non-specific interactions with the hydrocarbon spacer-arm assembly that any specific affinity was completely eclipsed. 3. It is concluded that, in practice, the general effectiveness of a general ligand may be considerably distorted and attenuated by the nature of the immobilization linkage. However, this attenuation can result in an increase in specific effectiveness, allowing dehydrogenases to be separated from one another in a manner unlikely to be feasible if the general effectiveness of the ligand remained intact. 4. The bio-affinity of the various derivatives for lactate dehydrogenase is correlated with the known structure of the NAD+-binding site of this enzyme. Problems

  9. Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast.

    PubMed

    Ortmayr, Karin; Nocon, Justyna; Gasser, Brigitte; Mattanovich, Diethard; Hann, Stephan; Koellensperger, Gunda

    2014-08-01

    The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed-phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose-6-phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain. PMID:24841212

  10. Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

    PubMed Central

    Tanner, J. J.; Tu, S. C.; Barbour, L. J.; Barnes, C. L.; Krause, K. L.

    1999-01-01

    The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H). PMID:10493573

  11. Isotope effect studies of the chemical mechanism of nicotinamide adenine dinucleotide malic enzyme from Crassula

    SciTech Connect

    Grissom, C.B.; Willeford, O.; Wedding, R.T.

    1987-05-05

    The /sup 13/C primary kinetic isotope effect on the decarboxylation of malate by nicotinamide adenine dinucleotide malic enzyme from Crassula argentea is 1.0199 +/- 0.0006 with proteo L-malate-2-H and 1.0162 +/- 0.0003 with malate-2-d. The primary deuterium isotope effect is 1.45 +/- 0.10 on V/K and 1.93 +/- 0.13 on V/sub max/. This indicates a stepwise conversion of malate to pyruvate and CO/sub 2/ with hydride transfer preceding decarboxylation, thereby suggesting a discrete oxaloacetate intermediate. This is in agreement with the stepwise nature of the chemical mechanism of other malic enzymes despite the Crassula enzyme's inability to reduce or decarboxylate oxaloacetate. Differences in morphology and allosteric regulation between enzymes suggest specialization of the Crassula malic enzyme for the physiology of crassulacean and acid metabolism while maintaining the catalytic events founds in malic enzymes from animal sources.

  12. Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate).

    PubMed Central

    Billington, Richard A; Thuring, Jan W; Conway, Stuart J; Packman, Len; Holmes, Andrew B; Genazzani, Armando A

    2004-01-01

    The pyridine nucleotide NAADP (nicotinic acid-adenine dinucleotide phosphate) has been shown to act as a Ca2+-releasing intracellular messenger in a wide variety of systems from invertebrates to mammals and has been implicated in a number of cellular processes. NAADP is structurally very similar to its precursor, the endogenous coenzyme NADP and while much is known about the reduced form of NADP, NADPH, it is not known whether NAADP can also exist in a reduced state. Here we report that NAADP can be reduced to NAADPH by endogenous cellular enzymes and that NAADPH is functionally inert at the NAADP receptor. These data suggest that NAADPH could represent a mechanism for rapidly inactivating NAADP in cells. PMID:14606955

  13. Conducting polymer and its composite materials based electrochemical sensor for Nicotinamide Adenine Dinucleotide (NADH).

    PubMed

    Omar, Fatin Saiha; Duraisamy, Navaneethan; Ramesh, K; Ramesh, S

    2016-05-15

    Nicotinamide Adenine Dinucleotide (NADH) is an important coenzyme in the human body that participates in many metabolic reactions. The impact of abnormal concentrations of NADH significantly causes different diseases in human body. Electrochemical detection of NADH using bare electrode is a challenging task especially in the presence of main electroactive interferences such as ascorbic acid (AA), uric acid (UA) and dopamine (DA). Modified electrodes have been widely explored to overcome the problems of poor sensitivity and selectivity occurred from bare electrodes. This review gives an overview on the progress of using conducting polymers, polyelectrolyte and its composites (co-polymer, carbonaceous, metal, metal oxide and clay) based modified electrodes for the sensing of NADH. In addition, developments on the fabrication of numerous conducting polymer composites based modified electrodes are clearly described. PMID:26774092

  14. Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

    PubMed Central

    Andreesen, Jan R.; Ljungdahl, Lars G.

    1974-01-01

    The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5′-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent Km at 55 C and pH 7.5 for formate was 2.27 × 10−4 M with NADP and 0.83 × 10−4 with methyl viologen as acceptor. The apparent Km for NADP was 1.09 × 10−4 M and for methyl viologen was 2.35 × 10−3 M. NADP showed substrate inhibition at 5 × 10−3 M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate. PMID:4154039

  15. A method of preparation and purification of (4R)-deuterated-reduced nicotinamide adenine dinucleotide phosphate.

    PubMed

    Jeong, S S; Gready, J E

    1994-09-01

    (4R)-Deuterated-reduced nicotinamide adenine dinucleotide phosphate, (4R)-[2H]NADPH, was prepared by reduction of NADP+ using an NADP(+)-dependent alcohol dehydrogenase (EC 1.1.1.2) from Thermoanaerobium brockii and isopropanol-d8 as substrate at 43 degrees C, pH 9. More than 80% of the product was identified as reduced cofactor by reverse-phase (ODS) HPLC, and a 1H NMR study showed that all of the reduced cofactor was (4R)-deuterated. Less than 10% of the product was oxidized cofactor, the remainder being impurities from the breakdown of the dinucleotide compound. Subsequent purification carried out by semipreparative reverse-phase HPLC with 0.1 M NaCl at pH 8.5 gave a compound of more than 96% purity. Separated (4R)-[2H]NADPH fractions were freeze-dried and the white solid was stored at 5 degrees C with desiccant. PMID:7810866

  16. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells.

    PubMed

    Greenhouse, W V; Lehninger, A L

    1977-11-01

    Measurements of respiration, CO2 and lactate production, and changes in the levels of various key metabolites of the glycolytic sequence and tricarboxylic acid cycle were made on five lines of rodent ascites tumor cells (two strains of Ehrlich ascites tumor cells, Krebs II carcinoma, AS-30D carcinoma, and L1210 cells) incubated aerobically in the presence of uniformly labeled D-[14C]glucose. From these data, as well as earlier evidence demonstrating that the reduced nicotinamide adenine dinucleotide (NADH) shuttle in these cells requires a transaminase step and is thus identified as the malate-aspartate shuttle (W.V.V. Greenhouse and A.L. Lehninger, Cancer Res., 36: 1392-1396, 1976), metabolic flux diagrams were constructed for the five cell lines. These diagrams show the relative rates of glycolysis, the tricarboxylic acid cycle, electron transport, and the malate-aspartate shuttle in these tumors. Large amounts of cytosolic NADH were oxidized by the mitochondrial respiratory chain via the NADH shuttle, comprising anywhere from about 20 to 80% of the total flow of reducing equivalents to oxygen in these tumors. Calculations of the sources of energy for adenosine triphosphate synthesis indicated that on the average about one-third of the respiratory adenosine triphosphate is generated by electron flow originating from cytosolic NADH via the malate-aspartate shuttle. PMID:198130

  17. Ligation-triggered fluorescent silver nanoclusters system for the detection of nicotinamide adenine dinucleotide.

    PubMed

    Cao, Zhijuan; Wang, Pei; Qiu, Xue; Lau, Choiwan; Lu, Jianzhong

    2014-03-01

    Herein, we demonstrate a novel silver nanocluster-based fluorescent system for the detection of nicotinamide adenine dinucleotide (NAD(+)), an important biological small molecule involved in a wide range of biological processes. A single-stranded dumbbell DNA probe was designed and used for the assay, which contained a nick in the stem, a poly-cytosine nucleotide loop close to 5' end as the template for the formation of highly fluorescent silver nanoclusters (Ag NCs) and another loop close to 3' end. Only in the presence of NAD(+), the probe was linked at 5' and 3' ends by Escherichia coli DNA ligase, which blocked the DNA polymerase-based extension reaction, ensuring the formation of fluorescent Ag NCs. This technique provided a logarithmic linear relationship in the range of 1 pM-500 nM with a detection limit of as low as 1 pM NAD(+), and exhibited high selectivity against its analogues, and was then successfully used for the detection of NAD(+) level in four kinds of cell homogenates. In addition, this new approach was conducted in an isothermal and homogeneous condition without the need of any thermal cycling, washing, and separation steps, making it very simple. Overall, this label-free protocol offers a promising alternative for the detection of NAD(+), taking advantage of specificity, sensitivity, cost-efficiency, and simplicity. PMID:24442015

  18. BRCA1 as a nicotinamide adenine dinucleotide (NAD)-dependent metabolic switch in ovarian cancer

    PubMed Central

    Li, Da; Chen, Na-Na; Cao, Ji-Min; Sun, Wu-Ping; Zhou, Yi-Ming; Li, Chun-Yan; Wang, Xiu-Xia

    2014-01-01

    Both hereditary factors (e.g., BRCA1) and nicotinamide adenine dinucleotide (NAD)-dependent metabolic pathways are implicated in the initiation and progression of ovarian cancer. However, whether crosstalk exists between BRCA1 and NAD metabolism remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation and promoter methylation) were accompanied by elevated levels of NAD; (ii) the knockdown or overexpression of BRCA1 was an effective way to induce an increase or decrease of nicotinamide phosphoribosyltransferase (Nampt)-related NAD synthesis, respectively; and (iii) BRCA1 expression patterns were inversely correlated with NAD levels in human ovarian cancer specimens. In addition, it is worth noting that: (i) NAD incubation induced increased levels of BRCA1 in a concentration-dependent manner; (ii) Nampt knockdown-mediated reduction in NAD levels was effective at inhibiting BRCA1 expression; and (iii) the overexpression of Nampt led to higher NAD levels and a subsequent increase in BRCA1 levels in primary ovarian cancer cells and A2780, HO-8910 and ES2 ovarian cancer cell lines. These results highlight a novel link between BRCA1 and NAD. Our findings imply that genetic (e.g., BRCA1 inactivation) and NAD-dependent metabolic pathways are jointly involved in the malignant progression of ovarian cancer. PMID:25486197

  19. 3-Picolyl Azide Adenine Dinucleotide as a Probe of Femtosecond to Picosecond Enzyme Dynamics

    PubMed Central

    Dutta, Samrat; Li, Yun-Liang; Rock, William; Houtman, Jon C. D.; Kohen, Amnon; Cheatum, Christopher M.

    2012-01-01

    Functionally relevant femtosecond to picosecond dynamics in enzyme active sites can be difficult to measure because of a lack of spectroscopic probes that can be located in the active site without altering the behavior of the enzyme. We have developed a new NAD+ analog 3-Picolyl Azide Adenine Dinucleotide (PAAD+), which has the potential to be a general spectroscopic probe for NAD-dependent enzymes. This analog is stable and binds in the active site of a typical NAD-dependent enzyme formate dehydrogenase (FDH) with similar characteristics to natural NAD+. It has an isolated infrared transition with high molar absorptivity that makes it suitable for observing enzyme dynamics using 2D IR spectroscopy. 2D IR experiments show that in aqueous solution, the analog undergoes complete spectral diffusion within hundreds of femtoseconds consistent with the water hydrogen bonding dynamics that would be expected. When bound to FDH in a binary complex, it shows picosecond fluctuations and a large static offset, consistent with previous studies of the binary complexes of this enzyme. These results show that PAAD+ is an excellent probe of local dynamics and that it should be a general tool for probing the dynamics of a wide range of NAD-dependent enzymes. PMID:22126535

  20. Preclinical evidence of mitochondrial nicotinamide adenine dinucleotide as an effective alarm parameter under hypoxia

    NASA Astrophysics Data System (ADS)

    Shi, Hua; Sun, Nannan; Mayevsky, Avraham; Zhang, Zhihong; Luo, Qingming

    2014-01-01

    Early detection of tissue hypoxia in the intensive care unit is essential for effective treatment. Reduced nicotinamide adenine dinucleotide (NADH) has been suggested to be the most sensitive indicator of tissue oxygenation at the mitochondrial level. However, no experimental evidence comparing the kinetics of changes in NADH and other physiological parameters has been provided. The aim of this study is to obtain the missing data in a systematic and reliable manner. We constructed four acute hypoxia models, including hypoxic hypoxia, hypemic hypoxia, circulatory hypoxia, and histogenous hypoxia, and measured NADH fluorescence, tissue reflectance, cerebral blood flow, respiration, and electrocardiography simultaneously from the induction of hypoxia until death. We found that NADH was not always the first onset parameter responding to hypoxia. The order of responses was mainly affected by the cause of hypoxia. However, NADH reached its alarm level earlier than the other monitored parameters, ranging from several seconds to >10 min. As such, we suggest that the NADH can be used as a hypoxia indicator, although the exact level that should be used must be further investigated. When the NADH alarm is detected, the body still has a chance to recover if appropriate and timely treatment is provided.

  1. Electrochemical oxidation of dihydronicotinamide adenine dinucleotide at nitrogen-doped carbon nanotube electrodes.

    PubMed

    Goran, Jacob M; Favela, Carlos A; Stevenson, Keith J

    2013-10-01

    Nitrogen-doped carbon nanotubes (N-CNTs) substantially lower the overpotential necessary for dihydronicotinamide adenine dinucleotide (NADH) oxidation compared to nondoped CNTs or traditional carbon electrodes such as glassy carbon (GC). We observe a 370 mV shift in the peak potential (Ep) from GC to CNTs and another 170 mV shift from CNTs to 7.4 atom % N-CNTs in a sodium phosphate buffer solution (pH 7.0) with 2.0 mM NADH (scan rate 10 mV/s). The sensitivity of 7.4 atom % N-CNTs to NADH was measured at 0.30 ± 0.04 A M(-1) cm(-2), with a limit of detection at 1.1 ± 0.3 μM and a linear range of 70 ± 10 μM poised at a low potential of -0.32 V (vs Hg/Hg2SO4). NADH fouling, known to occur to the electrode surface during NADH oxidation, was investigated by measuring both the change in Ep and the resulting loss of electrode sensitivity. NADH degradation, known to occur in phosphate buffer, was characterized by absorbance at 340 nm and correlated with the loss of NADH electroactivity. N-CNTs are further demonstrated to be an effective platform for dehydrogenase-based biosensing by allowing glucose dehydrogenase to spontaneously adsorb onto the N-CNT surface and measuring the resulting electrode's sensitivity to glucose. The glucose biosensor had a sensitivity of 0.032 ± 0.003 A M(-1) cm(-2), a limit of detection at 6 ± 1 μM, and a linear range of 440 ± 50 μM. PMID:23991631

  2. Nicotinic acid-adenine dinucleotide phosphate activates the skeletal muscle ryanodine receptor.

    PubMed Central

    Hohenegger, Martin; Suko, Josef; Gscheidlinger, Regina; Drobny, Helmut; Zidar, Andreas

    2002-01-01

    Calcium is a universal second messenger. The temporal and spatial information that is encoded in Ca(2+)-transients drives processes as diverse as neurotransmitter secretion, axonal outgrowth, immune responses and muscle contraction. Ca(2+)-release from intracellular Ca(2+) stores can be triggered by diffusible second messengers like Ins P (3), cyclic ADP-ribose or nicotinic acid-adenine dinucleotide phosphate (NAADP). A target has not yet been identified for the latter messenger. In the present study we show that nanomolar concentrations of NAADP trigger Ca(2+)-release from skeletal muscle sarcoplasmic reticulum. This was due to a direct action on the Ca(2+)-release channel/ryanodine receptor type-1, since in single channel recordings, NAADP increased the open probability of the purified channel protein. The effects of NAADP on Ca(2+)-release and open probability of the ryanodine receptor occurred over a similar concentration range (EC(50) approximately 30 nM) and were specific because (i) they were blocked by Ruthenium Red and ryanodine, (ii) the precursor of NAADP, NADP, was ineffective at equimolar concentrations, (iii) NAADP did not affect the conductance and reversal potential of the ryanodine receptor. Finally, we also detected an ADP-ribosyl cyclase activity in the sarcoplasmic reticulum fraction of skeletal muscle. This enzyme was not only capable of synthesizing cyclic GDP-ribose but also NAADP, with an activity of 0.25 nmol/mg/min. Thus, we conclude that NAADP is generated in the vicinity of type 1 ryanodine receptor and leads to activation of this ion channel. PMID:12102654

  3. Relationships between laser powers and photoacoustic signal intensities of flavin adenine dinucleotide and beta-carotene dissolved in solutions

    NASA Astrophysics Data System (ADS)

    Imakubo, Keiichi

    1994-10-01

    Ar ion laser-induced photoacoustic spectroscopy has been performed on 0.01 mu M flavin adenine dinucleotide in H2O and 0.01 mu M beta-carotene in n-hexane where the optical absorption spectroscopy is not applicable. On the basis of the linear relationships between laser powers and photoacoustic signal intensities up to 500 mW, it may be concluded that laser power ranging from 10 to 50 mW is required for the successful observation of photoacoustic signals without any photochemical or photobiological effects.

  4. In vivo native fluorescence spectroscopy and nicotinamide adinine dinucleotide/flavin adenine dinucleotide reduction and oxidation states of oral submucous fibrosis for chemopreventive drug monitoring

    NASA Astrophysics Data System (ADS)

    Sivabalan, Shanmugam; Vedeswari, C. Ponranjini; Jayachandran, Sadaksharam; Koteeswaran, Dornadula; Pravda, Chidambaranathan; Aruna, Prakasa Rao; Ganesan, Singaravelu

    2010-01-01

    Native fluorescence spectroscopy has shown potential to characterize and diagnose oral malignancy. We aim at extending the native fluorescence spectroscopy technique to characterize normal and oral submucous fibrosis (OSF) patients under pre- and post-treated conditions, and verify whether this method could also be considered in the monitoring of therapeutic prognosis noninvasively. In this study, 28 normal subjects and 28 clinically proven cases of OSF in the age group of 20 to 40 years are diagnosed using native fluorescence spectroscopy. The OSF patients are given dexamethasone sodium phosphate and hyaluronidase twice a week for 6 weeks, and the therapeutic response is monitored using fluorescence spectroscopy. The fluorescence emission spectra of normal and OSF cases of both pre- and post-treated conditions are recorded in the wavelength region of 350 to 600 nm at an excitation wavelength of 330 nm. The statistical significance is verified using discriminant analysis. The oxidation-reduction ratio of the tissue is also calculated using the fluorescence emission intensities of flavin adenine dinucleotide and nicotinamide adinine dinucleotide at 530 and 440 nm, respectively, and they are compared with conventional physical clinical examinations. This study suggests that native fluorescence spectroscopy could also be extended to OSF diagnosis and therapeutic prognosis.

  5. Comparison of nicotinamide adenine dinucleotide phosphate-induced immune responses against biotrophic and necrotrophic pathogens in Arabidopsis thaliana.

    PubMed

    Wang, Chenggang; Zhang, Xudong; Mou, Zhonglin

    2016-06-01

    The pyridine nucleotide nicotinamide adenine dinucleotide phosphate (NADP) is a universal coenzyme in anabolic reactions and also functions in intracellular signaling by serving as a substrate for production of the Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP). It has recently been shown that, in mammalian cells, cellular NADP can be released into the extracellular compartment (ECC) upon environmental stresses by active exocytosis or diffusion through transmembrane transporters in living cells or passive leakage across the membrane in dying cells. In the ECC, NADP can either serve as a substrate for production of NAADP or act directly on purinoceptors to activate transmembrane signaling. In the last several years, extracellular NADP has also been suggested to function in plant immune responses. Here, we compared exogenous NADP-induced immune responses against biotrophic and necrotrophic pathogens in the Arabidopsis thaliana ecotype Columbia and found that NADP addition induces salicylic acid-mediated defense signaling but not jasmonic acid/ethylene-mediated defense responses. These results suggest the specificity of exogenous NADP-activated signaling in plants. PMID:27031653

  6. Nicotinic Acid Adenine Dinucleotide Phosphate (Naadp+) Is an Essential Regulator of T-Lymphocyte Ca2+-Signaling

    PubMed Central

    Berg, Ingeborg; Potter, Barry V.L.; Mayr, Georg W.; Guse, Andreas H.

    2000-01-01

    Microinjection of human Jurkat T-lymphocytes with nicotinic acid adenine dinucleotide phosphate (NAADP+) dose-dependently stimulated intracellular Ca2+-signaling. At a concentration of 10 nM NAADP+ evoked repetitive and long-lasting Ca2+-oscillations of low amplitude, whereas at 50 and 100 nM, a rapid and high initial Ca2+-peak followed by trains of smaller Ca2+-oscillations was observed. Higher concentrations of NAADP+ (1 and 10 μM) gradually reduced the initial Ca2+-peak, and a complete self-inactivation of Ca2+-signals was seen at 100 μM. The effect of NAADP+ was specific as it was not observed with nicotinamide adenine dinucleotide phosphate. Both inositol 1,4,5-trisphosphate– and cyclic adenosine diphosphoribose–mediated Ca2+-signaling were efficiently inhibited by coinjection of a self-inactivating concentration of NAADP+. Most importantly, microinjection of a self-inactivating concentration of NAADP+ completely abolished subsequent stimulation of Ca2+-signaling via the T cell receptor/CD3 complex, indicating that a functional NAADP+ Ca2+-release system is essential for T-lymphocyte Ca2+-signaling. PMID:10931869

  7. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    PubMed

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors. PMID:26323301

  8. Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium.

    PubMed

    Yu, L; Wolin, M J

    1972-01-01

    A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction. PMID:4333382

  9. Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism.

    PubMed

    Meneely, Kathleen M; Lamb, Audrey L

    2007-10-23

    Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism. PMID:17900176

  10. The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

    SciTech Connect

    Long, C.M.; Rohrmann, G.F.; Merrill, G.F.

    2009-06-05

    Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

  11. Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release

    PubMed Central

    Trabbic, Christopher J.; Zhang, Fan; Walseth, Timothy F.; Slama, James T.

    2015-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a Ca2+ releasing intracellular second messenger in both mammals and echinoderms. We report that large functionalized substituents introduced at the nicotinic acid 5-position are recognized by the sea urchin receptor, albeit with a 20–500 fold loss in agonist potency. 5-(3-Azidopropyl)-NAADP was shown to release Ca2+ with an EC50 of 31 µM and to compete with NAADP for receptor binding with an IC50 of 56 nM. Attachment of charged groups to the nicotinic acid of NAADP is associated with loss of activity, suggesting that the nicotinate riboside moiety is recognized as a neutral zwitterion. Substituents (Br- and N3-) can be introduced at the 8-adenosyl position of NAADP while preserving high potency and agonist efficacy and an NAADP derivative substituted at both the 5-position of the nicotinic acid and at the 8-adenosyl position was also recognized although the agonist potency was significantly reduced. PMID:25826221

  12. Surface enhanced Raman scattering investigation of protein-bound flavin adenine dinucleotide structure

    NASA Astrophysics Data System (ADS)

    Maskevich, S. A.; Strekal, N. D.; Artsukevich, I. M.; Kivach, L. N.; Chernikevich, I. P.

    1995-04-01

    The SERS spectra of alcohol oxidase from Pichia pastoris adsorbed on a silver electrode were obtained. The similarities and differences of these spectra with the SERS spectrum of free flavin adenine dinucleiotide were considered. The dependence of relative intensity of 1258 cm -1 band from the electrode potential in the protein SERS spectra differed from that of free flavin. From the data on this band being sensitive to the protein-flavin interaction a suggestion was made about incomplete dissociation of flavin from the protein. This conclusion is confirmed both by the fluorescence data and the SERS data on alcohol oxidase purified from Candida boidinii. The results of the SERS investigation of the interaction between the substrate, ethanol and the cofactor, FAD, as well as between protein-bound cofactor with the substrate are presented. The problem of retaining the protein enzyme activity is discussed.

  13. Redox State of Flavin Adenine Dinucleotide Drives Substrate Binding and Product Release in Escherichia coli Succinate Dehydrogenase

    PubMed Central

    Cheng, Victor W.T.; Piragasam, Ramanaguru Siva; Rothery, Richard A.; Maklashina, Elena; Cecchini, Gary; Weiner, Joel H.

    2016-01-01

    The Complex II family of enzymes, comprising the respiratory succinate dehydrogenases and fumarate reductases, catalyze reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, the soluble fumarate reductases (e.g. that from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and the FAD was examined. The variants SdhA-R286A/K/Y and -H242A/Y, that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in assembly of a noncovalent FAD cofactor, which led to a significant decrease (−87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The “free” and “occupied” states of the active site were linked to the reduced and oxidized states of the FAD, respectively. Our data allows for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD. PMID:25569225

  14. Application of nicotin amide-adenine dinucleotide analogs for clinical enzymology: alcohol dehydrogenase activity in liver injury.

    PubMed

    Fujisawa, K; Kimura, A; Minato, S; Tamaoki, H; Mizushima, H

    1976-06-01

    The activities of alcohol dehydrogease(ADH) in serum and in the subcellular fractions of rat liver were determined with n-amyl alcohol or ethanol as substrate and thionicotinamide-adenine dinucleotide as coenzyme. It was found that the enzyme's activity ratio on the amyl alcohol and ethanol(A/E value) of serum and on the particulate fractions of the liver were different, but the A/E value of the soluble fraction was similar to that of serum. The A/E value of the particulate fractions were higher than that of the soluble fraction. From the results of experimental liver damage in the rat, it seems that estimation of the A/E value of ADH activity in serum is a useful parameter for the diagnosis of active liver injury. Since the A/E values of patients' sera differed from those of the normal subjects, the estimation of the A/E value of serum may give diagnostic information on liver injury, especially in chronic liver injury. PMID:179739

  15. Population Genetics of Aedes albopictus (Diptera: Culicidae) Invading Populations, Using Mitochondrial nicotinamide Adenine Dinucleotide Dehydrogenase Subunit 5 Sequences

    PubMed Central

    Usmani-Brown, Sahar; Cohnstaedt, Lee; Munstermann, Leonard E.

    2012-01-01

    Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito indigenous to Asia, now an invasive species worldwide, is an important vector for several arboviruses. Genetic analysis using the mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 5 (ND5) gene was carried out in populations from Cameroon (n = 50), Hawaii (n = 38), Italy (n = 20), the continental United States, Brazil, and its native range. Data for Brazil, the continental United States, and the native range was obtained from Birungi and Munstermann (2002). Direct sequencing was used to identity unique haplotypes. The limited phylogeographic partitioning of haplotypes with low levels of sequence divergence in both Cameroon and Hawaii was consistent with the population structure of Ae. albopictus in the United States and Brazil. Four new haplotypes were identified from the samples from Cameroon and Hawaii, adding to previously described haplotypes. Hawaii shared a haplotype with Cameroon that was unique to these two regions. Hawaii also had higher overall haplotype diversity than seen in previous continental United States, Brazil, or native range populations. Hawaiian, Cameroon, and Italian populations did not share haplotypes with Brazil, which validates the earlier mitochondrial DNA studies indicating a separate introduction of this species into Brazil. PMID:22544973

  16. The first step in polyethylene glycol degradation by sphingomonads proceeds via a flavoprotein alcohol dehydrogenase containing flavin adenine dinucleotide.

    PubMed

    Sugimoto, M; Tanabe, M; Hataya, M; Enokibara, S; Duine, J A; Kawai, F

    2001-11-01

    Several Sphingomonas spp. utilize polyethylene glycols (PEGs) as a sole carbon and energy source, oxidative PEG degradation being initiated by a dye-linked dehydrogenase (PEG-DH) that oxidizes the terminal alcohol groups of the polymer chain. Purification and characterization of PEG-DH from Sphingomonas terrae revealed that the enzyme is membrane bound. The gene encoding this enzyme (pegA) was cloned, sequenced, and expressed in Escherichia coli. The purified recombinant enzyme was vulnerable to aggregation and inactivation, but this could be prevented by addition of detergent. It is as a homodimeric protein with a subunit molecular mass of 58.8 kDa, each subunit containing 1 noncovalently bound flavin adenine dinucleotide but not Fe or Zn. PEG-DH recognizes a broad variety of primary aliphatic and aromatic alcohols as substrates. Comparison with known sequences revealed that PEG-DH belongs to the group of glucose-methanol-choline (GMC) flavoprotein oxidoreductases and that it is a novel type of flavoprotein alcohol dehydrogenase related (percent identical amino acids) to other, so far uncharacterized bacterial, membrane-bound, dye-linked dehydrogenases: alcohol dehydrogenase from Pseudomonas oleovorans (46%); choline dehydrogenase from E. coli (40%); L-sorbose dehydrogenase from Gluconobacter oxydans (38%); and 4-nitrobenzyl alcohol dehydrogenase from a Pseudomonas species (35%). PMID:11673442

  17. Redox state of flavin adenine dinucleotide drives substrate binding and product release in Escherichia coli succinate dehydrogenase.

    PubMed

    Cheng, Victor W T; Piragasam, Ramanaguru Siva; Rothery, Richard A; Maklashina, Elena; Cecchini, Gary; Weiner, Joel H

    2015-02-01

    The Complex II family of enzymes, comprising respiratory succinate dehydrogenases and fumarate reductases, catalyzes reversible interconversion of succinate and fumarate. In contrast to the covalent flavin adenine dinucleotide (FAD) cofactor assembled in these enzymes, soluble fumarate reductases (e.g., those from Shewanella frigidimarina) that assemble a noncovalent FAD cannot catalyze succinate oxidation but retain the ability to reduce fumarate. In this study, an SdhA-H45A variant that eliminates the site of the 8α-N3-histidyl covalent linkage between the protein and FAD was examined. Variants SdhA-R286A/K/Y and -H242A/Y that target residues thought to be important for substrate binding and catalysis were also studied. The variants SdhA-H45A and -R286A/K/Y resulted in the assembly of a noncovalent FAD cofactor, which led to a significant decrease (-87 mV or more) in its reduction potential. The variant enzymes were studied by electron paramagnetic resonance spectroscopy following stand-alone reduction and potentiometric titrations. The "free" and "occupied" states of the active site were linked to the reduced and oxidized states of FAD, respectively. Our data allow for a proposed model of succinate oxidation that is consistent with tunnel diode effects observed in the succinate dehydrogenase enzyme and a preference for fumarate reduction catalysis in fumarate reductase homologues that assemble a noncovalent FAD. PMID:25569225

  18. Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide-tetrazolium coupling reactions for quantitative l-lactate analysis.

    PubMed

    Kan, Shu-Chen; Chang, Wei-Feng; Lan, Min-Chi; Lin, Chia-Chi; Lai, Wei-Shiang; Shieh, Chwen-Jen; Hsiung, Kuang-Pin; Liu, Yung-Chuan

    2015-02-15

    In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2(-6)U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25U/μl), and NAD(+) (2μl, 1.5×10(-5)M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications. PMID:25454507

  19. Interaction of reduced nicotinamide adenine dinucleotide with an antifreeze protein from Dendroides canadensis: mechanistic implication of antifreeze activity enhancement

    PubMed Central

    Wen, Xin; Wang, Sen; Amornwittawat, Natapol; Houghton, Eric A.; Sacco, Michael A.

    2016-01-01

    Antifreeze proteins (AFPs) found in many organisms can noncolligatively lower the freezing point of water without altering the melting point. The difference between the depressed freezing point and the melting point, termed thermal hysteresis (TH), is usually a measure of the antifreeze activity of AFPs. Certain low molecular mass molecules and proteins can further enhance the antifreeze activity of AFPs. Interaction between an enhancer and arginine is known to play an important role in enhancing the antifreeze activity of an AFP from the beetle Dendroides canadensis (DAFP-1). Here, we examined the enhancement effects of several prevalent phosphate-containing coenzymes on the antifreeze activity of DAFP-1. β-Nicotinamide adenine dinucleotide (reduced) (NADH) is identified as the most efficient enhancer of DAFP-1, which increases the antifreeze activity of DAFP-1 by around 10 times. Examination of the enhancement abilities of a series of NADH analogs and various molecular fragments of NADH reveals that the modifications of nicotinamide generate a series of highly efficient enhancers, though none as effective as NADH itself, and the whole molecular structure of NADH is necessary for its highly efficient enhancement effect. We also demonstrated a 1:1 binding between DAFP-1 and NADH. The binding was characterized by high-performance liquid chromatography (HPLC) using the gel filtration method of Hummel and Dreyer. The data analysis suggests binding between DAFP-1 and NADH with a dissociation constant in the micromolar range. Interactions between DAFP-1 and NADH are discussed along with molecular mechanisms of enhancer action. PMID:22038809

  20. β-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury.

    PubMed

    Umapathy, Nagavedi Siddaramappa; Gonzales, Joyce; Fulzele, Sadanand; Kim, Kyung-mi; Lucas, Rudolf; Verin, Alexander Dimitrievich

    2012-06-01

    Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) occur in approximately 200,000 patients per year. Studies indicate that lung endothelium plays a significant role in ALI. The authors' recent in vitro studies demonstrate a novel mechanism of β-nicotinamide adenine dinucleotide (β-NAD)-induced protection against gram-positive (pneumolysin, PLY) and gram-negative (lipopolysaccharide, LPS) toxin-induced lung endothelial cell (EC) barrier dysfunction. The objective of the current study was to evaluate the protective effect of β-NAD against LPS-induced ALI in mice. C57BL/6J mice were randomly divided into 4 groups: vehicle, β-NAD, LPS, and LPS/β-NAD. After surgery, mice were allowed to recover for 24 hours. Evans blue dye-albumin (EBA) was given through the internal jugular vein 2 hours prior to the termination of the experiments. Upon sacrificing the animals, bronchoalveolar lavage fluid (BALF) was collected and the lungs were harvested. β-NAD treatment significantly attenuated the inflammatory response by means of reducing the accumulation of cells and protein in BALF, blunting the parenchymal neutrophil infiltration, and preventing capillary leak. In addition, the histological examination demonstrated decreased interstitial edema in the LPS/β-NAD specimens, as compared to the LPS-only specimens. The mRNA levels of the anti-inflammatory cytokines were up-regulated in the LPS group treated with β-NAD compared to the LPS-only-treated group. β-NAD treatment down-regulated the mRNA levels of the proinflammatory cytokines. These findings suggest that β-NAD could be investigated as a therapeutic option against bacterial toxin-induced lung inflammation and ALI in mice. PMID:22563684

  1. Induction of nicotinamide-adenine dinucleotide phosphate oxidase and apoptosis by biodegradable polymers in macrophages: implications for stents.

    PubMed

    Potnis, Pushya A; Tesfamariam, Belay; Wood, Steven C

    2011-06-01

    The drug-eluting stent platform has a limited surface area, and a polymer carrier matrix is coated to enable sufficient loading of drugs. The development of a suitable polymer has been challenging because it must exhibit biocompatibility with the intravascular milieu. The use of biodegradable polymers seems to be attractive because it enables drug release as it degrades and is eventually eliminated from the body leaving the permanent metallic stent polymer-free. The aim of this study was to investigate the biocompatibility of biodegradable polymers using the human monocyte cell line. Cultured monocytes differentiated into functional macrophages (THP-1) were incubated with various polymers including poly-L-lactide (PLA), polycaprolactone (PCL), or poly-D, L-lactide-co-glycolide (PLGA) for up to 5 days. Exposure of cells to the polymers resulted in macrophage-polymer adhesion and induced marked pro-oxidant species as measured by calcein AM uptake assay and flow cytometric analysis of 2',7'-dichlorofluorescin fluorescence, respectively. Real-time reverse-transcription polymerase chain reaction and Western blot analysis of expression of nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases revealed enhanced expression of NADPH oxidase subunits in response to PLA and PLGA compared with that of PCL. Flow cytometric analysis of fluorescein isothiocyanate-Annexin V and propium iodide-stained PLA and PGLA polymer-exposed THP-1 cells showed early and late apoptotic changes. Similarly, exposure to the PLA and PGLA polymers, but not to the PCL polymer, resulted in enhanced staining for cleaved poly(ADP-ribose) polymerase-1, a protein fragment produced by caspase cleavage. These results indicate that biodegradable polymers are associated with cell adhesion, NADPH oxidase-induced generation of reactive oxygen species and excess apoptosis. PMID:21436724

  2. A comparative cluster analysis of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry in the brains of amphibians.

    PubMed

    Pinelli, Claudia; Rastogi, Rakesh K; Scandurra, Anna; Jadhao, Arun G; Aria, Massimo; D'Aniello, Biagio

    2014-09-01

    Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) is a key enzyme in the synthesis of the gaseous neurotransmitter nitric oxide. We compare the distribution of NADPH-d in the brain of four species of hylid frogs. NADPH-d-positive fibers are present throughout much of the brain, whereas stained cell groups are distributed in well-defined regions. Whereas most brain areas consistently show positive neurons in all species, in some areas species-specific differences occur. We analyzed our data and those available for other amphibian species to build a matrix on NADPH-d brain distribution for a multivariate analysis. Brain dissimilarities were quantified by using the Jaccard index in a hierarchical clustering procedure. The whole brain dendrogram was compared with that of its main subdivisions by applying the Fowlkes-Mallows index for dendrogram similarity, followed by bootstrap replications and a permutation test. Despite the differences in the distribution map of the NADPH-d system among species, cluster analysis of data from the whole brain and hindbrain faithfully reflected the evolutionary history (framework) of amphibians. Dendrograms from the secondary prosencephalon, diencephalon, mesencephalon, and isthmus showed some deviation from the main scheme. Thus, the present analysis supports the major evolutionary stability of the hindbrain. We provide evidence that the NADPH-d system in main brain subdivisions should be cautiously approached for comparative purposes because specific adaptations of a single species could occur and may affect the NADPH-d distribution pattern in a brain subdivision. The minor differences in staining pattern of particular subdivisions apparently do not affect the general patterns of staining across species. PMID:24549578

  3. Exceptionally high glucose current on a hierarchically structured porous carbon electrode with "wired" flavin adenine dinucleotide-dependent glucose dehydrogenase.

    PubMed

    Tsujimura, Seiya; Murata, Kazuki; Akatsuka, Wataru

    2014-10-15

    This article introduces a carbon electrode designed to achieve efficient enzymatic electrolysis by exploiting a hierarchical pore structure based on macropores for efficient mass transfer and mesopores for high enzyme loading. Magnesium oxide-templated mesoporous carbon (MgOC, mean pore diameter 38 nm) was used to increase the effective specific surface area for enzyme immobilization. MgOC particles were deposited on a current collector by an electrophoretic deposition method to generate micrometer-scale macropores to improve the mass transfer of glucose and electrolyte (buffer) ions. To create a glucose bioanode, the porous-carbon-modified electrode was further coated with a biocatalytic hydrogel composed of a conductive redox polymer, deglycosylated flavin adenine dinucleotide-dependent glucose dehydrogenase (d-FAD-GDH), and a cross-linker. Carbohydrate chains on the peripheral surfaces of the FAD-GDH molecules were removed by periodate oxidation before cross-linking. The current density for the oxidation of glucose was 100 mA cm(-2) at 25 °C and pH 7, with a hydrogel loading of 1.0 mg cm(-2). For the same hydrogel composition and loading, the current density on the MgOC-modified electrode was more than 30 times higher than that on a flat carbon electrode. On increasing the solution temperature to 45 °C, the catalytic current increased to 300 mA cm(-2), with a hydrogel loading of 1.6 mg cm(-2). Furthermore, the stability of the hydrogel electrode was improved by using the mesoporous carbon materials; more than 95% of the initial catalytic current remained after a 220-day storage test in 4 °C phosphate buffer, and 80% was observed after 7 days of continuous operation at 25 °C. PMID:25244161

  4. Modulation of nicotinamide adenine dinucleotide and poly(adenosine diphosphoribose) metabolism by the synthetic "C" nucleoside analogs, tiazofurin and selenazofurin. A new strategy for cancer chemotherapy.

    PubMed Central

    Berger, N A; Berger, S J; Catino, D M; Petzold, S J; Robins, R K

    1985-01-01

    Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents. PMID:3919063

  5. Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

    PubMed Central

    Li, M; Dyda, F; Benhar, I; Pastan, I; Davies, D R

    1996-01-01

    The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles. Images Fig. 1 Fig. 3 PMID:8692916

  6. Simultaneous determination of purine nucleotides, their metabolites and beta-nicotinamide adenine dinucleotide in cerebellar granule cells by ion-pair high performance liquid chromatography.

    PubMed

    Giannattasio, Sergio; Gagliardi, Sara; Samaja, Michele; Marra, Ersilia

    2003-02-01

    The method described here allows the quantitative simultaneous determination of adenosine 5'-triphosphate, adenosine 5'-diphosphate, adenosine 5'-monophosphate, adenosine, guanosine 5'-triphosphate, guanosine 5'-diphosphate, guanosine, inosine 5'-monophosphate, inosine, uric acid, xanthine, hypoxanthine and beta-nicotinamide adenine dinucleotide by ion-pair high performance liquid chromatography. The chromatographic analysis requires 26 min per sample and allows the separation of the mentioned metabolites in a time as short as 16 min. Primary cultures of rat cerebellar granule cells were incubated in serum-free medium containing 25 mM KCl for 1.5-48 h and their acid extracts were injected onto column. Uric acid, inosine 5'-monophosphate, inosine, beta-nicotinamide adenine dinucleotide, adenosine, adenosine 5'-monophosphate, guanosine 5'-diphosphate, adenosine 5'-diphosphate, guanosine 5'-triphosphate and adenosine 5'-triphosphate were identified and quantified, while hypoxanthine, xanthine and guanosine were below the detection limit. This method makes use of a single-step sample pre-treatment procedure which allows a greater than 91% recovery of the compounds of interest and provides the assay of the metabolites of interest in little amounts of cell extracts. Therefore, this method is suitable to evaluate the energetic state in a variety of cell types, both under normal and dismetabolic conditions, such as after the induction of apoptosis or necrosis. PMID:12565687

  7. Facile synthesis of near infrared fluorescent trypsin-stabilized Ag nanoclusters with tunable emission for 1,4-dihydronicotinamide adenine dinucleotide and ethanol sensing.

    PubMed

    Liu, Siyu; Wang, Hui; Cheng, Zhen; Liu, Hongguang

    2015-07-30

    A facile chemical synthetic route was developed to prepare near-infrared fluorescent trypsin-stabilized Ag nanoclusters (Try-Ag NCs). The fluorescence emission wavelength of the produced Try-Ag NCs is tunable by simple adjusting pH value of the synthesis system, and the Try-Ag NCs offer a symmetric fluorescent excitation and emission peak. The fluorescence of Try-Ag NCs remains constant in the presence of various ions and molecules, and it can be effectively quenched by 1,4-dihydronicotinamide adenine dinucleotide (NADH) instead of its oxidized forms nicotinamide adenine dinucleotide (NAD(+)). This property enables the Try-Ag NCs to be a novel analytical platform to monitor biological reaction involved with NADH. In this work, the Try-Ag NCs was also applied to analyze ethanol based on the generation of NADH which was the product of NAD(+) and ethanol in the catalysis of alcohol dehydrogenase. And the proposed platform allowed ethanol to be determined in the range from 10 to 300 μmol/L with 5 μmol/L detection limit. PMID:26320647

  8. Oxidation of reduced cytosolic nicotinamide adenine dinucleotide by the malate-aspartate shuttle in the K-562 human leukemia cell line.

    PubMed

    López-Alarcón, L; Eboli, M L

    1986-11-01

    The activity of the malate-aspartate shuttle for the reoxidation of reduced cytosolic nicotinamide adenine dinucleotide (NADH) by mitochondria was studied in a line of human myeloid leukemia cells (K-562). The tumor cells showed mitochondrial reoxidation of cytosolic NADH, as evidenced by the accumulation of pyruvate, when incubated aerobically with L-lactate. The involvement of the respiratory chain in the reoxidation of cytosolic NADH was demonstrated by the action of rotenone, antimycin A, and oligomycin which strongly inhibited the formation of pyruvate from added L-lactate. Moreover, pyruvate production was greatly inhibited by the transaminase inhibitor, aminooxyacetate. Under glycolytic conditions, in the presence of aminooxyacetate, the rate of pyruvate production was also markedly inhibited, the rate of lactate accumulation was stimulated, and at 60 min the cytosolic NADH/nicotinamide adenine dinucleotide (NAD) ratio had increased progressively about 5-fold with respect to untreated cells. The maximal rate of the malate-aspartate shuttle has also been established by addition of arsenite to inhibit mitochondrial oxidation of the pyruvate formed from added L-lactate. PMID:3756905

  9. Reduction of mitomycin C is catalysed by human recombinant NRH:quinone oxidoreductase 2 using reduced nicotinamide adenine dinucleotide as an electron donating co-factor

    PubMed Central

    Jamieson, D; Tung, A T Y; Knox, R J; Boddy, A V

    2006-01-01

    NRH:Quinone Oxidoreductase 2 (NQO2) has been described as having no enzymatic activity with nicotinamide adenine dinucleotide (NADH) or NADPH as electron donating cosubstrates. Mitomycin C (MMC) is both a substrate for and a mechanistic inhibitor of the NQO2 homologue NQO1. NRH:quinone oxidoreductase 2 catalysed the reduction of MMC at pH 5.8 with NADH as a co-factor. This reaction results in species that inhibit the NQO2-mediated metabolism of CB1954. In addition, MMC caused an increase in DNA cross-links in a cell line transfected to overexpress NQO2 to an extent comparable to that observed with an isogenic NQO1-expressing cell line. These data indicate that NQO2 may contribute to the metabolism of MMC to cytotoxic species. PMID:17031400

  10. Biocomposite based on reduced graphene oxide film modified with phenothiazone and flavin adenine dinucleotide-dependent glucose dehydrogenase for glucose sensing and biofuel cell applications.

    PubMed

    Ravenna, Yehonatan; Xia, Lin; Gun, Jenny; Mikhaylov, Alexey A; Medvedev, Alexander G; Lev, Ovadia; Alfonta, Lital

    2015-10-01

    A novel composite material for the encapsulation of redox enzymes was prepared. Reduced graphene oxide film with adsorbed phenothiazone was used as a highly efficient composite for electron transfer between flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase and electrodes. Measured redox potential for glucose oxidation was lower than 0 V vs Ag/AgCl electrode. The fabricated biosensor showed high sensitivity of 42 mA M(-1) cm(-2), a linear range of glucose detection of 0.5-12 mM, and good reproducibility and stability as well as high selectivity for different interfering compounds. In a semibiofuel cell configuration, the hybrid film generated high power output of 345 μW cm(-2). These results demonstrate a promising potential for this composition in various bioelectronic applications. PMID:26334692

  11. Modulation of spontaneous transmitter release from the frog neuromuscular junction by interacting intracellular Ca(2+) stores: critical role for nicotinic acid-adenine dinucleotide phosphate (NAADP).

    PubMed Central

    Brailoiu, Eugen; Patel, Sandip; Dun, Nae J

    2003-01-01

    Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca(2+)-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca(2+)-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP(3)), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP(3), cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca(2+) stores with distinct Ca(2+) stores targeted by NAADP. PMID:12749764

  12. Conformational change in cytochrome P450 reductase adsorbed at a Au(110)—phosphate buffer interface induced by interaction with nicotinamide adenine dinucleotide phosphate

    NASA Astrophysics Data System (ADS)

    Smith, C. I.; Convery, J. H.; Harrison, P.; Khara, B.; Scrutton, N. S.; Weightman, P.

    2014-08-01

    Changes observed in the reflection anisotropy spectroscopy (RAS) profiles of monolayers of cytochrome P450 reductase adsorbed at Au(110)-electrolyte interfaces at 0.056 V following the addition of nicotinamide adenine dinucleotide phosphate (NADP+) are explained in terms of a simple model as arising from changes in the orientation of an isoalloxazine ring located in the flavin mononucleotide binding domain of the protein. The model also accounts for the changes observed in the RAS as the potential applied to the Au(110) surface is varied and suggests that differences in the dependence of the RAS profile of the adsorbed protein on the potential applied to the electrode in the absence and presence of NADP+ are explicable as arising from a competition between the applied potential acting to reduce the protein and the NADP+ to oxidize it.

  13. Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

    PubMed

    Ido, Yasuo

    2016-07-01

    Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate-activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C-peptide. My research and others' suggest that the SIRT1-liver kinase B1-AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. 'Diabetic memory' is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1-AMPK positive feedback through activating FOXO3. PMID:27181414

  14. Sleep Fragmentation in Mice Induces Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2-Dependent Mobilization, Proliferation, and Differentiation of Adipocyte Progenitors in Visceral White Adipose Tissue

    PubMed Central

    Khalyfa, Abdelnaby; Wang, Yang; Zhang, Shelley X.; Qiao, Zhuanhong; Abdelkarim, Amal; Gozal, David

    2014-01-01

    Background: Chronic sleep fragmentation (SF) without sleep curtailment induces increased adiposity. However, it remains unclear whether mobilization, proliferation, and differentiation of adipocyte progenitors (APs) occurs in visceral white adipose tissue (VWAT), and whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2) activity plays a role. Methods: Changes in VWAT depot cell size and AP proliferation were assessed in wild-type and Nox2 null male mice exposed to SF and control sleep (SC). To assess mobilization, proliferation, and differentiation of bone marrow mesenchymal stem cells (BM-MSC), Sca-1+ bone marrow progenitors were isolated from GFP+ or RFP+ mice, and injected intravenously to adult male mice (C57BL/6) previously exposed to SF or SC. Results: In comparison with SC, SF was associated with increased weight accrual at 3 w and thereafter, larger subcutaneous and visceral fat depots, and overall adipocyte size at 8 w. Increased global AP numbers in VWAT along with enhanced AP BrDU labeling in vitro and in vivo emerged in SF. Systemic injections of GFP+ BM-MSC resulted in increased AP in VWAT, as well as in enhanced differentiation into adipocytes in SF-exposed mice. No differences occurred between SF and SC in Nox2 null mice for any of these measurements. Conclusions: Chronic sleep fragmentation (SF) induces obesity in mice and increased proliferation and differentiation of adipocyte progenitors (AP) in visceral white adipose tissue (VWAT) that are mediated by increased Nox2 activity. In addition, enhanced migration of bone marrow mesenchymal stem cells from the systemic circulation into VWAT, along with AP differentiation, proliferation, and adipocyte formation occur in SF-exposed wild-type but not in oxidase 2 (Nox2) null mice. Thus, Nox2 may provide a therapeutic target to prevent obesity in the context of sleep disorders. Citation: Khalyfa A, Wang Y, Zhang SX, Qiao Z, Abdelkarim A, Gozal D. Sleep fragmentation in mice induces

  15. Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies

    SciTech Connect

    Kiick, D.M.; Harris, B.G.; Cook, P.F.

    1986-01-14

    The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. Results indicate that substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.

  16. Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes.

    PubMed

    Serrano, Ana; Frago, Susana; Velázquez-Campoy, Adrián; Medina, Milagros

    2012-01-01

    In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS. PMID:23203077

  17. A label-free fluorescence strategy for selective detection of nicotinamide adenine dinucleotide based on a dumbbell-like probe with low background noise.

    PubMed

    Chen, Xuexu; Lin, Chunshui; Chen, Yiying; Wang, Yiru; Chen, Xi

    2016-03-15

    In this work we developed a novel label-free fluorescence sensing approach for the detection of nicotinamide adenine dinucleotide (NAD(+)) based on a dumbbell-like DNA probe designed for both ligation reaction and digestion reaction with low background noise. SYBR Green I (SG I), a double-helix dye, was chosen as the readout fluorescence signal. In the absence of NAD(+), the ligation reaction did not occur, but the probe was digested to mononucleotides after the addition of exonuclease I (Exo I) and exonuclease I (Exo III), resulting in a weak fluorescence intensity due to the weak interaction between SG I and mononucleotides. In the presence of NAD(+), the DNA probe was ligated by Escherichia coli DNA ligase, blocking the digestion by Exo I and Exo III. As a result, SG I was intercalated into the stem part of the DNA dumbbell probe and fluorescence enhancement was achieved. This method was simple in design, fast to operate, with good sensitivity and selectivity which could discriminate NAD(+) from its analogs. PMID:26454831

  18. New insights into the analysis of the electrode kinetics of flavin adenine dinucleotide redox center of glucose oxidase immobilized on carbon electrodes.

    PubMed

    Simonov, Alexandr N; Grosse, Willo; Mashkina, Elena A; Bethwaite, Blair; Tan, Jeff; Abramson, David; Wallace, Gordon G; Moulton, Simon E; Bond, Alan M

    2014-03-25

    New insights into electrochemical kinetics of the flavin adenine dinucleotide (FAD) redox center of glucose-oxidase (GlcOx) immobilized on reduced graphene oxide (rGO), single- and multiwalled carbon nanotubes (SW and MWCNT), and combinations of rGO and CNTs have been gained by application of Fourier transformed AC voltammetry (FTACV) and simulations based on a range of models. A satisfactory level of agreement between experiment and theory, and hence establishment of the best model to describe the redox chemistry of FAD, was achieved with the aid of automated e-science tools. Although still not perfect, use of Marcus theory with a very low reorganization energy (≤0.3 eV) best mimics the experimental FTACV data, which suggests that the process is gated as also deduced from analysis of FTACV data obtained at different frequencies. Failure of the simplest models to fully describe the electrode kinetics of the redox center of GlcOx, including those based on the widely employed Laviron theory is demonstrated, as is substantial kinetic heterogeneity of FAD species. Use of a SWCNT support amplifies the kinetic heterogeneity, while a combination of rGO and MWCNT provides a more favorable environment for fast communication between FAD and the electrode. PMID:24571209

  19. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells*

    PubMed Central

    Arredouani, Abdelilah; Ruas, Margarida; Collins, Stephan C.; Parkesh, Raman; Clough, Frederick; Pillinger, Toby; Coltart, George; Rietdorf, Katja; Royle, Andrew; Johnson, Paul; Braun, Matthias; Zhang, Quan; Sones, William; Shimomura, Kenju; Morgan, Anthony J.; Lewis, Alexander M.; Chuang, Kai-Ting; Tunn, Ruth; Gadea, Joaquin; Teboul, Lydia; Heister, Paula M.; Tynan, Patricia W.; Bellomo, Elisa A.; Rutter, Guy A.; Rorsman, Patrik; Churchill, Grant C.; Parrington, John; Galione, Antony

    2015-01-01

    Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca2+ action potentials due to the activation of voltage-dependent Ca2+ channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca2+ release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca2+ from the endolysosomal system, resulting in localized Ca2+ signals. We show here that NAADP-mediated Ca2+ release from endolysosomal Ca2+ stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca2+ release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca2+ signals, and insulin secretion. Our findings implicate NAADP-evoked Ca2+ release from acidic Ca2+ storage organelles in stimulus-secretion coupling in β cells. PMID:26152717

  20. Deficiency of the iron-sulfur clusters of mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase (complex I) in an infant with congenital lactic acidosis.

    PubMed

    Moreadith, R W; Batshaw, M L; Ohnishi, T; Kerr, D; Knox, B; Jackson, D; Hruban, R; Olson, J; Reynafarje, B; Lehninger, A L

    1984-09-01

    We report the case of an infant with hypoglycemia, progressive lactic acidosis, an increased serum lactate/pyruvate ratio, and elevated plasma alanine, who had a moderate to profound decrease in the ability of mitochondria from four organs to oxidize pyruvate, malate plus glutamate, citrate, and other NAD+-linked respiratory substrates. The capacity to oxidize the flavin adenine dinucleotide-linked substrate, succinate, was normal. The most pronounced deficiency was in skeletal muscle, the least in kidney mitochondria. Enzymatic assays on isolated mitochondria ruled out defects in complexes II, III, and IV of the respiratory chain. Further studies showed that the defect was localized in the inner membrane mitochondrial NADH-ubiquinone oxidoreductase (complex I). When ferricyanide was used as an artificial electron acceptor, complex I activity was normal, indicating that electrons from NADH could reduce the flavin mononucleotide cofactor. However, electron paramagnetic resonance spectroscopy performed on liver submitochondrial particles showed an almost total loss of the iron-sulfur clusters characteristic of complex I, whereas normal signals were noted for other mitochondrial iron-sulfur clusters. This infant is presented as the first reported case of congenital lactic acidosis caused by a deficiency of the iron-sulfur clusters of complex I of the mitochondrial electron transport chain. PMID:6432847

  1. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Calcium Signaling and Arrhythmias in the Heart Evoked by β-Adrenergic Stimulation*♦

    PubMed Central

    Nebel, Merle; Schwoerer, Alexander P.; Warszta, Dominik; Siebrands, Cornelia C.; Limbrock, Ann-Christin; Swarbrick, Joanna M.; Fliegert, Ralf; Weber, Karin; Bruhn, Sören; Hohenegger, Martin; Geisler, Anne; Herich, Lena; Schlegel, Susan; Carrier, Lucie; Eschenhagen, Thomas; Potter, Barry V. L.; Ehmke, Heimo; Guse, Andreas H.

    2013-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2+-releasing second messenger known to date. Here, we report a new role for NAADP in arrhythmogenic Ca2+ release in cardiac myocytes evoked by β-adrenergic stimulation. Infusion of NAADP into intact cardiac myocytes induced global Ca2+ signals sensitive to inhibitors of both acidic Ca2+ stores and ryanodine receptors and to NAADP antagonist BZ194. Furthermore, in electrically paced cardiac myocytes BZ194 blocked spontaneous diastolic Ca2+ transients caused by high concentrations of the β-adrenergic agonist isoproterenol. Ca2+ transients were recorded both as increases of the free cytosolic Ca2+ concentration and as decreases of the sarcoplasmic luminal Ca2+ concentration. Importantly, NAADP antagonist BZ194 largely ameliorated isoproterenol-induced arrhythmias in awake mice. We provide strong evidence that NAADP-mediated modulation of couplon activity plays a role for triggering spontaneous diastolic Ca2+ transients in isolated cardiac myocytes and arrhythmias in the intact animal. Thus, NAADP signaling appears an attractive novel target for antiarrhythmic therapy. PMID:23564460

  2. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells.

    PubMed

    Arredouani, Abdelilah; Ruas, Margarida; Collins, Stephan C; Parkesh, Raman; Clough, Frederick; Pillinger, Toby; Coltart, George; Rietdorf, Katja; Royle, Andrew; Johnson, Paul; Braun, Matthias; Zhang, Quan; Sones, William; Shimomura, Kenju; Morgan, Anthony J; Lewis, Alexander M; Chuang, Kai-Ting; Tunn, Ruth; Gadea, Joaquin; Teboul, Lydia; Heister, Paula M; Tynan, Patricia W; Bellomo, Elisa A; Rutter, Guy A; Rorsman, Patrik; Churchill, Grant C; Parrington, John; Galione, Antony

    2015-08-28

    Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in β cells. PMID:26152717

  3. Increase of reduced nicotinamide adenine dinucleotide fluorescence lifetime precedes mitochondrial dysfunction in staurosporine-induced apoptosis of HeLa cells

    NASA Astrophysics Data System (ADS)

    Yu, Jia-Sin; Guo, Han-Wen; Wang, Chih-Hao; Wei, Yau-Huei; Wang, Hsing-Wen

    2011-03-01

    In vivo noninvasive detection of apoptosis represents a new tool that may yield a more definite diagnosis, a more accurate prognosis, and help improve therapies for human diseases. The intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) may be a potential optical biomarker for the apoptosis detection because NADH is involved in the respiration for the mitochondrial membrane potential (ΔΨ) formation and adenosine-5'-triphosphate (ATP) synthesis, and the depletion of ΔΨ and ATP level is the hallmark of apoptosis. We have previously observed the NADH fluorescence lifetime change is associated with staurosporine (STS)-induced mitochondria-mediated apoptosis. However, its relationship with mitochondrial functions such as ΔΨ, ATP, and oxygen consumption rate is not clear. In this study, we investigated this relationship. Our results indicate that the NADH fluorescence lifetime increased when ΔΨ and ATP levels were equal to or higher than their values of controls and decreased before the depletion of ΔΨ and ATP, and the oxygen consumption rate did not change. These findings suggest that the increased NADH fluorescence lifetime in STS-induced cell death occurred before the depletion of ΔΨ and ATP and activation of caspase 3, and was not simply caused by cellular metabolic change. Furthermore, the NADH fluorescence lifetime change is associated with the pace of apoptosis.

  4. Effects of Nicotinamide Adenine Dinucleotide (NAD(+)) and Diadenosine Tetraphosphate (Ap4A) on Electrical Activity of Working and Pacemaker Atrial Myocardium in Guinea Pigs.

    PubMed

    Pustovit, K B; Abramochkin, D V

    2016-04-01

    Effects of nucleotide polyphosphate compounds (nicotinamide adenine dinucleotide, NAD(+); diadenosine tetraphosphate, Ap4A) on the confi guration of action potentials were studied in isolated preparations of guinea pig sinoatrial node and right atrial appendage (auricle). In the working myocardium, NAD(+) and Ap4A in concentrations of 10(-5) and 10(-4) M had no effect on resting potential, but significantly reduced the duration of action potentials; the most pronounced decrease was found at 25% repolarization. In the primary pacemaker of the sinoatrial node, both concentrations of NAD(+) and Ap4A induced hyperpolarization and reduction in the rate of slow diastolic depolarization, but significant slowing of the sinus rhythm was produced by these substances only in the concentration of 10(-4) M. Moreover, AP shortening and marked acceleration of AP upstroke were observed in the pacemaker myocardium after application of polyphosphates. Comparative analysis of the effects of NAD(+) and Ap4A in the working and pacemaker myocardium drove us to a hypothesis on inhibitory effects of these substances on L-type calcium current accompanied by stimulation of one or several potassium currents, which induce enhancement of repolarization and hyperpolarization of membranes probably mediated by the activation of purine receptors. PMID:27165058

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

    PubMed

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

    2015-06-01

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

  6. Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

    PubMed

    Ali, Ramadan A; Camick, Christina; Wiles, Katherine; Walseth, Timothy F; Slama, James T; Bhattacharya, Sumit; Giovannucci, David R; Wall, Katherine A

    2016-02-26

    Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. PMID:26728458

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

    PubMed Central

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

    2015-01-01

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

  8. Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:Flavin Adenine Dinucleotide Oxidoreductase (TftC) of Burkholderia cepacia AC1100

    PubMed Central

    Gisi, Michelle R.; Xun, Luying

    2003-01-01

    Burkholderia cepacia AC1100 uses 2,4,5-trichlorophenoxyacetic acid, an environmental pollutant, as a sole carbon and energy source. Chlorophenol 4-monooxygenase is a key enzyme in the degradation of 2,4,5-trichlorophenoxyacetic acid, and it was originally characterized as a two-component enzyme (TftC and TftD). Sequence analysis suggests that they are separate enzymes. The two proteins were separately produced in Escherichia coli, purified, and characterized. TftC was an NADH:flavin adenine dinucleotide (FAD) oxidoreductase. A C-terminally His-tagged fusion TftC used NADH to reduce either FAD or flavin mononucleotide (FMN) but did not use NADPH or riboflavin as a substrate. Kinetic and binding property analysis showed that FAD was a better substrate than FMN. TftD was a reduced FAD (FADH2)-utilizing monooxygenase, and FADH2 was supplied by TftC. It converted 2,4,5-trichlorophenol to 2,5-dichloro-p-quinol and then to 5-chlorohydroxyquinol but converted 2,4,6-trichlorophenol only to 2,6-dichloro-p-quinol as the final product. TftD interacted with FADH2 and retarded its rapid oxidation by O2. A spectrum of possible TftD-bound FAD-peroxide was identified, indicating that the peroxide is likely the active oxygen species attacking the aromatic substrates. The reclassification of the two enzymes further supports the new discovery of FADH2-utilizing enzymes, which have homologues in the domains Bacteria and Archaea. PMID:12700257

  9. Oxidation of C1 Compounds by Particulate fractions from Methylococcus capsulatus: distribution and properties of methane-dependent reduced nicotinamide adenine dinucleotide oxidase (methane hydroxylase).

    PubMed Central

    Ribbons, D W

    1975-01-01

    Cell-free particulate fractions of extracts from the obligate methylotroph Methylococcus capsulatus catalyze the reduced nicotinamide adenine dinucleotide (NADH) and O2-dependent oxidation of methane (methane hydroxylase). The only oxidation product detected was formate. These preparations also catalyze the oxidation of methanol and formaldehyde to formate in the presence or absence of phenazine methosulphate with oxygen as the terminal electron acceptor. Methane hydroxylase activity cannot be reproducibly obtained from disintegrated cell suspensions even though the whole cells actively respired when methane was presented as a substrate. Varying the disintegration method or extraction medium had no significant effect on the activities obtained. When active particles were obtained, hydroxylase activity was stable at 0 C for days. Methane hydroxylase assays were made by measuring the methane-dependent oxidation of NADH by O2. In separate experiments, methane consumption and the accumulation of formate were also demonstrated. Formate is not oxidized by these particulate fractions. The effects of particle concentration, temperature, pH, and phosphate concentration on enzymic activity are described. Ethane is utilized in the presence of NADH and O2. The stoichiometric relationships of the reaction(s) with methane as substrate were not established since (i) the presumed initial product, methanol, is also oxidized to formate, and (ii) the contribution that NADH oxidase activity makes to the observed consumption of reactants could not be assessed in the presence of methane. Studies with known inhibitors of electron transport systems indicate that the path of electron flow from NADH to oxygen is different for the NADH oxidase, methane hydroxylase, and methanol oxidase activities. Images PMID:238946

  10. De-methylation of displacement loop of mitochondrial DNA is associated with increased mitochondrial copy number and nicotinamide adenine dinucleotide subunit 2 expression in colorectal cancer.

    PubMed

    Gao, Jinhang; Wen, Shilei; Zhou, Hongying; Feng, Shi

    2015-11-01

    DNA methylation occurs in the displacement loop (D-loop) region of mammals; however, D-loop regions of certain tumor tissue types were found to be de‑methylated. Whether hypomethylation of the D‑loop region is involved in the regulation of the mitochondrial DNA (mtDNA) copy number and nicotinamide adenine dinucleotide subunit 2 (ND‑2) expressions in colorectal cancer has remained elusive. In the present study, the association between methylation status of the D‑loop region, mtDNA copy number and ND‑2 expression was investigated in 65 colorectal cancer specimens and their corresponding non‑cancerous tissues. In addition, a de‑methylation experiment was performed on the Caco‑2 colorectal cancer cell line by using 5‑aza-2'-deoxycytidine (5‑Aza). The methylation rate of the D‑loop region in all 65 colorectal cancer tissues was markedly reduced when compared with that of their corresponding non‑cancerous tissues (13.8 vs. 81.5%; P<0.05). Furthermore, the methylation rate of the D‑loop region in colorectal cancer tissues was markedly decreased in clinicopathological stages III and IV compared with that in clinicopathological stages I and II (7.1 and 0% vs. 25 and 16%; P<0.05). In addition, the mean relative mtDNA copy number and ND‑2 expression in colorectal cancer tissues were increased compared with those in the corresponding non‑cancerous tissues. De‑methylation of the D‑loop region was associated with an elevated mtDNA copy number and an increased ND‑2 expression. Furthermore, the mtDNA copy number and ND‑2 expression in Caco‑2 cells were significantly increased after 5‑Aza treatment. In conclusion, de‑methylation of the D‑loop region is likely to be involved in the regulation of the mtDNA copy number and ND-2 expression. PMID:26323487

  11. Electrochemical synthesis and characterization of TiO2 nanoparticles and their use as a platform for flavin adenine dinucleotide immobilization and efficient electrocatalysis

    NASA Astrophysics Data System (ADS)

    Ashok Kumar, S.; Lo, Po-Hsun; Chen, Shen-Ming

    2008-06-01

    Here, we report the electrochemical synthesis of TiO2 nanoparticles (NPs) using the potentiostat method. Synthesized particles have been characterized by using x-ray diffraction (XRD) studies, atomic force microscopy (AFM) and scanning electron microscopy (SEM). The results revealed that the TiO2 film produced was mainly composed of rutile and that the particles are of a size in the range of 100 ± 50 nm. TiO2 NPs were used for the modification of a screen printed carbon electrode (SPE). The resulting TiO2 film coated SPE was used to immobilize flavin adenine dinucleotide (FAD). The flavin enzyme firmly attached onto the metal oxide surface and this modified electrode showed promising electrocatalytic activities towards the reduction of hydrogen peroxide (H2O2) in physiological conditions. The electrochemistry of FAD confined in the oxide film was investigated. The immobilized FAD displayed a pair of redox peaks with a formal potential of -0.42 V in pH 7.0 oxygen-free phosphate buffers at a scan rate of 50 mV s-1. The FAD in the nanostructured TiO2 film retained its bioactivity and exhibited excellent electrocatalytic response to the reduction of H2O2, based on which a mediated biosensor for H2O2 was achieved. The linear range for the determination of H2O2 was from 0.15 × 10-6 to 3.0 × 10-3 M with the detection limit of 0.1 × 10-6 M at a signal-to-noise ratio of 3. The stability and repeatability of the biosensor is also discussed.

  12. Amelioration of nicotinamide adenine dinucleotide phosphate-oxidase mediated stress reduces cell death after blast-induced traumatic brain injury.

    PubMed

    Lucke-Wold, Brandon P; Naser, Zachary J; Logsdon, Aric F; Turner, Ryan C; Smith, Kelly E; Robson, Matthew J; Bailes, Julian E; Lee, John M; Rosen, Charles L; Huber, Jason D

    2015-12-01

    A total of 1.7 million traumatic brain injuries (TBIs) occur each year in the United States, but available pharmacologic options for the treatment of acute neurotrauma are limited. Oxidative stress is an important secondary mechanism of injury that can lead to neuronal apoptosis and subsequent behavioral changes. Using a clinically relevant and validated rodent blast model, we investigated how nicotinamide adenine dinucleotide phosphate oxidase (Nox) expression and associated oxidative stress contribute to cellular apoptosis after single and repeat blast injuries. Nox4 forms a complex with p22phox after injury, forming free radicals at neuronal membranes. Using immunohistochemical-staining methods, we found a visible increase in Nox4 after single blast injury in Sprague Dawley rats. Interestingly, Nox4 was also increased in postmortem human samples obtained from athletes diagnosed with chronic traumatic encephalopathy. Nox4 activity correlated with an increase in superoxide formation. Alpha-lipoic acid, an oxidative stress inhibitor, prevented the development of superoxide acutely and increased antiapoptotic markers B-cell lymphoma 2 (t = 3.079, P < 0.05) and heme oxygenase 1 (t = 8.169, P < 0.001) after single blast. Subacutely, alpha-lipoic acid treatment reduced proapoptotic markers Bax (t = 4.483, P < 0.05), caspase 12 (t = 6.157, P < 0.001), and caspase 3 (t = 4.573, P < 0.01) after repetitive blast, and reduced tau hyperphosphorylation indicated by decreased CP-13 and paired helical filament staining. Alpha-lipoic acid ameliorated impulsive-like behavior 7 days after repetitive blast injury (t = 3.573, P < 0.05) compared with blast exposed animals without treatment. TBI can cause debilitating symptoms and psychiatric disorders. Oxidative stress is an ideal target for neuropharmacologic intervention, and alpha-lipoic acid warrants further investigation as a therapeutic for prevention of chronic neurodegeneration. PMID:26414010

  13. Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

    PubMed Central

    Eppink, M H; Boeren, S A; Vervoort, J; van Berkel, W J

    1997-01-01

    A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate. PMID:9352916

  14. Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells: Implications in Pulmonary Arterial Hypertension.

    PubMed

    Hood, Katie Y; Montezano, Augusto C; Harvey, Adam P; Nilsen, Margaret; MacLean, Margaret R; Touyz, Rhian M

    2016-09-01

    Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16α-hydroxyestrone (16αOHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)-induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen-Nox-dependent processes was studied in female Nox1(-/-) and Nox4(-/-) mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16αOHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-α, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16αOHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16αOHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid-related factor 2 activity and expression of nuclear factor erythroid-related factor 2-regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1(-/-) but not Nox4(-/-) mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1(-/-) mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid-related factor 2 whereby 16αOHE1 influences

  15. Nicotinamide Adenine Dinucleotide Phosphate Oxidase–Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells

    PubMed Central

    Hood, Katie Y.; Montezano, Augusto C.; Harvey, Adam P.; Nilsen, Margaret; MacLean, Margaret R.

    2016-01-01

    Estrogen and oxidative stress have been implicated in pulmonary arterial hypertension (PAH). Mechanisms linking these systems are elusive. We hypothesized that estrogen metabolite, 16α-hydroxyestrone (16αOHE1), stimulates nicotinamide adenine dinucleotide phosphate oxidase (Nox)–induced reactive oxygen species (ROS) generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMCs) and that in PAH aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of estrogen–Nox–dependent processes was studied in female Nox1−/− and Nox4−/− mice with PAH. PASMCs from control subjects (control hPASMCs) and PAH patients (PAH-hPASMCs) were exposed to estrogen and 16αOHE1 in the presence/absence of inhibitors of Nox, cytochrome P450 1B1, and estrogen receptors. Estrogen, through estrogen receptor-α, increased Nox-derived ROS and redox-sensitive growth in hPASMCs, with greater effects in PAH-hPASMCs versus control hPASMCs. Estrogen effects were inhibited by cytochrome P450 1B1 blockade. 16αOHE1 stimulated transient ROS production in hPASMCs, with sustained responses in PAH-hPASMCs. Basal expression of Nox1/Nox4 was potentiated in PAH-hPASMCs. In hPASMCs, 16αOHE1 increased Nox1 expression, stimulated irreversible oxidation of protein tyrosine phosphatases, decreased nuclear factor erythroid–related factor 2 activity and expression of nuclear factor erythroid–related factor 2–regulated antioxidant genes, and promoted proliferation. This was further amplified in PAH-hPASMCs. Nox1−/− but not Nox4−/− mice were protected against PAH and vascular remodeling. Our findings demonstrate that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth primarily through Nox1. Supporting this, in vivo studies exhibited protection against pulmonary hypertension and remodeling in Nox1−/− mice. This study provides new insights through Nox1/ROS and nuclear factor erythroid–related factor 2

  16. Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart.

    PubMed

    Capel, Rebecca A; Bolton, Emma L; Lin, Wee K; Aston, Daniel; Wang, Yanwen; Liu, Wei; Wang, Xin; Burton, Rebecca-Ann B; Bloor-Young, Duncan; Shade, Kai-Ting; Ruas, Margarida; Parrington, John; Churchill, Grant C; Lei, Ming; Galione, Antony; Terrar, Derek A

    2015-12-11

    Ca(2+)-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca(2+) responses in cardiac myocytes from Tpcn2(-/-) mice, unlike myocytes from wild-type (WT) mice. Ca(2+)/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca(2+) transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2(-/-) mice. Increases in amplitude of L-type Ca(2+) currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2(-/-) mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2(-/-) mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2(-/-) mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼ 25 nm). We propose that Ca(2+)-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias. PMID:26438825

  17. Biochemical Analysis of Recombinant AlkJ from Pseudomonas putida Reveals a Membrane-Associated, Flavin Adenine Dinucleotide-Dependent Dehydrogenase Suitable for the Biosynthetic Production of Aliphatic Aldehydes

    PubMed Central

    Kirmair, Ludwig

    2014-01-01

    The noncanonical alcohol dehydrogenase AlkJ is encoded on the alkane-metabolizing alk operon of the mesophilic bacterium Pseudomonas putida GPo1. To gain insight into the enzymology of AlkJ, we have produced the recombinant protein in Escherichia coli and purified it to homogeneity using His6 tag affinity and size exclusion chromatography (SEC). Despite synthesis in the cytoplasm, AlkJ was associated with the bacterial cell membrane, and solubilization with n-dodecyl-β-d-maltoside was necessary to liberate the enzyme. SEC and spectrophotometric analysis revealed a dimeric quaternary structure with stoichiometrically bound reduced flavin adenine dinucleotide (FADH2). The holoenzyme showed thermal denaturation at moderate temperatures around 35°C, according to both activity assay and temperature-dependent circular dichroism spectroscopy. The tightly bound coenzyme was released only upon denaturation with SDS or treatment with urea-KBr and, after air oxidation, exhibited the characteristic absorption spectrum of FAD. The enzymatic activity of purified AlkJ for 1-butanol, 1-hexanol, and 1-octanol as well as the n-alkanol derivative ω-hydroxy lauric acid methyl ester (HLAMe) was quantified in the presence of the artificial electron acceptors phenazine methosulfate (PMS) and 2,6-dichlorophenolindophenol (DCPIP), indicating broad substrate specificity with the lowest activity on the shortest alcohol, 1-butanol. Furthermore, AlkJ was able to accept as cosubstrates/oxidants the ubiquinone derivatives Q0 and Q1, also in conjunction with cytochrome c, which suggests coupling to the bacterial respiratory chain of this membrane-associated enzyme in its physiological environment. Using gas chromatographic analysis, we demonstrated specific biocatalytic conversion by AlkJ of the substrate HLAMe to the industrially relevant aldehyde, thus enabling the biotechnological production of 12-amino lauric acid methyl ester via subsequent enzymatic transamination. PMID:24509930

  18. Two-pore Channels (TPC2s) and Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) at Lysosomal-Sarcoplasmic Reticular Junctions Contribute to Acute and Chronic β-Adrenoceptor Signaling in the Heart*

    PubMed Central

    Capel, Rebecca A.; Bolton, Emma L.; Lin, Wee K.; Aston, Daniel; Wang, Yanwen; Liu, Wei; Wang, Xin; Burton, Rebecca-Ann B.; Bloor-Young, Duncan; Shade, Kai-Ting; Ruas, Margarida; Parrington, John; Churchill, Grant C.; Lei, Ming; Galione, Antony; Terrar, Derek A.

    2015-01-01

    Ca2+-permeable type 2 two-pore channels (TPC2) are lysosomal proteins required for nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca2+ release in many diverse cell types. Here, we investigate the importance of TPC2 proteins for the physiology and pathophysiology of the heart. NAADP-AM failed to enhance Ca2+ responses in cardiac myocytes from Tpcn2−/− mice, unlike myocytes from wild-type (WT) mice. Ca2+/calmodulin-dependent protein kinase II inhibitors suppressed actions of NAADP in myocytes. Ca2+ transients and contractions accompanying action potentials were increased by isoproterenol in myocytes from WT mice, but these effects of β-adrenoreceptor stimulation were reduced in myocytes from Tpcn2−/− mice. Increases in amplitude of L-type Ca2+ currents evoked by isoproterenol remained unchanged in myocytes from Tpcn2−/− mice showing no loss of β-adrenoceptors or coupling mechanisms. Whole hearts from Tpcn2−/− mice also showed reduced inotropic effects of isoproterenol and a reduced tendency for arrhythmias following acute β-adrenoreceptor stimulation. Hearts from Tpcn2−/− mice chronically exposed to isoproterenol showed less cardiac hypertrophy and increased threshold for arrhythmogenesis compared with WT controls. Electron microscopy showed that lysosomes form close contacts with the sarcoplasmic reticulum (separation ∼25 nm). We propose that Ca2+-signaling nanodomains between lysosomes and sarcoplasmic reticulum dependent on NAADP and TPC2 comprise an important element in β-adrenoreceptor signal transduction in cardiac myocytes. In summary, our observations define a role for NAADP and TPC2 at lysosomal/sarcoplasmic reticulum junctions as unexpected but major contributors in the acute actions of β-adrenergic signaling in the heart and also in stress pathways linking chronic stimulation of β-adrenoceptors to hypertrophy and associated arrhythmias. PMID:26438825

  19. Null mutation of the nicotinamide adenine dinucleotide phosphate-oxidase subunit p67phox protects the Dahl-S rat from salt-induced reductions in medullary blood flow and glomerular filtration rate.

    PubMed

    Evans, Louise C; Ryan, Robert P; Broadway, Elizabeth; Skelton, Meredith M; Kurth, Theresa; Cowley, Allen W

    2015-03-01

    Null mutations in the p67(phox) subunit of nicotinamide adenine dinucleotide phosphate-oxidase confer protection from salt sensitivity on Dahl salt-sensitive rats. Here, we track the sequential changes in medullary blood flow (MBF), glomerular filtration rate (GFR), urinary protein, and mean arterial pressure in SSp67(phox) null rats and wild-type littermates during 21 days of 4.0% NaCl high-salt (HS) diet. Optical fibers were implanted in the renal medulla and MBF was measured in conscious rats by laser Doppler flowmetry. Separate groups of rats were prepared with femoral venous catheters and GFR was measured by the transcutaneous assessment of fluorescein isothiocyanate-sinistrin disappearance curves. Mean arterial blood pressure was measured by telemetry. In wild-type rats, HS caused a rapid reduction in MBF, which was significantly lower than control values by HS day-6. Reduced MBF was associated with a progressive increase in mean arterial pressure, averaging 170±5 mm Hg by HS salt day-21. A significant reduction in GFR was evident on day-14 HS, after the onset of hypertension and reduced MBF. In contrast, HS had no significant effect on MBF in SSp67(phox) null rats and the pressor response to sodium was blunted, averaging 150±3 mm Hg on day-21 HS. GFR was maintained throughout the study and proteinuria was reduced. In summary, when p67(phox) is not functional in the salt-sensitive rats, HS does not cause reduced MBF and salt-sensitive hypertension is attenuated, and consequently renal injury is reduced and GFR is maintained. PMID:25489057

  20. Nicotinamide Adenine Dinucleotide Based Therapeutics, Update.

    PubMed

    Pankiewicz, K W; Petrelli, R; Singh, R; Felczak, K

    2015-01-01

    About 500 NAD (P)-dependent enzymes in the cell use NAD (P) as a cofactor or a substrate. This family of broadly diversified enzymes is crucial for maintaining homeostasis of all living organisms. The NAD binding domain of these enzymes is conserved and it was believed that NAD mimics would not be of therapeutic value due to lack of selectivity. Consequently, only mycophenolic acid which selectively binds at the cofactor pocket of NAD-dependent IMP-dehydrogenase (IMPDH) has been approved as an immunosuppressant. Recently, it became clear that the NAD (P)-binding domain was structurally much more diversified than anticipated and numerous highly potent and selective inhibitors of NAD (P) dependent enzymes have been reported. It is likely, that as in the case of protein kinases inhibitors, inhibitors of NAD (P)-dependent enzymes would find soon their way to the clinic. In this review, recent developments of selective inhibitors of NAD-dependent human IMPDH, as well as inhibitors of IMPDHs from parasites, and from bacterial sources are reported. Therapies against Cryptosporidium parvum and the development of new antibiotics that are on the horizon will be discussed. New inhibitors of bacterial NAD-ligases, NAD-kinases, NMN-adenylyl transferases, as well as phosphoribosyl transferases are also described. Although none of these compounds has yet to be approved, the progress in revealing and understanding crucial factors that might allow for designing more potent and efficient drug candidates is enormous and highly encouraging. PMID:26295463

  1. Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

    PubMed Central

    Montanaro, L.; Sperti, Simonetta

    1967-01-01

    1. Changes in protein fluorescence have been utilized in determining the stoicheiometry and dissociation constants of the complexes of diphtheria toxin with NADH2, NAD, NADPH2 and NADP. 2. The binding stoicheiometry is 2moles of NADH2 and 1mole of NADPH2/mole of diphtheria toxin. The binding sites for NADH2 appear to be equivalent and independent. 3. The toxin shows a higher affinity for the reduced than for the oxidized forms of the nucleotides. 4. Dissociation constants at 0·01I, pH7 and 25° are 0·7×10−6m for NADH2 and 0·45×10−6m for NADPH2. Dissociation constants increase with increasing ionic strength, indicating that the binding is mainly electrostatic. 5. Bound NADH2 and NADPH2 may be activated to fluoresce by the transfer of energy from the excited aromatic amino acids of the toxin. Activation and emission spectra of bound and free nucleotides are compared. 6. Since NAD and NADH2 are cofactors specifically required for the inhibition of protein synthesis by diphtheria toxin, the possible role of toxin–nucleotide complexes is discussed in this regard. PMID:4384596

  2. Bioluminescent Cell-Based NAD(P)/NAD(P)H Assays for Rapid Dinucleotide Measurement and Inhibitor Screening

    PubMed Central

    Leippe, Donna; Sobol, Mary; Vidugiris, Gediminas; Zhou, Wenhui; Meisenheimer, Poncho; Gautam, Prson; Wennerberg, Krister; Cali, James J.

    2014-01-01

    Abstract The central role of nicotinamide adenine dinucleotides in cellular energy metabolism and signaling makes them important nodes that link the metabolic state of cells with energy homeostasis and gene regulation. In this study, we describe the implementation of cell-based bioluminescence assays for rapid and sensitive measurement of those important redox cofactors. We show that the sensitivity of the assays (limit of detection ∼0.5 nM) enables the selective detection of total amounts of nonphosphorylated or phosphorylated dinucleotides directly in cell lysates. The total amount of NAD+NADH or NADP+NADPH levels can be detected in as low as 300 or 600 cells/well, respectively. The signal remains linear up to 5,000 cells/well with the maximum signal-to-background ratios ranging from 100 to 200 for NAD+NADH and from 50 to 100 for NADP+NADPH detection. The assays are robust (Z′ value >0.7) and the inhibitor response curves generated using a known NAD biosynthetic pathway inhibitor FK866 correlate well with the reported data. More importantly, by multiplexing the dinucleotide detection assays with a fluorescent nonmetabolic cell viability assay, we show that dinucleotide levels can be decreased dramatically (>80%) by FK866 treatment before changes in cell viability are detected. The utility of the assays to identify modulators of intracellular nicotinamide adenine dinucleotide levels was further confirmed using an oncology active compound library, where novel dinucleotide regulating compounds were identified. For example, the histone deacetylase inhibitor entinostat was a potent inhibitor of cellular nicotinamide adenine dinucleotides, whereas the selective estrogen receptor modulator raloxifene unexpectedly caused a twofold increase in cellular nicotinamide adenine dinucleotide levels. PMID:25506801

  3. Search for interstellar adenine

    NASA Astrophysics Data System (ADS)

    Chakrabarti, Sandip K.; Majumdar, Liton; Das, Ankan; Chakrabarti, Sonali

    2015-05-01

    It is long debated if pre-biotic molecules are indeed present in the interstellar medium. Despite substantial works pointing to their existence, pre-biotic molecules are yet to be discovered with a complete confidence. In this paper, our main aim is to study the chemical evolution of interstellar adenine under various circumstances. We prepare a large gas-grain chemical network by considering various pathways for the formation of adenine. Majumdar et al. (New Astron. 20:15, 2013) proposed that in the absence of adenine detection, one could try to trace two precursors of adenine, namely, HCCN and NH2CN. Recently Merz et al. (J. Phys. Chem. A 118:3637-3644, 2014), proposed another route for the formation of adenine in interstellar condition. They proposed two more precursor molecules. But it was not verified by any accurate gas-grain chemical model. Neither was it known if the production rate would be high or low. Our paper fills this important gap. We include this new pathways to find that the contribution through this pathways for the formation of Adenine is the most dominant one in the context of interstellar medium. We propose that observers may look for the two precursors (C3NH and HNCNH) in the interstellar media which are equally important for predicting abundances of adenine. We perform quantum chemical calculations to find out spectral properties of adenine and its two new precursor molecules in infrared, ultraviolet and sub-millimeter region. Our present study would be useful for predicting abundance of adenine.

  4. Fate of prebiotic adenine.

    PubMed

    Cohn, C A; Hansson, T K; Larsson, H S; Sowerby, S J; Holm, N G

    2001-01-01

    Equilibrium adsorption isotherm data for the purine base adenine has been obtained on several prebiotically relevant minerals by frontal analysis using water as a mobile phase. Adenine is far displaced toward adsorption on pyrite (FeS2), quartz (SiO2), and pyrrhotite (FeS), but somewhat less for magnetite (Fe3O4) and forsterite (Mg2SiO4). The prebiotic prevalence of these minerals would have allowed them to act as a sink for adenine; removal from the aqueous phase would confer protection from hydrolysis as well, establishing a nonequilibrium thermodynamic framework for increased adenine synthesis. Our results provide evidence that adsorption phenomena may have been critical for the primordial genetic architecture. PMID:12448980

  5. A DFT study of 2-aminopurine-containing dinucleotides: prediction of stacked conformations with B-DNA structure.

    PubMed

    Smith, Darren A; Holroyd, Leo F; van Mourik, Tanja; Jones, Anita C

    2016-05-25

    The fluorescence properties of dinucleotides incorporating 2-aminopurine (2AP) suggest that the simplest oligonucleotides adopt conformations similar to those found in duplex DNA. However, there is a lack of structural data for these systems. We report a density functional theory (DFT) study of the structures of 2AP-containing dinucleotides (deoxydinucleoside monophosphates), including full geometry optimisation of the sugar-phosphate backbone. Our DFT calculations employ the M06-2X functional for reliable treatment of dispersion interactions and include implicit aqueous solvation. Dinucleotides with 2AP in the 5'-position and each of the natural bases in the 3'-position are examined, together with the analogous 5'-adenine-containing systems. Computed structures are compared in detail with typical B-DNA base-step parameters, backbone torsional angles and sugar pucker, derived from crystallographic data. We find that 2AP-containing dinucleotides adopt structures that closely conform to B-DNA in all characteristic parameters. The structures of 2AP-containing dinucleotides closely resemble those of their adenine-containing counterparts, demonstrating the fidelity of 2AP as a mimic of the natural base. As a first step towards exploring the conformational heterogeneity of dinucleotides, we also characterise an imperfectly stacked conformation and one in which the bases are completely unstacked. PMID:27186599

  6. Photodissociation of dinucleotide ions in a storage ring

    NASA Astrophysics Data System (ADS)

    Worm, Esben S.; Andersen, Inge Hald; Andersen, Jens Ulrik; Holm, Anne I. S.; Hvelplund, Preben; Kadhane, Umesh; Nielsen, Steen Brøndsted; Poully, Jean-Christophe; Støchkel, Kristian

    2007-04-01

    The decay of protonated DNA dinucleotides, dA2+ , dG2+ , dT2+ , dC2+ and deprotonated ones, dA2- and dT2- , after 260nm photon absorption was measured in an electrostatic ion storage ring (A denotes adenine, G guanine, T thymine, and C cytosine). Fragmentation on the microsecond time scale was observed and assigned to statistical dissociation. Good fits to the decay spectra were obtained with a model based on microcanonical rate constants of the Arrhenius type with activation energies and preexponential factors for the dissociation that agree well with literature values. In accordance with results from other groups, dT2+ was found to have the longest lifetime among the cations. The importance of decay processes faster than the microsecond time scale is elucidated by a comparison between the total ion beam depletion and that due to the observed statistical decay. We find that such processes play a major role for all of the dinucleotides, being more than 25 times more probable than the microsecond statistical dissociation for dA2+ , dG2+ , and dC2+ , about 10 times for dT2+ , and between 2 and 6 times for dA2- and dT2- . For the cations, we ascribe these processes to nonstatistical dissociation prior to randomization of the excitation energy among all degrees of freedom whereas direct photoelectron detachment may play a role for the anions. Thus, our data indicate that the propensity for nonstatistical dissociation increases upon nucleobase protonation. Consistent with this trend, the propensity is less for dT2+ than for the other dinucleotide cations because the phosphoric acid group competes with thymine for the proton.

  7. Photoinduced Electron Transfer in DNA: Charge Shift Dynamics Between 8-Oxo-Guanine Anion and Adenine.

    PubMed

    Zhang, Yuyuan; Dood, Jordan; Beckstead, Ashley A; Li, Xi-Bo; Nguyen, Khiem V; Burrows, Cynthia J; Improta, Roberto; Kohler, Bern

    2015-06-18

    Femtosecond time-resolved IR spectroscopy is used to investigate the excited-state dynamics of a dinucleotide containing an 8-oxoguanine anion at the 5'-end and neutral adenine at the 3'-end. UV excitation of the dinucleotide transfers an electron from deprotonated 8-oxoguanine to its π-stacked neighbor adenine in less than 1 ps, generating a neutral 8-oxoguanine radical and an adenine radical anion. These species are identified by the excellent agreement between the experimental and calculated IR difference spectra. The quantum efficiency of this ultrafast charge shift reaction approaches unity. Back electron transfer from the adenine radical anion to the 8-oxguanine neutral radical occurs in 9 ps, or approximately 6 times faster than between the adenine radical anion and the 8-oxoguanine radical cation (Zhang, Y. et al. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 11612-11617). The large asymmetry in forward and back electron transfer rates is fully rationalized by semiclassical nonadiabatic electron transfer theory. Forward electron transfer is ultrafast because the driving force is nearly equal to the reorganization energy, which is estimated to lie between 1 and 2 eV. Back electron transfer is highly exergonic and takes place much more slowly in the Marcus inverted region. PMID:25660103

  8. Maximal dinucleotide and trinucleotide circular codes.

    PubMed

    Michel, Christian J; Pellegrini, Marco; Pirillo, Giuseppe

    2016-01-21

    We determine here the number and the list of maximal dinucleotide and trinucleotide circular codes. We prove that there is no maximal dinucleotide circular code having strictly less than 6 elements (maximum size of dinucleotide circular codes). On the other hand, a computer calculus shows that there are maximal trinucleotide circular codes with less than 20 elements (maximum size of trinucleotide circular codes). More precisely, there are maximal trinucleotide circular codes with 14, 15, 16, 17, 18 and 19 elements and no maximal trinucleotide circular code having less than 14 elements. We give the same information for the maximal self-complementary dinucleotide and trinucleotide circular codes. The amino acid distribution of maximal trinucleotide circular codes is also determined. PMID:26382231

  9. Oxidized and Reduced Nicotinamide Adenine Dinucleotide Phosphate Levels of Plants Hardened and Unhardened Against Chilling Injury

    PubMed Central

    Kuraishi, Susumu; Arai, Noriko; Ushijima, Tadahiro; Tazaki, Tadayoshi

    1968-01-01

    Pea plants (Pisum sativum L. var. Alaska) subjected to low temperature (5°) in the light acquired resistance against chilling injury. Unhardened plants maintained high NADP and low NADPH levels during illumination at 25° but hardened plants had low NADP and high NADPH levels in the light. When the unhardened plants were transferred to the dark room at 25°, their NADPH levels decreased immediately. On the other hand, hardened plants maintained a high NADPH level for a few hours even in the dark. PMID:16656757

  10. Studies on free and enzyme-bound nicotinamide adenine dinucleotide free radicals

    SciTech Connect

    Bielski, B.H.J.; Chan, P.C.

    1980-02-27

    The spectral and kinetic properties of the NAD free radical have been studied as a function of temperature and pH. The radical decays by second-order kinetics to an enzymatically inactive dimer (NAD)/sub 2/. At 23.5/sup 0/C and pH 7.3 the corresponding rate constant is k/sub 9/ = (7.72 +- 0.78) x 10/sup 7/ M/sup -1/s/sup -1/ with an activation energy E/sub a/ = 3.4 +- 0.4 kcal/mol. Upon attachment of the NAD radical to an enzyme active site, the radical becomes stabilized. The stabilization effect (ratio of the rate of NAD disappearance in the absence and presence of an enzyme) depends upon the nature of the enzyme and varies from 1.54 x 10/sup 2/ for alcohol dehydrogenase, 2.57 x 10/sup 2/ for malate dehydrogenase, 1.1 x 10/sup 3/ for lactate dehydrogenase, to 1.54 x 10/sup 4/ for glyceraldehyde-3-phosphate dehydrogenase. The observed second-order disappearance of enzyme-stabilized NAD is explained by a mechanism that is dependent upon the dissociation constant of the enzyme-NAD complex. 6 figures

  11. Glyceraldehyde-3-phosphate dehydrogenase-catalyzed chain oxidation of reduced nicotinamide adenine dinucleotide by perhydroxyl radicals

    SciTech Connect

    Chan, P.C.

    1980-02-10

    The chain oxidation of glyceraldehyde-3-phosphate dehydrogenase NADH by perhydroxyl radicals and propagated by molecular oxygen was studied by the xanthine-xanthine oxidase system, /sup 60/Co ..gamma..-ray, and pulse radiolysis. The chain length, amount of NADH oxidized per HO/sub 2/ generated, increases with increasing acidity of the medium and reaches a value of 73 at pH 5.0. The rate constant for the oxidation of the glyceraldehyde-3-phosphate dehydrogenase NADH complex by HO/sub 2/ was estimated to be 2 x 10/sup 7/ m/sup -1/s/sup -1/ at ambient temperatures (23-24/sup 0/C). Rate studies as a function of pH indicate that O/sub 2//sup -/ is unreactive toward the glyceraldehyde-3-phosphate dehydrogenase NADH complex. Other dehydrogenases (malate dehydrogenase, glutamate dehydrogenase, and isocitric dehydrogenase) studied showed no catalytic activity in the oxidation of NADH by HO/sub 2//O/sub 2//sup -/.

  12. Interaction of pigeon-liver nicotinamide-adenine dinucleotide kinase with cibacron blue F3GA.

    PubMed Central

    Apps, D K; Gleed, C D

    1976-01-01

    The interaction of pigeon liver NAD kinase with Cibacron Blue F3GA was investigated. By using steady-state rate measurements, spectrophotometric titration and chromatography of the enzyme on immobilized dye, it was shown that binding occurs at two nucleotide sites with different affinities, and also at a site distinct from the substrate-binding region. PMID:187176

  13. Predicting Flavin and Nicotinamide Adenine Dinucleotide-Binding Sites in Proteins Using the Fragment Transformation Method

    PubMed Central

    Lin, Yu-Feng; Chen, Jin-Yi

    2015-01-01

    We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified. PMID:26000290

  14. [Synthesis of nicotinamide adenine dinucleotide in the nuclei of pigeon erythrocytes].

    PubMed

    Nemchinskaia, V L; Makarova, T G; Mozhenok, T P; Braun, A D

    1975-08-01

    The nuclei of pigeon erythrocytes are capable of synthesizing NAD from nicotinamid-mononucleotides and ATP. Some data on the kinetics of NAD-pyrophosphorylase have been obtained: the optimal concentration of nuclei and the effect of various incubation time. The pretreatment of nuclei by Triton X-100, or by ultrasonics enhances NAD synthesis. The results suggest that cyclic 3',5'-AMP (Fluka) may have no effect on NAD synthesis. The control of the cell metabolism by NAD formation is considered. PMID:181878

  15. Aging-related nicotinamide adenine dinucleotide oxidase response to dietary supplementation: the French paradox revisited.

    PubMed

    Morré, D James; Morré, Dorothy M; Shelton, Thomas B

    2010-01-01

    Aging-related cell-surface NADH oxidase (arNOX)-specific activities increase with age between age 30 and ages 50-65. The protein is shed and circulates. Activity correlates with a number of aging-related disorders including low-density lipoprotein (LDL) oxidation as a precondition to atherosclerosis as well as oxidation of collagen and elastin as a major contributor to skin aging. arNOX inhibitors formulated for sustained release are capable of maintaining circulating arNOX at low levels with regular use as food supplements formulated with natural compounds. Among the best sources are certain culinary seasonings, all of which are ingredients used extensively in the French kitchen. Their regular use may contribute to an understanding of the nutritional basis for the French Paradox. PMID:19954304

  16. Modification of Metabolic Pattern by Variation of Nicotinamide Adenine Dinucleotide Phosphate Level 1

    PubMed Central

    Yamamoto, Yukio

    1969-01-01

    The experiments were designed to get some information on the metabolism controlled by variation of the NADP level, which is known to change with the variation of environmental factors. The exogenous NADP added to the mitochondria prepared from Vigna sesquipedalis cotyledons was associated with and/or penetrated into the mitochondria. The combined NADP served in the operation of the mitochondrial NADP-isocitric acid dehydrogenase. The variation of NADP level by exogenous NADP was observed to modify the rates of metabolic processes. The increase of exogenous NADP in Vigna hypocotyl slices lowered malic- and citric-acid contents and raised the α-ketoglutaric acid content. The incorporation of 14C from acetate-2-14C into lipid, organic acid, amino acid, was promoted with the exogenous NADP. The 14C-incorporation into glycolic acid, malic acid and glutamic acid was accelerated. In the mannitol homogenate of Vigna cotyledon, 14CO2 evolution and 14C-incorporation into lipid, sugar, and glycolic acid from acetate-2-14C were promoted with the exogenous NADP. Endogenous citric acid content was lowered by NADP, while malic acid content was increased. The activation of NADP-enzymes by NADP was discussed to be involved in these variations. PMID:16657076

  17. Automated genotyping of dinucleotide repeat markers

    SciTech Connect

    Perlin, M.W.; Hoffman, E.P. |

    1994-09-01

    The dinucleotide repeats (i.e., microsatellites) such as CA-repeats are a highly polymorphic, highly abundant class of PCR-amplifiable markers that have greatly streamlined genetic mapping experimentation. It is expected that over 30,000 such markers (including tri- and tetranucleotide repeats) will be characterized for routine use in the next few years. Since only size determination, and not sequencing, is required to determine alleles, in principle, dinucleotide repeat genotyping is easily performed on electrophoretic gels, and can be automated using DNA sequencers. Unfortunately, PCR stuttering with these markers generates not one band for each allele, but a pattern of bands. Since closely spaced alleles must be disambiguated by human scoring, this poses a key obstacle to full automation. We have developed methods that overcome this obstacle. Our model is that the observed data is generated by arithmetic superposition (i.e., convolution) of multiple allele patterns. By quantitatively measuring the size of each component band, and exploiting the unique stutter pattern associated with each marker, closely spaced alleles can be deconvolved; this unambiguously reconstructs the {open_quotes}true{close_quotes} allele bands, with stutter artifact removed. We used this approach in a system for automated diagnosis of (X-linked) Duchenne muscular dystrophy; four multiplexed CA-repeats within the dystrophin gene were assayed on a DNA sequencer. Our method accurately detected small variations in gel migration that shifted the allele size estimate. In 167 nonmutated alleles, 89% (149/167) showed no size variation, 9% (15/167) showed 1 bp variation, and 2% (3/167) showed 2 bp variation. We are currently developing a library of dinucleotide repeat patterns; together with our deconvolution methods, this library will enable fully automated genotyping of dinucleotide repeats from sizing data.

  18. Molybdopterin Dinucleotide Biosynthesis in Escherichia coli

    PubMed Central

    Neumann, Meina; Seduk, Farida; Iobbi-Nivol, Chantal; Leimkühler, Silke

    2011-01-01

    The molybdenum cofactor is modified by the addition of GMP or CMP to the C4′ phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP:molybdopterin guanylyltransferase MobA and the CTP:molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein. PMID:21081498

  19. Vertical Singlet Excitations on Adenine Dimer: A Time Dependent Density Functional Study

    NASA Astrophysics Data System (ADS)

    Crespo-Hernández, Carlos E.; Marai, Christopher N. J.

    2007-12-01

    The condense phase, excited state dynamics of the adenylyl(3'→5')adenine (ApA) dinucleotide has been previously studied using transient absorption spectroscopy with femtosecond time resolution (Crespo-Hernández et al. Chem. Rev. 104, 1977-2019 (2004)). An ultrafast and a long-lived component were observed with time constants of <1 ps and 60±16 ps, respectively. Comparison of the time constants measured for the dinucleotide with that for the adenine nucleotide suggested that the fast component observed in ApA could be assigned to monomer dynamics. The long-lived component observed in ApA was assigned to an excimer state that originates from a fraction of base stacked conformations present at the time of excitation. In this contribution, supermolecule calculations using the time dependent implementation of density functional theory is used to provide more insights on the origin of the initial Franck-Condon excitations. Monomer-like, localized excitations are observed for conformations having negligible base stacking interactions, whereas delocalized excitations are predicted for conformations with significant vertical base-base overlap.

  20. Solution conformation of 2-aminopurine (2-AP) dinucleotide determined by ultraviolet 2D fluorescence spectroscopy (UV-2D FS)

    PubMed Central

    Widom, Julia R.; Johnson, Neil P.; von Hippel, Peter H.; Marcus, Andrew H.

    2013-01-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analog of the nucleic acid base adenine. This was accomplished by extending two-dimensional fluorescence spectroscopy (2D FS) – a fluorescence-detected variation of 2D electronic spectroscopy – to excite molecular transitions in the ultraviolet (UV) regime. A collinear sequence of four ultrafast laser pulses centered at 323 nm was used to resonantly excite the coupled transitions of 2-AP dinucleotide. The phases of the optical pulses were continuously swept at kilohertz frequencies, and the ensuing nonlinear fluorescence was phase-synchronously detected at 370 nm. Upon optimization of a point-dipole coupling model to our data, we found that in aqueous buffer the 2-AP dinucleotide adopts an average conformation in which the purine bases are non-helically stacked (center-to-center distance R12 = 3.5 Å ± 0.5 Å, twist angle θ12 = 5° ± 5°), which differs from the conformation of such adjacent bases in duplex DNA. These experiments establish UV-2D FS as a method for examining the local conformations of an adjacent pair of fluorescent nucleotides substituted into specific DNA or RNA constructs, which will serve as a powerful probe to interpret, in structural terms, biologically significant local conformational changes within the nucleic acid framework of protein-nucleic acid complexes. PMID:24223491

  1. Adenine Aminohydrolase from Leishmania donovani

    PubMed Central

    Boitz, Jan M.; Strasser, Rona; Hartman, Charles U.; Jardim, Armando; Ullman, Buddy

    2012-01-01

    Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent Km of 15.4 μm, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Δaah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Δaah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Δaah lesion was also introduced into a conditionally lethal Δhgprt/Δxprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2′-deoxycoformycin. The Δaah/Δhgprt/Δxprt triple knock-out no longer required 2′-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani. PMID:22238346

  2. Bound anionic states of adenine

    SciTech Connect

    Haranczyk, Maciej; Gutowski, Maciej S; Li, Xiang; Bowen, Kit H

    2007-03-20

    Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases, are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the new-found anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The discovery of these valence anionic states of adenine was facilitated by the development of: (i) a new experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (ii) a new combinatorial/ quantum chemical approach for identification of the most stable tautomers of organic molecules. The computational portion of this work was supported by the: (i) Polish State Committee for Scientific Research (KBN) Grants: DS/8000-4-0140-7 (M.G.) and N204 127 31/2963 (M.H.), (ii) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.), and (iii) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic

  3. Formation of the imidazolides of dinucleotides under potentially prebiotic conditions

    NASA Technical Reports Server (NTRS)

    Sleeper, H. L.; Lohrmann, R.; Orgel, L. E.

    1978-01-01

    Imidazolides of dinucleotides such as ImpApA can be formed from the corresponding dinucleotides in a two-stage process, which gives up to 15% yields under potentially prebiotic conditions. First a solution of the dinucleotide and sodium trimetaphosphate is dried out at constant temperature and humidity. This produces polyphosphates such as p(n)ApA in excellent yield (greater than or equal to 80%). The products are dissolved in water, imidazole is added, and the solution is dried out again. This yields the 5'-phosphorimidazolides.

  4. Mononucleotide and dinucleotide frequencies, and codon usage in poliovirion RNA.

    PubMed Central

    Rothberg, P G; Wimmer, E

    1981-01-01

    The polio type 1 (Mahoney) RNA sequence (1) has been analyzed in terms of the distribution of its mononucleotides, dinucleotides and trinucleotides (codons). The distribution of adenosine in the sequence is nonuniform, being lower at the 5' end and higher at the 3' end. The dinucleotide CG is relatively rare and the dinucleotides UG and CA are relatively more common than expected. Codon usage is decidedly nonrandom. Codons containing CG are avoided and those ending in adenosine are favored. The asymmetric use of mononucleotides, dinucleotides and codons in polio RNA is unexplained at the present time although the lowered CG frequency may be the result of a DNA origin for polio RNA. PMID:6275352

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

    PubMed Central

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

    1969-01-01

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

  6. Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells.

    PubMed

    Yang, Tianle; Chan, Noel Yan-Ki; Sauve, Anthony A

    2007-12-27

    A new two-step methodology achieves stereoselective synthesis of beta-nicotinamide riboside and a series of related amide, ester, and acid nucleosides. Compounds were prepared through a triacetylated-nicotinate ester nucleoside, via coupling of either ethylnicotinate or phenylnicotinate with 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose. Nicotinamide riboside, nicotinic acid riboside, O-ethylnicotinate riboside, O-methylnicotinate riboside, and several N-alkyl derivatives increased NAD+ concentrations from 1.2-2.7-fold in several mammalian cell lines. These findings establish bioavailability and potent effects of these nucleosides in stimulating the increase of NAD+ concentrations in mammalian cells. PMID:18052316

  7. Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

    PubMed

    Dittrich, P

    1976-02-01

    NAD-specific "malic" enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO(4) (2-), suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn(2+), which cannot be replaced by NADP or Mg(2+). For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn(2+), and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 mumole/hr.mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO(2) for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  8. A COUPLED MICROSOMAL-ACTIVATING/EMBRYO CULTURE SYSTEM: TOXICITY OF REDUCED BETA-NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (NADPH)

    EPA Science Inventory

    An NADPH-dependent microsomal-activating system has been coupled to a rat embryo culture in vitro. No embryonic morphological abnormalities or decrease in final yolk sac or embryo DNA and protein contents occurred when 0.2 mM NADPH was used in this coupled system. In contrast, 1....

  9. Enzymatic production by tissue extracts of a metabolite of nicotinamide adenine dinucleotide with calcium-releasing ability

    SciTech Connect

    Tich, N.R.

    1989-01-01

    This research investigated the occurrence and characterization of the metabolite in mammalian tissues. In all mammalian tissues tested, including rabbit liver, heart, spleen, kidney, and brain, the factor to convert NAD into its active metabolite was present. The conversion exhibited many characteristics of an enzymatic process such as temperature sensitivity, concentration dependence and protease sensitivity. Production of the NAD metabolite occurred within a time frame of 15-45 minutes at 37{degree}C, depending upon the particular preparation. The metabolite was isolated using high performance liquid chromatography from all mammalian tissues. This purified metabolite was then tested for its effectiveness in releasing intracellular calcium in an intact cell by microinjecting it into unfertilized sea urchin eggs. These eggs undergo a massive morphological change upon fertilization which is dependent upon the release of calcium from inside the cell. Upon injection of the NAD metabolite into unfertilized eggs, this same morphological change was observed showing indirectly that the metabolite released intracellular calcium from an intact, viable cell. In addition, radioactive studies using {sup 45}Ca{sup 2+} loaded into permeabilized hepatocytes, indicated in preliminary studies that the NAD metabolite could also release calcium from intracellular stores of mammalian cells.

  10. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

    In this work we study deactivation processes in adenine oligomers after absorption of UV radiation using Quantum Mechanics combined with Molecular Mechanics (QM/MM). Correlated electronic structure methods appropriate for describing the excited states are used to describe a π-stacked dimer of adenine bases incorporated into (dA)20(dT)20. The results of these calculations reveal three different types of excited state minima which play a role in deactivation processes. Within this set of minima there are minima where the excited state is localized on one adenine (monomer-like) as well as minima where the excited state is delocalized on two adenines, forming different types of excimers and bonded excimers of varying but inter-related character. The proximity of their energies reveals that the minima can decay into one another along a flat potential energy surface dependent on the interbase separation. Additionally, analysis of the emissive energies and other physical properties, including theoretical anisotropy calculations, and comparison with fluorescence experiments, provides evidence that excimers play an important role in long-lived signals in adenine oligonucleotides while the subpicosecond decay is attributed to monomer-like minima. The necessity for a close approach of the nucleobases reveals that the deactivation mechanism is tied to macro-molecular motion. PMID:26536353

  11. The catalase activity of diiron adenine deaminase.

    PubMed

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

    2011-12-01

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

  12. Dinucleotide controlled null models for comparative RNA gene prediction

    PubMed Central

    Gesell, Tanja; Washietl, Stefan

    2008-01-01

    Background Comparative prediction of RNA structures can be used to identify functional noncoding RNAs in genomic screens. It was shown recently by Babak et al. [BMC Bioinformatics. 8:33] that RNA gene prediction programs can be biased by the genomic dinucleotide content, in particular those programs using a thermodynamic folding model including stacking energies. As a consequence, there is need for dinucleotide-preserving control strategies to assess the significance of such predictions. While there have been randomization algorithms for single sequences for many years, the problem has remained challenging for multiple alignments and there is currently no algorithm available. Results We present a program called SISSIz that simulates multiple alignments of a given average dinucleotide content. Meeting additional requirements of an accurate null model, the randomized alignments are on average of the same sequence diversity and preserve local conservation and gap patterns. We make use of a phylogenetic substitution model that includes overlapping dependencies and site-specific rates. Using fast heuristics and a distance based approach, a tree is estimated under this model which is used to guide the simulations. The new algorithm is tested on vertebrate genomic alignments and the effect on RNA structure predictions is studied. In addition, we directly combined the new null model with the RNAalifold consensus folding algorithm giving a new variant of a thermodynamic structure based RNA gene finding program that is not biased by the dinucleotide content. Conclusion SISSIz implements an efficient algorithm to randomize multiple alignments preserving dinucleotide content. It can be used to get more accurate estimates of false positive rates of existing programs, to produce negative controls for the training of machine learning based programs, or as standalone RNA gene finding program. Other applications in comparative genomics that require randomization of multiple

  13. Graphene-Enhanced Raman Scattering from the Adenine Molecules.

    PubMed

    Dolgov, Leonid; Pidhirnyi, Denys; Dovbeshko, Galyna; Lebedieva, Tetiana; Kiisk, Valter; Heinsalu, Siim; Lange, Sven; Jaaniso, Raivo; Sildos, Ilmo

    2016-12-01

    An enhanced Raman scattering from a thin layer of adenine molecules deposited on graphene substrate was detected. The value of enhancement depends on the photon energy of the exciting light. The benzene ring in the structure of adenine molecule suggests π-stacking of adenine molecule on top of graphene. So, it is proposed that the enhancement in the adenine Raman signal is explained by the resonance electron transfer from the Fermi level of graphene to the lowest unoccupied molecular orbital (LUMO) level of adenine. PMID:27075339

  14. Graphene-Enhanced Raman Scattering from the Adenine Molecules

    NASA Astrophysics Data System (ADS)

    Dolgov, Leonid; Pidhirnyi, Denys; Dovbeshko, Galyna; Lebedieva, Tetiana; Kiisk, Valter; Heinsalu, Siim; Lange, Sven; Jaaniso, Raivo; Sildos, Ilmo

    2016-04-01

    An enhanced Raman scattering from a thin layer of adenine molecules deposited on graphene substrate was detected. The value of enhancement depends on the photon energy of the exciting light. The benzene ring in the structure of adenine molecule suggests π-stacking of adenine molecule on top of graphene. So, it is proposed that the enhancement in the adenine Raman signal is explained by the resonance electron transfer from the Fermi level of graphene to the lowest unoccupied molecular orbital (LUMO) level of adenine.

  15. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

    SciTech Connect

    Lee, Seongmin; Verdine, Gregory L.

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases have been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.

  16. Excimer states in microhydrated adenine clusters.

    PubMed

    Smith, V R; Samoylova, E; Ritze, H-H; Radloff, W; Schultz, T

    2010-09-01

    We present femtosecond pump-probe mass and photoelectron spectra for adenine (A) and microhydrated A(m)(H(2)O)(n) clusters. Three distinct relaxation processes of photoexcited electronic states were distinguished: in unhydrated A, relaxation of the optically bright pipi* state occurred via the dark npi* state with respective lifetimes of <0.1 and 1.3 ps. In microhydrated clusters A(H(2)O)(n), relaxation via the npi* state is quenched by a faster relaxation process, probably involving pisigma* states. For the predominantly hydrogen-bonded adenine dimer (A(2)), excited state relaxation is dominated by monomer-like processes. When the adenine dimer is clustered with several water molecules, we observe a nanosecond lifetime from excimer states in pi-stacked clusters. From the electron spectra we estimate adiabatic ionization potentials of 8.32 eV (A), 8.27 eV (A(H(2)O)(1)), 8.19 eV (A(H(2)O)(2)), 8.10 eV (A(H(2)O)(3)), 8.18 eV (A(2)), and 8.0 eV (A(2)(H(2)O)(3-5)). PMID:20556283

  17. The catalase activity of diiron adenine deaminase

    SciTech Connect

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

    2011-12-01

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

  18. The Nature's Clever Trick for Making Cyclic Dinucleotide.

    PubMed

    Fang, Pengfei; Guo, Min

    2015-05-01

    Ever since their initial discovery few years ago, cyclic dinucleotides (cDNs), their biosynthesis, and their biological function have been in focus of intense research efforts. In this issue, Kato et al. (2015) present strong evidence that the key enzyme in cDN biosynthesis, DncV, is poised on a tipping point such that, given a nudge, the enzyme, can link the nucleotides into a distinct cyclic loop, leading to a specific innate immune response. PMID:25955098

  19. Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

    PubMed Central

    2015-01-01

    The cyclic dinucleotides cyclic 3′,5′-diguanylate (c-di-GMP) and cyclic 3′,5′-diadenylate (c-di-AMP) have emerged as key components of bacterial signal transduction networks. These closely related second messengers follow the classical general principles of nucleotide signaling by integrating diverse signals into regulatory pathways that control cellular responses to changing environments. They impact distinct cellular processes, with c-di-GMP having an established role in promoting bacterial adhesion and inhibiting motility and c-di-AMP being involved in cell wall metabolism, potassium homeostasis, and DNA repair. The involvement of c-dinucleotides in the physiology of the filamentous, nonmotile streptomycetes remained obscure until recent discoveries showed that c-di-GMP controls the activity of the developmental master regulator BldD and that c-di-AMP determines the level of the resuscitation-promoting factor A(RpfA) cell wall-remodelling enzyme. Here, I summarize our current knowledge of c-dinucleotide signaling in Streptomyces species and highlight the important roles of c-di-GMP and c-di-AMP in the biology of these antibiotic-producing, multicellular bacteria. PMID:26216850

  20. Background correction using dinucleotide affinities improves the performance of GCRMA

    PubMed Central

    Gharaibeh, Raad Z; Fodor, Anthony A; Gibas, Cynthia J

    2008-01-01

    Background High-density short oligonucleotide microarrays are a primary research tool for assessing global gene expression. Background noise on microarrays comprises a significant portion of the measured raw data, which can have serious implications for the interpretation of the generated data if not estimated correctly. Results We introduce an approach to calculate probe affinity based on sequence composition, incorporating nearest-neighbor (NN) information. Our model uses position-specific dinucleotide information, instead of the original single nucleotide approach, and adds up to 10% to the total variance explained (R2) when compared to the previously published model. We demonstrate that correcting for background noise using this approach enhances the performance of the GCRMA preprocessing algorithm when applied to control datasets, especially for detecting low intensity targets. Conclusion Modifying the previously published position-dependent affinity model to incorporate dinucleotide information significantly improves the performance of the model. The dinucleotide affinity model enhances the detection of differentially expressed genes when implemented as a background correction procedure in GeneChip preprocessing algorithms. This is conceptually consistent with physical models of binding affinity, which depend on the nearest-neighbor stacking interactions in addition to base-pairing. PMID:18947404

  1. Methods for detection of methyl-CpG dinucleotides

    DOEpatents

    Dunn, John J.

    2012-09-11

    The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

  2. Methods for detection of methyl-CpG dinucleotides

    DOEpatents

    Dunn, John J.

    2013-01-29

    The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

  3. Methods for detection of methyl-CpG dinucleotides

    DOEpatents

    Dunn, John J

    2013-11-26

    The invention provides methods for enriching methyl-CpG sequences from a DNA sample. The method makes use of conversion of cytosine residues to uracil under conditions in which methyl-cytosine residues are preserved. Additional methods of the invention enable to preservation of the context of me-CpG dinucleotides. The invention also provides a recombinant, full length and substantially pure McrA protein (rMcrA) for binding and isolation of DNA fragments containing the sequence 5'-C.sup.MeCpGG-3'. Methods for making and using the rMcrA protein, and derivatives thereof are provided.

  4. In vitro selection of adenine-dependent hairpin ribozymes.

    PubMed

    Meli, Marc; Vergne, Jacques; Maurel, Marie-Christine

    2003-03-14

    Adenine-dependent hairpin ribozymes were isolated by in vitro selection from a degenerated hairpin ribozyme population. Two new adenine-dependent ribozymes catalyze their own reversible cleavage in the presence of free adenine. Both aptamers have Mg(2+) requirements for adenine-assisted cleavage similar to the wild-type hairpin ribozyme. Cleavage kinetics studies in the presence of various other small molecules were compared. The data suggest that adenine does not induce RNA self-cleavage in the same manner for both aptamers. In addition, investigations of pH effects on catalytic rates show that both adenine-dependent aptamers are more active in basic conditions, suggesting that they use new acid/base catalytic strategies in which adenine could be involved directly. The discovery of hairpin ribozymes dependent on adenine for their reversible self-cleavage presents considerable biochemical and evolutionary interests because we show that RNA is able to use exogenous reactive molecules to enhance its own catalytic activity. Such a mechanism may have been a means by which the ribozymes of the RNA world enlarged their chemical repertoire. PMID:12519767

  5. Adenine adlayers on Cu(111): XPS and NEXAFS study.

    PubMed

    Tsud, Nataliya; Bercha, Sofiia; Ševčíková, Klára; Acres, Robert G; Prince, Kevin C; Matolín, Vladimír

    2015-11-01

    The adsorption of adenine on Cu(111) was studied by photoelectron and near edge x-ray absorption fine structure spectroscopy. Disordered molecular films were deposited by means of physical vapor deposition on the substrate at room temperature. Adenine chemisorbs on the Cu(111) surface with strong rehybridization of the molecular orbitals and the Cu 3d states. Annealing at 150 °C caused the desorption of weakly bonded molecules accompanied by formation of a short-range ordered molecular adlayer. The interface is characterized by the formation of new states in the valence band at 1.5, 7, and 9 eV. The present work complements and refines existing knowledge of adenine interaction with this surface. The coverage is not the main parameter that defines the adenine geometry and adsorption properties on Cu(111). Excess thermal energy can further rearrange the molecular adlayer and, independent of the initial coverage, the flat lying stable molecular adlayer is formed. PMID:26547179

  6. Adenine adlayers on Cu(111): XPS and NEXAFS study

    SciTech Connect

    Tsud, Nataliya; Bercha, Sofiia; Ševčíková, Klára; Matolín, Vladimír; Acres, Robert G.; Prince, Kevin C.

    2015-11-07

    The adsorption of adenine on Cu(111) was studied by photoelectron and near edge x-ray absorption fine structure spectroscopy. Disordered molecular films were deposited by means of physical vapor deposition on the substrate at room temperature. Adenine chemisorbs on the Cu(111) surface with strong rehybridization of the molecular orbitals and the Cu 3d states. Annealing at 150 °C caused the desorption of weakly bonded molecules accompanied by formation of a short-range ordered molecular adlayer. The interface is characterized by the formation of new states in the valence band at 1.5, 7, and 9 eV. The present work complements and refines existing knowledge of adenine interaction with this surface. The coverage is not the main parameter that defines the adenine geometry and adsorption properties on Cu(111). Excess thermal energy can further rearrange the molecular adlayer and, independent of the initial coverage, the flat lying stable molecular adlayer is formed.

  7. Depletion of CpG Dinucleotides in Papillomaviruses and Polyomaviruses: A Role for Divergent Evolutionary Pressures

    PubMed Central

    Upadhyay, Mohita; Vivekanandan, Perumal

    2015-01-01

    Background Papillomaviruses and polyomaviruses are small ds-DNA viruses infecting a wide-range of vertebrate hosts. Evidence supporting co-evolution of the virus with the host does not fully explain the evolutionary path of papillomaviruses and polyomaviruses. Studies analyzing CpG dinucleotide frequencies in virus genomes have provided interesting insights on virus evolution. CpG dinucleotide depletion has not been extensively studied among papillomaviruses and polyomaviruses. We sought to analyze the relative abundance of dinucleotides and the relative roles of evolutionary pressures in papillomaviruses and polyomaviruses. Methods We studied 127 full-length sequences from papillomaviruses and 56 full-length sequences from polyomaviruses. We analyzed the relative abundance of dinucleotides, effective codon number (ENC), differences in synonymous codon usage. We examined the association, if any, between the extent of CpG dinucleotide depletion and the evolutionary lineage of the infected host. We also investigated the contribution of mutational pressure and translational selection to the evolution of papillomaviruses and polyomaviruses. Results All papillomaviruses and polyomaviruses are CpG depleted. Interestingly, the evolutionary lineage of the infected host determines the extent of CpG depletion among papillomaviruses and polyomaviruses. CpG dinucleotide depletion was more pronounced among papillomaviruses and polyomaviruses infecting human and other mammals as compared to those infecting birds. Our findings demonstrate that CpG depletion among papillomaviruses is linked to mutational pressure; while CpG depletion among polyomaviruses is linked to translational selection. We also present evidence that suggests methylation of CpG dinucleotides may explain, at least in part, the depletion of CpG dinucleotides among papillomaviruses but not polyomaviruses. Conclusions The extent of CpG depletion among papillomaviruses and polyomaviruses is linked to the

  8. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase†

    PubMed Central

    Kamat, Siddhesh S.; Bagaria, Ashima; Kumaran, Desigan; Holmes-Hampton, Gregory P.; Fan, Hao; Sali, Andrej; Sauder, J. Michael; Burley, Stephen K.; Lindahl, Paul A.; Swaminathan, Subramanyam; Raushel, Frank M.

    2011-01-01

    Adenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine and ammonia. The enzyme isolated from Escherichia coli using standard expression conditions was low for the deamination of adenine (kcat = 2.0 s−1; kcat/Km = 2.5 × 103 M−1 s−1). However, when iron was sequestered with a metal chelator and the growth medium was supplemented with Mn2+ prior to induction, the purified enzyme was substantially more active for the deamination of adenine with values of kcat and kcat/Km of 200 s−1 and 5 × 105 M−1s−1, respectively. The apo-enzyme was prepared and reconstituted with Fe2+, Zn2+, or Mn2+. In each case, two enzyme-equivalents of metal were necessary for reconstitution of the deaminase activity. This work provides the first example of any member within the deaminase sub-family of the amidohydrolase superfamily (AHS) to utilize a binuclear metal center for the catalysis of a deamination reaction. [FeII/FeII]-ADE was oxidized to [FeIII/FeIII]-ADE with ferricyanide with inactivation of the deaminase activity. Reducing [FeIII/FeIII]-ADE with dithionite restored the deaminase activity and thus the di-ferrous form of the enzyme is essential for catalytic activity. No evidence for spin-coupling between metal ions was evident by EPR or Mössbauer spectroscopies. The three-dimensional structure of adenine deaminase from Agrobacterium tumefaciens (Atu4426) was determined by X-ray crystallography at 2.2 Å resolution and adenine was modeled into the active site based on homology to other members of the amidohydrolase superfamily. Based on the model of the adenine-ADE complex and subsequent mutagenesis experiments, the roles for each of the highly conserved residues were proposed. Solvent isotope effects, pH rate profiles and solvent viscosity were utilized to propose a chemical reaction mechanism and the identity of the rate limiting steps. PMID:21247091

  9. Induction of hepatoma carcinoma cell apoptosis through activation of the JNK-nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-ROS self-driven death signal circuit.

    PubMed

    Zeng, Ke-Wu; Song, Fang-Jiao; Wang, Ying-Hong; Li, Ning; Yu, Qian; Liao, Li-Xi; Jiang, Yong; Tu, Peng-Fei

    2014-10-28

    As an efficient method for inducing tumor cell apoptosis, ROS can be constantly formed and accumulated in NADPH oxidase overactivated-cells, resulting in further mitochondrial membrane damage and mitochondria-dependent apoptosis. In addition, JNK mitogen-activated protein kinase (JNK MAPK) signal also acts as a vital candidate pathway for inducing tumor cell apoptosis by targeting mitochondrial death pathway. However, the relationship between NADPH oxidase-ROS and JNK MAPK signal still remains unclear. Here, we discovered a novel self-driven signal circuit between NADPH oxidase-ROS and JNK MAPK, which was induced by a cytotoxic steroidal saponin (ASC) in hepatoma carcinoma cells. NADPH oxidase-dependent ROS production was markedly activated by ASC and directly led to JNK MAPK activation. Moreover, antioxidant, NADPH oxidase inhibitor and specific knock-out for p47 subunit of NADPH oxidase could effectively block NADPH oxidase-ROS-dependent JNK activation, suggesting that NADPH oxidase is an upstream regulator of JNK MAPK. Conversely, a specific JNK inhibitor could inhibit ASC-induced NADPH oxidase activation and down-regulate ROS levels as well, indicating that JNK might also regulate NADPH oxidase activity to some extent. These observations indicate that NADPH oxidase and JNK MAPK activate each other as a signal circuit. Furthermore, drug pretreatment experiments with ASC showed this signal circuit operated continuously via a self-driven mode and finally induced apoptosis in hepatoma carcinoma cells. Taken together, we provide a proof for inducing hepatoma carcinoma cell apoptosis by activating the JNK-NADPH oxidase-ROS-dependent self-driven signal circuit pathway. PMID:25064608

  10. Enhancement of photophysical and photosensitizing properties of flavin adenine dinucleotide by mutagenesis of the C-terminal extension of a bacterial flavodoxin reductase.

    PubMed

    Valle, Lorena; Abatedaga, Inés; Vieyra, Faustino E Morán; Bortolotti, Ana; Cortez, Néstor; Borsarelli, Claudio D

    2015-03-16

    The role of the mobile C-terminal extension present in Rhodobacter capsulatus ferredoxin-NADP(H) reductase (RcFPR) was evaluated using steady-state and dynamic spectroscopies for both intrinsic Trp and FAD in a series of mutants in the absence of NADP(H). Deletion of the six C-terminal amino acids beyond Ala266 was combined with the replacement A266Y to emulate the structure of plastidic reductases. Our results show that these modifications of the wild-type RcFPR produce subtle global conformational changes, but strongly reduce the local rigidity of the FAD-binding pocket, exposing the isoalloxazine ring to the solvent. Thus, the ultrafast charge-transfer quenching of (1) FAD* by the conserved Tyr66 residue was absent in the mutant series, producing enhancement of the excited singlet- and triplet-state properties of FAD. This work highlights the delicate balance of the specific interactions between FAD and the surrounding amino acids, and how the functionality and/or photostability of redox flavoproteins can be modified. PMID:25641205

  11. Activation of human neutrophil nicotinamide adenine dinucleotide phosphate, reduced (triphosphopyridine nucleotide, reduced) oxidase by arachidonic acid in a cell-free system.

    PubMed Central

    Curnutte, J T

    1985-01-01

    Sonicates from unstimulated human neutrophils produce no measurable superoxide since the superoxide-generating enzyme, NADPH oxidase, is inactive in these preparations. Previous attempts to activate the oxidase in disrupted cells with conventional neutrophil stimuli have been unsuccessful. This report describes a cell-free system in which arachidonic acid (82 microM) was able to activate superoxide generation that was dependent upon the presence of NADPH and the sonicate. For activation to occur, both the particulate and supernatant fractions of the sonicate must be present. Calcium ions, which are required for activation of intact neutrophils by arachidonate, were not necessary in the cell-free system. In quantitative terms, the superoxide-generating activity in the cell-free system could account for at least 20-50% of the superoxide rate observed in intact neutrophils stimulated with arachidonate. Sonicates from patients with chronic granulomatous disease (CGD) could not be activated by arachidonic acid in the cell-free system. In three patients representing both genetic forms of CGD, the defect appeared to reside in the particulate fraction. The soluble cofactor was normal in all three patients and could be used to activate normal neutrophil pellets in the presence of arachidonic acid. Thus, at least a portion of the activation mechanism in the neutrophil, that residing in the soluble phase, appeared to be normal in patients with CGD. PMID:2987311

  12. The role of phospholipids in the reduction of ubiquinone analogues by the mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone oxidoreductase complex.

    PubMed Central

    Ragan, C I

    1978-01-01

    The isolated NADH-ubiquinone oxidoreductase complex of bovine heart mitochondria reduces ubiquinone analogues by two pathways. One pathway is inhibited by rotenone, and reduction of quinones takes place in the lipid phase of the system. The other pathway is insensitive to rotenone and reduction takes place in the aqueous phase. The variation of rates of electron transpport with the chemical nature of the quinone analogue and the concentrations of both quinone and phospholipid can be rationalized in terms of partition of the quinone between the aqueous and lipid phases of the system. Thus one function of phospholipid associated with the enzyme appears to be to act as solvent for ubiquinone reduced by the rotenone-sensitive pathway. This proposal is supported by the kinetic behaviour of enzyme whose endogenous lipids have been replaced by (1,2)-dimyristoylsn-glycero-3-phosphocholine. Thus, under certain circumstances, the rotenone-sensitive reduction of ubiquinone-1 exhibited a substantial increase in activation energy below the phase-transition temperature of the synthetic lipid, whereas the reduction of other acceptors was unaffected. PMID:210762

  13. The lactate dehydrogenase--reduced nicotinamide--adenine dinucleotide--pyruvate complex. Kinetics of pyruvate binding and quenching of coeznyme fluorescence.

    PubMed

    Südi, J

    1974-04-01

    The stopped-flow kinetic studies described in this and the following paper (Südi, 1974) demonstrate that a Haldane-type description of the reversible lactate dehydrogenase reaction presents an experimentally feasible task. Combined results of these two papers yield numerical values for the six rate constants defined by the following equilibrium scheme, where E represents lactate dehydrogenase: [Formula: see text] The experiments were carried out at pH8.4 at a relatively low temperature (6.3 degrees C) with the pig heart enzyme. Identification of the above two intermediates and determination of the corresponding rate constants actually involve four series of independent observations in these studies, since (a) the reaction can be followed in both directions, and (b) both the u.v. absorption and the fluorescence of the coenzymes are altered in the reaction, and it is shown that these two spectral changes do not occur simultaneously. Kinetic observations made in the reverse direction are reported in this paper. It is demonstrated that the fluorescence of NADH can no longer be observed in the ternary complex E(NADH) (Pyr). Even though the oxidation-reduction reaction rapidly follows the formation of this complex, the numerical values of k(-4) (8.33x10(5)m(-1).s(-1)) and k(+4) (222s(-1)) are easily obtained from a directly observed second-order reaction step in which fluorescent but not u.v.-absorbing material is disappearing. U.v.-absorption measurements do not clearly resolve the subsequent oxidation-reduction step from the dissociation of lactate. It is shown that this must be due partly to the instrumental dead time, and partly to a low transient concentration of E(NAD+) (Lac) in the two-step sequential reaction in which the detectable disappearance of u.v.-absorbing material takes place. It is estimated that about one-tenth of the total change in u.v. absorption is due to a ;burst reaction' in which E(NAD+) (Lac) is produced, and this estimation yields, from k(obs.)=120s(-1), k(-2)=1200s(-1). PMID:4377095

  14. Enhanced Reduced Nicotinamide Adenine Dinucleotide electrocatalysis onto multi-walled carbon nanotubes-decorated gold nanoparticles and their use in hybrid biofuel cell

    NASA Astrophysics Data System (ADS)

    Aquino Neto, S.; Almeida, T. S.; Belnap, D. M.; Minteer, S. D.; De Andrade, A. R.

    2015-01-01

    We report the preparation of Au nanoparticles synthetized by different protocols and supported on the surface of multi-walled carbon nanotubes containing different functional groups, focusing on their electrochemical performance towards NADH oxidation, ethanol bioelectrocatalysis, and ethanol/O2 biofuel cell. We describe four different synthesis protocols: microwave-assisted heating, water-in-oil, and dendrimer-encapsulated nanoparticles using acid or thiol species in the extraction step. The physical characterization of the metallic nanoparticles indicated that both the synthetic protocol as well as the type of functional groups on the carbon nanotubes affect the final particle size (varying from 13.4 to 2.4 nm) and their distribution onto the carbon surface. Moreover, the electrochemical data indicated that these two factors also influence their performance toward the electrooxidation of NADH. We observed that the samples containing Au nanoparticles with smaller size leads to higher catalytic currents and also shifts the oxidation potential of the targeted reaction, which varied from 0.13 to -0.06 V vs Ag/AgCl. Ethanol/O2 biofuel cell tests indicated that the hybrid bioelectrodes containing smaller and better distributed Au nanoparticles on the surface of carbon nanotubes generates higher power output, confirming that the electrochemical regeneration of NAD+ plays an important role in the overall biofuel cell performance.

  15. Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP +) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

    NASA Astrophysics Data System (ADS)

    Polshakov, Vladimir I.; Biekofsky, Rodolfo R.; Birdsall, Berry; Feeney, James

    2002-01-01

    Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP +. The origins of the difference in binding affinity to DHFR between NADPH and NADP + are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in 1H and 15N chemical shift between NADPH and NADP + in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP + compared with NADPH results partly from NADP + having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP + being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.

  16. Nicotinamide Adenine Dinucleotide-specific “Malic” Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism 1

    PubMed Central

    Dittrich, Peter

    1976-01-01

    NAD-specific “malic” enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO42−, suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn2+, which cannot be replaced by NADP or Mg2+. For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn2+, and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 μmole/hr·mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO2 for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  17. Expression and characterization of ferredoxin and flavin adenine dinucleotide binding domains of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Blazyk, Jessica L; Lippard, Stephen J

    2002-12-31

    Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) catalyzes the selective oxidation of methane to methanol, the first step in the primary catabolic pathway of methanotrophic bacteria. A reductase (MMOR) mediates electron transfer from NADH through its FAD and [2Fe-2S] cofactors to the dinuclear non-heme iron sites housed in a hydroxylase (MMOH). The structurally distinct [2Fe-2S], FAD, and NADH binding domains of MMOR facilitated division of the protein into its functional ferredoxin (MMOR-Fd) and FAD/NADH (MMOR-FAD) component domains. The 10.9 kDa MMOR-Fd (MMOR residues 1-98) and 27.6 kDa MMOR-FAD (MMOR residues 99-348) were expressed and purified from recombinant Escherichia coli systems. The Fd and FAD domains have absorbance spectral features identical to those of the [2Fe-2S] and flavin components, respectively, of MMOR. Redox potentials, determined by reductive titrations that included indicator dyes, for the [2Fe-2S] and FAD cofactors in the domains are as follows: -205.2 +/- 1.3 mV for [2Fe-2S](ox/red), -172.4 +/- 2.0 mV for FAD(ox/sq), and -266.4 +/- 3.5 mV for FAD(sq/hq). Kinetic and spectral properties of intermediates observed in the reaction of oxidized MMOR-FAD (FAD(ox)) with NADH at 4 degrees C were established with stopped-flow UV-visible spectroscopy. Analysis of the influence of pH on MMOR-FAD optical spectra, redox potentials, and NADH reaction kinetics afforded pK(a) values for the semiquinone (FAD(sq)) and hydroquinone (FAD(hq)) MMOR-FAD species and two protonatable groups near the flavin cofactor. Electron transfer from MMOR-FAD(hq) to oxidized MMOR-Fd is extremely slow (k = 1500 M(-1) s(-1) at 25 degrees C, compared to 90 s(-1) at 4 degrees C for internal electron transfer between cofactors in MMOR), indicating that cofactor proximity is essential for efficient interdomain electron transfer. PMID:12501207

  18. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca2+ Signaling in Cardiac Hypertrophy Induced by β-Adrenergic Stimulation

    PubMed Central

    Shawl, Asif Iqbal; Im, Soo-Yeul; Nam, Tae-Sik; Lee, Sun-Hwa; Ko, Jae-Ki; Jang, Kyu Yoon; Kim, Donghee; Kim, Uh-Hyun

    2016-01-01

    Ca2+ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively. PMID:26959359

  19. Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium.

    PubMed Central

    Ragan, C I; Bloxham, D P

    1977-01-01

    1. NADH-ubiquinone-1 and NADH-menadione reductase activities of Complex I were inhibited by diphenyleneiodonium (apparent Ki 23 and 30 nmol/mg of protein respectively). Reduction of K3Fe(CN)6 and juglone was relatively unaffected. 2. Iodoniumdiphenyl and derivatives were much less effective inhibitors. Compounds with similar ring structures to diphenyleneiodonium, in particular dibenzofuran, were inhibitors of NADH-ubiquinone-1 oxidoreductase. 3. Diphenylene[125I]iodonium specifically labelled a polypeptide of mol.wt. 23500. Maximum incorporation was 1 mol/mol of Complex-I flavin or 1 mol/mol of the 23500-mol.wt. polypeptide. 4. The label associated with this polypeptide was of limited stability, especially at lower pH. 5. Complete inhibition of ubiquinone reduction was achieved when 1 mol of inhibitor was incorporated/mol of Complex-I flavin, but the relationship between inhibition and labelling was not linear. 6. No evidence for covalent interaction between diphenyleneiodonium and the phospholipids of Complex I was obtained. 7. Rotenone increased the apparent affinity of diphenyleneiodonium for the 23500-mol.wt. polypeptide without affecting the maximum incorporation. 8. The 23500-mol.wt. polypeptide was not solubilized by chaotropic agents. Prior treatment of Complex I with chaotropic agents or sodium dodecyl sulphate prevented incorporation of diphenyleneiodonium into this polypeptide. PMID:18140

  20. Flavin adenine dinucleotide and flavin mononucleotide metabolism in rat liver--the occurrence of FAD pyrophosphatase and FMN phosphohydrolase in isolated mitochondria.

    PubMed

    Barile, M; Brizio, C; De Virgilio, C; Delfine, S; Quagliariello, E; Passarella, S

    1997-11-01

    In order to gain some insight into mitochondrial flavin biochemistry, rat liver mitochondria essentially free of lysosomal and microsomal contamination were prepared and their capability to metabolise externally added and endogenous FAD and FMN tested both spectroscopically and via HPLC. The existence of two novel mitochondrial enzymes, namely FAD pyrophosphatase (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2), which catalyse FAD-->FMN and FMN-->riboflavin conversion, respectively, is shown. They differ from each other and from extramitochondrial enzymes, as judged by their pH profile and inhibitor sensitivity, and can be separated in a partial FAD pyrophosphatase purification. Digitonin titration and subfractionation experiments show that FAD pyrophosphatase is located in the outer mitochondrial membrane and FMN phosphohydrolase in the intermembrane space. Since these enzymes can metabolise endogenous FAD and FMN, which are made available by using both Triton X-100 and the effector oxaloacetate, a proposal is made that FAD pyrophosphatase and FMN phosphohydrolase play a major role in mitochondrial flavoprotein turnover. PMID:9395326

  1. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide.

    PubMed Central

    Schraufstatter, I U; Hinshaw, D B; Hyslop, P A; Spragg, R G; Cochrane, C G

    1986-01-01

    To determine the biochemical basis of the oxidant-induced injury of cells, we have studied early changes after exposure of P388D1 murine macrophages to hydrogen peroxide. Total intracellular NAD+ levels in P388D1 cells decreased with H2O2 concentrations of 40 microM or higher. Doses of H2O2 between 0.1 and 2.5 mM led to an 80% depletion of NAD within 20 min. With doses of H2O2 of 250 microM or lower, the fall in NAD and, as shown previously, ATP, was reversible. Higher doses of H2O2 that cause ultimate lysis of the cells, induced an irreversible depletion of NAD and ATP. Poly-ADP-ribose polymerase, a nuclear enzyme associated with DNA damage and repair, which catalyzes conversion of NAD to nicotinamide and protein-bound poly-ADP-ribose, was activated by exposure of the cells to concentrations of 40 microM H2O2 or higher. Activation of poly-ADP-ribose polymerase was also observed in peripheral lymphocytes incubated in the presence of phorbol myristate acetate-stimulated polymorphonuclear neutrophils. Examination of the possibility that DNA alteration was involved was performed by measurement of thymidine incorporation and determination of DNA single-strand breaks (SSB) in cells exposed to H2O2. H2O2 at 40 microM or higher inhibited DNA synthesis, and induced SSB within less than 30 s. These results suggest that DNA damage induced within seconds after addition of oxidant may lead to stimulation of poly-ADP-ribose polymerase, and a consequent fall in NAD. Excessive stimulation of poly-ADP-ribose polymerase leads to a fall in NAD sufficient to interfere with ATP synthesis. PMID:2937805

  2. Characterization of DNA methylation as a function of biological complexity via dinucleotide inter-distances.

    PubMed

    Paci, Giulia; Cristadoro, Giampaolo; Monti, Barbara; Lenci, Marco; Degli Esposti, Mirko; Castellani, Gastone C; Remondini, Daniel

    2016-03-13

    We perform a statistical study of the distances between successive occurrences of a given dinucleotide in the DNA sequence for a number of organisms of different complexity. Our analysis highlights peculiar features of the CG dinucleotide distribution in mammalian DNA, pointing towards a connection with the role of such dinucleotide in DNA methylation. While the CG distributions of mammals exhibit exponential tails with comparable parameters, the picture for the other organisms studied (e.g. fish, insects, bacteria and viruses) is more heterogeneous, possibly because in these organisms DNA methylation has different functional roles. Our analysis suggests that the distribution of the distances between CG dinucleotides provides useful insights into characterizing and classifying organisms in terms of methylation functionalities. PMID:26857665

  3. Cerulenin-mediated apoptosis is involved in adenine metabolic pathway

    SciTech Connect

    Chung, Kyung-Sook; Sun, Nam-Kyu; Lee, Seung-Hee; Lee, Hyun-Jee; Choi, Shin-Jung; Kim, Sun-Kyung; Song, Ju-Hyun; Jang, Young-Joo; Song, Kyung-Bin; Yoo, Hyang-Sook; Simon, Julian . E-mail: jsimon@fhcrc.org; Won, Misun . E-mail: misun@kribb.re.kr

    2006-10-27

    Cerulenin, a fatty acid synthase (FAS) inhibitor, induces apoptosis of variety of tumor cells. To elucidate mode of action by cerulenin, we employed the proteomics approach using Schizosaccharomyces pombe. The differential protein expression profile of S. pombe revealed that cerulenin modulated the expressions of proteins involved in stresses and metabolism, including both ade10 and adk1 proteins. The nutrient supplementation assay demonstrated that cerulenin affected enzymatic steps transferring a phosphoribosyl group. This result suggests that cerulenin accumulates AMP and p-ribosyl-s-amino-imidazole carboxamide (AICAR) and reduces other necessary nucleotides, which induces feedback inhibition of enzymes and the transcriptional regulation of related genes in de novo and salvage adenine metabolic pathway. Furthermore, the deregulation of adenine nucleotide synthesis may interfere ribonucleotide reductase and cause defects in cell cycle progression and chromosome segregation. In conclusion, cerulenin induces apoptosis through deregulation of adenine nucleotide biosynthesis resulting in nuclear division defects in S. pombe.

  4. Structural basis for removal of adenine mispaired with 8-oxoguanine by MutY adenine DNA glycosylase.

    PubMed

    Fromme, J Christopher; Banerjee, Anirban; Huang, Susan J; Verdine, Gregory L

    2004-02-12

    The genomes of aerobic organisms suffer chronic oxidation of guanine to the genotoxic product 8-oxoguanine (oxoG). Replicative DNA polymerases misread oxoG residues and insert adenine instead of cytosine opposite the oxidized base. Both bases in the resulting A*oxoG mispair are mutagenic lesions, and both must undergo base-specific replacement to restore the original C*G pair. Doing so represents a formidable challenge to the DNA repair machinery, because adenine makes up roughly 25% of the bases in most genomes. The evolutionarily conserved enzyme adenine DNA glycosylase (called MutY in bacteria and hMYH in humans) initiates repair of A*oxoG to C*G by removing the inappropriately paired adenine base from the DNA backbone. A central issue concerning MutY function is the mechanism by which A*oxoG mispairs are targeted among the vast excess of A*T pairs. Here we report the use of disulphide crosslinking to obtain high-resolution crystal structures of MutY-DNA lesion-recognition complexes. These structures reveal the basis for recognizing both lesions in the A*oxoG pair and for catalysing removal of the adenine base. PMID:14961129

  5. Mature Microsatellites: Mechanisms Underlying Dinucleotide Microsatellite Mutational Biases in Human Cells

    PubMed Central

    Baptiste, Beverly A.; Ananda, Guruprasad; Strubczewski, Noelle; Lutzkanin, Andrew; Khoo, Su Jen; Srikanth, Abhinaya; Kim, Nari; Makova, Kateryna D.; Krasilnikova, Maria M.; Eckert, Kristin A.

    2013-01-01

    Dinucleotide microsatellites are dynamic DNA sequences that affect genome stability. Here, we focused on mature microsatellites, defined as pure repeats of lengths above the threshold and unlikely to mutate below it in a single mutational event. We investigated the prevalence and mutational behavior of these sequences by using human genome sequence data, human cells in culture, and purified DNA polymerases. Mature dinucleotides (≥10 units) are present within exonic sequences of >350 genes, resulting in vulnerability to cellular genetic integrity. Mature dinucleotide mutagenesis was examined experimentally using ex vivo and in vitro approaches. We observe an expansion bias for dinucleotide microsatellites up to 20 units in length in somatic human cells, in agreement with previous computational analyses of germ-line biases. Using purified DNA polymerases and human cell lines deficient for mismatch repair (MMR), we show that the expansion bias is caused by functional MMR and is not due to DNA polymerase error biases. Specifically, we observe that the MutSα and MutLα complexes protect against expansion mutations. Our data support a model wherein different MMR complexes shift the balance of mutations toward deletion or expansion. Finally, we show that replication fork progression is stalled within long dinucleotides, suggesting that mutational mechanisms within long repeats may be distinct from shorter lengths, depending on the biochemistry of fork resolution. Our work combines computational and experimental approaches to explain the complex mutational behavior of dinucleotide microsatellites in humans. PMID:23450065

  6. Dinucleotide Weight Matrices for Predicting Transcription Factor Binding Sites: Generalizing the Position Weight Matrix

    PubMed Central

    Siddharthan, Rahul

    2010-01-01

    Background Identifying transcription factor binding sites (TFBS) in silico is key in understanding gene regulation. TFBS are string patterns that exhibit some variability, commonly modelled as “position weight matrices” (PWMs). Though convenient, the PWM has significant limitations, in particular the assumed independence of positions within the binding motif; and predictions based on PWMs are usually not very specific to known functional sites. Analysis here on binding sites in yeast suggests that correlation of dinucleotides is not limited to near-neighbours, but can extend over considerable gaps. Methodology/Principal Findings I describe a straightforward generalization of the PWM model, that considers frequencies of dinucleotides instead of individual nucleotides. Unlike previous efforts, this method considers all dinucleotides within an extended binding region, and does not make an attempt to determine a priori the significance of particular dinucleotide correlations. I describe how to use a “dinucleotide weight matrix” (DWM) to predict binding sites, dealing in particular with the complication that its entries are not independent probabilities. Benchmarks show, for many factors, a dramatic improvement over PWMs in precision of predicting known targets. In most cases, significant further improvement arises by extending the commonly defined “core motifs” by about 10bp on either side. Though this flanking sequence shows no strong motif at the nucleotide level, the predictive power of the dinucleotide model suggests that the “signature” in DNA sequence of protein-binding affinity extends beyond the core protein-DNA contact region. Conclusion/Significance While computationally more demanding and slower than PWM-based approaches, this dinucleotide method is straightforward, both conceptually and in implementation, and can serve as a basis for future improvements. PMID:20339533

  7. Detection of electronically equivalent tautomers of adenine base: DFT study

    SciTech Connect

    Siddiqui, Shamoon Ahmad; Bouarissa, Nadir; Rasheed, Tabish; Al-Assiri, M.S.; Al-Hajry, A.

    2014-03-01

    Graphical abstract: - Highlights: • DFT calculations have been performed on adenine and its rare tautomer Cu{sup 2+} complexes. • Interaction of A-Cu{sup 2+} and rA-Cu{sup 2+} complexes with AlN modified fullerene (C{sub 60}) have been studied briefly. • It is found that AlN modified C{sub 60} could be used as a nanoscale sensor to detect these two A-Cu{sup 2+} and rA-Cu{sup 2+} complexes. - Abstract: In the present study, quantum chemical calculations were carried out to investigate the electronic structures and stabilities of adenine and its rare tautomer along with their Cu{sup 2+} complexes. Density Functional Theory (B3LYP method) was used in all calculations. The two Cu{sup 2+} complexes of adenine have almost similar energies and electronic structures; hence, their chemical differentiation is very difficult. For this purpose, interactions of these complexes with AlN modified fullerene (C{sub 60}) have been studied. Theoretical investigations reveal that AlN-doped C{sub 60} may serve as a potentially viable nanoscale sensor for detection of the two Cu{sup 2+} complexes of adenine.

  8. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

    Köse, Kazım; Erol, Kadir; Özgür, Erdoğan; Uzun, Lokman; Denizli, Adil

    2016-10-01

    Cryogels are used effectively for many diverse applications in a variety of fields. The isolation or purification of RNA, one of the potential utilizations for cryogels, is crucial due to their vital roles such as encoding, decoding, transcription and translation, and gene expression. RNA principally exists within every living thing, but their tendency to denaturation easily is still the most challenging issue. Herein, we aimed to develop adenine incorporated polymeric cryogels as an alternative sorbent for cost-friendly and fast RNA purification with high capacity. For this goal, we synthesized the polymerizable derivative of adenine called as adenine methacrylate (AdeM) through the substitution reaction between adenine and methacryloyl chloride. Then, 2-hydroxyethyl methacrylate (HEMA)-based cryogels were prepared in a partially frozen aqueous medium by copolymerization of monomers, AdeM, and HEMA. The cryogels were characterized by using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), surface area measurements, thermogravimetric analysis (TGA), and swelling tests. RNA adsorption experiments were performed via batch system while varying different conditions including pH, initial RNA concentration, temperature, and interaction time. We achieved high RNA adsorption capacity of cryogels, with the swelling ratio around 510%, as 11.86mg/g. The cryogels might be reused at least five times without significant decrease in adsorption capacity. PMID:27434154

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

    SciTech Connect

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

    2011-12-31

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

  10. Effect of Adenine on Clozapine-induced Neutropenia in Patients with Schizophrenia: A Preliminary Study

    PubMed Central

    Takeuchi, Ippei; Kishi, Taro; Hanya, Manako; Uno, Junji; Fujita, Kiyoshi; Kamei, Hiroyuki

    2015-01-01

    Objective This study examined the utility of adenine for preventing clozapine-induced neutropenia. Methods This retrospective study examined the effect of adenine on clozapine-induced neutropenia in patients with treatment-resistant schizophrenia and was conducted at Okehazama Hospital in Japan from July 2010 to June 2013. Adenine was available for use from June 2011 onwards. Twenty-one patients started receiving clozapine treatment from July 2010 to April 2011 (the pre-adenine adoption group), and 47 patients started receiving it from May 2011 to June 2013 (the post-adenine adoption group). The effects of adenine were assessed based on changes in the patients’ leukocyte counts and the frequency of treatment discontinuation due to clozapine-induced neutropenia. Results Sixty-eight patients were treated with clozapine from July 2010 to June 2013. Of the 21 patients in the pre-adenine adoption group, 4 discontinued treatment due to clozapine-induced neutropenia, whereas only 2 of the 47 patients in the post-adenine adoption group discontinued treatment. The frequency of treatment discontinuation due to clozapine-induced neutropenia was significantly lower in post-adenine adoption group than in the pre-adenine adoption group (p=0.047). Conclusion Adenine decreased the frequency of treatment discontinuation due to clozapine-induced neutropenia. Our data suggest that combined treatment with clozapine and adenine is a safe and effective strategy against treatment-resistant schizophrenia. PMID:26243842

  11. Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

    SciTech Connect

    Weiss, P.M.; Urbauer, J.L.; Cleland, W.W. ); Gavva, S.R.; Harris, B.G.; Cook, P.F. )

    1991-06-11

    Deuterium isotope effects and {sup 13}C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the {sup 13}C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the {sup 13}C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a {beta}-secondary {sup 13}C isotope effect accompanies hydride transfer as a result of hyperconjugation of the {beta}-carboxyl of malate as the transition state for the hydride transfer step is approached.

  12. Excited-State Deactivation of Adenine by Electron-Driven Proton-Transfer Reactions in Adenine-Water Clusters: A Computational Study.

    PubMed

    Wu, Xiuxiu; Karsili, Tolga N V; Domcke, Wolfgang

    2016-05-01

    The reactivity of photoexcited 9H-adenine with hydrogen-bonded water molecules in the 9H-adenine-(H2 O)5 cluster is investigated by using ab initio electronic structure methods, focusing on the photoreactivity of the three basic sites of 9H-adenine. The energy profiles of excited-state reaction paths for electron/proton transfer from water to adenine are computed. For two of the three sites, a barrierless or nearly barrierless reaction path towards a low-lying S1 -S0 conical intersection is found. This reaction mechanism, which is specific for adenine in an aqueous environment, can explain the substantially shortened excited-state lifetime of 9H-adenine in water. Depending on the branching ratio of the nonadiabatic dynamics at the S1 -S0 conical intersection, the electron/proton transfer process can enhance the photostability of 9H-adenine in water or can lead to the generation of adenine-H(⋅) and OH(⋅) free radicals. Although the branching ratio is yet unknown, these findings indicate that adenine might have served as a catalyst for energy harvesting by water splitting in the early stages of the evolution of life. PMID:26833826

  13. Cyclic dinucleotides modulate human T-cell response through monocyte cell death.

    PubMed

    Tosolini, Marie; Pont, Frédéric; Verhoeyen, Els; Fournié, Jean-Jacques

    2015-12-01

    Cyclic dinucleotides, a class of microbial messengers, have been recently identified in bacteria, but their activity in humans remains largely unknown. Here, we have studied the function of cyclic dinucleotides in humans. We found that c-di-AMP and cGAMP, two adenosine-based cyclic dinucleotides, activated T lymphocytes in an unusual manner through monocyte cell death. c-di-AMP and cGAMP induced the selective apoptosis of human monocytes, and T lymphocytes were activated by the direct contact with these dying monocytes. The ensuing T-cell response comprised cell-cycle exit, phenotypic maturation into effector memory cells and proliferation arrest, but not cell death. This quiescence was transient since T cells remained fully responsive to further restimulation. Together, our results depict a novel activation pattern for human T lymphocytes: a transient quiescence induced by c-di-AMP- or cGAMP-primed apoptotic monocytes. PMID:26460927

  14. Copper-Adenine Complex Catalyst for O2 Production from

    NASA Astrophysics Data System (ADS)

    Vergne, Jacques; Bruston, F.; Calvayrac, R.; Grajcar, L.; Baron, M.-H.; Maurel, M.-C.

    The advent of oxygen-evolving photosynthesis is one of the central event in the development of life on earth. The early atmosphere has been midly reducing or neutral in overall redox balance and water photolysis by UV light can produce hydrogen peroxide. Before oxidation of water, intermediate stages are proposed in which H_2^O_2 was oxidized. The oxidation of H_2^O_2 to oxygen can be carried out by a modestly oxidizing species in which a metal-catalase like enzyme could extract electrons from H_2^O_2 producing the first oxygen-evolving complex. After what, modern photosynthesis with chlorophyll, to help transform H_2^O in O_2 was ready to come to light. In preliminary UV studies we were able to show that [Cu(adenine)2] system, containing copper coordinated to nitrogen activates H_2^O_2 disappearance. This was confirmed with the help of Raman and polarographic studies. Raman spectroscopy shows the formation of [Cu(adenine)2] complex in solution, quantifies H_2^O_2 consumption, polarography quantifies O_2 production. In both cases CuCl_2 addition entails H_2^O_2 disappearance. Without adenine, Cu_2^+ has only a weak catalytic effect. The molar activity of the [Cu(adenine)2] complex is much larger and concentration dependent. We emphasize that Cu(adenine)2 may have mimicked enzyme properties in the first stage of life evolution, in order to split H_2^O_2 into O_2 and H_2^O. Moreover, diluted copper and adenine, in small ephemeral prebiotic ponds , could have preserved biologically active entities from H_2^O_2 damage via dual properties: catalyzing H_2^O_2 disproportionation and also directly acting as a reductant complex. Finally, the present Mars surface is considered to be both reactive and embedded with oxydants. As it has been shown that the depth of diffusion for H_2^O_2 is less than 3 meters, it is important to study all the ways of H_2^O_2 consumption.

  15. Excited State Pathways Leading to Formation of Adenine Dimers.

    PubMed

    Banyasz, Akos; Martinez-Fernandez, Lara; Ketola, Tiia-Maaria; Muñoz-Losa, Aurora; Esposito, Luciana; Markovitsi, Dimitra; Improta, Roberto

    2016-06-01

    The reaction intermediate in the path leading to UV-induced formation of adenine dimers A═A and AA* is identified for the first time quantum mechanically, using PCM/TD-DFT calculations on (dA)2 (dA: 2'deoxyadenosine). In parallel, its fingerprint is detected in the absorption spectra recorded on the millisecond time-scale for the single strand (dA)20 (dA: 2'deoxyadenosine). PMID:27163876

  16. Dynamics and reactivity in Thermus aquaticus N6-adenine methyltransferase.

    PubMed

    Aranda, Juan; Zinovjev, Kirill; Roca, Maite; Tuñón, Iñaki

    2014-11-19

    M.TaqI is a DNA methyltransferase from Thermus aquaticus that catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the N6 position of an adenine, a process described only in prokaryotes. We have used full atomistic classical molecular dynamics simulations to explore the protein-SAM-DNA ternary complex where the target adenine is flipped out into the active site. Key protein-DNA interactions established by the target adenine in the active site are described in detail. The relaxed structure was used for a combined quantum mechanics/molecular mechanics exploration of the reaction mechanism using the string method. According to our free energy calculations the reaction takes place through a stepwise mechanism where the methyl transfer precedes the abstraction of the proton from the exocyclic amino group. The methyl transfer is the rate-determining step, and the obtained free energy barrier is in good agreement with the value derived from the experimental rate constant. Two possible candidates to extract the leftover proton have been explored: a water molecule found in the active site and Asn105, a residue activated by the hydrogen bonds formed through the amide hydrogens. The barrier for the proton abstraction is smaller when Asn105 acts as a base. The reaction mechanisms can be different in other N6-DNA-methyltransferases, as determined from the exploration of the reaction mechanism in the Asn105Asp M.TaqI mutant. PMID:25347783

  17. Isolation and characterizaton of dinucleotide microsatellites in greater amberjack, Seriola dumerili

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Thirteen nuclear-encoded dinucleotide microsatellites were characterized from a genomic DNA library of greater amberjack, Seriola dumerili. The microsatellites include 12 perfect-repeat motifs and one imperfect-repeat motif. The number of alleles at the 13 microsatellites among a sample of 29 fish...

  18. Stacking interactions in RNA and DNA: Roll-slide energy hyperspace for ten unique dinucleotide steps.

    PubMed

    Mukherjee, Sanchita; Kailasam, Senthilkumar; Bansal, Manju; Bhattacharyya, Dhananjay

    2015-03-01

    Understanding dinucleotide sequence directed structures of nuleic acids and their variability from experimental observation remained ineffective due to unavailability of statistically meaningful data. We have attempted to understand this from energy scan along twist, roll, and slide degrees of freedom which are mostly dependent on dinucleotide sequence using ab initio density functional theory. We have carried out stacking energy analysis in these dinucleotide parameter phase space for all ten unique dinucleotide steps in DNA and RNA using DFT-D by ωB97X-D/6-31G(2d,2p), which appears to satisfactorily explain conformational preferences for AU/AU step in our recent study. We show that values of roll, slide, and twist of most of the dinucleotide sequences in crystal structures fall in the low energy region. The minimum energy regions with large twist values are associated with the roll and slide values of B-DNA, whereas, smaller twist values correspond to higher stability to RNA and A-DNA like conformations. Incorporation of solvent effect by CPCM method could explain the preference shown by some sequences to occur in B-DNA or A-DNA conformations. Conformational preference of BII sub-state in B-DNA is preferentially displayed mainly by pyrimidine-purine steps and partly by purine-purine steps. The purine-pyrimidine steps show largest effect of 5-methyl group of thymine in stacking energy and the introduction of solvent reduces this effect significantly. These predicted structures and variabilities can explain the effect of sequence on DNA and RNA functionality. PMID:25257334

  19. Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates.

    PubMed

    Bayliss, Christopher D; Sweetman, Wendy A; Moxon, E Richard

    2004-05-01

    High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5' TA repeats located between the -10 and -35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5' AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5' AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5' TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5' TA-mediated pilin PV. PMID:15126452

  20. Influence of hydrogen bonding on the geometry of the adenine fragment

    NASA Astrophysics Data System (ADS)

    Słowikowska, Joanna Maria; Woźniak, Krzysztof

    1996-01-01

    The crystal structures of two adenine derivatives, N(6),9-dimethyl-8-butyladenine (I) and its hydrate (1 : 1) (II), have been determined by single-crystal X-ray diffraction. The geometrical features of both structures are discussed. The influence of protonation, substitution and hydrogen bond formation on the geometry of the adenine fragment was studied, based on data retrieved from the Cambridge Structural Database. Total correlation analysis showed mutual correlation between the structural parameters in the adenine ring system; partial correlation calculations for the adenine nucleoside fragments suggest intercorrelation between the parameters of the hydrogen bonding involved in base pairing and the N(adenine)-C(sugar) bond through the adenine fragment; few such correlations were found for fragments without the sugar substituent.

  1. A9145, a New Adenine-Containing Antifungal Antibiotic: Fermentation

    PubMed Central

    Boeck, L. D.; Clem, G. M.; Wilson, M. M.; Westhead, J. E.

    1973-01-01

    A9145 is a basic, water-soluble, antifungal antibiotic which is produced in a complex organic medium by Streptomyces griseolus. The metabolite has a molecular weight of 510, and contains adenine as well as sugar hydroxyl and amino groups. Although glucose, fructose, glucose polymers, and some long-chain fatty acid methyl esters supported biosynthesis, oils were superior, with cottonseed oil being preferred. Several ions and salts, especially Co2+, PO43−, and CaCO3, were stimulatory. Adenine, nucleosides, and some amino acids increased the accumulation of A9145 in shaken-flask fermentors. Enrichment of the culture medium with tyrosine afforded maximal enhancement of antibiotic production in both flask and tank fermentors. Control of the dissolved O2 level was also critical, the optimal concentration being 3 × 10−2 to 4.5 × 10−2 μmole of O2/ml. Optimization of various fermentation parameters increased antibiotic titers approximately 135-fold in shaken flask fermentors and 225-fold in stirred vessels. PMID:4208279

  2. PA0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine

    SciTech Connect

    Goble, A.M.; Swaminathan, S.; Zhang, Z.; Sauder, J. M.; Burley, S. K.; Raushel, F. M.

    2011-08-02

    Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with k{sub cat}/K{sub m} values that exceed 10{sup 5} M{sup -1} s{sup -1}. These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted ({beta}/{alpha}){sub 8} barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine, and hypoxanthine were also determined, thereby permitting identification of the residues responsible for coordinating the substrate and product.

  3. Pa0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine

    SciTech Connect

    A Goble; Z Zhang; J Sauder; S Burley; S Swaminathan; F Raushel

    2011-12-31

    Four proteins from NCBI cog1816, previously annotated as adenosine deaminases, have been subjected to structural and functional characterization. Pa0148 (Pseudomonas aeruginosa PAO1), AAur1117 (Arthrobacter aurescens TC1), Sgx9403e, and Sgx9403g have been purified and their substrate profiles determined. Adenosine is not a substrate for any of these enzymes. All of these proteins will deaminate adenine to produce hypoxanthine with k{sub cat}/K{sub m} values that exceed 10{sup 5} M{sup -1} s{sup -1}. These enzymes will also accept 6-chloropurine, 6-methoxypurine, N-6-methyladenine, and 2,6-diaminopurine as alternate substrates. X-ray structures of Pa0148 and AAur1117 have been determined and reveal nearly identical distorted ({beta}/{alpha}){sub 8} barrels with a single zinc ion that is characteristic of members of the amidohydrolase superfamily. Structures of Pa0148 with adenine, 6-chloropurine, and hypoxanthine were also determined, thereby permitting identification of the residues responsible for coordinating the substrate and product.

  4. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

    Bruschi, C. V.; Chuba, P. J.

    1988-01-01

    The expression of certain adenine biosynthetic mutations in the yeast Saccharomyces cerevisiae results in a red colony color. This phenomenon has historically provided an ideal genetic marker for the study of mutation, recombination, and aneuploidy in lower eukaryotes by classical genetic analysis. In this paper, it is reported that cells carrying ade1 and/or ade2 mutations exhibit primary fluorescence. Based on this observation, the nonselective enrichment of yeast cultures for viable adenine mutants by using the fluorescence-activated cell sorter has been achieved. The advantages of this approach over conventional genetic analysis of mutation, recombination, and mitotic chromosomal stability include speed and accuracy in acquiring data for large numbers of clones. By using appropriate strains, the cell sorter has been used for the isolation of both forward mutations and chromosomal loss events in S. cerevisiae. The resolving power of this system and its noninvasiveness can easily be extended to more complex organisms, including mammalian cells, in which analogous metabolic mutants are available.

  5. Identification of Prophages in Bacterial Genomes by Dinucleotide Relative Abundance Difference

    PubMed Central

    Srividhya, K. V.; Alaguraj, V.; Poornima, G.; Kumar, Dinesh; Singh, G. P.; Raghavenderan, L.; Katta, A. V. S. K. Mohan; Mehta, Preeti; Krishnaswamy, S.

    2007-01-01

    Background Prophages are integrated viral forms in bacterial genomes that have been found to contribute to interstrain genetic variability. Many virulence-associated genes are reported to be prophage encoded. Present computational methods to detect prophages are either by identifying possible essential proteins such as integrases or by an extension of this technique, which involves identifying a region containing proteins similar to those occurring in prophages. These methods suffer due to the problem of low sequence similarity at the protein level, which suggests that a nucleotide based approach could be useful. Methodology Earlier dinucleotide relative abundance (DRA) have been used to identify regions, which deviate from the neighborhood areas, in genomes. We have used the difference in the dinucleotide relative abundance (DRAD) between the bacterial and prophage DNA to aid location of DNA stretches that could be of prophage origin in bacterial genomes. Prophage sequences which deviate from bacterial regions in their dinucleotide frequencies are detected by scanning bacterial genome sequences. The method was validated using a subset of genomes with prophage data from literature reports. A web interface for prophage scan based on this method is available at http://bicmku.in:8082/prophagedb/dra.html. Two hundred bacterial genomes which do not have annotated prophages have been scanned for prophage regions using this method. Conclusions The relative dinucleotide distribution difference helps detect prophage regions in genome sequences. The usefulness of this method is seen in the identification of 461 highly probable loci pertaining to prophages which have not been annotated so earlier. This work emphasizes the need to extend the efforts to detect and annotate prophage elements in genome sequences. PMID:18030328

  6. 3-base periodicity in coding DNA is affected by intercodon dinucleotides

    PubMed Central

    Sánchez, Joaquín

    2011-01-01

    All coding DNAs exhibit 3-base periodicity (TBP), which may be defined as the tendency of nucleotides and higher order n-tuples, e.g. trinucleotides (triplets), to be preferentially spaced by 3, 6, 9 etc, bases, and we have proposed an association between TBP and clustering of same-phase triplets. We here investigated if TBP was affected by intercodon dinucleotide tendencies and whether clustering of same-phase triplets was involved. Under constant protein sequence intercodon dinucleotide frequencies depend on the distribution of synonymous codons. So, possible effects were revealed by randomly exchanging synonymous codons without altering protein sequences to subsequently document changes in TBP via frequency distribution of distances (FDD) of DNA triplets. A tripartite positive correlation was found between intercodon dinucleotide frequencies, clustering of same-phase triplets and TBP. So, intercodon C|A (where “|” indicates the boundary between codons) was more frequent in native human DNA than in the codon-shuffled sequences; higher C|A frequency occurred along with more frequent clustering of C|AN triplets (where N jointly represents A, C, G and T) and with intense CAN TBP. The opposite was found for C|G, which was less frequent in native than in shuffled sequences; lower C|G frequency occurred together with reduced clustering of C|GN triplets and with less intense CGN TBP. We hence propose that intercodon dinucleotides affect TBP via same-phase triplet clustering. A possible biological relevance of our findings is briefly discussed. PMID:21814388

  7. Adenine attenuates the Ca(2+) contraction-signaling pathway via adenine receptor-mediated signaling in rat vascular smooth muscle cells.

    PubMed

    Fukuda, Toshihiko; Kuroda, Takahiro; Kono, Miki; Hyoguchi, Mai; Tajiri, Satoshi; Tanaka, Mitsuru; Mine, Yoshinori; Matsui, Toshiro

    2016-09-01

    Our previous study demonstrated that adenine (6-amino-6H-purine) relaxed contracted rat aorta rings in an endothelial-independent manner. Although adenine receptors (AdeRs) are expressed in diverse tissues, aortic AdeR expression has not been ascertained. Thus, the aims of this study were to clarify the expression of AdeR in rat vascular smooth muscle cells (VSMCs) and to investigate the adenine-induced vasorelaxation mechanism(s). VSMCs were isolated from 8-week-old male Wistar-Kyoto rats and used in this study. Phosphorylation of myosin light chain (p-MLC) was measured by western blot. AdeR mRNA was detected by RT-PCR. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured by using Fura-2/AM. Vasorelaxant adenine (10-100 μM) significantly reduced p-MLC by angiotensin II (Ang II, 10 μM) in VSMCs (P < 0.05). We confirmed the expression of aortic AdeR mRNA and the activation of PKA in VSMCs through stimulation of AdeR by adenine by ELISA. Intracellular Ca(2+) concentration ([Ca(2+)]i) measurement demonstrated that adenine inhibits Ang II- and m-3M3FBS (PLC agonist)-induced [Ca(2+)]i elevation. In AdeR-knockdown VSMCs, PKA activation and p-MLC reduction by adenine were completely abolished. These results firstly demonstrated that vasorelaxant adenine can suppress Ca(2+) contraction signaling pathways via aortic AdeR/PKA activation in VSMCs. PMID:27318925

  8. Dynamic changes in nicotinamide pyridine dinucleotide content in normal human epidermal keratinocytes and their effect on retinoic acid biosynthesis

    SciTech Connect

    Pinkas-Sarafova, Adriana . E-mail: apinkassaraf@notes.cc.sunysb.edu; Markova, N.G. . E-mail: nmarkova@notes.cc.sunysb.edu; Simon, M. . E-mail: marsimon@notes.cc.sunysb.edu

    2005-10-21

    The function of many enzymes that regulate metabolism and transcription depends critically on the nicotinamide pyridine dinucleotides. To understand the role of NAD(P)(H) in physiology and pathophysiology, it is imperative to estimate both their amount and ratios in a given cell type. In human epidermis and in cultured epidermal keratinocytes, we found that the total dinucleotide content is in the low millimolar range. The dinucleotide pattern changes during proliferation and maturation of keratinocytes in culture. Differences in the concentrations of NAD(P)(H) of 1.5- to 12-fold were observed. This resulted in alteration of the NAD(P)H/NAD(P) ratio, which could impact the differential regulation of both transcriptional and metabolic processes. In support of this notion, we provide evidence that the two-step oxidation of retinol to retinoic acid, a nuclear hormone critical for epidermal homeostasis, can be regulated by the relative physiological amounts of the pyridine dinucleotides.

  9. Adenine, a hairpin ribozyme cofactor--high-pressure and competition studies.

    PubMed

    Ztouti, Myriam; Kaddour, Hussein; Miralles, Francisco; Simian, Christophe; Vergne, Jacques; Hervé, Guy; Maurel, Marie-Christine

    2009-05-01

    The RNA world hypothesis assumes that life arose from ancestral RNA molecules, which stored genetic information and catalyzed chemical reactions. Although RNA catalysis was believed to be restricted to phosphate chemistry, it is now established that the RNA has much wider catalytic capacities. In this respect, we devised, in a previous study, two hairpin ribozymes (adenine-dependent hairpin ribozyme 1 and adenine-dependent hairpin ribozyme 2) that require adenine as cofactor for their reversible self-cleavage. We have now used high hydrostatic pressure to investigate the role of adenine in the catalytic activity of adenine-dependent hairpin ribozyme 1. High-pressure studies are of interest because they make it possible to determine the volume changes associated with the reactions, which in turn reflect the conformational modifications and changes in hydration involved in the catalytic mechanism. They are also relevant in the context of piezophilic organisms, as well as in relation to the extreme conditions that prevailed at the origin of life. Our results indicate that the catalytic process involves a transition state whose formation is accompanied by a positive activation volume and release of water molecules. In addition, competition experiments with adenine analogs strongly suggest that exogenous adenine replaces the adenine present at the catalytic site of the wild-type hairpin ribozyme. PMID:19476496

  10. Characterization of photophysical and base-mimicking properties of a novel fluorescent adenine analogue in DNA

    PubMed Central

    Dierckx, Anke; Dinér, Peter; El-Sagheer, Afaf H.; Kumar, Joshi Dhruval; Brown, Tom; Grøtli, Morten; Wilhelmsson, L. Marcus

    2011-01-01

    To increase the diversity of fluorescent base analogues with improved properties, we here present the straightforward click-chemistry-based synthesis of a novel fluorescent adenine-analogue triazole adenine (AT) and its photophysical characterization inside DNA. AT shows promising properties compared to the widely used adenine analogue 2-aminopurine. Quantum yields reach >20% and >5% in single- and double-stranded DNA, respectively, and show dependence on neighbouring bases. Moreover, AT shows only a minor destabilization of DNA duplexes, comparable to 2-aminopurine, and circular dichroism investigations suggest that AT only causes minimal structural perturbations to normal B-DNA. Furthermore, we find that AT shows favourable base-pairing properties with thymine and more surprisingly also with normal adenine. In conclusion, AT shows strong potential as a new fluorescent adenine analogue for monitoring changes within its microenvironment in DNA. PMID:21278417

  11. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: spectroscopic and molecular docking investigations.

    PubMed

    Rajendiran, N; Thulasidhasan, J

    2015-06-01

    Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV-visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern-Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules. PMID:25754395

  12. Interaction of sulfanilamide and sulfamethoxazole with bovine serum albumin and adenine: Spectroscopic and molecular docking investigations

    NASA Astrophysics Data System (ADS)

    Rajendiran, N.; Thulasidhasan, J.

    2015-06-01

    Interaction between sulfanilamide (SAM) and sulfamethoxazole (SMO) with BSA and DNA base (adenine) was investigated by UV-visible, fluorescence, cyclic voltammetry and molecular docking studies. Stern-Volmer fluorescence quenching constant (Ka) suggests SMO is more quenched with BSA/adenine than that of SAM. The distance r between donor (BSA/adenine) and acceptor (SAM and SMO) was obtained according to fluorescence resonance energy transfer (FRET). The results showed that hydrophobic forces, electrostatic interactions, and hydrogen bonds played vital roles in the SAM and SMO with BSA/adenine binding interaction. During the interaction, sulfa drugs could insert into the hydrophobic pocket, where the non-radioactive energy transfer from BSA/adenine to sulfa drugs occurred with high possibility. Cyclic voltammetry results suggested that when the drug concentration is increased, the anodic electrode potential deceased. The docking method indicates aniline group is interacted with the BSA molecules.

  13. Ultraviolet absorption and luminescence of matrix-isolated adenine

    SciTech Connect

    Polewski, K.; Sutherland, J.; Zinger, D.; Trunk, J.

    2011-10-01

    We have investigated the absorption, the fluorescence and phosphorescence emission and the fluorescence lifetimes of adenine in low-temperature argon and nitrogen matrices at 15 K. Compared to other environments the absorption spectrum shows higher intensity at the shortest wavelengths, and a weak apparent absorption peak is observed at 280 nm. The resolved fluorescence excitation spectrum has five peaks at positions corresponding to those observed in the absorption spectrum. The position of the fluorescence maximum depends on the excitation wavelength. Excitation below 220 nm displays a fluorescence maximum at 305 nm, while for excitations at higher wavelengths the maximum occurs at 335 nm. The results suggest that multiple-emission excited electronic states are populated in low-temperature gas matrices. Excitation at 265 nm produces a phosphorescence spectrum with a well-resolved vibrational structure and a maximum at 415 nm. The fluorescence decays corresponding to excitation at increasing energy of each resolved band could be fit with a double exponential, with the shorter and longer lifetimes ranging from 1.7 to 3.3 ns and from 12 to 23 ns, respectively. Only for the excitation at 180 nm one exponential is required, with the calculated lifetimes of 3.3 ns. The presented results provide an experimental evidence of the existence of multiple site-selected excited electronic states, and may help elucidate the possible deexcitation pathways of adenine. The additional application of synchrotron radiation proved to result in a significant enhancement of the resolution and spectral range of the phenomena under investigation.

  14. Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA.

    PubMed

    Huang, Ke-Jing; Niu, De-Jun; Sun, Jun-Yong; Han, Cong-Hui; Wu, Zhi-Wei; Li, Yan-Li; Xiong, Xiao-Qin

    2011-02-01

    A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334V. The detection limit for individual determination of guanine and adenine was 5.0×10(-8)M and 2.5×10(-8)M (S/N=3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G+C)/(A+T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability. PMID:21050729

  15. Cosolute effect on crystallization of two dinucleotide complexes of bovine seminal ribonuclease from concentrated salt solutions

    NASA Astrophysics Data System (ADS)

    Sica, Filomena; Adinolfi, Salvatore; Vitagliano, Luigi; Zagari, Adriana; Capasso, Sante; Mazzarella, Lelio

    1996-10-01

    Two complexes of bovine seminal ribonuclease with dinucleotides, uridylyl(2'-5')adenosine (UpA) and 2'-deoxycytidylyl(3'-5')-2'-deoxyadenosine (d(CpA)), were crystallized under unusual conditions involving a liquid-liquid phase separation. This phenomenon was induced by adding small aliquots of organic cosolutes to highly concentrated ammonium sulfate solutions. The liquid-liquid interface acts as a source of nucleation centers for growth of large crystals. Among the cosolutes tested in these salt-mediated crystallizations, 2-methyl-2,4-pentanediol was found to be the most efficient.

  16. Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety.

    PubMed

    Shanmugasundaram, Muthian; Charles, Irudaya; Kore, Anilkumar R

    2016-03-15

    The first example of the synthesis of new dinucleotide cap analog containing propargyl group such as m(7,3'-)(O)(-propargyl)G[5']ppp[5']G is reported. The effect of propargyl cap analog with standard cap was evaluated with respect to their capping efficiency, in vitro T7 RNA polymerase transcription efficiency, and translation activity using cultured HeLa cells. It is noteworthy that propargyl cap analog outperforms standard cap by 3.1 fold in terms of translational properties. The propargyl cap analog forms a more stable complex with translation initiation factor eIF4E based on the molecular modeling studies. PMID:26899596

  17. Single Nucleotide Polymorphisms of Human STING Can Affect Innate Immune Response to Cyclic Dinucleotides

    PubMed Central

    Yi, Guanghui; Brendel, Volker P.; Shu, Chang; Li, Pingwei; Palanathan, Satheesh; Cheng Kao, C.

    2013-01-01

    The STING (stimulator of interferon genes) protein can bind cyclic dinucleotides to activate the production of type I interferons and inflammatory cytokines. The cyclic dinucleotides can be bacterial second messengers c-di-GMP and c-di-AMP, 3’5’-3’5’ cyclic GMP-AMP (3’3’ cGAMP) produced by Vibrio cholerae and metazoan second messenger 2’5’-3’5’ Cyclic GMP-AMP (2’3’ cGAMP). Analysis of single nucleotide polymorphism (SNP) data from the 1000 Genome Project revealed that R71H-G230A-R293Q (HAQ) occurs in 20.4%, R232H in 13.7%, G230A-R293Q (AQ) in 5.2%, and R293Q in 1.5% of human population. In the absence of exogenous ligands, the R232H, R293Q and AQ SNPs had only modest effect on the stimulation of IFN-β and NF-κB promoter activities in HEK293T cells, while HAQ had significantly lower intrinsic activity. The decrease was primarily due to the R71H substitution. The SNPs also affected the response to the cyclic dinucleotides. In the presence of c-di-GMP, the R232H variant partially decreased the ability to activate IFN-βsignaling, while it was defective for the response to c-di-AMP and 3’3’ cGAMP. The R293Q dramatically decreased the stimulatory response to all bacterial ligands. Surprisingly, the AQ and HAQ variants maintained partial abilities to activate the IFN-β signaling in the presence of ligands due primarily to the G230A substitution. Biochemical analysis revealed that the recombinant G230A protein could affect the conformation of the C-terminal domain of STING and the binding to c-di-GMP. Comparison of G230A structure with that of WT revealed that the conformation of the lid region that clamps onto the c-di-GMP was significantly altered. These results suggest that hSTING variation can affect innate immune signaling and that the common HAQ haplotype expresses a STING protein with reduced intrinsic signaling activity but retained the ability to response to bacterial cyclic dinucleotides. PMID:24204993

  18. Renoprotective effect of the xanthine oxidoreductase inhibitor topiroxostat on adenine-induced renal injury.

    PubMed

    Kamijo-Ikemori, Atsuko; Sugaya, Takeshi; Hibi, Chihiro; Nakamura, Takashi; Murase, Takayo; Oikawa, Tsuyoshi; Hoshino, Seiko; Hisamichi, Mikako; Hirata, Kazuaki; Kimura, Kenjiro; Shibagaki, Yugo

    2016-06-01

    The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice (n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model. PMID:27029427

  19. Dinucleotide repeat loci contribute highly informative genetic markers to the human chromosome 2 linkage map

    SciTech Connect

    Todd, S. ); Sherman, S.L. ); Naylor, S.L. )

    1993-06-01

    Microsatellite repeat loci can provide informative markers for genetic linkage. Currently, the human chromosome 2 genetic linkage map has very few highly polymorphic markers. Being such a large chromosome, it will require a large number of informative markers for the dense coverage desired to allow disease genes to be mapped quickly and accurately. Dinucleotide repeat loci from two anonymous chromosome 2 genomic DNA clones were sequenced so that oligonucleotide primers could be designed for amplifying each locus using the polymerase chain reaction (PCR). Five sets of PCR primers were also generated from nucleotide sequences in the GenBank Database of chromosome 2 genes containing dinucleotide repeats. In addition, one PCR primer pair was made that amplifies a restriction fragment length polymorphism on the TNP1 gene. These markers were placed on the CEPH genetic linkage map by screening the CEPH reference DNA panel with each primer set, combining these data with those of other markers previously placed on the map, and analyzing the combined data set using CRI-MAP and LINKAGE. The microsatellite loci are highly informative markers and the TNP1 locus, as expected, is only moderately informative. A map was constructed with 38 ordered loci (odds [ge] 1000:1) spanning 296 cM (male) and 476 cM (female) of chromosome 2 compared with 306 cM (male) and 529 cM (female) for a previous map of 20 markers. 32 refs., 2 figs., 3 tabs.

  20. Resolvase-catalysed reactions between res sites differing in the central dinucleotide of subsite I.

    PubMed Central

    Stark, W M; Grindley, N D; Hatfull, G F; Boocock, M R

    1991-01-01

    The resolvase-catalysed reaction between two res sites in a circular DNA substrate normally gives two circular recombination products linked in a two-noded catenane. Homology between the two res sites at the central overlap dinucleotide of subsite I is important for recombination. Reactions between res sites differing at one position in the central dinucleotide (AC X AT) gave a low yield of recombinants containing mismatched base-pairs, but gave large amounts of a non-recombinant four-noded knot. This result was predicted by a 'simple rotation' model for strand exchange. The mismatch is evidently recognized only after commitment to an initial 180 degrees rotation of the resolvase-linked DNA ends, and it induces a second 180 degrees rotation which restores correct base-pairing at the overlap, giving the four-noded product. Correct base-pairing is not essential for religation, but may be important for release of the products. Characteristic patterns of 4, 6, 8 and 10 node knots, or 4, 8, 12 and 16 node knots were obtained, depending on the reaction conditions and the resolvase. Two pathways for multiple rounds of rotation in 360 degrees steps are inferred. The results support a model for strand exchange by supercoil-directed subunit rotation within a resolvase tetramer. Images PMID:1655422

  1. DNA methylation on N6-adenine in C. elegans

    PubMed Central

    Greer, Eric Lieberman; Blanco, Mario Andres; Gu, Lei; Sendinc, Erdem; Liu, Jianzhao; Aristizábal-Corrales, David; Hsu, Chih-Hung; Aravind, L.; He, Chuan; Shi, Yang

    2015-01-01

    Summary In mammalian cells, DNA methylation on the 5th position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylation of histone H3K4me2 and 6mA, and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. PMID:25936839

  2. Adenine nucleotide translocator transports haem precursors into mitochondria.

    PubMed

    Azuma, Motoki; Kabe, Yasuaki; Kuramori, Chikanori; Kondo, Masao; Yamaguchi, Yuki; Handa, Hiroshi

    2008-01-01

    Haem is a prosthetic group for haem proteins, which play an essential role in oxygen transport, respiration, signal transduction, and detoxification. In haem biosynthesis, the haem precursor protoporphyrin IX (PP IX) must be accumulated into the mitochondrial matrix across the inner membrane, but its mechanism is largely unclear. Here we show that adenine nucleotide translocator (ANT), the inner membrane transporter, contributes to haem biosynthesis by facilitating mitochondrial accumulation of its precursors. We identified that haem and PP IX specifically bind to ANT. Mitochondrial uptake of PP IX was inhibited by ADP, a known substrate of ANT. Conversely, ADP uptake into mitochondria was competitively inhibited by haem and its precursors, suggesting that haem-related porphyrins are accumulated into mitochondria via ANT. Furthermore, disruption of the ANT genes in yeast resulted in a reduction of haem biosynthesis by blocking the translocation of haem precursors into the matrix. Our results represent a new model that ANT plays a crucial role in haem biosynthesis by facilitating accumulation of its precursors into the mitochondrial matrix. PMID:18728780

  3. Adenine nucleotides as allosteric effectors of PEA seed glutamine synthetase

    SciTech Connect

    Unkefer, P.J.; Knight, T.J.

    1986-05-01

    The energy charge in the plant cell has been proposed as a regulator of glutamine synthetase (GS) activity. The authors have shown that 2.1 moles of ..gamma..(/sup 32/P)-ATP were bound/mole subunits of purified pea seed GS during complete inactivation with methionine sulfoximine. Since GS has one active site per subunit, the second binding site provides the potential for allosteric regulation of GS by adenine nucleotides. The authors have investigated the inhibition of the ATP-dependent synthetic activity by ADP and AMP. ADP and AMP cannot completely inhibit GS; but ATP does overcome the inhibition by ADP and AMP as shown by plots of % inhibition vs inhibitor concentration. This indicates that inhibition of GS by ADP or AMP is not completely due to competitive inhibition. In the absence of ADP or AMP, double reciprocal plots for ATP are linear below 10 mM; however, in the presence of either ADP or AMP these pots are curvilinear downwards. The ratio of Vm/asymptote is less than 1. The Hill number for ATP in the absence of ADP or AMP is 0.93 but decreases with increasing ADP or AMP to a value of 0.28 with 10 mM ADP. These data are consistent with negative cooperativity by ADP and AMP. Thus, as the ADP/ATP or AMP/ATP ratios are increased GS activity decreases. This is consistent with regulation of GS activity by energy charge in planta.

  4. The Cellular Environment Stabilizes Adenine Riboswitch RNA Structure

    PubMed Central

    Tyrrell, Jillian; McGinnis, Jennifer L.; Weeks, Kevin M.; Pielak, Gary J.

    2016-01-01

    There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand-binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute timescale using SHAPE. The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg2+, the approximate Mg2+ concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg2+ concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg2+ alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations. PMID:24215455

  5. Spin-dependent electron transport in zinc- and manganese-doped adenine molecules

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2014-01-28

    The spin-dependent electron transport properties of zinc- and manganese-doped adenine molecules connected to zigzag graphene leads are studied in the zero bias regime using the non-equilibrium Green's function method. The conductance of the adenine molecule increased and became spin-dependent when a zinc or manganese atom was doped into the molecules. The effects of a transverse electric field on the spin-polarization of the transmitted electrons were investigated and the spin-polarization was controlled by changing the transverse electric field. Under the presence of a transverse electric field, both the zinc- and manganese-doped adenine molecules acted as spin-filters. The maximum spin-polarization of the manganese-doped adenine molecule was greater than the molecule doped with zinc.

  6. Identification of a mitochondrial ATP synthase-adenine nucleotide translocator complex in Leishmania.

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

    The ATP synthasome is a macromolecular complex consisting of ATP synthase, adenine nucleotide translocator and phosphate carrier. To determine if this complex is evolutionary old or young, we searched for its presence in Leishmania, a mitochondria containing protozoan which evolved from the main eukaryote line soon after eukaryotes split from prokaryotes. Sucrose gradient centrifugation showed that the distribution of ANT among the fractions coincided with the distribution of ATP synthase. In addition, ATP synthase co-precipitated with FLAG tagged and wild type adenine nucleotide translocator isolated with anti FLAG and anti adenine nucleotide translocator antibodies, respectively. These data indicate that the adenine nucleotide translocator interacted with the ATP synthase to form a stable structure referred to as the ATP synthasome. The presence of the ATP synthasome in Leishmania, an organism branching off the main line of eukaryotes early in the development of eukaryotes, as well as in higher eukaryotes suggests that the ATP synthasome is a phylogenetically ancient structure. PMID:17920025

  7. Adenine and guanine nucleotide metabolism during platelet storage at 22 degree C

    SciTech Connect

    Edenbrandt, C.M.; Murphy, S. )

    1990-11-01

    Adenine and guanine nucleotide metabolism of platelet concentrates (PCs) was studied during storage for transfusion at 22 +/- 2 degrees C over a 7-day period using high-pressure liquid chromatography. There was a steady decrease in platelet adenosine triphosphate (ATP) and adenosine diphosphate (ADP), which was balanced quantitatively by an increase in plasma hypoxanthine. As expected, ammonia accumulated along with hypoxanthine but at a far greater rate. A fall in platelet guanosine triphosphate (GTP) and guanosine diphosphate (GDP) paralleled the fall in ATP + ADP. When adenine was present in the primary anticoagulant, it was carried over into the PC and metabolized. ATP, GTP, total adenine nucleotides, and total guanine nucleotides declined more slowly in the presence of adenine than in its absence. With adenine, the increase in hypoxanthine concentration was more rapid and quantitatively balanced the decrease in adenine and platelet ATP + ADP. Plasma xanthine rose during storage but at a rate that exceeded the decline in GTP + GDP. When platelet ATP + ADP was labeled with 14C-adenine at the initiation of storage, half of the radioactivity was transferred to hypoxanthine (45%) and GTP + GDP + xanthine (5%) by the time storage was completed. The isotopic data were consistent with the presence of a radioactive (metabolic) and a nonradioactive (storage) pool of ATP + ADP at the initiation of storage with each pool contributing approximately equally to the decline in ATP + ADP during storage. The results suggested a continuing synthesis of GTP + GDP from ATP + ADP, explaining the slower rate of fall of GTP + GDP relative to the rate of rise of plasma xanthine. Throughout storage, platelets were able to incorporate 14C-hypoxanthine into both adenine and guanine nucleotides but at a rate that was only one fourth the rate of hypoxanthine accumulation.

  8. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study.

    PubMed

    Emel'yanenko, Vladimir N; Zaitsau, Dzmitry H; Shoifet, Evgeni; Meurer, Florian; Verevkin, Sergey P; Schick, Christoph; Held, Christoph

    2015-09-17

    The thermochemical properties available in the literature for adenine and cytosine are in disarray. A new condensed phase standard (p° = 0.1 MPa) molar enthalpy of formation at T = 298.15 K was measured by using combustion calorimetry. New molar enthalpies of sublimation were derived from the temperature dependence of vapor pressure measured by transpiration and by the quarz-crystal microbalance technique. The heat capacities of crystalline adenine and cytosine were measured by temperature-modulated DSC. Thermodynamic data on adenine and cytosine available in the literature were collected, evaluated, and combined with our experimental results. Thus, the evaluated collection of data together with the new experimental results reported here has helped to resolve contradictions in the available enthalpies of formation. A set of reliable thermochemical data is recommended for adenine and cytosine for further thermochemical calculations. Quantum-chemical calculations of the gas phase molar enthalpies of formation of adenine and cytosine have been performed by using the G4 method and results were in excellent agreement with the recommended experimental data. The standard molar entropies of formation and the standard molar Gibbs functions of formation in crystal and gas state have been calculated. Experimental vapor-pressure data measured in this work were used to estimate pure-component PC-SAFT parameters. This allowed modeling solubility of adenine and cytosine in water over the temperature interval 278-310 K. PMID:26317826

  9. Sequence-dependent folding landscapes of adenine riboswitch aptamers

    NASA Astrophysics Data System (ADS)

    Lin, Jong-Chin; Hyeon, Changbong; Thirumalai, D.

    Prediction of the functions of riboswitches requires a quantitative description of the folding landscape so that the barriers and time scales for the conformational change in the switching region in the aptamer can be estimated. Using a combination of all atom molecular dynamics and coarse-grained model simulations we studied the response of adenine (A) binding add and pbuE A-riboswitches to mechanical force. The two riboswitches contain a structurally similar three-way junction formed by three paired helices, P1, P2, and P3, but carry out different functions. Using pulling simulations, with structures generated in MD simulations, we show that after P1 rips the dominant unfolding pathway in add A-riboswitch is the rupture of P2 followed by unraveling of P3. In the pbuE A-riboswitch, after P1 unfolds P3 ruptures ahead of P2. The order of unfolding of the helices, which is in accord with single molecule pulling experiments, is determined by the relative stabilities of the individual helices. Our results show that the stability of isolated helices determines the order of assembly and response to force in these non-coding regions. We use the simulated free energy profile for pbuE A-riboswitch to estimate the time scale for allosteric switching, which shows that this riboswitch is under kinetic control lending additional support to the conclusion based on single molecule pulling experiments. A consequence of the stability hypothesis is that a single point mutation (U28C) in the P2 helix of the add A-riboswitch, which increases the stability of P2, would make the folding landscapes of the two riboswitches similar. This prediction can be tested in single molecule pulling experiments.

  10. Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

    Bollée, Guillaume; Dollinger, Cécile; Boutaud, Lucile; Guillemot, Delphine; Bensman, Albert; Harambat, Jérôme; Deteix, Patrice; Daudon, Michel; Knebelmann, Bertrand

    2010-01-01

    Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications. PMID:20150536

  11. Labeling of mitochondrial adenine nucleotides of bovine sperm

    SciTech Connect

    Cheetham, J.; Lardy, H.A.

    1986-05-01

    Incorporation of /sup 32/P/sub i/ into the adenine nucleotide pool of intact bovine spermatozoa utilizing endogenous substrates results in a specific activity (S.A.) ratio ATP/ADP of 0.3 to 0.5, suggesting compartmentation of nucleotide pools or a pathway for phosphorylation of AMP in addition to the myokinase reaction. Incubation of filipin-permeabilized cells with pyruvate, acetylcarnitine, or ..cap alpha..-ketoglutarate (..cap alpha..KG) resulted in ATP-ADP S.A. ratios of 0.5, 0.8, and 1.6, respectively, for mitochondrial nucleotides. However, when malate was included with pyruvate or acetylcarnitine, the ATP/ADP S.A. ratio increased by 400% to 2.0 for pyruvate/malate and by 290% to 2.8 for acetylcarnitine/malate, while the ATP/ADP ratio increased by less than 100% in both cases. These results may indicate that under conditions of limited flux through the citric acid cycle a pathway for phosphorylation of AMP from a precursor other than ATP exists or that ATP is compartmented within the mitochondrion. In the presence of uncoupler and oligomycin with ..cap alpha..KG, pyruvate/malate, or acetylcarnitine/malate, /sup 32/P/sub i/ is incorporated primarily into ATP, resulting in an ATP/ADP S.A. ratio of 4.0 for ..cap alpha..KG, 2.7 for pyruvate/malate, and 2.8 for acetylcarnitine/malate. These data are consistent with phosphorylation of ADP during substrate level phosphorylation in the citric acid cycle.

  12. Autosomal recessive chronic granulomatous disease caused by deletion at a dinucleotide repeat

    SciTech Connect

    Casimir, C.M.; Bu-Ghanim, H.N.; Rowe, P.; Segal, A.W. ); Rodaway, A.R.F.; Bentley, D.L. )

    1991-04-01

    Chronic granulomatous disease (CGD) is a rare inherited condition rendering neutrophils incapable of killing invading pathogens. This condition is due to the failure of a multicomponent microbicidal oxidase that normally yields a low-midpoint-potential b cytochrome (cytochrome b{sub 245}). Although defects in the X chromosome-linked cytochrome account for the majority of CGD patients, as many as 30% of CGD cases are due to an autosomal recessive disease. Of these, {gt}90% have been shown to be defective in the synthesis of a 47-kDa cytosolic component of the oxidase. The authors demonstrate here in three unrelated cases of autosomal recessive CGD that the identical underlying molecular lesion is a dinucleotide deletion at a GTGT tandem repeat, corresponding to the acceptor site of the first intron - exon junction. Slippage of the DNA duplex at this site may contribute to the high frequency of defects in this gene.

  13. Role of the intergenic dinucleotide in vesicular stomatitis virus RNA transcription.

    PubMed Central

    Barr, J N; Whelan, S P; Wertz, G W

    1997-01-01

    To investigate the role played by the intergenic dinucleotide sequence of the conserved vesicular stomatitis virus (VSV) gene junction in modulation of polymerase activity, we analyzed the RNA synthesis activities of bicistrionic genomic analogs that contained either the authentic N/P gene junction or gene junctions that had been altered to contain either the 16 possible dinucleotide combinations, single nucleotide intergenic sequences, or no intergenic sequence at all. Quantitative measurements of the amounts of upstream, downstream, and readthrough mRNAs that were transcribed by these mutant templates showed that the behavior of the viral polymerase was profoundly affected by the nucleotide sequence that it encountered as it traversed the gene junction, although the polymerase was able to accommodate a remarkable degree of sequence variation without altogether losing the ability to terminate and reinitiate transcription. Alteration or removal of the intergenic sequence such that the U tract responsible for synthesis of the upstream mRNA poly(A) tail was effectively positioned adjacent to the consensus downstream gene start signal resulted in almost complete abrogation of downstream mRNA synthesis, thus defining the intergenic sequence as an essential sequence element of the gene junction. Many genome analogs with altered intergenic sequences directed abundant synthesis of a readthrough transcript without correspondingly high levels of downstream mRNA, an observation inconsistent with the shunting model of VSV transcription, which suggests that polymerase molecules are prepositioned at gene junctions, awaiting a push from upstream. Instead, the findings of this study support a model of sequential transcription in which initiation of downstream mRNA can occur only following termination of the preceding transcript. PMID:9032308

  14. Sequence-dependent dynamics of duplex DNA: the applicability of a dinucleotide model.

    PubMed Central

    Okonogi, T M; Alley, S C; Reese, A W; Hopkins, P B; Robinson, B H

    2002-01-01

    The short-time (submicrosecond) bending dynamics of duplex DNA were measured to determine the effect of sequence on dynamics. All measurements were obtained from a single site on duplex DNA, using a single, site-specific modified base containing a rigidly tethered, electron paramagnetic resonance active spin probe. The observed dynamics are interpreted in terms of single-step sequence-dependent bending force constants, determined from the mean squared amplitude of bending relative to the end-to-end vector using the modified weakly bending rod model. The bending dynamics at a single site are a function of the sequence of the nucleotides constituting the duplex DNA. We developed and examined several dinucleotide-based models for flexibility. The models indicate that the dominant feature of the dynamics is best explained in terms of purine- and pyrimidine-type steps, although distinction is made among all 10 unique steps: It was found that purine-purine steps (which are the same as pyrimidine-pyrimidine steps) were near average in flexibility, but the pyrimidine-purine steps (5' to 3') were nearly twice as flexible, whereas purine-pyrimidine steps were more than half as flexible as average DNA. Therefore, the range of stepwise flexibility is approximately fourfold and is characterized by both the type of base pair step (pyrimidine/purine combination) and the identity of the bases within the pair (G, A, T, or C). All of the four models considered here underscore the complexity of the dependence of dynamics on DNA sequence with certain sequences not satisfactorily explainable in terms of any dinucleotide model. These findings provide a quantitative basis for interpreting the dynamics and kinetics of DNA-sequence-dependent biological processes, including protein recognition and chromatin packaging. PMID:12496111

  15. Assignment of the Gene for Adenine Phosphoribosyltransferase to Human Chromosome 16 by Mouse-Human Somatic Cell Hybridization

    PubMed Central

    Tischfield, Jay A.; Ruddle, Frank H.

    1974-01-01

    A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made in medium containing adenine and alanosine, an antimetabolite known to inhibit de novo adenylic acid biosynthesis. The mouse cells, unable to utilize exogenous adenine, were killed in this medium, but the hybrids proliferated as a consequence of their retaining the human aprt gene. The hybrids were then exposed to the adenine analogs 2,6-diaminopurine and 2-fluoroadenine to select for cells that had lost this gene. Before exposure to the adenine analogs, the expression of human adenine phosphoribosyltransferase by the hybrids was strongly associated only with the presence of human chromosome 16, and afterwards this was the only human chromosome consistently lost. This observation suggests that the human aprt gene can be assigned to chromosome 16. Images PMID:4129802

  16. Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

    PubMed Central

    Rein, T; Zorbas, H; DePamphilis, M L

    1997-01-01

    ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins. PMID:8972222

  17. DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

    PubMed Central

    Erova, Tatiana E.; Pillai, Lakshmi; Fadl, Amin A.; Sha, Jian; Wang, Shaofei; Galindo, Cristi L.; Chopra, Ashok K.

    2006-01-01

    Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (damAhSSU) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N6-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, PBAD promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam

  18. A computational study of adenine, uracil, and cytosine adsorption upon AlN and BN nano-cages

    NASA Astrophysics Data System (ADS)

    Baei, Mohammad T.; Taghartapeh, Mohammad Ramezani; Lemeski, E. Tazikeh; Soltani, Alireza

    Density-functional theory calculations are used to investigate the interaction of Al12N12 and B12N12 clusters with the adenine (A), uracil (U), and cytosine (C) molecules. The current calculations demonstrate that these hybrid adsorbent materials are able to adsorb the adenine, uracil, and cytosine molecules through exothermic processes. Our theoretical results reveal improvement in the adsorption of adenine, uracil, and cytosine on Al12N12 and B12N12. It is observed that B12N12 is highly sensitive to adenine, uracil, and cytosine compared with Al12N12 to serve as a biochemical sensor.

  19. Absolute effective cross sections of ionization of adenine and guanine molecules by electron impact

    NASA Astrophysics Data System (ADS)

    Shafranyosh, I. I.; Svida, Yu. Yu.; Sukhoviya, M. I.; Shafranyosh, M. I.; Minaev, B. F.; Baryshnikov, G. V.; Minaeva, V. A.

    2015-10-01

    Effective cross sections of the formation of positive ions of nitrous nucleic acids of adenine and guanine are determined by the crossed electron and molecular beam method in the energy interval from the threshold to 200 eV. It is found that the maximal value of the total cross section of adenine ionization is attained at an energy of 90 eV and is equal to (2.8 ± 0.6) × 10-15 cm2. The maximal value of the total cross section of guanine ionization is equal to (3.2 ± 0.7) × 10-15 cm2 and is observed at an energy of 88 eV. The energy ionization thresholds are determined, which amount to (8.8 ± 0.2) eV for adenine and to (8.3 ± 0.2) eV for guanine. The adenine and guanine mass spectra are measured. The absolute values of partial ionization cross sections of adenine and guanine molecules are determined.

  20. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu2+ complex

    NASA Astrophysics Data System (ADS)

    Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

    2016-01-01

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0 μmol L-1, with a correlation coefficient (R2) of 0.9994. The detection limit (3σ/k) was 0.046 μmol L-1, indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

  1. Regulation of photolyase in Escherichia coli K-12 during adenine deprivation.

    PubMed Central

    Alcorn, J L; Rupert, C S

    1990-01-01

    DNA photolyase, a DNA repair enzyme encoded by the phr gene of Escherichia coli, is normally regulated at 10 to 20 active molecules per cell. In purA mutants deprived of adenine, this amount increased sixfold within 2 h. Operon fusions placing lacZ under transcriptional control of phr promoters indicated no change in transcription rate during adenine deprivation, and gene fusions of phr with lacZ showed a nearly constant level of translation as well. Immunoblot analysis indicated that the total amount of photolyase protein remained constant during enzyme amplification. On the other hand, treatment of cells with chloramphenicol during the adenine deprivation prevented any increase. DNA regions lying 1.3 to 4.2 kb upstream of the phr coding sequences were necessary for this amplification to occur and for this purpose would function in trans. These results suggest that adenine deprivation leads to a posttranslational change, involving synthesis of protein encoded by sequences lying upstream of phr, which increases photolyase activity. The amplification in activity was found to be reversible, for when adenine was restored, the photolyase activity declined before cell growth resumed. Images PMID:2254263

  2. Spectroscopic investigation on cocrystal formation between adenine and fumaric acid based on infrared and Raman techniques

    NASA Astrophysics Data System (ADS)

    Du, Yong; Fang, Hong Xia; Zhang, Qi; Zhang, Hui Li; Hong, Zhi

    2016-01-01

    As an important component of double-stranded DNA, adenine has powerful hydrogen-bond capability, due to rich hydrogen bond donors and acceptors existing within its molecular structure. Therefore, it is easy to form cocrystal between adenine and other small molecules with intermolecular hydrogen-bond effect. In this work, cocrystal of adenine and fumaric acid has been characterized as model system by FT-IR and FT-Raman spectral techniques. The experimental results show that the cocrystal formed between adenine and fumaric acid possesses unique spectroscopical characteristic compared with that of starting materials. Density functional theory (DFT) calculation has been performed to optimize the molecular structures and simulate vibrational modes of adenine, fumaric acid and the corresponding cocrystal. Combining the theoretical and experimental vibrational results, the characteristic bands corresponding to bending and stretching vibrations of amino and carbonyl groups within cocrystal are shifted into lower frequencies upon cocrystal formation, and the corresponding bond lengths show some increase due to the effect of intermolecular hydrogen bonding. Different vibrational modes shown in the experimental spectra have been assigned based on the simulation DFT results. The study could provide experimental and theoretical benchmarks to characterize cocrystal formed between active ingredients and cocrystal formers and also the intermolecular hydrogen-bond effect within cocrystal formation process by vibrational spectroscopic techniques.

  3. Adenine: an important drug scaffold for the design of antiviral agents

    PubMed Central

    Wang, Changyuan; Song, Zhendong; Yu, Haiqing; Liu, Kexin; Ma, Xiaodong

    2015-01-01

    Adenine derivatives, in particular the scaffold bearing the acyclic nucleoside phosphonates (ANPS), possess significant antiviral and cytostatic activity. Till now, several effective adenine derivatives have been marketed for the treatment of HIV, HBV, CMV and other virus-infected diseases. These compounds are represented by tenofovir (PMPA), a medicine for both HIV and HBV, and adefovir as an anti-HBV agent. More than this, other analogs, such as GS9148, GS9131, and GS7340, are also well-known anti-viral agents that have been progressed to the clinical studies for their excellent activity. In general, the structures of these compounds include an adenine nucleobase linked to a phosphonate side chain. Considerable structural modifications on the scaffold itself and the peripheral sections were made. The structure-activity relationships (SARs) of this skeleton will provide valuable clues to identify more effective adenine derivatives as antiviral drugs. Here, we systematically summarized the SARs of the adenine derivatives, and gave important information for further optimizing this template. PMID:26579473

  4. Adenine phosphoribosyltransferase deficiency as a rare cause of renal allograft dysfunction.

    PubMed

    Kaartinen, Kati; Hemmilä, Ulla; Salmela, Kaija; Räisänen-Sokolowski, Anne; Kouri, Timo; Mäkelä, Satu

    2014-04-01

    Adenine phosphoribosyltransferase deficiency is a rare autosomal recessive disorder manifesting as urolithiasis or crystalline nephropathy. It leads to the generation of large amounts of poorly soluble 2,8-dihydroxyadenine excreted in urine, yielding kidney injury and in some patients, kidney failure. Early recognition of the disease, institution of xanthine analog therapy to block the formation of 2,8-dihydroxyadenine, high fluid intake, and low purine diet prevent CKD. Because of symptom variability and lack of awareness, however, the diagnosis is sometimes extremely deferred. We describe a patient with adenine phosphoribosyltransferase deficiency who was diagnosed during evaluation of a poorly functioning second kidney allograft. This report highlights the risk of renal allograft loss in patients with undiagnosed adenine phosphoribosyltransferase deficiency and the need for improved early detection of this disease. PMID:24459232

  5. Unique modification of adenine in genomic DNA of the marine cyanobacterium Trichodesmium sp. strain NIBB 1067.

    PubMed Central

    Zehr, J P; Ohki, K; Fujita, Y; Landry, D

    1991-01-01

    The genomic DNA of the marine nonheterocystous nitrogen-fixing cyanobacterium Trichodesmium sp. strain NIBB 1067 was found to be highly resistant to DNA restriction endonucleases. The DNA was digested extensively by the restriction enzyme DpnI, which requires adenine methylation for activity. The DNA composition, determined by high-performance liquid chromatography (HPLC), was found to be 69% AT. Surprisingly, it was found that a modified adenine which was not methylated at the usual N6 position was present and made up 4.7 mol% of the nucleosides in Trichodesmium DNA (15 mol% of deoxyadenosine). In order for adenine residues to be modified at this many positions, there must be many modifying enzymes or at least one of the modifying enzymes must have a degenerate recognition site. The reason(s) for this extensive methylation has not yet been determined but may have implications for the ecological success of this microorganism in nature. Images FIG. 1 FIG. 2 PMID:1657876

  6. Replacement of Tyr50 stacked on the si-face of the isoalloxazine ring of the flavin adenine dinucleotide prosthetic group modulates Bacillus subtilis ferredoxin-NADP(+) oxidoreductase activity toward NADPH.

    PubMed

    Seo, Daisuke; Naito, Hiroshi; Nishimura, Erika; Sakurai, Takeshi

    2015-08-01

    Ferredoxin-NAD(P)(+) oxidoreductases ([EC 1.18.1.2], [EC 1.18.1.3], FNRs) from green sulfur bacteria, purple non-sulfur bacteria and most of Firmicutes, such as Bacillus subtilis (BsFNR) are homo-dimeric flavoproteins homologous to bacterial NADPH-thioredoxin reductase. These FNRs contain two unique aromatic residues stacked on the si- and re-face of the isoalloxazine ring moiety of the FAD prosthetic group whose configurations are often found among other types of flavoproteins including plant-type FNR and flavodoxin, but not in bacterial NADPH-thioredoxin reductase. To investigate the role of the si-face Tyr50 residue in BsFNR, we replaced Tyr50 with Gly, Ser, and Trp and examined its spectroscopic properties and enzymatic activities in the presence of NADPH and ferredoxin (Fd) from B. subtilis (BsFd). The replacement of Tyr50 to Gly (Y50G), Ser (Y50S), and Trp (Y50W) in BsFNR resulted in a blue shift of the FAD transition bands. The Y50G and Y50S mutations enhanced the FAD fluorescence emission, whereas those of the wild type and Y50W mutant were quenched. All three mutants decreased thermal stabilities compared to wild type. Using a diaphorase assay, the k cat values for the Y50G and Y50S mutants in the presence of NADPH and ferricyanide were decreased to less than 5 % of the wild type activity. The Y50W mutant retained approximately 20 % reactivity in the diaphorase assay and BsFd-dependent cytochrome c reduction assay relative to wild type. The present results suggest that Tyr50 modulates the electronic properties and positioning of the prosthetic group. PMID:25698107

  7. Ultra-performance liquid chromatography tandem mass-spectrometry (uplc-ms/ms) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A novel, rapid and sensitive Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin a...

  8. One-step versus stepwise mechanism in protonated amino acid-promoted electron-transfer reduction of a quinone by electron donors and two-electron reduction by a dihydronicotinamide adenine dinucleotide analogue. Interplay between electron transfer and hydrogen bonding.

    PubMed

    Yuasa, Junpei; Yamada, Shunsuke; Fukuzumi, Shunichi

    2008-04-30

    Semiquinone radical anion of 1-(p-tolylsulfinyl)-2,5-benzoquinone (TolSQ(*-)) forms a strong hydrogen bond with protonated histidine (TolSQ(*-)/His x 2 H(+)), which was successfully detected by electron spin resonance. Strong hydrogen bonding between TolSQ(*-) and His x 2 H(+) results in acceleration of electron transfer (ET) from ferrocenes [R2Fc, R = C5H5, C5H4(n-Bu), C5H4Me] to TolSQ, when the one-electron reduction potential of TolSQ is largely shifted to the positive direction in the presence of His x 2 H(+). The rates of His x 2 H(+)-promoted ET from R2Fc to TolSQ exhibit deuterium kinetic isotope effects due to partial dissociation of the N-H bond in His x 2 H(+) at the transition state, when His x 2 H(+) is replaced by the deuterated compound (His x 2 D(+)-d6). The observed deuterium kinetic isotope effect (kH/kD) decreases continuously with increasing the driving force of ET to approach kH/kD = 1.0. On the other hand, His x 2 H(+) also promotes a hydride reduction of TolSQ by an NADH analogue, 9,10-dihydro-10-methylacridine (AcrH2). The hydride reduction proceeds via the one-step hydride-transfer pathway. In such a case, a large deuterium kinetic isotope effect is observed in the rate of the hydride transfer, when AcrH2 is replaced by the dideuterated compound (AcrD2). In sharp contrast to this, no deuterium kinetic isotope effect is observed, when His x 2 H(+) is replaced by His x 2 D(+)-d6. On the other hand, direct protonation of TolSQ and 9,10-phenanthrenequinone (PQ) also results in efficient reductions of TolSQH(+) and PQH(+) by AcrH2, respectively. In this case, however, the hydride-transfer reactions occur via the ET pathway, that is, ET from AcrH2 to TolSQH(+) and PQH(+) occurs in preference to direct hydride transfer from AcrH2 to TolSQH(+) and PQH(+), respectively. The AcrH2(*+) produced by the ET oxidation of AcrH2 by TolSQH(+) and PQH(+) was directly detected by using a stopped-flow technique. PMID:18386924

  9. Incorporation of L-[1-14C]leucine into protein by liver postmitochondrial supernatant: opposing effects of preincubated nicotinamide-adenine dinucleotide phosphate and 4-dimethylamino-3'-methylazobenzene.

    PubMed Central

    Madsen, N P; Labuc, J E

    1975-01-01

    Combination of preincubated drug-metabolizing medium containing NADP+ with a cell-free protein-synthesizing system resulted in marked stimulation of incorporation of L-[1-14C]leucine into protein. Addition of 4-dimethylamino-3'-methylazobenzene, present and previously preincubated in the drug-metabolizing medium, decreased this effect. PMID:239694

  10. Effects of increased mechanical work by isolated perfused rat heart during production or uptake of ketone bodies. Assessment of mitochondrial oxidized to reduced free nicotinamide-adenine dinucleotide ratios and oxaloacetate concentrations.

    PubMed Central

    Opie, L H; Owen, P

    1975-01-01

    Metabolic effects of increased mechanical work were studied by comparing isolated pumping rat hearts perfused by the atrial-filling technique with aortic-perfused non-pumping hearts perfused by the technique of Langendorff. The initial medium usually contained glucose (11 mm) and palmitate (0.6 mm bound to 0.1 mm albumin). During increased heart work (comparing pumping with non-pumping hearts) the uptake of oxygen and glucose increased threefold, but that of free fatty acids was unchanged. Tissue contents of alpha-oxoglutarate, NH4+, malate, lactate, pyruvate and Pi rose with increased heart work, but contents of ATP, phosphocreatine and citrate fell. Ketone bodies were produced with a ratio of beta-hydroxybutyrate/acetoacetate of about 3:1 in both pumping and non-pumping hearts but with higher net production rates in non-pumping hearts. When ketone bodies were added in relatively high concentrations (total 4 mm) to a glucose (11 mm) medium the medium, ratios of beta-hydroxybutyrate/acetoacetate were not steady even after 60 min of perfusion. The validity of calculating mitochondrial free NAD+/NADH ratios from the tissue contents of the reactants of the glutamate dehydrogenase system or the beta-hydroxybutyrate dehydrogenase system is assessed. The activities of these enzymes are considerably less in the rat heart than in the rat liver, introducing reservations into the application to the heart of the principles used by Williamson et al. (1967) for calculation of mitochondrial free NAD+/NADH ratios of liver mitochondria... PMID:173281

  11. Comparative study of spontaneous deamination of adenine and cytosine in unbuffered aqueous solution at room temperature

    NASA Astrophysics Data System (ADS)

    Wang, Shiliang; Hu, Anguang

    2016-06-01

    Adenine in unbuffered nanopure water at a concentration of 2 mM is completely deaminated (>99%) to hypoxanthine at room temperature in ca. 10 weeks, with an estimated half-life (t1/2) less than 10 days, about six orders of magnitude faster than previously reported. Cytosine is not deaminated under the same condition, even after 3 years. This is in contrast to previous observations that cytosine deaminates 20-40 times faster than adenine free base, in nucleoside, in nucleotide and in single-stranded DNA in buffered neutral aqueous solutions.

  12. Copper-catalyzed intramolecular cyclization of N-propargyl-adenine: synthesis of purine-fused tricyclics.

    PubMed

    Li, Ren-Long; Liang, Lei; Xie, Ming-Sheng; Qu, Gui-Rong; Niu, Hong-Ying; Guo, Hai-Ming

    2014-04-18

    A novel protocol to construct fluorescent purine-fused tricyclic products via intramolecular cyclization of N-propargyl-adenine has been developed. With CuBr as the catalyst, a series of purine-fused tricyclic products were obtained in good to excellent yields (19 examples, 75-89% yields). When R2 was a hydrogen atom in N-propargyl-adenines, the reactions only afforded the endocyclic double bond products. When R2 was an aryl group, the electron-donating groups favored the endocyclic double bond products, while the electron-withdrawing groups favored the exocyclic double bond products. PMID:24678722

  13. Efficient UV-induced charge separation and recombination in an 8-oxoguanine-containing dinucleotide.

    PubMed

    Zhang, Yuyuan; Dood, Jordan; Beckstead, Ashley A; Li, Xi-Bo; Nguyen, Khiem V; Burrows, Cynthia J; Improta, Roberto; Kohler, Bern

    2014-08-12

    During the early evolution of life, 8-oxo-7,8-dihydro-2'-deoxyguanosine (O) may have functioned as a proto-flavin capable of repairing cyclobutane pyrimidine dimers in DNA or RNA by photoinduced electron transfer using longer wavelength UVB radiation. To investigate the ability of O to act as an excited-state electron donor, a dinucleotide mimic of the FADH2 cofactor containing O at the 5'-end and 2'-deoxyadenosine at the 3'-end was studied by femtosecond transient absorption spectroscopy in aqueous solution. Following excitation with a UV pulse, a broadband mid-IR pulse probed vibrational modes of ground-state and electronically excited molecules in the double-bond stretching region. Global analysis of time- and frequency-resolved transient absorption data coupled with ab initio quantum mechanical calculations reveal vibrational marker bands of nucleobase radical ions formed by electron transfer from O to 2'-deoxyadenosine. The quantum yield of charge separation is 0.4 at 265 nm, but decreases to 0.1 at 295 nm. Charge recombination occurs in 60 ps before the O radical cation can lose a deuteron to water. Kinetic and thermodynamic considerations strongly suggest that all nucleobases can undergo ultrafast charge separation when π-stacked in DNA or RNA. Interbase charge transfer is proposed to be a major decay pathway for UV excited states of nucleic acids of great importance for photostability as well as photoredox activity. PMID:25071180

  14. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    NASA Astrophysics Data System (ADS)

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/.

  15. High Resolution Detection and Analysis of CpG Dinucleotides Methylation Using MBD-Seq Technology

    PubMed Central

    Lan, Xun; Adams, Christopher; Landers, Mark; Dudas, Miroslav; Krissinger, Daniel; Marnellos, George; Bonneville, Russell; Xu, Maoxiong; Wang, Junbai; Huang, Tim H.-M.; Meredith, Gavin; Jin, Victor X.

    2011-01-01

    Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution. PMID:21779396

  16. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks.

    PubMed

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named "DeepMethyl" to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/. PMID:26797014

  17. bis-Molybdopterin Guanine Dinucleotide Is Required for Persistence of Mycobacterium tuberculosis in Guinea Pigs

    PubMed Central

    Williams, Monique J.; Shanley, Crystal A.; Zilavy, Andrew; Peixoto, Blas; Manca, Claudia; Kaplan, Gilla; Orme, Ian M.; Mizrahi, Valerie

    2014-01-01

    Mycobacterium tuberculosis is able to synthesize molybdopterin cofactor (MoCo), which is utilized by numerous enzymes that catalyze redox reactions in carbon, nitrogen, and sulfur metabolism. In bacteria, MoCo is further modified through the activity of a guanylyltransferase, MobA, which converts MoCo to bis-molybdopterin guanine dinucleotide (bis-MGD), a form of the cofactor that is required by the dimethylsulfoxide (DMSO) reductase family of enzymes, which includes the nitrate reductase NarGHI. In this study, the functionality of the mobA homolog in M. tuberculosis was confirmed by demonstrating the loss of assimilatory and respiratory nitrate reductase activity in a mobA deletion mutant. This mutant displayed no survival defects in human monocytes or mouse lungs but failed to persist in the lungs of guinea pigs. These results implicate one or more bis-MGD-dependent enzymes in the persistence of M. tuberculosis in guinea pig lungs and underscore the applicability of this animal model for assessing the role of molybdoenzymes in this pathogen. PMID:25404027

  18. High resolution detection and analysis of CpG dinucleotides methylation using MBD-Seq technology.

    PubMed

    Lan, Xun; Adams, Christopher; Landers, Mark; Dudas, Miroslav; Krissinger, Daniel; Marnellos, George; Bonneville, Russell; Xu, Maoxiong; Wang, Junbai; Huang, Tim H-M; Meredith, Gavin; Jin, Victor X

    2011-01-01

    Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ∼100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution. PMID:21779396

  19. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks

    PubMed Central

    Wang, Yiheng; Liu, Tong; Xu, Dong; Shi, Huidong; Zhang, Chaoyang; Mo, Yin-Yuan; Wang, Zheng

    2016-01-01

    The hypo- or hyper-methylation of the human genome is one of the epigenetic features of leukemia. However, experimental approaches have only determined the methylation state of a small portion of the human genome. We developed deep learning based (stacked denoising autoencoders, or SdAs) software named “DeepMethyl” to predict the methylation state of DNA CpG dinucleotides using features inferred from three-dimensional genome topology (based on Hi-C) and DNA sequence patterns. We used the experimental data from immortalised myelogenous leukemia (K562) and healthy lymphoblastoid (GM12878) cell lines to train the learning models and assess prediction performance. We have tested various SdA architectures with different configurations of hidden layer(s) and amount of pre-training data and compared the performance of deep networks relative to support vector machines (SVMs). Using the methylation states of sequentially neighboring regions as one of the learning features, an SdA achieved a blind test accuracy of 89.7% for GM12878 and 88.6% for K562. When the methylation states of sequentially neighboring regions are unknown, the accuracies are 84.82% for GM12878 and 72.01% for K562. We also analyzed the contribution of genome topological features inferred from Hi-C. DeepMethyl can be accessed at http://dna.cs.usm.edu/deepmethyl/. PMID:26797014

  20. A multicopy dinucleotide marker that maps close to the spinal muscular atrophy gene

    SciTech Connect

    Burghes, A.H.M.; Ingraham, S.E.; Kote-Jarai, Z.; Carpten, J.D.; DiDonato, C.J. ); McLean, M.; Surh, L. ); Thompson, T.G.; McPherson, J.D. ); Ikeda, J.E. ); Wirth, B. )

    1994-05-15

    Spinal muscular atrophy (SMA) is a common autosomal recessive disorder resulting in loss of motor neurons. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S435-SMA-D5S557-5qter. The authors have isolated a new dinucleotide repeat marker, CATT1, that lies between these two closest markers. The marker CATT1 has 16 alleles and is highly polymorphic. The marker can have 1 to 4 (or more) copies per chromosome, giving rise to individuals with up to 8 (or more) alleles. All of the subloci map between the markers D5S557 and D5S435 and lie in close proximity to one another. The marker CATT1 is linked to the SMA gene with a lod score of Z[sub max] = 34.42 at [theta] = 0 and crosses all available recombinants. Certain alleles occurred more frequently in either the SMA or normal populations, indicating significant allelic association between CATT1 and the SMA locus. Haplotype analysis combining US and Canadian SMA families reveals that one haplotype group (VII) occurs significantly more frequently in the SMA population than in the normal. This confirms the allelic association of CATT1 with the SMA locus. 37 refs., 4 figs., 3 tabs.

  1. Alternate Polypurine Tracts (PPTs) Affect the Rous Sarcoma Virus RNase H Cleavage Specificity and Reveal a Preferential Cleavage following a GA Dinucleotide Sequence at the PPT-U3 Junction

    PubMed Central

    Chang, Kevin W.; Julias, John G.; Alvord, W. Gregory; Oh, Jangsuk; Hughes, Stephen H.

    2005-01-01

    Retroviral polypurine tracts (PPTs) serve as primers for plus-strand DNA synthesis during reverse transcription. The generation and removal of the PPT primer requires specific cleavages by the RNase H activity of reverse transcriptases; removal of the PPT primer defines the left end of the linear viral DNA. We replaced the endogenous PPT from RSVP(A)Z, a replication-competent shuttle vector based on Rous sarcoma virus (RSV), with alternate retroviral PPTs and the duck hepatitis B virus “PPT.” Viruses in which the endogenous RSV PPT was replaced with alternate PPTs had lower relative titers than the wild-type virus. 2-LTR circle junction analysis showed that the alternate PPTs caused significant decreases in the fraction of viral DNAs with complete (consensus) ends and significant increases in the insertion of part or all of the PPT at the 2-LTR circle junctions. The last two nucleotides in the 3′ end of the RSV PPT are GA. Examination of the (mis)cleavages of the alternate PPTs revealed preferential cleavages after GA dinucleotide sequences. Replacement of the terminal 3′ A of the RSV PPT with G caused a preferential miscleavage at a GA sequence spanning the PPT-U3 boundary, resulting in the deletion of the terminal adenine normally present at the 5′ end of the U3. A reciprocal G-to-A substitution at the 3′ end of the murine leukemia virus PPT increased the relative titer of the chimeric RSV-based virus and the fraction of consensus 2-LTR circle junctions. PMID:16227289

  2. Effects of adenine arabinoside on lymphocytes infected with Epstein-Barr virus.

    PubMed Central

    Benz, W C; Siegel, P J; Baer, J

    1978-01-01

    Low concentrations of adenine arabinoside inhibited growth of two Epstein-Barr virus producer cell lines in culture, while not significantly affecting a nonproducer cell line and a B-cell-negative line. These observations were extended to include freshly infected cells. Mitogen-stimulated human umbilical cord blood lymphocytes were unaffected by the drug at concentration levels that inhibited [3H]thymidine incorporation into the DNA of Epstein-Barr virus-stimulated cells. DNA synthesis in Epstein-Barr virus-superinfected Raji cells was also adversely affected by adenine arabinoside. However, these same low concentrations of adenine arabinoside in the triphosphate form produced less effect on DNA synthesis in nuclear systems and DNA polymerase assays than on growth or DNA synthesis in whole cells. Therefore the effects reported here of low concentrations of the drug on whole cells may be only in part related to DNA polymerase inhibition. The work reported here suggests that adenine arabinoside has multiple sites of action in infected cells. PMID:212577

  3. Phosphorus-31 NMR visibility and characterization of rat liver mitochondrial matrix adenine nucleotides

    SciTech Connect

    Hutson, S.M.; Berkich, D.; Williams, G.D.; LaNoue, K.F.; Briggs, R.W. )

    1989-05-16

    Compartmentation and NMR visibility of mitochondrial adenine nucleotides were quantitated in isolated rat liver mitochondria respiring on succinate and glutamate in vitro at 8 and 25{degree}C. Intra- and extramitochondrial nucleotides were discriminated by adding the chelator trans-1,2-diaminocyclohexane-N,N,N{prime},N{prime}-tetraacetic acid (CDTA). T{sub 1} values of about 0.2-0.3 s for magnesium-bound matrix nucleotides were determined. Adenine nucleotide T{sub 1} values were influenced by the ionic environment; only magnesium-free ATP T{sub 1}'s were affected by temperature. Intra- and extramitochondrial adenine nucleotide ratios were varied in ATP-loaded mitochondria with added ATP and phosphate using the mitochondrial inhibitors oligomycin and carboxyatractyloside, and adenine nucleotides were quantitated by using NMR and enzymatic analysis. There was good agreement between matrix ATP concentrations (magnesium-bound ATP) calculated by using NMR and standard biochemical techniques. Although matrix ADP could be detected by NMR, it was difficult to quantitate accurately by NMR. The data indicate that mitochondrial ATP is NMR-visible in isolated mitochondria in vitro.

  4. Assembly of an antiparallel homo-adenine DNA duplex by small-molecule binding.

    PubMed

    Persil, Ozgül; Santai, Catherine T; Jain, Swapan S; Hud, Nicholas V

    2004-07-21

    Molecules that reversibly bind DNA and trigger the formation of non-Watson-Crick secondary structures would be useful in the design of dynamic DNA nanostructures and as potential leads for new therapeutic agents. We demonstrate that coralyne, a small crescent-shaped molecule, promotes the formation of a duplex secondary structure from homo-adenine oligonucleotides. AFM studies reveal that the staggered alignment of homo-adenine oligonucleotides upon coralyne binding produces polymers of micrometers in length, but only 2 nm in height. A DNA duplex was also studied that contained eight A.A mismatches between two flanking 7-bp Watson-Crick helices. CD spectra confirm that the multiple A.A mismatches of this duplex bind coralyne in manner similar to that of homo-adenine oligonucleotides. Furthermore, the melting temperature of this hybrid duplex increases by 13 degrees C upon coralyne binding. These observations illustrate that the helical structure of the homo-adenine-coralyne duplex is compatible with the B-form DNA helix. PMID:15250704

  5. Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury

    PubMed Central

    Aguiar, Cristhiane Favero; Naffah-de-Souza, Cristiane; Castoldi, Angela; Corrêa-Costa, Matheus; Braga, Tárcio T; Naka, Érika L; Amano, Mariane T; Abate, Débora T R S; Hiyane, Meire I; Cenedeze, Marcos A; Filho, Alvaro Pacheco e Silva; Câmara, Niels O S

    2015-01-01

    Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration. PMID:26101952

  6. ON THE INTERACTION OF ADENINE WITH IONIZING RADIATION: MECHANISTICAL STUDIES AND ASTROBIOLOGICAL IMPLICATIONS

    SciTech Connect

    Evans, Nicholas L.; Ullrich, Susanne; Bennett, Chris J.; Kaiser, Ralf I.

    2011-04-01

    The molecular inventory available on the prebiotic Earth was likely derived from both terrestrial and extraterrestrial sources. A complete description of which extraterrestrial molecules may have seeded early Earth is therefore necessary to fully understand the prebiotic evolution which led to life. Galactic cosmic rays (GCRs) are expected to cause both the formation and destruction of important biomolecules-including nucleic acid bases such as adenine-in the interstellar medium within the ices condensed on interstellar grains. The interstellar ultraviolet (UV) component is expected to photochemically degrade gas-phase adenine on a short timescale of only several years. However, the destruction rate is expected to be significantly reduced when adenine is shielded in dense molecular clouds or even within the ices of interstellar grains. Here, biomolecule destruction by the energetic charged particle component of the GCR becomes important as it is not fully attenuated. Presented here are results on the destruction rate of the nucleobase adenine in the solid state at 10 K by energetic electrons, as generated in the track of cosmic ray particles as they penetrate ices. When both UV and energetic charged particle destructive processes are taken into account, the half-life of adenine within dense interstellar clouds is found to be {approx}6 Myr, which is on the order of a star-forming molecular cloud. We also discuss chemical reaction pathways within the ices to explain the production of observed species, including the formation of nitriles (R-C{identical_to}N), epoxides (C-O-C), and carbonyl functions (R-C=O).

  7. Quantum chemical benchmark study on 46 RNA backbone families using a dinucleotide unit.

    PubMed

    Kruse, Holger; Mladek, Arnost; Gkionis, Konstantinos; Hansen, Andreas; Grimme, Stefan; Sponer, Jiri

    2015-10-13

    We have created a benchmark set of quantum chemical structure-energy data denoted as UpU46, which consists of 46 uracil dinucleotides (UpU), representing all known 46 RNA backbone conformational families. Penalty-function-based restrained optimizations with COSMO TPSS-D3/def2-TZVP ensure a balance between keeping the target conformation and geometry relaxation. The backbone geometries are close to the clustering-means of their respective RNA bioinformatics family classification. High-level wave function methods (DLPNO-CCSD(T) as reference) and a wide-range of dispersion-corrected or inclusive DFT methods (DFT-D3, VV10, LC-BOP-LRD, M06-2X, M11, and more) are used to evaluate the conformational energies. The results are compared to the Amber RNA bsc0χOL3 force field. Most dispersion-corrected DFT methods surpass the Amber force field significantly in accuracy and yield mean absolute deviations (MADs) for relative conformational energies of ∼0.4-0.6 kcal/mol. Double-hybrid density functionals represent the most accurate class of density functionals. Low-cost quantum chemical methods such as PM6-D3H+, HF-3c, DFTB3-D3, as well as small basis set calculations corrected for basis set superposition errors (BSSEs) by the gCP procedure are also tested. Unfortunately, the presently available low-cost methods are struggling to describe the UpU conformational energies with satisfactory accuracy. The UpU46 benchmark is an ideal test for benchmarking and development of fast methods to describe nucleic acids, including force fields. PMID:26574283

  8. Differences in Electrostatic Potential Around DNA Fragments Containing Adenine and 8-oxo-Adenine. An Analysis Based on Regular Cylindrical Projection

    SciTech Connect

    Haranczyk, Maciej; Miller, John H; Gutowski, Maciej S

    2007-07-01

    Changes of electrostatic potential (EP) around the DNA molecule resulting from chemical modifications of nucleotides may play a role in enzymatic recognition of damaged sites. Effects of chemical modifications of nucleotides on the structure of DNA have been characterized through large scale density functional theory computations. Quantum mechanical structural optimizations of DNA fragments with three pairs of nucleotides and accompanying counteractions were performed with a B3LYP exchange-correlation functional and 6-31G** basis sets. The “intact” DNA fragment contained adenine in the middle layer, while the “damaged” fragment had the adenine replaced with 8-oxo-adenine. The electrostatic potential around these DNA fragments was projected on a cylindrical surface around the double helix. The two-dimensional maps of EP of the intact and damaged DNA fragments were analyzed to identify these modifications of EP that result from the occurrence of 8-oxo-adenine (8oA). It was found that distortions of a phosphate group neighboring 8oA and displacements of the accompanying countercation are clearly reflected in the EP maps. Helpful discussions Michel Dupuis are gratefully acknowledged. Authors wish to thank Marcel Swart for directing us to a compilation of van der Waals radii. This work was supported by the: (i) US DOE Office of Biological and Environmental Research, Low Dose Radiation Research Program (M.G. and M.H.), (ii) the Office of Science (BER), U. S. Department of Energy, Grant No. DE-FG03-02ER63470 (JHM), (iii) Polish State Committee for Scientific Research (KBN) Grant DS/8221-4-0140-6 (MG), (iv) European Social Funds (EFS) ZPORR/2.22/II/2.6/ARP/U/2/05 (M.H.). M.H. holds the Foundation for Polish Science (FNP) award for young scientists. The calculations were performed at the Academic Computer Center in Gdansk (TASK) and at the Molecular Science Computing Facility (MSCF) in the William R. Wiley Environmental Molecular Sciences Laboratory, a national

  9. Effect of l-Methionine and S-Adenosylmethionine on Growth of an Adenine Mutant of Saccharomyces cerevisiae

    PubMed Central

    Yall, Irving; Norrell, Stephen A.; Joseph, Ronald; Knudsen, Richard C.

    1967-01-01

    A pink, adenine-requiring yeast utilized adenine, hypoxanthine, or S-adenosylmethionine (SAM), in quantities up to 3 μmoles per 100 ml of medium, as equivalent sources of purine for cell growth, but not methylthioadenosine or S-adenosylhomocysteine. Utilization of SAM for growth was inhibited by the presence of l-methionine in quantities greater than 0.6 μmole per 100 ml of medium. However, 6 μmoles of l-methionine had no effect on growth when adenine or hypoxanthine was the source of purine. These sources also reversed the inhibitory effects of 6 μmoles of the amino acid on the utilization of SAM. The presence of 400 μmoles of the amino acid resulted in some inhibition of growth when the organisms were grown with adenine, hypoxanthine, or adenine plus SAM but had no effect on the total uptake of adenine-8-14C. Studies on the uptake of radioactivity from a mixture of SAM-adenine-8-14C and 3H-labeled SAM-methyl indicated that these components were taken into the cells at different rates which were altered by the presence of l-methionine. The fixation of 35S from 35S-labeled adenosylmethionine into the cells was inhibited by the presence of the amino acid. The cells synthesized and accumulated SAM in the presence of 400 μmoles of l-methionine plus adenine even when exogenous SAM was supplied. Approximately 47% of radioactivity fixed from exogenous SAM-adenine-8-14C and 12% from 3H-labeled SAM-methyl were found in reisolated SAM. PMID:6025443

  10. The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site.

    PubMed

    Knospe, Melanie; Müller, Christa E; Rosa, Patrizia; Abdelrahman, Aliaa; von Kügelgen, Ivar; Thimm, Dominik; Schiedel, Anke C

    2013-09-01

    The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194(5.40) and Leu201(5.47), were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that-in contrast to most other rhodopsin-like GPCRs-the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors. PMID:23413038

  11. iRSpot-PseDNC: identify recombination spots with pseudo dinucleotide composition.

    PubMed

    Chen, Wei; Feng, Peng-Mian; Lin, Hao; Chou, Kuo-Chen

    2013-04-01

    Meiotic recombination is an important biological process. As a main driving force of evolution, recombination provides natural new combinations of genetic variations. Rather than randomly occurring across a genome, meiotic recombination takes place in some genomic regions (the so-called 'hotspots') with higher frequencies, and in the other regions (the so-called 'coldspots') with lower frequencies. Therefore, the information of the hotspots and coldspots would provide useful insights for in-depth studying of the mechanism of recombination and the genome evolution process as well. So far, the recombination regions have been mainly determined by experiments, which are both expensive and time-consuming. With the avalanche of genome sequences generated in the postgenomic age, it is highly desired to develop automated methods for rapidly and effectively identifying the recombination regions. In this study, a predictor, called 'iRSpot-PseDNC', was developed for identifying the recombination hotspots and coldspots. In the new predictor, the samples of DNA sequences are formulated by a novel feature vector, the so-called 'pseudo dinucleotide composition' (PseDNC), into which six local DNA structural properties, i.e. three angular parameters (twist, tilt and roll) and three translational parameters (shift, slide and rise), are incorporated. It was observed by the rigorous jackknife test that the overall success rate achieved by iRSpot-PseDNC was >82% in identifying recombination spots in Saccharomyces cerevisiae, indicating the new predictor is promising or at least may become a complementary tool to the existing methods in this area. Although the benchmark data set used to train and test the current method was from S. cerevisiae, the basic approaches can also be extended to deal with all the other genomes. Particularly, it has not escaped our notice that the PseDNC approach can be also used to study many other DNA-related problems. As a user-friendly web-server, i

  12. Theoretical Study of Tautomerization Reactions for the Ground and First Excited Electronic States of Adenine

    NASA Technical Reports Server (NTRS)

    Salter, Latasha M.; Chaban, Galina M.; Kwak, Dochan (Technical Monitor)

    2002-01-01

    Geometrical structures and energetic properties for different tautomers of adenine are calculated in this study, using multi-configurational wave functions. Both the ground and the lowest singlet excited state potential energy surfaces are studied. Four tautomeric forms are considered, and their energetic order is found to be different on the ground and the excited state potential energy surfaces. Minimum energy reaction paths are obtained for hydrogen atom transfer (tautomerization) reactions in the ground and the lowest excited electronic states. It is found that the barrier heights and the shapes of the reaction paths are different for the ground and the excited electronic states, suggesting that the probability of such tautomerization reaction is higher on the excited state potential energy surface. This tautomerization process should become possible in the presence of water or other polar solvent molecules and should play an important role in the photochemistry of adenine.

  13. BII stability and base step flexibility of N6-adenine methylated GATC motifs.

    PubMed

    Karolak, Aleksandra; van der Vaart, Arjan

    2015-01-01

    The effect of N6-adenine methylation on the flexibility and shape of palindromic GATC sequences has been investigated by molecular dynamics simulations. Variations in DNA backbone geometry were observed, which were dependent on the degree of methylation and the identity of the bases. While the effect was small, more frequent BI to BII conversions were observed in the GA step of hemimethylated DNA. The increased BII population of the hemimethylated system positively correlated with increased stacking interactions between methylated adenine and guanine, while stacking interactions decreased at the TC step for the fully methylated strand. The flexibility of the AT and TC steps was marginally affected by methylation, in a fashion that was correlated with stacking interactions. The facilitated BI to BII conversion in hemimethylated strands might be of importance for SeqA selectivity and binding. PMID:26004863

  14. Role of vacuum ultraviolet (VUV) radiation in abiogenic synthesis of adenine nucleotides

    NASA Astrophysics Data System (ADS)

    Kuzicheva, E. A.; Simakov, M. B.; Mal'Ko, I. L.; Dodonova, N. Ya.; Gontareva, N. B.

    With the use of high performance liquid chromatography the products of abiogenic synthesis of adenine nucleotides in solid films were indentified and estimated quantitatively. The main products of photosynthesis appeared to be adenosine and deoxyadenosine monophosphates. Maximal yield of these products in case of adenosine has been 0.36 for 5'AMP, 0.41% for 2'(3')AMP, 0.20 for 2'3'cAMP in case of deoxyadenosine 0.13% for 5'dAMP, 0.15% for 3'dAMP, 0.24% for 3'5'cdAMP. The destruction of initial adenosine and deoxyadenosine by the end of the experiment was 10 and 15%, respectively. By the increasing of irradiation dose, 5'AMP and 5'dAMP synthesized in the cource of VUV photolysis were destructed up to adenine, its yield being 15% in both cases.

  15. Kinetic and mechanistic analysis of dinucleotide and oligonucleotide formation from the 5'-phosphorimidazolide of adenosine on Na(+)-montmorillonite

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1994-01-01

    The rate constants for the condensation reaction of the 5'-phosphorimidazolide of adenosine (ImpA) to form dinucleotides and oligonucleotides have been measured in the presence of Na(+)-volclay (a Na(+)-montmorillonite) in pH 8 aqueous solution at 25 degrees C. The rates of the reaction of ImpA with an excess of adenosine 5'-monophosphoramidate (NH2pA), P1,P2-diadenosine 5',5'-pyrophosphate (A5'ppA), or adenosine 5'-monophosphate (5'-AMP or pA) in the presence of the montmorillonite to form NH2pA3'pA, A5'ppA3'pA, and pA3'pA, respectively, were measured. Only 3',5'-linked products were observed. The magnitude of the rate constants decrease in the order NH2pA3'pA > A5'-ppA3'pA > pA3'pA. The binding of ImpA to montmorillonite was measured, and the adsorption isotherm was determined. The binding of ImpA to montmorillonite and the formation of higher oligonucleotides is not observed in the absence of salts. Mg2+ enhances binding and oligonucleotide formation more than Ca2+ and Na+. The rate constants for the oligonucleotide formation were determined from the reaction products formed from 10 to 40 mM ImpA in the presence of Na(+)-montmorillonite using the computer program SIMFIT. The magnitudes of the rate constants for the formation of oligonucleotides increased in the order 2-mer < 3-mer < 4-mer ... 7-mer. The rate constants for dinucleotide and trinucleotide formation are more than 1000 times larger than those measured in the absence of montmorillonite. The rate constants for the formation of dinucleotide, trinucleotide, and tetranucleotide are 41,2.6, and 3.7 times larger than those for the formation of oligo(G)s with a poly(C) template. The hydrolysis of ImpA was accelerated 35 times in the presence of the montmorillonite. The catalytic ability of montmorillonite to form dinucleotides and oligonucleotides is quantitatively evaluated and possible pathways for oligo(A) formation are proposed.

  16. Structure of STING bound to c-di-GMP Reveals the Mechanism of Cyclic Dinucleotide Recognition by the Immune System

    PubMed Central

    Shu, Chang; Yi, Guanghui; Watts, Tylan; Kao, C. Cheng; Li, Pingwei

    2012-01-01

    STING, stimulator of interferon genes, is an innate immune sensor of cyclic dinucleotides that regulates the induction of type I interferons. STING C-terminal domain forms a V-shaped dimer and binds a c-di-GMP molecule at the dimer interface through direct and solvent-mediated hydrogen bonds. The guanine bases of c-di-GMP stack against the phenolic rings of a conserved tyrosine residue. Mutations at the c-di-GMP binding surface reduce nucleotide binding and affect signaling. PMID:22728658

  17. Long-Range Charge Transport in Adenine-Stacked RNA:DNA Hybrids.

    PubMed

    Li, Yuanhui; Artés, Juan M; Hihath, Joshua

    2016-01-27

    An extremely important biological component, RNA:DNA can also be used to design nanoscale structures such as molecular wires. The conductance of single adenine-stacked RNA:DNA hybrids is rapidly and reproducibly measured using the break junction approach. The conductance decreases slightly over a large range of molecular lengths, suggesting that RNA:DNA can be used as an oligonucleotide wire. PMID:26596516

  18. MICROCALORIMETRIC STUDIES ON THE FORMATION OF MAGNESIUM COMPLEXES OF ADENINE NUCLEOTIDES

    PubMed Central

    Belaich, J. P.; Sari, J. C.

    1969-01-01

    Values for the thermodynamic quantities (ΔF, ΔH, ΔS) in reactions in which complexes of adenine nucleotides with magnesium ion (ATPMg--, ADPMg-, AMPMg) are formed have been obtained by a microcalorimetric technique by using an isothermic Calvet's apparatus. Experimental values measured at ionic strength μ = 0.2 indicate that complex formation reactions are driven by the entropic factor and that stability of complexes increases with length of the phosphate chain. PMID:5261047

  19. Synthesis of metal-adeninate frameworks with high separation capacity on C2/C1 hydrocarbons

    NASA Astrophysics Data System (ADS)

    He, Yan-Ping; Zhou, Nan; Tan, Yan-Xi; Wang, Fei; Zhang, Jian

    2016-06-01

    By introducing isophthalic acid or 2,5-thiophenedicarboxylic acid to assemble with adenine and cadmium salt, two isostructural and anionic porous metal-organic frameworks (1 and 2) possessing the novel (4,8)-connected sqc topology are presented here. 1 shows permanent porosity with Langmuir surface area of 770.1 m2/g and exhibits high separation capacity on C2/C1 hydrocarbons.

  20. Femtosecond decay dynamics of intact adenine and thymine base pairs in a supersonic jet.

    PubMed

    Kim, Nam Joon; Chang, Jinyoung; Kim, Hyung Min; Kang, Hyuk; Ahn, Tae Kyu; Heo, Jiyoung; Kim, Seong Keun

    2011-07-11

    We investigated the decay dynamics of the DNA base pairs adenine-adenine (A(2)), adenine-thymine (AT), and thymine-thymine (T(2)) produced in a supersonic jet by femtosecond (fs) time-resolved photoionization spectroscopy. The base pair was excited by a fs pump pulse at 267 nm and the population change of its excited state was monitored by non-resonant three-photon ionization using a fs probe pulse at 800 nm after a certain time delay. All of the transients recorded in the mass channel of the parent ion exhibited a tri-exponential decay, with time constants ranging from 100 fs to longer than 100 ps. Most of these time constants coincide well with the previous values deduced indirectly from the transients of protonated adenine (AH(+)) and thymine (TH(+)), which were assumed to be produced by fragmentation of the base-pair ions. Notably, for the transient of T(2), we observed a new decay component with a time constant of 2.3 ps, which was absent in the transient of TH(+). We suggest that the new decay component arises from the decay of stacked T(2) dimers that are mostly ionized to T(2)(+), whereas the decay signal recorded in the mass channel of TH(+) is merely from the relaxation of hydrogen-bonded T(2) dimers. From the amplitude of the new decay component, the population of the stacked T(2) dimers relative to the hydrogen-bonded dimers was estimated to be ∼2 % in the supersonic jet, which is about fifteen times higher than the theoretical value. PMID:21710523

  1. Structure-wise discrimination of adenine and guanine by proteins on the basis of their nonbonded interactions.

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

    We have analyzed the nonbonded interactions of the structurally similar moieties, adenine and guanine forming complexes with proteins. The results comprise (a) the amino acid-ligand atom preferences, (b) solvent accessibility of ligand atoms before and after complex formation with proteins, and (c) preferred amino acid residue atoms involved in the interactions. We have observed that the amino acid preferences involved in the hydrogen bonding interactions vary for adenine and guanine. The structural variation between the purine atoms is clearly reflected by their burial tendency in the solvent environment. Correlation of the mean amino acid preference values show the variation that exists between adenine and guanine preferences of all the amino acid residues. All our observations provide evidence for the discriminating nature of the proteins in recognizing adenine and guanine. PMID:25245205

  2. Ethanol-induced activation of adenine nucleotide turnover. Evidence for a role of acetate

    SciTech Connect

    Puig, J.G.; Fox, I.H.

    1984-09-01

    Consumption of alcohol causes hyperuricemia by decreasing urate excretion and increasing its production. Our previous studies indicate that ethanol administration increases uric acid production by increasing ATP degradation to uric acid precursors. To test the hypothesis that ethanol-induced increased urate production results from acetate metabolism and enhanced adenosine triphosphate turnover, we gave intravenous sodium acetate, sodium chloride and ethanol (0.1 mmol/kg per min for 1 h) to five normal subjects. Acetate plasma levels increased from 0.04 +/- 0.01 mM (mean +/- SE) to peak values of 0.35 +/- 0.07 mM and to 0.08 +/- 0.01 mM during acetate and ethanol infusions, respectively. Urinary oxypurines increased to 223 +/- 13% and 316 +/- 44% of the base-line values during acetate and ethanol infusions, respectively. Urinary radioactivity from the adenine nucleotide pool labeled with (8-14C) adenine increased to 171 +/- 27% and to 128 +/- 8% of the base-line values after acetate and ethanol infusions. These data indicate that both ethanol and acetate increase purine nucleotide degradation by enhancing the turnover of the adenine nucleotide pool. They support the hypothesis that acetate metabolism contributes to the increased production of urate associated with ethanol intake.

  3. Functional Linkage of Adenine Nucleotide Binding Sites in Mammalian Muscle 6-Phosphofructokinase*

    PubMed Central

    Brüser, Antje; Kirchberger, Jürgen; Kloos, Marco; Sträter, Norbert; Schöneberg, Torsten

    2012-01-01

    6-Phosphofructokinases (Pfk) are homo- and heterooligomeric, allosteric enzymes that catalyze one of the rate-limiting steps of the glycolysis: the phosphorylation of fructose 6-phosphate at position 1. Pfk activity is modulated by a number of regulators including adenine nucleotides. Recent crystal structures from eukaryotic Pfk revealed several adenine nucleotide binding sites. Herein, we determined the functional relevance of two adenine nucleotide binding sites through site-directed mutagenesis and enzyme kinetic studies. Subsequent characterization of Pfk mutants allowed the identification of the activating (AMP, ADP) and inhibitory (ATP, ADP) allosteric binding sites. Mutation of one binding site reciprocally influenced the allosteric regulation through nucleotides interacting with the other binding site. Such reciprocal linkage between the activating and inhibitory binding sites is in agreement with current models of allosteric enzyme regulation. Because the allosteric nucleotide binding sites in eukaryotic Pfk did not evolve from prokaryotic ancestors, reciprocal linkage of functionally opposed allosteric binding sites must have developed independently in prokaryotic and eukaryotic Pfk (convergent evolution). PMID:22474333

  4. Monitoring potential molecular interactions of adenine with other amino acids using Raman spectroscopy and DFT modeling.

    PubMed

    Singh, Shweta; Donfack, P; Srivastava, Sunil K; Singh, Dheeraj K; Materny, A; Asthana, B P; Mishra, P C

    2015-10-01

    We report on the modes of inter-molecular interaction between adenine (Ade) and the amino acids: glycine (Gly), lysine (Lys) and arginine (Arg) using Raman spectroscopy of binary mixtures of adenine and each of the three amino acids at varying molar ratios in the spectral region 1550-550 cm(-1). We focused our attention on certain specific changes in the Raman bands of adenine arising due to its interaction with the amino acids. While the changes are less apparent in the Ade/Gly system, in the Ade/Lys or Ade/Arg systems, significant changes are observed, particularly in the Ade Raman bands that involve the amino group moiety and the N7 and N1 atoms of the purine ring. The ν(N1-C6), ν(N1-C2), δ(C8-H) and δ(N7-C8-N9) vibrations at 1486, 1332, 1253 and 948 cm(-1) show spectral changes on varying the Ade to amino acid molar ratio, the extent of variation being different for the three amino acids. This observation suggests a specific interaction mode between Ade and Lys or Arg, which is due to the hydrogen bonding. The measured spectral changes provide a clear indication that the interaction of Ade depends strongly on the structures of the amino acids, especially their side chains. Density functional theory (DFT) calculations were carried out to elucidate the most probable interaction modes of Ade with the different amino acids. PMID:25985129

  5. Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells.

    PubMed

    Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

    2015-01-01

    Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process. PMID:26643504

  6. Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

    NASA Astrophysics Data System (ADS)

    Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

    2015-12-01

    Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process.

  7. Selective self-assembly of adenine-silver nanoparticles forms rings resembling the size of cells

    PubMed Central

    Choi, Sungmoon; Park, Soonyoung; Yang, Seon-Ah; Jeong, Yujin; Yu, Junhua

    2015-01-01

    Self-assembly has played critical roles in the construction of functional nanomaterials. However, the structure of the macroscale multicomponent materials built by the self-assembly of nanoscale building blocks is hard to predict due to multiple intermolecular interactions of great complexity. Evaporation of solvents is usually an important approach to induce kinetically stable assemblies of building blocks with a large-scale specific arrangement. During such a deweting process, we tried to monitor the possible interactions between silver nanoparticles and nucleobases at a larger scale by epifluorescence microscopy, thanks to the doping of silver nanoparticles with luminescent silver nanodots. ssDNA oligomer-stabilized silver nanoparticles and adenine self-assemble to form ring-like compartments similar to the size of modern cells. However, the silver ions only dismantle the self-assembly of adenine. The rings are thermodynamically stable as the drying process only enrich the nanoparticles-nucleobase mixture to a concentration that activates the self-assembly. The permeable membrane-like edge of the ring is composed of adenine filaments glued together by silver nanoparticles. Interestingly, chemicals are partially confined and accumulated inside the ring, suggesting that this might be used as a microreactor to speed up chemical reactions during a dewetting process. PMID:26643504

  8. Stability Constants of Mixed Ligand Complexes of Nickel(II) with Adenine and Some Amino Acids

    PubMed Central

    Türkel, Naciye

    2015-01-01

    Nickel is one of the essential trace elements found in biological systems. It is mostly found in nickel-based enzymes as an essential cofactor. It forms coordination complexes with amino acids within enzymes. Nickel is also present in nucleic acids, though its function in DNA or RNA is still not clearly understood. In this study, complex formation tendencies of Ni(II) with adenine and certain L-amino acids such as aspartic acid, glutamic acid, asparagine, leucine, phenylalanine, and tryptophan were investigated in an aqueous medium. Potentiometric equilibrium measurements showed that both binary and ternary complexes of Ni(II) form with adenine and the above-mentioned L-amino acids. Ternary complexes of Ni(II)-adenine-L-amino acids are formed by stepwise mechanisms. Relative stabilities of the ternary complexes are compared with those of the corresponding binary complexes in terms of Δlog10⁡K, log10⁡X, and % RS values. It was shown that the most stable ternary complex is Ni(II):Ade:L-Asn while the weakest one is Ni(II):Ade:L-Phe in aqueous solution used in this research. In addition, results of this research clearly show that various binary and ternary type Ni(II) complexes are formed in different concentrations as a function of pH in aqueous solution. PMID:26843852

  9. Identification and characterization of a novel plastidic adenine nucleotide uniporter from Solanum tuberosum.

    PubMed

    Leroch, Michaela; Kirchberger, Simon; Haferkamp, Ilka; Wahl, Markus; Neuhaus, H Ekkehard; Tjaden, Joachim

    2005-05-01

    Homologs of BT1 (the Brittle1 protein) are found to be phylogenetically related to the mitochondrial carrier family and appear to occur in both mono- and dicotyledonous plants. Whereas BT1 from cereals is probably involved in the transport of ADP-glucose, which is essential for starch metabolism in endosperm plastids, BT1 from a noncereal plant, Solanum tuberosum (StBT1), catalyzes an adenine nucleotide uniport when functionally integrated into the bacterial cytoplasmic membrane. Import studies into intact Escherichia coli cells harboring StBT1 revealed a narrow substrate spectrum with similar affinities for AMP, ADP, and ATP of about 300-400 mum. Transiently expressed StBT1-green fluorescent protein fusion protein in tobacco leaf protoplasts showed a plastidic localization of the StBT1. In vitro synthesized radioactively labeled StBT1 was targeted to the envelope membranes of isolated spinach chloroplasts. Furthermore, we showed by real time reverse transcription-PCR a ubiquitous expression pattern of the StBT1 in autotrophic and heterotrophic potato tissues. We therefore propose that StBT1 is a plastidic adenine nucleotide uniporter used to provide the cytosol and other compartments with adenine nucleotides exclusively synthesized inside plastids. PMID:15737999

  10. Chemical evolution: The mechanism of the formation of adenine under prebiotic conditions

    PubMed Central

    Roy, Debjani; Najafian, Katayoun; von Ragué Schleyer, Paul

    2007-01-01

    Fundamental building blocks of life have been detected extraterrestrially, even in interstellar space, and are known to form nonenzymatically. Thus, the HCN pentamer, adenine (a base present in DNA and RNA), was first isolated in abiogenic experiments from an aqueous solution of ammonia and HCN in 1960. Although many variations of the reaction conditions giving adenine have been reported since then, the mechanistic details remain unexplored. Our predictions are based on extensive computations of sequences of reaction steps along several possible mechanistic routes. H2O- or NH3-catalyzed pathways are more favorable than uncatalyzed neutral or anionic alternatives, and they may well have been the major source of adenine on primitive earth. Our report provides a more detailed understanding of some of the chemical processes involved in chemical evolution, and a partial answer to the fundamental question of molecular biogenesis. Our investigation should trigger similar explorations of the detailed mechanisms of the abiotic formation of the remaining nucleic acid bases and other biologically relevant molecules. PMID:17951429

  11. An experimental and theoretical vibrational study of interaction of adenine and thymine with artificial seawaters: A prebiotic chemistry experiment

    NASA Astrophysics Data System (ADS)

    Anizelli, Pedro R.; Baú, João P. T.; Nabeshima, Henrique S.; da Costa, Marcello F.; de Santana, Henrique; Zaia, Dimas A. M.

    Nucleic acid bases play important roles in living beings. Thus, their interaction with salts the prebiotic Earth could be an important issue for the understanding of origin of life. In this study, the effect of pH and artificial seawaters on the structure of adenine and thymine was studied via parallel determinations using FT-IR, Raman spectroscopy and theoretical calculations. Thymine and adenine lyophilized in solutions at basic and acidic conditions showed characteristic bands of the enol-imino tautomer due to the deprotonation and the hydrochloride form due to protonation, respectively. The interaction of thymine and adenine with different seawaters representative of different geological periods on Earth was also studied. In the case of thymine a strong interaction with Sr2+ promoted changes in the Raman and infrared spectra. For adenine changes in infrared and Raman spectra were observed in the presence of salts from all seawaters tested. The experimental results were compared to theoretical calculations, which showed structural changes due to the presence of ions Na+, Mg2+, Ca2+ and Sr2+ of artificial seawaters. For thymine the bands arising from C4dbnd C5 and C6dbnd O stretching were shifted to lower values, and for adenine, a new band at 1310 cm-1 was observed. The reactivity of adenine and thymine was studied by comparing changes in nucleophilicity and energy of the HOMO orbital.

  12. Ozone therapy ameliorates tubulointerstitial inflammation by regulating TLR4 in adenine-induced CKD rats.

    PubMed

    Chen, Zhiyuan; Liu, Xiuheng; Yu, Gang; Chen, Hui; Wang, Lei; Wang, Zhishun; Qiu, Tao; Weng, Xiaodong

    2016-06-01

    Tubulointerstitium inflammation is a common pathway aggravating chronic kidney disease (CKD) progression and the mechanism is partly associated with excessive activation of toll-like receptor 4 (TLR4) in tubulointerstitium. Ozone therapy is demonstrated to alleviate inflammation in some experiments. The aim of this study is to examine whether ozone therapy could ameliorate chronic tubulointerstitium inflammation by suppressing TLR4 in adenine-induced CKD rats. Sprague-Dawley rats were fed with 0.75% adenine-containing diet to induce CKD and tubulointerstitium inflammation injury. Ozone therapy (1.1 mg/kg) was simultaneously administrated by rectal insufflations (i.r.). After 4 weeks, serum and kidney samples were collected for detection. Renal function and systemic electrolyte were detected. Renal pathological changes were assessed by hematoxylin-eosin (H&E) staining and Masson trichrome (MT) staining. Immunohistochemistry, Western blot and Real-time PCR were applied to evaluate tubulointerstitium inflammation as well as the expression of TLR4 and phosphorylated nuclear factor kappa B P65 (p-NF-κB P65) in rats. The results showed ozone therapy improved serious renal insufficiency, systemic electrolyte disorder and tubulointerstitium morphology damages in adenine-induced CKD rats. In addition, ozone therapy suppressed excessive activation of TLR4 and p-NF-κB P65 in the tubulointerstitium of adenine-induced CKD rats, accompanied by the reduction of inflammation-related cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). The protein expression of TLR4 was positively correlated with the protein expression levels of MCP-1 (r = 0.7863, p < 0.01) and TNF-α (r = 0.7547, p < 0.01) in CKD rats. These findings indicated ozone therapy could attenuate tubulointerstitium inflammation injury in adenine-induced CKD rats and the mechanism might associate with the

  13. Undetectable levels of N6-methyl adenine in mouse DNA: Cloning and analysis of PRED28, a gene coding for a putative mammalian DNA adenine methyltransferase.

    PubMed

    Ratel, David; Ravanat, Jean-Luc; Charles, Marie-Pierre; Platet, Nadine; Breuillaud, Lionel; Lunardi, Joël; Berger, François; Wion, Didier

    2006-05-29

    Three methylated bases, 5-methylcytosine, N4-methylcytosine and N6-methyladenine (m6A), can be found in DNA. However, to date, only 5-methylcytosine has been detected in mammalian genomes. To reinvestigate the presence of m6A in mammalian DNA, we used a highly sensitive method capable of detecting one N6-methyldeoxyadenosine per million nucleosides. Our results suggest that the total mouse genome contains, if any, less than 10(3) m6A. Experiments were next performed on PRED28, a putative mammalian N6-DNA methyltransferase. The murine PRED28 encodes two alternatively spliced RNA. However, although recombinant PRED28 proteins are found in the nucleus, no evidence for an adenine-methyltransferase activity was detected. PMID:16684535

  14. 6MAP, a fluorescent adenine analogue, is a probe of base flipping by DNA photolyase.

    PubMed

    Yang, Kongsheng; Matsika, Spiridoula; Stanley, Robert J

    2007-09-01

    Cyclobutylpyrimidine dimers (CPDs) are formed between adjacent pyrimidines in DNA when it absorbs ultraviolet light. CPDs can be directly repaired by DNA photolyase (PL) in the presence of visible light. How PL recognizes and binds its substrate is still not well understood. Fluorescent nucleic acid base analogues are powerful probes of DNA structure. We have used the fluorescent adenine analogue 6MAP, a pteridone, to probe the local double helical structure of the CPD substrate when bound by photolyase. Duplex melting temperatures were obtained by both UV-vis absorption and fluorescence spectroscopies to ascertain the effect of the probe and the CPD on DNA stability. Steady-state fluorescence measurements of 6MAP-containing single-stranded and doubled-stranded oligos with and without protein show that the local region around the CPD is significantly disrupted. 6MAP shows a different quenching pattern compared to 2-aminopurine, another important adenine analogue, although both probes show that the structure of the complementary strand opposing the 5'-side of the CPD lesion is more destacked than that opposing the 3'-side in substrate/protein complexes. We also show that 6MAP/CPD duplexes are substrates for PL. Vertical excitation energies and transition dipole moment directions for 6MAP were calculated using time-dependent density functional theory. Using these results, the Förster resonance energy transfer efficiency between the individual adenine analogues and the oxidized flavin cofactor was calculated to account for the observed intensity pattern. These calculations suggest that energy transfer is highly efficient for the 6MAP probe and less so for the 2Ap probe. However, no experimental evidence for this process was observed in the steady-state emission spectra. PMID:17696385

  15. The effects of cyclic adenosine 3',5'-monophosphate and other adenine nucleotides on body temperature.

    PubMed Central

    Dascombe, M J; Milton, A S

    1975-01-01

    1. Adenosine 3',5'-monophosphate (cAMP), its dibutyryl derivative (Db-cAMP) and other adenine nucleotides have been micro-injected into the hypothalamic region of the unanaesthetized cat and the effects on body temperature, and on behavioural and autonomic thermoregulatory activities observed. 2. Db-cAMP and cAMP both produced hypothermia when applied to the pre-optic anterior hypothalamus. With Db-cAMP the hypothermia was shown to be dose dependent between 50 and 500 mug (0-096-0-96 mumole). 3. AMP, ADP and ATP also produced hypothermia when injected into the pre-optic anterior hypothalamus. 4. The order of relative potencies of the adenine nucleotides with respect both to the hypothermia produced and to the autonomic thermoregulatory effects observed were similar. Db-cAMP was most potent and cAMP least. 5. Micro-injection into the pre-optic anterior hypothalamus of many substances including saline produced in most cats a non-specific rise in body temperature apparently the result of tissue damage. Intraperitoneal injection of 4-acetamidophenol (paracetamol 50 mg/kg) reduced or abolished this febrile response. 6. The hypothermic effect of the adenine nucleotides has been compared with the effects produced in these same cats by micro-injections of noradrenaline, 5-hydroxytryptamine, a mixture of acetylcholine and physostigmine (1:1), EDTA and excess Ca2+ ions. 7. It is concluded that as Db-cAMP and cAMP both produce hypothermia, it is unlikely that endogenous cAMP in the pre-optic anterior hypothalamus mediates the hyperthermic responses to pyrogens and prostaglandins. PMID:170396

  16. The effects of cyclic adenosine 3',5'-monophosphate and other adenine nucleotides on body temperature.

    PubMed

    Dascombe, M J; Milton, A S

    1975-08-01

    1. Adenosine 3',5'-monophosphate (cAMP), its dibutyryl derivative (Db-cAMP) and other adenine nucleotides have been micro-injected into the hypothalamic region of the unanaesthetized cat and the effects on body temperature, and on behavioural and autonomic thermoregulatory activities observed. 2. Db-cAMP and cAMP both produced hypothermia when applied to the pre-optic anterior hypothalamus. With Db-cAMP the hypothermia was shown to be dose dependent between 50 and 500 mug (0-096-0-96 mumole). 3. AMP, ADP and ATP also produced hypothermia when injected into the pre-optic anterior hypothalamus. 4. The order of relative potencies of the adenine nucleotides with respect both to the hypothermia produced and to the autonomic thermoregulatory effects observed were similar. Db-cAMP was most potent and cAMP least. 5. Micro-injection into the pre-optic anterior hypothalamus of many substances including saline produced in most cats a non-specific rise in body temperature apparently the result of tissue damage. Intraperitoneal injection of 4-acetamidophenol (paracetamol 50 mg/kg) reduced or abolished this febrile response. 6. The hypothermic effect of the adenine nucleotides has been compared with the effects produced in these same cats by micro-injections of noradrenaline, 5-hydroxytryptamine, a mixture of acetylcholine and physostigmine (1:1), EDTA and excess Ca2+ ions. 7. It is concluded that as Db-cAMP and cAMP both produce hypothermia, it is unlikely that endogenous cAMP in the pre-optic anterior hypothalamus mediates the hyperthermic responses to pyrogens and prostaglandins. PMID:170396

  17. Fragmentation of the adenine and guanine molecules induced by electron collisions

    SciTech Connect

    Minaev, B. F. E-mail: boris@theochem.kth.se; Shafranyosh, M. I.; Svida, Yu. Yu; Sukhoviya, M. I.; Shafranyosh, I. I.; Baryshnikov, G. V.; Minaeva, V. A.

    2014-05-07

    Secondary electron emission is the most important stage in the mechanism of radiation damage to DNA biopolymers induced by primary ionizing radiation. These secondary electrons ejected by the primary electron impacts can produce further ionizations, initiating an avalanche effect, leading to genome damage through the energy transfer from the primary objects to sensitive biomolecular targets, such as nitrogenous bases, saccharides, and other DNA and peptide components. In this work, the formation of positive and negative ions of purine bases of nucleic acids (adenine and guanine molecules) under the impact of slow electrons (from 0.1 till 200 eV) is studied by the crossed electron and molecular beams technique. The method used makes it possible to measure the molecular beam intensity and determine the total cross-sections for the formation of positive and negative ions of the studied molecules, their energy dependences, and absolute values. It is found that the maximum cross section for formation of the adenine and guanine positive ions is reached at about 90 eV energy of the electron beam and their absolute values are equal to 2.8 × 10{sup −15} and 3.2 × 10{sup −15} cm{sup 2}, respectively. The total cross section for formation of the negative ions is 6.1 × 10{sup −18} and 7.6 × 10{sup −18} cm{sup 2} at the energy of 1.1 eV for adenine and guanine, respectively. The absolute cross-section values for the molecular ions are measured and the cross-sections of dissociative ionization are determined. Quantum chemical calculations are performed for the studied molecules, ions and fragments for interpretation of the crossed beams experiments.

  18. Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase.

    PubMed

    Miller, Danielle V; Brown, Anne M; Xu, Huimin; Bevan, David R; White, Robert H

    2016-06-01

    Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a kcat /KM of 3.2 × 10(7) and 3.0 × 10(7) s(- 1) M(- 1) , respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site-directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10-fold decrease in kcat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828-840. © 2016 Wiley Periodicals, Inc. PMID:26990095

  19. The effect of pi-stacking, h-bonding, and electrostatic interactions on the ionization energies of nucleic acid bases: adenine-adenine, thymine-thymine and adenine-thymine dimers

    SciTech Connect

    Bravaya, Ksenia B.; Kostko, Oleg; Ahmed, Musahid; Krylov, Anna I.

    2009-09-02

    A combined theoretical and experimental study of the ionized dimers of thymine and adenine, TT, AA, and AT, is presented. Adiabatic and vertical ionization energies(IEs) for monomers and dimers as well as thresholds for the appearance of the protonated species are reported and analyzed. Non-covalent interactions stronglyaffect the observed IEs. The magnitude and the nature of the effect is different for different isomers of the dimers. The computations reveal that for TT, the largestchanges in vertical IEs (0.4 eV) occur in asymmetric h-bonded and symmetric pi- stacked isomers, whereas in the lowest-energy symmetric h-bonded dimer the shiftin IEs is much smaller (0.1 eV). The origin of the shift and the character of the ionized states is different in asymmetric h-bonded and symmetric stacked isomers. Inthe former, the initial hole is localized on one of the fragments, and the shift is due to the electrostatic stabilization of the positive charge of the ionized fragment by thedipole moment of the neutral fragment. In the latter, the hole is delocalized, and the change in IE is proportional to the overlap of the fragments' MOs. The shifts in AAare much smaller due to a less effcient overlap and a smaller dipole moment. The ionization of the h-bonded dimers results in barrierless (or nearly barrierless) protontransfer, whereas the pi-stacked dimers relax to structures with the hole stabilized by the delocalization or electrostatic interactions.

  20. Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

    PubMed

    Cheng, Yi-Fang; Young, Guang-Huar; Lin, Jiun-Tsai; Jang, Hyun-Hwa; Chen, Chin-Chen; Nong, Jing-Yi; Chen, Po-Ku; Kuo, Cheng-Yi; Kao, Shao-Hsuan; Liang, Yao-Jen; Chen, Han-Min

    2015-01-01

    The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK. PMID:26544976

  1. Application of intron 9 and intron 25 dinucleotide repeats of the factor VIII gene for carrier diagnosis in haemophilia A.

    PubMed

    Venceslá, A; Baena, M; Fares Taie, L; Cornet, M; Baiget, M; Tizzano, E F

    2008-05-01

    We describe the usefulness of two dinucleotide repeats located in intron 9 and in intron 25 of the factor VIII gene for carrier diagnosis of haemophilia A. We analyzed 100 unrelated Spanish women and 34 women from haemophilia A (HA) families in whom known intragenic markers were unhelpful in determining their carrier status. The heterozygosity rate of intron 9 and intron 25 markers in the 100 control women was lower (0.28 and 0.38, respectively) than the values obtained with common markers routinely used in our laboratory. However, the application of intron 9 and intron 25 markers was effective in identifying the at-risk X chromosome in 11 of 34 (32%) of the uninformative women from HA families. The combined use of these repeats with current markers may facilitate the identification of the X chromosome in HA families for application in carrier, prenatal and pre-implantation diagnoses. PMID:18384354

  2. Synthesis, spectroscopic, structural and thermal characterizations of vanadyl(IV) adenine complex prospective as antidiabetic drug agent

    NASA Astrophysics Data System (ADS)

    El-Megharbel, Samy M.; Hamza, Reham Z.; Refat, Moamen S.

    2015-01-01

    The vanadyl(IV) adenine complex; [VO(Adn)2]ṡSO4; was synthesized and characterized. The molar conductivity of this complex was measured in DMSO solution that showed an electrolyte nature. Spectroscopic investigation of the green solid complex studied here indicate that the adenine acts as a bidentate ligand, coordinated to vanadyl(IV) ions through the nitrogen atoms N7 and nitrogen atom of amino group. Thus, from the results presented the vanadyl(IV) complex has square pyramid geometry. Further characterizations using thermal analyses and scanning electron techniques was useful. The aim of this paper was to introduce a new drug model for the diabetic complications by synthesized a novel mononuclear vanadyl(IV) adenine complex to mimic insulin action and reducing blood sugar level. The antidiabetic ability of this complex was investigated in STZ-induced diabetic mice. The results suggested that VO(IV)/adenine complex has antidiabetic activity, it improved the lipid profile, it improved liver and kidney functions, also it ameliorated insulin hormone and blood glucose levels. The vanadyl(IV) complex possesses an antioxidant activity and this was clear through studying SOD, CAT, MDA, GSH and methionine synthase. The current results support the therapeutic potentiality of vanadyl(IV)/adenine complex for the management and treatment of diabetes.

  3. Simultaneous Determination of Adenine and Guanine Using Cadmium Selenide Quantum Dots-Graphene Oxide Nanocomposite Modified Electrode.

    PubMed

    Kalaivani, Arumugam; Narayanan, Sangilimuthu Sriman

    2015-06-01

    A novel electrochemical sensor was fabricated by immobilizing Cadmium Selenide Quantum Dots (CdSe QDs)-Graphene Oxide (GO) nanocomposite on a paraffin wax impregnated graphite electrode (PIGE) and was used for the simultaneous determination of adenine and guanine. The CdSe QDs-GO nanocomposite was prepared by ultrasonication and was characterized with spectroscopic and microscopic techniques. The nanocomposite modified electrode was characterized by cyclic voltammetry (CV). The modified electrode showed excellent electrocatalytic activity towards the oxidative determination of adenine and guanine with a good peak separation of 0.31 V. This may be due to the high surface area and fast electron transfer kinetics of the nanocomposite. The modified electrode exhibited wide linear ranges from 0.167 μM to 245 μM for Guanine and 0.083 μM to 291 μM for Adenine with detection limits of 0.055 μM Guanine and 0.028 μM of Adenine (S/N = 3) respectively. Further, the modified electrode was used for the quantitative determination of adenine and guanine in herring sperm DNA with satisfactory results. The modified electrode showed acceptable selectivity, reproducibility and stability under optimal conditions. PMID:26369099

  4. Synthesis, spectroscopic, structural and thermal characterizations of vanadyl(IV) adenine complex prospective as antidiabetic drug agent.

    PubMed

    El-Megharbel, Samy M; Hamza, Reham Z; Refat, Moamen S

    2015-01-25

    The vanadyl(IV) adenine complex; [VO(Adn)2]⋅SO4; was synthesized and characterized. The molar conductivity of this complex was measured in DMSO solution that showed an electrolyte nature. Spectroscopic investigation of the green solid complex studied here indicate that the adenine acts as a bidentate ligand, coordinated to vanadyl(IV) ions through the nitrogen atoms N7 and nitrogen atom of amino group. Thus, from the results presented the vanadyl(IV) complex has square pyramid geometry. Further characterizations using thermal analyses and scanning electron techniques was useful. The aim of this paper was to introduce a new drug model for the diabetic complications by synthesized a novel mononuclear vanadyl(IV) adenine complex to mimic insulin action and reducing blood sugar level. The antidiabetic ability of this complex was investigated in STZ-induced diabetic mice. The results suggested that VO(IV)/adenine complex has antidiabetic activity, it improved the lipid profile, it improved liver and kidney functions, also it ameliorated insulin hormone and blood glucose levels. The vanadyl(IV) complex possesses an antioxidant activity and this was clear through studying SOD, CAT, MDA, GSH and methionine synthase. The current results support the therapeutic potentiality of vanadyl(IV)/adenine complex for the management and treatment of diabetes. PMID:25150436

  5. Metabolic fate of 14C-labelled nicotinamide and adenine in germinating propagules of the mangrove Bruguiera gymnorrhiza.

    PubMed

    Yin, Yuling; Watanabe, Shin; Ashihara, Hiroshi

    2012-01-01

    We studied the metabolic fate of [carbonyl-14C]nicotinamide and [8-(14)C]adenine in segments taken from young and developing leaves, stem, hypocotyls, and roots of a shoot-root type emerging propagule of the mangrove plant Bruguiera gymnorrhiza. Thin-layer chromatography was used together with a bioimaging analyser system. During 4 h of incubation, incorporation of radioactivity from [carbonyl-14C]nicotinamide into NAD and trigonelline was found in all parts of the propagules; the highest incorporation rates into NAD and trigonelline were found in newly emerged stem and young leaves, respectively. Radioactivity from [8-(14)C]adenine was distributed mainly in the salvage products (adenine nucleotides and RNA), and incorporation was less in catabolites (allantoin, allantoic acid, and CO2). Adenine salvage activity was higher in young leaves and stem than in hypocotyls and roots. Over a short time, the effect of 500 mM NaCl on nicotinamide and adenine metabolism indicated that NaCl inhibits both salvage and degradation activities in roots. PMID:22888538

  6. Comparative Study between Transcriptionally- and Translationally-Acting Adenine Riboswitches Reveals Key Differences in Riboswitch Regulatory Mechanisms

    PubMed Central

    Blouin, Simon; Heppell, Benoit; Bastet, Laurène; St-Pierre, Patrick; Massé, Eric; Lafontaine, Daniel A.

    2011-01-01

    Many bacterial mRNAs are regulated at the transcriptional or translational level by ligand-binding elements called riboswitches. Although they both bind adenine, the adenine riboswitches of Bacillus subtilis and Vibrio vulnificus differ by controlling transcription and translation, respectively. Here, we demonstrate that, beyond the obvious difference in transcriptional and translational modulation, both adenine riboswitches exhibit different ligand binding properties and appear to operate under different regulation regimes (kinetic versus thermodynamic). While the B. subtilis pbuE riboswitch fully depends on co-transcriptional binding of adenine to function, the V. vulnificus add riboswitch can bind to adenine after transcription is completed and still perform translation regulation. Further investigation demonstrates that the rate of transcription is critical for the B. subtilis pbuE riboswitch to perform efficiently, which is in agreement with a co-transcriptional regulation. Our results suggest that the nature of gene regulation control, that is transcription or translation, may have a high importance in riboswitch regulatory mechanisms. PMID:21283784

  7. Time evolution of the Infrared Laser Induced Breakdown Spectroscopy of DNA bases Guanine and Adenine

    NASA Astrophysics Data System (ADS)

    Diaz, L.; Rubio, L.; Camacho, J. J.

    2013-03-01

    Laser-Induced Breakdown Spectroscopy (LIBS) of DNA bases Guanine and Adenine was studied using a high-power CO2 pulsed laser ( λ=10.591 μm, τ FWHM=64 ns and fluences ranging from 25 to 70 J/cm2). The strong emission of the adenine and guanine plasma, collected using a high-resolution spectrometer, at medium-vacuum conditions (4 Pa) and at 1 mm from the target, exhibits excited molecular bands of CN (B2 Σ +-X2 Σ +) and excited neutral H and ionized N+ and C+. The medium-weak emission is due to excited species C2+, C3+, N, O, O+, O2+ and molecular band systems of C2(d3\\varPig{-}a3\\varPiu; D1\\varSigmau+{-}X1\\varSigmag+), OH(A2 Σ +-X2 Π), NH(A3 Π-X3 Σ -), CH(A2 Π-X2 Π), N2+(B2\\varSigmau+{-} X2\\varSigmag+) and N2(C3 Π u-B3 Π g). We focus our attention on the temporal evolution of different atomic/ionic and molecular species. The velocity distributions for various (different) species were obtained from time-of-flight (TOF) measurements. Intensities of some lines from C+ were used for determining electron temperature and their Stark-broadened profiles were employed to estimate the temporal evolution of electron density.

  8. Quantitative investigation of the poly-adenine DNA dissociation from the surface of gold nanoparticles

    NASA Astrophysics Data System (ADS)

    Lu, Weiwen; Wang, Lihua; Li, Jiang; Zhao, Yun; Zhou, Ziang; Shi, Jiye; Zuo, Xiaolei; Pan, Dun

    2015-05-01

    In recent years, poly adenine (polyA) DNA functionalized gold nanoparticles (AuNPs) free of modifications was fabricated with high density of DNA attachment and high hybridization ability similar to those of its thiolated counterpart. This nanoconjugate utilized poly adenine as an anchoring block for binding with the AuNPs surface thereby facilitated the appended recognition block a better upright conformation for hybridization, demonstrating its great potential to be a tunable plasmonic biosensor. It’s one of the key points for any of the practical applications to maintaining stable conjugation between DNA oligonucleotides and gold nanoparticles under various experimental treatments. Thus, in this research, we designed a simple but sensitive fluorescence turn-on strategy to systematically investigate and quantified the dissociation of polyA DNA on gold nanoparticles in diverse experimental conditions. DNA desorbed spontaneously as a function of elevated temperature, ion strength, buffer pH, organic solvents and keeping time. What’s more, evaluating this conjugate stability as affected by the length of its polyA anchor was another crucial aspect in our study. With the improved understanding from these results, we were able to control some of our experimental conditions to maintain a good stability of this kind of polyA DNA-AuNPs nanoconjugates.

  9. Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain

    PubMed Central

    Luo, Shukun; Tong, Liang

    2014-01-01

    Methylation of the N6 position of selected internal adenines (m6A) in mRNAs and noncoding RNAs is widespread in eukaryotes, and the YTH domain in a collection of proteins recognizes this modification. We report the crystal structure of the splicing factor YT521-B homology (YTH) domain of Zygosaccharomyces rouxii MRB1 in complex with a heptaribonucleotide with an m6A residue in the center. The m6A modification is recognized by an aromatic cage, being sandwiched between a Trp and Tyr residue and with the methyl group pointed toward another Trp residue. Mutations of YTH domain residues in the RNA binding site can abolish the formation of the complex, confirming the structural observations. These residues are conserved in the human YTH proteins that also bind m6A RNA, suggesting a conserved mode of recognition. Overall, our structural and biochemical studies have defined the molecular basis for how the YTH domain functions as a reader of methylated adenines. PMID:25201973

  10. Molecular basis for the recognition of methylated adenines in RNA by the eukaryotic YTH domain.

    PubMed

    Luo, Shukun; Tong, Liang

    2014-09-23

    Methylation of the N6 position of selected internal adenines (m(6)A) in mRNAs and noncoding RNAs is widespread in eukaryotes, and the YTH domain in a collection of proteins recognizes this modification. We report the crystal structure of the splicing factor YT521-B homology (YTH) domain of Zygosaccharomyces rouxii MRB1 in complex with a heptaribonucleotide with an m(6)A residue in the center. The m(6)A modification is recognized by an aromatic cage, being sandwiched between a Trp and Tyr residue and with the methyl group pointed toward another Trp residue. Mutations of YTH domain residues in the RNA binding site can abolish the formation of the complex, confirming the structural observations. These residues are conserved in the human YTH proteins that also bind m(6)A RNA, suggesting a conserved mode of recognition. Overall, our structural and biochemical studies have defined the molecular basis for how the YTH domain functions as a reader of methylated adenines. PMID:25201973

  11. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

    PubMed Central

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

    2015-01-01

    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs. PMID:26227585

  12. Effect of Electronic Excitation on Hydrogen Atom Transfer (Tautomerization) Reactions for the DNA Base Adenine

    NASA Technical Reports Server (NTRS)

    Chaban, Galina M.; Salter, Latasha M.; Kwak, Dochan (Technical Monitor)

    2002-01-01

    Geometrical structures and energetic properties for four different tautomers of adenine are calculated in this study, using multi-configurational wave functions. Both the ground and the lowest single excited state potential energy surface are studied. The energetic order of the tautomers on the ground state potential surface is 9H less than 7H less than 3H less than 1H, while on the excited state surface this order is found to be different: 3H less than 1H less than 9H less than 7H. Minimum energy reaction paths are obtained for hydrogen atom transfer (9 yields 3 tautomerization) reactions in the ground and the lowest excited electronic state. It is found that the barrier heights and the shapes of the reaction paths are different for the ground and the excited electronic state, suggesting that the probability of such tautomerization reaction is higher on the excited state potential energy surface. The barrier for this reaction in the excited state may become very low in the presence of water or other polar solvent molecules, and therefore such tautomerization reaction may play an important role in the solution phase photochemistry of adenine.

  13. Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design

    NASA Astrophysics Data System (ADS)

    Foller Larsen, Anders; Dumat, Blaise; Wranne, Moa S.; Lawson, Christopher P.; Preus, Søren; Bood, Mattias; Gradén, Henrik; Marcus Wilhelmsson, L.; Grøtli, Morten

    2015-07-01

    Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (ɛΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

  14. 3D Magnetically Ordered Open Supramolecular Architectures Based on Ferrimagnetic Cu/Adenine/Hydroxide Heptameric Wheels.

    PubMed

    Pérez-Aguirre, Rubén; Beobide, Garikoitz; Castillo, Oscar; de Pedro, Imanol; Luque, Antonio; Pérez-Yáñez, Sonia; Rodríguez Fernández, Jesús; Román, Pascual

    2016-08-01

    The present work provides two new examples of supramolecular metal-organic frameworks consisting of three-dimensional extended noncovalent assemblies of wheel-shaped heptanuclear [Cu7(μ-H2O)6(μ3-OH)6(μ-adeninato-κN3:κN9)6](2+) entities. The heptanuclear entity consists of a central [Cu(OH)6](4-) core connected to six additional copper(II) metal centers in a radial and planar arrangement through the hydroxides. It generates a wheel-shaped entity in which water molecules and μ-κN3:κN9 adeninato ligands bridge the peripheral copper atoms. The magnetic characterization indicates the central copper(II) center is anti-ferromagnetically coupled to external copper(II) centers, which are ferromagnetically coupled among them leading to an S = 5/2 ground state. The packing of these entities is sustained by π-π stacking interactions between the adenine nucleobases and by hydrogen bonds established among the hydroxide ligands, sulfate anions, and adenine nucleobases. The sum of both types of supramolecular interactions creates a rigid synthon that in combination with the rigidity of the heptameric entity generates an open supramolecular structure (40-50% of available space) in which additional sulfate and triethylammonium ions are located altogether with solvent molecules. These compounds represent an interesting example of materials combining both porosity and magnetic relevant features. PMID:27409976

  15. Theoretical study on the static and dynamic first-order hyperpolarisabilities of adenine tautomers

    NASA Astrophysics Data System (ADS)

    Alparone, Andrea

    2014-07-01

    Static and dynamic electronic and vibrational first-order hyperpolarisabilities (β) of the lowest energy neutral adenine tautomers (amine forms A7 and A9) were obtained in gaseous and aqueous phases by using Hartree-Fock, Møller-Plesset second-order and fourth-order perturbation theory (MP2 and MP4-SDQ) and conventional and long-range corrected density functional theory methods with the Dunning's correlation-consistent cc-pVDZ, aug-cc-pVDZ, aug-cc-pVTZ and d-aug-cc-pVDZ basis sets. Frequency-dependent properties were calculated at the characteristic wavelength of the Nd:YAG laser (1064 nm) for the second harmonic generation and electro-optical Pockels effect nonlinear optical processes. Solvent effects were introduced under the polarised continuum model approximation. The electronic βe values of the investigated isomers are noticeably affected by the theoretical level, basis set and solvation. In vacuum, the static and dynamic βe values of A9 are greater than the corresponding data of A7, whereas the contribution of the solvent significantly enhances the hyperpolarisabilities of the A7 tautomer, resulting in βe(A9)/βe(A7) ratios between 0.5 and 0.6. The vibrational hyperpolarisabilities of the adenine tautomers are quite close to each other.

  16. Probing ultrafast dynamics in adenine with mid-UV four-wave mixing spectroscopies.

    PubMed

    West, Brantley A; Womick, Jordan M; Moran, Andrew M

    2011-08-11

    Heterodyne-detected transient grating (TG) and two-dimensional photon echo (2DPE) spectroscopies are extended to the mid-UV spectral range in this investigation of photoinduced relaxation processes of adenine in aqueous solution. These experiments are the first to combine a new method for generating 25 fs laser pulses (at 263 nm) with the passive phase stability afforded by diffractive optics-based interferometry. We establish a set of conditions (e.g., laser power density, solute concentration) appropriate for the study of dynamics involving the neutral solute. Undesired solute photoionization is shown to take hold at higher peak powers of the laser pulses. Signatures of internal conversion and vibrational cooling dynamics are examined using TG measurements with signal-to-noise ratios as high as 350 at short delay times. In addition, 2DPE line shapes reveal correlations between excitation and emission frequencies in adenine, which reflect electronic and nuclear relaxation processes associated with particular tautomers. Overall, this study demonstrates the feasibility of techniques that will hold many advantages for the study of biomolecules whose lowest-energy electronic resonances are found in the mid-UV (e.g., DNA bases, amino acids). PMID:21756005

  17. Severity of cardiomyopathy associated with adenine nucleotide translocator-1 deficiency correlates with mtDNA haplogroup.

    PubMed

    Strauss, Kevin A; DuBiner, Lauren; Simon, Mariella; Zaragoza, Michael; Sengupta, Partho P; Li, Peng; Narula, Navneet; Dreike, Sandra; Platt, Julia; Procaccio, Vincent; Ortiz-González, Xilma R; Puffenberger, Erik G; Kelley, Richard I; Morton, D Holmes; Narula, Jagat; Wallace, Douglas C

    2013-02-26

    Mutations of both nuclear and mitochondrial DNA (mtDNA)-encoded mitochondrial proteins can cause cardiomyopathy associated with mitochondrial dysfunction. Hence, the cardiac phenotype of nuclear DNA mitochondrial mutations might be modulated by mtDNA variation. We studied a 13-generation Mennonite pedigree with autosomal recessive myopathy and cardiomyopathy due to an SLC25A4 frameshift null mutation (c.523delC, p.Q175RfsX38), which codes for the heart-muscle isoform of the adenine nucleotide translocator-1. Ten homozygous null (adenine nucleotide translocator-1(-/-)) patients monitored over a median of 6 years had a phenotype of progressive myocardial thickening, hyperalaninemia, lactic acidosis, exercise intolerance, and persistent adrenergic activation. Electrocardiography and echocardiography with velocity vector imaging revealed abnormal contractile mechanics, myocardial repolarization abnormalities, and impaired left ventricular relaxation. End-stage heart disease was characterized by massive, symmetric, concentric cardiac hypertrophy; widespread cardiomyocyte degeneration; overabundant and structurally abnormal mitochondria; extensive subendocardial interstitial fibrosis; and marked hypertrophy of arteriolar smooth muscle. Substantial variability in the progression and severity of heart disease segregated with maternal lineage, and sequencing of mtDNA from five maternal lineages revealed two major European haplogroups, U and H. Patients with the haplogroup U mtDNAs had more rapid and severe cardiomyopathy than those with haplogroup H. PMID:23401503

  18. Ultraviolet photolysis of adenine: Dissociation via the {sup 1}{pi}{sigma}{sup *} state

    SciTech Connect

    Nix, Michael G. D.; Devine, Adam L.; Cronin, Brid; Ashfold, Michael N. R.

    2007-03-28

    High resolution total kinetic energy release (TKER) spectra of the H atom fragments resulting from photodissociation of jet-cooled adenine molecules at 17 wavelengths in the range 280>{lambda}{sub phot}>214 nm are reported. TKER spectra obtained at {lambda}{sub phot}>233 nm display broad, isotropic profiles that peak at low TKER ({approx}1800 cm{sup -1}) and are largely insensitive to the choice of excitation wavelength. The bulk of these products is attributed to unintended multiphoton dissociation processes. TKER spectra recorded at {lambda}{sub phot}{<=}233 nm display additional fast structure, which is attributed to N{sub 9}-H bond fission on the {sup 1}{pi}{sigma}{sup *} potential energy surface (PES). Analysis of the kinetic energies and recoil anisotropies of the H atoms responsible for the fast structure suggests excitation to two {sup 1}{pi}{pi}{sup *} excited states (the {sup 1}L{sub a} and {sup 1}B{sub b} states) at {lambda}{sub phot}{approx}230 nm, both of which dissociate to yield H atoms together with ground state adeninyl fragments by radiationless transfer through conical intersections with the {sup 1}{pi}{sigma}{sup *} PES. Parallels with the photochemistry exhibited by other, smaller heteroaromatics (pyrrole, imidazole, phenol, etc.) are highlighted, as are inconsistencies between the present conclusions and those reached in two other recent studies of excited state adenine molecules.

  19. A role for adenine nucleotides in the sensing mechanism to purine starvation in Leishmania donovani.

    PubMed

    Martin, Jessica L; Yates, Phillip A; Boitz, Jan M; Koop, Dennis R; Fulwiler, Audrey L; Cassera, Maria Belen; Ullman, Buddy; Carter, Nicola S

    2016-07-01

    Purine salvage by Leishmania is an obligatory nutritional process that impacts both cell viability and growth. Previously, we have demonstrated that the removal of purines in culture provokes significant metabolic changes that enable Leishmania to survive prolonged periods of purine starvation. In order to understand how Leishmania sense and respond to changes in their purine environment, we have exploited several purine pathway mutants, some in which adenine and guanine nucleotide metabolism is uncoupled. While wild type parasites grow in any one of a variety of naturally occurring purines, the proliferation of these purine pathway mutants requires specific types or combinations of exogenous purines. By culturing purine pathway mutants in high levels of extracellular purines that are either permissive or non-permissive for growth and monitoring for previously defined markers of the adaptive response to purine starvation, we determined that adaptation arises from a surveillance of intracellular purine nucleotide pools rather than from a direct sensing of the extracellular purine content of the environment. Specifically, our data suggest that perturbation of intracellular adenine-containing nucleotide pools provides a crucial signal for inducing the metabolic changes necessary for the long-term survival of Leishmania in a purine-scarce environment. PMID:27062185

  20. Bacteriophage adenine methyltransferase: a life cycle regulator? Modelled using Vibrio harveyi myovirus like.

    PubMed

    Bochow, S; Elliman, J; Owens, L

    2012-11-01

    The adenine methyltransferase (DAM) gene methylates GATC sequences that have been demonstrated in various bacteria to be a powerful gene regulator functioning as an epigenetic switch, particularly with virulence gene regulation. However, overproduction of DAM can lead to mutations, giving rise to variability that may be important for adaptation to environmental change. While most bacterial hosts carry a DAM gene, not all bacteriophage carry this gene. Currently, there is no literature regarding the role DAM plays in life cycle regulation of bacteriophage. Vibrio campbellii strain 642 carries the bacteriophage Vibrio harveyi myovirus like (VHML) that has been proven to increase virulence. The complete genome sequence of VHML bacteriophage revealed a putative adenine methyltransferase gene. Using VHML, a new model of phage life cycle regulation, where DAM plays a central role between the lysogenic and lytic states, will be hypothesized. In short, DAM methylates the rha antirepressor gene and once methylation is removed, homologous CI repressor protein becomes repressed and non-functional leading to the switching to the lytic cycle. Greater understanding of life cycle regulation at the genetic level can, in the future, lead to the genesis of chimeric bacteriophage with greater control over their life cycle for their safe use as probiotics within the aquaculture industry. PMID:22681538

  1. Microwave-assisted stereospecific synthesis of novel tetrahydropyran adenine isonucleosides and crystal structures determination

    NASA Astrophysics Data System (ADS)

    Silva, Fábio P. L.; Cirqueira, Marilia L.; Martins, Felipe T.; Vasconcellos, Mário L. A. A.

    2013-11-01

    We describe in this article stereospecific syntheses for new isonucleosides analogs of adenine 5-7 from tosyl derivatives 2-4 accessing by microwave irradiations (50-80%). The adenine reacts entirely at the N(9) position. Compounds 2-4 were prepared in two steps from the corresponding alcohols 1, 8 and 9 (81-92%). These tetrahydropyrans alcohols 1, 8 and 9 are achiral (Meso compounds) and were prepared in two steps with complete control of 2,4,6-cis relative configuration by Prins cyclization reaction (60-63%) preceded by the Barbier reaction between allyl bromide with benzaldehyde, 4-fluorobenzaldehyde and 2-naphthaldehyde respectively under Lewis acid conditions (96-98%). The configurations and preferential conformations of 5-7 were determined by crystal structure of 6. These novel isonucleosides 5-7 present in silico potentiality to act as GPCR ligand, kinase inhibitor and enzyme inhibitor, evaluated by Molinspiration program, consistent with the expected antiviral and anticancer bioactivities.

  2. Valence anions in complexes of adenine and 9-methyladenine with formic acid: stabilization by intermolecular proton transfer.

    PubMed

    Mazurkiewicz, Kamil; Harańczyk, Maciej; Gutowski, Maciej; Rak, Janusz; Radisic, Dunja; Eustis, Soren N; Wang, Di; Bowen, Kit H

    2007-02-01

    Photoelectron spectra of adenine-formic acid (AFA(-)) and 9-methyladenine-formic acid (MAFA(-)) anionic complexes have been recorded with 2.540 eV photons. These spectra reveal broad features with maxima at 1.5-1.4 eV that indicate formation of stable valence anions in the gas phase. The neutral and anionic complexes of adenine/9-methyladenine and formic acid were also studied computationally at the B3LYP, second-order Møller-Plesset, and coupled-cluster levels of theory with the 6-31++G** and aug-cc-pVDZ basis sets. The neutral complexes form cyclic hydrogen bonds, and the most stable dimers are bound by 17.7 and 16.0 kcal/mol for AFA and MAFA, respectively. The theoretical results indicate that the excess electron in both AFA(-) and MAFA(-) occupies a pi* orbital localized on adenine/9-methyladenine, and the adiabatic stability of the most stable anions amounts to 0.67 and 0.54 eV for AFA(-) and MAFA(-), respectively. The attachment of the excess electron to the complexes induces a barrier-free proton transfer (BFPT) from the carboxylic group of formic acid to a N atom of adenine or 9-methyladenine. As a result, the most stable structures of the anionic complexes can be characterized as neutral radicals of hydrogenated adenine (9-methyladenine) solvated by a deprotonated formic acid. The BFPT to the N atoms of adenine may be biologically relevant because some of these sites are not involved in the Watson-Crick pairing scheme and are easily accessible in the cellular environment. We suggest that valence anions of purines might be as important as those of pyrimidines in the process of DNA damage by low-energy electrons. PMID:17263404

  3. Development of a new model for the induction of chronic kidney disease via intraperitoneal adenine administration, and the effect of treatment with gum acacia thereon.

    PubMed

    Al Za'abi, Mohammed; Al Busaidi, Mahfouda; Yasin, Javid; Schupp, Nicole; Nemmar, Abderrahim; Ali, Badreldin H

    2015-01-01

    Oral adenine (0.75% w/w in feed), is an established model for human chronic kidney disease (CKD). Gum acacia (GA) has been shown to be a nephroprotective agent in this model. Here we aimed at developing a new adenine-induced CKD model in rats via a systemic route (intraperitoneal, i.p.) and to test it with GA to obviate the possibility of a physical interaction between GA and adenine in the gut. Adenine was injected i.p. (50 or 100 mg/Kg for four weeks), and GA was given concomitantly in drinking water at a concentration of 15%, w/v. Several plasma and urinary biomarkers of oxidative stress were measured and the renal damage was assessed histopathologically. Adenine, at the two given i.p. doses, significantly reduced body weight, and increased relative kidney weight, water intake and urine output. It dose-dependently increased plasma and urinary inflammatory and oxidative stress biomarkers, and caused morphological and histological damage resembling that which has been reported with oral adenine. Concomitant treatment with GA significantly mitigated almost all the above measured indices. Administration of adenine i.p. induced CKD signs very similar to those induced by oral adenine. Therefore, this new model is quicker, more practical and accurate than the original (oral) model. GA ameliorates the CKD effects caused by adenine given i.p. suggesting that the antioxidant and anti-inflammatory properties possessed by oral GA are the main mechanism for its salutary action in adenine-induced CKD, an action that is independent of its possible interaction with adenine in the gut. PMID:25755826

  4. Development of a new model for the induction of chronic kidney disease via intraperitoneal adenine administration, and the effect of treatment with gum acacia thereon

    PubMed Central

    Al Za’abi, Mohammed; Al Busaidi, Mahfouda; Yasin, Javid; Schupp, Nicole; Nemmar, Abderrahim; Ali, Badreldin H

    2015-01-01

    Oral adenine (0.75% w/w in feed), is an established model for human chronic kidney disease (CKD). Gum acacia (GA) has been shown to be a nephroprotective agent in this model. Here we aimed at developing a new adenine-induced CKD model in rats via a systemic route (intraperitoneal, i.p.) and to test it with GA to obviate the possibility of a physical interaction between GA and adenine in the gut. Adenine was injected i.p. (50 or 100 mg/Kg for four weeks), and GA was given concomitantly in drinking water at a concentration of 15%, w/v. Several plasma and urinary biomarkers of oxidative stress were measured and the renal damage was assessed histopathologically. Adenine, at the two given i.p. doses, significantly reduced body weight, and increased relative kidney weight, water intake and urine output. It dose-dependently increased plasma and urinary inflammatory and oxidative stress biomarkers, and caused morphological and histological damage resembling that which has been reported with oral adenine. Concomitant treatment with GA significantly mitigated almost all the above measured indices. Administration of adenine i.p. induced CKD signs very similar to those induced by oral adenine. Therefore, this new model is quicker, more practical and accurate than the original (oral) model. GA ameliorates the CKD effects caused by adenine given i.p. suggesting that the antioxidant and anti-inflammatory properties possessed by oral GA are the main mechanism for its salutary action in adenine-induced CKD, an action that is independent of its possible interaction with adenine in the gut. PMID:25755826

  5. Comparative structural analysis of eubacterial 5S rRNA by oxidation of adenines in the N-1 position.

    PubMed Central

    Pieler, T; Schreiber, A; Erdmann, V A

    1984-01-01

    Adenines in free 5S rRNA from Escherichia coli, Bacillus stearothermophilus and Thermus thermophilus have been oxidized at their N-1 position using monoperphthalic acid. The determination of the number of adenine 1-N-oxides was on the basis of UV spectroscopic data of the intact molecule. Identification of the most readily accessible nucleotides by sequencing gel analysis reveals that they are located in conserved positions within loops, exposed hairpin loops and single-base bulge loops. Implications for the structure and function of 5S rRNA will be discussed on the basis of this comparative analysis. Images PMID:6201825

  6. Modeling the high-energy electronic state manifold of adenine: Calibration for nonlinear electronic spectroscopy

    SciTech Connect

    Nenov, Artur Giussani, Angelo; Segarra-Martí, Javier; Jaiswal, Vishal K.; Rivalta, Ivan; Cerullo, Giulio; Mukamel, Shaul; Garavelli, Marco E-mail: marco.garavelli@ens-lyon.fr

    2015-06-07

    Pump-probe electronic spectroscopy using femtosecond laser pulses has evolved into a standard tool for tracking ultrafast excited state dynamics. Its two-dimensional (2D) counterpart is becoming an increasingly available and promising technique for resolving many of the limitations of pump-probe caused by spectral congestion. The ability to simulate pump-probe and 2D spectra from ab initio computations would allow one to link mechanistic observables like molecular motions and the making/breaking of chemical bonds to experimental observables like excited state lifetimes and quantum yields. From a theoretical standpoint, the characterization of the electronic transitions in the visible (Vis)/ultraviolet (UV), which are excited via the interaction of a molecular system with the incoming pump/probe pulses, translates into the determination of a computationally challenging number of excited states (going over 100) even for small/medium sized systems. A protocol is therefore required to evaluate the fluctuations of spectral properties like transition energies and dipole moments as a function of the computational parameters and to estimate the effect of these fluctuations on the transient spectral appearance. In the present contribution such a protocol is presented within the framework of complete and restricted active space self-consistent field theory and its second-order perturbation theory extensions. The electronic excited states of adenine have been carefully characterized through a previously presented computational recipe [Nenov et al., Comput. Theor. Chem. 1040–1041, 295-303 (2014)]. A wise reduction of the level of theory has then been performed in order to obtain a computationally less demanding approach that is still able to reproduce the characteristic features of the reference data. Foreseeing the potentiality of 2D electronic spectroscopy to track polynucleotide ground and excited state dynamics, and in particular its expected ability to provide

  7. Modeling the high-energy electronic state manifold of adenine: Calibration for nonlinear electronic spectroscopy.

    PubMed

    Nenov, Artur; Giussani, Angelo; Segarra-Martí, Javier; Jaiswal, Vishal K; Rivalta, Ivan; Cerullo, Giulio; Mukamel, Shaul; Garavelli, Marco

    2015-06-01

    Pump-probe electronic spectroscopy using femtosecond laser pulses has evolved into a standard tool for tracking ultrafast excited state dynamics. Its two-dimensional (2D) counterpart is becoming an increasingly available and promising technique for resolving many of the limitations of pump-probe caused by spectral congestion. The ability to simulate pump-probe and 2D spectra from ab initio computations would allow one to link mechanistic observables like molecular motions and the making/breaking of chemical bonds to experimental observables like excited state lifetimes and quantum yields. From a theoretical standpoint, the characterization of the electronic transitions in the visible (Vis)/ultraviolet (UV), which are excited via the interaction of a molecular system with the incoming pump/probe pulses, translates into the determination of a computationally challenging number of excited states (going over 100) even for small/medium sized systems. A protocol is therefore required to evaluate the fluctuations of spectral properties like transition energies and dipole moments as a function of the computational parameters and to estimate the effect of these fluctuations on the transient spectral appearance. In the present contribution such a protocol is presented within the framework of complete and restricted active space self-consistent field theory and its second-order perturbation theory extensions. The electronic excited states of adenine have been carefully characterized through a previously presented computational recipe [Nenov et al., Comput. Theor. Chem. 1040-1041, 295-303 (2014)]. A wise reduction of the level of theory has then been performed in order to obtain a computationally less demanding approach that is still able to reproduce the characteristic features of the reference data. Foreseeing the potentiality of 2D electronic spectroscopy to track polynucleotide ground and excited state dynamics, and in particular its expected ability to provide

  8. Modeling the high-energy electronic state manifold of adenine: Calibration for nonlinear electronic spectroscopy

    NASA Astrophysics Data System (ADS)

    Nenov, Artur; Giussani, Angelo; Segarra-Martí, Javier; Jaiswal, Vishal K.; Rivalta, Ivan; Cerullo, Giulio; Mukamel, Shaul; Garavelli, Marco

    2015-06-01

    Pump-probe electronic spectroscopy using femtosecond laser pulses has evolved into a standard tool for tracking ultrafast excited state dynamics. Its two-dimensional (2D) counterpart is becoming an increasingly available and promising technique for resolving many of the limitations of pump-probe caused by spectral congestion. The ability to simulate pump-probe and 2D spectra from ab initio computations would allow one to link mechanistic observables like molecular motions and the making/breaking of chemical bonds to experimental observables like excited state lifetimes and quantum yields. From a theoretical standpoint, the characterization of the electronic transitions in the visible (Vis)/ultraviolet (UV), which are excited via the interaction of a molecular system with the incoming pump/probe pulses, translates into the determination of a computationally challenging number of excited states (going over 100) even for small/medium sized systems. A protocol is therefore required to evaluate the fluctuations of spectral properties like transition energies and dipole moments as a function of the computational parameters and to estimate the effect of these fluctuations on the transient spectral appearance. In the present contribution such a protocol is presented within the framework of complete and restricted active space self-consistent field theory and its second-order perturbation theory extensions. The electronic excited states of adenine have been carefully characterized through a previously presented computational recipe [Nenov et al., Comput. Theor. Chem. 1040-1041, 295-303 (2014)]. A wise reduction of the level of theory has then been performed in order to obtain a computationally less demanding approach that is still able to reproduce the characteristic features of the reference data. Foreseeing the potentiality of 2D electronic spectroscopy to track polynucleotide ground and excited state dynamics, and in particular its expected ability to provide

  9. A DNA-templated silver nanocluster probe for label-free, turn-on fluorescence-based screening of homo-adenine binding molecules.

    PubMed

    Park, Ki Soo; Park, Hyun Gyu

    2015-02-15

    A novel, label-free, turn-on fluorescence strategy to detect molecules that bind to adenine-rich DNA sequences has been developed. The probe employs DNA-templated silver nanoclusters (DNA-AgNCs) as the key detection component. The new strategy relies on the formation of non-Watson-Crick homo-adenine DNA duplex, triggered by strong interactions with homo-adenine binding molecules, which brings a guanine-rich sequence in one strand close to DNA-AgNCs located on the opposite strand. This phenomenon transforms weakly fluorescent AgNCs into highly emissive species that display bright red fluorescence. Finally, we have shown that the new fluorescence turn-on strategy can be employed to detect coralyne, the most representative homo-adenine binding molecule that triggers formation of a non-Watson-Crick homo-adenine DNA duplex. PMID:25441410

  10. Animal models of pediatric chronic kidney disease. Is adenine intake an appropriate model?

    PubMed

    Claramunt, Débora; Gil-Peña, Helena; Fuente, Rocío; Hernández-Frías, Olaya; Santos, Fernando

    2015-01-01

    Pediatric chronic kidney disease (CKD) has peculiar features. In particular, growth impairment is a major clinical manifestation of CKD that debuts in pediatric age because it presents in a large proportion of infants and children with CKD and has a profound impact on the self-esteem and social integration of the stunted patients. Several factors associated with CKD may lead to growth retardation by interfering with the normal physiology of growth plate, the organ where longitudinal growth rate takes place. The study of growth plate is hardly possible in humans and justifies the use of animal models. Young rats made uremic by 5/6 nephrectomy have been widely used as a model to investigate growth retardation in CKD. This article examines the characteristics of this model and analyzes the utilization of CKD induced by high adenine diet as an alternative research protocol. PMID:26522663

  11. [Absolute bioavailability of the adenine derivative VMA-99-82 possessing antiviral activity].

    PubMed

    Smirnova, L A; Suchkov, E A; Riabukha, A F; Kuznetsov, K A; Ozerov, A A

    2013-01-01

    Investigation of the main pharmacokinetic parameters of adenine derivative VMA-99-82 in rats showed large values of the half-life (T1/2 = 11.03 h) and the mean retention time of drug molecules in the organism (MRT = 9.53 h). A high rate of the drug concentration decrease in the plasma determines a small value of the area under the pharmacokinetic curve (AUC = 74.96 mg h/ml). The total distribution volume (V(d) = 10.61 l/kg) is 15.8 times greater than the volume of extracellular fluid in the body of rat, which is indicative of a high ability of VMA-99-82 to be distributed and accumulated in the organs and tissues. The absolute bioavailability of VMA-99-82 is 66%. PMID:24605425

  12. Prebiotic Synthesis of Adenine and Amino Acids Under Europa-like Conditions

    NASA Technical Reports Server (NTRS)

    Levy, Matthew; Miller, Stanley L.; Brinton, Karen; Bada, Jeffrey L.

    2003-01-01

    In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites. we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 year at -20 and -78 C. In addition the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20%. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be m der than previously thought.

  13. Prebiotic synthesis of adenine and amino acids under Europa-like conditions

    NASA Technical Reports Server (NTRS)

    Levy, M.; Miller, S. L.; Brinton, K.; Bada, J. L.

    2000-01-01

    In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites, we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 years at -20 and -78 degrees C. In addition, the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20 degrees C. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be wider than previously thought.

  14. Sites of adsorption of adenine, uracil, and their corresponding derivatives on sodium montmorillonite.

    PubMed

    Perezgasga, L; Serrato-Díaz, A; Negrón-Mendoza, A; De Pablo Galán, L; Mosqueira, F G

    2005-04-01

    Clay minerals are considered important to chemical evolution processes due to their properties, ancient origin, and wide distribution. To extend the knowledge of their role in the prebiotic epoch, the adsorption sites of adenine, adenosine, AMP, ADP, ATP, Poly A, uracil, uridine, UMP, UDP, UTP and Poly U on sodium montmorillonite are investigated. X-ray diffraction, ultraviolet and infrared spectroscopy studies indicate that these molecules distribute into the interlamellar channel and the edge of the clay crystals. Monomers are adsorbed predominantly in the interlamellar channel, whereas polymers adsorb along the crystal edges. Such behavior is discussed mainly in terms of bulk pH, pK(a) of the adsorbate, and Van der Waals interactions. PMID:16010992

  15. Dynamics of Excess-Electron Transfer through Alternating Adenine:Thymine Sequences in DNA.

    PubMed

    Lin, Shih-Hsun; Fujitsuka, Mamoru; Majima, Tetsuro

    2015-11-01

    This paper presents the results of an investigation into the sequence-dependent excess-electron transfer (EET) dynamics in DNA, which plays an important role in DNA damage/repair. There are many published studies on EET in consecutive adenine:thymine (A:T) sequences (Tn), but those in alternating A:T sequences (ATn) remain limited. Here, two series of functionalized DNA oligomers, Tn and ATn, were synthesized with a strongly electron-donating photosensitizer, a trimer of ethylenedioxythiophene (3 E), and an electron acceptor, diphenylacetylene (DPA). Laser flash photolysis experiments showed that the EET rate constant of AT3 is two times lower than that of T3 due to the lack of π-stacking of Ts in AT3. Thus, it was indicated that excess-electron hopping is affected by the interaction between LUMOs of nucleotides. PMID:26398266

  16. Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs

    PubMed Central

    Gilbert, Sunny D.; Reyes, Francis E.; Edwards, Andrea L.; Batey, Robert T.

    2009-01-01

    SUMMARY Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity. PMID:19523903

  17. External electric field promotes proton transfer in the radical cation of adenine-thymine

    NASA Astrophysics Data System (ADS)

    Zhang, Guiqing; Xie, Shijie

    2016-07-01

    According to pKa measurements, it has been predicted that proton transfer would not occur in the radical cation of adenine-thymine (A:T). However, recent theoretical calculations indicate that proton transfer takes place in the base pair in water below the room temperature. We have performed simulations of proton transfer in the cation of B-DNA stack composed of 10 A:T base pairs in water from 20 K to 300 K. Proton transfer occurs below the room temperature, meanwhile it could also be observed at the room temperature under the external electric field. Another case that interests us is that proton transfer bounces back after ˜300 fs from the appearance of proton transfer at low temperatures.

  18. The effects of tautomerization and protonation on the adenine-cytosine mismatches: a density functional theory study.

    PubMed

    Masoodi, Hamid Reza; Bagheri, Sotoodeh; Abareghi, Mahsa

    2016-06-01

    In the present work, we demonstrate the results of a theoretical study concerned with the question how tautomerization and protonation of adenine affect the various properties of adenine-cytosine mismatches. The calculations, in gas phase and in water, are performed at B3LYP/6-311++G(d,p) level. In gas phase, it is observed that any tautomeric form of investigated mismatches is more stabilized when adenine is protonated. As for the neutral mismatches, the mismatches containing amino form of cytosine and imino form of protonated adenine are more stable. The role of aromaticity on the stability of tautomeric forms of mismatches is investigated by NICS(1)ZZ index. The stability of mispairs decreases by going from gas phase to water. It can be explained using dipole moment parameter. The influence of hydrogen bonds on the stability of mismatches is examined by atoms in molecules and natural bond orbital analyses. In addition to geometrical parameters and binding energies, the study of the topological properties of electron charge density aids in better understanding of these mispairs. PMID:26198186

  19. Formation of Adenine from the Soft X-Ray Photo-Irradiation of N2-CH4 Ice

    NASA Astrophysics Data System (ADS)

    Pilling, S.; Andrade, D. P. P.; Neto, A. C.; Rittner, R.; de Brito, A. N.

    2010-04-01

    In this work, we present an experimental study of the chemical alteration produced by the interaction of soft X-rays (and secondary electrons) on Titan aerosol analogs producing prebiotic compounds such as adenine, one the constituents of the DNA molecule.

  20. [Corrective effect of trimethylglycine on the nicotinamide coenzyme and adenine nucleotide content of the tissues in experimental atherosclerosis].

    PubMed

    Zapadniuk, V I; Chekman, I S; Panteleĭmonova, T N; Tumanov, V A

    1986-01-01

    Experiments on adult rabbits with experimental atherosclerosis induced by cholesterol (0.25 g/kg for 90 days) showed that chronic administration of trimethylglycine (1.5 g/kg for 30 days) prevented a decrease of the liver and myocardium content of nicotinamide coenzymes and adenine nucleotides. PMID:3758334

  1. Mechanism of bracken fern carcinogenesis: evidence for H-ras activation via initial adenine alkylation by ptaquiloside.

    PubMed

    Prakash, A S; Pereira, T N; Smith, B L; Shaw, G; Seawright, A A

    1996-01-01

    Bracken fern (Pteridium spp.) causes cancer of the oesophagus and the urinary bladder in cattle and sheep. Ptaquiloside (PT) is believed to be the carcinogenic principle which alkylates DNA when activated to its unstable dienone form (APT) under alkaline conditions. In this report we present evidence for the presence of PT-DNA adducts in the ileum of bracken fem-fed calves using the 32P-postlabelling assay. H-ras mutations were also observed in the ileum using single strand conformation polymorphism (SSCP) technique. Mutations corresponding to adenine to pyrimidine transversions in the codon 61 of H-ras were identified by the cycle sequencing method. In vitro DNA alkylation studies showed that APT alkylated H-ras primarily at the adenines. In addition, the rate of depurination of alkylated adenine was sequence dependent. Investigation of DNA template activity using a plasmid DNA showed that DNA synthesis by T7 DNA polymerase was terminated by the presence of all alkylated bases but certain apurinic sites allowed the DNA synthesis to continue. These results suggest that initial alkylation of adenine by PT in codon 61 followed by depurination and error in DNA synthesis lead to activation of H-ras proto-oncogene. PMID:8946397

  2. Few-layer graphene sheets with embedded gold nanoparticles for electrochemical analysis of adenine

    PubMed Central

    Biris, Alexandru R; Pruneanu, Stela; Pogacean, Florina; Lazar, Mihaela D; Borodi, Gheorghe; Ardelean, Stefania; Dervishi, Enkeleda; Watanabe, Fumiya; Biris, Alexandru S

    2013-01-01

    This work describes the synthesis of few-layer graphene sheets embedded with various amounts of gold nanoparticles (Gr-Au-x) over an Aux/MgO catalytic system (where × = 1, 2, or 3 wt%). The sheet-like morphology of the Gr-Au-x nanostructures was confirmed by transmission electron microscopy and high resolution transmission electron microscopy, which also demonstrated that the number of layers within the sheets varied from two to seven. The sample with the highest percentage of gold nanoparticles embedded within the graphitic layers (Gr-Au-3) showed the highest degree of crystallinity. This distinct feature, along with the large number of edge-planes seen in high resolution transmission electron microscopic images, has a crucial effect on the electrocatalytic properties of this material. The reaction yields (40%–50%) and the final purity (96%–98%) of the Gr-Au-x composites were obtained by thermogravimetric analysis. The Gr-Au-x composites were used to modify platinum substrates and subsequently to detect adenine, one of the DNA bases. For the bare electrode, no oxidation signal was recorded. In contrast, all of the modified electrodes showed a strong electrocatalytic effect, and a clear peak for adenine oxidation was recorded at approximately +1.05 V. The highest increase in the electrochemical signal was obtained using a platinum/Gr-Au-3-modified electrode. In addition, this modified electrode had an exchange current density (I0, obtained from the Tafel plot) one order of magnitude higher than that of the bare platinum electrode, which also confirmed that the transfer of electrons took place more readily at the Gr-Au-3-modified electrode. PMID:23610521

  3. Chemical Synthesis, Crystal Structure and Enzymatic Evaluation of a Dinucleotide Spore Photoproduct Analogue Containing a Formacetal Linker

    PubMed Central

    Lin, Gengjie; Chen, Chun-Hsing; Pink, Maren; Pu, Jingzhi; Li, Lei

    2011-01-01

    Spore photoproduct (SP) is the exclusive DNA photodamage product found in bacterial endospores. Its photoformation and repair by a metal-loenzyme spore photoproduct lyase (SPL) composes the unique SP biochemistry. Despite the fact that the SP was discovered almost 50 years ago, its crystal structure is still unknown and the lack of structural information greatly hinders the study of SP biochemistry. Employing a formacetal linker and organic synthesis, we successfully prepared a dinucleotide SP isostere 5R-CH2SP, which contains a neutral CH2 moiety between the two thymine residues instead of a phosphate. The neutral linker dramatically facilitates the crystallization process, allowing us to obtain the crystal structure for this intriguing thymine dimer half a century after its discovery. Further ROESY spectroscopic, DFT computational, and enzymatic studies of this 5R-CH2SP compound prove that it possesses similar properties with the 5R-SP species, suggesting that the revealed structure truly reflects that of SP generated in Nature. PMID:21780208

  4. Population genetics of dinucleotide (dC-dA)n.(dG-dT)n polymorphisms in world populations.

    PubMed Central

    Deka, R; Jin, L; Shriver, M D; Yu, L M; DeCroo, S; Hundrieser, J; Bunker, C H; Ferrell, R E; Chakraborty, R

    1995-01-01

    We have characterized eight dinucleotide (dC-dA)n.(dG-dT)n repeat loci located on human chromosome 13q in eight human populations and in a sample of chimpanzees. Even though there is substantial variation in allele frequencies at each locus, at a given locus the most frequent alleles are shared by all human populations. The level of heterozygosity is reduced in isolated or small populations, such as the Pehuenche Indians of Chile, the Dogrib of Canada, and the New Guinea highlanders. On the other hand, larger average heterozygosities are observed in large and cosmopolitan populations, such as the Sokoto population from Nigeria and German Caucasians. Conformity with Hardy-Weinberg equilibrium is generally observed at these loci, unless (a) a population is isolated or small or (b) the repeat motif of the locus is not perfect (e.g., D13S197). Multilocus genotype probabilities at these microsatellite loci do not show departure from the independence rule, unless the loci are closely linked. The allele size distributions at these (CA)n loci do not follow a strict single-step stepwise-mutation model. However, this features does not compromise the ability to detect population affinities, when these loci are used simultaneously. The microsatellite loci examined here are present and, with the exception of the locus D13S197, are polymorphic in the chimpanzees, showing an overlapping distribution of allele sizes with those observed in human populations. Images Figure 1 PMID:7847383

  5. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    PubMed Central

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  6. Pioneer factor interactions and unmethylated CpG dinucleotides mark silent tissue-specific enhancers in embryonic stem cells.

    PubMed

    Xu, Jian; Pope, Scott D; Jazirehi, Ali R; Attema, Joanne L; Papathanasiou, Peter; Watts, Jason A; Zaret, Kenneth S; Weissman, Irving L; Smale, Stephen T

    2007-07-24

    Recent studies have suggested that, in ES cells, inactive genes encoding early developmental regulators possess bivalent histone modification domains and are therefore poised for activation. However, bivalent domains were not observed at typical tissue-specific genes. Here, we show that windows of unmethylated CpG dinucleotides and putative pioneer factor interactions mark enhancers for at least some tissue-specific genes in ES cells. The unmethylated windows expand in cells that express the gene and contract, disappear, or remain unchanged in nonexpressing tissues. However, in ES cells, they do not always coincide with common histone modifications. Genomic footprinting and chromatin immunoprecipitation demonstrated that transcription factor binding underlies the unmethylated windows at enhancers for the Ptcra and Alb1 genes. After stable integration of premethylated Ptcra enhancer constructs into the ES cell genome, the unmethylated windows readily appeared. In contrast, the premethylated constructs remained fully methylated and silent after introduction into Ptcra-expressing thymocytes. These findings provide initial functional support for a model in which pioneer factor interactions in ES cells promote the assembly of a chromatin structure that is permissive for subsequent activation, and in which differentiated tissues lack the machinery required for gene activation when these ES cell marks are absent. The enhancer marks may therefore represent important features of the pluripotent state. PMID:17640912

  7. Population genetics of dinucleotide (dC-dA)n.(dG-dT)n polymorphisms in world populations.

    PubMed

    Deka, R; Jin, L; Shriver, M D; Yu, L M; DeCroo, S; Hundrieser, J; Bunker, C H; Ferrell, R E; Chakraborty, R

    1995-02-01

    We have characterized eight dinucleotide (dC-dA)n.(dG-dT)n repeat loci located on human chromosome 13q in eight human populations and in a sample of chimpanzees. Even though there is substantial variation in allele frequencies at each locus, at a given locus the most frequent alleles are shared by all human populations. The level of heterozygosity is reduced in isolated or small populations, such as the Pehuenche Indians of Chile, the Dogrib of Canada, and the New Guinea highlanders. On the other hand, larger average heterozygosities are observed in large and cosmopolitan populations, such as the Sokoto population from Nigeria and German Caucasians. Conformity with Hardy-Weinberg equilibrium is generally observed at these loci, unless (a) a population is isolated or small or (b) the repeat motif of the locus is not perfect (e.g., D13S197). Multilocus genotype probabilities at these microsatellite loci do not show departure from the independence rule, unless the loci are closely linked. The allele size distributions at these (CA)n loci do not follow a strict single-step stepwise-mutation model. However, this features does not compromise the ability to detect population affinities, when these loci are used simultaneously. The microsatellite loci examined here are present and, with the exception of the locus D13S197, are polymorphic in the chimpanzees, showing an overlapping distribution of allele sizes with those observed in human populations. PMID:7847383

  8. Adenine Synthesis in Interstellar Space: Mechanisms of Prebiotic Pyrimidine-Ring Formation of Monocyclic HCN-Pentamers

    NASA Astrophysics Data System (ADS)

    Glaser, Rainer; Hodgen, Brian; Farrelly, Dean; McKee, Elliot

    2007-06-01

    The question whether the nucleobases can be synthesized in interstellar space is of fundamental significance in considerations of the origin of life. Adenine is formally the HCN pentamer, and experiments have demonstrated that adenine is formed under certain conditions by HCN pentamerization in gas, liquid, and condensed phases. Most mechanistic proposals invoke the intermediacy of the HCN tetramer AICN (4), and it is thought that adenine synthesis is completed by addition of the 5th HCN to 4 to form amidine 5 and subsequent pyrimidine cyclization. In this context, we have been studying the mechanism for prebiotic pyrimidine-ring formation of monocyclic HCN-pentamers with ab initio electronic structure theory. The calculations model gas phase chemistry, and the results primarily inform discussions of adenine synthesis in interstellar space. Purine formation requires tautomerization of 5 to the conjugated amidine 6 (via hydrogen-tunneling, thermally with H+ -catalysis, or by photolysis) or to keteneimine 7 (by photolysis). It was found that 5-(N'-formamidinyl)-1H-imidazole-4-carbonitrile (6) can serve as a substrate for proton-catalyzed purine formation under photolytic conditions and N-(4-(iminomethylene)-1H-imidazol-5(4H)-ylidene)formamidine (7) can serve as a substrate for uncatalyzed purine formation under photolytic conditions. The absence of any sizeable activation barrier for the cyclization of 7 to the (Z)-imino form of 9H-adenine (Z)-2 is quite remarkable, and it is this feature that allows for the formation of the purine skeleton from 7 without any further activation.

  9. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance. PMID:26946085

  10. Cyclic dinucleotide (c-di-GMP, c-di-AMP, and cGAMP) signalings have come of age to be inhibited by small molecules.

    PubMed

    Opoku-Temeng, Clement; Zhou, Jie; Zheng, Yue; Su, Jianmei; Sintim, Herman O

    2016-08-01

    Bacteria utilize nucleotide-based second messengers to regulate a myriad of physiological processes. Cyclic dinucleotides have emerged as central regulators of bacterial physiology, controlling processes ranging from cell wall homeostasis to virulence production, and so far over thousands of manuscripts have provided biological insights into c-di-NMP signaling. The development of small molecule inhibitors of c-di-NMP signaling has significantly lagged behind. Recent developments in assays that allow for high-throughput screening of inhibitors suggest that the time is right for a concerted effort to identify inhibitors of these fascinating second messengers. Herein, we review c-di-NMP signaling and small molecules that have been developed to inhibit cyclic dinucleotide-related enzymes. PMID:27339003

  11. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity

    PubMed Central

    Li, Wang; Li, Yong; Liu, Zhuoliang; Lin, Bin; Yi, Haibo; Xu, Feng; Nie, Zhou; Yao, Shouzhuo

    2016-01-01

    G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3′ end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3′ adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid–base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions. PMID:27422869

  12. Minor groove site coordination of adenine by platinum group metal ions: effects on basicity, base pairing, and electronic structure.

    PubMed

    Amantia, David; Price, Clayton; Shipman, Michelle A; Elsegood, Mark R J; Clegg, William; Houlton, Andrew

    2003-05-01

    Dithioether- or diamine-tethered adenine derivatives react with Pt(II), Pd(II), and Rh(III) ions to give N3-coordinated complexes of the types [MCl(SSN)](+) (M = Pt or Pd), [RhCl(3)(SSN)], or [RhCl(3)(NNN)] (where SSN = 1-(N9-adenine)-3,6-dithia-heptane or 1-(N9-adenine)-4,7-dithia-octane; NNN = ethylenediamine-N,9-ethyladenine). Single-crystal X-ray analysis confirms the nature of the metal-nucleobase interaction and highlights a conserved intermolecular hydrogen-bonding motif for all the complexes, irrespective of the metal-ion geometry. Coordination significantly reduces the basicity of the adeninyl group, as indicated by a pK(a) value of -0.16 for [PtCl(N3-1-(N9-adenine)-3,6-dithia-heptane)]BF(4), compared to a pK(a) value of 4.2 for 9-ethyladenine. The site of proton binding, N1 or N7, could not be unambiguously assigned from the (1)H NMR data, because of the similar effect on the chemical shifts of the H2 and H8 protons. Density functional calculations at the BP-LACVP level suggest N1 as the site of protonation for this type of complex. This is in contrast to the N7-protonation reported for [Pt(dien)(N3-6,6',9-trimethyladenine)](2+), as reported elsewhere (Meiser et al., Chem.-Eur. J. 1997, 3, 388). However, further electronic structure calculations in the gas phase reveal that the preferred site for protonation for N3-bound complexes is conformationally dependent. N3 coordination was also found to reduce the extent of base pairing between adenine and thymine in dimethylsulfoxide for the self-complementary complex [PtCl(L3)](+) (L3 = 1-(N9-adenine)-3,6-dithia-9-(N1-thymine)nonane), compared to that for the uncomplexed ligand. PMID:12716200

  13. Probing the reaction mechanism of spore photoproduct lyase (SPL) via diastereoselectively labeled dinucleotide SP TpT substrates

    PubMed Central

    Yang, Linlin; Lin, Gengjie; Liu, Degang; Dria, Karl J.; Telser, Joshua; Li, Lei

    2011-01-01

    5-thyminyl-5,6-dihydrothymine (commonly called spore photoproduct or SP) is the exclusive DNA photo-damage product in bacterial endospores. It is generated in the bacterial sporulation phase and repaired by a radical SAM enzyme, spore photoproduct lyase (SPL), at the early germination phase. SPL utilizes a special [4Fe-4S] cluster to reductively cleave S-adenosylmethionine (SAM) to generate a reactive 5′-dA radical. The 5′-dA radical is proposed to abstract one of the two H atoms at the C6 carbon of SP to initiate the repair process. Via organic synthesis and DNA photochemistry, we selectively labeled the 6-HproS or 6-HproR position with a deuterium in a dinucleotide SP TpT substrate. Monitoring the deuterium migration in enzyme catalysis (employing Bacillus subtilis SPL) revealed that it is the 6-HproR atom of SP that is abstracted by the 5′-dA radical. Surprisingly, the abstracted deuterium was not returned to the resulting TpT after enzymatic catalysis, an H atom from the aqueous buffer was incorporated into TpT instead. This result questions the currently hypothesized SPL mechanism which excludes the involvement of protein residue(s) in SPL reaction, suggesting that some protein residue(s), which is capable of exchanging a proton with the aqueous buffer, is involved in the enzyme catalysis. Moreover, evidence has been obtained for a possible SAM regeneration after each catalytic cycle; however, such a regeneration process is more complex than currently thought, with one or even more protein residues involved as well. These observations have enabled us to propose a modified reaction mechanism for this intriguing DNA repair enzyme. PMID:21671623

  14. A comparison of genetic map distance and linkage disequilibrium between 15 polymorphic dinucleotide repeat loci in two populations

    SciTech Connect

    Urbanek, M.; Goldman, D.; Long, J.C.

    1994-09-01

    Linkage disequilibrium has recently been used to map the diastrophic dysplasia gene in a Finnish sample. One advantage of this method is that the large pedigrees required by some other methods are unnecessary. Another advantage is that linkage disequilibrium mapping capitalizes on the cumulative history of recombination events, rather than those occurring within the sampled individuals. A potential limitation of linkage disequilibrium mapping is that linkage equilibrium is likely to prevail in all but the most isolated populations, e.g., those which have recently experienced founder effects or severe population bottlenecks. In order to test the method`s generality, we examined patterns of linkage disequilibrium between pairs of loci within a known genetic map. Two populations were analyzed. The first population, Navajo Indians (N=45), is an isolate that experienced a severe bottleneck in the 1860`s. The second population, Maryland Caucasians (N=45), is cosmopolitan. We expected the Navajo sample to display more linkage disequilibrium than the Caucasian sample, and possibly that the Navajo disequilibrium pattern would reflect the genetic map. Linkage disequilibrium coefficients were estimated between pairs of alleles at different loci using maximum likelihood. The genetic isolate structure of Navajo Indians is confirmed by the DNA typings. Heterozygosity is lower than in the Caucasians, and fewer different alleles are observed. However, a relationship between genetic map distance and linkage disequilibrium could be discerned in neither the Navajo nor the Maryland samples. Slightly more linkage disequilibrium was observed in the Navajos, but both data sets were characterized by very low disequilibrium levels. We tentatively conclude that linkage disequilibrium mapping with dinucleotide repeats will only be useful with close linkage between markers and diseases, even in very isolated populations.

  15. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine.

    PubMed

    Janowsky, Aaron; Tosh, Dilip K; Eshleman, Amy J; Jacobson, Kenneth A

    2016-04-01

    Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter

  16. Poly-adenine-based programmable engineering of gold nanoparticles for highly regulated spherical DNAzymes

    NASA Astrophysics Data System (ADS)

    Zhu, Dan; Pei, Hao; Chao, Jie; Su, Shao; Aldalbahi, Ali; Rahaman, Mostafizur; Wang, Lihua; Wang, Lianhui; Huang, Wei; Fan, Chunhai; Zuo, Xiaolei

    2015-11-01

    Enzyme complexes are assembled at the two-dimensional lipid membrane or prearranged on three-dimensional scaffolding proteins to regulate their catalytic activity in cells. Inspired by nature, we have developed gold nanoparticle-based spherical DNAzymes (SNAzymes) with programmably engineered activities by exploiting poly-adenine (polyA)-Au interactions. In a SNAzyme, AuNPs serve as the metal core, which is decorated with a functional shell of DNAzymes. Conventional thiolated DNAzyme-based assembly leads to disordered structures with suppressed activity. In contrast, by using an anchoring block of polyA tails, we find that the activity of SNAzymes can be programmably regulated. By using a polyA30 tail, SNAzymes demonstrated remarkably enhanced binding affinity compared to the thiolated DNAzyme-based assembly (~75-fold) or individual DNAzymes in the solution phase (~10-fold). More significantly, this increased affinity is directly translated to the sensitivity improvement in the SNAzyme-based lead sensor. Hence, this design of SNAzymes may provide new opportunities for developing biosensors and bioimaging probes for theranostic applications.Enzyme complexes are assembled at the two-dimensional lipid membrane or prearranged on three-dimensional scaffolding proteins to regulate their catalytic activity in cells. Inspired by nature, we have developed gold nanoparticle-based spherical DNAzymes (SNAzymes) with programmably engineered activities by exploiting poly-adenine (polyA)-Au interactions. In a SNAzyme, AuNPs serve as the metal core, which is decorated with a functional shell of DNAzymes. Conventional thiolated DNAzyme-based assembly leads to disordered structures with suppressed activity. In contrast, by using an anchoring block of polyA tails, we find that the activity of SNAzymes can be programmably regulated. By using a polyA30 tail, SNAzymes demonstrated remarkably enhanced binding affinity compared to the thiolated DNAzyme-based assembly (~75-fold) or

  17. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine

    PubMed Central

    Tosh, Dilip K.; Eshleman, Amy J.; Jacobson, Kenneth A.

    2016-01-01

    Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [125I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [3H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N6-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [125I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4′-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake

  18. Conversion of adenine to 5-amino-4-pyrimidinylimidazole caused by acetyl capping during solid phase oligonucleotide synthesis.

    PubMed

    Rodriguez, Andrew A; Cedillo, Isaiah; McPherson, Andrew K

    2016-08-01

    The acetyl capping reaction used throughout solid phase oligonucleotide synthesis is meant to minimize n-1 deletionmer impurities by terminating sequences that fail to couple to a phosphoramidite. However, the reaction is also responsible for the formation of a number of impurities. One capping-related impurity has an additional mass of 98amu from the parent oligonucleotide. The n+98 amu impurity was found to result from modification of an adenine nucleobase. The structure of the impurity was determined by preparation of an oligonucleotide enriched in n+98 amu, enzymatic digestion to individual nucleosides, isolation of the pure nucleoside+98 amu species, crystallization, and X-ray crystallographic analysis. The n+98 amu impurity is an oligonucleotide in which one adenine residue has been converted to 5-amino-4-pyrimidinylimidazole. The mechanism of formation of the impurity was investigated, and a mechanism is proposed. PMID:27353533

  19. The contribution of adenines in the catalytic core of 10-23 DNAzyme improved by the 6-amino group modifications.

    PubMed

    Zhu, Junfei; Li, Zhiwen; Wang, Qi; Liu, Yang; He, Junlin

    2016-09-15

    In the catalytic core of 10-23 DNAzyme, its five adenine residues are moderate conservative, but with highly conserved functional groups like 6-amino group and 7-nitrogen atom. It is this critical conservation that these two groups could be modified for better contribution. With 2'-deoxyadenosine analogues, several functional groups were introduced at the 6-amino group of the five adenine residues. 3-Aminopropyl substituent at 6-amino group of A15 resulted in a five-fold increase of kobs. More efficient DNAzymes are expected by delicate design of the linkage and the external functional groups for this 6-amino group of A15. With this modification approach, other functional groups or residues could be optimized for 10-23 DNAzyme. PMID:27506560

  20. Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

    PubMed Central

    Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

    2012-01-01

    SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

  1. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.

    NASA Technical Reports Server (NTRS)

    Yang, C. C.; Oro, J.

    1971-01-01

    Study of the formation of purines, pyrimidines, and other bases from CO, H2, and NH3 under conditions similar to those used in the Fischer-Tropsch process. It is found that industrial nickel/iron alloy catalyzes the synthesis of adenine, guanine, cytosine, and other nitrogenous compounds from mixtures of CO, H2, and NH3 at temperatures of about 600 C. Sufficient sample was accumulated to isolate as solid products adenine, guanine, and cytosine, which were identified by infrared spectrophotometry. In the absence of nickel/iron catalyst, at 650 C, or in the presence of this catalyst, at 450 C, no purines or pyrimidines were synthesized. These results confirm and extend some of the work reported by Kayatsu et al. (1968).

  2. Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification.

    PubMed

    Iyer, Lakshminarayan M; Zhang, Dapeng; Aravind, L

    2016-01-01

    While N(6) -methyladenosine (m(6) A) is a well-known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well-studied 5-methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction-modification and counter-restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m(6) A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m(6) A-binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m(6) A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m(6) A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract. PMID:26660621

  3. DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles.

    PubMed

    Liu, Pei; Wang, Dandan; Zhou, Yunlei; Wang, Haiyan; Yin, Huanshun; Ai, Shiyun

    2016-06-15

    DNA methyltransferase (MTase) has received a large amount of attention due to its catalyzation of DNA methylation in both eukaryotes and prokaryotes, which has a close relationship to cancer and bacterial diseases. Herein, a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine (polyA) DNA with a gold nanoparticles functioned glassy carbon electrode (AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue (MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in real biological matrices and inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery. PMID:26807517

  4. Tools for DNA adenine methyltransferase identification analysis of nuclear organization during C. elegans development.

    PubMed

    Sharma, Rahul; Ritler, Dominic; Meister, Peter

    2016-04-01

    C. elegans has recently emerged as a valuable model to understand the link between nuclear organization and cell fate, by combining microscopy approaches, genome-wide mapping techniques with advanced genetics. Crucial to these analyses are techniques to determine the genome-wide interaction pattern of proteins with DNA. Chromatin immunoprecipitation has proven valuable but it requires considerable amounts of starting material. This is sometimes difficult to achieve, in particular for specific genotypes (balanced strains, different sexes, severe phenotypes…). As an alternative to ChIP, DNA adenine methyltransferase identification by sequencing (DamID-seq) was recently shown to be able to characterize binding sites in single mammalian cells. Additionally, DamID can be achieved for cell-type specific analysis by expressing Dam fusion proteins under tissue specific promoters in a controlled manner. In this report, we present a user-friendly pipeline to analyse DamID-seq data in C. elegans. Based upon this pipeline, we provide a comparative analysis of libraries generated with different starting material and discuss important library features. Moreover, we introduce an adaptation of an imaging based tool to visualize in vivo the cell-specific tridimensional binding pattern of any protein of interest. genesis 54:151-159, 2016. © 2016 Wiley Periodicals, Inc. PMID:26845390

  5. Identification of the active oligomeric state of an essential adenine DNA methyltransferase from Caulobacter crescentus.

    PubMed

    Shier, V K; Hancey, C J; Benkovic, S J

    2001-05-01

    Caulobacter crescentus contains one of the two known prokaryotic DNA methyltransferases that lacks a cognate endonuclease. This endogenous cell cycle regulated adenine DNA methyltransferase (CcrM) is essential for C. crescentus cellular viability. DNA methylation catalyzed by CcrM provides an obligatory signal for the proper progression through the cell cycle. To further our understanding of the regulatory role played by CcrM, we sought to investigate its biophysical properties. In this paper we employed equilibrium ultracentrifugation, velocity ultracentrifugation, and chemical cross-linking to show that CcrM is dimeric at physiological concentrations. However, surface plasmon resonance experiments in the presence of S-adenosyl-homocysteine evince that CcrM binds as a monomer to a defined hemi-methylated DNA substrate containing the canonical methylation sequence, GANTC. Initial velocity experiments demonstrate that dimerization of CcrM does not affect DNA methylation. Collectively, these findings suggest that CcrM is active as a monomer and provides a possible in vivo role for dimerization as a means to stabilize CcrM from premature catabolism. PMID:11278726

  6. Content of Adenine Nucleotides and Orthophosphate in Exporting and Importing Mature Maize Leaves 1

    PubMed Central

    Eschrich, Walter; Fromm, Joerg

    1985-01-01

    Events of reactivation by re-illumination were studied in predarkened detached mature maize leaves, which were arranged as distal sources and proximal sinks; the latter were kept in CO2-free atmosphere and were either illuminated or darkened. Adenine nucleotide (AdN) content and orthophosphate (Pi) concentrations were measured 10 minutes, 30 minutes, and 2, 7, and 14 hours after the onset of re-illumination. For comparison, mature leaves attached to the plant were analyzed. The sum of AdN increased up to 7 hours of re-illumination, then dark sinks and their sources showed decreasing amounts of AdN, while the increase continued up to 14 hours in sources and illuminated sinks. In leaves attached to the plant, no further increase in AdN level followed the 7-hour mark. The amount of individual AdN (ATP, ADP, AMP) differed considerably in sources and sinks of the detached leaves. Although both the source supplying the illuminated sink and the source supplying the dark sink were treated the same, they showed striking differences in AdN contents. Such relations were also observed, when ATP/ADP ratios and Pi concentrations were compared. The influence a sink can exert on its source suggests a participation of the physiological events in the sink on the regulation of AdN and Pi metabolism in the source. PMID:16664246

  7. PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth.

    PubMed

    Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

  8. Release of adenine nucleotide metabolites by toxic concentrations of cardiac glycosides.

    PubMed

    Bernauer, W

    1994-01-01

    In isolated perfused guinea-pig hearts the effect of toxic concentrations of cardiac glycosides on the release of the adenine nucleotide metabolites adenosine, inosine, hypoxanthine, xanthine, and uric acid was investigated. Digoxin concentrations of 0.03-1 mumol.l-1 produced moderate to severe tachyarrhythmias. Large amounts of metabolites were released by concentrations of 0.1 mumol.l-1, and higher. Occurrence of glycoside-induced ventricular fibrillation was associated with a particularly high release. Metabolite release was also obtained when fibrillation was elicited electrically in normal control hearts, or in hearts receiving simultaneously a marginally toxic digoxin concentration (0.03 mumol.l-1). Digoxin-induced tachyarrhythmias and metabolite release were almost completely prevented by a high potassium concentration in the coronary perfusion fluid (8.1 mmol.l-1). The antiarrhythmic effect was also obtained with lidocaine (60 mumol.l-1), but the release was only partially antagonized. Similar results concerning arrhythmias and metabolite release as with digoxin were obtained with ouabain. The findings suggest that the decrease in myocardial ATP observed in glycoside-intoxicated heart preparations is partly due to the loss of nucleotide precursor substances. Moreover, it appears likely that liberated adenosine in the interstitium of severely intoxicated heart preparations reaches pharmacologically effective concentrations. PMID:7826306

  9. PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

    PubMed Central

    Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

  10. Analysis of functional coupling: mitochondrial creatine kinase and adenine nucleotide translocase.

    PubMed

    Vendelin, Marko; Lemba, Maris; Saks, Valdur A

    2004-07-01

    The mechanism of functional coupling between mitochondrial creatine kinase (MiCK) and adenine nucleotide translocase (ANT) in isolated heart mitochondria is analyzed. Two alternative mechanisms are studied: 1), dynamic compartmentation of ATP and ADP, which assumes the differences in concentrations of the substrates between intermembrane space and surrounding solution due to some diffusion restriction and 2), direct transfer of the substrates between MiCK and ANT. The mathematical models based on these possible mechanisms were composed and simulation results were compared with the available experimental data. The first model, based on a dynamic compartmentation mechanism, was not sufficient to reproduce the measured values of apparent dissociation constants of MiCK reaction coupled to oxidative phosphorylation. The second model, which assumes the direct transfer of substrates between MiCK and ANT, is shown to be in good agreement with experiments--i.e., the second model reproduced the measured constants and the estimated ADP flux, entering mitochondria after the MiCK reaction. This model is thermodynamically consistent, utilizing the free energy profiles of reactions. The analysis revealed the minimal changes in the free energy profile of the MiCK-ANT interaction required to reproduce the experimental data. A possible free energy profile of the coupled MiCK-ANT system is presented. PMID:15240503

  11. Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD.

    PubMed

    Mishima, Eikan; Fukuda, Shinji; Shima, Hisato; Hirayama, Akiyoshi; Akiyama, Yasutoshi; Takeuchi, Yoichi; Fukuda, Noriko N; Suzuki, Takehiro; Suzuki, Chitose; Yuri, Akinori; Kikuchi, Koichi; Tomioka, Yoshihisa; Ito, Sadayoshi; Soga, Tomoyoshi; Abe, Takaaki

    2015-08-01

    The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment. PMID:25525179

  12. Divalent phosphate is a counterion for carboxyatractyloside-insensitive adenine nucleotide transport in rat liver mitochondria

    SciTech Connect

    Nosek, M.T.; Aprille, J.R.

    1986-05-01

    Unidirectional, carboxyatractyloside(CAT)-insensitive adenine nucleotide (AdN) fluxes have been studied in isolated rat liver mitochondria (mito). Previous work has shown that ATP x Mg transport in one direction is coupled to ATP x Mg or P/sub i/ transport in the opposite direction. The purpose of this study was to determine whether divalent HPO/sub 4//sup 2 -/ or monovalent H/sub 2/PO/sub 4//sup -/ is the transported phosphate species. The authors used the monofluorophosphate (PO/sub 3/F/sup 2 -/) and difluorophosphate (PO/sub 2/F/sub 2//sup -/) analogues as potential counterions forAdN efflux. After a preincubation on ice with /sup 14/C-ADP to label the matrix AdN, efflux was measured at 30/sup 0/C, pH 7.4, in 225mM sucrose, 10mM KCl, 5mM MgCl/sub 2/, 5mM glutamate, 5mM malate, 10mM Tris, 0.5mM P/sub i/, 1mM ATP, and 5..mu..M CAT. With no other additions efflux was -0.62 +/- 0.20 nmole/minute/mg protein. The data supports the hypothesis that divalent but not monovalent phosphate can act as a counterion for ATPx Mg transport over this CAT-insensitive carrier.

  13. Excess electron trapping in duplex DNA: long range transfer via stacked adenines.

    PubMed

    Black, Paul J; Bernhard, William A

    2012-11-01

    An understanding of charge transfer (CT) in DNA lies at the root of assessing the risks and benefits of exposure to ionizing radiation. Energy deposition by high-energy photons and fast-charged particles creates holes and excess electrons (EEs) in DNA, and the subsequent reactions determine the complexity of DNA damage and ultimately the risk of disease. Further interest in CT comes from the possibility that hole transfer, excess electron transfer (EET), or both in DNA might be used to develop nanoscale circuits. To study EET in DNA, EPR spectroscopy was used to determine the distribution of EE trapping by oligodeoxynucleotides irradiated and observed at 4 K. Our results indicate that stretches of consecutive adenine bases on the same strand serve as an ideal conduit for intrastrand EET in duplex DNA at 4 K. Specifically, we show that A is an efficient trap for EE at 4 K if, and only if, the A strand of the duplex does not contain one of the other three bases. If there is a T, C, or G on the A strand, then trapping occurs at T or C instead of A. This holds true for stretches up to 32 A's. Whereas T competes effectively against A for the EE, it does not compete effectively against C. Long stretches of T pass the majority of EE to C. Our results show that AT stretches channel EE to cytosine, an end point with significance to both radiation damage and the photochemical repair of pyrimidine dimers. PMID:23067129

  14. Electron impact fragmentation of adenine: partial ionization cross sections for positive fragments

    NASA Astrophysics Data System (ADS)

    van der Burgt, Peter J. M.; Finnegan, Sinead; Eden, Samuel

    2015-07-01

    Using computer-controlled data acquisition we have measured mass spectra of positive ions for electron impact on adenine, with electron energies up to 100 eV. Ion yield curves for 50 ions have been obtained and normalized by comparing their sum to the average of calculated total ionization cross sections. Appearance energies have been determined for 37 ions; for 20 ions for the first time. All appearance energies are consistent with the fragmentation pathways identified in the literature. Second onset energies have been determined for 12 fragment ions (for 11 ions for the first time), indicating the occurrence of more than one fragmentation process e.g. for 39 u (C2HN+) and 70 u (C2H4N3+). Matching ion yield shapes (118-120 u, 107-108 u, 91-92 u, and 54-56 u) provide new evidence supporting closely related fragmentation pathways and are attributed to hydrogen rearrangement immediately preceding the fragmentation. We present the first measurement of the ion yield curve of the doubly charged parent ion (67.5 u), with an appearance energy of 23.5 ± 1.0 eV. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

  15. High-mobility Group Box-1 Protein Promotes Granulomatous Nephritis in Adenine-induced nephropathy

    PubMed Central

    Oyama, Yoko; Hashiguchi, Teruto; Taniguchi, Noboru; Tancharoen, Salunya; Uchimura, Tomonori; Biswas, Kamal K.; Kawahara, Ko-ichi; Nitanda, Takao; Umekita, Yoshihisa; Lotz, Martin; Maruyama, Ikuro

    2011-01-01

    Granulomatous nephritis can be triggered by diverse factors and results in kidney failure. However, despite accumulating data about granulomatous inflammation, pathogenetic mechanisms in nephritis remain unclear. The DNA-binding high-mobility group box-1 protein (HMGB1) initiates and propagates inflammation when released by activated macrophages, functions as an “alarm cytokine” signaling tissue damage. In this study, we demonstrated elevated HMGB1 expression in renal granulomas in rats with crystal-induced granulomatous nephritis caused by feeding an adenine-rich diet. HMGB1 levels were also raised in urine and serum, as well as monocyte chemoattractant protein-1 (MCP-1), a mediator of granulomatous inflammation. Injection of HMGB1 worsened renal function and upregulated MCP-1 in rats with crystal-induced granulomatous nephritis. HMGB1 also induced MCP-1 secretion through mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) pathways in rat renal tubular epithelial cells in vitro. Hmgb1+/− mice with crystal-induced nephritis displayed reduced MCP-1 expression in the kidneys and in urine and the number of macrophages in the kidneys was significantly decreased. We conclude that HMGB1 is a new mediator involved in crystal-induced nephritis that amplifies granulomatous inflammation in a cycle where MCP-1 attracts activated macrophages, resulting in excessive and sustained HMGB1 release. HMGB1 could be a novel target for inhibiting chronic granulomatous diseases. PMID:20231821

  16. Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

    PubMed Central

    Iyer, Lakshminarayan M.; Zhang, Dapeng

    2015-01-01

    While N6‐methyladenosine (m6A) is a well‐known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well‐studied 5‐methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction‐modification and counter‐restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m6A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m6A‐binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m6A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m6A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract. PMID:26660621

  17. Poly-adenine-based programmable engineering of gold nanoparticles for highly regulated spherical DNAzymes.

    PubMed

    Zhu, Dan; Pei, Hao; Chao, Jie; Su, Shao; Aldalbahi, Ali; Rahaman, Mostafizur; Wang, Lihua; Wang, Lianhui; Huang, Wei; Fan, Chunhai; Zuo, Xiaolei

    2015-11-28

    Enzyme complexes are assembled at the two-dimensional lipid membrane or prearranged on three-dimensional scaffolding proteins to regulate their catalytic activity in cells. Inspired by nature, we have developed gold nanoparticle-based spherical DNAzymes (SNAzymes) with programmably engineered activities by exploiting poly-adenine (polyA)-Au interactions. In a SNAzyme, AuNPs serve as the metal core, which is decorated with a functional shell of DNAzymes. Conventional thiolated DNAzyme-based assembly leads to disordered structures with suppressed activity. In contrast, by using an anchoring block of polyA tails, we find that the activity of SNAzymes can be programmably regulated. By using a polyA30 tail, SNAzymes demonstrated remarkably enhanced binding affinity compared to the thiolated DNAzyme-based assembly (∼75-fold) or individual DNAzymes in the solution phase (∼10-fold). More significantly, this increased affinity is directly translated to the sensitivity improvement in the SNAzyme-based lead sensor. Hence, this design of SNAzymes may provide new opportunities for developing biosensors and bioimaging probes for theranostic applications. PMID:26498866

  18. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase*

    PubMed Central

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L.

    2015-01-01

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  19. Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes.

    PubMed

    Cahn, J K B; Baumschlager, A; Brinkmann-Chen, S; Arnold, F H

    2016-01-01

    NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli. PMID:26512129

  20. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase.

    PubMed

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L

    2015-07-10

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  1. Molecular dynamics simulations of creatine kinase and adenine nucleotide translocase in mitochondrial membrane patch.

    PubMed

    Karo, Jaanus; Peterson, Pearu; Vendelin, Marko

    2012-03-01

    Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies. PMID:22241474

  2. Dispensability of the [4Fe-4S] cluster in novel homologues of adenine glycosylase MutY.

    PubMed

    Trasviña-Arenas, Carlos H; Lopez-Castillo, Laura M; Sanchez-Sandoval, Eugenia; Brieba, Luis G

    2016-02-01

    7,8-Dihydro-8-deoxyguanine (8oG) is one of the most common oxidative lesions in DNA. DNA polymerases misincorporate an adenine across from this lesion. Thus, 8oG is a highly mutagenic lesion responsible for G:C→T:A transversions. MutY is an adenine glycosylase, part of the base excision repair pathway that removes adenines, when mispaired with 8oG or guanine. Its catalytic domain includes a [4Fe-4S] cluster motif coordinated by cysteinyl ligands. When this cluster is absent, MutY activity is depleted and several studies concluded that the [4Fe-4S] cluster motif is an indispensable component for DNA binding, substrate recognition and enzymatic activity. In the present study, we identified 46 MutY homologues that lack the canonical cysteinyl ligands, suggesting an absence of the [4Fe-4S] cluster. A phylogenetic analysis groups these novel MutYs into two different clades. One clade is exclusive of the order Lactobacillales and another clade has a mixed composition of anaerobic and microaerophilic bacteria and species from the protozoan genus Entamoeba. Structural modeling and sequence analysis suggests that the loss of the [4Fe-4S] cluster is compensated by a convergent solution in which bulky amino acids substitute the [4Fe-4S] cluster. We functionally characterized MutYs from Lactobacillus brevis and Entamoeba histolytica as representative members from each clade and found that both enzymes are active adenine glycosylases. Furthermore, chimeric glycosylases, in which the [4Fe-4S] cluster of Escherichia coli MutY is replaced by the corresponding amino acids of LbY and EhY, are also active. Our data indicates that the [4Fe-4S] cluster plays a structural role in MutYs and evidences the existence of alternative functional solutions in nature. PMID:26613369

  3. ADENYLATE ENERGY CHARGE AND ADENINE NUCLEOTIDE MEASUREMENTS AS INDICATORS OF STRESS IN THE MUSSEL, MYTILUS EDULIS, TREATED WITH DREDGED MATERIAL UNDER LABORATORY CONDITIONS

    EPA Science Inventory

    Adenylate energy charge is an indication of the amount of energy available to an organism from the adenylate pool. t is calculated from measured concentrations of three adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP...

  4. Overoxidized polypyrrole/graphene nanocomposite with good electrochemical performance as novel electrode material for the detection of adenine and guanine.

    PubMed

    Gao, Yan-Sha; Xu, Jing-Kun; Lu, Li-Min; Wu, Li-Ping; Zhang, Kai-Xin; Nie, Tao; Zhu, Xiao-Fei; Wu, Yao

    2014-12-15

    Most conducting polymer/graphene composites have excellent electrical conductivity. However, the background currents of these composites modified electrodes are much larger. In order to improve the sensitivities of these methods, it is necessary to decrease the background signal. In this paper, porous structure films of overoxidized polypyrrole/graphene (PPyox/GR) have been electrochemically coated onto glassy carbon electrode (GCE) and successfully utilized as an efficient electrode material for the quantitive detection of adenine and guanine, two of the most important components of DNA and RNA. The permselective polymer coatings with low background current could improve the selectivity and sensitivity of microelectrodes for the electropositive purine bases. The GRs into these polymers would further improve sensitivity by increasing the electroactive surface area. The electrochemical sensor can be applied to the quantification of adenine and guanine with a linear range covering 0.06-100 µM and 0.04-100 µM, and a low detection limit of 0.02 μM and 0.01 μM, respectively. More importantly, the proposed method was applied to quantify adenine and guanine in calf thymus DNA with satisfactory results. PMID:25022509

  5. Comparison of the structural and dynamic effects of 5-methylcytosine and 5-chlorocytosine in a CpG dinucleotide sequence†

    PubMed Central

    Theruvathu, Jacob A.; Yin, Whitney; Pettitt, B. Montgomery; Sowers, Lawrence C.

    2014-01-01

    Inflammation-mediated reactive molecules can result in an array of oxidized and halogenated DNA damage products including 5-chlorocytosine (ClC). Previous studies have shown that ClC can mimic 5-methylcytosine (mC) and act as a fraudulent epigenetic signal, promoting the methylation of previously unmethylated DNA sequences. Although the 5-halouracils are good substrates for base excision repair, no repair activity has yet been identified for ClC. Due to the apparent biochemical similarities of mC and ClC, we have investigated the effects of mC and ClC substitution on oligonucleotide structure and dynamics. In this study, we have constructed oligonucleotide duplexes containing C, ClC and mC within a CpG dinucleotide. The thermal and thermodynamic stability of these duplexes are found to be experimentally indistinguishable. Crystallographic structures of duplex oligonucleotides containing mC and ClC were determined to 1.2 and 1.9 Å, respectively. Both duplexes are B-form and are superimposable on a previously determined structure of a cytosine-containing duplex with RMSD of approximately 0.25 Å. NMR solution studies indicate that all duplexes containing cytosine or the cytosine analogs are normal B-form, and no structural perturbations are observed surrounding the site of each substitution. The magnitude of the base-stacking induced upfield shifts for non-exchangeable base proton resonances are similar for each of the duplexes examined, indicating that neither mC nor ClC significantly alter base stacking interactions. The ClC analog is paired with G in an apparently normal geometry; however the G-imino proton of the ClC-G base pair resonates to higher field relative to mC-G or C-G, indicating a weaker imino hydrogen bond. Using selective 15N-enrichment and isotope-edited NMR, we observe that the amino group of ClC rotates at roughly half the rate of the corresponding amino groups of the C-G or mC-G base pairs. The altered chemical shifts of hydrogen bonding proton

  6. Coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

    SciTech Connect

    Ipata, P.L.; Mura, U.; Camici, M.; Giovannitti, M.P.

    1987-08-01

    A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H/sub 2/O----adenosine + Pi (5'-nucleotidase); adenosine + H/sub 2/O----inosine + NH/sub 3/ (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into (/sup 14/C)inosine via the same combined reaction. Tissue extracts are incubated with excess (/sup 14/C)adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

  7. A new microplatform based on titanium dioxide nanofibers/graphene oxide nanosheets nanocomposite modified screen printed carbon electrode for electrochemical determination of adenine in the presence of guanine.

    PubMed

    Arvand, Majid; Ghodsi, Navid; Zanjanchi, Mohammad Ali

    2016-03-15

    The current techniques for determining adenine have several shortcomings such as high cost, high time consumption, tedious pretreatment steps and the requirements for highly skilled personnel often restrict their use in routine analytical practice. This paper describes the development and utilization of a new nanocomposite consisting of titanium dioxide nanofibers (TNFs) and graphene oxide nanosheets (GONs) for screen printed carbon electrode (SPCE) modification. The synthesized GONs and TNFs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). The modified electrode (TNFs/GONs/SPCE) was used for electrochemical characterization of adenine. The TNFs/GONs/SPCE exhibited an increase in peak current and the electron transfer kinetics and decrease in the overpotential for the oxidation reaction of adenine. Using differential pulse voltammetry (DPV), the prepared sensor showed good sensitivity for determining adenine in two ranges from 0.1-1 and 1-10 μM, with a detection limit (DL) of 1.71 nM. Electrochemical studies suggested that the TNFs/GONs/SPCE provided a synergistic augmentation on the voltammetric behavior of electrochemical oxidation of adenine, which was indicated by the improvement of anodic peak current and a decrease in anodic peak potential. The amount of adenine in pBudCE4.1 plasmid was determined via the proposed sensor and the result was in good compatibility with the sequence data of pBudCE4.1 plasmid. PMID:26556182

  8. Modulation by adenine nucleotides of epileptiform activity in the CA3 region of rat hippocampal slices

    PubMed Central

    Ross, F M; Brodie, M J; Stone, T W

    1998-01-01

    Hippocampal slices (450 μm) generate epileptiform bursts of an interictal nature when perfused with a zero magnesium medium containing 4-aminopyridine (50 μM). The effect of adenine nucleotides on this activity was investigated.ATP and adenosine depressed this epileptiform activity in a concentration-dependent manner, with both purines being equipotent at concentrations above 10 μM.Adenosine deaminase 0.2 u ml−1, a concentration that annuls the effect of adenosine (50 μM), did not significantly alter the depression of activity caused by ATP (50 μM).8-Cyclopentyl-1, 3-dimethylxanthine (CPT), an A1 receptor antagonist, enhanced the discharge rate significantly and inhibited the depressant effect of both ATP and adenosine such that the net effect of ATP or adenosine plus CPT was excitatory.Several ATP analogues were also tested: α, β-methyleneATP (α, β-meATP), 2-methylthioATP (2-meSATP) and uridine triphosphate (UTP). Only α, β-meATP (10 μM) produced an increase in the frequency of spontaneous activity which suggests a lack of involvement of P2Y or P2U receptors.Suramin and pyridoxalphosphate-6-azophenyl-2′, 4′-disulphonic acid (PPADS), P2 receptor antagonists, failed to inhibit the depression produced by ATP (50 μM). The excitatory effect of α, β-meATP (10 μM) was inhibited by suramin (50 μM) and PPADS (5 μM).ATP therefore depresses epileptiform activity in this model in a manner which is not consistent with the activation of known P1 or P2 receptors, suggesting the involvement of a xanthine-sensitive nucleotide receptor. The results are also indicative of an excitatory P2X receptor existing in the hippocampal CA3 region. PMID:9484856

  9. Inhibition of the adenine nucleotide translocator by N-acetyl perfluorooctane sulfonamides in vitro

    SciTech Connect

    O'Brien, Timothy M. Oliveira, Paulo J.; Wallace, Kendall B.

    2008-03-01

    N-alkyl perfluorooctane sulfonamides have been widely used as surfactants on fabrics and papers, fire retardants, and anti-corrosion agents, among many other commercial applications. The global distribution and environmental persistence of these compounds has generated considerable interest regarding potential toxic effects. We have previously reported that perfluorooctanesulfonamidoacetate (FOSAA) and N-ethylperfluorooctanesulfonamidoacetate (N-EtFOSAA) induce the mitochondrial permeability transition (MPT) in vitro. In this study we tested the hypothesis that FOSAA and N-EtFOSAA interact with the adenine nucleotide translocator (ANT) resulting in a functional inhibition of the translocator and induction of the MPT. Respiration and membrane potential of freshly isolated liver mitochondria from Sprague-Dawley rats were measured using an oxygen electrode and a tetraphenylphosphonium-selective (TPP{sup +}) electrode, respectively. Mitochondrial swelling was measured spectrophotometrically. The ANT ligands bongkregkic acid (BKA) and carboxyatractyloside (cATR) inhibited uncoupling of mitochondrial respiration caused by 10 {mu}M N-EtFOSAA, 40 {mu}M FOSAA, and the positive control 8 {mu}M oleic acid. ADP-stimulated respiration and depolarization of mitochondrial membrane potential were inhibited by cATR, FOSAA, N-EtFOSAA, and oleic acid, but not by FCCP. BKA inhibited calcium-dependent mitochondrial swelling induced by FOSAA, N-EtFOSAA, and oleic acid. Seventy-five micromolar ADP also inhibited swelling induced by the test compounds, but cATR induced swelling was not inhibited by ADP. Results of this investigation indicate that N-acetyl perfluorooctane sulfonamides interact directly with the ANT to inhibit ADP translocation and induce the MPT, one or both of which may account for the metabolic dysfunction observed in vivo.

  10. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue.

    PubMed

    Stanley, Robert J; Hou, Zhanjia; Yang, Aiping; Hawkins, Mary E

    2005-03-01

    6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes. PMID:16851408

  11. Vacuum-Ultraviolet photoionization studies of the microhydrationof DNA bases (Guanine, Cytosine, Adenine and Thymine)

    SciTech Connect

    Belau, L.; Wilson, K.R.; Leone, S.R.; Musahid, Ahmed

    2007-01-22

    In this work, we report on a photoionization study of the microhydration of the four DNA bases. Gas-phase clusters of water with DNA bases [guanine (G), cytosine (C), adenine (A), and thymine (T)] are generated via thermal vaporization of the bases and expansion of the resultant vapor in a continuous supersonic jet expansion of water seeded in Ar. The resulting clusters are investigated by single-photon ionization with tunable vacuum-ultraviolet synchrotron radiation and mass analyzed using reflectron mass spectrometry. Photoionization efficiency (PIE) curves are recorded for the DNA bases and the following water (W) clusters: G, GW{sub n} (n = 1-3); C, CW{sub n} (n = 1-3); A, AW{sub n} (n = 1,2); and T, TW{sub n} (n = 1-3). Appearance energies (AE) are derived from the onset of these PIE curves (all energies in eV): G (8.1 {+-} 0.1), GW (8.0 {+-} 0.1), GW{sub 2} (8.0 {+-} 0.1), and GW{sub 3} (8.0); C (8.65 {+-} 0.05), CW (8.45 {+-} 0.05), CW{sub 2} (8.4 {+-} 0.1), and CW{sub 3} (8.3 {+-} 0.1); A (8.30 {+-} 0.05), AW (8.20 {+-} 0.05), and AW{sub 2} (8.1 {+-} 0.1); T (8.90 {+-} 0.05); and TW (8.75 {+-} 0.05), TW{sub 2} (8.6 {+-} 0.1), and TW{sub 3} (8.6 {+-} 0.1). The AEs of the DNA bases decrease slightly with the addition of water molecules (up to three) but do not converge to values found for photoinduced electron removal from DNA bases in solution.

  12. Adenine nucleoside diphosphates block adaptation of mechanoelectrical transduction in hair cells.

    PubMed

    Gillespie, P G; Hudspeth, A J

    1993-04-01

    By adapting to sustained stimuli, hair cells in the internal ear retain their sensitivity to minute transient displacements. Because one model for adaptation asserts that this process is mediated by a myosin isozyme, we reasoned that we should be able to arrest adaptation by interfering with myosin's ATPase cycle though introduction of ADP into hair cells. During tight-seal, whole-cell recordings of transduction currents in cells isolated from bullfrog (Rana catesbeiana) sacculus, dialysis with 5-25 mM ADP gave variable results. In half of the cells examined, the rate of adaptation remained unchanged or even increased; adaptation was blocked in the remaining cells. Because we suspected that the variable effect of ADP resulted from the conversion of ADP to ATP by adenylate kinase, we employed the ADP analog adenosine 5'-[beta-thio]diphosphate (ADP[beta S]), which is not a substrate for adenylate kinase. Adaptation consistently disappeared in the presence of 1-10 mM ADP[beta S]; in addition, the transduction channels' open probability at rest grew from approximately 0.1 to 0.8 or more. Both effects could be reversed by 2 mM ATP. When used in conjunction with the adenylate kinase inhibitor P1,P5-bis(5'-adenosyl) pentaphosphate (Ap5A), ADP had effects similar to those of ADP[beta S]. These results suggest that adaptation by hair cells involves adenine nucleotides, and they lend support to the hypothesis that the adaptation process is powered by a myosin motor. PMID:8464880

  13. Deficiency in the mouse mitochondrial adenine nucleotide translocator isoform 2 gene is associated with cardiac noncompaction.

    PubMed

    Kokoszka, Jason E; Waymire, Katrina G; Flierl, Adrian; Sweeney, Katelyn M; Angelin, Alessia; MacGregor, Grant R; Wallace, Douglas C

    2016-08-01

    The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. PMID:27048932

  14. Synthesis and in vivo evaluation of prodrugs of 9-[2-(phosphonomethoxy)ethoxy]adenine.

    PubMed

    Serafinowska, H T; Ashton, R J; Bailey, S; Harnden, M R; Jackson, S M; Sutton, D

    1995-04-14

    A number of esters and amides of the anti-HIV nucleotide analogue 9-[2-(phosphonomethoxy)-ethoxy]adenine (1) have been synthesized as potential prodrugs and evaluated for oral bioavailability in mice. Dialkyl esters 17-20 were prepared via a Mitsunobu coupling of alcohols 8-11 with 9-hydroxypurine 12 whereas (acyloxy)alkyl esters 25-33 and bis-[(alkoxycarbonyl)methyl] and bis(amidomethyl) esters 34-39 were obtained by reaction of 1 with a suitable alkylating agent. Phosphonodichloridate chemistry was employed for the preparation of dialkyl and diaryl esters 42-65, and bis(phosphonoamidates) 66 and 67. Following oral administration to mice, most of the dialkyl esters 17-20 were well-absorbed and then converted to the corresponding monoesters, but minimal further metabolism to 1 occurred. Bis[(pivaloyloxy)methyl] ester 25 displayed an oral bioavailability of 30% that was 15-fold higher than the bioavailability observed after dosing of 1. Methyl substitution at the alpha carbon of the bis[(pivaloyloxy)methyl] ester 25 (33) increased the oral bioavailability of 1 to 74%. Some of the diaryl esters also showed improved absorption properties in comparison with that of 1. In particular, the crystalline hydrochloride salt of diphenyl ester 55 was well-absorbed and efficiently converted to the parent compound with an oral bioavailability of 50%. On the basis of these results as well as the physicochemical properties of the prodrugs and their stability in mouse duodenal contents, the hydrochloride salt of diphenyl ester 55 was identified as the preferred prodrug of 1. PMID:7731022

  15. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function.

    PubMed

    Reynafarje, B; Lehninger, A L

    1978-10-01

    This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP(4-) (out) for ADP(3-) (in) + 0.5 phosphate(2-) (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated. PMID:283393

  16. Two Adenine Nucleotide Translocase Paralogues Involved in Cell Proliferation and Spermatogenesis in the Silkworm Bombyx mori

    PubMed Central

    Sugahara, Ryohei; Jouraku, Akiya; Nakakura, Takayo; Kusakabe, Takahiro; Yamamoto, Takenori; Shinohara, Yasuo; Miyoshi, Hideto; Shiotsuki, Takahiro

    2015-01-01

    Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4. PMID:25742135

  17. Mitochondrial permeability transition as induced by cross-linking of the adenine nucleotide translocase.

    PubMed

    Zazueta, C; Reyes-Vivas, H; Zafra, G; Sánchez, C A; Vera, G; Chávez, E

    1998-04-01

    Mitochondrial permeability transition is caused by the opening of a transmembrane pore whose chemical nature has not been well established yet. The present work was aimed to further contribute to the knowledge of the membrane entity comprised in the formation of the non-specific channel. The increased permeability was established by analyzing the inability of rat kidney mitochondria to take up and accumulate Ca2+, as well as their failure to build up a transmembrane potential, after the cross-linking of membrane proteins by copper plus ortho-phenanthroline. To identify the cross-linked proteins, polyacrylamide gel electrophoresis was performed. The results are representative of at least three separate experiments. It is indicated that 30 microM Cu2+ induced the release of 4.3 nmol Ca2+ per mg protein. However, in the presence of 100 microM ortho-phenanthroline only 2 microM Cu2+ was required to attain the total release of the accumulated Ca2+; it should be noted that such a reaction is not inhibited by cyclosporin. The increased permeability corresponds to cross-linking of membrane proteins in which approximately 4 nmol thiol groups per mg protein appear to be involved. Such a linking process is inhibited by carboxyatractyloside. By using the fluorescent probe eosin-5-maleimide the label was found in a cross-linking 60 kDa dimer of two 30 kDa monomers. From the data presented it is concluded that copper-o-phenanthroline induces the intermolecular cross-linking of the adenine nucleotide translocase which in turn is converted to non-specific pore. PMID:9675885

  18. Development and Evaluation of Solid Lipid Nanoparticles of N-6-Furfuryl Adenine for Prevention of Photoaging.

    PubMed

    Goindi, Shishu; Guleria, Ankita; Aggarwal, Nidhi

    2015-10-01

    N-6-furfuryl adenine (N6FA) also known as "kinetin" is a biologically active natural phytochemical. It belongs to the category of cytokinins, the natural plant growth hormones that promote cell division and play role in cell differentiation. Overall, N6FA aids in increasing the plant's life span. Human cells also contain.small quantities of N6FA. Scientists are trying to understand its function in humans. N6FA is being investigated for its properties such as antiplatelet, antioxidant, antiproliferative and anti-aging effects on human cells. The aim of the present investigation was to prepare solid lipid nanoparticle (SLN) based topical formulations of N6FA and to evaluate its efficacy against ultraviolet (UV) radiation induced skin photodamage. SLNs were prepared by hot microemulsion technique and optimized for the type and concentration of lipid and surfactant(s). The optimized SLN formulation was characterized in terms of particle size, drug entrapment efficiency, zeta potential and pH; evaluated for stability, spreadability, ex-vivo skin permeation and photoprotective effects against UV induced skin damage. The cumulative amount of drug permeated through mice skin using SLNs was 3 folds higher than from conventional cream base. The results of biochemical and histopathological investigations of skin treated with N6FA loaded SLNs clearly demonstrated the efficacy of optimized formulation in preventing photodamage (lesions, ulcers and changes in skin integrity) due to chronic UV exposure. The effects were comparable with widely used marketed formulation, Garnier wrinkle lift anti-aging cream. Results suggested that N6FA incorporated into SLNs may provide therapeutic as well as cosmeceutical benefits. PMID:26502637

  19. Sodium thiosulfate protects brain in rat model of adenine induced vascular calcification.

    PubMed

    Subhash, N; Sriram, R; Kurian, Gino A

    2015-11-01

    Vascular bed calcification is a common feature of ends stage renal disease that may lead to a complication in cardiovascular and cerebrovascular beds, which is a promoting cause of myocardial infarction, stroke, dementia and aneurysms. Sodium thiosulfate (STS) due to its multiple properties such as antioxidant and calcium chelation has been reported to prevent vascular calcification in uremic rats, without mentioning its impact on cerebral function. Moreover, the previous studies have not explored the effect of STS on the mitochondrial dysfunction, one of the main pathophysiological features associated with the disease and the main site for STS metabolism. The present study addresses this limitation by using a rat model where 0.75% adenine was administered to induce vascular calcification and 400 mg/kg b wt. of STS was given as preventive and curative agent. The blood and urine chemistries along with histopathology of aorta confirms the renal protective effect of STS in two modes of administration. The brain oxidative stress assessment was made through TBARS level, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, found to be in the near normal level. STS administration not only reduced the mitochondrial oxidative stress (measured by TBARS, SOD, GPx and CAT) but also preserved the mitochondrial respiratory enzyme activities (NADH dehydrogenase, Succinate dehydrogenase and Malate dehydrogenase) and its physiology (measured by P/O ratio and RCR). In fact, the protective effect of STS was prominent, when it was administered as a curative agent, where low H2S and high thiosulfate level was observed along with low cystathionine β synthase activity, confirms thiosulfate mediated renal protection. In conclusion, STS when given after induction of calcification is protective to the brain by preserving its mitochondria, compared to the treatment given concomitantly. PMID:26363090

  20. Adenine Nucleotide Translocase 4 Is Expressed Within Embryonic Ovaries and Dispensable During Oogenesis

    PubMed Central

    Lim, Chae Ho; Brower, Jeffrey V.; Resnick, James L.; Oh, S. Paul

    2015-01-01

    Adenine nucleotide translocase (Ant) facilitates the exchange of adenosine triphosphate across the mitochondrial inner membrane and plays a critical role for bioenergetics in eukaryotes. Mice have 3 Ant paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5), and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. We previously identified that Ant4 was expressed exclusively in testicular germ cells in adult mice and essential for spermatogenesis and subsequently male fertility. Further investigation into the process of spermatogenesis revealed that Ant4 was particularly highly expressed during meiotic prophase I and indispensable for normal progression of leptotene spermatocytes to the stages thereafter. In contrast, the expression and roles of Ant4 in female germ cells have not previously been elucidated. Here, we demonstrate that the Ant4 gene is expressed during embryonic ovarian development during which meiotic prophase I occurs. We confirmed embryonic ovary-specific Ant4 expression using a bacterial artificial chromosome transgene. In contrast to male, however, Ant4 null female mice were fertile although the litter size was slightly decreased. They showed apparently normal ovarian development which was morphologically indistinguishable from the control animals. These data indicate that Ant4 is a meiosis-specific gene expressed during both male and female gametogenesis however indispensable only during spermatogenesis and not oogenesis. The differential effects of Ant4 depletion within the processes of male and female gametogenesis may be explained by meiosis-specific inactivation of the X-linked Ant2 gene in male, a somatic paralog of the Ant4 gene. PMID:25031318

  1. The recurrent causal mutation for osteogenesis imperfecta type V occurs at a highly methylated CpG dinucleotide within the IFITM5 gene

    PubMed Central

    Corradi, Massimiliano; Monti, Elena; Venturi, Giacomo; Gandini, Alberto; Mottes, Monica; Antoniazzi, Franco

    2014-01-01

    Recent studies have identified the molecular defect underlying autosomal dominant osteogenesis imperfecta (OI) type V. Unlike all other OI types, which are characterized by high genetic heterogeneity, OI type V appears consistently associated to a unique de novo C>T transition within the 5′ UTR of the IFITM5 gene. Although the precise frequency of OI type V is not known, this recurrent base substitution may well represent a mutational hotspot in the human genome. We show that it occurs at a CpG dinucleotide that is highly methylated in several tissues and particularly in the sperm DNA, suggesting a mutational mechanism common to other de novo recurrent dominant mutations.

  2. Modelling mutational and selection pressures on dinucleotides in eukaryotic phyla –selection against CpG and UpA in cytoplasmically expressed RNA and in RNA viruses

    PubMed Central

    2013-01-01

    Background Loss of CpG dinucleotides in genomic DNA through methylation-induced mutation is characteristic of vertebrates and plants. However, these and other eukaryotic phyla show a range of other dinucleotide frequency biases with currently uncharacterized underlying mutational or selection mechanisms. We developed a parameterized Markov process to identify what neighbour context-dependent mutations best accounted for patterns of dinucleotide frequency biases in genomic and cytoplasmically expressed mRNA sequences of different vertebrates, other eukaryotic groups and RNA viruses that infect them. Results Consistently, 11- to 14-fold greater frequencies of the methylation-associated mutation of C to T upstream of G (depicted as C→T,G) than other transitions best modelled dinucleotide frequencies in mammalian genomic DNA. However, further mutations such as G→T,T (5-fold greater than the default transversion rate) were required to account for the full spectrum of dinucleotide frequencies in mammalian sequence datasets. Consistent with modeling predictions for these two mutations, instability of both CpG and CpT dinucleotides was identified through SNP frequency analysis of human DNA sequences. Different sets of context-dependent mutations were modelled in other eukaryotes with non-methylated genomic DNA. In contrast to genomic DNA, best-fit models of dinucleotide frequencies in transcribed RNA sequences expressed in the cytoplasm from all organisms were dominated by mutations that eliminated UpA dinucleotides, observations consistent with cytoplasmically driven selection for mRNA stability. Surprisingly, mRNA sequences from organisms with methylated genomes showed evidence for additional selection against CpG through further context-dependent mutations (eg. C→A,G). Similar mutation or selection processes were identified among single-stranded mammalian RNA viruses; these potentially account for their previously described but unexplained under-representations of

  3. Dual role for adenine nucleotides in the regulation of the atrial natriuretic peptide receptor, guanylyl cyclase-A.

    PubMed

    Foster, D C; Garbers, D L

    1998-06-26

    The ability to both sensitize and desensitize a guanylyl cyclase receptor has not been previously accomplished in a broken cell or membrane preparation. The guanylyl cyclase-A (GC-A) receptor is known to require both atrial natriuretic peptide (ANP) and an adenine nucleotide for maximal cyclase activation. When membranes from NIH 3T3 cells stably overexpressing GC-A were incubated with ATP, AMPPNP, or ATPgammaS, only ATPgammaS dramatically potentiated ANP-dependent cyclase activity. When the membranes were incubated with ATPgammaS and then washed, GC-A now became sensitive to ANP/AMPPNP stimulation, suggestive that thiophosphorylation had sensitized GC-A to ligand and adenine nucleotide binding. Consistent with this hypo- thesis, the ATPgammaS effects were both time- and concentration-dependent. Protein phosphatase stability of thiophosphorylation (ATPgammaS) relative to phosphorylation (ATP) appeared to explain the differential effects of the two nucleotides since microcystin, beta-glycerol phosphate, or okadaic acid coincident with ATP or ATPgammaS effectively sensitized GC-A to ligand stimulation over prolonged periods of time in either case. GC-A was phosphorylated in the presence of [gamma32P]ATP, and the magnitude of the phosphorylation was increased by the addition of microcystin. Thus, the phosphorylation of GC-A correlates with the acquisition of ligand sensitivity. The establishment of an in vitro system to sensitize GC-A demonstrates that adenine nucleotides have a daul function in the regulation of GC-A through both phosphorylation of and binding to regulatory sites. PMID:9632692

  4. High-spin ferric ions in Saccharomyces cerevisiae vacuoles are reduced to the ferrous state during adenine-precursor detoxification.

    PubMed

    Park, Jinkyu; McCormick, Sean P; Cockrell, Allison L; Chakrabarti, Mrinmoy; Lindahl, Paul A

    2014-06-24

    The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) Fe(III) complexes with polyphosphate-related ligands. Some Fe(III) oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS Fe(III). Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 μM Fe(III) citrate) accumulated two major NHHS Fe(II) species as the vacuolar NHHS Fe(III) species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS Fe(II) complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS Fe(III) with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS Fe(III) species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated. PMID:24919141

  5. Investigation of the C-terminal domain of the bacterial DNA-(adenine N6)-methyltransferase CcrM.

    PubMed

    Maier, Johannes A H; Albu, Razvan F; Jurkowski, Tomasz P; Jeltsch, Albert

    2015-12-01

    CcrM-related DNA-(adenine N6)-methyltransferases play very important roles in the biology of Caulobacter crescentus and other alpha-proteobacteria. These enzymes methylate GANTC sequences, but the molecular mechanism by which they recognize their target sequence is unknown. We carried out multiple sequence alignments and noticed that CcrM enzymes contain a conserved C-terminal domain (CTD) which is not present in other DNA-(adenine N6)-methyltransferases and we show here that deletion of this part abrogates catalytic activity and DNA binding of CcrM. A mutational study identified 7 conserved residues in the CTD (out of 13 tested), mutation of which led to a strong reduction in catalytic activity. All of these mutants showed altered DNA binding, but no change in AdoMet binding and secondary structure. Some mutants exhibited reduced DNA binding, but others showed an enhanced DNA binding. Moreover, we show that CcrM does not specifically bind to DNA containing GANTC sequences. Taken together, these findings suggest that the specific CcrM-DNA complex undergoes a conformational change, which is endergonic but essential for catalytic activity and this step is blocked by some of the mutations. Moreover, our data indicate that the CTD of CcrM is involved in DNA binding and recognition. This suggests that the CTD functions as target recognition domain of CcrM and, therefore, CcrM can be considered the first example of a δ-type DNA-(adenine N6)-methyltransferase identified so far. PMID:26475175

  6. Nephrogenic systemic fibrosis-like effects of magnetic resonance imaging contrast agents in rats with adenine-induced renal failure.

    PubMed

    Fretellier, Nathalie; Bouzian, Nejma; Parmentier, Nadège; Bruneval, Patrick; Jestin, Gaëlle; Factor, Cécile; Mandet, Chantal; Daubiné, Florence; Massicot, France; Laprévote, Olivier; Hollenbeck, Claire; Port, Marc; Idée, Jean-Marc; Corot, Claire

    2013-01-01

    Nephrogenic systemic fibrosis (NSF) is a scleroderma-like disease associated with prior administration of certain gadolinium chelates (GCs). NSF occurs in patients with severe renal failure. The purpose of this study was to set up a rat model of GC-associated NSF to elucidate the mechanism of this devastating disease. Firstly, after characterization of the model, male Wistar rats received a 0.75% adenine-enriched diet for 8, 14, or 16 days to obtain various degrees of renal failure. Rats received five consecutive daily iv injections of saline or gadodiamide (2.5 mmol/kg/day). Secondly, the safety profile and in vivo propensity to dissociate of all categories of marketed GCs (gadoterate, gadobutrol, gadobenate, gadopentetate, and gadodiamide) were compared in rats receiving adenine-enriched diet for 16 days. Serial skin biopsies were performed for blinded histopathological study. Total Gd concentration in tissues was measured by Inductively Coupled Plasma Mass Spectrometry. Relaxometry was used to evaluate the presence of dissociated Gd in skin and bone. Gadodiamide-induced high mortality and skin lesions (dermal fibrosis, calcification, and inflammation) were related to adenine diet duration. No skin lesions were observed with other molecules. Unlike macrocyclic GCs, gadodiamide, gadopentetate, and gadobenate gradually increased the r(1) relaxivity value, consistent with in vivo dissociation and release of soluble Gd (or, in the case of gadobenate, the consequence of protein binding). Gadodiamide-induced cutaneous and systemic toxicity depended on baseline renal function. We demonstrate in vivo dissociation of linear GCs, gadodiamide, and gadopentetate, whereas macrocyclic agents remained stable over the study period. PMID:22977165

  7. Various cytosine/adenine permease homologues are involved in the toxicity of 5-fluorocytosine in Saccharomyces cerevisiae.

    PubMed

    Paluszynski, John P; Klassen, Roland; Rohe, Matthias; Meinhardt, Friedhelm

    2006-07-15

    5-Fluorocytosine (5-FC), a medically applied antifungal agent (Ancotil), is also active against the model organism Saccharomyces cerevisiae. 5-FC uptake in S. cerevisiae was considered to be mediated by the FCY2-encoded cytosine/adenine permease. By applying a highly sensitive assay, a low-level but dose-dependent toxicity of 5-FC in fcy2 mutants was detected, whereas cells deficient in the cytosine deaminase (encoded by FCY1), which is essential for intracellular conversion of 5-FC to 5-fluorouracil, display strong dose-independent resistance. Thus, an alternative, Fcy2-independent access pathway for 5-FC exists in S. cerevisiae. A genome-wide search for cytosine permease homologues identified two uncharacterized candidate genes, designated FCY21 and FCY22, both of which exhibit highest similarity to FCY2. Disruption of either FCY21 or FCY22 resulted in strains displaying low-level resistance, indicating the functional involvement of both gene products in 5-FC toxicity. When mutations in FCY21 or FCY22 were combined with the FCY2 disruption, both double mutants displayed stronger resistance when compared to the FCY2 mutant alone. Disruptions in all three permease genes consequently conferred the highest degree of resistance, not only towards 5-FC but also to the toxic adenine analogon 8-azaadenine. As residual 5-FC sensitivity was, however, even detectable in the fcy2 fcy21 fcy22 mutant, we analysed the relevance of other FCY2 homologues, i.e. TPN1, FUR4, DAL4, FUI1 and yOR071c, for 5-FC toxicity. Among these, Tpn1, Fur4 and the one encoded by yOR071c were found to contribute significantly to 5-FC toxicity, thus revealing alternative entry routes for 5-FC via other cytosine/adenine permease homologues. PMID:16845689

  8. Quantitative Subtractively Normalized Interfacial Fourier Transform Infrared Reflection Spectroscopy Study of the Adsorption of Adenine on Au(111) Electrodes.

    PubMed

    Prieto, Francisco; Su, Zhangfei; Leitch, J Jay; Rueda, Manuela; Lipkowski, Jacek

    2016-04-26

    Quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was used to determine the molecular orientation and identify the metal-molecular interactions responsible for the adsorption of adenine from the bulk electrolyte solution onto the surface of the Au(111) electrode. The recorded p-polarized IR spectra of the adsorbed species were subtracted from the collected s-polarized IR spectra to remove the IR contributions of the vibrational bands of the desorbed molecules that are located within the thin layer cavity of the spectroelectrochemical cell. The intense IR band around 1640 cm(-1), which is assigned to the pyrimidine ring stretching vibrations of the C5-C6 and C6-N10 bonds, and the IR band at 1380 cm(-1), which results from a combination of the ring stretching vibration of the C5-C7 bond and the in-plane CH bending vibration, were selected for the quantitative analysis measurements. The transition dipoles of these bands were evaluated by DFT calculations. Their orientations differed by 85 ± 5°. The tilt angles of adsorbed adenine molecules were calculated from the intensity of these two vibrations at different potentials. The results indicate that the molecular plane is tilted at an angle of 40° with respect to the surface normal of the electrode and rotates by 16° around its normal axis with increasing electrode potential. This orientation results from the chemical interaction between the N10 and gold atoms coupled with the π-π parallel stacking interactions between the adjacent adsorbed molecules. Furthermore, the changes in the molecular plane rotation with the electric field suggests that the N1 atom of adenine must also participate in the interaction between the molecule and metal. PMID:27040121

  9. Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate.

    PubMed

    Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís

    2016-05-23

    The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D-UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter-base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long-lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the (1) Lb , S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D-UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm(-1) in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D-UV spectroscopy to disentangle the photophysics of multichromophoric systems. PMID:27113273

  10. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

  11. Regioselective Palmitoylation of 9-(2,3-Dihydroxy- propyl)adenine Catalyzed by a Glycopolymer-enzyme Conjugate.

    PubMed

    Brabcová, Jana; Blažek, Jiří; Krečmerová, Marcela; Vondrášek, Jiří; Palomo, Jose M; Zarevúcka, Marie

    2016-01-01

    The enzymatic regioselective monopalmitoylation of racemic 9-(2,3-dihydroxypropyl)- adenine (DHPA), an approved antiviral agent, has been performed by an immobilized form of Candida antarctica B lipase (CAL-B) using a 4:1 DMF/hexane mixture as the reaction medium. To improve the chemical yield of the desired monopalmitoylation reaction, solid-phase chemical modifications of the lipase were evaluated. The reaction yield was successfully increased obtaining 100% product after a second treatment of the product solution with fresh immobilised chemically glycosylated-CAL-B. PMID:27196879

  12. Synthesis and biological properties of prodrugs of (S)-3-(adenin-9-yl)-2-(phosphonomethoxy)propanoic acid.

    PubMed

    Kaiser, Martin Maxmilian; Poštová-Slavětínská, Lenka; Dračínský, Martin; Lee, Yu-Jen; Tian, Yang; Janeba, Zlatko

    2016-01-27

    The lack of antiviral activity of recently described (S)-3-(adenin-9-yl)-2-(phosphonomethoxy)propanoic acid, or (S)-CPMEA in brief, has been speculated to possibly be due to the increased hydrophilicity of the molecule and, thus, by its limited cellular permeability. Efficient syntheses of novel lipophilic prodrugs of (S)-CPMEA masking either the carboxylic group or preferably both the phosphonate and carboxylic moieties, have been developed in order to increase bioavailability of the parent compound. Two prodrugs of (S)-CPMEA, namely phosphonate bis-amidate 15 and phenyloxy amidate 16, exhibited pan-genotypic anti-HCV activity at submicromolar concentrations. PMID:26706348

  13. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  14. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  15. High-mobility group box-1 protein in adenine-induced chronic renal failure and the influence of gum arabic thereon.

    PubMed

    Ali, B H; Al Za'abi, M; Al Shukaili, A; Nemmar, A

    2015-01-01

    Pathogenesis of adenine-induced chronic renal failure may involve inflammatory, immunological and/or oxidant mechanisms. Gum arabic (GA) is a complex polysaccharide that acts as an anti-oxidant which can modulate inflammatory and/or immunological processes. Therefore, we tested here the effect of GA treatment (15 % in the drinking water for 4 weeks) in plasma and urine of rats, on a novel cytokine that has been shown to be pro-inflammatory, viz, DNA-binding high-mobility group box-1 protein (HMGB1). Adenine (0.75 % in the feed, 4 weeks) significantly increased indoxyl sulphate, urea and creatinine concentrations in plasma, and significantly decreased the creatinine clearance. GA significantly abated these effects. The concentrations of HMGB1 in urine before the start of the experiment were similar in all four groups. However, 24 h after the last treatment, adenine treatment increased significantly the concentration of HMGB1 when compared with the control. GA treatment did not affect the HMGB1 concentration in urine. Moreover, the concentration of HMGB1 in plasma obtained 24 h after the last treatment in rats treated with adenine was drastically reduced compared with the control group. This may explain its significant rise in urine. In conclusion, HMGB1 can be considered a potentially useful biomarker in adenine induced CRF and its treatment. PMID:25194125

  16. Reactivity of nitrogen atoms in adenine and (Ade)2Cu complexes towards ribose and 2-furanmethanol: Formation of adenosine and kinetin.

    PubMed

    Nashalian, Ossanna; Yaylayan, Varoujan A

    2017-01-15

    To explore the interaction of nucleosides and nucleobases in the context of the Maillard reaction and to identify the selectivity of purine nitrogen atoms towards various electrophiles, model systems composed of adenine or adenosine, glycine, ribose and/or 2-furanmethanol (with and without copper) were studied in aqueous solutions heated at 110°C for 2h and subsequently analyzed by ESI/qTOF/MS/MS in addition to isotope labelling techniques. The results indicated that ribose selectively formed mono-ribosylated N(6) adenine, but in the presence of (Ade)2Cu complex the reaction mixture generated mono-, di- and tri-substituted sugar complexes and their hydrolysis products of mono-ribosylated N(6) and N(9) adenine adducts and di-ribosylated N(6,9) adenine. Furthermore, the reaction of 2-furanmethanol with adenine in the presence of ribose generated kinetin and its isomer, while its reaction with adenosine generated kinetin riboside, as confirmed by comparing the MS/MS profiles of these adducts to those of commercial standards. PMID:27542499

  17. A weak pulsed magnetic field affects adenine nucleotide oscillations, and related parameters in aggregating Dictyostelium discoideum amoebae.

    PubMed

    Davies, E; Olliff, C; Wright, I; Woodward, A; Kell, D

    1999-02-01

    A model eukaryotic cell system was used to explore the effect of a weak pulsed magnetic field (PMF) on time-varying physiological parameters. Dictyostelium discoideum cells (V12 strain) were exposed to a pulsed magnetic field (PMF) of flux density 0.4 mT, generated via air-cored coils in trains of 2 ms pulses gated at 20 ms. This signal is similar to those used to treat non-uniting fractures. Samples were taken over periods of 20 min from harvested suspensions of amoebae during early aggregation phase, extracted and derivatised for HPLC fluorescent assay of adenine nucleotides. Analysis of variance showed a significant athermal damping effect (P < 0.002, n = 22) of the PMF on natural adenine nucleotide oscillations and some consistent changes in phase relationships. The technique of nonlinear dielectric spectroscopy (NLDS) revealed a distinctive effect of PMF, caffeine and EGTA in modulating the cellular harmonic response to an applied weak signal. Light scattering studies also showed altered frequency response of cells to PMF, EGTA and caffeine. PMF caused a significant reduction of caffeine induced cell contraction (P < 0.0006, n = 19 by paired t-test) as shown by Malvern particle size analyser, suggesting that intracellular calcium may be involved in mediating the effect of the PMF. PMID:10228582

  18. Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae

    PubMed Central

    Rubio-Texeira, Marta; Varnum, James M; Bieganowski, Pawel; Brenner, Charles

    2002-01-01

    Background The FHIT gene is lost early in the development of many tumors. Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression. Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit. Which substrates are most important for Fhit signaling remain unknown. Results Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2. Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent. Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells. Conclusions The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2. PMID:12028594

  19. Localization of a hole on an adenine-thymine radical cation in B-form DNA in water.

    PubMed

    Kravec, S M; Kinz-Thompson, C D; Conwell, E M

    2011-05-19

    A quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulation has been carried out using CP2K for a hole introduced into a B-form DNA molecule consisting of 10 adenine-thymine (A/T) pairs in water. At the beginning of the simulation, the hole wave function is extended over several adenines. Within 20-25 fs, the hole wave function contracts so that it is localized on a single A. At 300 K, it stays on this A for the length of the simulation, several hundred fs, with the wave function little changed. In a range of temperatures below 300 K, proton transfer from A to T is seen to take place within the A/T occupied by the hole; it is completed by ∼40 fs after the contraction. We show that the contraction is due to polarization of the water by the hole. This polarization also plays a role in the proton transfer. Implications for transport are considered. PMID:21491917

  20. Bud endophytes of Scots pine produce adenine derivatives and other compounds that affect morphology and mitigate browning of callus cultures.

    PubMed

    Pirttilä, Anna Maria; Joensuu, Päivi; Pospiech, Helmut; Jalonen, Jorma; Hohtola, Anja

    2004-06-01

    Endophytes are found in meristematic bud tissues of Scots pine (Pinus sylvestris L.) especially prior to growth, which would suggest their involvement in growth of the bud. To test this hypothesis, production of phytohormones by two bacterial (Methylobacterium extorquens, Pseudomonas synxantha) and one fungal endophyte (Rhodotorula minuta) was studied by mass spectrometry. The most common gibberellins, auxins, or cytokinins were not detected in the fractions studied. Instead, M. extorquens and R. minuta produced adenine derivatives that may be used as precursors in cytokinin biosynthesis. A plant tissue culture medium was conditioned with the endophytes, and pine tissue cultures were started on the media. Tetracycline inhibited callus production, which was restored on the endophyte-conditioned media. In addition, conditioning mitigated browning of the Scots pine explants. However, a decrease in tissue size was observed on the endophyte-conditioned media. Addition of adenosine monophosphate in the plant culture medium restored callus production and increased growth of the tissues, but had no effect on browning. Therefore, production of adenine ribosides by endophytes may play some role in the morphological effect observed in the pine tissues. PMID:15153198

  1. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence

    PubMed Central

    James, Allison E.; Rogovskyy, Artem S.; Crowley, Michael A.; Bankhead, Troy

    2016-01-01

    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence. PMID:27195796

  2. Modified Iterative Extended Hueckel. 2: Application to the interaction of Na(+), Na(+)(aq.), Mg(+)-2(aq.) with adenine and thymine

    NASA Technical Reports Server (NTRS)

    Aronowitz, S.; Macelroy, R.; Chang, S.

    1980-01-01

    Modified Iterative Extended Hueckel, which includes explicit effective internuclear and electronic interactions, is applied to the study of the energetics of Na(+),Mg(+), Na(+) (aqueous), and Mg(+2) (aqueous) ions approaching various possible binding sites on adenine and thymine. Results for the adenine + ion and thymine + ion are in good qualitative agreement with ab initio work on analogous systems. Energy differences between competing sites are in excellent agreement. Hydration appears to be a critical factor in determining favorable binding sites. That the adenine Nl and N3 sites cannot displace a water molecule from the hydrated cation indicates that they are not favorable binding sites in aqueous media. Of those sites investigated, 04 was the most favorable binding site on the thymine for the bare Na(+). However, the 02 site was the most favorable binding site for either hydrated cation.

  3. Erythro-9-(2-hydroxy-3-nonyl) Adenine alone and in combination with 9-beta-D-arabinofuranosyladenine in treatment of systemic herpesvirus infections in mice.

    PubMed Central

    Shannon, W M; Arnett, G; Schabel, F M; North, T W; Cohen, S S

    1980-01-01

    Although the antiviral activity of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenosine deaminase inhibitor, against herpes simplex virus type 1 in cell culture was readily confirmed, the compound was found to be totally ineffective in the treatment of experimentally induced systemic herpes simplex virus type 1 infections in Swiss mice. Data were obtained, however, which clearly indicated that the antiviral potency of 9-beta-D-arabinofuranosyladenine in vivo could be enhanced by the co-administration of low, nontoxic doses of erythro-9-(2-hydroxy-3-nonyl)adenine. PMID:6255863

  4. Molecular diagnosis of Prader-Willi syndrome: Parent-of-origin dependent methylation sites and non-isotopic detection of (CA){sub n} dinucleotide repeat polymorphisms

    SciTech Connect

    Lerer, I.; Meiner, V.; Pashut-Lavon, I.; Abeliovich, D.

    1994-08-01

    We describe our experience in the molecular diagnosis of 22 patients suspected of Prader-Willi syndrome (PWS) using a DNA probe PW71 (D15S63) which detects a parent-of-origin specific methylated site in the PWS critical region. The cause of the syndrome was determined as deletion or uniparental disomy according to the segregation of (CA){sub n} dinucleotide repeat polymorphisms of the PWS/AS region and more distal markers of chromosome 15. In 10 patients the clinical diagnosis was confirmed by the segregation of (CA){sub n}, probably due to paternal microdeletion in the PWs critical region which did not include the loci D15S97, D15S113, GABRB3, and GABRA5. This case demonstrates the advantage of the DNA probe PW71 in the diagnosis of PWS. 31 refs., 2 figs., 3 tabs.

  5. A new dinucleotide repeat polymorphism at the telomere of chromosome 21q reveals a significant difference between male and female rates of recombination

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.

    1995-08-01

    We have used a half-YAC containing the human chromosome 21 long-arm telomere to clone, map, and characterize a new dinucleotide repeat polymorphism (D21S1575) close to 21qter. The marker is <120 kb from the telomeric (TTAGGG){sub n} sequences and is the most distal highly polymorphic marker on chromosome 21q. This marker has a heterozygosity of 71% because of a variable (TA){sub n} repeat embedded within a long interspersed element (LINE) element. Genotyping of the CEPH families and linkage analysis provided a more accurate determination of the full length of the chromosome 21 genetic map. A highly significant difference was detected between male and female recombination rates in the telomeric region: in the most telomeric 2.3 Mb of chromosome 21q, recombination was only observed in male meioses. 35 refs., 4 figs., 2 tabs.

  6. Preliminary evidence for an association of a dinucleotide repeat polymorphism at the MAOA gene with early onset alcoholism/substance abuse

    SciTech Connect

    Vanyukov, M.M.; Moss, H.B.; Tarter, R.E.

    1995-04-24

    An association between the liability to early onset alcoholism/substance abuse and a recently discovered dinucleotide repeat length polymorphism at the MAOA gene (MAOCA-1) was examined using polymerase chain reaction (PCR). A significant correlation between the presence/absence of the disorder and the length of the MAOCA-1 repeat was found in males, but not females, with {open_quotes}long{close_quotes} alleles (repeat length above 115 bp) associated with both increased risk for the disorder and lower age of onset of substance abuse. These preliminary data suggest that further exploration of the relationship between the MAOA gene and behavioral traits in an expanded sample is warranted. 22 refs., 1 fig., 3 tabs.

  7. Tissue-specific methylation of individual CpG dinucleotides in the 5{prime} upstream region of the mouse catalase gene (Cas-1)

    SciTech Connect

    Pillay, I.L.; Singh, S.M.

    1994-09-01

    The intracellular antioxidant enzyme, catalase, is encoded by a gene whose level of expression in different organisms, including humans, varies with tissue-type. The {open_quotes}TATA-less{close_quotes} 5{prime} upstream region of the catalase gene, in mice and humans, contains a CpG island. Such CG-rich regions are target sites for cytosine methylation and have been implicated in tissue-specific gene expression. However, the methylation status of individual CpG dinucleotides and their significance in gene expression has not been established. A 275 bp fragment within the 5{prime} region of Cas-1 was evaluated for CpG methylation. HpaII digestion of genomic DNA, followed by polymerase chain reaction amplification (HpaII-PCR), suggests that at least one of three CCGG is not methylated in nine different somatic tissues that express this enzyme at various levels. In contrast, all three CCGG sites are methylated in DNA from sperm and spleen. Further examination of the methylation specificity of individual CCGG sites was conducted using sodium bisulfite modification of genomic DNA followed by HPaII-PCR. Sodium bisulfite modifies non-methylated cytosines to uracils, changing a CG to a TG dinucleotide. This nucleotide substitution eliminates HpaII sites and allows the methylation status of each of the CCGG sites to be assessed. The ability to discern the number and combination of methylated sites within the 5{prime} region of a gene permits the determination of a possible correlation between differential methylation patterns and temporal/spatial gene regulation. Analysis of differential methylation, using the mouse catalase gene as a model, provides further insight into CpG methylation as one mechanism of mammalian gene regulation.

  8. Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

    PubMed Central

    Opie, L. H.; Mansford, K. R. L.; Owen, Patricia

    1971-01-01

    1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (`working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [14C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and 14CO2). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue

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

    PubMed Central

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

    1974-01-01

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

  10. Hydrogen peroxide formation photoinduced by near-UV radiation in aqueous solutions of adenine derivatives at 77 K

    NASA Astrophysics Data System (ADS)

    Lozinova, T. A.; Lobanov, A. V.; Lander, A. V.

    2015-08-01

    An estimate of the content of free radicals in aqueous solutions of adenosine (Ado), adenosine-5'-diphosphate (ADP) and guanosine-5'-monophosphate (GMP) irradiated with near-UV radiation at 77 K is obtained by interpreting EPR spectra. It is established that in the presence of NaCl (0.1 M), the total number of peroxyl radicals O{2/-·} and HO{2/·} in samples of the studied compounds was 15-45% of the total quantity of produced free radicals and was affected by the conditions of exposure. The estimates are compared with the results from hydrogen peroxide (H2O2) determination in the same samples after thawing. Although the number of peroxyl radicals in the samples of adenine derivatives (A) and GMP are comparable, the formation of H2O2 is observed only in the case of A derivatives, but not in GMP. Possible reasons for these differences are discussed.

  11. Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Zhi, Xuan; Han, Baoqin; Sui, Xianxian; Hu, Rui; Liu, Wanshun

    2015-02-01

    The effects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigated in vivo and in vitro. Chitosan were hydrolyzed using chitosanase at pH 6-7 and 37° for 24 h to obtain LMWC. In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For the in vivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the control group, indicating that the rat chronic renal failure model worked successfully. The results after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100 mg kg-1 d-1.

  12. Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation

    SciTech Connect

    Stephansen, Anne B.; King, Sarah B.; Li, Wei-Li; Kunin, Alice; Yokoi, Yuki; Minoshima, Yusuke; Takayanagi, Toshiyuki; Neumark, Daniel M.

    2015-09-14

    Dipole bound (DB) and valence bound (VB) anions of binary iodide-adenine complexes have been studied using one-color and time-resolved photoelectron imaging at excitation energies near the vertical detachment energy. The experiments are complemented by quantum chemical calculations. One-color spectra show evidence for two adenine tautomers, the canonical, biologically relevant A9 tautomer and the A3 tautomer. In the UV-pump/IR-probe time-resolved experiments, transient adenine anions can be formed by electron transfer from the iodide. These experiments show signals from both DB and VB states of adenine anions formed on femto- and picosecond time scales, respectively. Analysis of the spectra and comparison with calculations suggest that while both the A9 and A3 tautomers contribute to the DB signal, only the DB state of the A3 tautomer undergoes a transition to the VB anion. The VB anion of A9 is higher in energy than both the DB anion and the neutral, and the VB anion is therefore not accessible through the DB state. Experimental evidence of the metastable A9 VB anion is instead observed as a shape resonance in the one-color photoelectron spectra, as a result of UV absorption by A9 and subsequent electron transfer from iodide into the empty π-orbital. In contrast, the iodide-A3 complex constitutes an excellent example of how DB states can act as doorway state for VB anion formation when the VB state is energetically available.

  13. Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation

    NASA Astrophysics Data System (ADS)

    Stephansen, Anne B.; King, Sarah B.; Yokoi, Yuki; Minoshima, Yusuke; Li, Wei-Li; Kunin, Alice; Takayanagi, Toshiyuki; Neumark, Daniel M.

    2015-09-01

    Dipole bound (DB) and valence bound (VB) anions of binary iodide-adenine complexes have been studied using one-color and time-resolved photoelectron imaging at excitation energies near the vertical detachment energy. The experiments are complemented by quantum chemical calculations. One-color spectra show evidence for two adenine tautomers, the canonical, biologically relevant A9 tautomer and the A3 tautomer. In the UV-pump/IR-probe time-resolved experiments, transient adenine anions can be formed by electron transfer from the iodide. These experiments show signals from both DB and VB states of adenine anions formed on femto- and picosecond time scales, respectively. Analysis of the spectra and comparison with calculations suggest that while both the A9 and A3 tautomers contribute to the DB signal, only the DB state of the A3 tautomer undergoes a transition to the VB anion. The VB anion of A9 is higher in energy than both the DB anion and the neutral, and the VB anion is therefore not accessible through the DB state. Experimental evidence of the metastable A9 VB anion is instead observed as a shape resonance in the one-color photoelectron spectra, as a result of UV absorption by A9 and subsequent electron transfer from iodide into the empty π-orbital. In contrast, the iodide-A3 complex constitutes an excellent example of how DB states can act as doorway state for VB anion formation when the VB state is energetically available.

  14. Flow injection determination of adenine at trace level based on luminol-K2Cr2O7 chemiluminescence in a micellar medium.

    PubMed

    Erbao, Liu; Bingchun, Xue

    2006-05-01

    A novel flow injection chemiluminescence (CL) analysis method for the determination of adenine in the presence of sodium dodecylbenzene sulfonate (SDBS) surfactant micelles is described. This method is based on the luminescent properties of the luminol-K2Cr2O7-adenine in alkaline medium sensitized by SDBS. The optimized experimental conditions were evaluated and the possible mechanism was discussed by examining CL emission spectrum and the effect of various free radical scavengers on CL emission intensity. The CL increase is linearly related to the concentration of adenine in the range of 2.92x10(-6) to 4.38x10(-10) mol l-1 with a detection limit of 2.46x10(-10) mol l-1 (S/N=3). The relative standard deviation for 2.92x10(-7) mol l-1 samples was 1.67% (n=12). The proposed method has been applied to the determination of adenine in human serum. PMID:16413727

  15. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    PubMed

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-01-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. PMID:26132002

  16. Metal-organic frameworks and β-cyclodextrin-based composite electrode for simultaneous quantification of guanine and adenine in a lab-on-valve manifold.

    PubMed

    Wang, Yang; Chen, Huanhuan; Wu, Yichun; Ge, Huali; Ye, Guiqin; Hu, Xiaoya

    2014-12-01

    In this work, a novel chemically modified electrode is constructed based on metal-organic frameworks and β-cyclodextrin (Cu3(BTC)2/β-CD, BTC = benzene-1,3,5-tricarboxylate) composite material. The electrode was used for simultaneous determination of guanine and adenine in a sequential injection lab-on-valve format and exhibited sensitive responses to guanine and adenine oxidation due to the π-π stacking interaction of Cu3(BTC)2 and the inclusion behavior of β-CD. The analytical performance was assessed with respect to the supporting electrolyte and its pH, accumulation time and accumulation potential, and the fluid flow rates. Under optimal conditions, linear calibration ranges for both guanine and adenine were from 1.0 × 10(-7) to 1.0 × 10(-5) mol L(-1), and detection limits (S/N = 3) were found to be 5.2 × 10(-8) and 2.8 × 10(-8) mol L(-1), respectively. The proposed sensor showed advantages of high sensitivity, simple sample preparation protocol, enhanced throughput and good reproducibility. Finally, the practical application of the proposed sensor has been performed for the determination of guanine and adenine in real samples with satisfactory results. PMID:25299249

  17. Endogenously elevated bilirubin modulates kidney function and protects from circulating oxidative stress in a rat model of adenine-induced kidney failure

    PubMed Central

    Boon, Ai-Ching; Lam, Alfred K.; Gopalan, Vinod; Benzie, Iris F.; Briskey, David; Coombes, Jeff S.; Fassett, Robert G.; Bulmer, Andrew C.

    2015-01-01

    Mildly elevated bilirubin is associated with a reduction in the presence and progression of chronic kidney disease and related mortality, which may be attributed to bilirubin’s antioxidant properties. This study investigated whether endogenously elevated bilirubin would protect against adenine-induced kidney damage in male hyperbilirubinaemic Gunn rats and littermate controls. Animals were orally administered adenine or methylcellulose solvent (vehicle) daily for 10 days and were then monitored for 28 days. Serum and urine were assessed throughout the protocol for parameters of kidney function and antioxidant/oxidative stress status and kidneys were harvested for histological examination upon completion of the study. Adenine-treated animals experienced weight-loss, polyuria and polydipsia; however, these effects were significantly attenuated in adenine-treated Gunn rats. No difference in the presence of dihydroadenine crystals, lymphocytic infiltration and fibrosis were noted in Gunn rat kidneys versus controls. However, plasma protein carbonyl and F2-isoprostane concentrations were significantly decreased in Gunn rats versus controls, with no change in urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine or kidney tissue F2-isoprostane concentrations. These data indicated that endogenously elevated bilirubin specifically protects from systemic oxidative stress in the vascular compartment. These data may help to clarify the protective relationship between bilirubin, kidney function and cardiovascular mortality in clinical investigations. PMID:26498893

  18. DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments.

    PubMed

    Wu, Feinan; Olson, Brennan G; Yao, Jie

    2016-01-01

    The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq. PMID:26862720

  19. Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers.

    PubMed

    Spagnoletta, Anna; De Santis, Aurelio; Palmieri, Ferdinando; Genchi, Giuseppe

    2002-12-01

    The adenine nucleotide carrier from Jerusalem artichoke (Helianthus tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5'-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. Vmax of the reconstituted ATP/ATP exchange was determined to be 0.53 micromol/min per mg protein at 25 degrees C. The half-saturation constant Km and the corresponding inhibition constant Ki were 20.4 microM for ATP and 45 microM for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30 degrees C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots. PMID:12678438

  20. ATP production from adenine by a self-coupling enzymatic process: high-level accumulation under ammonium-limited conditions.

    PubMed

    Maruyama, A; Fujio, T

    2001-03-01

    To improve ATP production from adenine, we optimized cultivation and reaction conditions for the ATP producing strain, Corynebacterium ammoniagenes KY13510. In the conventional method, 28% NH4OH has been used both to adjust pH during cultivation and reaction, and to provide nitrogen for cell growth. In the ATP-producing reaction, high concentrations of inorganic phosphate and magnesium ion are needed, which form magnesium ammonium phosphate (MgNH4PO4) precipitate. To keep inorganic phosphate and magnesium ions soluble in the reaction mixture, it was indispensable to add phytic acid as a chelating agent of divalent metal ions. Under such conditions, 37 mg/ml (61.2 mM) ATP was accumulated in 13 h (Appl. Microbiol. Biotechnol. 21, 143 1985). If ammonium ion was depleted from the reaction mixture to avoid MgNH4 PO4 formation, we expected that there was no need to add phytic acid and ATP accumulation might be improved. Therefore, we obtained the cultured broth of C. ammoniagenes KY13510 strain with low ammonium ion content (less than 1 mg/ml as NH3) by the method that a part of alkali solution (28% NH4OH) for pH control was replaced with 10 N KOH. Using this culture broth, ATP producing reaction was done in 2-liter jar fermentor, controlling the pH of the reaction mixture with 10 N KOH. Under these conditions, the rate of ATP accumulation improved greatly, and 70.6 mg/ml (117 mM) ATP was accumulated in 28 h. The molar conversion ratio from adenine to ATP was about 82%. Phytic acid was slightly inhibitory to ATP formation under these ammonium-limited conditions. PMID:11330681

  1. A Poly Adenine-Mediated Assembly Strategy for Designing Surface-Enhanced Resonance Raman Scattering Substrates in Controllable Manners.

    PubMed

    Zhu, Ying; Jiang, Xiangxu; Wang, Houyu; Wang, Siyi; Wang, Hui; Sun, Bin; Su, Yuanyuan; He, Yao

    2015-07-01

    In this article, we introduce a Poly adenine (Poly A)-assisted fabrication method for rationally designing surface-enhanced resonance Raman scattering (SERRS) substrates in controllable and reliable manners, enabling construction of core-satellite SERRS assemblies in both aqueous and solid phase (e.g., symmetric core (Au)-satellite (Au) nanoassemblies (Au-Au NPs), and asymmetric Ag-Au NPs-decorated silicon wafers (Ag-Au NPs@Si)). Of particular significance, assembly density is able to be controlled by varying the length of the Poly A block (e.g., 10, 30, and 50 consecutive adenines at the 5' end of DNA sequence, Poly A10/A30/A50), producing the asymmetric core-satellite nanoassemblies with adjustable surface density of Au NPs assembly on core NPs surface. Based on quantitative interrogation of the relationship between SERRS performance and assemble density, the Ag-Au NPs@Si featuring the strongest SERRS enhancement factor (EF ≈ 10(7)) and excellent reproducibility can be achieved under optimal conditions. We further employ the resultant Ag-Au NPs@Si as a high-performance SERRS sensing platform for the selective and sensitive detection of mercury ions (Hg(2+)) in a real system, with a low detection limit of 100 fM, which is ∼5 orders of magnitude lower than the United States Environmental Protection Agency (USEPA)-defined limit (10 nM) in drinkable water. These results suggest the Poly A-mediated assembly method as new and powerful tools for designing high-performance SERRS substrates with controllable structures, facilitating improvement of sensitivity, reliability, and reproducibility of SERRS signals. PMID:26028356

  2. Differences between cystic fibrosis transmembrane conductance regulator and HisP in the interaction with the adenine ring of ATP.

    PubMed

    Berger, A L; Welsh, M J

    2000-09-22

    The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is a member of the ATP-binding cassette transporter family. The most conserved features of this family are the nucleotide-binding domains. As in other members of this family, these domains bind and hydrolyze ATP; in CFTR this opens and closes the channel pore. The recent crystal structures of related bacterial transporters show that an aromatic residue interacts with the adenine ring of ATP to stabilize nucleotide binding. CFTR contains six aromatic residues that are candidates to coordinate the nucleotide base. We mutated each to cysteine and examined the functional consequences. None of the mutations disrupted channel function or the ability to discriminate between ATP, GTP, and CTP. We also applied [2-(triethylammonium)ethyl] methanethiosulfonate to covalently modify the introduced cysteines. The mutant channels CFTR-F429C, F430C, F433C, and F1232C showed no difference from wild-type CFTR, indicating that either the residues were not accessible to modification, or cysteine modification did not affect function. Although modification inactivated CFTR-Y1219C more rapidly than wild-type CFTR, and inactivation of CFTR-F446C was nucleotide-dependent; failure of these mutations to alter gating suggested that Tyr(1219) and Phe(446) were not important for nucleotide binding. The results suggest that ATP binding may not involve the coordination of the adenine ring by an aromatic residue analogous to that in some bacterial transporters. Taken together with earlier work, this study points to a model in which most of the binding energy for ATP is contributed by the phosphate groups. PMID:10893239

  3. Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate.

    PubMed Central

    Hutton, J C; Penn, E J; Peshavaria, M

    1983-01-01

    The concentrations of Zn2+, Ca2+, Mg2+, Pi and adenine nucleotides were determined in insulin-secretory granules prepared from a transplantable rat insulinoma. Differential and density-gradient centrifugation analyses revealed that Zn2+ in this tissue was principally localized in the secretory granule, a second major fraction being found in association with cytosolic proteins. Pi was principally recovered in the latter fraction, whereas Ca2+ and Mg2+ were more widely distributed. Intragranular ion-distribution experiments suggested that Zn2+ was complexed mainly to insulin and its precursor forms and remained in the granule in an insoluble state. The Zn2+/insulin ratio (0.54) was greater than that expected for insulin molecules having two centrally co-ordinated Zn2+ atoms/hexamer, but less than the maximal Zn2+-binding capacity of the molecule. Most of the granular Ca2+, Mg2+ and Pi was released in a soluble form when granules were disrupted by sonication. Simulation in vitro of the ionic composition of the granule suggested that up to 90% of its Ca2+ was complexed to Pi and adenine nucleotides. Granular macromolecules also bound Ca2+, as shown by equilibrium-dialysis studies of granule lysates. However, such binding was displaced by Mg2+. Examination of the efflux of Ca2+ from granules incubated in iso-osmotic suspensions at 37 degrees C suggested that the passive permeability of the granule membrane to Ca2+ was very low. Nevertheless, more than 50% of the granular Ca2+ was rapidly released in an ionized form on hypo-osmotic or detergent-induced disruption of the granule membrane. This may represent a potentially mobilizable pool of Ca2+ in vivo. PMID:6344863

  4. Maturation of multisensory integration in the superior colliculus: Expression of nitric oxide synthase and neurofilament SMI-32

    PubMed Central

    Fuentes-Santamaria, Veronica; McHaffie, John G.; Stein, Barry E.

    2010-01-01

    Nitric oxide (NO) containing (nitrergic) interneurons are well-positioned to convey the cortical influences that are crucial for multisensory integration in superior colliculus (SC) output neurons. However, it is not known whether nitrergic interneurons are in this position early in life, and might, therefore, also play a role in the functional maturation of this circuit. In the present study, we investigated the postnatal developmental relationship between these two populations of neurons using B-nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH) histochemistry and SMI-32 immunocytochemistry to label presumptive interneurons and output neurons, respectively. SMI-32 immunostained neurons were proved to mature and retained immature anatomical features until approximately 8 postnatal weeks. In contrast, nitrergic interneurons developed more rapidly. They had achieved their adult-like anatomy by 4 postnatal weeks and were in a position to influence the dendritic elaboration of output neurons. It is this dendritic substrate through which much of the cortico-collicular influence is expressed. Double-labeling experiments showed that the dendritic and axonal processes of nitrergic interneurons already apposed the somata and dendrites of SMI-32 labeled neurons even at the earliest age examined. The results suggest that nitrergic interneurons play a role in refining the cortico-collicular projection patterns that are believed to be essential for SC output neurons to engage in multisensory integration and to support normal orientation responses to cross-modal stimuli. PMID:18486108

  5. Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB.

    PubMed

    Li, Deyu; Delaney, James C; Page, Charlotte M; Yang, Xuedong; Chen, Alvin S; Wong, Cintyu; Drennan, Catherine L; Essigmann, John M

    2012-05-30

    The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and α-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo. PMID:22512456

  6. Evaluation of the immune response and protective efficacy of Schistosoma mansoni Cathepsin B in mice using CpG dinucleotides as adjuvant.

    PubMed

    Ricciardi, Alessandra; Dalton, John P; Ndao, Momar

    2015-01-01

    Schistosomiasis is the most important human helminth infection due to its impact on public health. Worldwide, schistosomiasis is estimated to infect at least 200 million individuals while 700 million are at risk. The clinical manifestations are chronic and significantly decrease an individual's quality of life. Infected individuals suffer from long-term organ pathologies including fibrosis which eventually leads to organ failure. The development of a vaccine against this parasitic disease would contribute to a long-lasting decrease in disease spectrum and transmission. Our group has chosen to target Schistosoma mansoni Cathepsin B as a prospective vaccine candidate. The recombinant protein was tested in the presence of synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides, which are Toll-like receptor 9 agonists known to stimulate a Th1 response. This formulation conferred a 59% decrease in worm burden as well as a reduction in egg burden. Hepatic egg burden and intestinal egg burden were decreased by 56% and 54% respectively. Immunizations with the formulation elicited robust production of Sm-Cathepsin B specific antibodies, both IgG1 and IgG2c but with the latter predominating. Furthermore, splenocytes isolated from the immunized animals, compared to control animals, had increased secretion levels of key Th1 cytokines, IFN-γ and TNF-α, as well as the chemokine CCL5 when stimulated with recombinant Sm-Cathepsin B. These results highlight the potential of Sm-Cathepsin B/CpG as a vaccine candidate against schistosomiasis. PMID:25448114

  7. Deep intron elements mediate nested splicing events at consecutive AG dinucleotides to regulate alternative 3' splice site choice in vertebrate 4.1 genes.

    PubMed

    Parra, Marilyn K; Gallagher, Thomas L; Amacher, Sharon L; Mohandas, Narla; Conboy, John G

    2012-06-01

    Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3' splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explored intrasplicing in other normal and disease gene contexts and found conservation of intrasplicing through vertebrate evolution. In the paralogous 4.1B gene, we identified ∼120 kb upstream of E2 an ultradistal intraexon, iE(B), that mediates intrasplicing by promoting two intricately coupled splicing events that ensure selection of a weak distal acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iE(B) in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iE(B) function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant 4.1R gene lacking E2 through E4, we showed that aberrant splicing is consistent with iE(R)-mediated intrasplicing at the first available exons downstream of iE(R), namely, alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference for nested 3' splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing at a subset of alternative 3' splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity. PMID:22473990

  8. Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation

    PubMed Central

    Chery, Jessica; Siggers, Trevor; Boor, Sonia; Bliss, Jacob; Liu, Wei; Jogl, Gerwald; Rohs, Remo; Singh, Nadia D.; Bulyk, Martha L.; Tolstorukov, Michael Y.; Larschan, Erica

    2016-01-01

    Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression. PMID:27414415

  9. Fluorescence polarization-based method with bisulfite conversion-specific one-label extension for quantification of single CpG dinucleotide methylation.

    PubMed

    Li, Shufen; Wang, Zhongju; Zhou, Lin; Luo, Fu; Zhao, Cunyou

    2015-07-01

    To quantify the methylation at individual CpG dinucleotide sites in large biological or clinical samples, we developed a bisulfite conversion-specific one-label extension (BS-OLE) method using visualization by fluorescence polarization (FP) measurement of methylation at single CpG sites in small amounts of genomic DNA. Genomic DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil leaving 5-methylcytosine unaltered, and BS-PCR was used to generate DNA template containing target CpG sites. BS-OLE uses a BS-primer hybridized immediately upstream of the target CpG site being examined and then fluorescent dCTP or dUTP is incorporated into the methylated (CpG) or unmethylated (TpG) form of the target site through single-nucleotide chain extension, yielding an FP ratio between the fluorescent dCTP- and dUTP-incorporated products as a measure of methylation. This provides stable estimates of the methylation level of human genomic DNA and of a 250-bp plasmid DNA segment containing a single TCGA TaqI cleavage site, in accordance with the results of a combined bisulfite restriction analysis method. We used BS-OLE to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA methyltransferase inhibitor 5-aza-dC. BS-OLE is well suited to high-throughput multi-sample applications in biological and medical studies. PMID:26334496

  10. The rates of G:C[yields]T:A and G:C[yields]C:G transversions at CpG dinucleotides in the human factor IX gene

    SciTech Connect

    Ketterling, R.P.; Vielhaber, E.; Sommer, S.S. )

    1994-05-01

    The authors have identified eight independent transversions at CpG in 290 consecutive families with hemophilia B. These eight transversions account for 16.3% of all independent transversions in the sample, yet the expected frequency of CpG transversions at random in the factor IX gene is only 2.6% (P<0.1). The aggregate data suggest that the two types of CpG transversions (G:C[yields]T:A and G:C[yields]C:G) possess similar mutation rates (24.8 [times] 10[sup [minus]10] and 20.6 [times] 10[sup [minus]10], respectively), which are about fivefold greater than the comparable rates for transversions at non-CpG dinucleotides. The enhancement of transversions at CpG suggest that the model by which mutations occur at CpG may need to be reevaluated. The relationship, if any, between deamination of 5-methyl cytosine and enhancement of transversions at CpG remains to be defined. 28 refs., 2 tabs.

  11. Rheological kinetics of thermo-sensitive supramolecular assemblies from poly( N-isopropyl acrylamide) and adenine-functionalized poly(ethylene oxide) stabilized by complementary multiple hydrogen bonds

    NASA Astrophysics Data System (ADS)

    Cui, Hui-Wang; Kuo, Shiao-Wei

    2014-05-01

    In this study, we synthesized a poly( N-isopropylacrylamide) (PNIPAm) through the polymerization of N-isopropylacrylamide in distilled water with azodiisobutyronitrile as the initiator and a bisadenine-functionalized poly(ethylene oxide) (A-PEO-A) from the reaction of adenine with a difunctionalized toluenesulfonyl-PEO. When blended together in distilled water, PNIPAm and A-PEO-A formed supramolecular aggregates stabilized through complementary multiple hydrogen bonds between the amide groups of PNIPAm and the adenine units of A-PEO-A. Agrawal integral equation and rheometry revealed the rheological kinetics of supramolecular assemblies, which were influenced significantly by the spherical micelles, large associated aggregates of spherical micelles, network structures, and toroid structures formed in aqueous solutions.

  12. The adenosine deaminase inhibitor erythro-9-[2-hydroxyl-3-nonyl]-adenine decreases intestinal permeability and protects against experimental sepsis: a prospective, randomised laboratory investigation

    PubMed Central

    Kayhan, Nalan; Funke, Benjamin; Conzelmann, Lars Oliver; Winkler, Harald; Hofer, Stefan; Steppan, Jochen; Schmidt, Heinfried; Bardenheuer, Hubert; Vahl, Christian-Friedrich; Weigand, Markus A

    2008-01-01

    Introduction The treatment of septic conditions in critically ill patients is still one of medicine's major challenges. Cyclic nucleotides, adenosine and its receptors play a pivotal role in the regulation of inflammatory responses and in limiting inflammatory tissue destruction. The aim of this study was to verify the hypothesis that adenosine deaminase-1 and cyclic guanosine monophosphate-stimulated phosphodiesterase inhibition by erythro-9-[2-hydroxyl-3-nonyl]-adenine could be beneficial in experimental endotoxicosis/sepsis. Method We used two established animal models for endotoxicosis and sepsis. Twenty-four male Wistar rats that had been given intravenous endotoxin (Escherichia coli lipopolysaccharide) were treated with either erythro-9-[2-hydroxyl-3-nonyl]-adenine infusion or 0.9% saline during a study length of 120 minutes. Sepsis in 84 female C57BL/6 mice was induced by caecal ligation and puncture. Animals were treated with repeated erythro-9-[2-hydroxyl-3-nonyl]-adenine injections after 0, 12 and 24 hours or 4, 12 and 24 hours for delayed treatment. Results In endotoxaemic rats, intestinal production of hypoxanthine increased from 9.8 +/- 90.2 μmol/l at baseline to 411.4 +/- 124.6 μmol/l and uric acid formation increased from 1.5 +/- 2.3 mmol/l to 13.1 +/- 2.7 mmol/l after 120 minutes. In endotoxaemic animals treated with erythro-9-[2-hydroxyl-3-nonyl]-adenine, we found no elevation of adenosine metabolites. The lactulose/L-rhamnose ratio (14.3 versus 4.2 in control animals; p = 2.5 × 10-7) reflects a highly permeable small intestine and through the application of erythro-9-[2-hydroxyl-3-nonyl]-adenine, intestinal permeability could be re-established. The lipopolysaccharide animals had decreased L-rhamnose/3-O-methyl-D-glucose urine excretion ratios. Erythro-9-[2-hydroxyl-3-nonyl]-adenine reduced this effect. The mucosa damage score of the septic animals was higher compared with control and therapy animals (p < 0.05). Septic shock induction by caecal

  13. The effect of antidepressive drugs and some related compounds on the levels of adenine nucleotides, inorganic phosphate and phosphocreatine in the rat brain

    PubMed Central

    Lewis, J. J.; Van Petten, G. R.

    1963-01-01

    The effects upon levels of adenine nucleotides, phosphocreatine and inorganic phosphate of iproniazid, isoniazid, phenelzine, pheniprazine, tranylcypromine, harmine, imipramine, amitriptyline, orphenadrine, diphenhydramine and cocaine have been studied. With the exception of harmine and diphenhydramine, each of these compounds increased the brain level of adenosine triphosphate and, with the exception of imipramine and cocaine, the level of adenosine diphosphate decreased. Harmine had no effect on levels of adenine nucleotides and, in the case of diphenhydramine, the level of adenosine diphosphate increased and the level of adenosine triphosphate tended to decrease. There appears to be a relationship between the ability of the drugs to cause behavioural signs of central nervous stimulation and to produce an increase in the adenosine triphosphate/diphosphate ratio. This effect may be a factor in the action of antidepressive drugs. PMID:19108178

  14. Theoretical Investigation on the Substituent Effect of Halogen Atoms at the C8 Position of Adenine: Relative Stability, Vibrational Frequencies, and Raman Spectra of Tautomers.

    PubMed

    Chen, Yan-Li; Wu, De-Yin; Tian, Zhong-Qun

    2016-06-16

    We have theoretically investigated the substituent effect of adenine at the C8 position with a substituent X = H, F, Cl, and Br by using the density functional theory (DFT) at the B3LYP/6-311+G(d, p) level. The aim is to study the substituent effect of halogen atoms on the relative stability, vibrational frequencies, and solvation effect of tautomers. Our calculated results show that for substituted adenine molecules the N9H8X tautomer to be the most stable structure in gas phase at the present theoretical level. Here N9H8X denotes the hydrogen atom binds to the N9 position of imidazole ring and X denotes H, F, Cl, and Br atoms. The influence of the induced attraction of the fluorine substituent is significantly larger than chlorine and bromine ones. The halogen substituent effect has a significant influence on changes of vibrational frequencies and Raman intensities. PMID:27243104

  15. [Dependence of creatine kinase and glycogen synthetase activities of skeletal muscles on state of adenine nucleotide phosphorylation and cAMP metabolism].

    PubMed

    Iakovlev, N N; Chagovets, N R; Maksimova, L V

    1980-01-01

    Changes in the contents of adenine nucleotides, creatine phosphate, inorganic phosphate, creatine, glucose-6-phosphate and glycogen and the activity of adenylate cyclase, creatine kinase, glycogen phosphorylase 31:51-AMP-phosphodiesterase and glycogen synthetase in muscles and of blood catecholamines were studied in adult rats before loading, immediately after the cessation of the muscular activity, and at rest. Adenine nucleotides are established to play a regulatory role in catabolic and anabolic processes nucleotides are established to play a regulatory role in catabolic and anabolic processes related to the muscular activity. It is established that compensation and supercompensation of the working losses of muscular creatine phosphate and glycogen are due to activation of anabolic processes under conditions of higher phosphorylation of the adenylic system. PMID:6247797

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

    PubMed Central

    Cusack, N. J.; Planker, M.

    1979-01-01

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

  17. Multi-pathway excited state relaxation of adenine oligomers in aqueous solution: a joint theoretical and experimental study.

    PubMed

    Banyasz, Akos; Gustavsson, Thomas; Onidas, Delphine; Changenet-Barret, Pascale; Markovitsi, Dimitra; Improta, Roberto

    2013-03-11

    The singlet excited states of adenine oligomers, model systems widely used for the understanding of the interaction of ultraviolet radiation with DNA, are investigated by fluorescence spectroscopy and time-dependent (TD) DFT calculations. Fluorescence decays, fluorescence anisotropy decays, and time-resolved fluorescence spectra are recorded from the femtosecond to the nanosecond timescales for single strand (dA)20 in aqueous solution. These experimental observations and, in particular, the comparison of the fluorescence behavior upon UVC and UVA excitation allow the identification of various types of electronic transitions with different energy and polarization. Calculations performed for up to five stacked 9-methyladenines, taking into account the solvent, show that different excited states are responsible for the absorption in the UVC and UVA spectral domains. Independently of the number of bases, bright excitons may evolve toward two types of excited dimers having π-π* or charge-transfer character, each one distinguished by its own geometry and spectroscopic signature. According to the picture arising from the joint experimental and theoretical investigation, UVC-induced fluorescence contains contribution from 1) exciton states with a different degree of localization, decaying within a few ps, 2) "neutral" excited dimers decaying on the sub-nanosecond timescale, being the dominant species, and 3) charge-transfer states decaying on the nanosecond timescale. The majority of the photons emitted upon UVA excitation are related to charge-transfer states. PMID:23335234

  18. The silencing of adenine nucleotide translocase isoform 1 induces oxidative stress and programmed cell death in ADF human glioblastoma cells.

    PubMed

    Lena, Annalisa; Rechichi, Mariarosa; Salvetti, Alessandra; Vecchio, Donatella; Evangelista, Monica; Rainaldi, Giuseppe; Gremigni, Vittorio; Rossi, Leonardo

    2010-07-01

    Adenine nucleotide translocases (ANTs) are multitask proteins involved in several aspects of cell metabolism, as well as in the regulation of cell death/survival processes. We investigated the role played by ANT isoforms 1 and 2 in the growth of a human glioblastoma cell line (ADF cells). The silencing of ANT2 isoform, by small interfering RNA, did not produce significant changes in ADF cell viability. By contrast, the silencing of ANT1 isoform strongly reduced ADF cell viability by inducing a non-apoptotic cell death process resembling paraptosis. We demonstrated that cell death induced by ANT1 depletion cannot be ascribed to the loss of the ATP/ADP exchange function of this protein. By contrast, our findings indicate that ANT1-silenced cells experience oxidative stress, thus allowing us to hypothesize that the effect of ANT1-silencing on ADF is mediated by the loss of the ANT1 uncoupling function. Several studies ascribe a pro-apoptotic role to ANT1 as a result of the observation that ANT1 overexpression sensitizes cells to mitochondrial depolarization or to apoptotic stimuli. In the present study, we demonstrate that, despite its pro-apoptotic function at a high expression level, the reduction of ANT1 density below a physiological baseline impairs fundamental functions of this protein in ADF cells, leading them to undertake a cell death process. PMID:20528917

  19. How the CCA-Adding Enzyme Selects Adenine over Cytosine at Position 76 of tRNA

    SciTech Connect

    B Pan; Y Xiong; T Steitz

    2011-12-31

    CCA-adding enzymes [ATP(CTP):tRNA nucleotidyltransferases] add CCA onto the 3' end of transfer RNA (tRNA) precursors without using a nucleic acid template. Although the mechanism by which cytosine (C) is selected at position 75 of tRNA has been established, the mechanism by which adenine (A) is selected at position 76 remains elusive. Here, we report five cocrystal structures of the enzyme complexed with both a tRNA mimic and nucleoside triphosphates under catalytically active conditions. These structures suggest that adenosine 5'-monophosphate is incorporated onto the A76 position of the tRNA via a carboxylate-assisted, one-metal-ion mechanism with aspartate 110 functioning as a general base. The discrimination against incorporation of cytidine 5'-triphosphate (CTP) at position 76 arises from improper placement of the {alpha} phosphate of the incoming CTP, which results from the interaction of C with arginine 224 and prevents the nucleophilic attack by the 3' hydroxyl group of cytidine75.

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

    PubMed

    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. PMID:25605536

  1. Genetic mapping of human heart-skeletal muscle adenine nucleotide translocator and its relationship to the facioscapulohumeral muscular dystrophy locus

    SciTech Connect

    Haraguchi, Y.; Chung, A.B.; Torroni, A.; Stepien, G.; Shoffner, J.M.; Costigan, D.A.; Polak, M.; Wasmuth, J.J.; Altherr, M.R.; Winokur, S.T.

    1993-05-01

    The mitochondrial heart-skeletal muscle adenine nucleotide translocator (ANT1) was regionally mapped to 4q35-qter using somatic cell hybrids containing deleted chromosome 4. The regional location was further refined through family studies using ANT1 intron and promoter nucleotide polymorphisms recognized by the restriction endonucleases MboII, NdeI, and HaeIII. Two alleles were found, each at a frequency of 0.5. The ANT1 locus was found to be closely linked to D4S139, D4S171, and the dominant skeletal muscle disease locus facioscapulohumeral muscular dystrophy (FSHD). A crossover that separated D4S171 and ANT1 from D4S139 was found. Since previous studies have established the chromosome 4 map order as centromere-D4S171-D4S139-FSHD, it was concluded that ANT1 is located on the side of D4S139, that is opposite from FSHD. This conclusion was confirmed by sequencing the exons and analyzing the transcripts of ANT1 from several FSHD patients and finding no evidence of aberration. 35 refs., 5 figs., 1 tab.

  2. Thermodynamic Potential for the Abiotic Synthesis of Adenine, Cytosine, Guanine, Thymine, Uracil, Ribose, and Deoxyribose in Hydrothermal Systems

    NASA Astrophysics Data System (ADS)

    Larowe, Douglas E.; Regnier, Pierre

    2008-10-01

    The thermodynamic potential for the abiotic synthesis of the five common nucleobases (adenine, cytosine, guanine, thymine, and uracil) and two monosaccharides (ribose and deoxyribose) from formaldehyde and hydrogen cyanide has been quantified under temperature, pressure, and bulk composition conditions that are representative of hydrothermal systems. The activities of the precursor molecules (formaldehyde and hydrogen cyanide) required to evaluate the thermodynamics of biomolecule synthesis were computed using the concentrations of aqueous N2, CO, CO2 and H2 reported in the modern Rainbow hydrothermal system. The concentrations of precursor molecules that can be synthesized are strongly dependent on temperature with larger concentrations prevailing at lower temperatures. Similarly, the thermodynamic drive to synthesize nucleobases, ribose and deoxyribose varies considerably as a function of temperature: all of the biomolecules considered in this study are thermodynamically favored to be synthesized throughout the temperature range from 0°C to between 150°C and 250°C, depending on the biomolecule. Furthermore, activity diagrams have been generated to illustrate that activities in the range of 10-2- 10-6 for nucleobases, ribose and deoxyribose can be in equilibrium with a range of precursor molecule activities at 150°C and 500 bars. The results presented here support the notion that hydrothermal systems could have played a fundamental role in the origin of life, and can be used to plan and constrain experimental investigation of the abiotic synthesis of nucleic-acid related biomolecules.

  3. In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to alpha 1-adrenoceptor stimulation.

    PubMed Central

    Shinozuka, K; Hashimoto, M; Masumura, S; Bjur, R A; Westfall, D P; Hattori, K

    1994-01-01

    1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature. PMID:7889273

  4. Adenine derivatives as phosphate-activating groups for the regioselective formation of 3',5'-linked oligoadenylates on montmorillonite: possible phosphate-activating groups for the prebiotic synthesis of RNA

    NASA Technical Reports Server (NTRS)

    Prabahar, K. J.; Ferris, J. P.

    1997-01-01

    Methyladenine and adenine N-phosphoryl derivatives of adenosine 5'-monophosphate (5'-AMP) and uridine 5'-monophosphate (5'-UMP) are synthesized, and their structures are elucidated. The oligomerization reactions of the adenine derivatives of 5'-phosphoramidates of adenosine on montmorillonite are investigated. 1-Methyladenine and 3-methyladenine derivatives on montmorillonite yielded oligoadenylates as long as undecamer, and the 2-methyladenine and adenine derivatives on montmorillonite yielded oligomers up to hexamers and pentamers, respectively. The 1-methyladenine derivative yielded linear, cyclic, and A5'ppA-derived oligonucleotides with a regioselectivity for the 3',5'-phosphodiester linkages averaging 84%. The effect of pKa and amine structure of phosphate-activating groups on the montmorillonite-catalyzed oligomerization of the 5'-phosphoramidate of adenosine are discussed. The binding and reaction of methyladenine and adenine N-phosphoryl derivatives of adenosine are described.

  5. Proton-impact ionization cross sections of adenine measured at 0.5 and 2.0 MeV by electron spectroscopy

    SciTech Connect

    Iriki, Y.; Kikuchi, Y.; Imai, M.; Itoh, A.

    2011-11-15

    Double-differential ionization cross sections (DDCSs) of vapor-phase adenine molecules (C{sub 5}H{sub 5}N{sub 5}) by 0.5- and 2.0-MeV proton impact have been measured by the electron spectroscopy method. Electrons ejected from adenine were analyzed by a 45 Degree-Sign parallel-plate electrostatic spectrometer over an energy range of 1.0-1000 eV at emission angles from 15 Degree-Sign to 165 Degree-Sign . Single-differential cross sections (SDCSs) and total ionization cross sections (TICSs) were also deduced. It was found from the Platzman plot, defined as SDCSs divided by the classical Rutherford knock-on cross sections per target electron, that the SDCSs at higher electron energies are proportional to the total number of valence electrons (50) of adenine, while those at low-energy electrons are highly enhanced due to dipole and higher-order interactions. The present results of TICS are in fairly good agreement with recent classical trajectory Monte Carlo calculations, and moreover, a simple analytical formula gives nearly equivalent cross sections in magnitude at the incident proton energies investigated.

  6. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model.

    PubMed

    Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang

    2016-07-01

    Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules. PMID:27394096

  7. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model

    NASA Astrophysics Data System (ADS)

    Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang

    2016-07-01

    Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

  8. Adenine-DNA adducts derived from the highly tumorigenic dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not

    PubMed Central

    Kropachev, Konstantin; Kolbanovskiy, Marina; Liu, Zhi; Cai, Yuqin; Zhang, Lu; Schwaid, Adam G.; Kolbanovskiy, Alexander; Ding, Shuang; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2013-01-01

    The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N2-dG) and adenine (N6-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N2-dG linkage site is ~ 35 times more susceptible to NER dual incisions than the stereochemically identical N6-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar, but somewhat smaller effect (factor of ~15) is observed. The striking resistance of the bulky N6-dA in contrast to the modest to good susceptibilities of the N2-dG adducts to NER are interpreted in terms of the balance between lesion-induced DNA-distorting and DNA-stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA. PMID:23570232

  9. Proton-impact ionization cross sections of adenine measured at 0.5 and 2.0 MeV by electron spectroscopy

    NASA Astrophysics Data System (ADS)

    Iriki, Y.; Kikuchi, Y.; Imai, M.; Itoh, A.

    2011-11-01

    Double-differential ionization cross sections (DDCSs) of vapor-phase adenine molecules (C5H5N5) by 0.5- and 2.0-MeV proton impact have been measured by the electron spectroscopy method. Electrons ejected from adenine were analyzed by a 45∘ parallel-plate electrostatic spectrometer over an energy range of 1.0-1000 eV at emission angles from 15∘ to 165∘. Single-differential cross sections (SDCSs) and total ionization cross sections (TICSs) were also deduced. It was found from the Platzman plot, defined as SDCSs divided by the classical Rutherford knock-on cross sections per target electron, that the SDCSs at higher electron energies are proportional to the total number of valence electrons (50) of adenine, while those at low-energy electrons are highly enhanced due to dipole and higher-order interactions. The present results of TICS are in fairly good agreement with recent classical trajectory Monte Carlo calculations, and moreover, a simple analytical formula gives nearly equivalent cross sections in magnitude at the incident proton energies investigated.

  10. Topical delivery of DNA oligonucleotide to induce p53 generation in the skin via thymidine dinucleotide (pTT)-encapsulated liposomal carrier

    PubMed Central

    Fang, Yi-Ping

    2011-01-01

    Introduction Transcription factor p53 has a powerful tumor suppressing function that is associated with many cancers. Since the molecular weight of p53 is 53 kDa, it is difficult to transport across cell membranes. Thymidine dinucleotide (pTT) is an oligonucleotide that can activate the p53 transcription factor and trigger the signal transduction cascade. However, the negative charge and high water solubility of pTT limit its transport through cellular membranes, thereby preventing it from reaching its target in the nucleus. A suitable delivery carrier for pTT is currently not available. Objective The purpose of this study was to employ a nanoscale liposomal carrier to resolve the delivery problem, and increase the bioavailability and efficiency of pTT. Methodology The approach was to employ liposomes to deliver pTT and then evaluate the particle size and zeta potential by laser light scattering (LLS), and permeation properties of pTT in vitro in a Franz diffusion assembly, and in vivo in a murine model using confocal laser scanning microscopy (CLSM). Results We found that dioleoylphosphatidylethanolamine (DOPE) combined with cholesterol 3 sulfate (C3S) were the best ingredients to achieve an average desired vehicle size of 133.6 ± 2.8 nm, a polydispersity index (PDI, representing the distribution of particle sizes) of 0.437, and a zeta potential of −93.3 ± 1.88. An in vitro penetration study showed that the liposomal carrier was superior to the free form of pTT at 2–24 hours. CLSM study observed that the penetration depth of pTT reached the upper epidermis and potential of penetration maintained up to 24 hours. Conclusion These preliminary data demonstrate that nanosized DOPE/C3S liposomes can be exploited as a potential carrier of drugs for topical use in treating skin diseases. PMID:22267922

  11. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots.

    PubMed

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao

    2016-08-16

    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements. PMID:27410247

  12. Toward high-throughput genotyping: dynamic and automatic software for manipulating large-scale genotype data using fluorescently labeled dinucleotide markers.

    PubMed

    Li, J L; Deng, H; Lai, D B; Xu, F; Chen, J; Gao, G; Recker, R R; Deng, H W

    2001-07-01

    To efficiently manipulate large amounts of genotype data generated with fluorescently labeled dinucleotide markers, we developed a Microsoft database management system, named. offers several advantages. First, it accommodates the dynamic nature of the accumulations of genotype data during the genotyping process; some data need to be confirmed or replaced by repeat lab procedures. By using, the raw genotype data can be imported easily and continuously and incorporated into the database during the genotyping process that may continue over an extended period of time in large projects. Second, almost all of the procedures are automatic, including autocomparison of the raw data read by different technicians from the same gel, autoadjustment among the allele fragment-size data from cross-runs or cross-platforms, autobinning of alleles, and autocompilation of genotype data for suitable programs to perform inheritance check in pedigrees. Third, provides functions to track electrophoresis gel files to locate gel or sample sources for any resultant genotype data, which is extremely helpful for double-checking consistency of raw and final data and for directing repeat experiments. In addition, the user-friendly graphic interface of renders processing of large amounts of data much less labor-intensive. Furthermore, has built-in mechanisms to detect some genotyping errors and to assess the quality of genotype data that then are summarized in the statistic reports automatically generated by. The can easily handle >500,000 genotype data entries, a number more than sufficient for typical whole-genome linkage studies. The modules and programs we developed for the can be extended to other database platforms, such as Microsoft SQL server, if the capability to handle still greater quantities of genotype data simultaneously is desired. PMID:11435414

  13. Antimutagenic activity of oxidase enzymes

    SciTech Connect

    Agabeili, R.A.

    1986-11-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity.

  14. Standard role for a conserved aspartate or more direct involvement in deglycosylation? An ONIOM and MD investigation of adenine-DNA glycosylase.

    PubMed

    Kellie, Jennifer L; Wilson, Katie A; Wetmore, Stacey D

    2013-12-01

    8-Oxoguanine (OG) is one of the most frequently occurring forms of DNA damage and is particularly deleterious since it forms a stable Hoogsteen base pair with adenine (A). The repair of an OG:A mispair is initiated by adenine-DNA glycosylase (MutY), which hydrolyzes the sugar-nucleobase bond of the adenine residue before the lesion is processed by other proteins. MutY has been proposed to use a two-part chemical step involving protonation of the adenine nucleobase, followed by SN1 hydrolysis of the glycosidic bond. However, differences between a recent (fluorine recognition complex, denoted as the FLRC) crystal structure and the structure on which most mechanistic conclusions have been based to date (namely, the lesion recognition complex or LRC) raise questions regarding the mechanism used by MutY and the discrete role of various active-site residues. The present work uses both molecular dynamics (MD) and quantum mechanical (ONIOM) models to compare the active-site conformational dynamics in the two crystal structures, which suggests that only the understudied FLRC leads to a catalytically competent reactant. Indeed, all previous computational studies on MutY have been initiated from the LRC structure. Subsequently, for the first time, various mechanisms are examined with detailed ONIOM(M06-2X:PM6) reaction potential energy surfaces (PES) based on the FLRC structure, which significantly extends the mechanistic picture. Specifically, our work reveals that the reaction proceeds through a different route than the commonly accepted mechanism and the catalytic function of various active-site residues (Geobacillus stearothermophilus numbering). Specifically, contrary to proposals based on the LRC, E43 is determined to solely be involved in the initial adenine protonation step and not the deglycosylation reaction as the general base. Additionally, a novel catalytic role is proposed for Y126, whereby this residue plays a significant role in stabilizing the highly charged

  15. Effect of Base Stacking on the Acid-Base Properties of the Adenine Cation Radical [A•+] in Solution: ESR and DFT Studies

    PubMed Central

    Adhikary, Amitava; Kumar, Anil; Khanduri, Deepti

    2015-01-01

    In this study, the acid–base properties of the adenine cation radical are investigated by means of experiment and theory. Adenine cation radical (A•+) is produced by one-electron oxidation of dAdo and of the stacked DNA-oligomer (dA)6 by Cl2•− in aqueous glass (7.5 M LiCl in H2O and in D2O) and investigated by ESR spectroscopy. Theoretical calculations and deuterium substitution at C8–H and N6–H in dAdo aid in our assignments of structure. We find the pKa value of A•+ in this system to be ca. 8 at 150 K in seeming contradiction to the accepted value of ≤ 1 at ambient temperature. However, upon thermal annealing to ≥160 K, complete deprotonation of A•+ occurs in dAdo in these glassy systems even at pH ca. 3. A•+ found in (dA)6 at 150 K also deprotonates on thermal annealing. The stability of A•+ at 150 K in these systems is attributed to charge delocalization between stacked bases. Theoretical calculations at various levels (DFT B3LYP/6-31G*, MPWB95, and HF-MP2) predict binding energies for the adenine stacked dimer cation radical of 12 to 16 kcal/mol. Further DFT B3LYP/6-31G* calculations predict that, in aqueous solution, monomeric A•+ should deprotonate spontaneously (a predicted pKa of ca. −0.3 for A•+). However, the charge resonance stabilized dimer AA•+ is predicted to result in a significant barrier to deprotonation and a calculated pKa of ca. 7 for the AA•+ dimer which is 7 pH units higher than the monomer. These theoretical and experimental results suggest that A•+ isolated in solution and A•+ in adenine stacks have highly differing acid–base properties resulting from the stabilization induced by hole delocalization within adenine stacks. PMID:18611019

  16. Hypothesis on Skeletal Muscle Aging: Mitochondrial Adenine Nucleotide Translocator Decreases Reactive Oxygen Species Production While Preserving Coupling Efficiency

    PubMed Central

    Diolez, Philippe; Bourdel-Marchasson, Isabelle; Calmettes, Guillaume; Pasdois, Philippe; Detaille, Dominique; Rouland, Richard; Gouspillou, Gilles

    2015-01-01

    Mitochondrial membrane potential is the major regulator of mitochondrial functions, including coupling efficiency and production of reactive oxygen species (ROS). Both functions are crucial for cell bioenergetics. We previously presented evidences for a specific modulation of adenine nucleotide translocase (ANT) appearing during aging that results in a decrease in membrane potential - and therefore ROS production—but surprisingly increases coupling efficiency under conditions of low ATP turnover. Careful study of the bioenergetic parameters (oxidation and phosphorylation rates, membrane potential) of isolated mitochondria from skeletal muscles (gastrocnemius) of aged and young rats revealed a remodeling at the level of the phosphorylation system, in the absence of alteration of the inner mitochondrial membrane (uncoupling) or respiratory chain complexes regulation. We further observed a decrease in mitochondrial affinity for ADP in aged isolated mitochondria, and higher sensitivity of ANT to its specific inhibitor atractyloside. This age-induced modification of ANT results in an increase in the ADP concentration required to sustain the same ATP turnover as compared to young muscle, and therefore in a lower membrane potential under phosphorylating—in vivo—conditions. Thus, for equivalent ATP turnover (cellular ATP demand), coupling efficiency is even higher in aged muscle mitochondria, due to the down-regulation of inner membrane proton leak caused by the decrease in membrane potential. In the framework of the radical theory of aging, these modifications in ANT function may be the result of oxidative damage caused by intra mitochondrial ROS and may appear like a virtuous circle where ROS induce a mechanism that reduces their production, without causing uncoupling, and even leading in improved efficiency. Because of the importance of ROS as therapeutic targets, this new mechanism deserves further studies. PMID:26733871

  17. Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

    PubMed Central

    Mampel, Teresa; Viñas, Octavi

    2016-01-01

    Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows. PMID:26842067

  18. Comparison of the Antiviral Effects of 5-Methoxymethyl-deoxyuridine with 5-Iododeoxyuridine, Cytosine Arabinoside, and Adenine Arabinoside

    PubMed Central

    Babiuk, Lorne A.; Meldrum, Blair; Gupta, V. Sagar; Rouse, Barry T.

    1975-01-01

    The antiviral activity of 5-methoxymethyl-2′-deoxyuridine (MMUdR) was compared with that of 5-iodo-2′-deoxyuridine (IUdR), cytosine arabinoside (Ara-C), and adenine arabinoside (Ara-A). At concentrations of 2 to 4 μg/ml, MMUdR was inhibitory to herpes simplex virus type 1, but concentrations as high as 128 μg/ml were not inhibitory to three other herpesviruses tested (equine rhinopneumonitis virus, murine cytomegalovirus, and feline rhinopneumonitis virus) or to vaccinia virus. The other nucleosides, in contrast, were inhibitory at similar concentrations (1 to 8 μg/ml) against all viruses tested. The inhibition of HSV-1 by MMUdR appeared to be the result of interference with virus replication rather than the result of drug toxicity to host cells. The drug was not toxic to host cells at 100 times the antiviral concentrations, and pretreatment of host cells with high concentrations of MMUdR had no effect on subsequent virus replication. Combination of MMUdR with either IUdR, Ara-A, or Ara-C gave an enhanced antiviral effect, suggesting that the mechanism of action of MMUdR is different from that of the other three drugs. Antiviral indexes were calculated for each compound and were found to be >250, 80, 40, and 8 for MMUdR, IUdR, Ara-A, and Ara-C, respectively. These were defined as the minimum dose at which toxicity was observed microscopically divided by the dose which reduced plaque numbers by 50%. PMID:1239978

  19. A QM/QTAIM microstructural analysis of the tautomerisationviathe DPT of the hypoxanthine·adenine nucleobase pair

    NASA Astrophysics Data System (ADS)

    Brovarets', Ol'ha O.; Zhurakivsky, Roman O.; Hovorun, Dmytro M.

    2014-08-01

    We provide a pathway for the tautomerisation of the biologically important hypoxanthine.adenine (Hyp.Ade) nucleobase pair (Cs) formed by the keto tautomer of the Hyp and the amino tautomer of the Ade into the Hyp*.Ade* base pair (Cs) formed by the enol tautomer of the Hyp and the imino tautomer of the Ade by applying quantum-mechanical calculations and Bader's Quantum Theory of Atoms in Molecules analysis. It was found out that the dipole active Hyp.Ade↔Hyp*.Ade* tautomerisation occurs via the asynchronous concerted double proton transfer (DPT) through the TSHyp.Ade↔Hyp*.Ade* (Cs). Based on the sweeps of the energies of the intermolecular H-bonds along the intrinsic reaction coordinate, it was established that the N6H...O6 H-bond (5.40) is cooperative with the N1H...N1 H-bond (6.99) in the Hyp.Ade base pair, as well as the O6H...N6 H-bond (11.50) is cooperative with the N1H...N1 H-bond (7.28 kcal.mol-1) in the Hyp*.Ade* base pair, mutually strengthening each other. The Hyp*.Ade* base pair possesses an extremely short lifetime 2.68.10-14 s, which is predetermined by the negative value of the Gibbs free energy of the reverse barrier of its tautomerisation, and all of the six low-frequency intermolecular vibrations cannot develop during this period of time. Consequently, the Hyp.Ade→Hyp*.Ade* DPT tautomerisation cannot serve as a source of the rare tautomers of the bases.

  20. The role of Glu-60 in the specificity of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) towards dinucleotides, poly(A) and RNA.

    PubMed Central

    Bastyns, K; Froeyer, M; Volckaert, G; Engelborghs, Y

    1994-01-01

    A computer model of the complex between G2'p5'G and barnase, the recombinant ribonuclease of Bacillus amyloliquefaciens, was constructed, based on the known structure of the complex RNAase T1.G2'p5'G. This model suggests that the conserved residue Glu-60 plays an important role in the specificity of barnase for guanosine. A barnase mutant was therefore made in which Glu-60 was replaced by Gln. This mutation increases the Km for the dinucleotides GpC and GpA, by a factor of 10, but does not change the kcat. For ApA, the kcat/Km decreases by a similar factor, but the individual parameters could not be determined. The mutation, however, has no influence on the kcat and the Km of barnase action towards RNA and poly(A). This demonstrates that the interactions between the substrate and the residue at position 60 must be different in the case of ApA and poly(A). For RNA, this conclusion is also likely, but not absolutely certain, because barnase/RNA might be a Briggs-Haldane type enzyme/substrate pair. Therefore, if the effect of the mutation were limited to an increase of the dissociation rate constant of the substrate (k-1), this would not be evident in Km or kcat/Km. In view of the clear cut situation with poly(A), the pH profile for and the effect of salt concentration on the kinetic parameters of the mutant barnase were studied for this substrate. The influence of salt on the Km can be interpreted via the linked function concept and shows a cooperative dissociation of 7-10 counterions upon poly(A) binding. The binding of the substrate is strongly reduced at high pH, and the pKa involved decreases strongly at high salt concentrations. Poly(A) and RNA show a pH dependency of their absorbance spectrum, indicating a pH-dependent change of base stacking, which may influence the catalytic parameters. PMID:7516656

  1. Interaction of tRNA with tRNA (guanosine-1)methyltransferase: binding specificity determinants involve the dinucleotide G36pG37 and tertiary structure.

    PubMed

    Redlak, M; Andraos-Selim, C; Giege, R; Florentz, C; Holmes, W M

    1997-07-22

    The sequence G37pG36 is present in all tRNA species recognized and methylated by the Escherichia coli modification enzyme tRNA (guanosine-1)methyltransferase. We have examined whether this dinucleotide sequence provides the base specific recognition signal for this enzyme and have assessed the role of the remaining tRNA in recognition. E. coli tRNAHis and yeast tRNAAsp were substituted with G at positions 36 and 37 and were found to be excellent substrates for methylation. This suggested that the general tRNA structure can be specifically bound by the enzyme. In addition, heterologous tRNA species including fully modified tRNA1Leu are excellent inhibitors of tRNA1Leu transcript methylation. Analyses of structural variants of yeast tRNAAsp and E. coli tRNA1Leu demonstrate clearly that the core tertiary structures of tRNA are required for recognition and that G37 must be in the correct position in space relative to important contacts elsewhere in the molecule. This latter conclusion was reached because the addition of one to three stacked base pairs in the anticodon stem of tRNA1Leu dramatically alters activity. In this case, the G37 base is rotated away from the correct position in space relative to other tRNA contact sites. The acceptor stem structure is required for optimal activity since deletion of three or five base pairs is detrimental to activity; however, specific base sequence may not be important because (i) the addition of three stacked base pairs of different sequence had little effect on activity and (ii) heterologous tRNAs with little or no sequence homology in the acceptor stem are excellent substrates. Both poly G and GpG are potent and specific inhibitors of enzyme activity and are minimal substrates which can be methylated, forming m1G. Taken together, these studies suggest that 1MGT can bind the general tRNA structure and that the crucial base-pair contacts are G37 and G36. PMID:9220956

  2. Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Shanak, Siba; Helms, Volkhard

    2014-12-01

    Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

  3. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

    PubMed

    Witteveldt, Jeroen; Martin-Gans, Marion; Simmonds, Peter

    2016-05-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  4. Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing

    PubMed Central

    Witteveldt, Jeroen; Martin-Gans, Marion

    2016-01-01

    Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs. PMID:26953209

  5. The Characterization of Escherichia coli CpdB as a Recombinant Protein Reveals that, besides Having the Expected 3´-Nucleotidase and 2´,3´-Cyclic Mononucleotide Phosphodiesterase Activities, It Is Also Active as Cyclic Dinucleotide Phosphodiesterase

    PubMed Central

    López-Villamizar, Iralis; Cabezas, Alicia; Pinto, Rosa María; Canales, José; Ribeiro, João Meireles; Cameselle, José Carlos; Costas, María Jesús

    2016-01-01

    Endogenous cyclic diadenylate phosphodiesterase activity was accidentally detected in lysates of Escherichia coli BL21. Since this kind of activity is uncommon in Gram-negative bacteria, its identification was undertaken. After partial purification and analysis by denaturing gel electrophoresis, renatured activity correlated with a protein identified by fingerprinting as CpdB (cpdB gene product), which is annotated as 3´-nucleotidase / 2´,3´-cyclic-mononucleotide phosphodiesterase, and it is synthesized as a precursor protein with a signal sequence removable upon export to the periplasm. It has never been studied as a recombinant protein. The coding sequence of mature CpdB was cloned and expressed as a GST fusion protein. The study of the purified recombinant protein, separated from GST, confirmed CpdB annotation. The assay of catalytic efficiencies (kcat/Km) for a large substrate set revealed novel CpdB features, including very high efficiencies for 3´-AMP and 2´,3´-cyclic mononucleotides, and previously unknown activities on cyclic and linear dinucleotides. The catalytic efficiencies of the latter activities, though low in relative terms when compared to the major ones, are far from negligible. Actually, they are perfectly comparable to those of the ‘average’ enzyme and the known, bona fide cyclic dinucleotide phosphodiesterases. On the other hand, CpdB differs from these enzymes in its extracytoplasmic location and in the absence of EAL, HD and DHH domains. Instead, it contains the domains of the 5´-nucleotidase family pertaining to the metallophosphoesterase superfamily, although CpdB lacks 5´-nucleotidase activity. The possibility that the extracytoplasmic activity of CpdB on cyclic dinucleotides could have physiological meaning is discussed. PMID:27294396

  6. Pleiotropic effects of the yeast Sal1 and Aac2 carriers on mitochondrial function via an activity distinct from adenine nucleotide transport

    PubMed Central

    Kucejova, Blanka; Li, Li; Wang, Xiaowen; Giannattasio, Sergio; Chen, Xin Jie

    2009-01-01

    In Saccharomyces cerevisiae, SAL1 encodes a Ca2+-binding mitochondrial carrier. Disruption of SAL1 is synthetically lethal with the loss of a specific function associated with the Aac2 isoform of the ATP/ADP translocase. This novel activity of Aac2 is defined as the V function (for Viability of aac2 sal1 double mutant), which is independent of the ATP/ADP exchange activity required for respiratory growth (the R function). We found that co-inactivation of SAL1 and AAC2 leads to defects in mitochondrial translation and mitochondrial DNA (mtDNA) maintenance. Additionally, sal1Δ exacerbates the respiratory deficiency and mtDNA instability of ggc1Δ, shy1Δ and mtg1Δ mutants, which are known to reduce mitochondrial protein synthesis or protein complex assembly. The V function is complemented by the human Short Ca2+-binding Mitochondrial Carrier (SCaMC) protein, SCaMC-2, a putative ATP-Mg/Pi exchangers on the inner membrane. However, mitochondria lacking both Sal1p and Aac2p are not depleted of adenine nucleotides. The Aac2R252I and Aac2R253I variants mutated at the R252-254 triplet critical for nucleotide transport retain the V function. Likewise, Sal1p remains functionally active when the R479I and R481I mutations were introduced into the structurally equivalent R479-T480-R481 motif. Finally, we found that the naturally occurring V-R+ Aac1 isoform of adenine nucleotide translocase partially gains the V function at the expense of the R function by introducing the mutations P89L and A96V. Thus, our data support the view that the V function is independent of adenine nucleotide transport associated with Sal1p and Aac2p and this evolutionarily conserved activity affects multiple processes in mitochondria. PMID:18431598

  7. Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases*

    PubMed Central

    Boitz, Jan M.; Ullman, Buddy

    2010-01-01

    Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice. PMID:20363738

  8. Can an Excess Electron Localise on a Purine Moiety in the Adenine-thymine Watson-Crick Base Pair? A Computational Study

    SciTech Connect

    Mazurkiewicz, Kamil; Haranczyk, Maciej; Gutowski, Maciej S.; Rak, Janusz

    2007-04-17

    The electron affinity and the propensity to electron-induced proton transfer (PT) of hydrogen-bonded complexes between the Watson–Crick adenine–thymine pair (AT) and simple organic acid (HX), attached to adenine in the Hoogsteen-type configuration, were studied at the B3LYP/6-31+G** level. Although the carboxyl group is deprotonated at physiological pH, its neutral form, COOH, resembles the peptide bond or the amide fragment in the side chain of asparagine (Asn) or glutamine (Gln). Thus, these complexes mimic the interaction between the DNA environment (e.g., proteins) and nucleobase pairs incorporated in the biopolymer. Electron attachment is thermodynamically feasible and adiabatic electron affinities range from 0.41 to 1.28 eV, while the vertical detachment energies of the resulting anions span the range of 0.39 –2.88 eV. Low-energy activation barriers separate the anionic minima: aHX(AT) from the more stable single-PT anionic geometry, aHX(AT)-SPT, and aHX(AT)-SPT from the double-PT anionic geometry, aHX(AT)-DPT. Interaction between the adenine of the Watson–Crick AT base pair with an acidic proton donor probably counterbalances the larger EA of isolated thymine, as SOMO is almost evenly delocalized over both types of nucleic bases in the aHX(AT) anions. Moreover, as a result of PT the excess electron localizes entirely on adenine. Thus, in DNA interacting with its physiological environment, damage induced by low-energy electrons could begin, contrary to the current view, with the formation of purine anions, which are not formed in isolated DNA because of the greater stability of anionic pyrimidines.

  9. Adsorption of adenine, cytosine, thymine, and uracil on sulfide-modified montmorillonite: FT-IR, Mössbauer and EPR spectroscopy and X-ray diffractometry studies.

    PubMed

    Carneiro, Cristine E A; Berndt, Graciele; de Souza Junior, Ivan G; de Souza, Cláudio M D; Paesano, Andrea; da Costa, Antonio C S; di Mauro, Eduardo; de Santana, Henrique; Zaia, Cássia T B V; Zaia, Dimas A M

    2011-10-01

    In the present work the interactions of nucleic acid bases with and adsorption on clays were studied at two pHs (2.00, 7.00) using different techniques. As shown by Mössbauer and EPR spectroscopies and X-ray diffractometry, the most important finding of this work is that nucleic acid bases penetrate into the interlayer of the clays and oxidize Fe(2+) to Fe(3+), thus, this interaction cannot be regarded as a simple physical adsorption. For the two pHs the order of the adsorption of nucleic acid bases on the clays was: adenine ≈ cytosine > thymine > uracil. The adsorption of adenine and cytosine on clays increased with decreasing of the pH. For unaltered montmorillonite this result could be explained by electrostatic forces between adenine/cytosine positively charged and clay negatively charged. However for montmorillonite modified with Na(2)S, probably van der Waals forces also play an important role since both adenine/cytosine and clay were positively charged. FT-IR spectra showed that the interaction between nucleic acid bases and clays was through NH(+) or NH (2) (+) groups. X-ray diffractograms showed that nucleic acid bases adsorbed on clays were distributed into the interlayer surface, edge sites and external surface functional groups (aluminol, silanol) EPR spectra showed that the intensity of the line g ≈ 2 increased probably because the oxidation of Fe(2+) to Fe(3+) by nucleic acid bases and intensity of the line g = 4.1 increased due to the interaction of Fe(3+) with nucleic acid bases. Mössbauer spectra showed a large decreased on the Fe(2+) doublet area of the clays due to the reaction of nucleic acid bases with Fe(2+). PMID:21717172

  10. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

    PubMed

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

    2015-04-14

    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein. PMID:25790177

  11. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    PubMed

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

  12. Adenine Nucleotide Analogues Locked in a Northern Methanocarba Conformation: Enhanced Stability and Potency as P2Y1 Receptor Agonists

    PubMed Central

    Ravi, R. Gnana; Kim, Hak Sung; Servos, Jörg; Zimmermann, Herbert; Lee, Kyeong; Maddileti, Savitri; Boyer, José L.; Harden, T. Kendall; Jacobson, Kenneth A.

    2016-01-01

    Preference for the Northern (N) ring conformation of the ribose moiety of nucleotide 5′-triphosphate agonists at P2Y1, P2Y2, P2Y4, and P2Y11 receptors, but not P2Y6 receptors, was established using a ring-constrained methanocarba (a 3.1.0-bicyclohexane) ring as a ribose substitute (Kim et al. J. Med. Chem. 2002, 45, 208–218.). We have now combined the ring-constrained (N)-methanocarba modification of adenine nucleotides with other functionalities known to enhance potency at P2 receptors. The potency of the newly synthesized analogues was determined in the stimulation of phospholipase C through activation of turkey erythrocyte P2Y1 or human P2Y1 and P2Y2 receptors stably expressed in astrocytoma cells. An (N)-methanocarba-2-methylthio-ADP analogue displayed an EC50 at the hP2Y1 receptor of 0.40 nM and was 55-fold more potent than the corresponding triphosphate and 16-fold more potent than the riboside 5′-diphosphate. 2-Cl–(N)-methanocarba-ATP and its N6-Me analogue were also highly selective, full agonists at P2Y1 receptors. The (N)-methanocarba-2-methylthio and 2-chloromonophosphate analogues were full agonists exhibiting micromolar potency at P2Y1 receptors, while the corresponding ribosides were inactive. Although β,γ-methylene-ATP was inactive at P2Y receptors, β,γ-methylene-(N)-methanocarba-ATP was a potent hP2Y1 receptor agonist with an EC50 of 160 nM and was selective versus hP2Y2 and hP2Y4 receptors. The rates of hydrolysis of Northern (N) and Southern (S) methanocarba analogues of AMP by rat 5′-ectonucleotidase were negligible. The rates of hydrolysis of the corresponding triphosphates by recombinant rat NTPDase1 and 2 were studied. Both isomers were hydrolyzed by NTPDase 1 at about half the rate of ATP hydrolysis. The (N) isomer was hardly hydrolyzed by NTPDase 2, while the (S) isomer was hydrolyzed at one-third of the rate of ATP hydrolysis. This suggests that new, more stable and selective nucleotide agonists may be designed on the basis of

  13. Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase.

    PubMed

    Parker, W B; Allan, P W; Waud, W R; Hong, J S; Sorscher, E J

    2011-06-01

    The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT. PMID:21394111

  14. Acidity and complex formation studies of 3-(adenine-9-yl)-propionic and 3-(thymine-1-yl)-propionic acids in ethanol-water media

    NASA Astrophysics Data System (ADS)

    Hammud, Hassan H.; El Shazly, Shawky; Sonji, Ghassan; Sonji, Nada; Bouhadir, Kamal H.

    2015-05-01

    The ligands 3-(adenine-9-yl)propionic acid (AA) and 3-(thymine-1-yl)propionic acid (TA) were prepared by N9-alkylation of adenine and N1-alkylation of thymine with ethylacrylate in presence of a base catalyst, followed by acid hydrolysis of the formed ethyl esters to give the corresponding propionic acid derivatives. The products were characterized by spectral methods (FTIR, 1H NMR and 13C NMR), which confirm their structures. The dissociation constants of ligands, were potentiometrically determined in 0.3 M KCl at 20-50 °C temperature range. The work was extended to study complexation behavior of AA and TA with various biologically important divalent metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Mn2+ and Pb2+) in 50% v/v water-ethanol medium at four different temperatures, keeping ionic strength constant (0.3 M KCl). The order of the stability constants of the formed complexes decreases in the sequence Cu2+ > Pb2+ > Zn2+ > Ni2+ > Co2+ > Mn2+ > Cd2+ for both ligands. The effect of temperature was also studied and the corresponding thermodynamic functions (ΔG, ΔH, ΔS) were derived and discussed. The formation of metal complexes has been found to be spontaneous, and the stability constants were dependant markedly on the basicity of the ligands.

  15. A new regulatory principle for in vivo biochemistry: pleiotropic low affinity regulation by the adenine nucleotides--illustrated for the glycolytic enzymes of Saccharomyces cerevisiae.

    PubMed

    Mensonides, Femke I C; Bakker, Barbara M; Cremazy, Frederic; Messiha, Hanan L; Mendes, Pedro; Boogerd, Fred C; Westerhoff, Hans V

    2013-09-01

    Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction. PMID:23856461

  16. Quantitative trait locus mapping in chickens by selective DNA pooling with dinucleotide microsatellite markers by using purified DNA and fresh or frozen red blood cells as applied to marker-assisted selection.

    PubMed

    Lipkin, E; Fulton, J; Cheng, H; Yonash, N; Soller, M

    2002-03-01

    Many large, half-sib sire families are an integral component of chicken genetic improvement programs. These family structures include a sufficient number of individuals for mapping quantitative trait loci (QTL) at high statistical power. However, realizing this statistical power through individual or selective genotyping is yet too costly to be feasible under current genotyping methodologies. Genotyping costs can be greatly reduced through selective DNA pooling, involving densitometric estimates of marker allele frequencies in pooled DNA samples. When using dinucleotide microsatellite markers, however, such estimates are often confounded by overlapping "shadow" bands and can be confounded further by differential amplification of alleles. In the present study a shadow correction procedure provided accurate densitometric estimates of allele frequency for dinucleotide microsatellite markers in pools made from chicken purified DNA samples, fresh blood samples, and frozen-thawed blood samples. In a retrospective study, selective DNA pooling with thawed blood samples successfully identified two QTL previously shown by selective genotyping to affect resistance in chickens to Marek's disease. It is proposed that use of selective DNA pooling can provide relatively low-cost mapping and use in marker-assisted selection of QTL that affect production traits in chickens. PMID:11902402

  17. Mechanisms and energetics for N-glycosidic bond cleavage of protonated adenine nucleosides: N3 protonation induces base rotation and enhances N-glycosidic bond stability.

    PubMed

    Wu, R R; Rodgers, M T

    2016-06-21

    Our previous gas-phase infrared multiple photon dissociation action spectroscopy study of protonated 2'-deoxyadenosine and adenosine, [dAdo+H](+) and [Ado+H](+), found that both N3 and N1 protonated conformers are populated with the N3 protonated ground-state conformers predominant in the experiments. Therefore, N-glycosidic bond dissociation mechanisms of N3 and N1 protonated [dAdo+H](+) and [Ado+H](+) and the associated quantitative thermochemical values are investigated here using both experimental and theoretical approaches. Threshold collision-induced dissociation (TCID) of [dAdo+H](+) and [Ado+H](+) with Xe is studied using guided ion beam tandem mass spectrometry techniques. For both systems, N-glycosidic bond cleavage reactions are observed as the major dissociation pathways resulting in production of protonated adenine or elimination of neutral adenine. Electronic structure calculations are performed at the B3LYP/6-311+G(d,p) level of theory to probe the potential energy surfaces (PESs) for N-glycosidic bond cleavage of [dAdo+H](+) and [Ado+H](+). Relative energetics of the reactants, transition states, intermediates and products along the PESs for N-glycosidic bond cleavage are determined at the B3LYP/6-311+G(2d,2p), B3LYP-GD3BJ/6-311+G(2d,2p), and MP2(full)/6-311+G(2d,2p) levels of theory. The predicted N-glycosidic bond dissociation mechanisms for the N3 and N1 protonated species differ. Base rotation of the adenine residue enables formation of a strong N3H(+)O5' hydrogen-bonding interaction that stabilizes the N3 protonated species and its glycosidic bond. Comparison between experiment and theory indicates that the N3 protonated species determine the threshold energies, as excellent agreement between the measured and B3LYP computed activation energies (AEs) and reaction enthalpies (ΔHrxns) for N-glycosidic bond cleavage of the N3 protonated species is found. PMID:27240654

  18. Oligonucleotides, part 5+: synthesis and fluorescence studies of DNA oligomers d(AT)5 containing adenines covalently linked at C-8 with dansyl fluorophore.

    PubMed Central

    Singh, D; Kumar, V; Ganesh, K N

    1990-01-01

    The synthesis of oligodeoxynucleotides d(AT)5 in which specific adenines are linked at C-8 position with dansyl fluorophores via a variable polymethylene spacer chain are reported. This was achieved by a strategy involving prelabelling at the monomeric stage followed by solid phase assembly of oligonucleotides to obtain regiospecifically labeled oligonucleotides. Several mono and polydansyl d(AT)5 derivatives in which the fluorophore is linked via ethylene, tetramethylene and hexamethylene spacer arms were synthesised for a systematic study of their fluorescence characteristics. It was observed that (i) enhancements in fluorescence intensity and emission quantum yields are seen due to multiple labelling, (ii) the magnitude of enhancements are related to labelling configuration and (iii) quenching efficiency is minimal with shorter and rigid spacer arms. The results may aid rational design of multiple fluorescent DNA probes for nonradioactive detection of nucleic acids. PMID:2356124

  19. A possible prebiotic synthesis of purine, adenine, cytosine, and 4(3H)-pyrimidinone from formamide: implications for the origin of life.

    PubMed

    Saladino, R; Crestini, C; Costanzo, G; Negri, R; Di Mauro, E

    2001-05-01

    The synthesis of prebiotic molecules is a major problem in chemical evolution as well as in any origin-of-life theory. We report here a plausible new prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide under catalytic conditions. In the presence of CaCO(3) and different inorganic oxides, namely silica, alumine, kaolin, and zeolite (Y type), neat formamide undergoes the formation of purine, adenine, cytosine, and 4(3H)-pyrimidinone, from acceptable to good yields. The role of catalysts showed to be not limited to the improvement of the yield but it is also relevant in providing a high selectivity in the products distribution. PMID:11377183

  20. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    SciTech Connect

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. )

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  1. Supra­molecular hydrogen-bonding patterns in the N(9)—H protonated and N(7)—H tautomeric form of an N6-benzoyl­adenine salt: N 6-benzoyl­adeninium nitrate

    PubMed Central

    Karthikeyan, Ammasai; Jeeva Jasmine, Nithianantham; Thomas Muthiah, Packianathan; Perdih, Franc

    2016-01-01

    In the title molecular salt, C12H10N5O+·NO3 −, the adenine unit has an N 9-protonated N(7)—H tautomeric form with non-protonated N1 and N3 atoms. The dihedral angle between the adenine ring system and the phenyl ring is 51.10 (10)°. The typical intra­molecular N7—H⋯O hydrogen bond with an S(7) graph-set motif is also present. The benzoyl­adeninium cations also form base pairs through N—H⋯O and C—H⋯N hydrogen bonds involving the Watson–Crick face of the adenine ring and the C and O atoms of the benzoyl ring of an adjacent cation, forming a supra­molecular ribbon with R 2 2(9) rings. Benzoyl­adeninum cations are also bridged by one of the oxygen atoms of the nitrate anion, which acts as a double acceptor, forming a pair of N—H⋯O hydrogen bonds to generate a second ribbon motif. These ribbons together with π–π stacking inter­actions between the phenyl ring and the five- and six-membered adenine rings of adjacent mol­ecules generate a three-dimensional supra­molecular architecture. PMID:26958373

  2. Effects of extending the computational model on DNA-protein T-shaped interactions: the case of adenine-histidine dimers.

    PubMed

    Rutledge, Lesley R; Navarro-Whyte, Lex; Peterson, Terri L; Wetmore, Stacey D

    2011-11-17

    The MP2/6-31G*(0.25) π-π or π(+)-π T-shaped (edge-to-face) interactions between neutral or protonated histidine and adenine were considered using computational models of varying size to determine the effects of the protein and DNA backbones on the preferred dimer structure and binding strength. The overall consequences of the backbones are reasonably subtle for the neutral adenine-histidine T-shaped dimers. Furthermore, the minor changes in the binding strengths of these dimers upon model extension arise from additional (attractive) backbone-π (bb-π) contacts and changes in the preferred π-π orientations, which is verified by the quantum theory of atoms in molecules (QTAIM). Since the binding strength of the extended dimer equals the sum of the individual backbone-π and π-π contributions, the π-π component is not appreciably affected by polarization of the ring upon inclusion of the biological backbone. In contrast, the larger effect of the backbone on the protonated histidine dimers cannot simply be predicted as the sum of changes in the π-π and bb-π components regardless of the dimer type or model. This suggests, and QTAIM qualitatively supports, that the magnitude of the π(+)-π contribution changes, which is likely due to alterations in the electrostatic landscape of the monomer rings upon inclusion of the biological backbone that largely affect T-shaped dimers. These findings differ from those previously reported for (neutral) π-π stacked and (metallic) cation-π interactions, which highlights the distinct properties of each (π-π, π(+)-π, and cation-π) classification of noncovalent interaction. Furthermore, these results emphasize the importance of considering backbone-π interactions when analyzing contacts that appear in experimental crystal structures and cautions the use of truncated models when evaluating the magnitude of the π(+)-π contribution present in large biological complexes. PMID:21648440

  3. Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

    PubMed Central

    Korotkov, Sergey M.

    2016-01-01

    There we made available information about the effects of the adenine nucleotide translocase (ANT) ‘c’ conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as thiol reagent (4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca2+-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25–75 mM TlNO3, 125 mM KNO3, and 100 µM Ca2+. The fixers and DIDS, in comparison to Ca2+ alone, greatly increased A540 decline and the rate of Ca2+-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in ‘m’ conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript “To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria” (Korotkov et al., 2016 [1]). PMID:27054168

  4. Mineral bone disorder in chronic kidney disease: head-to-head comparison of the 5/6 nephrectomy and adenine models

    PubMed Central

    2014-01-01

    Background Experimental models are important to the understanding of the pathophysiology of, as well as the effects of therapy on, certain diseases. In the case of chronic kidney disease-mineral bone disorder, there are currently two models that are used in evaluating the disease: 5/6 nephrectomy (Nx) and adenine-induced renal failure (AIRF). However, the two models have never been compared in studies using animals maintained under similar conditions. Therefore, we compared these two models, focusing on the biochemical, bone histomorphometry, and vascular calcification aspects. Methods Wistar rats, initially fed identical diets, were divided into two groups: those undergoing 5/6 Nx (5/6Nx group) and those that were switched to an adenine-enriched diet (AIRF group). After 9 weeks, animals were sacrificed, and we conducted biochemical and bone histomorphometry analyses, as well as assessing vascular calcification. Results At sacrifice, the mean body weight was higher in the 5/6Nx group than in the AIRF group, as was the mean blood pressure. No differences were seen regarding serum phosphate, ionized calcium, intact parathyroid hormone (PTH), or fibroblast growth factor 23 (FGF23). However, creatinine clearance was lower and fractional excretion of phosphate (FeP) was higher in the AIRF group rats, which also had a more severe form of high-turnover bone disease. Vascular calcification, as evaluated through von Kossa staining, was not observed in any of the animals. Conclusions Overt vascular calcification was not seen in either model as applied in this study. Under similar conditions of diet and housing, the AIRF model produces a more severe form of bone disease than does 5/6 Nx. This should be taken into account when the choice is made between these models for use in preclinical studies. PMID:24885705

  5. Adenine Nucleotide Translocase Is Acetylated in Vivo in Human Muscle: Modeling Predicts a Decreased ADP Affinity and Altered Control of Oxidative Phosphorylation

    PubMed Central

    2015-01-01

    Proteomics techniques have revealed that lysine acetylation is abundant in mitochondrial proteins. This study was undertaken (1) to determine the relationship between mitochondrial protein acetylation and insulin sensitivity in human skeletal muscle, identifying key acetylated proteins, and (2) to use molecular modeling techniques to understand the functional consequences of acetylation of adenine nucleotide translocase 1 (ANT1), which we found to be abundantly acetylated. Eight lean and eight obese nondiabetic subjects had euglycemic clamps and muscle biopsies for isolation of mitochondrial proteins and proteomics analysis. A number of acetylated mitochondrial proteins were identified in muscle biopsies. Overall, acetylation of mitochondrial proteins was correlated with insulin action (r = 0.60; P < 0.05). Of the acetylated proteins, ANT1, which catalyzes ADP–ATP exchange across the inner mitochondrial membrane, was acetylated at lysines 10, 23, and 92. The extent of acetylation of lysine 23 decreased following exercise, depending on insulin sensitivity. Molecular dynamics modeling and ensemble docking simulations predicted the ADP binding site of ANT1 to be a pocket of positively charged residues, including lysine 23. Calculated ADP–ANT1 binding affinities were physiologically relevant and predicted substantial reductions in affinity upon acetylation of lysine 23. Insertion of these derived binding affinities as parameters into a complete mathematical description of ANT1 kinetics predicted marked reductions in adenine nucleotide flux resulting from acetylation of lysine 23. Therefore, acetylation of ANT1 could have dramatic physiological effects on ADP–ATP exchange. Dysregulation of acetylation of mitochondrial proteins such as ANT1 therefore could be related to changes in mitochondrial function that are associated with insulin resistance. PMID:24884163

  6. Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

    PubMed Central

    Seib, Kate L.; Jen, Freda E.-C.; Tan, Aimee; Scott, Adeana L.; Kumar, Ritesh; Power, Peter M.; Chen, Li-Tzu; Wu, Hsing-Ju; Wang, Andrew H.-J.; Hill, Dorothea M. C.; Luyten, Yvette A.; Morgan, Richard D.; Roberts, Richard J.; Maiden, Martin C. J.; Boitano, Matthew; Clark, Tyson A.; Korlach, Jonas; Rao, Desirazu N.; Jennings, Michael P.

    2015-01-01

    Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGYm6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-ACm6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CCm6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGYm6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGCm6AGG-3′ sites, to 100% at 5′-ACGTm6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching. PMID:25845594

  7. Differential Expression of Adenine Nucleotide Converting Enzymes in Mitochondrial Intermembrane Space: A Potential Role of Adenylate Kinase Isozyme 2 in Neutrophil Differentiation

    PubMed Central

    Tanimura, Ayako; Horiguchi, Taigo; Miyoshi, Keiko; Hagita, Hiroko; Noma, Takafumi

    2014-01-01

    Adenine nucleotide dynamics in the mitochondrial intermembrane space (IMS) play a key role in oxidative phosphorylation. In a previous study, Drosophila adenylate kinase isozyme 2 (Dak2) knockout was reported to cause developmental lethality at the larval stage in Drosophila melanogaster. In addition, two other studies reported that AK2 is a responsible gene for reticular dysgenesis (RD), a human disease that is characterized by severe combined immunodeficiency and deafness. Therefore, mitochondrial AK2 may play an important role in hematopoietic differentiation and ontogenesis. Three additional adenine nucleotide metabolizing enzymes, including mitochondrial creatine kinases (CKMT1 and CKMT2) and nucleoside diphosphate kinase isoform D (NDPK-D), have been found in IMS. Although these kinases generate ADP for ATP synthesis, their involvement in RD remains unclear and still an open question. In this study, mRNA and protein expressions of these mitochondrial kinases were firstly examined in mouse ES cells, day 8 embryos, and 7-week-old adult mice. It was found that their expressions are spatiotemporally regulated, and Ak2 is exclusively expressed in bone marrow, which is a major hematopoietic tissue in adults. In subsequent experiments, we identified increased expression of both AK2 and CKMT1 during macrophage differentiation and exclusive production of AK2 during neutrophil differentiation using HL-60 cells as an in vitro model of hematopoietic differentiation. Furthermore, AK2 knockdown specifically inhibited neutrophil differentiation without affecting macrophage differentiation. These data suggest that AK2 is indispensable for neutrophil differentiation and indicate a possible causative link between AK2 deficiency and neutropenia in RD. PMID:24587121

  8. Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M

    2016-06-01

    There we made available information about the effects of the adenine nucleotide translocase (ANT) 'c' conformation fixers (phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside) as well as thiol reagent (4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS)) on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling) and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4]), as well as an increase in Ca(2+)-induced safranin fluorescence (F485/590, arbitrary units), showed a dissipation in the inner membrane potential (ΔΨmito), in experiments with energized rat liver mitochondria, injected into the buffer containing 25-75 mM TlNO3, 125 mM KNO3, and 100 µM Ca(2+). The fixers and DIDS, in comparison to Ca(2+) alone, greatly increased A540 decline and the rate of Ca(2+)-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide) fixing the ANT in 'm' conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript "To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria" (Korotkov et al., 2016 [1]). PMID:27054168

  9. Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on cape cod

    USGS Publications Warehouse

    Metge, D.W.; Brooks, M.H.; Smith, R.L.; Harvey, R.W.

    1993-01-01

    Changes in adenylate energy charge (EC(A)) and in total adenine nucleotides (A(T)) and DNA content (both normalized to the abundance of free- living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. EC(A)s (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated EC(A)s (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although EC(A) and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture. These data suggest that the DNA content of groundwater bacteria may be more sensitive than their A(T) to the degree of carbon loading, which may have significant ramifications in the use of nucleic acids and adenine nucleotides for estimating the metabolic status of bacterial communities within more highly contaminated aquifers.

  10. The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication

    PubMed Central

    Atkinson, Nicky J.; Witteveldt, Jeroen; Evans, David J.; Simmonds, Peter

    2014-01-01

    Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1–1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-β, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host–pathogen interactions. PMID:24470146

  11. Crystal structures and kinetic properties of enoyl-acyl carrier protein reductase I from Candidatus Liberibacter asiaticus

    PubMed Central

    Jiang, Ling; Gao, Zengqiang; Li, Yanhua; Wang, Shennan; Dong, Yuhui

    2014-01-01

    Huanglongbing (HLB) is a destructive citrus disease. The leading cause of HLB is Candidatus Liberibacter asiaticus. Fatty acid biosynthesis is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterial agents. Enoyl−acyl carrier protein reductase (also called ENR or FabI and a product of the fabI gene) is an enzyme required in a critical step of bacterial fatty acid biosynthesis and has attracted attention as a target of novel antimicrobial agents. We determined the crystal structures of FabI from Ca. L. asiaticus in its apoform as well as in complex with b-nicotinamide adenine dinucleotide (NAD) at 1.7 and 2.7 Å resolution, respectively, to facilitate the design and screening of small molecule inhibitors of FabI. The monomeric ClFabI is highly similar to other known FabI structures as expected; however, unlike the typical tetramer, ClFabI exists as a hexamer in crystal, whereas as dimer in solution, on the other hand, the substrate binding loop which always disordered in apoform FabI structures is ordered in apo-ClFabI. Interestingly, the structure of ClFabI undergoes remarkable conformational change in the substrate-binding loop in the presence of NAD. We conclude that the signature sequence motif of FabI can be considered as Gly-(Xaa)5-Ser-(Xaa)n-Val-Tyr-(Xaa)6-Lys-(Xaa)n-Thr instead of Tyr-(Xaa)6-Lys. We have further identified isoniazid as a competitive inhibitor with NADH. PMID:24407918

  12. Crystal structures and kinetic properties of enoyl-acyl carrier protein reductase I from Candidatus Liberibacter asiaticus.

    PubMed

    Jiang, Ling; Gao, Zengqiang; Li, Yanhua; Wang, Shennan; Dong, Yuhui

    2014-04-01

    Huanglongbing (HLB) is a destructive citrus disease. The leading cause of HLB is Candidatus Liberibacter asiaticus. Fatty acid biosynthesis is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterial agents. Enoyl-acyl carrier protein reductase (also called ENR or FabI and a product of the fabI gene) is an enzyme required in a critical step of bacterial fatty acid biosynthesis and has attracted attention as a target of novel antimicrobial agents. We determined the crystal structures of FabI from Ca. L. asiaticus in its apoform as well as in complex with b-nicotinamide adenine dinucleotide (NAD) at 1.7 and 2.7 Å resolution, respectively, to facilitate the design and screening of small molecule inhibitors of FabI. The monomeric ClFabI is highly similar to other known FabI structures as expected; however, unlike the typical tetramer, ClFabI exists as a hexamer in crystal, whereas as dimer in solution, on the other hand, the substrate binding loop which always disordered in apoform FabI structures is ordered in apo-ClFabI. Interestingly, the structure of ClFabI undergoes remarkable conformational change in the substrate-binding loop in the presence of NAD. We conclude that the signature sequence motif of FabI can be considered as Gly-(Xaa)5-Ser-(Xaa)n-Val-Tyr-(Xaa)6-Lys-(Xaa)n-Thr instead of Tyr-(Xaa)6-Lys. We have further identified isoniazid as a competitive inhibitor with NADH. PMID:24407918

  13. Excited state evolution of DNA stacked adenines resolved at the CASPT2//CASSCF/Amber level: from the bright to the excimer state and back.

    PubMed

    Conti, Irene; Nenov, Artur; Höfinger, Siegfried; Flavio Altavilla, Salvatore; Rivalta, Ivan; Dumont, Elise; Orlandi, Giorgio; Garavelli, Marco

    2015-03-21

    Deactivation routes of bright ππ* (La) and excimer charge transfer (CT) states have been mapped for two stacked quantum mechanical (CASPT2//CASSCF) adenines inside a solvated DNA double strand decamer (poly(dA)·poly(dT)) described at the molecular mechanics level. Calculations show that one carbon (C2) puckering is a common relaxation coordinate for both the La and CT paths. By mapping the lowest crossing regions between La and CT states, together with the paths connecting the two states, we conclude that at least one CT state can be easily accessible. The lowest-lying conical intersections between ground state (GS) and CT states have been fully characterized in a realistic DNA environment for the first time. We show that the path to reach this crossing region from the CT minima involves high barriers that are not consistent with experimental data lifetimes. Instead, the multiexponential decay recorded in DNA, including the longest (ca. 100 picoseconds) lifetime component detected in oligomeric single- and double-stranded systems, is compatible with both intra-monomer relaxation processes along the La deactivation path (involving small barriers) and the population of the excimer (CT) state that behaves as a trap. In the latter case, deactivation is feasible only going back to the La state by following its preferred decay coordinate. PMID:25695904

  14. An adenine nucleotide translocase (ANT) gene from Apostichopus japonicus; molecular cloning and expression analysis in response to lipopolysaccharide (LPS) challenge and thermal stress.

    PubMed

    Liu, Qiu-Ning; Chai, Xin-Yue; Tu, Jie; Xin, Zhao-Zhe; Li, Chao-Feng; Jiang, Sen-Hao; Zhou, Chun-Lin; Tang, Bo-Ping

    2016-02-01

    The adenine nucleotide translocases (ANTs) play a vital role in energy metabolism via ADP/ATP exchange in eukaryotic cells. Apostichopus japonicus (Echinodermata: Holothuroidea) is an important economic species in China. Here, a cDNA representing an ANT gene of A. japonicus was isolated and characterized from respiratory tree and named AjANT. The full-length AjANT cDNA is 1924 bp, including a 5'-untranslated region (UTR) of 38 bp, 3'-UTR of 980 bp and an open reading frame (ORF) of 906 bp encoding a polypeptide of 301 amino acids. The protein contains three homologous repeat Mito_carr domains (Pfam00153). The deduced AjANT protein sequence has 49-81% in comparison to ANT proteins from other individuals. The predicted tertiary structure of AjANT protein is highly similar to animal ANT proteins. Phylogenetic analysis showed that the AjANT is closely related to Holothuroidea ANT genes. Real-time quantitative reverse transcription-PCR (qPCR) analysis showed that AjANT expression is higher in the respiratory tree than in other examined tissues. After thermal stress or LPS challenge, expression of AjANT was significantly fluctuant compared to the control. These results suggested that changes in the expression of ANT gene might be involved in immune defense and in protecting A. japonicus against thermal stress. PMID:26706223

  15. To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M; Konovalova, Svetlana A; Brailovskaya, Irina V; Saris, Nils-Erik L

    2016-04-01

    The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds. PMID:26835787

  16. Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: Potential implications for methylation-independent transcriptional repression

    SciTech Connect

    Horton, John R.; Zhang, Xing; Blumenthal, Robert M.; Cheng, Xiaodong

    2015-04-06

    DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify a DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). All together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.

  17. Structures of Escherichia coli DNA adenine methyltransferase (Dam) in complex with a non-GATC sequence: Potential implications for methylation-independent transcriptional repression

    DOE PAGESBeta

    Horton, John R.; Zhang, Xing; Blumenthal, Robert M.; Cheng, Xiaodong

    2015-04-06

    DNA adenine methyltransferase (Dam) is widespread and conserved among the γ-proteobacteria. Methylation of the Ade in GATC sequences regulates diverse bacterial cell functions, including gene expression, mismatch repair and chromosome replication. Dam also controls virulence in many pathogenic Gram-negative bacteria. An unexplained and perplexing observation about Escherichia coli Dam (EcoDam) is that there is no obvious relationship between the genes that are transcriptionally responsive to Dam and the promoter-proximal presence of GATC sequences. Here, we demonstrate that EcoDam interacts with a 5-base pair non-cognate sequence distinct from GATC. The crystal structure of a non-cognate complex allowed us to identify amore » DNA binding element, GTYTA/TARAC (where Y = C/T and R = A/G). This element immediately flanks GATC sites in some Dam-regulated promoters, including the Pap operon which specifies pyelonephritis-associated pili. In addition, Dam interacts with near-cognate GATC sequences (i.e. 3/4-site ATC and GAT). All together, these results imply that Dam, in addition to being responsible for GATC methylation, could also function as a methylation-independent transcriptional repressor.« less

  18. A study of fast and metastable dissociations of adenine-thymine binary-base oligonucleotides by using positive-ion MALDI-TOF mass spectrometry.

    PubMed

    Chan, T W Dominic; Fung, Y M Eva; Li, Y C Leo

    2002-09-01

    In the present study, fast and metastable dissociations of a number of adenine-thymine binary-base oligonucleotides under the conditions of UV matrix-assisted laser desorption/ionization mass spectrometry were investigated. 2-Aminobenzoic acid/ammonium fluoride (ABA/NH4F) matrix system was used. The spectra obtained under metastable and fast dissociation conditions exhibit distinctive dissociation products. From the post-source-decay analysis, all oligonucleotides underwent predominantly metastable dissociations at the 3' C-O linkages to form [a(n)-B]+ and w(n)+ complimentary ion series. Based on the present results, the so-called "[wn+80]+" ions were postulated to be the complimentary [Z(8-n)AH]+ ions rather than the expected phosphate rearrangement products. In addition, these oligonucleotides were found to generate fast dissociation products of b(n)+, d(N)+, w(N)+ and y(N)+ ions through backbone cleavages at 5' C-O, 5' O-P, 3' C-O and 3' P-O linkages, respectively. Product ion series formed under PSD conditions were not observed. The implications of this mutually exclusive occurrence of the two sets of fragment ions under fast and metastable conditions using ABA/NH4F matrix would be discussed. A model of ion activation under UV-MALDI conditions was also proposed. PMID:12322953

  19. Adenine nucleotide translocator isoforms 1 and 2 are differently distributed in the mitochondrial inner membrane and have distinct affinities to cyclophilin D.

    PubMed Central

    Vyssokikh, M Y; Katz, A; Rueck, A; Wuensch, C; Dörner, A; Zorov, D B; Brdiczka, D

    2001-01-01

    Different isoforms of the adenine nucleotide translocase (ANT) are expressed in a tissue-specific manner. It was assumed that ANT-1 and ANT-2 co-exist in every single mitochondrion and might be differently distributed within the membrane structures that constitute the peripheral inner membrane or the crista membrane. To discriminate between ANT originating from peripheral or from cristal inner membranes we made use of the fact that complexes between porin, the outer-membrane pore protein, and the ANT can be generated. Such complexes between porin and the ANT in the peripheral inner membrane were induced in rat heart mitochondria and isolated from rat brain and kidney. Using ANT-isotype-specific antibodies and sequence analysis of the N-terminal end, it was discovered that the peripheral inner membrane contained ANT-1 and ANT-2, whereas the cristal membrane contained exclusively ANT-2. Cyclophilin was co-purified with the porin-ANT complexes, whereas it was absent in the crista-derived ANT. This suggested that ANT-1 might have a higher affinity for cyclophilin. This specific intra-mitochondrial distribution of the two ANT isotypes and cyclophilin D suggests specific functions of the peripheral and crista-forming parts of the inner membrane and the two ANT isotypes therein. PMID:11513733

  20. Gender and chronological age affect erythrocyte membrane oxidative indices in citrate phosphate dextrose adenine-formula 1 (CPDA-1) blood bank storage condition.

    PubMed

    Erman, Hayriye; Aksu, Uğur; Belce, Ahmet; Atukeren, Pınar; Uzun, Duygu; Cebe, Tamer; Kansu, Ahmet D; Gelişgen, Remisa; Uslu, Ezel; Aydın, Seval; Çakatay, Ufuk

    2016-07-01

    It is well known that in vitro storage lesions lead to membrane dysfunction and decreased number of functional erythrocytes. As erythrocytes get older, in storage media as well as in peripheral circulation, they undergo a variety of biochemical changes. In our study, the erythrocytes with different age groups in citrate phosphate dextrose adenine-formula 1 (CPDA-1) storage solution were used in order to investigate the possible effect of gender factor on oxidative damage. Oxidative damage biomarkers in erythrocyte membranes such as ferric reducing antioxidant power, pro-oxidant-antioxidant balance, protein-bound advance glycation end products, and sialic acid were analyzed. Current study reveals that change in membrane redox status during blood-bank storage condition also depends on both gender depended homeostatic factors and the presence of CPDA-1. During the storage period in CPDA-1, erythrocytes from the male donors are mostly affected by free radical-mediated oxidative stress but erythrocytes obtained from females are severely affected by glyoxidative stress. PMID:27045670

  1. B Cell Lymphoma (Bcl)-2 Protein Is the Major Determinant in bcl-2 Adenine-Uridine-rich Element Turnover Overcoming HuR Activity*

    PubMed Central

    Ghisolfi, Laura; Calastretti, Angela; Franzi, Sara; Canti, Gianfranco; Donnini, Martino; Capaccioli, Sergio; Nicolin, Angelo; Bevilacqua, Annamaria

    2009-01-01

    In the 3′-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins. PMID:19520857

  2. B cell lymphoma (Bcl)-2 protein is the major determinant in bcl-2 adenine-uridine-rich element turnover overcoming HuR activity.

    PubMed

    Ghisolfi, Laura; Calastretti, Angela; Franzi, Sara; Canti, Gianfranco; Donnini, Martino; Capaccioli, Sergio; Nicolin, Angelo; Bevilacqua, Annamaria

    2009-07-31

    In the 3'-untranslated region, the destabilizing adenine-uridine (AU)-rich elements (AREs) control the expression of several transcripts through interactions with ARE-binding proteins (AUBPs) and RNA degradation machinery. Although the fundamental role for AUBPs and associated factors in eliciting ARE-dependent degradation of cognate mRNAs has been recently highlighted, the molecular mechanisms underlying the specific regulation of individual mRNA turnover have not yet been fully elucidated. Here we focused on the post-transcriptional regulation of bcl-2 mRNA in human cell lines under different conditions and genetic backgrounds. In the context of an AUBPs silencing approach, HuR knockdown reduced the expression of endogenous bcl-2, whereas unexpectedly, a bcl-2 ARE-reporter transcript increased significantly, suggesting that HuR expression has opposite effects on endogenous and ectopic bcl-2 ARE. Moreover, evidence was provided for the essential, specific and dose-dependent role of the Bcl-2 protein in regulating the decay kinetics of its own mRNA, as ascertained by a luciferase reporter system. Altogether, the data support a model whereby the Bcl-2 protein is the major determinant of its own ARE-dependent transcript half-life in living cells and its effect overcomes the activity of ARE-binding proteins. PMID:19520857

  3. Stabilization of cellular mRNAs and up-regulation of proteins by oligoribonucleotides homologous to the Bcl2 adenine-uridine rich element motif.

    PubMed

    Bevilacqua, Annamaria; Ghisolfi, Laura; Franzi, Sara; Maresca, Giovanna; Gherzi, Roberto; Capaccioli, Sergio; Nicolin, Angelo; Canti, Gianfranco

    2007-02-01

    Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs. PMID:17077270

  4. The importance of helix P1 stability for structural pre-organization and ligand binding affinity of the adenine riboswitch aptamer domain

    PubMed Central

    Nozinovic, Senada; Reining, Anke; Kim, Yong-Boum; Noeske, Jonas; Schlepckow, Kai; Wöhnert, Jens; Schwalbe, Harald

    2014-01-01

    We report here an in-depth characterization of the aptamer domain of the transcriptional adenine-sensing riboswitch (pbuE) by NMR and fluorescence spectroscopy. By NMR studies, the structure of two aptamer sequences with different lengths of the helix P1, the central element involved in riboswitch conformational switching, was characterized. Hydrogen-bond interactions could be mapped at nucleotide resolution providing information about secondary and tertiary structure, structure homogeneity and dynamics. Our study reveals that the elongation of helix P1 has pronounced effects not only on the local but on the global structure of the apo aptamer domain. The structural differences induced by stabilizing helix P1 were found to be linked to changes of the ligand binding affinity as revealed from analysis of kinetic and thermodynamic data obtained from stopped-flow fluorescence studies. The results provide new insight into the sequence-dependent fine tuning of the structure and function of purine-sensing riboswitches. PMID:24921630

  5. Determination of guanine and adenine by high-performance liquid chromatography with a self-fabricated wall-jet/thin-layer electrochemical detector at a glassy carbon electrode.

    PubMed

    Zhou, Yaping; Yan, Hongling; Xie, Qingji; Yao, Shouzhuo

    2015-03-01

    A sensitive wall-jet/thin-layer amperometric electrochemical detector (ECD) coupled to high-performance liquid chromatography (HPLC) was developed for simultaneous determination of guanine (G) and adenine (A). The analytes were detected at a glassy carbon electrode (GCE) and the HPLC-ECD calibration curves showed good linearity (R(2)>0.997) under optimized conditions. Limits of detection for G and A are 0.6 nM and 1.4 nM (S/N=3), respectively, which are lower than those obtained with an UV-vis detector and a commercial electrochemical detector. We have successfully applied this HPLC-ECD to assess the contents of G and A in hydrochloric acid-digested calf thymus double-stranded DNA. In addition, we compared in detail the analysis of G and A by cyclic voltammetry (CV) and by the HPLC-ECD system on both bare GCE and electroreduced graphene oxide (ERGO) modified GCE. We found that the adsorption of G and A on the electrode surfaces can vary their anodic CV peaks and the competitive adsorption of G and A on the limited sites of the electrode surfaces can cause crosstalk effects on their anodic CV peak signals, but the HPLC-ECD system is insensitive to such electrode-adsorption and can give more reliable analytical results. PMID:25618679

  6. Application of the measurement of oxidized pyridine dinucleotides with high-performance liquid chromatography-fluorescence detection to assay the uncoupled oxidation of NADPH by neuronal nitric oxide synthase.

    PubMed

    Pálfi, Melinda; Halász, Attila Sándor; Tábi, Tamás; Magyar, Kálmán; Szöko, Eva