Science.gov

Sample records for adenine dinucleotide nad

  1. The chemistry of nicotinamide adenine dinucleotide (NAD) analogues containing C-nucleosides related to nicotinamide riboside.

    PubMed

    Pankiewicz, Krzysztof W; Watanabe, Kyoichi A; Lesiak-Watanabe, Krystyna; Goldstein, Barry M; Jayaram, Hiremagalur N

    2002-04-01

    Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

  2. 5' End Nicotinamide Adenine Dinucleotide Cap in Human Cells Promotes RNA Decay through DXO-Mediated deNADding.

    PubMed

    Jiao, Xinfu; Doamekpor, Selom K; Bird, Jeremy G; Nickels, Bryce E; Tong, Liang; Hart, Ronald P; Kiledjian, Megerditch

    2017-03-09

    Eukaryotic mRNAs generally possess a 5' end N7 methyl guanosine (m(7)G) cap that promotes their translation and stability. However, mammalian mRNAs can also carry a 5' end nicotinamide adenine dinucleotide (NAD(+)) cap that, in contrast to the m(7)G cap, does not support translation but instead promotes mRNA decay. The mammalian and fungal noncanonical DXO/Rai1 decapping enzymes efficiently remove NAD(+) caps, and cocrystal structures of DXO/Rai1 with 3'-NADP(+) illuminate the molecular mechanism for how the "deNADding" reaction produces NAD(+) and 5' phosphate RNA. Removal of DXO from cells increases NAD(+)-capped mRNA levels and enables detection of NAD(+)-capped intronic small nucleolar RNAs (snoRNAs), suggesting NAD(+) caps can be added to 5'-processed termini. Our findings establish NAD(+) as an alternative mammalian RNA cap and DXO as a deNADding enzyme modulating cellular levels of NAD(+)-capped RNAs. Collectively, these data reveal that mammalian RNAs can harbor a 5' end modification distinct from the classical m(7)G cap that promotes rather than inhibits RNA decay.

  3. Partial purification of nicotinamide adenine dinucleotide (NAD) pyrophosphatase from Salmonella typhimurium

    SciTech Connect

    Putt, M.M.; Foster, J.W.; Kasvinsky, P.J.

    1987-05-01

    NAD is an extremely important compound in cellular physiology. In the pyridine nucleotide cycle of S. typhimurium NAD pyrophosphatase, located in the inner membrane, carries out the cleavage of NAD prior to the transport of nicotinamide mononucleotide (NMN) into the cell. The partial purification of this enzyme is reported here. A cell suspension of S. typhimurium was passed twice through a French pressure cell, centrifugated at 5000 xg, and at 200,000 xg, for 1 hr. The pellet containing the crude membrane fraction was extracted with a novel detergent extraction using the differential solubility of NAD pyrophosphatase at various concentrations of the non-ionic detergent n-octyl glucoside (nOG). Extraction of the membrane fraction with 0.5% nOG in the presence of 10mM MgCl/sub 2/ removed 60% of the protein with no loss in activity. A second extraction with 2% nOG and 10mM MgCl/sub 2/ removed 20% of the protein and 71% of the activity from the membrane fraction. Ammonium sulfate fractionation at 45 to 50% sat. gave a partially purified enzyme preparation having a specific activity of about 2500 units/mg with a 94% recovery compared to the crude extract. One unit of activity is the cleavage of 1 nmole /sup 14/C NAD to /sup 14/C NMN per minute. The enzyme appears to have a MW of 200,000 on Sephacryl S-200, is temperature labile, and stabilized by 1mM Mg/sup + +/ and storage at -70/sup 0/.

  4. Protective effect of nicotinamide adenine dinucleotide (NAD(+)) against spinal cord ischemia-reperfusion injury via reducing oxidative stress-induced neuronal apoptosis.

    PubMed

    Xie, Lei; Wang, Zhenfei; Li, Changwei; Yang, Kai; Liang, Yu

    2017-02-01

    As previous studies demonstrate that oxidative stress and apoptosis play crucial roles in ischemic pathogenesis and nicotinamide adenine dinucleotide (NAD(+)) treatment attenuates oxidative stress-induced cell death among primary neurons and astrocytes as well as significantly reduce cerebral ischemic injury in rats. We used a spinal cord ischemia injury (SCII) model in rats to verify our hypothesis that NAD(+) could ameliorate oxidative stress-induced neuronal apoptosis. Adult male rats were subjected to transient spinal cord ischemia for 60min, and different doses of NAD(+) were administered intraperitoneally immediately after the start of reperfusion. Neurological function was determined by Basso, Beattie, Bresnahan (BBB) scores. The oxidative stress level was assessed by superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. The degree of apoptosis was analyzed by deoxyuridinetriphosphate nick-end labeling (TUNEL) staining and protein levels of cleaved caspase-3 and AIF (apoptosis inducing factor). The results showed that NAD(+) at 50 or 100mg/kg significantly decreased the oxidative stress level and neuronal apoptosis in the spinal cord of ischemia-reperfusion rats compared with saline, as accompanied with the decreased oxidative stress, NAD(+) administration significantly restrained the neuronal apoptosis after ischemia injury while improved the neurological and motor function. These findings suggested that NAD(+) might protect against spinal cord ischemia-reperfusion via reducing oxidative stress-induced neuronal apoptosis.

  5. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

    PubMed

    Mukherjee, Sarmistha; Chellappa, Karthikeyani; Moffitt, Andrea; Ndungu, Joan; Dellinger, Ryan W; Davis, James G; Agarwal, Beamon; Baur, Joseph A

    2017-02-01

    The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR.

  6. Novel concept of enzyme selective nicotinamide adenine dinucleotide (NAD)-modified inhibitors based on enzyme taxonomy from the diphosphate conformation of NAD.

    PubMed

    Fujii, Mikio; Kitagawa, Yasuyuki; Iida, Shui; Kato, Keisuke; Ono, Machiko

    2015-11-15

    The dihedral angle θ of the diphosphate part of NAD(P) were investigated to distinguish the differences in the binding-conformation of NAD(P) to enzymes and to create an enzyme taxonomy. Furthermore, new inhibitors with fixed dihedral angles showed that enzymes could recognize the differences in the dihedral angle θ. We suggest the taxonomy and the dihedral angle θ are important values for chemists to consider when designing inhibitors and drugs that target enzymes.

  7. Regulation of the Nicotinamide Adenine Dinucleotide- and Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutamate Dehydrogenases of Saccharomyces cerevisiae

    PubMed Central

    Roon, Robert J.; Even, Harvey L.

    1973-01-01

    Saccharomyces cerevisiae contains two distinct l-glutamate dehydrogenases. These enzymes are affected in a reciprocal fashion by growth on ammonia or dicarboxylic amino acids as the nitrogen source. The specific activity of the nicotinamide adenine dinucleotide phosphate (NADP) (anabolic) enzyme is highest in ammonia-grown cells and is reduced in cells grown on glutamate or aspartate. Conversely, the specific activity of the nicotinamide adenine dinucleotide (NAD) (catabolic) glutamate dehydrogenase is highest in cells grown on glutamate or aspartate and is much lower in cells grown on ammonia. The specific activity of both enzymes is very low in nitrogen-starved yeast. Addition of the ammonia analogue methylamine to the growth medium reduces the specific activity of the NAD-dependent enzyme and increases the specific activity of the NADP-dependent enzyme. PMID:4147647

  8. 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

  9. 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

  10. 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.

  11. A label-free fluorescence DNA probe based on ligation reaction with quadruplex formation for highly sensitive and selective detection of nicotinamide adenine dinucleotide.

    PubMed

    Zhao, Jingjin; Zhang, Liangliang; Jiang, Jianhui; Shen, Guoli; Yu, Ruqin

    2012-05-11

    A simple label-free fluorescent sensing scheme for sensitive and selective detection of nicotinamide adenine dinucleotide (NAD(+)) has been developed based on DNA ligation reaction with ligand-responsive quadruplex formation. This approach can detect 0.5 nM NAD(+) with high selectivity against other NAD(+) analogs.

  12. Naturally Occurring β-Nicotinamide Adenine Dinucleotide-Independent Avibacterium paragallinarum Isolate in Peru.

    PubMed

    Falconi-Agapito, Francesca; Saravia, Luis E; Flores-Pérez, Aldo; Fernández-Díaz, Manolo

    2015-06-01

    The β-nicotinamide adenine dinucleotide (NAD) requirement has been considered to be essential for the isolation of the causal agent of infectious coryza, Avibacterium paragallinarum. Nevertheless, NAD-independent reports from South Africa and Mexico dismissed this paradigm. It is now accepted that both NAD-dependent and NAD-independent agents are able to cause infectious coryza and thus belong to the species A. paragallinarum. Here, we report for the first time in Peru a NAD-independent isolate from broiler chickens with typical signs of infectious coryza that have received a trivalent inactivated vaccine against infectious coryza. The isolate was identified based on its morphology, biochemical and serologic tests, and PCR results. Partial 16S rRNA gene sequence analysis confirmed the isolate as A. paragallinarum. There have been no cases of NAD-independent A. paragallinarum isolates reported in South America. Increasing reports around the world highlight not only the need to reconsider the in vitro nutritional requirements of this species for its correct isolation but also the cross-protection conferred by commercial infectious coryza vaccines against NAD-independent isolates.

  13. Nicotinamide Adenine Dinucleotide Protects against Spinal Cord Ischemia Reperfusion Injury-Induced Apoptosis by Blocking Autophagy

    PubMed Central

    Yu, Sifei; Wang, Zhenfei; Yang, Kai; Liu, Zhuochao

    2017-01-01

    The role of autophagy, neuroprotective mechanisms of nicotinamide adenine dinucleotide (NAD+), and their relationship in spinal cord ischemic reperfusion injury (SCIR) was assessed. Forty-eight Sprague-Dawley rats were divided into four groups: sham, ischemia reperfusion (I/R), 10 mg/kg NAD+, and 75 mg/kg NAD+. Western blotting, immunofluorescence, and immunohistochemistry were used to assess autophagy and apoptosis. Basso, Beattie, and Bresnahan (BBB) scores were used to assess neurological function. Expression levels of Beclin-1, Atg12-Atg5, LC3B-II, cleaved caspase 3, and Bax were upregulated in the I/R group and downregulated in the 75 mg/kg NAD+ group; p-mTOR, p-AKT, p62, and Bcl-2 were downregulated in the I/R group and upregulated in the 75 mg/kg NAD+ group. Numbers of LC3B-positive, caspase 3-positive, Bax-positive, and TUNEL-positive cells were significantly increased in the I/R group and decreased in the 75 mg/kg NAD+ group. The mean integrated option density of Bax increased and that of Nissl decreased in the I/R group, and it decreased and increased, respectively, in the 75 mg/kg NAD+ group. BBB scores significantly increased in the 75 mg/kg NAD+ group relative to the I/R group. No difference was observed between I/R and 10 mg/kg NAD+ groups for these indicators. Therefore, excessive and sustained autophagy aggravates SCIR; administration of NAD+ alleviates injury. PMID:28367271

  14. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy.

    PubMed

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

    Axonal degeneration occurs in many neurodegenerative diseases and after traumatic injury and is a self-destructive program independent from programmed cell death. Previous studies demonstrated that overexpression of nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) or exogenous application of nicotinamide adenine dinucleotide (NAD) can protect axons of cultured dorsal root ganglion (DRG) neurons from degeneration caused by mechanical or neurotoxic injury. In mammalian cells, NAD can be synthesized from multiple precursors, including tryptophan, nicotinic acid, nicotinamide, and nicotinamide riboside (NmR), via multiple enzymatic steps. To determine whether other components of these NAD biosynthetic pathways are capable of delaying axonal degeneration, we overexpressed each of the enzymes involved in each pathway and/or exogenously administered their respective substrates in DRG cultures and assessed their capacity to protect axons after axotomy. Among the enzymes tested, Nmnat1 had the strongest protective effects, whereas nicotinamide phosphoribosyl transferase and nicotinic acid phosphoribosyl transferase showed moderate protective activity in the presence of their substrates. Strong axonal protection was also provided by Nmnat3, which is predominantly located in mitochondria, and an Nmnat1 mutant localized to the cytoplasm, indicating that the subcellular location of NAD production is not crucial for protective activity. In addition, we showed that exogenous application of the NAD precursors that are the substrates of these enzymes, including nicotinic acid mononucleotide, nicotinamide mononucleotide, and NmR, can also delay axonal degeneration. These results indicate that stimulation of NAD biosynthetic pathways via a variety of interventions may be useful in preventing or delaying axonal degeneration.

  15. Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide

    NASA Astrophysics Data System (ADS)

    Pan, Fu-shih; Chen, Stephen; Mintzer, Robert A.; Chen, Chin-Tu; Schumacker, Paul

    1991-03-01

    It is of fundamental importance for biological scientists to assess cellular energetics. Under aerobic conditions, the tricarboxylic acid cycle (TCA cycle) is coupled with the mitochondrial electron cascade pathway to provide the cell with energy. The nicotinamide adenine dinucleotide-conjugated pair (NAD and NADH) is the coenzyme in numerous important biomedical reactions which include several important dehydrogenase reactions in the TCA cycle. Based on Le Chatelier's principle, NADH will accumulate when this energy production mechanism is impaired. The relative amounts of NAD and NADH in a cell are defined as the redox state of the cell (Williamson et.al. 1967) which provides a valuable index of cellular energetics. The sum of the amounts of NAD and NADH in a cell may be assumed to be constant during a finite time; therefore, a reliable means of measuring the NADH concentration would provide us with a useful indicator of tissue viability. Traditionally, the quantities of NADH and NAD may be measured by chemical assay methods. We can avoid these tediois analyses by exploiting the significant difference between the ultraviolet absorption spectra of this redox pair. However, because of the opacity of biological samples and the interference of other biochemicals that also absorb ultraviolet radiation, measurement of NADH and NAD+ concentrations in vivo by absorption spectroscopy is not feasible.

  16. Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner

    PubMed Central

    Preyat, N; Rossi, M; Kers, J; Chen, L; Bertin, J; Gough, P J; Le Moine, A; Rongvaux, A; Van Gool, F; Leo, O

    2016-01-01

    Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD+) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD+ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD+-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD+ does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors. PMID:26001219

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

    PubMed

    Mericskay, Mathias

    2016-03-01

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

  18. Study on nicotinamide adenine dinucleotide adsorbed at nano-boehmite/water and nano-corundum/water interfaces.

    PubMed

    Li, Li; Xie, Yanfang; Wang, Yanping; Yang, Xiaodi; Chen, Rong Fu; Shen, Ren Fang

    2013-02-01

    In this study, the adsorption behaviors of nicotinamide adenine dinucleotide (NAD(+)) on nano-boehmite (γ-AlOOH) and nano-corundum (γ-Al(2)O(3)) surfaces were investigated. The results showed that NAD(+) was predominantly adsorbed at the boehmite/water and corundum/water interfaces in outer-sphere fashions by electrostatic interaction between NAD(+) phosphate and surface hydroxyl groups. However, the features of ATR-FTIR spectra suggested that some minor inner-sphere complex should be considered at low pH conditions on corundum surface, which was consistent with the effect of NAD(+) on dissolution rate of corundum. In addition, the adsorption data well fitted with Langmuir and Freundlich isotherms on the boehmite and corundum surfaces, respectively. Also, the Gibbs adsorption energy was negative on the boehmite surface, which indicated that the adsorption behavior was spontaneous.

  19. Evidence for two-step binding of reduced nicotinamide-adenine dinucleotide to aldehyde dehydrogenase.

    PubMed Central

    MacGibbon, A K; Buckley, P D; Blackwell, L F

    1977-01-01

    The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate. Images Fig. 1. Fig. 2. Fig. 3. Fig. 5. PMID:21657

  20. Flavin Adenine Dinucleotide Structural Motifs: From Solution to Gas Phase

    PubMed Central

    2015-01-01

    Flavin adenine dinucleotide (FAD) is involved in important metabolic reactions where the biological function is intrinsically related to changes in conformation. In the present work, FAD conformational changes were studied in solution and in gas phase by measuring the fluorescence decay time and ion-neutral collision cross sections (CCS, in a trapped ion mobility spectrometer, TIMS) as a function of the solvent conditions (i.e., organic content) and gas-phase collisional partner (i.e., N2 doped with organic molecules). Changes in the fluorescence decay suggest that FAD can exist in four conformations in solution, where the abundance of the extended conformations increases with the organic content. TIMS-MS experiments showed that FAD can exist in the gas phase as deprotonated (M = C27H31N9O15P2) and protonated forms (M = C27H33N9O15P2) and that multiple conformations (up to 12) can be observed as a function of the starting solution for the [M + H]+ and [M + Na]+molecular ions. In addition, changes in the relative abundances of the gas-phase structures were observed from a “stack” to a “close” conformation when organic molecules were introduced in the TIMS cell as collision partners. Candidate structures optimized at the DFT/B3LYP/6-31G(d,p) were proposed for each IMS band, and results showed that the most abundant IMS band corresponds to the most stable candidate structure. Solution and gas-phase experiments suggest that the driving force that stabilizes the different conformations is based on the interaction of the adenine and isoalloxazine rings that can be tailored by the “solvation” effect created with the organic molecules. PMID:25222439

  1. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829.

    PubMed

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy

    2016-06-01

    The present study was conducted to investigate a new pathway for the degradation of nicotinamide adenine dinucleotide (NAD) by Penicillium brevicompactum NRC 829 extracts. Enzymes involved in the hydrolysis of NAD, i.e. alkaline phosphatase, aminohydrolase and glycohydrolase were determined. Alkaline phosphatase was found to catalyse the sequential hydrolysis of two phosphate moieties of NAD molecule to nicotinamide riboside plus adenosine. Adenosine was then deaminated by aminohydrolase to inosine and ammonia. While glycohydrolase catalyzed the hydrolysis of the nicotinamide-ribosidic bond of NAD+ to produce nicotinamide and ADP-ribose in equimolar amounts, enzyme purification through a 3-step purification procedure revealed the existence of two peaks of alkaline phosphatases, and one peak contained deaminase and glycohydrolase activities. NAD deaminase was purified to homogeneity as estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with an apparent molecular mass of 91 kDa. Characterization and determination of some of NAD aminohydrolase kinetic properties were conducted due to its biological role in the regulation of cellular NAD level. The results also revealed that NAD did not exert its feedback control on nicotinamide amidase produced by P. brevicompactum.

  2. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay.

    PubMed

    Ummarino, Simone; Mozzon, Massimo; Zamporlini, Federica; Amici, Adolfo; Mazzola, Francesca; Orsomando, Giuseppe; Ruggieri, Silverio; Raffaelli, Nadia

    2017-04-15

    Nicotinamide riboside, the most recently discovered form of vitamin B3, and its phosphorylated form nicotinamide mononucleotide, have been shown to be potent supplements boosting intracellular nicotinamide adenine dinucleotide (NAD) levels, thus preventing or ameliorating metabolic and mitochondrial diseases in mouse models. Here we report for the first time on the simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and NAD in milk by means of a fluorometric, enzyme-coupled assay. Application of this assay to milk from different species revealed that the three vitamers were present in human and donkey milk, while being selectively distributed in the other milks. Human milk was the richest source of nicotinamide mononucleotide. Overall, the three vitamers accounted for a significant fraction of total vitamin B3 content. Pasteurization did not affect the bovine milk content of nicotinamide riboside, whereas UHT processing fully destroyed the vitamin. In human milk, NAD levels were significantly affected by the lactation time.

  3. A Nicotinamide Adenine Dinucleotide Dispersed Multi-walled Carbon Nanotubes Electrode for Direct and Selective Electrochemical Detection of Uric Acid.

    PubMed

    Chen, Yan; Li, Yiwei; Ma, Yaohong; Meng, Qingjun; Yan, Yan; Shi, Jianguo

    2015-01-01

    A nanocomposite platform built with multi-walled carbon nanotubes (MWCNTs) and nicotinamide adenine dinucleotide (NAD(+)) via a noncovalent interaction between the large π systems in NAD(+) molecules and MWCNTs on a glassy carbon substrate was successfully developed for the sensitive and selective detection of uric acid (UA) in the presence of ascorbic acid (AA), dopamine (DA). NAD(+) has an adenine subunit and a nicotinamide subunit, which enabled interaction with the purine subunit of UA through a strong π-π interaction to enhance the specificity of UA. Compared with a bare glassy carbon electrode (GCE) and MWCNTs/GCE, the MWCNTs-NAD(+)/GCE showed a low background current and a remarkable enhancement of the oxidation peak current of UA. Using differential pulse voltammetry (DPV), a high sensitivity for the determination of UA was explored for the MWCNTs-NAD(+) modified electrode. A linear relationship between the DPV peak current of UA and its concentration could be obtained in the range of 0.05 - 10 μM with the detection limit as low as 10 nM (S/N = 3). This present strategy provides a novel and promising platform for the detection of UA in human urine and serum samples.

  4. Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg.

    PubMed

    Walseth, Timothy F; Lin-Moshier, Yaping; Jain, Pooja; Ruas, Margarida; Parrington, John; Galione, Antony; Marchant, Jonathan S; Slama, James T

    2012-01-20

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [(32)P-5-azido]nicotinic acid adenine dinucleotide phosphate ([(32)P-5N(3)]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [(32)P-5N(3)]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N(3)-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [(32)P-5N(3)]NAADP binding was saturable and displayed high affinity (K(d) ∼10 nM) in both binding and photolabeling experiments. [(32)P-5N(3)]NAADP photolabeling was irreversible in a high K(+) buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [(32)P-5N(3)]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs.

  5. Properties of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum

    PubMed Central

    Li, Lan-Fun; Ljungdahl, Lars; Wood, Harland G.

    1966-01-01

    Li, Lan-Fun (Western Reserve University School of Medicine, Cleveland, Ohio), Lars Ljungdahl, and Harland G. Wood. Properties of nicotinamide adenine dinucleotide phosphate-dependent formate dehydrogenase from Clostridium thermoaceticum. J. Bacteriol. 92: 405–412. 1966.—A nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase has been isolated from C. thermoaceticum. The enzyme is very sensitive to oxygen and requires sulfhydryl compounds for activity. The apparent Km at 50 C and pH 7.0 for NADP is 5.9 × 10−5m and for formate, 2.2 × 10−4m. The enzyme is most active at about 60 C and at pH values between 7.0 and 9.0. The enzyme catalyzes an exchange between C14O2 and formate, which requires NADP, but net synthesis of formate from CO2 and reduced nicotinamide adenine dinucleotide phosphate could not be demonstrated. The reaction does not involve ferredoxin. PMID:16562128

  6. Tissue-specific regulation of sirtuin and nicotinamide adenine dinucleotide biosynthetic pathways identified in C57Bl/6 mice in response to high-fat feeding.

    PubMed

    Drew, Janice E; Farquharson, Andrew J; Horgan, Graham W; Williams, Lynda M

    2016-11-01

    The sirtuin (SIRT)/nicotinamide adenine dinucleotide (NAD) system is implicated in development of type 2 diabetes (T2D) and diet-induced obesity, a major risk factor for T2D. Mechanistic links have not yet been defined. SIRT/NAD system gene expression and NAD/NADH levels were measured in liver, white adipose tissue (WAT) and skeletal muscle from mice fed either a low-fat diet or high-fat diet (HFD) for 3 days up to 16 weeks. An in-house custom-designed multiplex gene expression assay assessed all 7 mouse SIRTs (SIRT1-7) and 16 enzymes involved in conversion of tryptophan, niacin, nicotinamide riboside and metabolic precursors to NAD. Significantly altered transcription was correlated with body weight, fat mass, plasma lipids and hormones. Regulation of the SIRT/NAD system was associated with early (SIRT4, SIRT7, NAPRT1 and NMNAT2) and late phases (NMNAT3, NMRK2, ABCA1 and CD38) of glucose intolerance. TDO2 and NNMT were identified as markers of HFD consumption. Altered regulation of the SIRT/NAD system in response to HFD was prominent in liver compared with WAT or muscle. Multiple components of the SIRTs and NAD biosynthetic enzymes network respond to consumption of dietary fat. Novel molecular targets identified above could direct strategies for dietary/therapeutic interventions to limit metabolic dysfunction and development of T2D.

  7. 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.

  8. Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach.

    PubMed

    Zeng, Lingfei; Shin, Woong-Hee; Zhu, Xiaolei; Park, Sung Hoon; Park, Chiwook; Tao, W Andy; Kihara, Daisuke

    2017-02-03

    Protein-ligand interaction plays a critical role in regulating the biochemical functions of proteins. Discovering protein targets for ligands is vital to new drug development. Here, we present a strategy that combines experimental and computational approaches to identify ligand-binding proteins in a proteomic scale. For the experimental part, we coupled pulse proteolysis with filter-assisted sample preparation (FASP) and quantitative mass spectrometry. Under denaturing conditions, ligand binding affected protein stability, which resulted in altered protein abundance after pulse proteolysis. For the computational part, we used the software Patch-Surfer2.0. We applied the integrated approach to identify nicotinamide adenine dinucleotide (NAD)-binding proteins in the Escherichia coli proteome, which has over 4200 proteins. Pulse proteolysis and Patch-Surfer2.0 identified 78 and 36 potential NAD-binding proteins, respectively, including 12 proteins that were consistently detected by the two approaches. Interestingly, the 12 proteins included 8 that are not previously known as NAD binders. Further validation of these eight proteins showed that their binding affinities to NAD computed by AutoDock Vina are higher than their cognate ligands and also that their protein ratios in the pulse proteolysis are consistent with known NAD-binding proteins. These results strongly suggest that these eight proteins are indeed newly identified NAD binders.

  9. Physical Separation of Streptococcal Nicotinamide Adenine Dinucleotide Glycohydrolase from Streptolysin O

    PubMed Central

    Shany, S.; Grushoff, Phyllis S.; Bernheimer, Alan W.

    1973-01-01

    Streptococcal nicotinamide adenine dinucleotide glycohydrolase (NADase) with a molecular weight of about 55,000 and an isoelectric pH of 8.55 was isolated from crude streptolysin O (SLO) preparations. NADase differed from SLO in size, charge, and immunological behavior. Streptococcal NADase is considered to have no role in the hemolytic process because it has no hemolytic activity; conversely, partially purified SLO showed no NADase activity. The hemolytic activity of crude SLO was completely inhibited by anti-tetanolysin, whereas the NADase activity in the same reaction mixture was unaffected. Experiments involving double diffusion in agar also demonstrated immunological nonidentity of the two proteins. Images PMID:4357989

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

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

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

  11. 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.

  12. Release of beta-nicotinamide adenine dinucleotide upon stimulation of postganglionic nerve terminals in blood vessels and urinary bladder.

    PubMed

    Smyth, Lisa M; Bobalova, Janette; Mendoza, Michael G; Lew, Christy; Mutafova-Yambolieva, Violeta N

    2004-11-19

    Chemical signaling in autonomic neuromuscular transmission involves agents that function as neurotransmitters and/or neuromodulators. Using high performance liquid chromatography techniques with fluorescence and electrochemical detection we observed that, in addition to ATP and norepinephrine (NE), electrical field stimulation (EFS, 4-16 Hz, 0.1-0.3 ms, 15 V, 60-120 s) of isolated vascular and non-vascular preparations co-releases a previously unidentified compound with apparent nucleotide or nucleoside structure. Extensive screening of more than 25 nucleotides and nucleosides followed by detailed peak identification revealed that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in tissue superfusates upon EFS of canine mesenteric artery (CMA), canine urinary bladder, and murine urinary bladder in the amounts of 7.1 +/- 0.7, 26.5 +/- 4.5, and 15.1 +/- 3.2 fmol/mg of tissue, respectively. Smaller amounts of the beta-NAD metabolites cyclic adenosine 5'-diphosphoribose (cADPR) and ADPR were also present in the superfusates collected during EFS of CMA (2.5 +/- 0.9 and 5.8 +/- 0.8 fmol/mg of tissue, respectively), canine urinary bladder (1.8 +/- 0.5 and 9.0 +/- 6.0 fmol/mg of tissue, respectively), and murine urinary bladder (1.4 +/- 0.1 and 6.2 +/- 2.4 fmol/mg of tissue, respectively). The three nucleotides were also detected in the samples collected before EFS (0.2-1.6 fmol/mg of tissue). Exogenous beta-NAD, cADPR, and ADPR (all 100 nm) reduced the release of NE in CMA at 16 Hz from 27.8 +/- 6.0 fmol/mg of tissue to 15.5 +/- 5.0, 12 +/- 3.0, and 10.0 +/- 4.0 fmol/mg of tissue, respectively. In conclusion, we detected constitutive and nerve-evoked overflow of beta-NAD, cADPR, and ADPR in vascular and non-vascular smooth muscles, beta-NAD being the prevailing compound. These substances modulate the release of NE, implicating novel nucleotide mechanisms of autonomic nervous system control of smooth muscle.

  13. 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

  14. 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.

  15. Conformational behavior of flavin adenine dinucleotide: conserved stereochemistry in bound and free states.

    PubMed

    Kuppuraj, Gopi; Kruise, Dennis; Yura, Kei

    2014-11-26

    Metabolic enzymes utilize the cofactor flavin adenine dinucleotide (FAD) to catalyze essential biochemical reactions. Because these enzymes have been implicated in disease pathways, it will be necessary to target them via FAD-based structural analogues that can either activate/inhibit the enzymatic activity. To achieve this, it is important to explore the conformational space of FAD in the enzyme-bound and free states. Herein, we analyze X-ray crystallographic data of the enzyme-bound FAD conformations and sample conformations of the molecule in explicit water by molecular dynamics (MD) simulations. Enzyme-bound FAD conformations segregate into five distinct groups based on dihedral angle principal component analysis (PCA). A notable feature in the bound FADs is that the adenine base and isoalloxazine ring are oppositely oriented relative to the pyrophosphate axis characterized by near trans hypothetical dihedral angle "δV" values. Not surprisingly, MD simulations in water show final compact but not perfectly stacked ring structures in FAD. Simulation data did not reveal noticeable changes in overall conformational dynamics of the dinucleotide in reduced and oxidized forms and in the presence and/or absence of ions. During unfolding-folding dynamics, the riboflavin moiety is more flexible than the adenosine monophosphate group in the molecule. Conversely, the isoalloxazine ring is more stable than the variable adenine base. The pyrophosphate group depicts an unusually highly organized fluctuation illustrated by its dihedral angle distribution. Conformations sampled from enzymes and MD are quantified. The extent to which the protein shifts the distribution from the unbound state is discussed in terms of prevalent FAD shapes and dihedral angle population.

  16. Cytoprotection of pyruvic acid and reduced beta-nicotinamide adenine dinucleotide against hydrogen peroxide toxicity in neuroblastoma cells.

    PubMed

    Mazzio, Elizabeth A; Soliman, Karam F A

    2003-05-01

    Elevated production of hydrogen peroxide (H2O2) in the central nervous system has been implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, ischemic reperfusion, stroke, and Alzheimer's disease. Pyruvic acid has a critical role in energy metabolism and a capability to nonenzymatically decarboxylate H2O2 into H2O. This study examined the effects of glycolytic regulation of pyruvic acid on H2O2 toxicity in murine neuroblastoma cells. Glycolytic energy substrates including D-(+)-glucose, D-(-) fructose and the adenosine transport blocker dipyridamole, were not effective in providing protection against H2O2 toxicity, negating energy as a factor. On the other hand, pyruvic acid completely prevented H2O2 toxicity, restoring the loss of ATP and cell viability. H2O2 toxicity was also attenuated by D-fructose 1,6 diphosphate (FBP), phospho (enol) pyruvate (PEP), niacinamide, beta-nicotinamide adenine dinucleotide (beta-NAD+), and reduced form (beta-NADH). Both FBP and PEP exerted positive kinetic effects on pyruvate kinase (PK) activity. Interestingly, only pyruvic acid and beta-NADH exhibited powerful stoichiometric H2O2 antioxidant properties. Further, beta-NADH may exert positive effects on PK activity. Subsequent pyruvic acid accumulation can lead to the recycling of beta-NAD+ through lactate dehydrogenase and beta-NADH through glyceraldehyde-3-phosphate dehydrogenase. It was concluded from these studies that intracellular pyruvic acid and beta-NADH appear to act in concert through glycolysis, to enhance H2O2 intracellular antioxidant capacity in neuroblastoma cells. Future research will be required to examine whether similar effects are observed in primary neuronal culture or intact tissue.

  17. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system

    PubMed Central

    Cordiglieri, Chiara; Odoardi, Francesca; Zhang, Bo; Nebel, Merle; Kawakami, Naoto; Klinkert, Wolfgang E. F.; Lodygin, Dimtri; Lühder, Fred; Breunig, Esther; Schild, Detlev; Ulaganathan, Vijay Kumar; Dornmair, Klaus; Dammermann, Werner; Potter, Barry V. L.; Guse, Andreas H.

    2010-01-01

    Nicotinic acid adenine dinucleotide phosphate represents a newly identified second messenger in T cells involved in antigen receptor-mediated calcium signalling. Its function in vivo is, however, unknown due to the lack of biocompatible inhibitors. Using a recently developed inhibitor, we explored the role of nicotinic acid adenine dinucleotide phosphate in autoreactive effector T cells during experimental autoimmune encephalomyelitis, the animal model for multiple sclerosis. We provide in vitro and in vivo evidence that calcium signalling controlled by nicotinic acid adenine dinucleotide phosphate is relevant for the pathogenic potential of autoimmune effector T cells. Live two photon imaging and molecular analyses revealed that nicotinic acid adenine dinucleotide phosphate signalling regulates T cell motility and re-activation upon arrival in the nervous tissues. Treatment with the nicotinic acid adenine dinucleotide phosphate inhibitor significantly reduced both the number of stable arrests of effector T cells and their invasive capacity. The levels of pro-inflammatory cytokines interferon-gamma and interleukin-17 were strongly diminished. Consecutively, the clinical symptoms of experimental autoimmune encephalomyelitis were ameliorated. In vitro, antigen-triggered T cell proliferation and cytokine production were evenly suppressed. These inhibitory effects were reversible: after wash-out of the nicotinic acid adenine dinucleotide phosphate antagonist, the effector T cells fully regained their functions. The nicotinic acid derivative BZ194 induced this transient state of non-responsiveness specifically in post-activated effector T cells. Naïve and long-lived memory T cells, which express lower levels of the putative nicotinic acid adenine dinucleotide phosphate receptor, type 1 ryanodine receptor, were not targeted. T cell priming and recall responses in vivo were not reduced. These data indicate that the nicotinic acid adenine dinucleotide phosphate

  18. 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.

  19. Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase.

    PubMed Central

    Hartmans, S; van der Werf, M J; de Bont, J A

    1990-01-01

    By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed. PMID:2339888

  20. 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.

  1. 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.

  2. 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.

  3. Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

    PubMed Central

    Todo, T; Kim, S T; Hitomi, K; Otoshi, E; Inui, T; Morioka, H; Kobayashi, H; Ohtsuka, E; Toh, H; Ikenaga, M

    1997-01-01

    Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase. PMID:9016626

  4. 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.

  5. Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation.

    PubMed

    Nakama, Mitsuo; Murakami, Yuhko; Tanaka, Hiroshi; Nakata, Satoru

    2012-03-01

    Skin pigmentation is caused by various physical and chemical factors. It might also be influenced by changes in the physiological function of skin with aging. Nicotinamide adenine dinucleotide (NADH) dehydrogenase is an enzyme related to the mitochondrial electron transport system and plays a key role in cellular energy production. It has been reported that the functional decrease in this system causes Parkinson's disease. Another study reports that the amount of NADH dehydrogenase in heart and skeletal muscle decreases with aging. A similar decrease in the skin would probably affect its physiological function. However, no reports have examined the age-related change in levels of NADH dehydrogenase in human skin. In this study, we investigated this change and its effect on skin pigmentation using cultured human epidermal keratinocytes. The mRNA expression of NDUFA1, NDUFB7, and NDUFS2, subunits of NADH dehydrogenase, and its activity were significantly decreased in late passage keratinocytes compared to early passage cells. Conversely, the mRNA expression of melanocyte-stimulating cytokines, interleukin-1 alpha and endothelin 1, was increased in late passage cells. On the other hand, the inhibition of NADH dehydrogenase upregulated the mRNA expression of melanocyte-stimulating cytokines. Moreover, the level of NDUFB7 mRNA was lower in pigmented than in nonpigmented regions of skin in vivo. These results suggest the decrease in NADH dehydrogenase with aging to be involved in skin pigmentation.

  6. Eco-synthesis of graphene and its use in dihydronicotinamide adenine dinucleotide sensing.

    PubMed

    Amouzadeh Tabrizi, Mahmoud; Jalilzadeh Azar, Somayeh; Nadali Varkani, Javad

    2014-09-01

    In this paper, we report a green and eco-friendly approach to synthesize reduced graphene oxide (rGO) via a mild hydrothermal process using malt as a reduced agent. The proposed method is based on the reduction of graphene oxide (GO) in malt solution by making use of the reducing capability of phenolic compounds contained in malt solution. The obtained rGO was characterized by atomic force microscopy (AFM), ultraviolet-visible (UV-vis) absorption spectroscopy, X-ray diffraction spectroscopy (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Electrochemical impedance spectroscopy analysis revealed that the charge transfer resistance of rGO modified glassy carbon (GC) electrode was much lower than that of the GC electrode. The electrochemical behavior of dihydronicotinamide adenine dinucleotide (NADH) on rGO modified GC electrode was investigated by cyclic voltammetry and amperometry. Electrochemical experiments indicated that rGO/GC electrode exhibited excellent electrocatalytic activity toward the NADH, which can be attributed to excellent electrical conductivity and high specific surface area of the rGO composite. The resulting biosensor showed highly sensitive amperometric response to NADH with a low detection limit (0.33μM).

  7. Dynamic and static quenching of 1,N6-ethenoadenine fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3-(indol-3-yl) propyl) adenine.

    PubMed Central

    Gruber, B A; Leonard, N J

    1975-01-01

    For nicotinamide 1,N6-ethenoadenine dinucleotide (epsilonNAD+), the fluorescent analog of NAD+, in neutral aqueous solution the quantum yield has been determined to be 0.028 and the fluorescent lifetime, 2.1 nsec. Simultaneous determination of quantum yields and lifetimes of epsilonNAD+ and of the "half molecule" epsilonAMP allows the calculation of the percentage of stacked and open conformations of the dinucleotide. At 25 degrees in neutral aqueous solution there is 45 +/- 5% of stacked forms. The value of the fluorescent impurities, especially those containing the epsilon-adenosine moiety, and a purification procedure using high performance liquid chromatography was devised to obtain fluorescently homogeneous preparations. In order to study the effect on epsilon-adenosine fluorescence caused by the possible close proximity of a tryptophan in a polypeptide chain or protein, we have prepared 1,N6-etheno-9-[3-(indol-3-yl)propyl]adenine (epsilonAde9-C3-Ind3), a model compound in which indole is used as a neutral substitute for tryptophan. Fluorescence studies on epsilonAde9-C3-Ind3 show that the formation of an intramolecular complex results in complete quenching of the epsilon-adenine fluorescence. It is therefore predictable that positioning of the epsilon-adenosine of any fluorescent coenzyme moiety (e.q., epsilonATP, epsilonADP) in close proximity to a tryptophan in a protein will result in complete fluorescence quenching of the former. PMID:172889

  8. Enhancement of anaerobic degradation of azo dye with riboflavin and nicotinamide adenine dinucleotide harvested by osmotic lysis of wasted fermentation yeasts.

    PubMed

    Victral, Davi M; Dias, Heitor R A; Silva, Silvana Q; Baeta, Bruno E L; Aquino, Sérgio F

    2017-02-01

    The study presented here aims at identifying the source of redox mediators (riboflavin), electron carriers nicotinamide adenine dinucleotide (NAD) and carbon to perform decolorization of azo dye under anaerobic conditions after osmotic shock pretreatment of residual yeast from industrial fermentation. Pretreatment conditions were optimized by Doehlert experiment, varying NaCl concentration, temperature, yeast density and time. After the optimization, the riboflavin concentration in the residual yeast lysate (RYL) was 46% higher than the one present in commercial yeast extract. Moreover, similar NAD concentration was observed in both extracts. Subsequently, two decolorization experiments were performed, that is, a batch experiment (48 h) and a kinetic experiment (102 h). The results of the batch experiment showed that the use of the RYL produced by the optimized method increased decolorization rates and led to color removal efficiencies similar to those found when using the commercial extract (∼80%) and from 23% to 50% higher when compared to the control (without redox mediators). Kinetics analysis showed that methane production was also higher in the presence of yeast extract and RYL, and biogas was mostly generated after stabilization of color removal. In all kinetics experiments the azo dye degradation followed the pseudo-second-order model, which suggested that there was a concomitant adsorption/degradation of the dye on the biomass cell surface. Therefore, results showed the possibility of applying the pretreated residual yeast to improve color removal under anaerobic conditions, which is a sustainable process.

  9. 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

  10. The Responses of Isolated Plant Mitochondria to External Nicotinamide Adenine Dinucleotide 1

    PubMed Central

    Soole, Kathleen L.; Dry, Ian B.; Wiskich, Joseph T.

    1986-01-01

    The effects of added NAD on substrate oxidation by turnip (Brassica rapa L.) and beetroot (Beta vulgaris L.) mitochondria were investigated. State 3 malate and 2-oxoglutarate oxidation rates with turnip mitochondria were stimulated 25 to 40% by external NAD. Following NAD-depletion this stimulation by NAD was increased to 70 to 80%. With purified beetroot mitochondria, state 3 malate and 2-oxoglutarate oxidation rates were only marginally increased (10-15%) by the addition of NAD but after NAD-depletion treatments this stimulation increased to 55%. The effect of added NAD on oxidation rates could be reduced by preloading mitochondria with NAD in the presence of succinate. Oxidation rates were found to be most sensitive to the addition of external NAD when rotenone was present. The uptake of external NAD into beetroot mitochondria appeared to be composed of both an active and a diffusive component. The active component displayed saturation kinetics with an approximate Km of 0.105 ± 0.046 millimolar. These results provide further evidence, reported previously with potato mitochondria, that NAD can move across the inner membrane of plant mitochondria. They are particularly significant with respect to beetroot mitochondria which in contrast to other plant mitochondria, have not demonstrated any response to added NAD. PMID:16664861

  11. 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.

  12. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger in muscarinic receptor-induced contraction of guinea pig trachea.

    PubMed

    Aley, Parvinder K; Singh, Nisha; Brailoiu, G Cristina; Brailoiu, Eugen; Churchill, Grant C

    2013-04-19

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is increasingly being demonstrated to be involved in calcium signaling in many cell types and species. Although it has been shown to play a role in smooth muscle cell contraction in several tissues, nothing is known about its possible role in tracheal smooth muscle, a muscle type that is clinically relevant to asthma. To determine whether NAADP functions as a second messenger in tracheal smooth muscle contraction, we used the criteria set out by Sutherland for a molecule to be designated a second messenger. We report that NAADP satisfies all five criteria as follows. First, the NAADP antagonist Ned-19 inhibited contractions in tracheal rings and calcium increases in isolated smooth muscle cells induced by the muscarinic agonist carbachol. Second, NAADP increased cytosolic calcium in isolated cells when microinjected and was blocked by Ned-19. Third, tracheal homogenates could synthesize NAADP by base exchange from exogenous NADP and nicotinic acid and metabolize exogenous NAADP to nicotinic acid adenine dinucleotide by a 2'-phosphatase. Fourth, carbachol induced a rapid and transient increase in endogenous NAADP levels. Fifth, tracheal homogenates contained NAADP-binding sites of high affinity. Taken together, these data demonstrate that NAADP functions as a second messenger in tracheal smooth muscle, and therefore, steps in the NAADP signaling pathway might provide possible new drug targets.

  13. 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.

  14. 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.

  15. Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula polymorpha requires the cofactor flavin adenine dinucleotide.

    PubMed Central

    Evers, M E; Titorenko, V I; van der Klei, I J; Harder, W; Veenhuis, M

    1994-01-01

    The peroxisomal flavoprotein alcohol oxidase (AO) is an octamer (600 kDa) consisting of eight identical subunits, each of which contains one flavin adenine dinucleotide molecule as a cofactor. Studies on a riboflavin (Rf) auxotrophic mutant of the yeast Hansenula polymorpha revealed that limitation of the cofactor led to drastic effects on AO import and assembly as well as peroxisome proliferation. Compared to wild-type control cells Rf-limitation led to 1) reduced levels of AO protein, 2) reduced levels of correctly assembled and activated AO inside peroxisomes, 3) a partial inhibition of peroxisomal protein import, leading to the accumulation of precursors of matrix proteins in the cytosol, and 4) a significant increase in peroxisome number. We argue that the inhibition of import may result from the saturation of a peroxisomal molecular chaperone under conditions that normal assembly of a major matrix protein inside the target organelle is prevented. Images PMID:7803851

  16. 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.

  17. Mutations in adenine-binding pockets enhance catalytic properties of NAD(P)H-dependent enzymes

    PubMed Central

    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

  18. Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase.

    PubMed

    Moye, W S; Amuro, N; Rao, J K; Zalkin, H

    1985-07-15

    The yeast GDH1 gene encodes NADP-dependent glutamate dehydrogenase. This gene was isolated by complementation of an Escherichia coli glutamate auxotroph. NADP-dependent glutamate dehydrogenase was overproduced 6-10-fold in Saccharomyces cerevisiae bearing GDH1 on a multicopy plasmid. The nucleotide sequence of the 1362-base pair coding region and 5' and 3' flanking sequences were determined. Transcription start sites were located by S1 nuclease mapping. Regulation of GDH1 was not maintained when the gene was present on a multicopy plasmid. Protein secondary structure predictions identified a region with potential to form the dinucleotide-binding domain. The amino acid sequences of the yeast and Neurospora crassa enzymes are 63% conserved. Unlike the N. crassa gene, yeast GDH1 has no introns.

  19. Electron-transfer studies with a new flavin adenine dinucleotide dependent glucose dehydrogenase and osmium polymers of different redox potentials.

    PubMed

    Zafar, Muhammad Nadeem; Wang, Xiaoju; Sygmund, Christoph; Ludwig, Roland; Leech, Dónal; Gorton, Lo

    2012-01-03

    A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration.

  20. Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin II-induced hyperpermeability in endothelial cells.

    PubMed

    Ying, Chen-Jiang; Xu, Jin-Wen; Ikeda, Katsumi; Takahashi, Kyoko; Nara, Yasuo; Yamori, Yukio

    2003-10-01

    Out-of-control reactive oxygen species (ROS) signaling is one of the key events in the pathogenesis of endothelial dysfunction and essential hypertension. We observed that tea polyphenols decreased the production of ROS via regulation of the protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in bovine carotid artery endothelial cells (BCAECs). Both green tea polyphenols (GTP) and black tea polyphenols (BTP) down-regulated the expression of NADPH oxidase subunits p22phox and p67phox while up-regulating catalase expression (p < 0.05, respectively). Pre-treatment with GTP or BTP for 24 h significantly decreased the superoxide anion level (p < 0.05) and permeable fluorescence intensities in Ang II-stimulated BCAECs. A decrease in cell permeability was also observed by pre-treatment with diphenylene iodonium chloride (DPI) or vitamin E (p < 0.05, respectively). The result demonstrates that tea polyphenols alleviate angiotensin (Ang) II-induced hyperpermeability mainly by decreasing ROS production. Our results suggest that tea polyphenols regulate ROS-related protein expression and may be beneficial in preventing endothelial cell dysfunction and development of cardiovascular diseases, including hypertension.

  1. 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

  2. Degradation of pentachlorophenol by a novel peroxidase-catalyzed process in the presence of reduced nicotinamide adenine dinucleotide.

    PubMed

    Li, Haitao; Li, Yuping; Cao, Hongbin; Li, Xingang; Zhang, Yi

    2011-03-01

    A novel horseradish peroxidase (HRP)-catalyzed H₂O₂ process in the presence of reduced nicotinamide adenine dinucleotide (NADH) was applied to remove aqueous pentachlorophenol (PCP). Parameters (pH, H₂O₂ concentration, HRP activity and NADH dosage) on PCP removal were investigated. It was found that initial 0.05mM PCP was removed by 98% in HRP-NADH-H₂O₂ system at pH 5.0 and 30°C for 1h. Addition of O₂ in HRP-NADH-H₂O₂ system enhanced the removal rate of PCP due to promoting hydroxyl radicals (.OH) and superoxide anion radical (.O₂⁻) generation, which were confirmed by electron paramagnetic resonance (EPR)-spin trapping method. PCP removal efficiency decreased when .O₂⁻ and H₂O₂ were scavenged by superoxide dismutase and catalase in HRP-NADH-O₂ system, indicating that .OH/.O₂⁻ played a great role in the degradation of PCP. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that octachlorinated dibenzodioxin (OCDD) in residual solution was reduced after treated by the HRP-NADH-O₂ process, resulting in lower toxicity of treated solution than conventional enzymatic process. Two enzymatic-catalysis pathways were proposed for PCP removal in HRP-NADH-H₂O₂/O₂ system: (i) OH/.O₂⁻ free radical oxidation (ii) conventional phenoxy polymerization.

  3. A computational comparison of electron transfer from reduced ferredoxin to flavin adenine dinucleotide and a gold electrode.

    PubMed

    Walch, Stephen P; Komadina, Jason D; Prinz, Fritz B

    2009-05-21

    We have carried out calculations of the electronic structure of ferredoxin and of the electronic coupling matrix element Hif for electron transfer from reduced ferredoxin to flavin adenine dinucleotide (FAD) and to cluster models of the Au111 surface and a Au111 surface with a mercaptopyridene self-assembled monolayer (SAM). We conclude, based on Hif2, that a gold electrode is approximately 14 times less efficient as an electron acceptor than FAD and that the mercaptopyridine SAM enhances electron transfer. The magnitude of Hif is large enough for these systems that the weak coupling limit approximations may no longer be valid. However, the barrier to electron transfer in the strong coupling limit is computed to be small due to minimal geometry change between oxidized and reduced ferredoxin. MD simulations of the interaction of ferredoxin and protonated pyridine within a water solvation box indicate that the protonated pyridine does strongly orient the ferredoxin, favoring electron transfer as compared to a bare gold surface, where we speculate the orientation of the ferredoxin may be more random.

  4. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    PubMed

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  5. Localization of nicotinamide adenine dinucleotide phosphate-diaphorase activity in electrosensory and electromotor systems of a gymnotiform teleost, Apteronotus leptorhynchus.

    PubMed

    Turner, R W; Moroz, L L

    1995-05-29

    The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity was determined in electrosensory and electromotor systems of the weakly electric gymnotiform teleost Apteronotus leptorhynchus as an indicator of putative nitric oxide synthase-containing cells. NADPH-d activity was detected in electroreceptors and in afferent nerves of both ampullary and type I and type II tuberous organs. All cell bodies within the anterior lateral line nerve ganglion were positive for NADPH-d activity, as were the primary afferent axons and termination fields in the medullary electrosensory lateral line lobe. In the corpus cerebelli and valvula cerebelli, NADPH-d label was present in Purkinje cell somata, mossy fiber synaptic glomeruli, granule cells, and parallel fibers. In the midbrain, NADPH-d activity was apparent in layer VIIIB of the torus semicircularis dorsalis and in electrosensory laminae of the optic tectum. NADPH-d was particularly associated with diencephalic electrosensory and electromotor nuclei, including the prepacemaker nucleus, the nucleus subelectrosensorius, and the central posterior nucleus of the thalamus. Intense NADPH-d activity was present in pacemaker and relay cells of the medullary pacemaker nucleus but was absent from a novel class of smaller cells in this structure. Relay cell axons and spinal electromotor neurons and their axons within the electric organ were positive for NADPH-d activity. These results indicate that putative nitric oxide synthase-containing neurons in Apteronotus are localized preferentially to electrosensory and electromotor structures, suggesting a role for nitric oxide in determining the activity of cells involved in detecting or generating weakly electric fields.

  6. Electrochemical behavior of flavin adenine dinucleotide adsorbed onto carbon nanotube and nitrogen-doped carbon nanotube electrodes.

    PubMed

    Goran, Jacob M; Stevenson, Keith J

    2013-11-05

    Flavin adenine dinucleotide (FAD) is a cofactor for many enzymes, but also an informative redox active surface probe for electrode materials such as carbon nanotubes (CNTs) and nitrogen-doped CNTs (N-CNTs). FAD spontaneously adsorbs onto the surface of CNTs and N-CNTs, displaying Langmuir adsorption characteristics. The Langmuir adsorption model provides a means of calculating the electroactive surface area (ESA), the equilibrium constant for the adsorption and desorption processes (K), and the Gibbs free energy of adsorption (ΔG°). Traditional ESA measurements based on the diffusional flux of a redox active molecule to the electrode surface underestimate the ESA of porous materials because pores are not penetrated. Techniques such as gas adsortion (BET) overestimate the ESA because it includes both electroactive and inactive areas. The ESA determined by extrapolation of the Langmuir adsorption model with the electroactive surface probe FAD will penetrate pores and only include electroactive areas. The redox activity of adsorbed FAD also displays a strong dependency on pH, which provides a means of determining the pKa of the surface confined species. The pKa of FAD decreases as the nitrogen content in the CNTs increases, suggesting a decreased hydrophobicity of the N-CNT surface. FAD desorption at N-CNTs slowly transforms the main FAD surface redox reaction with E1/2 at -0.84 V into two new, reversible, surface confined redox reactions with E1/2 at -0.65 and -0.76 V (vs Hg/Hg2SO4), respectively (1.0 M sodium phosphate buffer pH = 6.75). This is the first time these redox reactions have been observed. The new surface confined redox reactions were not observed during FAD desorption from nondoped CNTs.

  7. Changes of collagen and nicotinamide adenine dinucleotide in human cancerous and normal prostate tissues studied using native fluorescence spectroscopy with selective excitation wavelength

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Wang, Wubao; Tang, Guichen; Alfano, Robert R.

    2010-07-01

    The fluorescence spectra of human cancerous and normal prostate tissues obtained by the selective excitation wavelength of 340 nm were measured. The contributions of principle biochemical components to tissue fluorescence spectra were investigated using the method of multivariate curve resolution with alternating least squares. The results show that there is a reduced contribution from the emission of collagen and increased contribution from nicotinamide adenine dinucleotide (NADH) in cancerous tissues as compared with normal tissue. This difference is attributed to the changes of relative contents of NADH and collagen during cancer development. This research may present a potential native biomarker for prostate cancer detection.

  8. NAD+ and Sirtuins in Aging and Disease

    PubMed Central

    Imai, Shin-ichiro; Guarente, Leonard

    2014-01-01

    Nicotinamide adenine dinucleotide (NAD+) is a classical coenzyme mediating many redox reactions. NAD+ also plays an important role in the regulation of NAD+-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD+ biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD+ levels decline during the aging process and may be an Achilles’ heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD+ by supplementing NAD+ intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD+ intermediate supplementation may be an effective anti-aging intervention, providing hope to aging societies worldwide. PMID:24786309

  9. NAD+ and sirtuins in aging and disease.

    PubMed

    Imai, Shin-ichiro; Guarente, Leonard

    2014-08-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a classical coenzyme mediating many redox reactions. NAD(+) also plays an important role in the regulation of NAD(+)-consuming enzymes, including sirtuins, poly-ADP-ribose polymerases (PARPs), and CD38/157 ectoenzymes. NAD(+) biosynthesis, particularly mediated by nicotinamide phosphoribosyltransferase (NAMPT), and SIRT1 function together to regulate metabolism and circadian rhythm. NAD(+) levels decline during the aging process and may be an Achilles' heel, causing defects in nuclear and mitochondrial functions and resulting in many age-associated pathologies. Restoring NAD(+) by supplementing NAD(+) intermediates can dramatically ameliorate these age-associated functional defects, counteracting many diseases of aging, including neurodegenerative diseases. Thus, the combination of sirtuin activation and NAD(+) intermediate supplementation may be an effective antiaging intervention, providing hope to aging societies worldwide.

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

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

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

  11. Urea induced unfolding dynamics of flavin adenine dinucleotide (FAD): spectroscopic and molecular dynamics simulation studies from femto-second to nanosecond regime.

    PubMed

    Sengupta, Abhigyan; Singh, Reman K; Gavvala, Krishna; Koninti, Raj Kumar; Mukherjee, Arnab; Hazra, Partha

    2014-02-20

    Here, we investigate the effect of urea in the unfolding dynamics of flavin adenine dinucleotide (FAD), an important enzymatic cofactor, through steady state, time-resolved fluorescence spectroscopic and molecular dynamics (MD) simulation studies. Steady state results indicate the possibility of urea induced unfolding of FAD, inferred from increasing emission intensity of FAD with urea. The TCSPC and up-conversion results suggest that the stack-unstack dynamics of FAD severely gets affected in the presence of urea and leads to an increase in the unstack conformation population from 15% in pure water to 40% in 12 M urea. Molecular dynamics simulation was employed to understand the nature of the interaction between FAD and urea at the molecular level. Results depict that urea molecules replace many of the water molecules around adenine and isoalloxazine rings of FAD. However, the major driving force for the stability of this unstack conformations arises from the favorable stacking interaction of a significant fraction of the urea molecules with adenine and isoalloxazine rings of FAD, which overcomes the intramolecular stacking interaction between themselves observed in pure water.

  12. NAD+ metabolism in health and disease.

    PubMed

    Belenky, Peter; Bogan, Katrina L; Brenner, Charles

    2007-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid.

  13. 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.

  14. 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

    2015-10-06

    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.

  15. 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.

  16. 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.

  17. Differential effect of pH upon cyclic-ADP-ribose and nicotinate-adenine dinucleotide phosphate-induced Ca2+ release systems.

    PubMed Central

    Chini, E N; Liang, M; Dousa, T P

    1998-01-01

    We investigated the pH dependence and the effects of thimerosal and dithiothreitol (DTT) upon the Ca2+ release induced by cADP-ribose (cADPR) and nicotinate-adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates. Both Ca2+ release triggered by cADPR and the binding of [3H]cADPR to sea urchin egg homogenates were decreased by alkalization of the assay media from pH 7.2 to 8.9. In contrast, NAADP-triggered Ca2+ release was not influenced by changes in pH. The Ca2+ release induced by cADPR was potentiated by thimerosal and inhibited by DTT, but neither thimerosal nor DTT had any effect upon the Ca2+ release induced by NAADP. We conclude that cADPR-sensitive Ca2+-release mechanisms are dependent on pH of the assay media and are sensitive to thiol group modification. On the other hand, these functional properties are not shared by NAADP-regulated Ca2+ channels. PMID:9794787

  18. 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.

  19. Detection, distribution, and organohalogen compound discovery implications of the reduced flavin adenine dinucleotide-dependent halogenase gene in major filamentous actinomycete taxonomic groups.

    PubMed

    Gao, Peng; Huang, Ying

    2009-07-01

    Halogenases have been shown to play a significant role in biosynthesis and introducing the bioactivity of many halogenated secondary metabolites. In this study, 54 reduced flavin adenine dinucleotide (FADH(2))-dependent halogenase gene-positive strains were identified after the PCR screening of a large collection of 228 reference strains encompassing all major families and genera of filamentous actinomycetes. The wide distribution of this gene was observed to extend to some rare lineages with higher occurrences and large sequence diversity. Subsequent phylogenetic analyses revealed that strains containing highly homologous halogenases tended to produce halometabolites with similar structures, and halogenase genes are likely to propagate by horizontal gene transfer as well as vertical inheritance within actinomycetes. Higher percentages of halogenase gene-positive strains than those of halogenase gene-negative ones contained polyketide synthase genes and/or nonribosomal peptide synthetase genes or displayed antimicrobial activities in the tests applied, indicating their genetic and physiological potentials for producing secondary metabolites. The robustness of this halogenase gene screening strategy for the discovery of particular biosynthetic gene clusters in rare actinomycetes besides streptomycetes was further supported by genome-walking analysis. The described distribution and phylogenetic implications of the FADH(2)-dependent halogenase gene present a guide for strain selection in the search for novel organohalogen compounds from actinomycetes.

  20. Exploring NAD+ metabolism in host-pathogen interactions.

    PubMed

    Mesquita, Inês; Varela, Patrícia; Belinha, Ana; Gaifem, Joana; Laforge, Mireille; Vergnes, Baptiste; Estaquier, Jérôme; Silvestre, Ricardo

    2016-03-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a vital molecule found in all living cells. NAD(+) intracellular levels are dictated by its synthesis, using the de novo and/or salvage pathway, and through its catabolic use as co-enzyme or co-substrate. The regulation of NAD(+) metabolism has proven to be an adequate drug target for several diseases, including cancer, neurodegenerative or inflammatory diseases. Increasing interest has been given to NAD(+) metabolism during innate and adaptive immune responses suggesting that its modulation could also be relevant during host-pathogen interactions. While the maintenance of NAD(+) homeostatic levels assures an adequate environment for host cell survival and proliferation, fluctuations in NAD(+) or biosynthetic precursors bioavailability have been described during host-pathogen interactions, which will interfere with pathogen persistence or clearance. Here, we review the double-edged sword of NAD(+) metabolism during host-pathogen interactions emphasizing its potential for treatment of infectious diseases.

  1. Electrochemical synthesis and characterization of TiO(2) nanoparticles and their use as a platform for flavin adenine dinucleotide immobilization and efficient electrocatalysis.

    PubMed

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

    2008-06-25

    Here, we report the electrochemical synthesis of TiO(2) 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 TiO(2) film produced was mainly composed of rutile and that the particles are of a size in the range of 100 ± 50 nm. TiO(2) NPs were used for the modification of a screen printed carbon electrode (SPE). The resulting TiO(2) 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 (H(2)O(2)) 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 TiO(2) film retained its bioactivity and exhibited excellent electrocatalytic response to the reduction of H(2)O(2), based on which a mediated biosensor for H(2)O(2) was achieved. The linear range for the determination of H(2)O(2) 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.

  2. 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

  3. Spinal Cord Injury Leads to Hyperoxidation and Nitrosylation of Skeletal Muscle Ryanodine Receptor-1 Associated with Upregulation of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4.

    PubMed

    Liu, Xin-Hua; Harlow, Lauren; Graham, Zachary A; Bauman, William A; Cardozo, Christopher

    2017-02-27

    Spinal cord injury (SCI) results in marked atrophy and dysfunction of skeletal muscle. There are currently no effective treatments for SCI-induced muscle atrophy or the dysfunction of the remaining muscle tissue. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (Nox4) produces reactive oxygen species (ROS) in sarcoplasmic reticulum (SR) and has been identified as an important O2 sensor in skeletal muscle. Ryanodine receptors (RyRs) are calcium (Ca(2+)) channels that are responsible for Ca(2+) release from SR. In skeletal muscle, type1 RyR (RyR1) is predominantly functional. RyR1 is regulated by multiple proteins, including calstabin1, which assures that they close appropriately once contraction has ceased. RyR1 function is also regulated by oxidation and redox-dependent cysteine nitrosylation. Excessive oxidation/nitrosylation of RyR1 is associated with dissociation of calstabin1 and reduced muscle force generation. However, whether Nox4 levels in skeletal muscle are elevated or whether RyR1 is oxidized or nitrosylated after SCI has not been determined. In this study, we examined Nox4 expression, oxidation/nitrolysation status, and association of calstabin1 with RyR1 in skeletal muscle derived from rats that were subjected to T4 complete transection (SCI), and observed elevated expression of Nox4 messenger RNA and protein in muscle after SCI associated with enhanced binding of Nox4 to RyR1, increased oxidation and nitrosylation of RyR1, and dissociation of calstabin1 from RyR1 in SCI rat muscle. Our data suggest that RyR1 dysfunction resulting from excessive oxidation/nitrosylation may contribute to reduced specific force after SCI and suggest that Nox4 may be the source of ROS responsible for increased oxidation and nitrosylation of RyR1.

  4. A novel flavin adenine dinucleotide (FAD) containing d-lactate dehydrogenase from the thermoacidophilic crenarchaeota Sulfolobus tokodaii strain 7: purification, characterization and expression in Escherichia coli.

    PubMed

    Satomura, Takenori; Kawakami, Ryushi; Sakuraba, Haruhiko; Ohshima, Toshihisa

    2008-07-01

    Dye-linked D-lactate dehydrogenase activity was found in the crude extract of a continental thermoacidophilic crenarchaeota, Sulfolobus tokodaii strain 7, and was purified 375-fold through four sequential chromatography steps. With a molecular mass of about 93 kDa, this enzyme was a homodimer comprised of identical subunits with molecular masses of about 48 kDa. The enzyme retained its full activity after incubation at 80 degrees C for 10 min and after incubation at pHs ranging from 6.5 to 10.0 for 30 min at 50 degrees C. The preferred substrate for this enzyme was D-lactate, with 2,6-dichloroindophenol serving as the electron acceptor. Using high-performance liquid chromatography (HPLC), the enzyme's prosthetic group was determined to be flavin adenine dinucleotide (FAD). Its N-terminal amino acid sequence was MLEGIEYSQGEEREDFVGFKIKPKI. Using that sequence and previously reported genome information, the gene encoding the enzyme (ST0649) was identified. It was subsequently cloned and expressed in Escherichia coli and found to encode a polypeptide of 440 amino acids with a calculated molecular weight of 49,715. The amino acid sequence of this dye-linked D-lactate dehydrogenase showed higher homology (39% identity) with that of a glycolate oxidase subunit homologue from Archaeoglobus fulgidus, but less similarity (32% identity) to D-lactate dehydrogenase from A. fulgidus. Taken together, our findings indicate that the dye-linked D-lactate dehydrogenase from S. tokodaii is a novel type of FAD containing D-lactate dehydrogenase.

  5. Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

    PubMed Central

    Xun, Luying; Sandvik, Erik R.

    2000-01-01

    4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH2)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH2 in vitro, HpaB was able to use FADH2 and O2 for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH2 for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH2-utilizing monooxygenase. FADH2 generated by Fre was rapidly oxidized by O2 to form H2O2 in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH2 and transitorily protected it from rapid autoxidation by O2. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O2 for FADH2 utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH2 produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH2 was autoxidized by O2, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH2-utilizing monooxygenase that uses FADH2 as a substrate rather than as a cofactor. PMID:10653707

  6. A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca2+ Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat

    PubMed Central

    Lee, Jeong Hoon; Ha, Jeong Mi

    2015-01-01

    Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca2+]. However, the dye usage is intrinsically limited, as the dyes require ultraviolet (UV) excitation, which can also generate great interference, mainly from nicotinamide adenine dinucleotide (NADH) autofluorescence. Specifically, this limitation causes serious problems for the quantitative measurement of mitochondrial [Ca2+], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca2+] because the majority of NADH is located in the mitochondria. The emission intensity ratio of two different excitation wavelengths must be constant when the fluorescent dye concentration is the same. In accordance with this principle, we developed a novel online method that corrected NADH and Fura-2-FF interference. We simultaneously measured multiple parameters, including NADH, [Ca2+], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca2+] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca2+] concentration was 1.03 µM. This 1 µM cytosolic Ca2+ could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca2+] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca2+. Our method solved the problem of NADH signal contamination during the use of Fura-2 analogs, and therefore the method may be useful when NADH interference is expected. PMID:26170742

  7. The distribution of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) in the medulla oblongata, spinal cord, cranial and spinal nerves of frog, Microhyla ornata.

    PubMed

    Jadhao, Arun G; Biswas, Saikat P; Bhoyar, Rahul C; Pinelli, Claudia

    2017-04-01

    Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians.

  8. Enhancing NAD+ salvage metabolism is neuroprotective in a PINK1 model of Parkinson's disease

    PubMed Central

    Lehmann, Susann; Loh, Samantha H. Y.

    2017-01-01

    ABSTRACT Familial forms of Parkinson's disease (PD) caused by mutations in PINK1 are linked to mitochondrial impairment. Defective mitochondria are also found in Drosophila models of PD with pink1 mutations. The co-enzyme nicotinamide adenine dinucleotide (NAD+) is essential for both generating energy in mitochondria and nuclear DNA repair through NAD+-consuming poly(ADP-ribose) polymerases (PARPs). We found alterations in NAD+ salvage metabolism in Drosophila pink1 mutants and showed that a diet supplemented with the NAD+ precursor nicotinamide rescued mitochondrial defects and protected neurons from degeneration. Additionally, a mutation of Parp improved mitochondrial function and was neuroprotective in the pink1 mutants. We conclude that enhancing the availability of NAD+ by either the use of a diet supplemented with NAD+ precursors or the inhibition of NAD+-dependent enzymes, such as PARPs, which compete with mitochondria for NAD+, is a viable approach to preventing neurotoxicity associated with mitochondrial defects. PMID:28011627

  9. Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD(+) Metabolites.

    PubMed

    Trammell, Samuel Aj; Brenner, Charles

    2013-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a coenzyme for hydride transfer reactions and a substrate for sirtuins and other NAD(+)-consuming enzymes. The abundance of NAD (+), NAD(+) biosynthetic intermediates, and related nucleotides reflects the metabolic state of cells and tissues. High performance liquid chromatography (HPLC) followed by ultraviolet-visible (UV-Vis) spectroscopic analysis of NAD(+) metabolites does not offer the specificity and sensitivity necessary for robust quantification of complex samples. Thus, we developed a targeted, quantitative assay of the NAD(+) metabolome with the use of HPLC coupled to mass spectrometry. Here we discuss NAD(+) metabolism as well as the technical challenges required for reliable quantification of the NAD(+) metabolites. The new method incorporates new separations and improves upon a previously published method that suffered from the problem of ionization suppression for particular compounds.

  10. Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae

    PubMed Central

    Walters, Robert W.; Matheny, Tyler; Mizoue, Laura S.; Rao, Bhalchandra S.; Muhlrad, Denise; Parker, Roy

    2017-01-01

    RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5′ nicotinamide-adenine dinucleotide (NAD+) RNA in bacteria. Whether 5′ NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5′ NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae. NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5′ end processed. These results define an additional 5′ RNA cap structure in eukaryotes and raise the possibility that this 5′ NAD+ cap could modulate RNA stability and translation on specific subclasses of mRNAs. PMID:28031484

  11. 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

  12. Effect of Exogenous Extracellular Nicotinamide Adenine Dinucleotide (NAD⁺) on Bioelectric Activity of the Pacemaker and Conduction System of the Heart.

    PubMed

    Pustovit, K B; Kuz'min, V S; Sukhova, G S

    2015-06-01

    In rat sinoatrial node, NAD(+) (10 μM) reduced the rate of spontaneous action potentials, duration of action potentials, and the velocity of slow diastolic depolarization, but the rate of action potential front propagation increases. In passed rabbit Purkinje fibers, NAD(+) (10 μM) reduced the duration of action potentials. Under conditions of spontaneous activity of Purkinje fibers, NAD(+) reduced the fi ring rate and the rate of slow diastolic depolarization. The effects of extracellular NAD(+) on bioelectric activity of the pacemaker (sinoatrial node) and conduction system of the heart (Purkinje fibers) are probably related to activation of P1 and P2 purinoceptors.

  13. Altered behavioral and metabolic circadian rhythms in mice with disrupted NAD+ oscillation

    PubMed Central

    Sahar, Saurabh; Nin, Veronica; Barbosa, Maria Thereza; Chini, Eduardo Nunes; Sassone-Corsi, Paolo

    2011-01-01

    The Intracellular levels of nicotinamide adenine dinucleotide (NAD+) are rhythmic and controlled by the circadian clock. However, whether NAD+ oscillation in turn contributes to circadian physiology is not fully understood. To address this question we analyzed mice mutated for the NAD+ hydrolase CD38. We found that rhythmicity of NAD+ was altered in the CD38-deficient mice. The high, chronic levels of NAD+ results in several anomalies in circadian behavior and metabolism. CD38-null mice display a shortened period length of locomotor activity and alteration in the rest-activity rhythm. Several clock genes and, interestingly, genes involved in amino acid metabolism were deregulated in CD38-null livers. Metabolomic analysis identified alterations in the circadian levels of several amino acids, specifically tryptophan levels were reduced in the CD38-null mice at a circadian time paralleling with elevated NAD+ levels. Thus, CD38 contributes to behavioral and metabolic circadian rhythms and altered NAD+ levels influence the circadian clock. PMID:21937766

  14. Preventing NAD+ Depletion Protects Neurons against Excitotoxicity

    PubMed Central

    Liu, Dong; Pitta, Michael; Mattson, Mark P.

    2008-01-01

    Neurons are excitable cells that require large amounts of energy to support their survival and functions and are therefore prone to excitotoxicity, which involves energy depletion. By examining bioenergetic changes induced by glutamate, we found that the cellular nicotinamide adenine dinucleotide (NAD+) level is a critical determinant of neuronal survival. The bioenergetic effects of mitochondrial uncoupling and caloric restriction were also examined in cultured neurons and rodent brain. 2, 4-dinitrophenol (DNP) is a chemical mitochondrial uncoupler that stimulates glucose uptake and oxygen consumption on cultured neurons, which accelerates oxidation of NAD(P)H to NAD+ in mitochondria. The NAD+-dependent histone deacetylase sirtulin 1 (SIRT1) and glucose transporter 1 (GLUT1) mRNA are upregulated mouse brain under caloric restriction. To examine whether NAD+ mediates neuroprotective effects, nicotinamide, a precursor of NAD+ and inhibitor of SIRT1 and poly (ADP-ribose) polymerase 1 (PARP1) (two NAD+-dependent enzymes), was employed. Nicotinamide attenuated excitotoxic death and preserved cellular NAD+ levels to support SIRT1 and PARP 1 activities. Our findings suggest that mild mitochondrial uncoupling and caloric restriction exert hormetic effects by stimulating bioenergetics in neurons thereby increasing tolerance of neurons to metabolic stress. PMID:19076449

  15. Dehydrogenation of androsterone by purified 3α-hydroxy steroid-dependent nicotinamide–adenine dinucleotide (phosphate)-transhydrogenating enzyme of rat liver

    PubMed Central

    Pietruszko, Regina; Baron, D. N.

    1965-01-01

    1. An enzyme from rat liver, catalysing 3α-hydroxy steroid-dependent NAD(P) transhydrogenation and NAD-linked and NADP-linked dehydrogenation of 3α-hydroxy steroids, has been purified 100-fold by chromatography on DEAE-cellulose and calcium phosphate gel. 2. No separation of these activities into different protein fractions has been achieved. 3. The properties of the enzyme in catalysing NAD-linked and NADP-linked dehydrogenation have been compared, with androsterone as substrate. Differences were found in pH optima, affinity for coenzyme and steroid, equilibrium constants and effects of salts. 4. NAD-linked dehydrogenation is inhibited by NADPH2 but is protected from this inhibition by chloride, which alone is itself an inhibitor. 5. The relevance of these findings to the problem of the number of enzymes involved in catalysis of 3α-hydroxy steroid-dependent transhydrogenation is discussed. PMID:4378709

  16. Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH.

    PubMed

    Yuan, Yufeng; Huang, Kehan; Chang, Mengfang; Qin, Cuifang; Zhang, Sanjun; Pan, Haifeng; Chen, Yan; Xu, Jianhua

    2016-02-01

    Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD(+) (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD(+) concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD(+) and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I395/I550), fitting by a single-exponential decay function, can efficiently detect various NAD(+) levels from 100 to 4000 μM, as well as label NAD(+)/NADH (reduced form of NAD) ratios in the range of 1-50.

  17. Sequence-motif Detection of NAD(P)-binding Proteins: Discovery of a Unique Antibacterial Drug Target

    NASA Astrophysics Data System (ADS)

    Hua, Yun Hao; Wu, Chih Yuan; Sargsyan, Karen; Lim, Carmay

    2014-09-01

    Many enzymes use nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate (NAD(P)) as essential coenzymes. These enzymes often do not share significant sequence identity and cannot be easily detected by sequence homology. Previously, we determined all distinct locally conserved pyrophosphate-binding structures (3d motifs) from NAD(P)-bound protein structures, from which 1d sequence motifs were derived. Here, we aim to establish the precision of these 3d and 1d motifs to annotate NAD(P)-binding proteins. We show that the pyrophosphate-binding 3d motifs are characteristic of NAD(P)-binding proteins, as they are rarely found in nonNAD(P)-binding proteins. Furthermore, several 1d motifs could distinguish between proteins that bind only NAD and those that bind only NADP. They could also distinguish between NAD(P)-binding proteins from nonNAD(P)-binding ones. Interestingly, one of the pyrophosphate-binding 3d and corresponding 1d motifs was found only in enoyl-acyl carrier protein reductases, which are enzymes essential for bacterial fatty acid biosynthesis. This unique 3d motif serves as an attractive novel drug target, as it is conserved across many bacterial species and is not found in human proteins.

  18. Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of NAD synthetase from methicillin-resistant Staphylococcus aureus.

    PubMed

    Arbade, Gajanan Kashinathrao; Srivastava, Sandeep Kumar

    2015-06-01

    Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. The prevalence of multidrug-resistant S. aureus strains in both hospital and community settings makes it imperative to characterize new drug targets to combat S. aureus infections. In this context, enzymes involved in NAD metabolism and synthesis are significant drug targets as NAD is a central player in several cellular processes. NAD synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide, making it a crucial intermediate enzyme linked to the biosynthesis of several amino acids, purine and pyrimidine nucleotides, coenzymes and antibiotics.

  19. Effects of NAD at purine receptors in isolated blood vessels.

    PubMed

    Alefishat, E; Alexander, S P H; Ralevic, V

    2015-03-01

    Nicotinamide adenine dinucleotide (NAD) belongs to the family of naturally occurring adenine dinucleotides, best known for their various intracellular roles. However, there is evidence that they can also be released from cells to act as novel extracellular signalling molecules. Relatively little is known about the extracellular actions of NAD, especially in the cardiovascular system. The present study investigated the actions of NAD in the rat thoracic aorta, porcine coronary artery and porcine mesenteric arteries, mounted in organ baths for isometric tension recording. In the rat thoracic aorta and porcine coronary artery, NAD caused endothelium-independent concentration-dependent vasorelaxations which were unaffected by palmitoylCoA, a P2Y1 receptor antagonist, but which were blocked by CGS15943, a non-selective adenosine receptor antagonist. In the porcine coronary artery, NAD-evoked relaxations were abolished by SCH58261, a selective A2A receptor antagonist. In the rat thoracic aorta, NAD-evoked relaxations were attenuated by A2A receptor antagonism with SCH58261 but were unaffected by an A2B receptor antagonist, MRS1754. In contrast, in the porcine mesenteric artery, NAD-evoked endothelium-independent contractions, which were unaffected by a P2 receptor antagonist, suramin, or by NF449, a P2X1 receptor antagonist, but were attenuated following P2X receptor desensitisation with αβ-meATP. In conclusion, the present results show that NAD can alter vascular tone through actions at purine receptors in three different arteries from two species; its molecular targets differ according to the type of blood vessel.

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

    PubMed

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

    2017-03-24

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

  1. Nmnat3 Is Dispensable in Mitochondrial NAD Level Maintenance In Vivo.

    PubMed

    Yamamoto, Masashi; Hikosaka, Keisuke; Mahmood, Arshad; Tobe, Kazuyuki; Shojaku, Hideo; Inohara, Hidenori; Nakagawa, Takashi

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD) is an essential co-enzyme mediating various enzymatic reactions. Mitochondrial NAD particularly occupies a considerable amount of total NAD in cells, and serves as a co-enzyme in tricarboxylic acid cycle (TCA cycle), β-oxidation, and oxidative phosphorylation. Despite the importance of mitochondrial NAD, its synthesis pathway remains unknown. It has been proposed that NAD synthesis enzyme, Nmnat3, was localized in mitochondria, but its physiological relevance to the metabolism in mitochondria was not fully elucidated. Previously, we have reported that murine Nmnat3 protein was strongly expressed in the cytoplasm of mature erythrocytes, in which mitochondria were absent, and Nmnat3-deficient mice (Nmnat3-KO mice) exhibited splenomegaly and hemolytic anemia due to reduced NAD levels in mature erythrocytes. These results challenged the role of Nmnat3 in mitochondrial NAD synthesis. In this study, we demonstrated that mitochondrial NAD levels in various tissues, except for red blood cells, were unchanged in Nmnat3-KO mice. We also analyzed the metabolites in glycolysis and TCA cycle and found that there were no differences between Nmnat3-KO and WT mice. In addition, the aged Nmnat3-KO mice had comparable NAD levels to that observed in WT mice. Our results indicated that Nmnat3 is dispensable in the maintenance of mitochondrial NAD levels, and that other NAD regulatory pathways may exist in mitochondria.

  2. 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.

  3. Molecular characterization of Fasciola hepatica and phylogenetic analysis based on mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I and cytochrome oxidase subunit I) genes from the North-East of Iran

    PubMed Central

    Reaghi, Saber; Haghighi, Ali; Harandi, Majid Fasihi; Spotin, Adel; Arzamani, Kourosh; Rouhani, Soheila

    2016-01-01

    Aim: Fascioliasis is one of the most zoonotic diseases with global extension. As the epidemiological distribution of Fasciola may lead to various genetic patterns of the parasite, the aim of this study is to identify Fasciola hepatica based on spermatogenesis, and phylogenetic analysis using mitochondrial (nicotiamide adenine dinucleotide dehydrogenase subunit I [ND1] and cytochrome oxidase subunit I) gene marker. Materials and Methods: In this study, 90 F. hepatica collected from 30 cattle at slaughterhouse located in three different geographical locations in the North-East of Iran were evaluated based on spermatogenetic ability and internal transcribed spacer 1 gene restriction fragment length polymorphism pattern. Genetic diversity and phylogenetic relationship using mtDNA gene marker for the isolates from the North-East of Iran, and other countries were then analyzed. Results: Partial sequences of mtDNA showed eight haplotypes in both genes. The phylogenic analysis using neighbor joining as well as maximum likelihood methods showed similar topologies of trees. Pairwise fixation index between different F. hepatica populations calculated from the nucleotide data set of ND1 gene are statistically significant and show the genetic difference. Conclusion: F. hepatica found in this region of Iran has different genetic structures through the other Fasciola populations in the world. PMID:27733809

  4. NAD+ maintenance attenuates light induced photoreceptor degeneration Δ

    PubMed Central

    Bai, Shi; Sheline, Christian T.

    2013-01-01

    Light-induced retinal damage (LD) occurs after surgery or sun exposure. We previously showed that zinc (Zn2+) accumulated in photoreceptors and RPE cells after LD but prior to cell death, and pyruvate or nicotinamide attenuated the resultant death perhaps by restoring nicotinamide adenine dinucleotide (NAD+) levels. We first examined the levels of NAD+ and the efficacy of pyruvate or nicotinamide in oxidative toxicities using primary retinal cultures. We next manipulated NAD+ levels in vivo and tested the affect on LD to photoreceptors and RPE. NAD+ levels cycle with a 24-h rhythm in mammals, which is affected by the feeding schedule. Therefore, we tested the affect of increasing NAD+ levels on LD by giving nicotinamide, inverting the feeding schedule, or using transgenic mice which overexpress cytoplasmic nicotinamide mononucleotide adenyl-transferase-1 (cytNMNAT1), an NAD+ synthetic enzyme. Zn2+ accumulation was also assessed in culture and in retinal sections. Retinas of light damaged animals were examined by OCT and plastic sectioning, and retinal NAD levels were measured. Day fed, or nicotinamide treated rats showed less NAD+ loss, and LD compared to night fed rats or untreated rats without changing the Zn2+ staining pattern. CytNMNAT1 showed less Zn2+ staining, NAD+ loss, and cell death after LD. In conclusion, intense light, Zn2+ and oxidative toxicities caused an increase in Zn2+, NAD+ loss, and cell death which were attenuated by NAD+ restoration. Therefore, NAD+ levels play a protective role in LD-induced death of photoreceptors and RPE cells. PMID:23274583

  5. HIV-1 trans activator of transcription protein elicits mitochondrial hyperpolarization and respiratory deficit, with dysregulation of complex IV and nicotinamide adenine dinucleotide homeostasis in cortical neurons.

    PubMed

    Norman, John P; Perry, Seth W; Kasischke, Karl A; Volsky, David J; Gelbard, Harris A

    2007-01-15

    HIV-1 causes a common, progressive neurological disorder known as HIV-associated dementia (HAD). The prevalence of this disorder has increased despite the use of highly active antiretroviral therapy, and its underlying pathogenesis remains poorly understood. However, evidence suggests that some aspects of HAD may be reversible. To model the reversible aspects of HAD, we have used the HIV-1 neurotoxin trans activator of transcription protein (Tat) to investigate nonlethal changes in cultured neurons. Exposure of rodent cortical neurons to sublethal concentrations of Tat elicits mitochondrial hyperpolarization. In this study, we used the cationic lipophilic dye rhodamine 123 to confirm this observation, and then performed follow-up studies to examine the mechanism involved. In intact neurons, we found Tat elicited a rapid drop in internal mitochondrial pH, and addition of Tat to purified mitochondrial extracts inhibited complex IV of the electron transport chain. To correlate enzyme activity in mitochondrial extracts with results in intact cells, we measured neuronal respiration following Tat exposure. Cortical neurons demonstrated decreased respiration upon Tat treatment, consistent with inhibition of complex IV. We examined mitochondrial Ca(2+) homeostasis using a mitochondrial targeted enhanced yellow fluorescent protein-calmodulin construct. We detected a decrease in mitochondrial calcium concentration following exposure to Tat. Finally, we measured the energy intermediate NAD(P)H after Tat treatment, and found a 20% decrease in the autofluorescence. Based on these findings, we suggest that decreased NADPH and calcium concentration contribute to subsequent respiratory decline after exposure to Tat, with detrimental effects on neuronal signaling.

  6. Biofuel cell anode: NAD +/glucose dehydrogenase-coimmobilized ketjenblack electrode

    NASA Astrophysics Data System (ADS)

    Miyake, T.; Oike, M.; Yoshino, S.; Yatagawa, Y.; Haneda, K.; Kaji, H.; Nishizawa, M.

    2009-09-01

    We have studied the coimmobilization of glucose dehydrogenase (GDH) and its cofactor, oxidized nicotinamide adenine dinucleotide (NAD +), on a ketjenblack (KB) electrode as a step toward a biofuel cell anode that works without mediators. A KB electrode was first treated with a sulfuric acid/nitric acid/water mixture to lower the overvoltage for NADH oxidation, and was next chemically modified with NAD + and GDH. The improved GDH/NAD +/KB electrode is found to oxidize glucose around 0 V vs. Ag/AgCl. A biofuel cell constructed with a bilirubin oxidase-immobilized KB cathode showed a maximum power density of 52 μW/cm 2 at 0.3 V.

  7. Assimilation of NAD(+) precursors in Candida glabrata.

    PubMed

    Ma, Biao; Pan, Shih-Jung; Zupancic, Margaret L; Cormack, Brendan P

    2007-10-01

    The yeast pathogen Candida glabrata is a nicotinamide adenine dinucleotide (NAD(+)) auxotroph and its growth depends on the environmental supply of vitamin precursors of NAD(+). C. glabrata salvage pathways defined in this article allow NAD(+) to be synthesized from three compounds - nicotinic acid (NA), nicotinamide (NAM) and nicotinamide riboside (NR). NA is salvaged through a functional Preiss-Handler pathway. NAM is first converted to NA by nicotinamidase and then salvaged by the Preiss-Handler pathway. Salvage of NR in C. glabrata occurs via two routes. The first, in which NR is phosphorylated by the NR kinase Nrk1, is independent of the Preiss-Handler pathway. The second is a novel pathway in which NR is degraded by the nucleosidases Pnp1 and Urh1, with a minor role for Meu1, and ultimately converted to NAD(+) via the nicotinamidase Pnc1 and the Preiss-Handler pathway. Using C. glabrata mutants whose growth depends exclusively on the external NA or NR supply, we also show that C. glabrata utilizes NR and to a lesser extent NA as NAD(+) sources during disseminated infection.

  8. New approach to biosensing of co-enzyme nicotinamide adenine dinucleotide (NADH) by incorporation of neutral red in aluminum doped nanostructured ZnO thin films.

    PubMed

    V T, Fidal; T S, Chandra

    2017-01-04

    Biosensing of NADH on bare electrodes has drawbacks such as high over-potential and poisoning during the oxidation reaction. To overcome this challenge a different approach has been undertaken by incorporating neutral red (NR) in Al doped ZnO (AZO) thin films using one-pot chemical bath deposition (CBD). The surface morphology of the films was hexagonal nanorods along the c-axis, perpendicular to the substrate. The thickness of the thin films were ranging from 400 to 3000nm varying dependent on time of deposition (30 to 150min). The average diameter of the nanorods was larger in the presence of neutral red (NR-AZO) with ~300nm in contrast to its absence (AZO) with ~200nm. The density of the packing of nanorods was dependent on the citrate concentration used during deposition. Control over the dopant concentration in the films was achieved by varying the area of Al foil used in the deposition solution. The selected area diffraction (SAED) and X-ray diffraction (XRD) indicated 002 plane of orientation in the nanorods. FTIR and FT-Raman analysis revealed conserved structure of NR and AZO. Chronoamperometric (CA) analysis showed a sensitivity of 0.45μAcm(-2)mM(-1) and LoD of 22μM within the range 0.075-4mM of NADH. The biological sensing of NADH was validated by physical adsorption of NAD(+) dependent-lactate dehydrogenase (LDH) on NR-AZO. CA showed sensitivity of 0.56μAcm(-2)mM(-1) and LoD for lactate was 27μM in the range of 0.1-1mM of lactate. Further validation with real-time serum sample shows that LDH/NR-AZO correlates with the clinical values. The distinction in this study is that the organic mediator like neutral red has been incorporated into the grain structure of the ZnO thin film whereas other study with the mediators have only attempted surface functionalization. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

  9. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment

    PubMed Central

    Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F.

    2015-01-01

    The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer’s, Parkinson’s and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD+) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD+ followed by decreased ATP production, and are completely rescued by treatment with NAD+ or its precursor nicotinamide because of restoration of physiological NAD+ levels. Toxic prion protein-induced NAD+ depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD+. Intranasal NAD+ treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD+ starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD+ replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases. PMID:25678560

  10. Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment.

    PubMed

    Zhou, Minghai; Ottenberg, Gregory; Sferrazza, Gian Franco; Hubbs, Christopher; Fallahi, Mohammad; Rumbaugh, Gavin; Brantley, Alicia F; Lasmézas, Corinne I

    2015-04-01

    The mechanisms of neuronal death in protein misfolding neurodegenerative diseases such as Alzheimer's, Parkinson's and prion diseases are poorly understood. We used a highly toxic misfolded prion protein (TPrP) model to understand neurotoxicity induced by prion protein misfolding. We show that abnormal autophagy activation and neuronal demise is due to severe, neuron-specific, nicotinamide adenine dinucleotide (NAD(+)) depletion. Toxic prion protein-exposed neuronal cells exhibit dramatic reductions of intracellular NAD(+) followed by decreased ATP production, and are completely rescued by treatment with NAD(+) or its precursor nicotinamide because of restoration of physiological NAD(+) levels. Toxic prion protein-induced NAD(+) depletion results from PARP1-independent excessive protein ADP-ribosylations. In vivo, toxic prion protein-induced degeneration of hippocampal neurons is prevented dose-dependently by intracerebral injection of NAD(+). Intranasal NAD(+) treatment of prion-infected sick mice significantly improves activity and delays motor impairment. Our study reveals NAD(+) starvation as a novel mechanism of autophagy activation and neurodegeneration induced by a misfolded amyloidogenic protein. We propose the development of NAD(+) replenishment strategies for neuroprotection in prion diseases and possibly other protein misfolding neurodegenerative diseases.

  11. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.

    PubMed

    Bogan, Katrina L; Brenner, Charles

    2008-01-01

    Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research.

  12. 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

  13. Toward productive aging: SIRT1, systemic NAD biosynthesis, and the NAD world.

    PubMed

    Imai, Shin-ichiro

    2010-11-01

    In our current society, achieving "productive aging," which aims to keep people as productive as possible with good health and spirit for their private and social activities as they grow older, will be important to maximize the duration of high quality of life and to promote a stable economy and solvent social security in healthcare systems. To achieve this goal, we have been studying mechanisms of mammalian aging and longevity, focusing on the physiological importance of the mammalian nicotinamide adenine dinucleotide (NAD)--dependent protein deacetylase SIRT1 and systemic NAD biosynthesis mediated by nicotinamide phosphoribosyltransferase for the regulation of metabolism and aging. Through these studies, we have recently proposed a comprehensive concept of a novel systemic regulatory network for metabolism and aging, named "NAD World." This new concept of NAD World also conveys ideas of functional hierarchy and frailty for the induction of aging in mammals. Details of the NAD World and its implications to age-associated metabolic diseases such as type 2 diabetes will be discussed.

  14. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs.

    PubMed

    Cahová, Hana; Winz, Marie-Luise; Höfer, Katharina; Nübel, Gabriele; Jäschke, Andres

    2015-03-19

    A distinctive feature of prokaryotic gene expression is the absence of 5'-capped RNA. In eukaryotes, 5',5'-triphosphate-linked 7-methylguanosine protects messenger RNA from degradation and modulates maturation, localization and translation. Recently, the cofactor nicotinamide adenine dinucleotide (NAD) was reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signalling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. The unknown identity of NAD-modified RNAs has so far precluded functional analyses. Here we identify NAD-linked RNAs from bacteria by chemo-enzymatic capture and next-generation sequencing (NAD captureSeq). Among those identified, specific regulatory small RNAs (sRNAs) and sRNA-like 5'-terminal fragments of certain mRNAs are particularly abundant. Analogous to a eukaryotic cap, 5'-NAD modification is shown in vitro to stabilize RNA against 5'-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. The nudix phosphohydrolase NudC decaps NAD-RNA and thereby triggers RNase-E-mediated RNA decay, while being inactive against triphosphate-RNA. In vivo, ∼13% of the abundant sRNA RNAI is NAD-capped in the presence, and ∼26% in the absence, of functional NudC. To our knowledge, this is the first description of a cap-like structure and a decapping machinery in bacteria.

  15. New Therapeutic Concept of NAD Redox Balance for Cisplatin Nephrotoxicity

    PubMed Central

    Oh, Gi-Su; Kim, Hyung-Jin; Shen, AiHua; Lee, Su-Bin; Yang, Sei-Hoon; Shim, Hyeok; Cho, Eun-Young; Kwon, Kang-Beom; Kwak, Tae Hwan; So, Hong-Seob

    2016-01-01

    Cisplatin is a widely used chemotherapeutic agent for the treatment of various tumors. In addition to its antitumor activity, cisplatin affects normal cells and may induce adverse effects such as ototoxicity, nephrotoxicity, and peripheral neuropathy. Various mechanisms such as DNA adduct formation, mitochondrial dysfunction, oxidative stress, and inflammatory responses are closely associated with cisplatin-induced nephrotoxicity; however, the precise mechanism remains unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Recent studies have demonstrated associations between disturbance in intracellular NAD+ levels and clinical progression of various diseases through the production of reactive oxygen species and inflammation. Furthermore, we demonstrated that reduction of the intracellular NAD+/NADH ratio is critically involved in cisplatin-induced kidney damage through inflammation and oxidative stress and that increase of the cellular NAD+/NADH ratio suppresses cisplatin-induced kidney damage by modulation of potential damage mediators such as oxidative stress and inflammatory responses. In this review, we describe the role of NAD+ metabolism in cisplatin-induced nephrotoxicity and discuss a potential strategy for the prevention or treatment of cisplatin-induced adverse effects with a particular focus on NAD+-dependent cellular pathways. PMID:26881219

  16. Physiological and pathophysiological roles of NAMPT and NAD metabolism.

    PubMed

    Garten, Antje; Schuster, Susanne; Penke, Melanie; Gorski, Theresa; de Giorgis, Tommaso; Kiess, Wieland

    2015-09-01

    Nicotinamide phosphoribosyltransferase (NAMPT) is a regulator of the intracellular nicotinamide adenine dinucleotide (NAD) pool. NAD is an essential coenzyme involved in cellular redox reactions and is a substrate for NAD-dependent enzymes. In various metabolic disorders and during ageing, levels of NAD are decreased. Through its NAD-biosynthetic activity, NAMPT influences the activity of NAD-dependent enzymes, thereby regulating cellular metabolism. In addition to its enzymatic function, extracellular NAMPT (eNAMPT) has cytokine-like activity. Abnormal levels of eNAMPT are associated with various metabolic disorders. NAMPT is able to modulate processes involved in the pathogenesis of obesity and related disorders such as nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM) by influencing the oxidative stress response, apoptosis, lipid and glucose metabolism, inflammation and insulin resistance. NAMPT also has a crucial role in cancer cell metabolism, is often overexpressed in tumour tissues and is an experimental target for antitumour therapies. In this Review, we discuss current understanding of the functions of NAMPT and highlight progress made in identifying the physiological role of NAMPT and its relevance in various human diseases and conditions, such as obesity, NAFLD, T2DM, cancer and ageing.

  17. A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase.

    PubMed

    Bembenek, Michael E; Kuhn, Eric; Mallender, William D; Pullen, Lester; Li, Ping; Parsons, Thomas

    2005-10-01

    NAD synthetase is responsible for the conversion of nicotinic acid adenine dinucleotide to nicotinamide adenine dinucleotide. This reaction provides a biosynthetic route of the coenzyme and, thus, a source of cellular reducing equivalents. Alterations in the oxidative reductive potential of the cell have been implicated as a contributing factor in many disease states. Thus, this enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a coupled enzyme assay format for the measurement of recombinant human NAD synthetase by employing lactate dehydrogenase in a cycling/amplification reaction linked ultimately to the fluorescence generation of resorufin from resazurin via diaphorase. We present kinetics of the reaction of NAD synthetase in the coupled assay format, optimization conditions, and inhibition of the reaction by gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimethyl-5,5'-bis(1-methylethyl)-[2,2'- binaphthalene]-8,8'-dicarboxaldehyde] and illustrate the robustness of the assay by demonstrating 384-well microtiter plate uniformity statistics. Collectively, our results show that the assay method is both robust and well suited for this class of enzymes involved in the NAD+ biosynthetic pathway.

  18. 8-Thioalkyl-adenosine derivatives inhibit Listeria monocytogenes NAD kinase through a novel binding mode.

    PubMed

    Paoletti, Julie; Assairi, Liliane; Gelin, Muriel; Huteau, Valérie; Nahori, Marie-Anne; Dussurget, Olivier; Labesse, Gilles; Pochet, Sylvie

    2016-11-29

    Increased resistance of pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials. Biosynthetic pathways of several cofactors important for bacterial growth, such as nicotinamide adenine dinucleotide phosphate (NADP), have been proposed as a promising source of antibiotic targets. Nicotinamide adenine dinucleotide kinases (NADK; EC 2.7.1.23) are attractive for inhibitor development, since they catalyze the phosphorylation of NAD to NADP, which is an essential step of NADP metabolism. We previously synthesized diadenosine derivatives that inhibited NADK from two human pathogens, Listeria monocytogenes and Staphylococcus aureus, in the micromolar range. They behave as NAD mimics with the 5',5'-diphosphate group substituted by a 8,5' thioglycolic bridge. In an attempt to improve inhibitory potency, we designed new NAD mimics based on a single adenosine moiety harboring a larger derivatization attached to the C8 position and a small group at the 5' position. Here we report the synthesis of a series of 8-thioalkyl-adenosine derivatives containing various aryl and heteroaryl moieties and their evaluation as inhibitors of L. monocytogenes NADK1, S. aureus NADK and their human counterpart. Novel, sub-micromolar inhibitors of LmNADK1 were identified. Surprisingly, most LmNADK1 inhibitors demonstrated a high selectivity index against the close staphylococcal ortholog and the human NADK. Structural characterization of enzyme-inhibitor complexes revealed the original binding mode of these novel NAD mimics.

  19. Identification of UshA as a major enzyme for NAD degradation in Escherichia coli.

    PubMed

    Wang, Lei; Zhou, Yongjin J; Ji, Debin; Lin, Xinping; Liu, Yuxue; Zhang, Yixin; Liu, Wujun; Zhao, Zongbao K

    2014-05-10

    Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH are essential cofactors for many redox biocatalysts. Because these cofactors are consumed in stoichiometric amounts, whole-cell biocatalysts have been routinely employed in order to reduce the costs. To further improve the efficacy of redox biocatalysts, it is essential to maintain the stability of nicotinamide cofactors, for which it is attractive to block degradation pathways for NAD(H). While the biosynthesis of NAD(H) has been well studied, it is less understood how NAD(H) are degraded. Here we demonstrated that UshA was a major periplasmic enzyme for NAD degradation in Escherichia coli. Purified recombinant UshA showed high pyrophosphatase activity with the catalytic efficiencies for hydrolysis of NAD and NADH at 3.7μM(-1)s(-1) and 1.4μM(-1)s(-1), respectively. Deletion of the ushA gene from the chromosome led to faster cell growth and improved extracellular NAD stability by 3-fold under conditions similar to whole-cell biocatalysis. These results significantly enriched our understanding on NAD metabolism, and should facilitate many applications including designing more robust redox biocatalysts.

  20. Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

    PubMed

    Hsu, Chia George; Burkholder, Thomas J

    2016-12-01

    The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.

  1. Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues.

    PubMed

    Mori, Valerio; Amici, Adolfo; Mazzola, Francesca; Di Stefano, Michele; Conforti, Laura; Magni, Giulio; Ruggieri, Silverio; Raffaelli, Nadia; Orsomando, Giuseppe

    2014-01-01

    NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.

  2. Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique.

    PubMed

    Balan, Irina S; Fiskum, Gary; Kristian, Tibor

    2010-02-26

    A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD(+) that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD(+) glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration.

  3. Visualization and quantification of NAD(H) in brain sections by a novel histo-enzymatic nitrotetrazolium blue staining technique

    PubMed Central

    Balan, Irina S.; Fiskum, Gary; Kristian, Tibor

    2010-01-01

    A histo-enzymatic technique for visualizing and quantifying endogenous NAD(H) in brain tissue was developed, based on coupled enzymatic cycling reactions that reduce nitrotetrazolium blue chloride to produce formazan. Conditions were used where the endogenous level of nicotinamide adenine dinucleotides (NAD(H)) was the rate limiting factor for formazan production. Spontaneous degradation of NAD+ that occurs during incubation of thawed tissue was minimized by the addition of nicotinamide mononucleotide, an inhibitor of NAD+ glycohydrolases. Cryostat sections of brains obtained from rats immediately after decapitation and at 30 min later were used to determine the effects of ischemia alone on brain NAD(H) levels and neuroanatomic distribution. The ischemic insult resulted in a greater than 50% decline in the rate of formazan generation in the CA1 pyramidal neuronal layer of the hippocampus and in the parietal cortex and striatum, but not in the CA3 and dentate gyrus (DG) subregions of the hippocampus. The ischemia-induced changes in NAD(H) levels were confirmed by utilizing spectrofluorimetric measurements of NAD(H) present in perchloric acid extracts of brain samples. This new histo-enzymatic technique is suitable for visualizing and quantifying relative NAD(H) levels in the brain. This assay could prove useful in identifying region-selective NAD(H) catabolism that may contribute to neurodegeneration. PMID:20036220

  4. Detection of cerebral NAD(+) by in vivo (1)H NMR spectroscopy.

    PubMed

    de Graaf, Robin A; Behar, Kevin L

    2014-07-01

    Nicotinamide adenine dinucleotide (NAD(+)) plays a central role in cellular metabolism both as a coenzyme for electron-transfer enzymes as well as a substrate for a wide range of metabolic pathways. In the current study NAD(+) was detected on rat brain in vivo at 11.7T by 3D localized (1)H MRS of the NAD(+) nicotinamide protons in the 8.7-9.5 ppm spectral region. Avoiding water perturbation was critical to the detection of NAD(+) as strong, possibly indirect cross-relaxation between NAD(+) and water would lead to a several-fold reduction of the NAD(+) intensity in the presence of water suppression. Water perturbation was minimized through the use of localization by adiabatic spin-echo refocusing (LASER) in combination with frequency-selective excitation. The NAD(+) concentration in the rat cerebral cortex was determined at 296 ± 28 μm, which is in good agreement with recently published (31) P NMR-based results as well as results from brain extracts in vitro (355 ± 34 μm). The T1 relaxation time constants of the NAD(+) nicotinamide protons as measured by inversion recovery were 280 ± 65 and 1136 ± 122 ms in the absence and presence of water inversion, respectively. This confirms the strong interaction between NAD(+) nicotinamide and water protons as observed during water suppression. The T2 relaxation time constants of the NAD(+) nicotinamide protons were determined at 60 ± 13 ms after confounding effects of scalar coupling evolution were taken into account. The simplicity of the MR sequence together with the robustness of NAD(+) signal detection and quantification makes the presented method a convenient choice for studies on NAD(+) metabolism and function. As the method does not critically rely on magnetic field homogeneity and spectral resolution it should find immediate applications in rodents and humans even at lower magnetic fields.

  5. Occurrence of cold-labile NAD-specific glutamate dehydrogenase in Bacillus species.

    PubMed

    Jahns, T

    1992-09-15

    A nicotinamide adenine dinucleotide-specific glutamate dehydrogenase (NAD-GluDH; EC 1.4.1.3) inactivated by incubation at low temperatures was detected in several species of the genus Bacillus, including strains of B. cereus, B. laterosporus, B. lentus, B. panthotenicus, B. pasteurii, B. sphaericus, B. stearothermophilus, B. subtilis and B. thuringiensis. Incubation of cell-free extracts of these strains at 0 degrees C resulted in an 80-100% inactivation of NAD-GluDH activity within 120 min. The addition of 20% glycerol protected the enzyme from this inactivation in the cold. Strains of B. fastidiosus, B. licheniformis, B. macerans, B. megaterium and B. pumilus were found to lack NAD-GluDH activity.

  6. Monocyte-derived extracellular Nampt-dependent biosynthesis of NAD(+) protects the heart against pressure overload.

    PubMed

    Yano, Masamichi; Akazawa, Hiroshi; Oka, Toru; Yabumoto, Chizuru; Kudo-Sakamoto, Yoko; Kamo, Takehiro; Shimizu, Yu; Yagi, Hiroki; Naito, Atsuhiko T; Lee, Jong-Kook; Suzuki, Jun-ichi; Sakata, Yasushi; Komuro, Issei

    2015-11-02

    Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD(+)) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD(+) by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD(+) concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD(+), were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD(+) levels and NAD(+)-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD(+) against heart failure.

  7. Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro.

    PubMed

    Wang, Shengjun; Yang, Xue; Lin, Youting; Qiu, Xiaoxue; Li, Hui; Zhao, Xiuhe; Cao, Lili; Liu, Xuewu; Pang, Yuejiu; Wang, Xuping; Chi, Zhaofu

    2013-10-16

    Intense poly(ADP-ribose) polymerase-1 (PARP-1) activation was implicated as a major cause of caspase-independent cell death in the hippocampal neuronal culture (HNC) model of acute acquired epilepsy (AE). The molecular mechanisms are quite complicated. The linkage among neuronal death, cellular nicotinamide adenine dinucleotide (NAD) levels, apoptosis-inducing factor (AIF) translocation, SIRT1 expression and activity were investigated here. The results showed that PARP-1 over-activation caused by Mg²⁺-free stimuli led to cellular NAD depletion which could block AIF translocation from mitochondria to nucleus and attenuate neuronal death. Also, SIRT1 deacetylase activity was reduced by Mg²⁺-free treatment, accompanied by elevated ratio of neuronal death, which could be rescued by NAD repletion. These data demonstrated that cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated epileptic neuronal death in the HNC model of acute AE.

  8. Diabetes and the control of pyruvate dehydrogenase in rat heart mitochondria by concentration ratios of adenosine triphosphate/adenosine diphosphate, of reduced/oxidized nicotinamide-adenine dinucleotide and of acetyl-coenzyme A/coenzyme A.

    PubMed Central

    Kerbey, A L; Radcliffe, P M; Randle, P J

    1977-01-01

    1. The proportion of active (dephosphorylated) pyruvate dehydrogenase in rat heart mitochondria was correlated with total concentration ratios of ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. These metabolites were measured with ATP-dependent and NADH-dependent luciferases. 2. Increase in the concentration ratio of NADH/NAD+ at constant [ATP]/[ADP] and [acetyl-CoA]/[CoA] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between mitochondria incubated with 0.4mM- or 1mM-succinate and mitochondria incubated with 0.4mM-succinate+/-rotenone. 3. Increase in the concentration ratio acetyl-CoA/CoA at constant [ATP]/[ADP] and [NADH][NAD+] was associated with increased phosphorylation and inactivation of pyruvate dehydrogenase. This was based on comparison between incubations in 50 micrometer-palmitotoyl-L-carnitine and in 250 micrometer-2-oxoglutarate +50 micrometer-L-malate. 4. These findings are consistent with activation of the pyruvate dehydrogenase kinase reaction by high ratios of [NADH]/[NAD+] and of [acetyl-CoA]/[CoA]. 5. Comparison between mitochondria from hearts of diabetic and non-diabetic rats shows that phosphorylation and inactivation of pyruvate dehydrogenase is enhanced in alloxan-diabetes by some factor other than concentration ratios of ATP/ADP, NADH/NAD+ or acetyl-CoA/CoA. PMID:196589

  9. Secondary NAD+ deficiency in the inherited defect of glutamine synthetase.

    PubMed

    Hu, Liyan; Ibrahim, Khalid; Stucki, Martin; Frapolli, Michele; Shahbeck, Noora; Chaudhry, Farrukh A; Görg, Boris; Häussinger, Dieter; Penberthy, W Todd; Ben-Omran, Tawfeg; Häberle, Johannes

    2015-11-01

    Glutamine synthetase (GS) deficiency is an ultra-rare inborn error of amino acid metabolism that has been described in only three patients so far. The disease is characterized by neonatal onset of severe encephalopathy, low levels of glutamine in blood and cerebrospinal fluid, chronic moderate hyperammonemia, and an overall poor prognosis in the absence of an effective treatment. Recently, enteral glutamine supplementation was shown to be a safe and effective therapy for this disease but there are no data available on the long-term effects of this intervention. The amino acid glutamine, severely lacking in this disorder, is central to many metabolic pathways in the human organism and is involved in the synthesis of nicotinamide adenine dinucleotide (NAD(+)) starting from tryptophan or niacin as nicotinate, but not nicotinamide. Using fibroblasts, leukocytes, and immortalized peripheral blood stem cells (PBSC) from a patient carrying a GLUL gene point mutation associated with impaired GS activity, we tested whether glutamine deficiency in this patient results in NAD(+) depletion and whether it can be rescued by supplementation with glutamine, nicotinamide or nicotinate. The present study shows that congenital GS deficiency is associated with NAD(+) depletion in fibroblasts, leukocytes and PBSC, which may contribute to the severe clinical phenotype of the disease. Furthermore, it shows that NAD(+) depletion can be rescued by nicotinamide supplementation in fibroblasts and leukocytes, which may open up potential therapeutic options for the treatment of this disorder.

  10. Dunnione ameliorates cisplatin ototoxicity through modulation of NAD(+) metabolism.

    PubMed

    Kim, Hyung-Jin; Pandit, Arpana; Oh, Gi-Su; Shen, AiHua; Lee, Su-Bin; Khadka, Dipendra; Lee, SeungHoon; Shim, Hyeok; Yang, Sei-Hoon; Cho, Eun-Young; Kwak, Tae Hwan; Choe, Seong-Kyu; Park, Raekil; So, Hong-Seob

    2016-03-01

    Ototoxicity is an important issue in patients receiving cisplatin chemotherapy. Numerous studies have demonstrated that cisplatin-induced ototoxicity is related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as an important regulator of energy metabolism and cellular homeostasis. Here, we demonstrate that the levels and activities of sirtuin-1 (SIRT1) are suppressed by the reduction of intracellular NAD(+) levels in cisplatin-mediated ototoxicity. We provide evidence that the decreases in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) polymerase-1 (PARP-1) activation and microRNA-34a levels through cisplatin-mediated p53 activation aggravate the associated ototoxicity. Furthermore, we show that the induction of cellular NAD(+) levels using dunnione, which targets intracellular NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity.

  11. One-electron transfer reactions of the couple NAD. /NADH. [Pulse radiolysis

    SciTech Connect

    Grodkowski, J.; Neta, P.; Carlson, B.W.; Miller, L.

    1983-08-04

    One-electron transfer reactions involving nicotinamide-adenine dinucleotide in its oxidized and reducd forms (NAD./NADH) were studied by pulse radiolysis in aqueous solutions. One-electron oxidation of NADH by various phenoxyl radicals and phenothiazine cation radicals was found to take place with rate constants in the range of 10/sup 5/ to 10/sup 8/ M/sup -1/ s/sup -1/, depending on the redox potential of the oxidizing species. In all cases, NAD. is formed quantitatively with no indication for the existence of the protonated form (NADH/sup +/.). The spectrum of NAD., as well as the rates of oxidation of NADH by phenoxyl and by (chlorpromazine)/sup +/. were independent of pH between pH 4.5 and 13.5. Reaction of deuterated NADH indicated only a small kinetic isotope effect. All these findings point to an electron transfer mechanism. On the other hand, attempts to observe the reverse electron transfer, i.e., one-electron reduction of NAD. to NADH by radicals such as semiquinones, showed that k was less than 10/sup 4/ to 10/sup 5/ M/sup -1/ s/sup -1/, so that it was unobservable. Consequently, it was not possible to achieve equilibrium conditions which would have permitted the direct measurement of the redox potential for NAD./NADH. One-electron reduction of NAD. appears to be an unlikely process. 1 table.

  12. ARTD1 (PARP1) activation and NAD(+) in DNA repair and cell death.

    PubMed

    Fouquerel, Elise; Sobol, Robert W

    2014-11-01

    Nicotinamide adenine dinucleotide, NAD(+), is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD(+) and NADH). NAD(+) is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalyzed by the sirtuins (SIRTs) which use NAD(+) as a substrate. In this review, we highlight the significance of NAD(+) catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by ARTD1 activation and energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked, are dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischaemia/reperfusion.

  13. Roles of NAD in Protection of Axon against Degeneration via SIRT1 Pathways.

    PubMed

    Zhang, Jing; Guo, Wei-Hua; Qi, Xiao-Xia; Li, Gui-Bao; Hu, Yan-Lai; Wu, Qi; Ding, Zhao-Xi; Li, Hong-Yu; Hao, Jing; Sun, Jin-Hao

    2016-04-30

    Axonal degeneration is a common pathological change of neurogenical disease which often arises before the neuron death. But it had not found any effective method to protect axon from degeneration. In this study we intended to confirm the protective effect of nicotinamide adenine dinucleotide (NAD), investigate the optimal administration dosage and time of NAD, and identify the relationship between silence signal regulating factor 1 (SIRT1) and axonal degeneration. An axonal degeneration model was established using dorsal root ganglion (DRG) neurons injured by vincristine to observe the protective effects of NAD to the injured axons. In addition, the potential contribution of the SIRT1 in axonal degeneration was also investigated. Through the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, immunochemistry staining, axons counting and length measuring, transmission electron microscope (TEM) observation, we demonstrated that NAD played an important role in preventing axonal degeneration. Further study revealed that the expression of SIRT1 and phosphorylated Akt1 (p-Akt1) was up-regulated when NAD was added into the culturing medium. Taking together, our results demonstrated that NAD might delay the axonal degeneration through SIRT1/Akt1 pathways.

  14. Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

    SciTech Connect

    Puzon, Geoffrey J.; Huang, Yan C.; Dohnalkova, Alice; Xun, Luying

    2008-06-01

    Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)3 precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD+)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD+-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD+-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD+ with a doubling time of 17 h, and even more slowly on the NAD+-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD+ dissociated from the NAD+-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.

  15. Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD+ homeostasis and contributes to longevity

    PubMed Central

    TSANG, Felicia; LIN, Su-Ju

    2016-01-01

    Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast, Saccharomyces cerevisiae, and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD+ (nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NAD+ precursor) homeostasis. Maintaining adequate NAD+ pools has been shown to play key roles in life span extension, but factors regulating NAD+ metabolism and homeostasis are not completely understood. Recently, NAD+ metabolism was also linked to the phosphate (Pi)-sensing PHO pathway in yeast. Canonical PHO activation requires Pi-starvation. Interestingly, NAD+ depletion without Pi-starvation was sufficient to induce PHO activation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel with PHO signaling components to regulate NR/NAD+ homeostasis. These studies suggest that NAD+ metabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of PHO, PKA, TOR and Sch9 pathways was reported to potentially affect NAD+ metabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NAD+ homeostasis, and cross-talk among nutrient sensing pathways and NAD+ homeostasis. PMID:27683589

  16. NAD+ treatment can prevent rotenone-induced increases in DNA damage, Bax levels and nuclear translocation of apoptosis-inducing factor in differentiated PC12 cells.

    PubMed

    Hong, Yunyi; Nie, Hui; Wei, Xunbin; Fu, Shen; Ying, Weihai

    2015-04-01

    Nicotinamide adenine dinucleotide (NAD(+)) plays critical roles in energy metabolism, mitochondrial functions, calcium homeostasis and immunological functions. Our previous studies have found that NAD(+) administration can profoundly decrease ischemic brain injury and traumatic brain injury. Our recent study has also provided first direct evidence indicating that NAD(+) treatment can decrease cellular apoptosis, while the mechanisms underlying this protective effect remain unclear. In our current study, we determined the effects of NAD(+) treatment on several major factors in apoptosis and necrosis, including levels of Bax and nuclear translocation of apoptosis-inducing factor (AIF), as well as levels of DNA double-strand breaks (DSBs) and intracellular ATP in rotenone-treated differentiated PC12 cells. We found that NAD(+) treatment can markedly attenuate the rotenone-induced increases in the levels of Bax and nuclear translocation of AIF in the cells. We further found that NAD(+) treatment can significantly attenuate the rotenone-induced increase in the levels of DSBs and decrease in the intracellular ATP levels. Collectively, our study has suggested mechanisms underlying the preventive effects of NAD(+) on apoptosis, which has highlighted the therapeutic potential of NAD(+) for decreasing apoptotic changes in multiple major diseases.

  17. Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme.

    PubMed

    Avalos, José L; Bever, Katherine M; Wolberger, Cynthia

    2005-03-18

    Sir2 enzymes form a unique class of NAD(+)-dependent deacetylases required for diverse biological processes, including transcriptional silencing, regulation of apoptosis, fat mobilization, and lifespan regulation. Sir2 activity is regulated by nicotinamide, a noncompetitive inhibitor that promotes a base-exchange reaction at the expense of deacetylation. To elucidate the mechanism of nicotinamide inhibition, we determined ternary complex structures of Sir2 enzymes containing nicotinamide. The structures show that free nicotinamide binds in a conserved pocket that participates in NAD(+) binding and catalysis. Based on our structures, we engineered a mutant that deacetylates peptides by using nicotinic acid adenine dinucleotide (NAAD) as a cosubstrate and is inhibited by nicotinic acid. The characteristics of the altered specificity enzyme establish that Sir2 enzymes contain a single site that participates in catalysis and nicotinamide regulation and provides additional insights into the Sir2 catalytic mechanism.

  18. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

    PubMed

    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.

  19. NAD+ Attenuates Bilirubin-Induced Hyperexcitation in the Ventral Cochlear Nucleus by Inhibiting Excitatory Neurotransmission and Neuronal Excitability

    PubMed Central

    Liang, Min; Yin, Xin-Lu; Wang, Lu-Yang; Yin, Wei-Hai; Song, Ning-Ying; Shi, Hai-Bo; Li, Chun-Yan; Yin, Shan-Kai

    2017-01-01

    Nicotinamide adenine dinucleotide (NAD+) is an important molecule with extensive biological functions in various cellular processes, including protection against cell injuries. However, little is known regarding the roles of NAD+ in neuronal excitation and excitotoxicity associated with many neurodegenerative disorders and diseases. Using patch-clamp recordings, we studied its potential effects on principal neurons in the ventral cochlear nucleus (VCN), which is particularly vulnerable to bilirubin excitotoxicity. We found that NAD+ effectively decreased the size of evoked excitatory postsynaptic currents (eEPSCs), increased paired-pulse ratio (PPR) and reversed the effect of bilirubin on eEPSCs, implicating its inhibitory effects on the presynaptic release probability (Pr). Moreover, NAD+ not only decreased the basal frequency of miniature EPSCs (mEPSCs), but also reversed bilirubin-induced increases in the frequency of mEPSCs without affecting their amplitude under either condition. Furthermore, we found that NAD+ decreased the frequency of spontaneous firing of VCN neurons as well as bilirubin-induced increases in firing frequency. Whole-cell current-clamp recordings showed that NAD+ could directly decrease the intrinsic excitability of VCN neurons in the presence of synaptic blockers, suggesting NAD+ exerts its actions in both presynaptic and postsynaptic loci. Consistent with these observations, we found that the latency of the first postsynaptic spike triggered by high-frequency train stimulation of presynaptic afferents (i.e., the auditory nerve) was prolonged by NAD+. These results collectively indicate that NAD+ suppresses presynaptic transmitter release and postsynaptic excitability, jointly weakening excitatory neurotransmission. Our findings provide a basis for the exploration of NAD+ for the prevention and treatment of bilirubin encephalopathy and excitotoxicity associated with other neurological disorders. PMID:28217084

  20. Unprecedented head-to-head right-handed cross-links between the antitumor bis(mu-N,N'-di-p-tolylformamidinate) dirhodium(II,II) core and the dinucleotide d(ApA) with the adenine bases in the rare imino form.

    PubMed

    Chifotides, Helen T; Dunbar, Kim R

    2007-10-17

    Reactions of the anticancer active compound cis-[Rh2(DTolF)2(CH3CN)6](BF4)2 with 9-ethyladenine (9-EtAdeH) or the dinucleotide d(ApA) proceed with bridging adenine bases in the rare imino form (A*), spanning the Rh-Rh bond at equatorial positions via N7/N6. The inflection points for the pH-dependent H2 and H8 NMR resonance curves of cis-[Rh2(DTolF)2(9-EtAdeH)2](BF4)2 correspond to N1H deprotonation of the metal-stabilized rare imino tautomer, which takes place at pKa approximately 7.5 in CD3CN-d3, a considerably reduced value as compared to that of the imino form of 9-EtAdeH. Similarly, coordination of the metal atoms to the N7/N6 adenine sites in Rh2(DTolF)2{d(ApA)} induces formation of the rare imino tautomer of the bases with a concomitant substantial decrease in the basicity of the N1H sites (pKa approximately 7.0 in CD3CN-d3), as compared to the imino form of the free dinucleotide. The presence of the adenine bases in the rare imino form, due to bidentate metalation of the N6/N7 sites, is further corroborated by DQF-COSY H2/N1H and ROE N1H/N6H cross-peaks in the 2D NMR spectra of Rh2(DTolF)2{d(ApA)} in CD3CN-d3 at -38 degrees C. Due to the N7/N6 bridging mode of the adenine bases in Rh2(DTolF)2{d(ApA)}, only the anti orientation of the imino tautomer is possible. The imino form A* of adenine in DNA may result in AT-->CG transversions or AT-->GC transitions, which can eventually lead to lethal mutations. The HH arrangement of the bases in Rh2(DTolF)2{d(ApA)} is indicated by the H8/H8 NOE cross-peaks in the 2D ROESY NMR spectrum, whereas the formamidinate bridging groups dictate the presence of one right-handed conformer HH1R in solution. Complete characterization of Rh2(DTolF)2{d(ApA)} by 2D NMR spectroscopy and molecular modeling supports the presence of the HH1R conformer, anti orientation of both sugar residues about the glycosyl bonds, and N-type conformation for the 5'-A base.

  1. Structural Basis for NADH/NAD+ Redox Sensing by a Rex Family Repressor

    SciTech Connect

    McLaughlin, K.J.; Soares, A.; Strain-Damerell, C. M.; Xie, K.; Brekasis, D.; Pagent, M. S. B.; Kielkopf, C. L.

    2010-05-28

    Nicotinamide adenine dinucleotides have emerged as key signals of the cellular redox state. Yet the structural basis for allosteric gene regulation by the ratio of reduced NADH to oxidized NAD{sup +} is poorly understood. A key sensor among Gram-positive bacteria, Rex represses alternative respiratory gene expression until a limited oxygen supply elevates the intracellular NADH:NAD{sup +} ratio. Here we investigate the molecular mechanism for NADH/NAD{sup +} sensing among Rex family members by determining structures of Thermus aquaticus Rex bound to (1) NAD{sup +}, (2) DNA operator, and (3) without ligand. Comparison with the Rex/NADH complex reveals that NADH releases Rex from the DNA site following a 40{sup o} closure between the dimeric subunits. Complementary site-directed mutagenesis experiments implicate highly conserved residues in NAD-responsive DNA-binding activity. These rare views of a redox sensor in action establish a means for slight differences in the nicotinamide charge, pucker, and orientation to signal the redox state of the cell.

  2. beta-NAD is a novel nucleotide released on stimulation of nerve terminals in human urinary bladder detrusor muscle.

    PubMed

    Breen, Leanne T; Smyth, Lisa M; Yamboliev, Ilia A; Mutafova-Yambolieva, Violeta N

    2006-02-01

    Endogenous nucleotides with extracellular functions may be involved in the complex neural control of human urinary bladder (HUB). Using HPLC techniques with fluorescence detection, we observed that in addition to ATP and its metabolites ADP, AMP and adenosine, electrical field stimulation (EFS; 4-16 Hz, 0.1 ms, 15 V, 60 s) of HUB detrusor smooth muscle coreleases novel nucleotide factors, which produce etheno-1N(6)-ADP-ribose (eADPR) on etheno-derivatization at high temperature. A detailed HPLC fraction analysis determined that nicotinamide adenine dinucleotide (beta-NAD+; 7.0 +/- 0.7 fmol/mg tissue) is the primary nucleotide that contributes to the formation of eADPR. The tissue superfusates collected during EFS also contained the beta-NAD+ metabolite ADPR (0.35 +/- 0.2 fmol/mg tissue) but not cyclic ADPR (cADPR). HUB failed to degrade nicotinamide guanine dinucleotide (NGD+), a specific substrate of ADP ribosyl cyclase, suggesting that the activity of this enzyme in the HUB is negligible. The EFS-evoked release of beta-NAD+ was frequency dependent and is reduced in the presence of tetrodotoxin (TTX; 0.3 micromol/l), omega-conotoxin GVIA (50 nmol/l), and botulinum neurotoxin A (BoNT/A; 100 nmol/l), but remained unchanged in the presence of guanethidine (3 micromol/l), omega-agatoxin IVA (50 nmol/l), or charbachol (1 micromol/l). Capsaicin (10 micromol/l) increased both the resting and EFS-evoked overflow of beta-NAD+. Exogenous beta-NAD+ (1 micromol/l) reduced both the frequency and amplitude of spontaneous contractions. In conclusion, we detected nerve-evoked overflow of beta-NAD+ and ADPR in HUB. The beta-NAD(+)/ADPR system may constitute a novel inhibitory extracellular nucleotide mechanism of neural control of the human bladder.

  3. Physiology-based kinetic modeling of neuronal energy metabolism unravels the molecular basis of NAD(P)H fluorescence transients

    PubMed Central

    Berndt, Nikolaus; Kann, Oliver; Holzhütter, Hermann-Georg

    2015-01-01

    Imaging of the cellular fluorescence of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) is one of the few metabolic readouts that enable noninvasive and time-resolved monitoring of the functional status of mitochondria in neuronal tissues. Stimulation-induced transient changes in NAD(P)H fluorescence intensity frequently display a biphasic characteristic that is influenced by various molecular processes, e.g., intracellular calcium dynamics, tricarboxylic acid cycle activity, the malate–aspartate shuttle, the glycerol-3-phosphate shuttle, oxygen supply or adenosine triphosphate (ATP) demand. To evaluate the relative impact of these processes, we developed and validated a detailed physiologic mathematical model of the energy metabolism of neuronal cells and used the model to simulate metabolic changes of single cells and tissue slices under different settings of stimulus-induced activity and varying nutritional supply of glucose, pyruvate or lactate. Notably, all experimentally determined NAD(P)H responses could be reproduced with one and the same generic cellular model. Our computations reveal that (1) cells with quite different metabolic status may generate almost identical NAD(P)H responses and (2) cells of the same type may quite differently contribute to aggregate NAD(P)H responses recorded in brain slices, depending on the spatial location within the tissue. Our computational approach reconciles different and sometimes even controversial experimental findings and improves our mechanistic understanding of the metabolic changes underlying live-cell NAD(P)H fluorescence transients. PMID:25899300

  4. Nicotinamide riboside kinase structures reveal new pathways to NAD+.

    PubMed

    Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

    2007-10-02

    The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

  5. Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

    SciTech Connect

    Sporty, J; Lin, S; Kato, M; Ognibene, T; Stewart, B; Turteltaub, K; Bench, G

    2009-02-18

    Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD

  6. Crystal structures of complexes of NAD{sup +}-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 with formate

    SciTech Connect

    Filippova, E. V. Polyakov, K. M.; Tikhonova, T. V.; Stekhanova, T. N.; Boiko, K. M.; Sadykhov, I. G.; Tishkov, V. I.; Popov, V. O.; Labru, N.

    2006-07-15

    Formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 catalyzes oxidation of formate to NI{sub 2} with the coupled reduction of nicotinamide adenine dinucleotide (NAD{sup +}). The three-dimensional structures of the apo form (the free enzyme) and the holo form (the ternary FDH-NAD{sup +}-azide complex) of FDH have been established earlier. In the present study, the structures of FDH complexes with formate are solved at 2.19 and 2.28 A resolution by the molecular replacement method and refined to the R factors of 22.3 and 20.5%, respectively. Both crystal structures contain four protein molecules per asymmetric unit. These molecules form two dimers identical to the dimer of the apo form of FDH. Two possible formatebinding sites are found in the active site of the FDH structure. In the complexes the sulfur atom of residue Cys354 exists in the oxidized state.

  7. A novel modified carbon paste electrode based on NiO/CNTs nanocomposite and (9, 10-dihydro-9, 10-ethanoanthracene-11, 12-dicarboximido)-4-ethylbenzene-1, 2-diol as a mediator for simultaneous determination of cysteamine, nicotinamide adenine dinucleotide and folic acid.

    PubMed

    Karimi-Maleh, Hassan; Biparva, Pourya; Hatami, Mehdi

    2013-10-15

    A carbon paste electrode (CPE) modified with (9, 10-dihydro-9, 10-ethanoanthracene-11, 12-dicarboximido)-4-ethylbenzene-1, 2-diol (DEDE) and NiO/CNTs nanocomposite was used for the sensitive voltammetric determination of cysteamine (CA), nicotinamide adenine dinucleotide (NADH) and folic acid (FA) for the first time. The synthesized materials were characterized with different methods such as XRD, cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and square wave voltammetry (SWV). The modified electrode exhibited a potent and persistent electron mediating behavior followed by well-separated oxidation peaks of CA, NADH and FA. The peak currents were linearly dependent on CA, NADH and FA concentrations using square wave voltammetry (SWV) method in the ranges of 0.01-250, 1.0-500, and 3.0-550 µmol L⁻¹, with detection limits of 0.007, 0.6, and 0.9 µmol L⁻¹, respectively. The modified electrode was used for the determination of CA, NADH and FA in biological and pharmaceutical samples.

  8. Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

    SciTech Connect

    Sporty, J; Kabir, M M; Turteltaub, K; Ognibene, T; Lin, S; Bench, G

    2008-01-10

    A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

  9. PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection.

    PubMed

    Tran, Mei T; Zsengeller, Zsuzsanna K; Berg, Anders H; Khankin, Eliyahu V; Bhasin, Manoj K; Kim, Wondong; Clish, Clary B; Stillman, Isaac E; Karumanchi, S Ananth; Rhee, Eugene P; Parikh, Samir M

    2016-03-24

    The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischaemia. Acute kidney injury (AKI) affects 3% of all hospitalized patients. Here we show that the mitochondrial biogenesis regulator, PGC1α, is a pivotal determinant of renal recovery from injury by regulating nicotinamide adenine dinucleotide (NAD) biosynthesis. Following renal ischaemia, Pgc1α(-/-) (also known as Ppargc1a(-/-)) mice develop local deficiency of the NAD precursor niacinamide (NAM, also known as nicotinamide), marked fat accumulation, and failure to re-establish normal function. Notably, exogenous NAM improves local NAD levels, fat accumulation, and renal function in post-ischaemic Pgc1α(-/-) mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulate the effects of NAM supplementation, including more local NAD and less fat accumulation with better renal function after ischaemia. PGC1α coordinately upregulates the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuates the de novo pathway. NAM enhances NAD via the enzyme NAMPT and augments production of the fat breakdown product β-hydroxybutyrate, leading to increased production of prostaglandin PGE2 (ref. 5), a secreted autacoid that maintains renal function. NAM treatment reverses established ischaemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-hydroxybutyrate signalling or prostaglandin production similarly abolishes PGC1α-dependent renoprotection. Given the importance of mitochondrial health in ageing and the function of metabolically active organs, the results implicate NAM and NAD as key effectors for achieving PGC1α-dependent stress resistance.

  10. A steady-state-kinetic model for formaldehyde dehydrogenase from human liver. A mechanism involving NAD+ and the hemimercaptal adduct of glutathione and formaldehyde as substrates and free glutathione as an allosteric activator of the enzyme.

    PubMed Central

    Uotila, L; Mannervik, B

    1979-01-01

    The steady-state kinetics of formaldehyde dehydrogenase from human liver have been explored. Non-linearities were obtained in v-versus-v[S] plots. It was necessary and sufficient to consider two reactants of the equilibrium mixture of formaldehyde, glutathione and their hemimercaptal adduct for a complete description of the kinetics. A random sequential reaction scheme is proposed in which adduct and beta-NAD+ are the substrates. In addition, glutathione can bind to an allosteric regulatory site and only the glutathione-containing enzyme is considered productive. Various alternative reaction models were examined but no simple alterative was superior to the model chosen. The discrimination was largely based on results of non-linear regression analysis. Several S-substituted glutathione derivatives were tested as activators or inhibitors of the enzyme, but all were without effect. Thio-NAD+, nicotinamide--hypoxanthine dinucleotide and 3-acetylpyridine-adenine dinucleotide could substitute for beta-NAD+ as the nucleotide substrate. alpha-NAD+ and ADP-ribose were competitive inhibitors with respect to beta-NAD+ and non-competitive with glutathione and the adduct. When used simultaneously, the inhibitors were linear competitive versus each other, indicating a single nucleotide-binding site or, if more than one, non-co-operative binding sites. PMID:220952

  11. The structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo NAD(+) content in OPI-induced teratogenesis in chickens.

    PubMed

    Seifert, Josef

    2016-05-01

    The objective of this study was to determine the structural requirements of organophosphorus insecticides (OPI) for reducing chicken embryo nicotinamide adenine dinucleotide (NAD(+)) content in OPI-induced teratogenesis and compare them with those needed for OPI inhibition of yolk sac membrane kynurenine formamidase (KFase), the proposed primary target for OPI teratogens in chicken embryos. The comparative molecular field analysis (COMFA) of three-dimensional quantitative structure-activity relationship (3D QSAR) revealed the electrostatic and steric fields as good predictors of OPI structural requirements to reduce NAD(+) content in chicken embryos. The dominant electrostatic interactions were localized at nitrogen-1, nitrogen-3, nitrogen of 2-amino substituent of the pyrimidinyl of pyrimidinyl phosphorothioates, and at the oxygen of crotonamide carbonyl in crotonamide phosphates. Bulkiness of the substituents at carbon-6 of the pyrimidinyls and/or N-substituents of crotonamides was the steric structural component that contributed to superiority of those OPI for reducing embryonic NAD(+) levels. Both electrostatic and steric requirements are similar to those defined in our previous study for OPI inhibition of chicken embryo yolk sac membrane KFase. The findings of this study provide another piece of evidence for the cause-and-effect relationship between yolk sac membrane KFase inhibition and reduced embryo NAD(+) content in NAD-associated OPI-induced teratogenesis in chickens.

  12. Augmentation of NAD+ by NQO1 attenuates cisplatin-mediated hearing impairment

    PubMed Central

    Kim, H-J; Oh, G-S; Shen, A; Lee, S-B; Choe, S-K; Kwon, K-B; Lee, S; Seo, K-S; Kwak, T H; Park, R; So, H-S

    2014-01-01

    Cisplatin (cis-diaminedichloroplatinum-II) is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of cellular energy metabolism and homeostasis. Here, we demonstrate for the first time that, in cisplatin-mediated ototoxicity, the levels and activities of SIRT1 are suppressed by the reduction of intracellular NAD+ levels. We provide evidence that the decrease in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) transferase (PARP)-1 activation and microRNA-34a through p53 activation aggravates cisplatin-mediated ototoxicity. Moreover, we show that the induction of cellular NAD+ levels using β-lapachone (β-Lap), whose intracellular target is NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD+ levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity. PMID:24922076

  13. Gene transfers shaped the evolution of de novo NAD+ biosynthesis in eukaryotes.

    PubMed

    Ternes, Chad M; Schönknecht, Gerald

    2014-09-01

    NAD(+) is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD(+) biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD(+) biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD(+) biosynthesis in eukaryotes was shaped by numerous gene transfers.

  14. NAD(+)-dependent SIRT1 deactivation has a key role on ischemia-reperfusion-induced apoptosis.

    PubMed

    Cattelan, Arianna; Ceolotto, Giulio; Bova, Sergio; Albiero, Mattia; Kuppusamy, Maniselvan; De Martin, Sara; Semplicini, Andrea; Fadini, Gian Paolo; de Kreutzenberg, Saula Vigili; Avogaro, Angelo

    2015-07-01

    Ischemia-reperfusion (IR) leads to severe organ injury and dysfunction. Sirtuins (SIRTs) are a family of histone deacetylases (HDACs) that require nicotinamide adenine dinucleotide (NAD(+)) for the deacetylation reaction. SIRTs play a major role in counteracting cellular stress and apoptosis. This study aimed to investigate the mechanisms of heart protection against apoptosis by SIRTs and the molecular pathways involved in SIRTs regulation and function in a rat model of IR injury. Hearts of male Wistar-Kyoto rats were subjected to 30-min ischemia followed by reperfusion up to 6h. IR increased cardiomyocyte apoptosis; the cleavage of caspase 3, induced a transient upregulation of SIRT1 and downregulation of SIRT6 expression, but decreased SIRT1 activity and reduced NAD(+) content. IR also increased forkhead box protein O1 (FoxO1) expression and FoxO1 binding to SIRT1 promoter region. Resveratrol restored SIRT1 activity and NAD(+) level by an AMPK-dependent mechanism, reduced cardiomyocyte apoptosis, and attenuated caspase 3 cleavage via heat shock factor-1 deacetylation and heat shock protein (HSP) expression upregulation. Our data show new potential molecular mechanisms of up and downstream regulation of SIRT1 in IR. The interplay among FoxO1, SIRT1, NAD(+), AMPK, HSP, and SIRT6 depicts a complex molecular network that protects the heart from apoptosis during IR and may be susceptible to therapeutic interventions.

  15. Dunnione ameliorates cisplatin-induced small intestinal damage by modulating NAD(+) metabolism.

    PubMed

    Pandit, Arpana; Kim, Hyung-Jin; Oh, Gi-Su; Shen, AiHua; Lee, Su-Bin; Khadka, Dipendra; Lee, SeungHoon; Shim, Hyeok; Yang, Sei-Hoon; Cho, Eun-Young; Kwon, Kang-Beom; Kwak, Tae Hwan; Choe, Seong-Kyu; Park, Raekil; So, Hong-Seob

    2015-11-27

    Although cisplatin is a widely used anticancer drug for the treatment of a variety of tumors, its use is critically limited because of adverse effects such as ototoxicity, nephrotoxicity, neuropathy, and gastrointestinal damage. Cisplatin treatment increases oxidative stress biomarkers in the small intestine, which may induce apoptosis of epithelial cells and thereby elicit damage to the small intestine. Nicotinamide adenine dinucleotide (NAD(+)) is a cofactor for various enzymes associated with cellular homeostasis. In the present study, we demonstrated that the hyper-activation of poly(ADP-ribose) polymerase-1 (PARP-1) is closely associated with the depletion of NAD(+) in the small intestine after cisplatin treatment, which results in downregulation of sirtuin1 (SIRT1) activity. Furthermore, a decrease in SIRT1 activity was found to play an important role in cisplatin-mediated small intestinal damage through nuclear factor (NF)-κB p65 activation, facilitated by its acetylation increase. However, use of dunnione as a strong substrate for the NADH:quinone oxidoreductase 1 (NQO1) enzyme led to an increase in intracellular NAD(+) levels and prevented the cisplatin-induced small intestinal damage correlating with the modulation of PARP-1, SIRT1, and NF-κB. These results suggest that direct modulation of cellular NAD(+) levels by pharmacological NQO1 substrates could be a promising therapeutic approach for protecting against cisplatin-induced small intestinal damage.

  16. Evidence for a Direct Effect of the NAD+ Precursor Acipimox on Muscle Mitochondrial Function in Humans

    PubMed Central

    van de Weijer, Tineke; Phielix, Esther; Bilet, Lena; Williams, Evan G.; Ropelle, Eduardo R.; Bierwagen, Alessandra; Livingstone, Roshan; Nowotny, Peter; Sparks, Lauren M.; Paglialunga, Sabina; Szendroedi, Julia; Havekes, Bas; Moullan, Norman; Pirinen, Eija; Hwang, Jong-Hee; Schrauwen-Hinderling, Vera B.; Hesselink, Matthijs K.C.; Auwerx, Johan

    2015-01-01

    Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD+) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD+ precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m2) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD+ levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD+ boosters can also directly affect skeletal muscle mitochondrial function in humans. PMID:25352640

  17. Properties of NAD (P) H azoreductase from alkaliphilic red bacteria Aquiflexum sp. DL6.

    PubMed

    Misal, Santosh A; Lingojwar, Devendra P; Gawai, Kachru R

    2013-12-01

    Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K m and V max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.

  18. Regulation of the intersubunit ammonia tunnel in Mycobacterium tuberculosis glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    Chuenchor, Watchalee; Doukov, Tzanko I.; Resto, Melissa; Chang, Andrew; Gerratana, Barbara

    2012-08-31

    Glutamine-dependent NAD{sup +} synthetase is an essential enzyme and a validated drug target in Mycobacterium tuberculosis (mtuNadE). It catalyses the ATP-dependent formation of NAD{sup +} from NaAD{sup +} (nicotinic acid-adenine dinucleotide) at the synthetase active site and glutamine hydrolysis at the glutaminase active site. An ammonia tunnel 40 {angstrom} (1 {angstrom} = 0.1 nm) long allows transfer of ammonia from one active site to the other. The enzyme displays stringent kinetic synergism; however, its regulatory mechanism is unclear. In the present paper, we report the structures of the inactive glutaminase C176A variant in an apo form and in three synthetase-ligand complexes with substrates (NaAD{sup +}/ATP), substrate analogue {l_brace}NaAD{sup +}/AMP-CPP (adenosine 5'-[{alpha},{beta}-methylene]triphosphate){r_brace} and intermediate analogues (NaAD{sup +}/AMP/PPi), as well as the structure of wild-type mtuNadE in a product complex (NAD{sup +}/AMP/PPi/glutamate). This series of structures provides snapshots of the ammonia tunnel during the catalytic cycle supported also by kinetics and mutagenesis studies. Three major constriction sites are observed in the tunnel: (i) at the entrance near the glutaminase active site; (ii) in the middle of the tunnel; and (iii) at the end near the synthetase active site. Variation in the number and radius of the tunnel constrictions is apparent in the crystal structures and is related to ligand binding at the synthetase domain. These results provide new insight into the regulation of ammonia transport in the intermolecular tunnel of mtuNadE.

  19. Unique ligation properties of eukaryotic NAD+-dependent DNA ligase from Melanoplus sanguinipes entomopoxvirus.

    PubMed

    Lu, Jing; Tong, Jie; Feng, Hong; Huang, Jianmin; Afonso, Claudio L; Rock, Dan L; Barany, Francis; Cao, Weiguo

    2004-09-01

    The eukaryotic Melanoplus sanguinipes entomopoxvirus (MsEPV) genome reveals a homologous sequence to eubacterial nicotinamide adenine dinucleotide (NAD(+))-dependent DNA ligases [J. Virol. 73 (1999) 533]. This 522-amino acid open reading frame (ORF) contains all conserved nucleotidyl transferase motifs but lacks the zinc finger motif and BRCT domain found in conventional eubacterial NAD(+) ligases. Nevertheless, cloned MsEPV ligase seals DNA nicks in a NAD(+)-dependent fashion, while adenosine 5'-monophosphate (ATP) cannot serve as an adenylation cofactor. The ligation activity of MsEPV ligase requires Mg(2+) or Mn(2+). MsEPV ligase seals sticky ends efficiently, but has little activity on 1-nucleotide gap or blunt-ended DNA substrates even in the presence of polyethylene glycol. In comparison, bacterial NAD(+)-dependent ligases seal blunt-ended DNA substrates in the presence of polyethylene glycol. MsEPV DNA ligase readily joins DNA nicks with mismatches at either side of the nick junction, except for mismatches at the nick junction containing an A base in the template strand (A/A, G/A, and C/A). MsEPV NAD(+)-dependent DNA ligase can join DNA probes on RNA templates, a unique property that distinguishes this enzyme from other conventional bacterial NAD(+) DNA ligases. T4 ATP-dependent DNA ligase shows no detectable mismatch ligation at the 3' side of the nick but substantial 5' T/G mismatch ligation on an RNA template. In contrast, MsEPV ligase joins mismatches at the 3' side of the nick more frequently than at the 5' side of the nick on an RNA template. The complementary specificities of these two enzymes suggest alternative primer design for genomic profiling approaches that use allele-specific detection directly from RNA transcripts.

  20. Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Aneba, Swida; Mateasik, Anton; Chorvat, Dusan; Comte, Blandine

    2013-06-01

    Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

  1. Environment Dictates Dependence on Mitochondrial Complex I for NAD+ and Aspartate Production and Determines Cancer Cell Sensitivity to Metformin.

    PubMed

    Gui, Dan Y; Sullivan, Lucas B; Luengo, Alba; Hosios, Aaron M; Bush, Lauren N; Gitego, Nadege; Davidson, Shawn M; Freinkman, Elizaveta; Thomas, Craig J; Vander Heiden, Matthew G

    2016-11-08

    Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism.

  2. The mechanism of RNA 5' capping with NAD+, NADH and desphospho-CoA

    SciTech Connect

    Bird, Jeremy G.; Zhang, Yu; Tian, Yuan; Panova, Natalya; Barvík, Ivan; Greene, Landon; Liu, Min; Buckley, Brian; Krásný, Libor; Lee, Jeehiun K.; Kaplan, Craig D.; Ebright, Richard H.; Nickels, Bryce E.

    2016-07-06

    The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.

  3. 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.

  4. Nicotinamide improves glucose metabolism and affects the hepatic NAD-sirtuin pathway in a rodent model of obesity and type 2 diabetes.

    PubMed

    Yang, Soo Jin; Choi, Jung Mook; Kim, Lisa; Park, Se Eun; Rhee, Eun Jung; Lee, Won Young; Oh, Ki Won; Park, Sung Woo; Park, Cheol-Young

    2014-01-01

    Nicotinic acid (NA) and nicotinamide (NAM) are major forms of niacin and exert their physiological functions as precursors of nicotinamide adenine dinucleotide (NAD). Sirtuins, which are NAD-dependent deacetylases, regulate glucose and lipid metabolism and are implicated in the pathophysiology of aging, diabetes, and hepatic steatosis. The aim of this study was to investigate the effects of two NAD donors, NA and NAM, on glucose metabolism and the hepatic NAD-sirtuin pathway. The effects were investigated in OLETF rats, a rodent model of obesity and type 2 diabetes. OLETF rats were divided into five groups: (1) high fat (HF) diet, (2) HF diet and 10 mg NA/kg body weight (BW)/day (NA 10), (3) HF diet and 100 mg NA/kg BW/day (NA 100), (4) HF diet and 10 mg NAM/kg BW/day (NAM 10), and (5) HF diet and 100 mg NAM/kg BW/day (NAM 100). NA and NAM were delivered via drinking water for four weeks. NAM 100 treatment affected glucose control significantly, as shown by lower levels of accumulative area under the curve during oral glucose tolerance test, serum fasting glucose, serum fasting insulin, and homeostasis model assessment of insulin resistance, and higher levels of serum adiponectin. With regard to NAD-sirtuin pathway, intracellular nicotinamide phosphoribosyltransferase, NAD, the NAD/NADH ratio, Sirt1, 2, 3, and 6 mRNA expressions, and Sirt1 activity all increased in livers of NAM 100-treated rats. These alterations were accompanied by the increased levels of proliferator-activated receptor gamma, coactivator 1 alpha and mitochondrial DNA. The effect of NA treatment was less evident than that of NAM 100. These results demonstrate that NAM is more effective than NA on the regulation of glucose metabolism and the NAD-sirtuin pathway, which may relate to the altered mitochondrial biogenesis.

  5. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein.

    PubMed Central

    Huang, Shaohui; Heikal, Ahmed A; Webb, Watt W

    2002-01-01

    Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique. PMID:11964266

  6. The mechanism of RNA 5′ capping with NAD+, NADH and desphospho-CoA.

    PubMed

    Bird, Jeremy G; Zhang, Yu; Tian, Yuan; Panova, Natalya; Barvík, Ivan; Greene, Landon; Liu, Min; Buckley, Brian; Krásný, Libor; Lee, Jeehiun K; Kaplan, Craig D; Ebright, Richard H; Nickels, Bryce E

    2016-07-21

    The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of ‘epitranscriptomic’ gene regulation. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate ‘cap’ in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated ‘ab initio capping’ may occur in all organisms.

  7. Differential release of β-NAD+ and ATP upon activation of enteric motor neurons in primate and murine colons

    PubMed Central

    DURNIN, LEONIE; SANDERS, KENTON M.; MUTAFOVA-YAMBOLIEVA, VIOLETA N.

    2012-01-01

    Background The purinergic component of enteric inhibitory neurotransmission is important for normal motility in the gastrointestinal (GI) tract. Controversies exist about the purine(s) responsible for inhibitory responses in GI muscles: adenosine 5′-triphosphate (ATP) has been assumed to be the purinergic neurotransmitter released from enteric inhibitory motor neurons, however recent studies demonstrate that β-nicotinamide adenine dinucleotide (β-NAD+) and ADP-ribose mimic the inhibitory neurotransmitter better than ATP in primate and murine colons. The study was designed to clarify the sources of purines in colons of Cynomolgus monkeys and C57BL/6 mice. Methods HPLC with fluorescence detection was used to analyze purines released by stimulation of nicotinic acetylcholine receptors (nAChR) and serotonergic 5-HT3 receptors (5-HT3R), known to be present on cell bodies and dendrites of neurons within the myenteric plexus. Key Results nAChR or 5-HT3R agonists increased overflow of ATP and β-NAD+ from tunica muscularis of monkey and murine colon. The agonists did not release purines from circular muscles of monkey colon lacking myenteric ganglia. Agonist-evoked overflow of β-NAD+, but not ATP, was inhibited by tetrodotoxin (0.5 μM) or ω-conotoxin GVIA (50 nM), suggesting that β-NAD+ release requires nerve action potentials and junctional mechanisms known to be critical for neurotransmission. ATP was likely released from nerve cell bodies in myenteric ganglia and not from nerve terminals of motor neurons. Conclusions & Inferences These results support the conclusion that ATP is not a motor neurotransmitter in the colon and are consistent with the hypothesis that β-NAD+, or its metabolites, serve as the purinergic inhibitory neurotransmitter. PMID:23279315

  8. NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms.

    PubMed

    Love, Nick R; Pollak, Nadine; Dölle, Christian; Niere, Marc; Chen, Yaoyao; Oliveri, Paola; Amaya, Enrique; Patel, Sandip; Ziegler, Mathias

    2015-02-03

    Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD(+) kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca(2+)/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

  9. Niacin-mediated Gene Expression and Role of NiaR as a Transcriptional Repressor of niaX, nadC, and pnuC in Streptococcus pneumoniae

    PubMed Central

    Afzal, Muhammad; Kuipers, Oscar P.; Shafeeq, Sulman

    2017-01-01

    NAD (Nicotinamide Adenine Dinucleotide) biosynthesis is vital for bacterial physiology and plays an important role in cellular metabolism. A naturally occurring vitamin B complex, niacin (nicotinic acid), is a precursor of coenzymes NAD and NADP. Here, we study the impact of niacin on global gene expression of Streptococcus pneumoniae D39 and elucidate the role of NiaR as a transcriptional regulator of niaX, nadC, and pnuC. Transcriptome comparison of the D39 wild-type grown in chemically defined medium (CDM) with 0 to 10 mM niacin revealed elevated expression of various genes, including niaX, nadC, pnuC, fba, rex, gapN, pncB, gap, adhE, and adhB2 that are putatively involved in the transport and utilization of niacin. Niacin-dependent expression of these genes is confirmed by promoter lacZ-fusion studies. Moreover, the role of transcriptional regulator NiaR in the regulation of these genes is explored by DNA microarray analysis. Our transcriptomic comparison of D39 ΔniaR to D39 wild-type revealed that the transcriptional regulator NiaR acts as a transcriptional repressor of niaX, pnuC, and nadC. NiaR-dependent regulation of niaX, nadC, and pnuC is further confirmed by promoter lacZ-fusion studies. The putative operator site of NiaR (5′-TACWRGTGTMTWKACASYTRWAW-3′) in the promoter regions of niaX, nadC, and pnuC is predicted and further confirmed by promoter mutational experiments. PMID:28337428

  10. Antidepressants may lead to a decrease in niacin and NAD in patients with poor dietary intake.

    PubMed

    Viljoen, Margaretha; Swanepoel, Annie; Bipath, Priyesh

    2015-03-01

    The term niacin is the generic name for the two compounds nicotinic acid and nicotinamide, the major dietary precursors for two important coenzymes, nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP. Niacin is important for the maintenance of cellular integrity and energy production and is involved in more than 500 intracellular reactions. Deficiencies of niacin may contribute to neuropsychiatric and neurodegenerative disorders. Patients who develop nutritional deficiencies as a result of poor dietary intake, especially inadequate intake of proteins and vitamins, could potentially suffer from niacin deficiency and NAD depletion. However, de novo synthesis of niacin and NAD in the kynurenine pathway of tryptophan metabolism may compensate for impaired dietary intake. The rate of synthesis of NAD and niacin from tryptophan oxidation depends on the induction of the enzyme indoleamine 2,3-dioxygenase (IDO) by pro-inflammatory cytokines such as interferon-gamma. Niacin synthesis is not limited by a decrease in tryptophan and excessive IDO activity may therefore lead to a decline in tryptophan levels. Antidepressants have an anti-inflammatory effect, including reduction of interferon-gamma and therefore inhibition of IDO, the rate-limiting enzyme of the kynurenine pathway. In theory, this could account for increased serotonin as more tryptophan becomes available for serotonin synthesis. However, the downside may be that less NAD and niacin are synthesised downstream, which could exacerbate common psychiatric problems. It is our hypothesis that patients with poor dietary intake, who are treated with antidepressants, are at risk of developing niacin/NAD deficiency with possible development of associated neuropsychiatric symptoms. We therefore propose that niacin supplementation be considered in patients with inadequate diets who are treated with antidepressants. We believe that if this does not happen, a subclinical niacin deficiency may result

  11. The NAD(+) salvage pathway modulates cancer cell viability via p73.

    PubMed

    Sharif, T; Ahn, D-G; Liu, R-Z; Pringle, E; Martell, E; Dai, C; Nunokawa, A; Kwak, M; Clements, D; Murphy, J P; Dean, C; Marcato, P; McCormick, C; Godbout, R; Gujar, S A; Lee, P W K

    2016-04-01

    The involvement of the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway in cancer cell survival is poorly understood. Here we show that the NAD(+) salvage pathway modulates cancer cell survival through the rarely mutated tumour suppressor p73. Our data show that pharmacological inhibition or knockdown of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme in the NAD(+) salvage pathway, enhances autophagy and decreases survival of cancer cells in a p53-independent manner. Such NAMPT inhibition stabilizes p73 independently of p53 through increased acetylation and decreased ubiquitination, resulting in enhanced autophagy and cell death. These effects of NAMPT inhibition can be effectively reversed using nicotinamide mononucleotide (NMN), the enzymatic product of NAMPT. Similarly, knockdown of p73 also decreases NAMPT inhibition-induced autophagy and cell death, whereas overexpression of p73 alone enhances these effects. We show that the breast cancer cell lines (MCF-7, MDA-MB-231 and MDA-MB-468) harbour significantly higher levels of NAMPT and lower levels of p73 than does the normal cell line (MCF-10A), and that NAMPT inhibition is cytotoxic exclusively to the cancer cells. Furthermore, data from 176 breast cancer patients demonstrate that higher levels of NAMPT and lower levels of p73 correlate with poorer patient survival, and that high-grade tumours have significantly higher NAMPT/p73 mRNA ratios. Therefore, the inverse relationship between NAMPT and p73 demonstrable in vitro is also reflected from the clinical data. Taken together, our studies reveal a new NAMPT-p73 nexus that likely has important implications for cancer diagnosis, prognosis and treatment.

  12. Predicted Role of NAD Utilization in the Control of Circadian Rhythms during DNA Damage Response

    PubMed Central

    Luna, Augustin; McFadden, Geoffrey B.; Aladjem, Mirit I.; Kohn, Kurt W.

    2015-01-01

    The circadian clock is a set of regulatory steps that oscillate with a period of approximately 24 hours influencing many biological processes. These oscillations are robust to external stresses, and in the case of genotoxic stress (i.e. DNA damage), the circadian clock responds through phase shifting with primarily phase advancements. The effect of DNA damage on the circadian clock and the mechanism through which this effect operates remains to be thoroughly investigated. Here we build an in silico model to examine damage-induced circadian phase shifts by investigating a possible mechanism linking circadian rhythms to metabolism. The proposed model involves two DNA damage response proteins, SIRT1 and PARP1, that are each consumers of nicotinamide adenine dinucleotide (NAD), a metabolite involved in oxidation-reduction reactions and in ATP synthesis. This model builds on two key findings: 1) that SIRT1 (a protein deacetylase) is involved in both the positive (i.e. transcriptional activation) and negative (i.e. transcriptional repression) arms of the circadian regulation and 2) that PARP1 is a major consumer of NAD during the DNA damage response. In our simulations, we observe that increased PARP1 activity may be able to trigger SIRT1-induced circadian phase advancements by decreasing SIRT1 activity through competition for NAD supplies. We show how this competitive inhibition may operate through protein acetylation in conjunction with phosphorylation, consistent with reported observations. These findings suggest a possible mechanism through which multiple perturbations, each dominant during different points of the circadian cycle, may result in the phase advancement of the circadian clock seen during DNA damage. PMID:26020938

  13. Head to Head Comparison of Short-Term Treatment with the NAD+ Precursor Nicotinamide Mononucleotide (NMN) and 6 Weeks of Exercise in Obese Female Mice

    PubMed Central

    Uddin, Golam M.; Youngson, Neil A.; Sinclair, David A.; Morris, Margaret J.

    2016-01-01

    Obesity is well known to be a major cause of several chronic metabolic diseases, which can be partially counteracted by exercise. This is due, in part, to an upregulation of mitochondrial activity through increased nicotinamide adenine dinucleotide (NAD+). Recent studies have shown that NAD+ levels can be increased by using the NAD+ precursor, nicotinamide mononucleotide (NMN) leading to the suggestion that NMN could be a useful intervention in diet related metabolic disorders. In this study we compared the metabolic, and especially mitochondrial-associated, effects of exercise and NMN in ameliorating the consequences of high-fat diet (HFD) induced obesity in mice. Sixty female 5 week old C57BL6/J mice were allocated across five groups: Chow sedentary: CS; Chow exercise: CEX; HFD sedentary: HS; HFD NMN: HNMN; HFD exercise: HEX (12/group). After 6 weeks of diet, exercise groups underwent treadmill exercise (15 m/min for 45 min), 6 days per week for 6 weeks. NMN or vehicle (500 mg/kg body weight) was injected (i.p.) daily for the last 17 days. No significant alteration in body weight was observed in response to exercise or NMN. The HFD significantly altered adiposity, glucose tolerance, plasma insulin, NADH levels and citrate synthase activity in muscle and liver. HEX and HNMN groups both showed significantly improved glucose tolerance compared to the HS group. NAD+ levels were increased significantly both in muscle and liver by NMN whereas exercise increased NAD+ only in muscle. Both NMN and exercise ameliorated the HFD-induced reduction in liver citrate synthase activity. However, exercise, but not NMN, ameliorated citrate synthase activity in muscle. Overall these data suggest that while exercise and NMN-supplementation can induce similar reversal of the glucose intolerance induced by obesity, they are associated with tissue-specific effects and differential alterations to mitochondrial function in muscle and liver. PMID:27594836

  14. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    PubMed

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity.

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

    PubMed

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

    1995-01-01

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

  16. NAD(+) metabolism: Bioenergetics, signaling and manipulation for therapy.

    PubMed

    Yang, Yue; Sauve, Anthony A

    2016-12-01

    We survey the historical development of scientific knowledge surrounding Vitamin B3, and describe the active metabolite forms of Vitamin B3, the pyridine dinucleotides NAD(+) and NADP(+) which are essential to cellular processes of energy metabolism, cell protection and biosynthesis. The study of NAD(+) has become reinvigorated by new understandings that dynamics within NAD(+) metabolism trigger major signaling processes coupled to effectors (sirtuins, PARPs, and CD38) that reprogram cellular metabolism using NAD(+) as an effector substrate. Cellular adaptations include stimulation of mitochondrial biogenesis, a process fundamental to adjusting cellular and tissue physiology to reduced nutrient availability and/or increased energy demand. Several mammalian metabolic pathways converge to NAD(+), including tryptophan-derived de novo pathways, nicotinamide salvage pathways, nicotinic acid salvage and nucleoside salvage pathways incorporating nicotinamide riboside and nicotinic acid riboside. Key discoveries highlight a therapeutic potential for targeting NAD(+) biosynthetic pathways for treatment of human diseases. A recent emergence of understanding that NAD(+) homeostasis is vulnerable to aging and disease processes has stimulated testing to determine if replenishment or augmentation of cellular or tissue NAD(+) can have ameliorative effects on aging or disease phenotypes. This experimental approach has provided several proofs of concept successes demonstrating that replenishment or augmentation of NAD(+) concentrations can provide ameliorative or curative benefits. Thus NAD(+) metabolic pathways can provide key biomarkers and parameters for assessing and modulating organism health.

  17. 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.

  18. 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.

  19. Evolution of function in the "two dinucleotide binding domains" flavoproteins.

    PubMed

    Ojha, Sunil; Meng, Elaine C; Babbitt, Patricia C

    2007-07-01

    Structural and biochemical constraints force some segments of proteins to evolve more slowly than others, often allowing identification of conserved structural or sequence motifs that can be associated with substrate binding properties, chemical mechanisms, and molecular functions. We have assessed the functional and structural constraints imposed by cofactors on the evolution of new functions in a superfamily of flavoproteins characterized by two-dinucleotide binding domains, the "two dinucleotide binding domains" flavoproteins (tDBDF) superfamily. Although these enzymes catalyze many different types of oxidation/reduction reactions, each is initiated by a stereospecific hydride transfer reaction between two cofactors, a pyridine nucleotide and flavin adenine dinucleotide (FAD). Sequence and structural analysis of more than 1,600 members of the superfamily reveals new members and identifies details of the evolutionary connections among them. Our analysis shows that in all of the highly divergent families within the superfamily, these cofactors adopt a conserved configuration optimal for stereospecific hydride transfer that is stabilized by specific interactions with amino acids from several motifs distributed among both dinucleotide binding domains. The conservation of cofactor configuration in the active site restricts the pyridine nucleotide to interact with FAD from the re-side, limiting the flow of electrons from the re-side to the si-side. This directionality of electron flow constrains interactions with the different partner proteins of different families to occur on the same face of the cofactor binding domains. As a result, superimposing the structures of tDBDFs aligns not only these interacting proteins, but also their constituent electron acceptors, including heme and iron-sulfur clusters. Thus, not only are specific aspects of the cofactor-directed chemical mechanism conserved across the superfamily, the constraints they impose are manifested in the

  20. Giardia duodenalis GlSir2.2, homolog of SIRT1, is a nuclear-located and NAD(+)-dependent deacethylase.

    PubMed

    Wang, Yun-Hua; Zheng, Guo-Xia; Li, Ya-Jie

    2016-10-01

    Sir2 family proteins are highly conserved and catalyze Nicotinamide Adenine Dinucleotide (NAD(+))-dependent protein deacetylation reaction that regulates multiple cellular processes. Little is known about Sir2 family proteins in Giardia. In this research, Sir2 homologs of Giardia were Phylogenetically analyzed. GL50803_10707 (GlSIR2.2) showed strong homology to SIRT1 and was the only parasite SIRT1 homolog being reported to date. Recombinant GlSIR2.2 (rGlSIR2.2) was expressed and purified. The renaturied recombinant protein showed a typical NAD-dependent protein deacetylase activity that could be inhibited by nicotinamide, with IC50 of 4.47 mM rGlSIR2.2 displayed deacetylase activity under varied NAD(+), with Km, kcat and kcat/Km values of 31.71 μM, 1.4 × 10(-3) s(-1), and 4.42 × 10(-5) μM(-1) s(-1). Similarly, the steady-state kinetic parameters with varied ZMAL, yielded Km, kcat and kcat/Km values of 96.89 μM, 4.7 × 10(-3) s(-1), and 4.85 × 10(-5) μM(-1) s(-1). Anti-rGlSIR2.2 serum was used to probe subcellular localization of GlSIR2.2 and strong staining was found predominantly in the nucleus. So we demonstrated that GlSIR2.2 was a SIRT1-like, nuclear-located, NAD(+)-dependent deacetylase. This is the first report of deacetylase activity of Sir2 family protein in Giardia.

  1. Spectrally and time-resolved study of NAD(P)H autofluorescence in cardiac myocytes from human biopsies

    NASA Astrophysics Data System (ADS)

    Cheng, Y.; Chorvat, D., Jr.; Poirier, N.; Miró, J.; Dahdah, N.; Chorvatova, A.

    2007-09-01

    Rejection of transplanted hearts remains an important reason for death of transplanted children. Finding diagnostic tools for its detection can therefore improve the prognosis in this population of patients. Endomyocardial biopsy (EMB) by cardiac catheterization is currently accepted as the "gold standard" for the diagnosis of rejection. Here, we investigate new approach to monitor mitochondrial metabolic state of cardiac cells using spectrally-resolved autofluorescence lifetime detection of nicotinamide adenine dinucleotide (phosphate), or NAD(P)H, the principal electron donor in mitochondrial oxidative energy metabolism responsible for vital ATP supply of cardiomyocytes. NAD(P)H autofluorescence is long used for non-invasive fluorescent probing the metabolic state of the heart. In this contribution we report dynamic characteristics of NAD(P)H fluorescence decays in living human cardiomyocytes from EMB, following excitation by UV-pulsed laser diode and detection by spectrally-resolved time-correlated single photon counting. At least a 3-exponential decay model, with 0.5-0.7 ns, 1.9-2.4 ns and 9.0-15.0 ns lifetimes, is necessary to describe cardiomyocyte autofluorescence in human cells. When gathered data were compared to those recorded under same conditions in rats, autofluorescence in human hearts was found significantly lower in comparison to rat ones. Rotenone, the inhibitor of the Complex I of the respiratory chain, increased the fluorescence in human cardiac cells, making them more comparable to experimental rat model. These results suggest that human cardiac cells are more metabolically active than the rat ones in the same conditions. Presented work proposes a new tool for evaluation of oxidative metabolism changes in transplanted hearts.

  2. Malic enzyme: Tritium isotope effects with alternative dinucleotide substrates and divalent metal ions

    SciTech Connect

    Karsten, W.E.; Harris, B.G.; Cook, P.F. )

    1992-01-01

    The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion dependent oxidative decarboxylation of L-malate to yield pyruvate, carbon dioxide and NADH. Multiple isotope effect studies suggest a stepwise chemical mechanism with hydride transfer from L-malate to NAD occurring first to form oxalacetate, followed by decarboxylation. Utilizing L-malate-2-T, tritium V/K isotope effects have been determined for the hydride transfer step using a variety of alternative dinucleotide substrates and divalent metal ions. Combination of these data with deuterium isotope effects data and previously determined [sup 13]C isotope effects has allowed the calculation of intrinsic isotope effects for the malic enzyme catalyzed reaction. The identity of both the dinucleotide substrate and divalent metal ion has an effect of the size of the intrinsic isotope effect for hydride transfer.

  3. MNADK, a Long-Awaited Human Mitochondrion-Localized NAD Kinase.

    PubMed

    Zhang, Ren

    2015-08-01

    Nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP, play essential roles in numerous cellular processes in all organisms. NADP maintains a pool of its reducing equivalent, NADPH, which regenerates cellular oxidative defense systems to counteract oxidative damages. Mitochondria represent a major source of oxidative stress, because the majority of superoxide, a reactive oxygen species, is generated from the mitochondrial respiratory chain. Therefore, as universal electron carriers in cellular electron transfer reactions, the pyridine nucleotides are required by mitochondria for both antioxidant protection and biosynthetic pathways. The NAD kinase (NADK) is the sole NADP biosynthetic enzyme. Because NADP is membrane-impermeable, eukaryotes need compartment-specific NADKs for different organelles. Consistently, in both yeast and plants, three compartment-specific NADKs have been identified. In contrast, even though the first human NADK, a cytosolic one, was identified in 2001, the identity of a hypothesized mitochondrial NADK remained elusive, until a recent discovery that the uncharacterized human gene C5ORF33 encodes a mitochondrion-localized NADK, referred to as MNADK. Three groups have characterized MNADK functions based on distinct systems involving yeast, mouse, and human studies, from aspects of both in vitro and in vivo evidence. MNADK is a mitochondrial NADK that is enriched and nutritionally-regulated in mouse liver, and a MNADK-deficient patient exhibits symptoms characteristic of mitochondrial disease. The identification of MNADK provides a key clue to the mechanism involved in mitochondrial NADPH production and the maintenance of redox balance in mammalian cells. The roles of MNADK in physiological and pathological processes have yet to be discovered.

  4. Adenine formation without HCN.

    PubMed

    Merz, Kenneth M; Aguiar, Eduardo C; da Silva, Joao Bosco P

    2014-05-22

    From a historic point of view adenine was always presumed to be the product of HCN pentamerization. In this work a new mechanism for adenine synthesis in the gas phase without HCN is proposed. The concept of retrosynthetic analysis was employed to create a tautomer of adenine, which can be reached from previously observed interstellar molecules C3NH and HNCNH and its isomer H2NCN. MP2/6-311++G(2d,2p) calculations were performed to calculate the Gibbs free energy of the minimum and the transition state (TS) structures involved in the six step mechanism. This new mechanism requires a smaller number of steps, the reaction energy is twice as exergonic, and the rate determining TS is lower in energy than the corresponding ones proposed elsewhere in the literature.

  5. NadN and e (P4) are essential for utilization of NAD and nicotinamide mononucleotide but not nicotinamide riboside in Haemophilus influenzae.

    PubMed

    Kemmer, G; Reilly, T J; Schmidt-Brauns, J; Zlotnik, G W; Green, B A; Fiske, M J; Herbert, M; Kraiss, A; Schlör, S; Smith, A; Reidl, J

    2001-07-01

    Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5'-nucleotidase activity. The e (P4) protein is also shown to have NMN 5'-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.

  6. Enzymology of mammalian NAD metabolism in health and disease.

    PubMed

    Magni, Giulio; Orsomando, Giuseppe; Raffelli, Nadia; Ruggieri, Silverio

    2008-05-01

    Mounting evidence attests to the paramount importance of the non-redox NAD functions. Indeed, NAD homeostasis is related to the free radicals-mediated production of reactive oxygen species responsible for irreversible cellular damage in infectious disease, diabetes, inflammatory syndromes, neurodegeneration and cancer. Because the cellular redox status depends on both the absolute concentration of pyridine dinucleotides and their respective ratios of oxidized and reduced forms (i.e., NAD/NADH and NADP/NADPH), it is conceivable that an altered regulation of the synthesis and degradation of NAD impairs the cell redox state and likely contributes to the mechanisms underlying the pathogenesis of the above mentioned diseases. Taking into account the recent appearance in the literature of comprehensive reviews covering different aspects of the significance of NAD metabolism, with particular attention to the enzymes involved in NAD cleavage, this monograph includes the most recent results on NAD biosynthesis in mammals and humans. Due to recent findings on nicotinamide riboside as a nutrient, its inclusion under "niacins" is proposed. Here, the enzymes involved in the de novo and reutilization pathways are overviewed.

  7. Using Intrinsic Flavoprotein and NAD(P)H Imaging to Map Functional Circuitry in the Main Olfactory Bulb

    PubMed Central

    Puche, Adam C.; Munger, Steven D.

    2016-01-01

    Neurons exhibit strong coupling of electrochemical and metabolic activity. Increases in intrinsic fluorescence from either oxidized flavoproteins or reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] in the mitochondria have been used as an indicator of neuronal activity for the functional mapping of neural circuits. However, this technique has not been used to investigate the flow of olfactory information within the circuitry of the main olfactory bulb (MOB). We found that intrinsic flavoprotein fluorescence signals induced by electrical stimulation of single glomeruli displayed biphasic responses within both the glomerular (GL) and external plexiform layers (EPL) of the MOB. Pharmacological blockers of mitochondrial activity, voltage-gated Na+ channels, or ionotropic glutamate receptors abolished stimulus-dependent flavoprotein responses. Blockade of GABAA receptors enhanced the amplitude and spatiotemporal spread of the flavoprotein signals, indicating an important role for inhibitory neurotransmission in shaping the spread of neural activity in the MOB. Stimulus-dependent spread of fluorescence across the GL and EPL displayed a spatial distribution consistent with that of individual glomerular microcircuits mapped by neuroanatomic tract tracing. These findings demonstrated the feasibility of intrinsic fluorescence imaging in the olfactory systems and provided a new tool to examine the functional circuitry of the MOB. PMID:27902689

  8. Facile photoreduction of graphene oxide by an NAD(P)H model: Hantzsch 1,4-dihydropyridine.

    PubMed

    Zhang, Hui-Hui; Liu, Qiang; Feng, Ke; Chen, Bin; Tung, Chen-Ho; Wu, Li-Zhu

    2012-05-29

    To make "clean" reduced GO sheets in high quality and in large scale, a natural reduced nicotinamine adenine dinucleotide NAD(P)H model, Hantzsch 1,4-dihydropyridine (HEH), is used as a mild organic photoreductant in this work. Benefiting from the intense absorption of HEH in the range of 300-420 nm, the graphene oxide (GO) can be readily reduced by HEH under UV light irradiation (λ > 320 nm) to afford single or few-layer reduced graphene oxide at room temperature. Studies on reduction extent reveal that both irradiation time and concentration ratio of HEH to GO are important for effective reduction of GO under UV light. The as-prepared photochemically reduced graphene oxide (PRGO) dispersion is stable without the need for any polymeric or surfactant stabilizers. Simply by extraction treatment, the "clean" PRGO sheets can be obtained in large quantities, and its conductivity approaches to 4680 S·m(-1) that is the highest value reported by photochemical approaches so far.

  9. Alteration of hydrogen metabolism of ldh-deleted Enterobacter aerogenes by overexpression of NAD+-dependent formate dehydrogenase.

    PubMed

    Lu, Yuan; Zhao, Hongxin; Zhang, Chong; Lai, Qiheng; Wu, Xi; Xing, Xin-Hui

    2010-03-01

    The NAD+-dependent formate dehydrogenase FDH1 gene (fdh1), cloned from Candida boidinii, was expressed in the ldh-deleted mutant of Enterobacter aerogenes IAM1183 strain. The plasmid of pCom10 driven by the PalkB promoter was used to construct the fdh1 expression system and thus introduce a new dihydronicotinamide adenine dinucleotide (NADH) regeneration pathway from formate in the ldh-deleted mutant. The knockout of NADH-consuming lactate pathway affected the whole cellular metabolism, and the hydrogen yield increased by 11.4% compared with the wild strain. Expression of fdh1 in the ldh-deleted mutant caused lower final cell concentration and final pH after 16 h cultivation, and finally resulted in 86.8% of increase in hydrogen yield per mole consumed glucose. The analysis of cellular metabolites and estimated redox state balance in the fdhl-expressed strain showed that more excess of reducing power was formed by the rewired NADH regeneration pathway, changing the metabolic distribution and promoting the hydrogen production.

  10. Regulation of Brain Glucose Metabolic Patterns by Protein Phosphorlyation and Drug Therapy

    DTIC Science & Technology

    2007-03-30

    either nicotinamide adenine dinucleotide phosphate (NADPH) or ribose. Both pathways are anaerobic, yet unlike glycolysis the pentose pathway shunt does...molecule of glucose to two molecules of pyruvate, generate two molecules of ATP, and reduce two molecules of nicotinamide adenine dinucleotide (NADH...dichloroactetate; LDH, lactate dehydrogenase; PDC, pyruvate dehydrogenase complex; NAD(P)H, nicotinamide adenine dinucleotide (phosphate); pyruvate dehydrogenase

  11. 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.

  12. 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.

  13. Dinucleotide circular codes and bijective transformations.

    PubMed

    Fimmel, Elena; Giannerini, Simone; Gonzalez, Diego Luis; Strüngmann, Lutz

    2015-12-07

    The presence of circular codes in mRNA coding sequences is postulated to be involved in informational mechanisms aimed at detecting and maintaining the normal reading frame during protein synthesis. Most of the recent research is focused on trinucleotide circular codes. However, also dinucleotide circular codes are important since dinucleotides are ubiquitous in genomes and associated to important biological functions. In this work we adopt the group theoretic approach used for trinucleotide codes in Fimmel et al. (2015) to study dinucleotide circular codes and highlight their symmetry properties. Moreover, we characterize such codes in terms of n-circularity and provide a graph representation that allows to visualize them geometrically. The results establish a theoretical framework for the study of the biological implications of dinucleotide circular codes in genomic sequences.

  14. Repertoires of the nucleosome-positioning dinucleotides.

    PubMed

    Bettecken, Thomas; Trifonov, Edward N

    2009-11-02

    It is generally accepted that the organization of eukaryotic DNA into chromatin is strongly governed by a code inherent in the genomic DNA sequence. This code, as well as other codes, is superposed on the triplets coding for amino acids. The history of the chromatin code started three decades ago with the discovery of the periodic appearance of certain dinucleotides, with AA/TT and RR/YY giving the strongest signals, all with a period of 10.4 bases. Every base-pair stack in the DNA duplex has specific deformation properties, thus favoring DNA bending in a specific direction. The appearance of the corresponding dinucleotide at the distance 10.4 xn bases will facilitate DNA bending in that direction, which corresponds to the minimum energy of DNA folding in the nucleosome. We have analyzed the periodic appearances of all 16 dinucleotides in the genomes of thirteen different eukaryotic organisms. Our data show that a large variety of dinucleotides (if not all) are, apparently, contributing to the nucleosome positioning code. The choice of the periodical dinucleotides differs considerably from one organism to another. Among other 10.4 base periodicities, a strong and very regular 10.4 base signal was observed for CG dinucleotides in the genome of the honey bee A. mellifera. Also, the dinucleotide CG appears as the only periodical component in the human genome. This observation seems especially relevant since CpG methylation is well known to modulate chromatin packing and regularity. Thus, the selection of the dinucleotides contributing to the chromatin code is species specific, and may differ from region to region, depending on the sequence context.

  15. Maximal dinucleotide comma-free codes.

    PubMed

    Fimmel, Elena; Strüngmann, Lutz

    2016-01-21

    The problem of retrieval and maintenance of the correct reading frame plays a significant role in RNA transcription. Circular codes, and especially comma-free codes, can help to understand the underlying mechanisms of error-detection in this process. In recent years much attention has been paid to the investigation of trinucleotide circular codes (see, for instance, Fimmel et al., 2014; Fimmel and Strüngmann, 2015a; Michel and Pirillo, 2012; Michel et al., 2012, 2008), while dinucleotide codes had been touched on only marginally, even though dinucleotides are associated to important biological functions. Recently, all maximal dinucleotide circular codes were classified (Fimmel et al., 2015; Michel and Pirillo, 2013). The present paper studies maximal dinucleotide comma-free codes and their close connection to maximal dinucleotide circular codes. We give a construction principle for such codes and provide a graphical representation that allows them to be visualized geometrically. Moreover, we compare the results for dinucleotide codes with the corresponding situation for trinucleotide maximal self-complementary C(3)-codes. Finally, the results obtained are discussed with respect to Crick׳s hypothesis about frame-shift-detecting codes without commas.

  16. Adenine phosphoribosyltransferase deficiency.

    PubMed

    Bollée, Guillaume; Harambat, Jérôme; Bensman, Albert; Knebelmann, Bertrand; Daudon, Michel; Ceballos-Picot, Irène

    2012-09-01

    Complete adenine phosphoribosyltransferase (APRT) deficiency is a rare inherited metabolic disorder that leads to the formation and hyperexcretion of 2,8-dihydroxyadenine (DHA) into urine. The low solubility of DHA results in precipitation of this compound and the formation of urinary crystals and stones. The disease can present as recurrent urolithiasis or nephropathy secondary to crystal precipitation into renal parenchyma (DHA nephropathy). The diagnostic tools available-including stone analysis, crystalluria, and APRT activity measurement-make the diagnosis easy to confirm when APRT deficiency is suspected. However, the disease can present at any age, and the variability of symptoms can present a diagnostic challenge to many physicians. The early recognition and treatment of APRT deficiency are of crucial importance for preventing irreversible loss of renal function, which still occurs in a non-negligible proportion of cases. This review summarizes the genetic and metabolic mechanisms underlying stone formation and renal disease, along with the diagnosis and management of APRT deficiency.

  17. The potential regulatory roles of NAD(+) and its metabolism in autophagy.

    PubMed

    Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

    2016-04-01

    (Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases.

  18. 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

  19. Grafted Azure A modified electrodes as disposable β-nicotinamide adenine dinucleotide sensors.

    PubMed

    Revenga-Parra, M; Gómez-Anquela, C; García-Mendiola, T; Gonzalez, E; Pariente, F; Lorenzo, E

    2012-10-17

    We report the in situ generation of aryl diazonium cations of Azure A, a redox-active phenothiazine dye, by reaction between the corresponding aromatic aminophenyl group and sodium nitrite in 0.1 M HCl. The subsequent electrochemical reduction of these dye diazonium salts gives rise to conductive electrografted films onto screen-printed carbon (SPC) electrodes. The resulting Azure A films have a very stable and reversible electrochemical response and exhibit potent and persistent electrocatalytic behavior toward NADH oxidation. We have optimized the electrografting conditions in order to obtain SPC modified electrodes with high and stable electrocatalytic response. The kinetic of the reaction between the NADH and the redox active centers in the Azure A film has been characterized using cyclic voltammetry and single step chronoamperometry. The catalytic currents were proportional to the concentration of NADH giving rise to linear calibration plots up to a concentration of 0.5 mM with a detection limit of 0.57±0.03 μM and a sensitivity of 9.48 A mol cm(-2) μM(-1). The precision of chronoamperometric determinations was found to be 2.3% for five replicate determinations of 3.95 μM NADH. The great stability of such modified electrodes makes them ideal for their application in the development of biosensing platforms based on dehydrogenases.

  20. Discovery of bacterial NAD+-dependent DNA ligase inhibitors: optimization of antibacterial activity.

    PubMed

    Stokes, Suzanne S; Huynh, Hoan; Gowravaram, Madhusudhan; Albert, Robert; Cavero-Tomas, Marta; Chen, Brendan; Harang, Jenna; Loch, James T; Lu, Min; Mullen, George B; Zhao, Shannon; Liu, Ce-Feng; Mills, Scott D

    2011-08-01

    Optimization of adenosine analog inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. Antibacterial activity against Streptococcus pneumoniae and Staphylococcus aureus was improved by modification of the 2-position substituent on the adenine ring and 3'- and 5'-substituents on the ribose. Compounds with logD values 1.5-2.5 maximized potency and maintained drug-like physical properties.

  1. Spectral decomposition of NAD(P)H fluorescence components recorded by multi-wavelength fluorescence lifetime spectroscopy in living cardiac cells

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Mateasik, Anton; Chorvat, Dusan, Jr.

    2013-12-01

    We report a novel analytical approach to identify individual components of a cell’s endogenous fluorescence, recorded by spectrally-resolved time-correlated single photon counting (TCSPC). Time-resolved area-normalized emission spectroscopy (TRANES) and principal component analysis (PCA) were applied to estimate the number of spectral components after metabolic modulation of cardiac cells following excitation with a 375 nm picosecond laser. Linear unmixing of TCSPC data spectrally decomposed individual components in living cells, while using characteristics of endogenously fluorescing molecules in solvents as a reference spectral database. Our data demonstrate the presence of three individual components, corresponding to the nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in organic and inorganic solvents and to the residual flavoprotein fluorescence. The presented analytical approach offers a new alternative for the spectral separation of multi-wavelength fluorescence lifetime spectroscopy data to the conventional analysis, and opens a new possibility for the use of pattern recognition for fast resolution of components in 2D fluorescence lifetime microscopy images.

  2. Solution conformation of 2-aminopurine dinucleotide determined by ultraviolet two-dimensional fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

    We have observed the conformation-dependent electronic coupling between the monomeric subunits of a dinucleotide of 2-aminopurine (2-AP), a fluorescent analogue 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 ± 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.

  3. Cloning and Expression of ntnD, Encoding a Novel NAD(P)+-Independent 4-Nitrobenzyl Alcohol Dehydrogenase from Pseudomonas sp. Strain TW3

    PubMed Central

    James, Keith D.; Hughes, Michelle A.; Williams, Peter A.

    2000-01-01

    Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)+ independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)+-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization. PMID:10809692

  4. 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.

  5. Protein modification by adenine propenal.

    PubMed

    Shuck, Sarah C; Wauchope, Orrette R; Rose, Kristie L; Kingsley, Philip J; Rouzer, Carol A; Shell, Steven M; Sugitani, Norie; Chazin, Walter J; Zagol-Ikapitte, Irene; Boutaud, Olivier; Oates, John A; Galligan, James J; Beavers, William N; Marnett, Lawrence J

    2014-10-20

    Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. N(ε)-Oxopropenyllysine, a lysine-lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is N(ε)-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA's ability to bind to a DNA substrate.

  6. Trypanosoma brucei adenine-phosphoribosyltransferases mediate adenine salvage and aminopurinol susceptibility but not adenine toxicity.

    PubMed

    Lüscher, Alexandra; Lamprea-Burgunder, Estelle; Graf, Fabrice E; de Koning, Harry P; Mäser, Pascal

    2014-04-01

    African trypanosomes, like all obligate parasitic protozoa, cannot synthesize purines de novo and import purines from their hosts to build nucleic acids. The purine salvage pathways of Trypanosoma brucei being redundant, none of the involved enzymes is likely to be essential. Nevertheless they can be of pharmacological interest due to their role in activation of purine nucleobase or nucleoside analogues, which only become toxic when converted to nucleotides. Aminopurine antimetabolites, in particular, are potent trypanocides and even adenine itself is toxic to trypanosomes at elevated concentrations. Here we report on the T. brucei adenine phosphoribosyltransferases TbAPRT1 and TbAPRT2, encoded by the two genes Tb927.7.1780 and Tb927.7.1790, located in tandem on chromosome seven. The duplication is syntenic in all available Trypanosoma genomes but not in Leishmania. While TbAPRT1 is cytosolic, TbAPRT2 possesses a glycosomal targeting signal and co-localizes with the glycosomal marker aldolase. Interestingly, the distribution of glycosomal targeting signals among trypanosomatid adenine phosphoribosyltransferases is not consistent with their phylogeny, indicating that the acquisition of adenine salvage to the glycosome happened after the radiation of Trypanosoma. Double null mutant T. brucei Δtbaprt1,2 exhibited no growth phenotype but no longer incorporated exogenous adenine into the nucleotide pool. This, however, did not reduce their sensitivity to adenine. The Δtbaprt1,2 trypanosomes were resistant to the adenine isomer aminopurinol, indicating that it is activated by phosphoribosyl transfer. Aminopurinol was about 1000-fold more toxic to bloodstream-form T. brucei than the corresponding hypoxanthine isomer allopurinol. Aminopurinol uptake was not dependent on the aminopurine permease P2 that has been implicated in drug resistance.

  7. Quantum-chemical study of interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-nucleobases.

    PubMed

    Mikulski, Damian; Szeląg, Małgorzata; Molski, Marcin

    2011-12-01

    Trans-resveratrol, a natural phytoalexin present in red wine and grapes, has gained considerable attention because of its antiproliferative, chemopreventive and proapoptotic activity against human cancer cells. The accurate quantum-chemical computations based on the density functional theory (DFT) and ab initio second-order Møller-Plesset perturbation method (MP2) have been performed for the first time to study interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-derived nitrogenous bases: adenine, guanine, cytosine and thymine in vacuum and water medium. This compound is found to show high affinity to nitrogenous bases and guanine-thymine dinucleotide. The electrostatic interactions from intermolecular hydrogen bonding increase the stability of complexes studied. In particular, significantly strong hydrogen bonds between 4'-H atom of trans-resveratrol and imidazole nitrogen as well as carbonyl oxygen atoms of nucleobases studied stabilize these systems. The stabilization energies computed reveal that the negatively charged trans-resveratrol-dinucleotide complex is more energetically stable in water medium than in vacuum. MP2 method gives more reliable and significantly high values of stabilization energy of trans-resveratrol-dinucleotide, trans-resveratrol-guanine and trans-resveratrol-thymine complexes than B3LYP exchange-correlation functional because it takes into account London dispersion energy. According to the results, in the presence of trans-resveratrol the 3'-5' phosphodiester bond in dinucleotide can be cleaved and the proton from 4'-OH group of trans-resveratrol migrates to the 3'-O atom of dinucleotide. It is concluded that trans-resveratrol is able to break the DNA strand. Hence, the findings obtained help understand antiproliferative and anticancer properties of this polyphenol.

  8. 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.

  9. Was adenine the first purine?

    NASA Technical Reports Server (NTRS)

    Schwartz, Alan W.; Bakker, C. G.

    1989-01-01

    Oligomerization of HCN (1 molar) in the presence of added formaldehyde (0.5 molar) produced an order of magnitude more 8-hydroxymethyladenine than adenine or any other biologically significant purine. This result suggests that on the prebiotic earth, nucleoside analogs may have been synthesized directly in more complex mixtures of HCN with other aldehydes.

  10. Base-modified NAD and AMP derivatives and their activity against bacterial DNA ligases.

    PubMed

    Pergolizzi, Giulia; Cominetti, Marco M D; Butt, Julea N; Field, Robert A; Bowater, Richard P; Wagner, Gerd K

    2015-06-14

    We report the chemical synthesis and conformational analysis of a collection of 2-, 6- and 8-substituted derivatives of β-NAD(+) and AMP, and their biochemical evaluation against NAD(+)-dependent DNA ligases from Escherichia coli and Mycobacterium tuberculosis. Bacterial DNA ligases are validated anti-microbial targets, and new strategies for their inhibition are therefore of considerable scientific and practical interest. Our study includes several pairs of β-NAD(+) and AMP derivatives with the same substitution pattern at the adenine base. This has enabled the first direct comparison of co-substrate and inhibitor behaviour against bacterial DNA ligases. Our results suggest that an additional substituent in position 6 or 8 of the adenine base in β-NAD(+) is detrimental for activity as either co-substrate or inhibitor. In contrast, substituents in position 2 are not only tolerated, but appear to give rise to a new mode of inhibition, which targets the conformational changes these DNA ligases undergo during catalysis. Using a molecular modelling approach, we highlight that these findings have important implications for our understanding of ligase mechanism and inhibition, and may provide a promising starting point for the rational design of a new class of inhibitors against NAD(+)-dependent DNA ligases.

  11. 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

  12. Onset of chiral adenine surface growth.

    PubMed

    Capitán, María Jose; Álvarez, Jesús; Wang, Yang; Otero, Roberto; Alcamí, Manuel; Martín, Fernando; Miranda, Rodolfo

    2013-10-07

    The structure and stability of adenine crystals and thin layers has been studied by using scanning tunneling microscopy, X-ray diffraction, and density functional theory calculations. We have found that adenine crystals can be grown in two phases that are energetically quasi-degenerate, the structure of which can be described as a pile-up of 2D adenine planes. In each plane, the structure can be described as an aggregation of adenine dimers. Under certain conditions, kinetic effects can favor the growth of the less stable phase. These results have been used to understand the growth of adenine thin films on gold under ultra-high vacuum conditions. We have found that the grown phase corresponds to the α-phase, which is composed of stacked prochiral planes. In this way, the adenine nanocrystals exhibit a surface that is enantiopure. These results could open new insight into the applications of adenine in biological, medical, and enantioselective or pharmaceutical fields.

  13. Supramolecular polymer formation by cyclic dinucleotides and intercalators affects dinucleotide enzymatic processing

    PubMed Central

    Nakayama, Shizuka; Zhou, Jie; Zheng, Yue; Szmacinski, Henryk; Sintim, Herman O

    2016-01-01

    Background: Cyclic dinucleotides form supramolecular aggregates with intercalators, and this property could be utilized in nanotechnology and medicine. Methods & results: Atomic force microscopy and electrophoretic mobility shift assays were used to show that cyclic diguanylic acid (c-di-GMP) forms G-wires in the presence of intercalators. The average fluorescence lifetime of thiazole orange, when bound to c-di-GMP was greater than when bound to DNA G-quadruplexes or dsDNA. The stability of c-di-GMP supramolecular polymers is dependent on both the nature of the cation present and the intercalator. C-di-GMP or cyclic diadenylic acid/intercalator complexes are more resistant to cleavage by YybT, a phosphodiesterase, than the uncomplexed nucleotides. Conclusion: Cleavage of bacterial cyclic dinucleotides could be slowed down via complexation with small molecules and that this could be utilized for diverse applications in nanotechnology and medicine. PMID:28031943

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

    PubMed

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

    2013-02-01

    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.

  15. Boosting NAD to spare hearing.

    PubMed

    Brenner, Charles

    2014-12-02

    Ex vivo experiments have strangely shown that inhibition or stimulation of NAD metabolism can be neuroprotective. In this issue of Cell Metabolism, Brown et al. (2014) demonstrate that cochlear NAD is diminished by deafening noise but protected by nicotinamide riboside or WldS mutation. Hearing protection by nicotinamide riboside depends on Sirt3.

  16. Bioorganometallic chemistry: biocatalytic oxidation reactions with biomimetic nad+/nadh co-factors and [cp*rh(bpy)h]+ for selective organic synthesis

    SciTech Connect

    Lutz, Jochen; Hollman, Frank; Ho, The Vinh; Schnyder, Adrian; Fish, Richard H.; Schmid, Andreas

    2004-03-09

    The biocatalytic, regioselective hydroxylation of 2-hydroxybiphenyl to the corresponding catechol was accomplished utilizing the monooxygenase 2-hydroxybiphenyl 3-monooxygenase (HbpA). The necessary natural nicotinamide adenine dinucleotide (NAD{sup +}) co-factor for this biocatalytic process was replaced by a biomimetic co-factor, N-benzylnicotinamide bromide, 1a. The interaction between the flavin (FAD) containing HbpA enzyme and the corresponding biomimetic NADH compound, N-benzyl-1,4-dihdronicotinamide, 1b, for hydride transfers, was shown to readily occur. The in situ recycling of the reduced NADH biomimic 1b from 1a was accomplished with [Cp*Rh(bpy)H](Cl); however, productive coupling of this regeneration reaction to the enzymatic hydroxylation reaction was not totally successful, due to a deactivation process concerning the HbpA enzyme peripheral groups; i.e., -SH or -NH{sub 2} possibly reacting with the precatalyst, [Cp*Rh(bpy)(H{sub 2}O)](Cl){sub 2}, and thus inhibiting the co-factor regeneration process. The deactivation mechanism was studied, and a promising strategy of derivatizing these peripheral -SH or -NH{sub 2} groups with a polymer containing epoxide was successful in circumventing the undesired interaction between HbpA and the precatalyst. This latter strategy allowed tandem co-factor regeneration using 1a or 2a, [Cp*Rh(bpy)(H2O)](Cl){sub 2}, and formate ion, in conjunction with the polymer bound, FAD containing HbpA enzyme to provide the catechol product.

  17. 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.

  18. Role of NAD-linked glutamate dehydrogenase in nitrogen metabolism in Saccharomyces cerevisiae.

    PubMed Central

    Miller, S M; Magasanik, B

    1990-01-01

    We cloned GDH2, the gene that encodes the NAD-linked glutamate dehydrogenase in the yeast Saccharomyces cerevisiae, by purifying the enzyme, making polyclonal antibodies to it, and using the antibodies to screen a lambda gt11 yeast genomic library. A yeast strain with a deletion-disruption allele of GDH2 which replaced the wild-type gene grew very poorly with glutamate as a nitrogen source, but growth improved significantly when the strain was also provided with adenine or other nitrogenous compounds whose biosynthesis requires glutamine. Our results indicate that the NAD-linked glutamate dehydrogenase catalyzes the major, but not sole, pathway for generation of ammonia from glutamate. We also isolated yeast mutants that lacked glutamate synthase activity and present evidence which shows that normally NAD-linked glutamate dehydrogenase is not involved in glutamate biosynthesis, but that if the enzyme is overexpressed, it may function reversibly in intact cells. PMID:1975578

  19. 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.

  20. Adenine and adenosine salvage in Leishmania donovani.

    PubMed

    Boitz, Jan M; Ullman, Buddy

    2013-08-01

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

  1. Bound Anionic States of Adenine

    SciTech Connect

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

    2007-03-20

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. 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 newfound 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 new valence states observed here, unlike the dipole-bound state, could exist in condensed phases and might be relevant to radiobiological damage. The discovery of these valence anionic states of adenine was facilitated by the development of (i) an experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (it) a combinatorial/quantum chemical approach for identification of the most stable tautomers of organic molecules.

  2. 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

  3. NAD deamidation "a new reaction" by an enzyme from Aspergillus terreus DSM 826.

    PubMed

    Elzainy, Tahany A; Ali, Thanaa H

    2005-02-01

    NAD deamidation is a non-previously recognized reaction. This reaction has been found to be catalyzed by extracts of Aspergillus terreus DSM 826. Conversion of NAD to the biosynthetic intermediate, deamido NAD, by these extracts, at the optimum pH and temperature did not exceed about 55 of the amount of the substrate added. Completion of the reaction was achieved when the extracts were pre-heated at 50 degrees C for 15 min in absence of the substrate. In a very similar manner, the extracts catalyzed hydrolytic cleavage of the amide linkages of different biomolecules such as nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, L-glutamine, L-asparagine and acetamide. Polyacrylamide was also deamidated under the same conditions. In addition, complete dephosphorylation of the dinucleotide molecule was also effected by the same extracts. Separation of the NAD deamidating enzyme from the NAD dephosphorylating enzyme was achieved on using either DEAE - Sephadex A-25 or Sephadex G-200 column chromatography. The obtained phosphohydrolase-free-deamidase showed optimum activity at pH 8 of 0.1 M phosphate buffer and 50 degrees C. It exhibited broad substrate specificity and hyperbolic substrate saturation kinetics. It was isosterically inhibited by the product of its activity and this inhibition was prevented by heating the extracts at 50 degrees C for 15 min. Its activity was not affected in presence of sodium fluoride, partially inhibited in presence of magnesium chloride and was retained in the freezer for some months.

  4. The role of nicotinamide–adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide–adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary

    PubMed Central

    Flint, A. P. F.; Denton, R. M.

    1970-01-01

    1. Superovulated rat ovary was found to contain high activities of NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD–malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP–citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP–malate dehydrogenase and NADP–isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective Km values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP–malate dehydrogenase or NADP–isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed. PMID:4393612

  5. Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor.

    PubMed

    Bairagya, Hridoy R; Mishra, Deepak K; Mukhopadhyay, Bishnu P; Sekar, K

    2014-01-01

    Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10 ns simulation of the IMP-NAD(+) complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD(+). Three conserved water molecules (W1, W, and W1') in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD(+)) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP-NAD(+)-enzyme complexes and their recognition to NAD(+), some covalent modification at carboxamide group of di-nucleotide (NAD(+)) has been made by substituting the -CONH2group by -CONHNH2 (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.

  6. High-resolution crystal structures of the photoreceptor glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with three and four-bound NAD molecules.

    PubMed

    Baker, Bo Y; Shi, Wuxian; Wang, Benlian; Palczewski, Krzysztof

    2014-11-01

    Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (G3P) into 1,3-diphosphoglycerate (BGP) in the presence of the NAD cofactor. GAPDH is an important drug target because of its central role in glycolysis, and nonglycolytic processes such as nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis. Recent studies found that GAPDH participates in the development of diabetic retinopathy and its progression after the cessation of hyperglycemia. Here, we report two structures for native bovine photoreceptor GAPDH as a homotetramer with differing occupancy by NAD, bGAPDH(NAD)4 , and bGAPDH(NAD)3 . The bGAPDH(NAD)4 was solved at 1.52 Å, the highest resolution for GAPDH. Structural comparison of the bGAPDH(NAD)4 and bGAPDH(NAD)3 models revealed novel details of conformational changes induced by cofactor binding, including a loop region (residues 54-56). Structure analysis of bGAPDH confirmed the importance of Phe34 in NAD binding, and demonstrated that Phe34 was stabilized in the presence of NAD but displayed greater mobility in its absence. The oxidative state of the active site Cys149 residue is regulated by NAD binding, because this residue was found oxidized in the absence of dinucleotide. The distance between Cys149 and His176 decreased upon NAD binding and Cys149 remained in a reduced state when NAD was bound. These findings provide an important structural step for understanding the mechanism of GAPDH activity in vision and its pathological role in retinopathies.

  7. 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

  8. Electrocatalytic oxidation of dihydronicotineamide adenine dinucleotide on gold electrode modified with catechol-terminated alkanethiol self-assembly.

    PubMed

    Nakano, Koji; Ohkubo, Kimihiko; Taira, Hiroaki; Takagi, Makoto; Imato, Toshihiko

    2008-06-30

    Synthesis of a mercaptoundecaneamide derivative having a terminus of catechol is described. FT-IR spectroscopic characterization showed that the new molecular entry simply undergoes molecular self-assembly on Au substrate surfaces promoting intra- and intermolecular hydrogen bonds to form well-packed monolayers. Cyclic voltammetric (CV) measurements on the monolayer-modified Au electrode revealed that the surface adlayer possesses specific electrochemical activity due to the reversible catechol/o-quinone redox reaction having characteristics of a surface process and also pH-dependence in its formal potential (59 mV per pH). Detailed analysis of CVs gave fundamental electrochemical parameters including the electroactive surface coverage (0.20-0.24 nmol cm(-2)), the transfer coefficients (0.24 in oxidation and 0.81 in reduction), and also the electron transfer rate constant (1.10-2.76 s(-1)). These data were almost consistent to those seen in literature. We have also found that the catechol monolayer modified electrode exhibits an electrocatalytic function in NADH oxidation. That is, the faradaic current appeared reinforcingly at around the same potential where catechol function is oxidized in the monolayer and increased with an increase in the NADH concentration from 1 to 5 mM, and then reached to a plateau indicating a catalyzed reaction pathway. Detailed analyses revealed that the present system could be characterized by its weak stability of the intermediate compound formed and prompt reaction rate compared with the previously reported chemically modified electrode (CME) systems. We think this type of achievement should be important for the basics of biosensors that rely on dehydrogenase enzymes.

  9. The ascorbic acid-dependent oxidation of reduced nicotinamide–adenine dinucleotide by ciliary and retinal microsomes

    PubMed Central

    Heath, H.; Fiddick, Rosemary

    1965-01-01

    1. The presence of an ascorbic acid-dependent NADH oxidation in ocular tissues has been established. Subcellular fractionation revealed that the enzyme is localized in the microsomes. The distribution of the enzyme in some ocular tissues has been determined; microsomes from the ciliary processes and the retina have comparable activities, which are much higher than those from the cornea or lens. 2. NADPH cannot replace NADH, and cysteine, reduced glutathione, ergothioneine and dehydroascorbic acid cannot be substituted for ascorbic acid in the reaction. The rate of NADH oxidation was greatly increased in the presence of cucumber ascorbate oxidase, and the enzyme appears to be NADH–monodehydroascorbate transhydrogenase. 3. Cytochrome b5 is present in retinal microsomes. 4. The enzyme is inhibited by p-chloromercuribenzoate and iodoacetate, but not by cyanide, Amytal or malonate. 5. High concentrations of chloroquine cause a partial inhibition of the reaction, probably owing to interaction of this compound with the enzyme thiol groups. Low concentrations of Diamox, comparable with those attained in tissues during therapy with this drug, bring about partial inhibition of the reaction. Eserine, cortisone, hydrocortisone, 11-deoxycorticosterone and dexamethasone have no effect on the rate of oxidation. 6. The possible role of ascorbic acid and NADH–monodehydroascorbate transhydrogenase in the formation of aqueous humour and secretory mechanisms is discussed. PMID:14345883

  10. Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs.

    PubMed Central

    Mewies, M.; McIntire, W. S.; Scrutton, N. S.

    1998-01-01

    The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge. PMID:9514256

  11. Alternative splicing and differential expression of two transcripts of nicotine adenine dinucleotide phosphate oxidase B gene from Zea mays.

    PubMed

    Lin, Fan; Zhang, Yun; Jiang, Ming-Yi

    2009-03-01

    With the exception of rice, little is known about the existence of respiratory burst oxidase homolog (rboh) gene in cereals. The present study reports the cloning and analysis of a novel rboh gene, termed ZmrbohB, from maize (Zea mays L.). The full-length cDNA of ZmrbohB encodes a 942 amino acid protein containing all of the respiratory burst oxidase homolog catalytically critical motifs. Alternative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-alpha and -beta. Spliced transcript ZmrbohB-beta retains an unspliced intron 11 that carries a premature termination codon and probably leads to nonsense-mediated mRNA decay. Expression analysis showed that two splice isoforms were differentially expressed in various tissues and at different developmental stages, and the major product was ZmrbohB-alpha. The transcripts of ZmrbohB-alpha accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold (4 degrees C), heat (40 degrees C), UV and salinity stress. In addition, several abiotic stimuli also affected the alternative splicing pattern of ZmrbohB except wounding. These results provide new insight into roles in the expression regulation of plant rboh genes and suggest that ZmrbohB gene may play a role in response to environmental stresses.

  12. The purification and properties of the respiratory-chain reduced nicotinamide–adenine dinucleotide dehydrogenase of Torulopsis utilis

    PubMed Central

    Tottmar, S. O. C.; Ragan, C. I.

    1971-01-01

    1. An NADH–ferricyanide reductase activity has been isolated from the respiratory chain of Torulopsis utilis by using detergents. The isolated enzyme contains non-haem iron, acid-labile sulphide and FMN in the molar proportions 27.5:28.4:1. The preparation is free of FAD and largely free of cytochrome. 2. The enzyme catalyses ferricyanide reduction by NADPH at about 1% of the rate with NADH, and reacts poorly with acceptors other than ferricyanide. The rates of reduction of some acceptors are, as percentages of the rate with ferricyanide: menadione, 0.35%; lipoate, 0.01%; cytochrome c, 0.065%; dichlorophenolindophenol, 0.35%; ubiquinone-1, 0.08%. 3. Several properties of submitochondrial particles of T. utilis (non-haem iron, acid-labile sulphide, FMN and an NADH-reducible electron-paramagnetic-resonance signal) were found to co-purify with the NADH–ferricyanide reductase activity. Thus about 70% of the FMN and, within the limits of accuracy of the experiments, 100% of the non-haem iron and acid-labile sulphide of submitochondrial particles derived from T. utilis cells grown under conditions of glycerol limitation (but relatively low iron availability) can be attributed to the NADH–ferricyanide reductase. 4. It was also shown that the component of submitochondrial particles specifically bleached at 460nm by NADH [species 1 of Ragan & Garland (1971)] co-purifies with the NADH–ferricyanide reductase. 5. This successful purification of an NADH dehydrogenase from T. utilis forms a starting point for investigating the molecular properties of phenotypically modified mitochondrial NADH oxidation pathways that lack energy conservation between NADH and the cytochromes. PMID:4399788

  13. Noncompetitive and irreversible inhibition of xanthine oxidase by benzimidazole analogues acting at the functional flavin adenine dinucleotide cofactor.

    PubMed

    Skibo, E B

    1986-07-29

    Benzimidazole derivatives possessing a leaving group in the 2 alpha-position and either 4,7-dione, 4,7-diol, or 4,7-dimethoxy substituents were examined as inhibitors of buttermilk xanthine oxidase. The quinone and hydroquinone derivatives are not inhibitors of xanthine-oxygen reductase activity, even though the latter is a powerful alkylating agent. The methoxylated hydroquinones are linear noncompetitive inhibitors, the best of which is the 2 alpha-bromo analogue (Ki = 46 microM). During xanthine-oxygen reductase activity, the 2 alpha-bromo analogue irreversibly traps the reduced enzyme. Formation of a C(4a) adduct of the reduced functional FAD cofactor is postulated on the basis of UV-visible spectral evidence and reconstitution of the enzyme after removal of the altered FAD. A probable sequence of events is reversible binding at or near the reduced cofactor followed by adduct formation. It is concluded that potent tight binding inhibitors could be designed that act at the FAD cofactor rather than the purine active site.

  14. Analysing two dinucleotide repeats of FVIII gene in Iranian population.

    PubMed

    Rabbani, B; Rezaeian, A; Khanahmad, H; Bagheri, R; Kamali, E; Zeinali, S

    2007-11-01

    Using dinucleotide repeats for carrier detection and prenatal diagnosis of haemophilia A patients, led us to find different alleles and their frequencies in Iranian population. Polymerase chain reaction (PCR) amplification of two short tandem repeat (STR) loci of factor VIII (FVIII) gene was performed, and the PCR products were resolved on 10% native polyacrylamide gel, and samples were analysed with sequenced DNA markers made of PCR cloning of the dinucleotide FVIII gene fragments. Seven different alleles were observed for intron 13 STR, having 18-24 (CA) repeating units and five alleles for intron 22 STR having 24-28 repeating units of (CACT). Bands produced during dinucleotide study were defined in detail so this could improve the genotyping of heterozygotes and homozygotes. Conformational band produced were characterized to specify the dinucleotide pattern. Our results confirm the Hardy-Weinberg proportions of the heterozygosity rate of the 85 analysed individuals. The observed heterozygosity rate for intron 13 and 22 was 52% and 59% respectively. Our data also indicate that our population is closer to caucasians than to any other populations. Finding different dinucleotide repeat alleles and their frequencies has made it possible to identify carriers and provide prenatal diagnosis with more confidence. This allows antenatal diagnosis to be performed in the vast majority of carriers.

  15. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  16. Converting molecular information of redox coenzymes via self-assembly.

    PubMed

    Morikawa, Masa-aki; Kimizuka, Nobuo

    2012-11-21

    β-Nicotinamide adenine dinucleotide (NAD(+)) and its reduced form NADH specifically interact with a cyanine dye in aqueous media, giving distinct spectral and nanostructural characteristics to which molecular information of constituent coenzymes are converted via self-assembly.

  17. On the origin of multiexponential fluorescence decays from 2-aminopurine-labeled dinucleotides

    NASA Astrophysics Data System (ADS)

    Remington, Jacob M.; Philip, Abbey M.; Hariharan, Mahesh; Kohler, Bern

    2016-10-01

    The fluorescent probe 2-aminopurine (2Ap) has been used for decades to study local conformational fluctuations in DNA. Steady-state and time-resolved measurements of 2Ap fluorescence have been used to predict specific conformational states through suitable modeling of the quenching of the fluorescence of a 2Ap residue incorporated site-specifically into a DNA strand. The success of this approach has been limited by a lack of understanding of the precise factors responsible for the complex, multiexponential decays observed experimentally. In this study, dinucleotides composed of 2Ap and adenine were studied by the time-correlated single-photon counting technique to investigate the causes of heterogeneous emission kinetics. Contrary to previous reports, we argue that emission from 2Ap that is stacked with a neighboring base contributes negligibly to the emission signals recorded more than 50 ps after excitation, which are instead dominated by emission from unstacked 2Ap. We find that the decay kinetics can be modeled using a continuous lifetime distribution, which arises from the inherent distance dependence of electron transfer rates without the need to postulate a small number of discrete states with decay times derived from multiexponential fits. These results offer a new perspective on the quenching of 2Ap fluorescence and expand the information that can be obtained from experiments.

  18. On the origin of multiexponential fluorescence decays from 2-aminopurine-labeled dinucleotides.

    PubMed

    Remington, Jacob M; Philip, Abbey M; Hariharan, Mahesh; Kohler, Bern

    2016-10-21

    The fluorescent probe 2-aminopurine (2Ap) has been used for decades to study local conformational fluctuations in DNA. Steady-state and time-resolved measurements of 2Ap fluorescence have been used to predict specific conformational states through suitable modeling of the quenching of the fluorescence of a 2Ap residue incorporated site-specifically into a DNA strand. The success of this approach has been limited by a lack of understanding of the precise factors responsible for the complex, multiexponential decays observed experimentally. In this study, dinucleotides composed of 2Ap and adenine were studied by the time-correlated single-photon counting technique to investigate the causes of heterogeneous emission kinetics. Contrary to previous reports, we argue that emission from 2Ap that is stacked with a neighboring base contributes negligibly to the emission signals recorded more than 50 ps after excitation, which are instead dominated by emission from unstacked 2Ap. We find that the decay kinetics can be modeled using a continuous lifetime distribution, which arises from the inherent distance dependence of electron transfer rates without the need to postulate a small number of discrete states with decay times derived from multiexponential fits. These results offer a new perspective on the quenching of 2Ap fluorescence and expand the information that can be obtained from experiments.

  19. 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.

  20. NAD(+), sirtuins, and cardiovascular disease.

    PubMed

    Borradaile, Nica M; Pickering, J Geoffrey

    2009-01-01

    Cardiovascular disease (CVD) is the most prevalent disease worldwide and there is intense interest in pharmaceutical approaches to reduce the burden of this chronic, aging-related condition. The sirtuin (SIRT) family of NAD(+)-dependent protein deacetylases and ADP-ribosyltransferases have emerged as exciting targets for CVD management that can impact the cardiovascular system both directly and indirectly, the latter by modulating whole body metabolism. SIRT1-4 regulate the activities of a variety of transcription factors, coregulators, and enzymes that improve metabolic control in adipose tissue, liver, skeletal muscle, and pancreas, particularly during obesity and aging. SIRT1 and 7 can control myocardial development and resist stress- and aging-associated myocardial dysfunction through the deacetylation of p53 and forkhead box O1 (FoxO1). By modulating the activity of endothelial nitric oxide synthase (eNOS), FoxO1, and p53, and the expression of angiotensin II type 1 receptor (AT1R), SIRT1 also promotes vasodilatory and regenerative functions in endothelial and smooth muscle cells of the vascular wall. Given the array of potentially beneficial effects of SIRT activation on cardiovascular health, interest in developing specific SIRT agonists is well-substantiated. Because SIRT activity depends on cellular NAD+ availability, enzymes involved in NAD+ biosynthesis, including nicotinamide phosphoribosyltransferase (Nampt), may also be valuable pharmaceutical targets for managing CVD. Herein we review the actions of the SIRT proteins on the cardiovascular system and consider the potential of modulating SIRT activity and NAD+ availability to control CVD.

  1. Adenine auxotrophy--be aware: some effects of adenine auxotrophy in Saccharomyces cerevisiae strain W303-1A.

    PubMed

    Kokina, Agnese; Kibilds, Juris; Liepins, Janis

    2014-08-01

    Adenine auxotrophy is a commonly used genetic marker in haploid yeast strains. Strain W303-1A, which carries the ade2-1 mutation, is widely used in physiological and genetic research. Yeast extract-based rich medium contains a low level of adenine, so that adenine is often depleted before glucose. This could affect the cell physiology of adenine auxotrophs grown in rich medium. The aim of our study was to assess the effects of adenine auxotrophy on cell morphology and stress physiology. Our results show that adenine depletion halts cell division, but that culture optical density continues to increase due to cell swelling. Accumulation of trehalose and a coincident 10-fold increase in desiccation stress tolerance is observed in adenine auxotrophs after adenine depletion, when compared to prototrophs. Under adenine starvation, long-term survival of W303-1A is lower than during carbon starvation, but higher than during leucine starvation. We observed drastic adenine-dependent changes in cell stress physiology, suggesting that results may be biased when adenine auxotrophs are grown in rich media without adenine supplementation.

  2. 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

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Vitamins and aging: pathways to NAD+ synthesis.

    PubMed

    Denu, John M

    2007-05-04

    Recent genetic evidence reveals additional salvage pathways for NAD(+) synthesis. In this issue, Belenky et al. (2007) report that nicotinamide riboside, a new NAD(+) precursor, regulates Sir2 deacetylase activity and life span in yeast. The ability of nicotinamide riboside to enhance life span does not depend on calorie restriction.

  10. Exploration of Excited State Deactivation Pathways of Adenine Monohydrates.

    PubMed

    Chaiwongwattana, Sermsiri; Sapunar, Marin; Ponzi, Aurora; Decleva, Piero; Došlić, Nađa

    2015-10-29

    Binding of a single water molecule has a dramatic effect on the excited state lifetime of adenine. Here we report a joint nonadiabatic dynamics and reaction paths study aimed at understanding the sub-100 fs lifetime of adenine in the monohydrates. Our nonadiabatic dynamics simulations, performed using the ADC(2) electronic structure method, show a shortening of the excited state lifetime in the monohydrates with respect to bare adenine. However, the computed lifetimes were found to be significantly longer that the observed one. By comparing the reaction pathways of several excited state deactivation processes in adenine and adenine monohydrates, we show that electron-driven proton transfer from water to nitrogen atom N3 of the adenine ring may be the process responsible for the observed ultrafast decay. The inaccessibility of the electron-driven proton transfer pathway to trajectory-based nonadiabatic dynamics simulation is discussed.

  11. Mitochondrial diaphorases as NAD+ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition

    PubMed Central

    Kiss, Gergely; Konrad, Csaba; Pour-Ghaz, Issa; Mansour, Josef J.; Németh, Beáta; Starkov, Anatoly A.; Adam-Vizi, Vera; Chinopoulos, Christos

    2014-01-01

    Substrate-level phosphorylation mediated by succinyl-CoA ligase in the mitochondrial matrix produces high-energy phosphates in the absence of oxidative phosphorylation. Furthermore, when the electron transport chain is dysfunctional, provision of succinyl-CoA by the α-ketoglutarate dehydrogenase complex (KGDHC) is crucial for maintaining the function of succinyl-CoA ligase yielding ATP, preventing the adenine nucleotide translocase from reversing. We addressed the source of the NAD+ supply for KGDHC under anoxic conditions and inhibition of complex I. Using pharmacologic tools and specific substrates and by examining tissues from pigeon liver exhibiting no diaphorase activity, we showed that mitochondrial diaphorases in the mouse liver contribute up to 81% to the NAD+ pool during respiratory inhibition. Under these conditions, KGDHC's function, essential for the provision of succinyl-CoA to succinyl-CoA ligase, is supported by NAD+ derived from diaphorases. Through this process, diaphorases contribute to the maintenance of substrate-level phosphorylation during respiratory inhibition, which is manifested in the forward operation of adenine nucleotide translocase. Finally, we show that reoxidation of the reducible substrates for the diaphorases is mediated by complex III of the respiratory chain.—Kiss, G., Konrad, C., Pour-Ghaz, I., Mansour, J. J., Németh, B., Starkov, A. A., Adam-Vizi, V., Chinopoulos, C. Mitochondrial diaphorases as NAD+ donors to segments of the citric acid cycle that support substrate-level phosphorylation yielding ATP during respiratory inhibition. PMID:24391134

  12. Identification of the NAD(P)H binding site of eukaryotic UDP-galactopyranose mutase.

    PubMed

    Dhatwalia, Richa; Singh, Harkewal; Solano, Luis M; Oppenheimer, Michelle; Robinson, Reeder M; Ellerbrock, Jacob F; Sobrado, Pablo; Tanner, John J

    2012-10-31

    UDP-galactopyranose mutase (UGM) plays an essential role in galactofuranose biosynthesis in microorganisms by catalyzing the conversion of UDP-galactopyranose to UDP-galactofuranose. The enzyme has gained attention recently as a promising target for the design of new antifungal, antitrypanosomal, and antileishmanial agents. Here we report the first crystal structure of UGM complexed with its redox partner NAD(P)H. Kinetic protein crystallography was used to obtain structures of oxidized Aspergillus fumigatus UGM (AfUGM) complexed with NADPH and NADH, as well as reduced AfUGM after dissociation of NADP(+). NAD(P)H binds with the nicotinamide near the FAD isoalloxazine and the ADP moiety extending toward the mobile 200s active site flap. The nicotinamide riboside binding site overlaps that of the substrate galactopyranose moiety, and thus NADPH and substrate binding are mutually exclusive. On the other hand, the pockets for the adenine of NADPH and uracil of the substrate are distinct and separated by only 6 Å, which raises the possibility of designing novel inhibitors that bind both sites. All 12 residues that contact NADP(H) are conserved among eukaryotic UGMs. Residues that form the AMP pocket are absent in bacterial UGMs, which suggests that eukaryotic and bacterial UGMs have different NADP(H) binding sites. The structures address the longstanding question of how UGM binds NAD(P)H and provide new opportunities for drug discovery.

  13. Detection and characterisation of NAD(P)H-diaphorase activity in Dictyostelium discoideum cells (Protozoa)

    PubMed Central

    Amaroli, A.; Chessa, M.G.

    2012-01-01

    In Dictyostelium discoideum (D. discoideum), compounds generating nitric oxide (NO) inhibit its aggregation and differentiation without altering cyclic guanosine monophosphate (cGMP) production. They do it by preventing initiation of cyclic adenosine monophosphate (cAMP) pulses. Furthermore, these compounds stimulate adenosine diphosphate (ADP)-ribosylation of a 41 kDa cytosolic protein and regulate the glyceraldehyde-3-phospate dehydrogenase activity. Yet, although D. discoideum cells produce NO at a relatively constant rate at the onset of their developmental cycle, there is still no evidence of the presence of nitric oxide synthase (NOS) enzymes. In this work, we detect the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in D. discoideum and we characterise it by specific inhibitors and physical-chemical conditions that allegedly distinguish between NOS-related and -unrelated NADPH-d activity. PMID:23361243

  14. The relationship between periodic dinucleotides and the nucleosomal DNA deformation revealed by normal mode analysis

    NASA Astrophysics Data System (ADS)

    Wang, Debby D.; Yan, Hong

    2011-12-01

    Nucleosomes, which contain DNA and proteins, are the basic unit of eukaryotic chromatins. Polymers such as DNA and proteins are dynamic, and their conformational changes can lead to functional changes. Periodic dinucleotide patterns exist in nucleosomal DNA chains and play an important role in the nucleosome structure. In this paper, we use normal mode analysis to detect significant structural deformations of nucleosomal DNA and investigate the relationship between periodic dinucleotides and DNA motions. We have found that periodic dinucleotides are usually located at the peaks or valleys of DNA and protein motions, revealing that they dominate the nucleosome dynamics. Also, a specific dinucleotide pattern CA/TG appears most frequently.

  15. CpG Dinucleotide Frequencies Reveal the Role of Host Methylation Capabilities in Parvovirus Evolution

    PubMed Central

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James

    2013-01-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to “fractional” methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses. PMID:24109231

  16. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution.

    PubMed

    Upadhyay, Mohita; Samal, Jasmine; Kandpal, Manish; Vasaikar, Suhas; Biswas, Banhi; Gomes, James; Vivekanandan, Perumal

    2013-12-01

    Parvoviruses are rapidly evolving viruses that infect a wide range of hosts, including vertebrates and invertebrates. Extensive methylation of the parvovirus genome has been recently demonstrated. A global pattern of methylation of CpG dinucleotides is seen in vertebrate genomes, compared to "fractional" methylation patterns in invertebrate genomes. It remains unknown if the loss of CpG dinucleotides occurs in all viruses of a given DNA virus family that infect host species spanning across vertebrates and invertebrates. We investigated the link between the extent of CpG dinucleotide depletion among autonomous parvoviruses and the evolutionary lineage of the infected host. We demonstrate major differences in the relative abundance of CpG dinucleotides among autonomous parvoviruses which share similar genome organization and common ancestry, depending on the infected host species. Parvoviruses infecting vertebrate hosts had significantly lower relative abundance of CpG dinucleotides than parvoviruses infecting invertebrate hosts. The strong correlation of CpG dinucleotide depletion with the gain in TpG/CpA dinucleotides and the loss of TpA dinucleotides among parvoviruses suggests a major role for CpG methylation in the evolution of parvoviruses. Our data present evidence that links the relative abundance of CpG dinucleotides in parvoviruses to the methylation capabilities of the infected host. In sum, our findings support a novel perspective of host-driven evolution among autonomous parvoviruses.

  17. Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

    PubMed

    Abdelwahab, Heba; Robinson, Reeder; Rodriguez, Pedro; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

    2016-09-15

    l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

  18. An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells.

    PubMed

    Whitney, John C; Quentin, Dennis; Sawai, Shin; LeRoux, Michele; Harding, Brittany N; Ledvina, Hannah E; Tran, Bao Q; Robinson, Howard; Goo, Young Ah; Goodlett, David R; Raunser, Stefan; Mougous, Joseph D

    2015-10-22

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD(+) and NADP(+). Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

  19. Effect of ouabain on metabolic oxidative state in living cardiomyocytes evaluated by time-resolved spectroscopy of endogenous NAD(P)H fluorescence

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Elzwiei, Fathia; Mateasik, Anton; Chorvat, Dusan

    2012-10-01

    Time-resolved spectrometry of endogenous nicotinamide dinucleotide phosphate [NAD(P)H] fluorescence is a useful method to evaluate metabolic oxidative state in living cells. Ouabain is a well-known pharmaceutical drug used in the treatment of cardiovascular disease, the effects of which on myocardial metabolism were recently demonstrated. Mechanisms implicated in these actions are still poorly understood. We investigate the effect of ouabain on the metabolic oxidative state of living cardiac cells identified by time-resolved fluorescence spectroscopy of mitochondrial NAD(P)H. Spectral unmixing is used to resolve individual NAD(P)H fluorescence components. Ouabain decreased the integral intensity of NAD(P)H fluorescence, leading to a reduced component amplitudes ratio corresponding to a change in metabolic state. We also noted that lactate/pyruvate, affecting the cytosolic NADH gradient, increased the effect of ouabain on the component amplitudes ratio. Cell oxidation levels, evaluated as the percentage of oxidized NAD(P)H, decreased exponentially with rising concentrations of the cardiac glycoside. Ouabain also stimulated the mitochondrial NADH production. Our study sheds a new light on the role that ouabain plays in the regulation of metabolic state, and presents perspective on a noninvasive, pharmaceutical approach for testing the effect of drugs on the mitochondrial metabolism by means of time-resolved fluorescence spectroscopy in living cells.

  20. Dinucleotide Composition in Animal RNA Viruses Is Shaped More by Virus Family than by Host Species.

    PubMed

    Di Giallonardo, Francesca; Schlub, Timothy E; Shi, Mang; Holmes, Edward C

    2017-04-15

    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide compositions of viruses should match those of their host species. If this is upheld, it may then be possible to use dinucleotide composition to predict the true host species of viruses sampled in metagenomic surveys. However, it is also clear that different taxonomic groups of viruses tend to have distinctive patterns of dinucleotide composition that may be independent of host species. To determine the relative strength of the effect of host versus virus family in shaping dinucleotide composition, we performed a comparative analysis of 20 RNA virus families from 15 host groupings, spanning two animal phyla and more than 900 virus species. In particular, we determined the odds ratios for the 16 possible dinucleotides and performed a discriminant analysis to evaluate the capability of virus dinucleotide composition to predict the correct virus family or host taxon from which it was isolated. Notably, while 81% of the data analyzed here were predicted to the correct virus family, only 62% of these data were predicted to their correct subphylum/class host and a mere 32% to their correct mammalian order. Similarly, dinucleotide composition has a weak predictive power for different hosts within individual virus families. We therefore conclude that dinucleotide composition is generally uniform within a virus family but less well reflects that of its host species. This has obvious implications for attempts to accurately predict host species from virus genome sequences alone.IMPORTANCE Determining the processes that shape virus genomes is central to understanding virus evolution and emergence. One question of particular importance is why nucleotide and dinucleotide frequencies differ so markedly between viruses. In particular, it is currently unclear whether host species or virus family has the biggest impact on dinucleotide frequencies and

  1. Evidence for two NAD kinases in Salmonella typhimurium.

    PubMed Central

    Cheng, W.; Roth, J. R.

    1994-01-01

    The electron-carrying cofactor NADP is formed by phosphorylation of NAD. A strategy for the isolation of NAD kinase mutants revealed two classes of temperature-sensitive mutations, nadF and nadG, mapping at min 13 and 72 of the Salmonella chromosome. Both mutant types grew on nutrient broth at both 30 and 42 degrees C but on minimal medium showed a temperature-sensitive growth defect which was not corrected by any of the single nutritional supplements tested. A nadF deletion mutant grew on nutrient broth but not on minimal medium. A double mutant with the nadF deletion and a nadG(Ts) mutation showed temperature-sensitive growth on all media. We propose that Salmonella typhimurium has two NAD kinases, one encoded by the nadF and one by the nadG gene. This is supported by the fact that temperature-sensitive mutants of both genes produce kinase activity with altered heat stability. Results suggest that either one of two NAD kinases is sufficient for growth on rich medium, but that both are needed for growth on minimal media. Enzyme assays show that the nadF gene is responsible for about 70% of total NAD kinase activity, and that the nadG gene dictates the remaining 30%. While testing nutritional phenotypes of nadF and nadG mutants, we found that the biosynthetic intermediate, quinolinic acid (QA) inhibited growth of nadF mutants on nutrient broth. This suggested that the NadG enzyme might be inhibited by QA. Enzyme assays demonstrated that QA inhibits the NadG but not the NadF enzyme. This suggests the existence of a regulatory mechanism which controls NADP levels. PMID:8021211

  2. Adenine suppresses IgE-mediated mast cell activation.

    PubMed

    Silwal, Prashanta; Shin, Keuna; Choi, Seulgi; Kang, Seong Wook; Park, Jin Bong; Lee, Hyang-Joo; Koo, Suk-Jin; Chung, Kun-Hoe; Namgung, Uk; Lim, Kyu; Heo, Jun-Young; Park, Jong Il; Park, Seung-Kiel

    2015-06-01

    Nucleobase adenine is produced by dividing human lymphoblasts mainly from polyamine synthesis and inhibits immunological functions of lymphocytes. We investigated the anti-allergic effect of adenine on IgE-mediated mast cell activation in vitro and passive cutaneous anaphylaxis (PCA) in mice. Intraperitoneal injection of adenine to IgE-sensitized mice attenuated IgE-mediated PCA reaction in a dose dependent manner, resulting in a median effective concentration of 4.21 mg/kg. In mast cell cultures, only adenine among cytosine, adenine, adenosine, ADP and ATP dose-dependently suppressed FcɛRI (a high affinity receptor for IgE)-mediated degranulation with a median inhibitory concentration of 1.6mM. It also blocked the production of LTB4, an inflammatory lipid mediator, and inflammatory cytokines TNF-α and IL-4. In addition, adenine blocked thapsigargin-induced degranulation which is FcɛRI-independent but shares FcɛRI-dependent signaling events. Adenine inhibited the phosphorylation of signaling molecules important to FcɛRI-mediated allergic reactions such as Syk, PLCγ2, Gab2, Akt, and mitogen activated protein kinases ERK and JNK. From this result, we report for the first time that adenine inhibits PCA in mice and allergic reaction by inhibiting FcɛRI-mediated signaling events in mast cells. Therefore, adenine may be useful for the treatment of mast cell-mediated allergic diseases. Also, the upregulation of adenine production may provide another mechanism for suppressing mast cell activity especially at inflammatory sites.

  3. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

    Demidov, V V; Potaman, V N; Solyanina, I P; Trofimov, V I

    1995-03-01

    We have investigated in detail radiation and thermal stabilities and transformations of adenosine mono- and triphosphates in liquid and frozen solid aqueous solutions within a wide range of absorbed radiation dose (up to 75 kGy) and temperature (up to 160 degrees C). Dephosphorylation is the main pathway of high temperature hydrolysis of adenine nucleotides. Basic thermodynamic and kinetic parameters of this process have been determined. Radiolysis of investigated compounds at room temperature results in scission of N-glycosidic bond with a radiation yield about of 1 mol/100 eV. Solution freezing significantly enhances radiation stability of nucleotides as well as other biomolecules. This circumstance is essential in the discussion of panspermia concepts.

  4. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences.

    PubMed

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-03-03

    NAD is an essential metabolite that exists in NAD(+) or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD(+)/NADH redox state and modulating cellular signaling processes through the activity of the NAD(+)-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD(+) and NADH contents and the NAD(+)/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD(+), total NAD contents, and NAD(+)/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

  5. In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

    PubMed Central

    Zhu, Xiao-Hong; Lu, Ming; Lee, Byeong-Yeul; Ugurbil, Kamil; Chen, Wei

    2015-01-01

    NAD is an essential metabolite that exists in NAD+ or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD+/NADH redox state and modulating cellular signaling processes through the activity of the NAD+-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD+ and NADH contents and the NAD+/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD+, total NAD contents, and NAD+/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ. PMID:25730862

  6. Adenine oxidation by pyrite-generated hydroxyl radicals.

    PubMed

    Cohn, Corey A; Fisher, Shawn C; Brownawell, Bruce J; Schoonen, Martin Aa

    2010-04-26

    Cellular exposure to particulate matter with concomitant formation of reactive oxygen species (ROS) and oxidization of biomolecules may lead to negative health outcomes. Evaluating the particle-induced formation of ROS and the oxidation products from reaction of ROS with biomolecules is useful for gaining a mechanistic understanding of particle-induced oxidative stress. Aqueous suspensions of pyrite particles have been shown to form hydroxyl radicals and degrade nucleic acids. Reactions between pyrite-induced hydroxyl radicals and nucleic acid bases, however, remain to be determined. Here, we compared the oxidation of adenine by Fenton-generated (i.e., ferrous iron and hydrogen peroxide) hydroxyl radicals to adenine oxidation by hydroxyl radicals generated in pyrite aqueous suspensions. Results show that adenine oxidizes in the presence of pyrite (without the addition of hydrogen peroxide) and that the rate of oxidation is dependent on the pyrite loading. Adenine oxidation was prevented by addition of either catalase or ethanol to the pyrite/adenine suspensions, which implies that hydrogen peroxide and hydroxyl radicals are causing the adenine oxidation. The adenine oxidation products, 8-oxoadenine and 2-hydroxyadenine, were the same whether hydroxyl radicals were generated by Fenton or pyrite-initiated reactions. Although nucleic acid bases are unlikely to be directly exposed to pyrite particles, the formation of ROS in the vicinity of cells may lead to oxidative stress.

  7. Discrepancy variation of dinucleotide microsatellite repeats in eukaryotic genomes.

    PubMed

    Gao, Huan; Cai, Shengli; Yan, Binlun; Chen, Baiyao; Yu, Fei

    2009-01-01

    To address whether there are differences of variation among repeat motif types and among taxonomic groups, we present here an analysis of variation and correlation of dinucleotide microsatellite repeats in eukaryotic genomes. Ten taxonomic groups were compared, those being primates, mammalia (excluding primates and rodentia), rodentia, birds, fish, amphibians and reptiles, insects, molluscs, plants and fungi, respectively. The data used in the analysis is from the literature published in the Journal of Molecular Ecology Notes. Analysis of variation reveals that there are no significant differences between AC and AG repeat motif types. Moreover, the number of alleles correlates positively with the copy number in both AG and AC repeats. Similar conclusions can be obtained from each taxonomic group. These results strongly suggest that the increase of SSR variation is almost linear with the increase of the copy number of each repeat motif. As well, the results suggest that the variability of SSR in the genomes of low-ranking species seem to be more than that of high-ranking species, excluding primates and fungi.

  8. Butyrate influences intracellular levels of adenine and adenine derivatives in the fungus Penicillium restrictum.

    PubMed

    Zutz, Christoph; Chiang, Yi Ming; Faehnrich, Bettina; Bacher, Markus; Hellinger, Roland; Kluger, Bernhard; Wagner, Martin; Strauss, Joseph; Rychli, Kathrin

    2017-04-01

    Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians including the inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence that butyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation. Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linked to its toxicity at a high concentration. In fungi there are indications that butyrate induces the production of secondary metabolites potentially via inhibition of histone deacetylases. However, information about the influence of butyrate on growth, primary metabolite production and metabolism, besides lipid catabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as a susceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activity was detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrate ranging from low (0.001mM) to high (30mM), potentially toxic, concentrations on biomass and antimicrobial activity. Butyrate at high concentrations (3 and 30mM) significantly reduced the fungal biomass. In contrast P. restrictum treated with 0.03mM of butyrate showed the highest antimicrobial activity. We isolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictum cellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivate adenosine. Production of all three compounds was increased at low butyrate concentrations. Furthermore we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP, an important signalling molecule in fungi and various organisms. In conclusion butyrate treatment increases the intracellular levels of adenine and its respective derivatives.

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

    PubMed

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

    2015-11-07

    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.

  10. 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.

  11. Intermolecular band dispersion in quasi-one-dimensional adenine assemblies.

    PubMed

    Wang, Ying; Fleurence, Antoine; Yamada-Takamura, Yukiko; Friedlein, Rainer

    2011-12-07

    Highly-ordered, hydrated adenine multilayer films grown on the surface of highly-oriented pyrolytic graphite, HOPG(0001), display extended electronic states, affording anisotropic band-like charge transport along the π-π stacking direction.

  12. A three-state model for the photophysics of adenine.

    PubMed

    Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio Carlos

    2006-08-25

    An ab initio theoretical study at the CASPT2 level is reported on minimum energy reaction paths, state minima, transition states, reaction barriers, and conical intersections on the potential energy hypersurfaces of two tautomers of adenine: 9H- and 7H-adenine. The obtained results led to a complete interpretation of the photophysics of adenine and derivatives, both under jet-cooled conditions and in solution, within a three-state model. The ultrafast subpicosecond fluorescence decay measured in adenine is attributed to the low-lying conical intersection (gs/pipi* La)(CI), reached from the initially populated 1(pipi* La) state along a path which is found to be barrierless only in 9H-adenine, while for the 7H tautomer the presence of an intermediate plateau corresponding to an NH2-twisted conformation may explain the absence of ultrafast decay in 7-substituted compounds. A secondary picosecond decay is assigned to a path involving switches towards two other states, 1(pipi* Lb) and 1(npi*), ultimately leading to another conical intersection with the ground state, (gs/npi*), with a perpendicular disposition of the amino group. The topology of the hypersurfaces and the state properties explain the absence of secondary decay in 9-substituted adenines in water in terms of the higher position of the 1(npi*) state and also that the 1(pipi* Lb) state of 7H-adenine is responsible for the observed fluorescence in water. A detailed discussion comparing recent experimental and theoretical findings is given. As for other nucleobases, the predominant role of a pipi*-type state in the ultrafast deactivation of adenine is confirmed.

  13. Free [NADH]/[NAD(+)] regulates sirtuin expression.

    PubMed

    Gambini, Juan; Gomez-Cabrera, Mari Carmen; Borras, Consuelo; Valles, Soraya L; Lopez-Grueso, Raul; Martinez-Bello, Vladimir E; Herranz, Daniel; Pallardo, Federico V; Tresguerres, Jesus A F; Serrano, Manuel; Viña, Jose

    2011-08-01

    Sirtuins are deacetylases involved in metabolic regulation and longevity. Our aim was to test the hypothesis that they are subjected to redox regulation by the [NADH]/[NAD(+)] ratio. We used NIH3T3 fibroblasts in culture, Drosophila fed with or without ethanol and exercising rats. In all three models an increase in [NADH]/[NAD(+)] came up with an increased expression of sirtuin mRNA and protein. PGC-1α (a substrate of sirtuins) protein level was significantly increased in fibroblasts incubated with lactate and pyruvate but this effect was lost in fibroblasts obtained from sirtuin-deficient mice. We conclude that the expression of sirtuins is subject to tight redox regulation by the [NADH]/[NAD(+)] ratio, which is a major sensor for metabolite availability conserved from invertebrates to vertebrates.

  14. Renal reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase-mediated metabolism of the carcinogen N-(4-(5-nitro-2-furyl)-2-thiazolyl)acetamide

    SciTech Connect

    Mattammal, M.B.; Zenser, T.V.; Palmier, M.O.; Davis, B.B.

    1985-01-01

    N-(4-(5-Nitro-2-furyl)-2-thiazolyl)acetamide (NFTA) metabolism was examined in vitro using microsomes prepared from rat liver and renal cortex and from rabbit liver and renal cortex and outer and inner medulla. NFTA nitroreduction was observed with each tissue. Three mol of NADPH were used per mol of NFTA reduced. Substrate and inhibitor specificity suggested that the microsomal nitroreduction was due to NADPH:cytochrome c reductase. Metabolite(s) formed bound to protein, RNA, DNA, and synthetic polyribonucleotides. Maximum covalent binding was seen with polyguanylic acid. A guanosine-NFTA adduct was isolated. Binding was inhibited by sulfhydryl compounds and vitamin E. The (/sup 14/C)NFTA:glutathione or (/sup 3/H)glutathione:NFTA conjugates obtained from microsomal incubations showed identical chromatographic properties as the product obtained by the reaction of synthetic N-hydroxy-NFTA with (/sup 3/H)glutathione. Structures of synthetic N-hydroxy-NFTA and the microsomal reduction product 1-(4-(2-acetylaminothiazolyl))-3-cyano-1-propanone were established by mass spectrometry. The latter reduction product did not bind macromolecules. These results suggest that renal NADPH:cytochrome c reductase reduces NFTA to an N-hydroxy-NFTA intermediate that binds nucleophilic sites on macromolecules.

  15. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, convalesce behavior and biochemistry of hypertension induced vascular dementia in rats.

    PubMed

    Sharma, Bhupesh; Singh, Nirmal

    2013-02-01

    Cognitive disorders are likely to increase over the coming years (5-10). Vascular dementia (VaD) has heterogeneous pathology and is a challenge for clinicians. Current Alzheimer's disease drugs have had limited clinical efficacy in treating VaD and none have been approved by major regulatory authorities specifically for this disease. Role of iNOS and NADPH-oxidase has been reported in various pathological conditions but there role in hypertension (Hypt) induced VaD is still unclear. This research work investigates the salutiferous effect of aminoguanidine (AG), an iNOS inhibitor and 4'-hydroxy-3'-methoxyacetophenone (HMAP), a NADPH oxidase inhibitor in Hypt induced VaD in rats. Deoxycorticosterone acetate-salt (DOCA-S) hypertension has been used for development of VaD in rats. Morris water-maze was used for testing learning and memory. Vascular system assessment was done by testing endothelial function. Mean arterial blood pressure (MABP), oxidative stress [aortic superoxide anion, serum and brain thiobarbituric acid reactive species (TBARS) and brain glutathione (GSH)], nitric oxide levels (serum nitrite/nitrate) and cholinergic activity (brain acetyl cholinesterase activity-AChE) were also measured. DOCA-S treated rats have shown increased MABP with impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate & brain GSH levels along with increase in serum & brain TBARS, and brain AChE activity. AG as well as HMAP significantly convalesce Hypt induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that AG, an iNOS inhibitor and HMAP, a NADPH-oxidase inhibitor may be considered as potential agents for the management of Hypt induced VaD.

  16. Effect of sulphate-limited growth on mitochondrial electron transfer and energy conservation between reduced nicotinamide–adenine dinucleotide and the cytochromes in Torulopsis utilis

    PubMed Central

    Haddock, B. A.; Garland, P. B.

    1971-01-01

    1. Conditions have been established for the sulphate-limited growth of Torulopsis utilis in continuous culture. 2. Mitochondria prepared from sulphate-limited cells lack both piericidin A sensitivity and the first energy-conservation site (site 1). Sensitivity to antimycin A or cyanide and the second and third energy-conservation sites were apparently unaffected by sulphate-limited growth. 3. Aerobic incubation for 8h of sulphate-limited cells with a low concentration of sulphate (50μm or less) resulted in the recovery of mitochondrial piericidin A sensitivity and site 1. The use of higher concentrations of sulphate (250μm or more) still resulted in the recovery of mitochondrial piericidin A sensitivity and site 1, but also resulted in the appearance of a non-phosphorylating oxidase, which mediated oxidation of the respiratory chain at about the level of cytochrome b in an antimycin A- and cyanide-insensitive manner. Both this alternative route and the conventional normal route of respiration were shown to coexist and to intercommunicate at the level of cytochrome b. 4. Low-temperature spectroscopy failed to identify any new respiratory component to explain the alternative route. 5. The apparent affinity of the alternative route for oxygen was similar to that for the conventional route through cytochrome oxidase, namely half-maximal activity at 0.1μm-oxygen or less. 6. The non-haem iron concentration of submitochondrial particles was unaffected by sulphate limitation, whereas the acid-labile sulphide concentration was lowered tenfold. Marked increases (between four- and 30-fold) in the acid-labile sulphide concentration of submitochondrial particles were observed in sulphate-limited cells after aerobic incubation with various concentrations of sulphate. The lowest increase (fourfold) was observed without added sulphate, the highest (30-fold) with 1.0mm added sulphate. 7. The ratio of non-haem iron to acid-labile sulphide in submitochondrial particles varied with different growth conditions from a maximum of 15.0 to a minimum of 0.72. It is suggested that analytical measurements of non-haem iron are an inadequate guide to the concentration of iron–sulphur protein in complex systems. 8. The effects of sulphate-limited growth on site 1 and piericidin sensitivity are interpreted to indicate a role for iron–sulphur protein in these properties. 9. The aerobic incubation of sulphate-limited cells with cycloheximide resulted in the recovery by mitochondria of site 1 but not of piericidin sensitivity. 10. The appearance of the alternative route for cyanide- and antimycin-A (but not piericidin A-) insensitive respiration on incubating sulphate-limited cells with sulphate concentrations higher than 250μm indicates that the alternative route involves an iron–sulphur protein. PMID:4399517

  17. 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.

  18. The role of Val-265 for flavin adenine dinucleotide (FAD) binding in pyruvate oxidase: FTIR, kinetic, and crystallographic studies on the enzyme variant V265A.

    PubMed

    Wille, Georg; Ritter, Michaela; Weiss, Manfred S; König, Stephan; Mäntele, Werner; Hübner, Gerhard

    2005-04-05

    In pyruvate oxidase (POX) from Lactobacillus plantarum, valine 265 participates in binding the cofactor FAD and is responsible for the strained conformation of its isoalloxazine moiety that is visible in the crystal structure of POX. The contrasting effects of the conservative amino acid exchange V265A on the enzyme's catalytic properties, cofactor affinity, and protein structure were investigated. The most prominent effect of the exchange was observed in the 2.2 A crystal structure of the mutant POX. While the overall structures of the wild-type and the variant are similar, flavin binding in particular is clearly different. Local disorder at the isoalloxazine binding site prevents modeling of the complete FAD cofactor and two protein loops of the binding site. Only the ADP moiety shows well-defined electron density, indicating an "anchor" function for this part of the molecule. This notion is corroborated by competition experiments where ADP was used to displace FAD from the variant enzyme. Despite the fact that the affinity of FAD binding in the variant is reduced, the catalytic properties are very similar to the wild-type, and the redox potential of the bound flavin is the same for both proteins. The rate of electron transfer toward the flavin during turnover is reduced to one-third compared to the wild-type, but k(cat) remains unchanged. Redox-triggered FTIR difference spectroscopy of free FAD shows the nu(C(10a)=N(1)) band at 1548 cm(-)(1). In POX-V265A, this band is found at 1538 cm(-)(1) and thus shifted less strongly than in wild-type POX where it is found at 1534 cm(-)(1). Taking these observations together, the conservative exchange V265A in POX has a surprisingly small effect on the catalytic properties of the enzyme, whereas the effect on the three-dimensional structure is rather big.

  19. A Mutation in the Flavin Adenine Dinucleotide-Dependent Oxidoreductase FOXRED1 Results in Cell-Type-Specific Assembly Defects in Oxidative Phosphorylation Complexes I and II

    PubMed Central

    Zurita Rendón, Olga; Antonicka, Hana; Horvath, Rita

    2016-01-01

    Complex I (NADH ubiquinone oxidoreductase) is a large multisubunit enzyme that catalyzes the first step in oxidative phosphorylation (OXPHOS). In mammals, complex I biogenesis occurs in a stepwise manner, a process that requires the participation of several nucleus-encoded accessory proteins. The FAD-dependent oxidoreductase-containing domain 1 (FOXRED1) protein is a complex I assembly factor; however, its specific role in the assembly pathway remains poorly understood. We identified a homozygous missense mutation, c.1308 G→A (p.V421M) in FOXRED1 in a patient who presented with epilepsy and severe psychomotor retardation. A patient myoblast line showed a severe reduction in complex I, associated with the accumulation of subassemblies centered around ∼340 kDa, and a milder decrease in complex II, all of which were rescued by retroviral expression of wild-type FOXRED1. Two additional assembly factors, AIFM1 and ACAD9, coimmunoprecipitated with FOXRED1, and all were associated with a 370-kDa complex I subassembly that, together with a 315-kDa subassembly, forms the 550-kDa subcomplex. Loss of FOXRED1 function prevents efficient formation of this midassembly subcomplex. Although we could not identify subassemblies of complex II, our results establish that FOXRED1 function is both broader than expected, involving the assembly of two flavoprotein-containing OXPHOS complexes, and cell type specific. PMID:27215383

  20. 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

  1. 7 CFR 614.15 - Implementation of final NAD determinations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 6 2014-01-01 2014-01-01 false Implementation of final NAD determinations. 614.15 Section 614.15 Agriculture Regulations of the Department of Agriculture (Continued) NATURAL RESOURCES... Implementation of final NAD determinations. (a) No later than 30 days after a NAD determination becomes a...

  2. 7 CFR 614.15 - Implementation of final NAD determinations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 6 2013-01-01 2013-01-01 false Implementation of final NAD determinations. 614.15 Section 614.15 Agriculture Regulations of the Department of Agriculture (Continued) NATURAL RESOURCES... Implementation of final NAD determinations. (a) No later than 30 days after a NAD determination becomes a...

  3. Lupin nad9 and nad6 genes and their expression: 5' termini of the nad9 gene transcripts differentiate lupin species.

    PubMed

    Rurek, Michał; Nuc, Katarzyna; Raczyńska, Katarzyna Dorota; Augustyniak, Halina

    2003-10-02

    The mitochondrial nad9 and nad6 genes were analyzed in four lupin species: Lupinus luteus, Lupinus angustifolius, Lupinus albus and Lupinus mutabilis. The nucleotide sequence of these genes confirmed their high conservation, however, higher number of nucleotide substitution was observed in the L. albus genes. Southern hybridizations confirmed the presence of single copy number of these genes in L. luteus, L. albus and L. angustifolius. The expression of nad9 and nad6 genes was analyzed by Northern in different tissue types of analyzed lupin species. Transcription analyses of the two nad genes displayed single predominant mRNA species of about 0.6 kb in L. luteus and L. angustifolius. The L. albus transcripts were larger in size. The nad9 and nad6 transcripts were modified by RNA editing at 8 and 11 positions, in L. luteus and L. angustifolius, respectively. The gene order, rps3-rpl16-nad9, found in Arabidopsis thaliana is also conserved in L. luteus and L. angustifolius mitochondria. L. luteus and L. angustifolius showed some variability in the sequence of the nad9 promoter region. The last feature along with the differences observed in nad9 mRNA 5' termini of two lupins differentiate L. luteus and L. angustifolius species.

  4. Mature microsatellites: mechanisms underlying dinucleotide microsatellite mutational biases in human cells.

    PubMed

    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

    2013-03-01

    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.

  5. Implications of Dna-Nanostructures by Hoogsteen-Dinucleotides on Transcription Factor Binding

    NASA Astrophysics Data System (ADS)

    Wanke, Dierk; Brand, Luise H.; Fischer, Nina M.; Peschke, Florian; Kilian, Joachim; Berendzen, Kenneth W.

    2013-01-01

    Recent findings showed that non-harmonic DNA-nanostructures are formed by Hoogsteen (HG) dinucleotides in vivo. In contrast to Waston-Crick (WC) base pairing, the purine base component is flipped from anti- to syn-conformation. This change consequently alters the width of the DNA-helix, the sizes of minor and major groove and biophysical properties, such as the melting temperature. Three dinucleotides (CA, TG and TA) have been identified that form stable HG conformations. Functional data and structural models imply that transcription factors specifically bind DNA-motifs that consist of both HG and WC base pairs - especially at the topological transition between HG and WC dinucleotides. We could show that most know cis -regulatory elements contain at least one HG dinucleotide. In addition, we focused our work on human promoter sequences that encode gene regulatory information within double stranded DNA. We compared occurrences of HG dinucleotides to all 16 dinucleotides. These ratios differed most in sequences closer to gene transcripts, where the promoters are located. These findings imply that transcription factors might explicitly recognize their DNA-motifs in regulatory promoter sequences that exhibit HG nanostructure islands.

  6. High-resolution structures of Thermus thermophilus enoyl-acyl carrier protein reductase in the apo form, in complex with NAD+ and in complex with NAD+ and triclosan

    PubMed Central

    Otero, José M.; Noël, Ann-Josée; Guardado-Calvo, Pablo; Llamas-Saiz, Antonio L.; Wende, Wolfgang; Schierling, Benno; Pingoud, Alfred; van Raaij, Mark J.

    2012-01-01

    Enoyl-acyl carrier protein reductase (ENR; the product of the fabI gene) is an important enzyme that is involved in the type II fatty-acid-synthesis pathway of bacteria, plants, apicomplexan protozoa and mitochondria. Harmful pathogens such as Mycobacterium tuberculosis and Plasmodium falciparum use the type II fatty-acid-synthesis system, but not mammals or fungi, which contain a type I fatty-acid-synthesis pathway consisting of one or two multifunctional enzymes. For this reason, specific inhibitors of ENR are attractive antibiotic candidates. Triclosan, a broad-range antibacterial agent, binds to ENR, inhibiting fatty-acid synthesis. As humans do not have an ENR enzyme, they are not affected. Here, high-resolution structures of Thermus thermophilus (Tth) ENR in the apo form, bound to NAD+ and bound to NAD+ plus triclosan are reported. Differences from and similarities to other known ENR structures are reported; in general, the structures are very similar. The cofactor-binding site is also very similar to those of other ENRs and, as reported for other species, triclosan leads to greater ordering of the loop that covers the cofactor-binding site, which, together with the presence of triclosan itself, presumably provides tight binding of the dinucleotide, preventing cycling of the cofactor. Differences between the structures of Tth ENR and other ENRs are the presence of an additional β-sheet at the N-terminus and a larger number of salt bridges and side-chain hydrogen bonds. These features may be related to the high thermal stability of Tth ENR. PMID:23027736

  7. Plant Defensins NaD1 and NaD2 Induce Different Stress Response Pathways in Fungi

    PubMed Central

    Dracatos, Peter M.; Payne, Jennifer; Di Pietro, Antonio; Anderson, Marilyn A.; Plummer, Kim M.

    2016-01-01

    Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (An∆pmtA, An∆pmtB, and An∆pmtC). An∆pmtA was resistant to both defensins, while An∆pmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2. PMID:27598152

  8. Kidney Disease in Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

    Runolfsdottir, Hrafnhildur Linnet; Palsson, Runolfur; Sch. Agustsdottir, Inger M.; Indridason, Olafur S.; Edvardsson, Vidar O.

    2015-01-01

    Background Adenine phosphoribosyltransferase (APRT) deficiency is a purine metabolism disorder causing kidney stones and chronic kidney disease (CKD). The course of nephrolithiasis and CKD has not been well characterized. The objective of this study was to examine long-term kidney outcomes in patients with APRT deficiency. Study Design An observational cohort study. Setting & Participants All patients enrolled in the APRT Deficiency Registry of the Rare Kidney Stone Consortium. Outcomes Kidney stones, acute kidney injury (AKI), stage of CKD and kidney failure, estimated glomerular filtration rate (eGFR) and changes in eGFR. Measurements Serum creatinine and eGFR calculated using creatinine-based equations. Results Of 53 patients, 30 (57%) were female and median age at diagnosis was 37.0 (range, 0.6–67.9) years. The median duration of follow-up was 10.3 (range, 0.0–31.5) years. At diagnosis, kidney stones had developed in 29 patients (55%) and 20 (38%) had CKD stages 3–5, including 11 patients (21%) with stage 5. At latest follow-up, 33 patients (62%) had had kidney stones; 18 (34%), AKI; and 22 (42%), CKD stage 3–5. Of the 14 (26%) patients with CKD stage 5, 12 had initiated renal replacement therapy. Kidney stones recurred in 18 of 33 patients (55%). The median eGFR slope was −0.38 (range, −21.99 to 1.42) mL/min/1.73 m2 per year in patients receiving treatment with xanthine dehydrogenase inhibitor and −5.74 (range, −75.8 to −0.10) mL/min/1.73 m2 per year in those not treated prior to the development of stage 5 CKD (p=0.001). Limitations Use of observational registry data. Conclusions Progressive CKD and AKI episodes are major features of APRT deficiency, while nephrolithiasis is the most common presentation. Advanced CKD without history of kidney stones is more prevalent than previously reported. Our data suggest that timely therapy may retard CKD progression. PMID:26724837

  9. [Study of some pharmacological properties of a new adenine derivative].

    PubMed

    Iasnetsov; Ozerov, A A; Motin, V G; Iasnetsov, Vik V; Karsanova, S K; Ivanov, Iu V; Chel'naia, N A

    2014-01-01

    It is established that the new compound, 9-[2-(4-isopropylphenoxy)ethyl]adenine (9-IPE-adenine) in a dose of 10 mg/kg per day produces neuroprotective effect in rats with brain ischemia model. 9-IPE-adenine decreased the neurologic deficiency 1.2 times more effectively (p < 0.05) than the reference drug mexidol in analogous dose, and had equal effect with this drug at 25 mg/kg per day on the neurologic deficiency and survival of animals. Electrophysiological studies in hippocampal slices in rats showed that 9-IPE-adenine depressed orthodromic population spikes in CA1 area by 42 ± 4%. Non-competitive antagonist of NMDA receptor complex MK-801, in contrast to D-AP5 (competitive NMDA receptor antagonist) and CNQX (competitive AMPA receptor antagonist), enhanced the depressive effect of the new drug more than two times. These ese results are indicative of the ability of 9-IPE-adenine to modulate the ion channel of NMDA receptor complex.

  10. DNA adenine hypomethylation leads to metabolic rewiring in Deinococcus radiodurans.

    PubMed

    Shaiwale, Nayana S; Basu, Bhakti; Deobagkar, Deepti D; Deobagkar, Dileep N; Apte, Shree K

    2015-08-03

    The protein encoded by DR_0643 gene from Deinococcus radiodurans was shown to be an active N-6 adenine-specific DNA methyltransferase (Dam). Deletion of corresponding protein reduced adenine methylation in the genome by 60% and resulted in slow-growth phenotype. Proteomic changes induced by DNA adenine hypomethylation were mapped by two-dimensional protein electrophoresis coupled with mass spectrometry. As compared to wild type D. radiodurans cells, at least 54 proteins were differentially expressed in Δdam mutant. Among these, 39 metabolic enzymes were differentially expressed in Δdam mutant. The most prominent change was DNA adenine hypomethylation induced de-repression of pyruvate dehydrogenase complex, E1 component (aceE) gene resulting in 10 fold increase in the abundance of corresponding protein. The observed differential expression profile of metabolic enzymes included increased abundance of enzymes involved in fatty acid and amino acid degradation to replenish acetyl Co-A and TCA cycle intermediates and diversion of phosphoenolpyruvate and pyruvate into amino acid biosynthesis, a metabolic rewiring attempt by Δdam mutant to restore energy generation via glycolysis-TCA cycle axis. This is the first report of DNA adenine hypomethylation mediated rewiring of metabolic pathways in prokaryotes.

  11. Theoretical study on absorption and emission spectra of adenine analogues.

    PubMed

    Liu, Hongxia; Song, Qixia; Yang, Yan; Li, Yan; Wang, Haijun

    2014-04-01

    Fluorescent nucleoside analogues have attracted much attention in studying the structure and dynamics of nucleic acids in recent years. In the present work, we use theoretical calculations to investigate the structural and optical properties of four adenine analogues (termed as A1, A2, A3, and A4), and also consider the effects of aqueous solution and base pairing. The results show that the fluorescent adenine analogues can pair with thymine to form stable H-bonded WC base pairs. The excited geometries of both adenine analogues and WC base pairs are similar to the ground geometries. The absorption and emission maxima of adenine analogues are greatly red shifted compared with nature adenine, the oscillator strengths of A1 and A2 are stronger than A3 and A4 in both absorption and emission spectra. The calculated low-energy peaks in the absorption spectra are in good agreement with the experimental data. In general, the aqueous solution and base pairing can slightly red-shift both the absorption and emission maxima, and can increase the oscillator strengths of absorption spectra, but significantly decrease the oscillator strengths of A3 in emission spectra.

  12. The human NAD metabolome: Functions, metabolism and compartmentalization

    PubMed Central

    Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

    2015-01-01

    Abstract The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD+ and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools. PMID:25837229

  13. The human NAD metabolome: Functions, metabolism and compartmentalization.

    PubMed

    Nikiforov, Andrey; Kulikova, Veronika; Ziegler, Mathias

    2015-01-01

    The metabolism of NAD has emerged as a key regulator of cellular and organismal homeostasis. Being a major component of both bioenergetic and signaling pathways, the molecule is ideally suited to regulate metabolism and major cellular events. In humans, NAD is synthesized from vitamin B3 precursors, most prominently from nicotinamide, which is the degradation product of all NAD-dependent signaling reactions. The scope of NAD-mediated regulatory processes is wide including enzyme regulation, control of gene expression and health span, DNA repair, cell cycle regulation and calcium signaling. In these processes, nicotinamide is cleaved from NAD(+) and the remaining ADP-ribosyl moiety used to modify proteins (deacetylation by sirtuins or ADP-ribosylation) or to generate calcium-mobilizing agents such as cyclic ADP-ribose. This review will also emphasize the role of the intermediates in the NAD metabolome, their intra- and extra-cellular conversions and potential contributions to subcellular compartmentalization of NAD pools.

  14. Dynamics of NAD-metabolism: everything but constant.

    PubMed

    Opitz, Christiane A; Heiland, Ines

    2015-12-01

    NAD, as well as its phosphorylated form, NADP, are best known as electron carriers and co-substrates of various redox reactions. As such they participate in approximately one quarter of all reactions listed in the reaction database KEGG. In metabolic pathway analysis, the total amount of NAD is usually assumed to be constant. That means that changes in the redox state might be considered, but concentration changes of the NAD moiety are usually neglected. However, a growing number of NAD-consuming reactions have been identified, showing that this assumption does not hold true in general. NAD-consuming reactions are common characteristics of NAD(+)-dependent signalling pathways and include mono- and poly-ADP-ribosylation of proteins, NAD(+)-dependent deacetylation by sirtuins and the formation of messenger molecules such as cyclic ADP-ribose (cADPR) and nicotinic acid (NA)-ADP (NAADP). NAD-consuming reactions are thus involved in major signalling and gene regulation pathways such as DNA-repair or regulation of enzymes central in metabolism. All known NAD(+)-dependent signalling processes include the release of nicotinamide (Nam). Thus cellular NAD pools need to be constantly replenished, mostly by recycling Nam to NAD(+). This process is, among others, regulated by the circadian clock, causing complex dynamic changes in NAD concentration. As disturbances in NAD homoeostasis are associated with a large number of diseases ranging from cancer to diabetes, it is important to better understand the dynamics of NAD metabolism to develop efficient pharmacological invention strategies to target this pathway.

  15. 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.

  16. Adenine and 2-aminopurine: paradigms of modern theoretical photochemistry.

    PubMed

    Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio C

    2006-06-06

    Distinct photophysical behavior of nucleobase adenine and its constitutional isomer, 2-aminopurine, has been studied by using quantum chemical methods, in particular an accurate ab initio multiconfigurational second-order perturbation theory. After light irradiation, the efficient, ultrafast energy dissipation observed for nonfluorescent 9H-adenine is explained here by the nonradiative internal conversion process taking place along a barrierless reaction path from the initially populated 1(pipi* La) excited state toward a low-lying conical intersection (CI) connected with the ground state. In contrast, the strong fluorescence recorded for 2-aminopurine at 4.0 eV with large decay lifetime is interpreted by the presence of a minimum in the 1(pipi* La) hypersurface lying below the lowest CI and the subsequent potential energy barrier required to reach the funnel to the ground state. Secondary deactivation channels were found in the two systems related to additional CIs involving the 1(pipi* Lb) and 1(npi*) states. Although in 9H-adenine a population switch between both states is proposed, in 7H-adenine this may be perturbed by a relatively larger barrier to access the 1(npi*) state, and, therefore, the 1(pipi* Lb) state becomes responsible for the weak fluorescence measured in aqueous adenine at approximately 4.5 eV. In contrast to previous models that explained fluorescence quenching in adenine, unlike in 2-aminopurine, on the basis of the vibronic coupling of the nearby 1(pipi*) and 1(npi*) states, the present results indicate that the 1(npi*) state does not contribute to the leading photophysical event and establish the prevalence of a model based on the CI concept in modern photochemistry.

  17. Negative ion formation in potassium-adenine collisions

    NASA Astrophysics Data System (ADS)

    Chunha, T.; Mendes, M.; Ferreira da Silva, F.; García, G.; Limáo Vieira, P.

    2016-09-01

    We have devoted experimental studies to time-of-flight negative ion formation in electron transfer experiments from neutral potassium atoms with neutral adenine molecules1. Total partial cross sections have been obtained as a function of the collision energy, together with branching ratios for the most relevant fragment anions. Additional set of measurements in adenine derivatives have been performed in order to probe the role of negative ions as well as to probe whether site- and bond-selective excision is also a prevalent mechanism within electron transfer in atom-molecule collision experiments.

  18. NADS - Nuclear and Atomic Data System

    SciTech Connect

    McKinley, Michael S.; Beck, Bret; McNabb, Dennis

    2005-05-24

    We have developed NADS (Nuclear and Atomic Data System), a web-based graphical interface for viewing pointwise and grouped cross sections and distributions. Our implementation is a client / server model. The client is a Java applet that displays the graphical interface, which has interactive 2-D, 3-D, and 4-D plots and tables. The server, which can serve and perform computations of the data, has been implemented in Python using the FUDGE package developed by Bret Beck at LLNL. Computational capabilities include algebraic manipulation of nuclear evaluated data in databases such as LLNL's ENDL-99, ENDF/B-V, and ENDF/B-VI, as well as user data. Processed data used in LLNL's transport codes are accessible as well. NADS is available from http://nuclear.llnl.gov/.

  19. NADS - Nuclear And Atomic Data System

    SciTech Connect

    McKinley, M S; Beck, B; McNabb, D

    2004-09-17

    We have developed NADS (Nuclear and Atomic Data System), a web-based graphical interface for viewing pointwise and grouped cross-sections and distributions. Our implementation is a client / server model. The client is a Java applet that displays the graphical interface, which has interactive 2-D, 3-D, and 4-D plots and tables. The server, which can serve and perform computations the data, has been implemented in Python using the FUDGE package developed by Bret Beck at LLNL. Computational capabilities include algebraic manipulation of nuclear evaluated data in databases such as LLNL's ENDL-99, ENDF/B-V and ENDF/B-VI as well as user data. Processed data used in LLNL's transport codes are accessible as well. NADS is available from http://nuclear.llnl.gov/

  20. Purification of NAD(+) glycohydrolase from human serum.

    PubMed

    Coşkun, Ozlem; Nurten, Rüstem

    2013-07-01

    In the present study, NAD(+) glycohydrolase was purified from serum samples collected from healthy individuals using ammonium sulfate fractionation, Affi-Gel blue (Cibacron Blue F3GA) affinity chromatography, Sephadex G-100 column chromatography and isoelectric focusing. The final step was followed by a second Sephadex G-100 column chromatography assay in order to remove the ampholytes from the isoelectric focusing step. In terms of enhancement of specific activity, the NAD(+) glycohydrolase protein was purified ∼480-fold, with a yield of 1% compared with the initial serum fraction. The purified fraction appeared to be homogeneous, with a molecular weight of 39 kDa, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and also corresponded to the soluble (monomeric) form of surface antigen CD38.

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

    PubMed Central

    Whittam, R.; Wiley, J. S.

    1968-01-01

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

  2. Nrt1 and Tna1-independent export of NAD+ precursor vitamins promotes NAD+ homeostasis and allows engineering of vitamin production.

    PubMed

    Belenky, Peter; Stebbins, Rebecca; Bogan, Katrina L; Evans, Charles R; Brenner, Charles

    2011-05-11

    NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.

  3. Coupling of NAD+ biosynthesis and nicotinamide ribosyl transport: characterization of NadR ribonucleotide kinase mutants of Haemophilus influenzae.

    PubMed

    Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

    2005-07-01

    Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate.

  4. Coupling of NAD+ Biosynthesis and Nicotinamide Ribosyl Transport: Characterization of NadR Ribonucleotide Kinase Mutants of Haemophilus influenzae

    PubMed Central

    Merdanovic, Melisa; Sauer, Elizabeta; Reidl, Joachim

    2005-01-01

    Previously, we characterized a pathway necessary for the processing of NAD+ and for uptake of nicotinamide riboside (NR) in Haemophilus influenzae. Here we report on the role of NadR, which is essential for NAD+ utilization in this organism. Different NadR variants with a deleted ribonucleotide kinase domain or with a single amino acid change were characterized in vitro and in vivo with respect to cell viability, ribonucleotide kinase activity, and NR transport. The ribonucleotide kinase mutants were viable only in a nadV+ (nicotinamide phosphoribosyltransferase) background, indicating that the ribonucleotide kinase domain is essential for cell viability in H. influenzae. Mutations located in the Walker A and B motifs and the LID region resulted in deficiencies in both NR phosphorylation and NR uptake. The ribonucleotide kinase function of NadR was found to be feedback controlled by NAD+ under in vitro conditions and by NAD+ utilization in vivo. Taken together, our data demonstrate that the NR phosphorylation step is essential for both NR uptake across the inner membrane and NAD+ synthesis and is also involved in controlling the NAD+ biosynthesis rate. PMID:15968050

  5. Regulation of NAD biosynthetic enzymes modulates NAD-sensing processes to shape mammalian cell physiology under varying biological cues.

    PubMed

    Ruggieri, Silverio; Orsomando, Giuseppe; Sorci, Leonardo; Raffaelli, Nadia

    2015-09-01

    In addition to its role as a redox coenzyme, NAD is a substrate of various enzymes that split the molecule to either catalyze covalent modifications of target proteins or convert NAD into biologically active metabolites. The coenzyme bioavailability may be significantly affected by these reactions, with ensuing major impact on energy metabolism, cell survival, and aging. Moreover, through the activity of the NAD-dependent deacetylating sirtuins, NAD behaves as a beacon molecule that reports the cell metabolic state, and accordingly modulates transcriptional responses and metabolic adaptations. In this view, NAD biosynthesis emerges as a highly regulated process: it enables cells to preserve NAD homeostasis in response to significant NAD-consuming events and it can be modulated by various stimuli to induce, via NAD level changes, suitable NAD-mediated metabolic responses. Here we review the current knowledge on the regulation of mammalian NAD biosynthesis, with focus on the relevant rate-limiting enzymes. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

  6. Base excision repair of tandem modifications in a methylated CpG dinucleotide.

    PubMed

    Sassa, Akira; Çağlayan, Melike; Dyrkheeva, Nadezhda S; Beard, William A; Wilson, Samuel H

    2014-05-16

    Cytosine methylation and demethylation in tracks of CpG dinucleotides is an epigenetic mechanism for control of gene expression. The initial step in the demethylation process can be deamination of 5-methylcytosine producing the TpG alteration and T:G mispair, and this step is followed by thymine DNA glycosylase (TDG) initiated base excision repair (BER). A further consideration is that guanine in the CpG dinucleotide may become oxidized to 7,8-dihydro-8-oxoguanine (8-oxoG), and this could affect the demethylation process involving TDG-initiated BER. However, little is known about the enzymology of BER of altered in-tandem CpG dinucleotides; e.g. Tp8-oxoG. Here, we investigated interactions between this altered dinucleotide and purified BER enzymes, the DNA glycosylases TDG and 8-oxoG DNA glycosylase 1 (OGG1), apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligases. The overall TDG-initiated BER of the Tp8-oxoG dinucleotide is significantly reduced. Specifically, TDG and DNA ligase activities are reduced by a 3'-flanking 8-oxoG. In contrast, the OGG1-initiated BER pathway is blocked due to the 5'-flanking T:G mispair; this reduces OGG1, AP endonuclease 1, and DNA polymerase β activities. Furthermore, in TDG-initiated BER, TDG remains bound to its product AP site blocking OGG1 access to the adjacent 8-oxoG. These results reveal BER enzyme specificities enabling suppression of OGG1-initiated BER and coordination of TDG-initiated BER at this tandem alteration in the CpG dinucleotide.

  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. Extreme Vulnerability of IDH1 Mutant Cancers to NAD+ Depletion.

    PubMed

    Tateishi, Kensuke; Wakimoto, Hiroaki; Iafrate, A John; Tanaka, Shota; Loebel, Franziska; Lelic, Nina; Wiederschain, Dmitri; Bedel, Olivier; Deng, Gejing; Zhang, Bailin; He, Timothy; Shi, Xu; Gerszten, Robert E; Zhang, Yiyun; Yeh, Jing-Ruey J; Curry, William T; Zhao, Dan; Sundaram, Sudhandra; Nigim, Fares; Koerner, Mara V A; Ho, Quan; Fisher, David E; Roider, Elisabeth M; Kemeny, Lajos V; Samuels, Yardena; Flaherty, Keith T; Batchelor, Tracy T; Chi, Andrew S; Cahill, Daniel P

    2015-12-14

    Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered a profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy, and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.

  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. Catalytic Mechanism and Three-Dimensional Structure of Adenine Deaminase

    SciTech Connect

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

    2011-03-22

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

  11. Relationship between periodic dinucleotides and the nucleosome structure revealed by alpha shape modeling

    NASA Astrophysics Data System (ADS)

    Zhou, Weiqiang; Yan, Hong

    2010-04-01

    As the fundamental repeating units in eukaryotic chromatin, nucleosomes play an important role in many biological processes. For this reason, the study of the structure of nucleosomes may help to reveal some of the crucial principals of these processes. In our research, we have used alpha shapes to model nucleosome structure and discovered that the periodic DNA dinucleotides AA, TT and GC occupy special positions in nucleosome structure with one nucleotide inside and the other outside the nucleosome surface. This structural feature and other dinucleotide characteristics can provide useful information for the study of nucleosome positioning.

  12. Mechanistic Links Between PARP, NAD, and Brain Inflammation After TBI

    DTIC Science & Technology

    2015-10-01

    efficacy of veliparib and NAD as agents for suppressing inflammation and improving outcomes after traumatic brain injury. The animal models include...1 AWARD NUMBER: W81XWH-13-2-0091 TITLE: Mechanistic Links Between PARP, NAD, and Brain Inflammation After TBI PRINCIPAL INVESTIGATOR...COVERED 25 Sep 2014 - 24 Sep 2015 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Mechanistic Links Between PARP, NAD, and Brain Inflammation After TBI 5b. GRANT

  13. In vitro adenine nucleotide catabolism in African catfish spermatozoa.

    PubMed

    Zietara, Marek S; Słomińska, Ewa; Rurangwa, Eugene; Ollevier, Frans; Swierczyński, Julian; Skorkowski, Edward F

    2004-08-01

    It has been shown recently that African catfish (Clarias gariepinus) spermatozoa possess relatively low ATP content and low adenylate energy charge (AEC). One of the possible explanations for this phenomenon is that the spermatozoa actively catabolize adenine nucleotides. A relatively high rate of such catabolism could then contribute to the low ATP concentration and low adenylate energy charge observed in the spermatozoa in vitro. To check this hypothesis, we investigated ATP content and adenine nucleotide catabolism in African catfish spermatozoa stored at 4 degrees C in the presence of glycine as an energetic substrate. Our results indicate that the storage of African catfish sperm at 4 degrees C in the presence of glycine causes time-dependent ATP depletion. In contrast to ATP, the AMP content increases significantly during the same period of sperm storage, while the ADP increases only slightly. Moreover, a significant increase of inosine and hypoxanthine content was also found. Hypoxanthine was accumulated in the storage medium, but xanthine was found neither in spermatozoa nor in the storage medium. It indicates that hypoxanthine is not converted to xanthine, probably due to lack of xanthine oxidase activity in catfish spermatozoa. Present results suggest that adenine nucleotides may be converted to hypoxanthine according to the following pathway: ATP-->ADP-->AMP (adenosine/IMP)-->inosine-->hypoxanthine. Moreover, hypoxanthine seems to be the end product of adenine nucleotide catabolism in African catfish spermatozoa. In conclusion, our results suggest that a relatively high rate of adenine nucleotide catabolism contributes to the low ATP concentration and low adenylate energy charge observed in African catfish spermatozoa in vitro.

  14. NAD(+) biosynthesis and salvage--a phylogenetic perspective.

    PubMed

    Gossmann, Toni I; Ziegler, Mathias; Puntervoll, Pål; de Figueiredo, Luis F; Schuster, Stefan; Heiland, Ines

    2012-09-01

    NAD is best known as an electron carrier and a cosubstrate of various redox reactions. However, over the past 20 years, NAD(+) has been shown to be a key signaling molecule that mediates post-translational protein modifications and serves as precursor of ADP-ribose-containing messenger molecules, which are involved in calcium mobilization. In contrast to its role as a redox carrier, NAD(+)-dependent signaling processes involve the release of nicotinamide (Nam) and require constant replenishment of cellular NAD(+) pools. So far, very little is known about the evolution of NAD(P) synthesis in eukaryotes. In the present study, genes involved in NAD(P) metabolism in 45 species were identified and analyzed with regard to similarities and differences in NAD(P) synthesis. The results show that the Preiss-Handler pathway and NAD(+) kinase are present in all organisms investigated, and thus seem to be ancestral routes. Additionally, two pathways exist that convert Nam to NAD(+); we identified several species that have apparently functional copies of both biosynthetic routes, which have been thought to be mutually exclusive. Furthermore, our findings suggest the parallel phylogenetic appearance of Nam N-methyltransferase, Nam phosphoribosyl transferase, and poly-ADP-ribosyltransferases.

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

    PubMed

    Verdin, Eric

    2015-12-04

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

  16. Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content.

    PubMed

    Nam, Tae-Sik; Park, Kwang-Hyun; Shawl, Asif Iqbal; Kim, Byung-Ju; Han, Myung-Kwan; Kim, Youngho; Moss, Joel; Kim, Uh-Hyun

    2014-06-06

    NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.

  17. Capillary zone electrophoresis with field enhanced sample stacking as a tool for targeted metabolome analysis of adenine nucleotides and coenzymes in Paracoccus denitrificans.

    PubMed

    Musilová, Jindra; Sedlácek, Vojtech; Kucera, Igor; Glatz, Zdenek

    2009-07-01

    The main aim of this work was to demonstrate the applicability of capillary zone electrophoresis in combination with field enhanced sample stacking in targeted metabolome analyses of adenine nucleotides--AMP, ADP, ATP, coenzymes NAD(+), NADP(+) and their reduced forms in Paracoccus denitrificans. Sodium carbonate/hydrogencarbonate buffer (100 mM, pH 9.6) with the addition of beta-CD at a concentration of 10 mM was found to be an effective BGE for their separation within 20 min. Besides this, special attention was paid to the development of the procedure for the extraction of specific metabolites from the bacterium P. denitrificans. This procedure was not only optimised to achieve the highest metabolite yields but also to obtain a sample that was fully compatible with the online preconcetration strategy used. The developed methodology was finally applied in a study of the bacterium P. denitrificans at various stages of the active respiratory chain.

  18. Improved predictive test for MEN2, using flanking dinucleotide repeats and RFLPs

    SciTech Connect

    Howe, J.R.; Lairmore, T.C.; Mishra, S.K.; Shenshen Dou; Veile, R.; Wells, S.A. Jr.; Donis-Keller, H. )

    1992-12-01

    Gene(s) for the autosomal dominant endocrine cancer syndromes, multiple endocrine neoplasia type 2A (MEN2A), multiple endocrine neoplasia type 2B (MEN2B), and familial medullary thyroid carcinoma (MTC1) all map to the pericentromeric region of chromosome 10. Predictive testing for the inheritance of mutant alleles in individuals at risk for these disorders has been limited by the availability of highly informative and closely linked flanking markers. The authors describe the development of eight new markers, including two PCR-based dinucleotide repeat polymorphisms and six RFLPs that flank the disease loci. One of the dinucleotide repeat markers (sJRH-1) derives from the RBP3 locus on 10q11.2 and has a PIC of .88. The other dinucleotide repeat (sTCL-1) defines a new locus, D10S176, that maps by in situ hybridization to 10p11.2 and has a PIC of .68. The authors have constructed a new genetic linkage map of the pericentromeric region of chromosome 10, on the basis of 13 polymorphisms at six loci, which places the MEN2A locus between the dinucleotide repeat markers, with odds of 5,750:1 over the next most likely position. Using this set of markers, predictive genetic testing of 130 at-risk individuals from six families segregating MEN2A revealed that 95% were jointly informative with flanking markers, representing a significant improvement in genetic testing capabilities. 42 refs., 6 figs., 3 tabs.

  19. Study on the oxidation form of adenine in phosphate buffer solution.

    PubMed

    Song, Yuan-Zhi; Zhou, Jian-Feng; Zhu, Feng-Xia; Ye, Yong; Xie, Ji-Min

    2010-07-01

    The oxidation of adenine in phosphate buffer solution is investigated using square-wave voltammetry and in situ UV spectroelectrochemistry. The geometry of adenine and the derivatives optimized at DFTB3LYP-6-31G (d, p)-PCM level is in agreement with the crystal structure, and the imitated UV spectra of adenine and the product at electrode are consistent with the in situ UV spectra. The relationship between the electrochemical property and the molecular structure is also discussed. The experimental and theoretical results show that the adenine oxidation origins from the neutral adenine.

  20. An interbacterial NAD(P)+ glycohydrolase toxin requires elongation factor Tu for delivery to target cells

    DOE PAGES

    Whitney, John C.; Quentin, Dennis; Sawai, Shin; ...

    2015-10-08

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). Thesemore » proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Lastly, visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.« less

  1. 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.

  2. Agents for replacement of NAD+/NADH system in enzymatic reactions

    DOEpatents

    Fish, Richard H.; Kerr, John B.; Lo, Christine H.

    2004-04-06

    Novel agents acting as co-factors for replacement of NAD(P).sup.+ /NAD(P)H co-enzyme systems in enzymatic oxido-reductive reactions. Agents mimicking the action of NAD(P).sup.+ /NAD(P)H system in enzymatic oxidation/reduction of substrates into reduced or oxidized products. A method for selection and preparation of the mimicking agents for replacement of NAD(P).sup.+ /NAD(P)H system and a device comprising co-factors for replacement of NAD(P).sup.+ /NAD(P)H system.

  3. NAD+ and vitamin B3: from metabolism to therapies.

    PubMed

    Sauve, Anthony A

    2008-03-01

    The role of NAD(+) metabolism in health and disease is of increased interest as the use of niacin (nicotinic acid) has emerged as a major therapy for treatment of hyperlipidemias and with the recognition that nicotinamide can protect tissues and NAD(+) metabolism in a variety of disease states, including ischemia/reperfusion. In addition, a growing body of evidence supports the view that NAD(+) metabolism regulates important biological effects, including lifespan. NAD(+) exerts potent effects through the poly(ADP-ribose) polymerases, mono-ADP-ribosyltransferases, and the recently characterized sirtuin enzymes. These enzymes catalyze protein modifications, such as ADP-ribosylation and deacetylation, leading to changes in protein function. These enzymes regulate apoptosis, DNA repair, stress resistance, metabolism, and endocrine signaling, suggesting that these enzymes and/or NAD(+) metabolism could be targeted for therapeutic benefit. This review considers current knowledge of NAD(+) metabolism in humans and microbes, including new insights into mechanisms that regulate NAD(+) biosynthetic pathways, current use of nicotinamide and nicotinic acid as pharmacological agents, and opportunities for drug design that are directed at modulation of NAD(+) biosynthesis for treatment of human disorders and infections.

  4. 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

    2016-06-02

    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).

  5. High resolution dissociative electron attachment to gas phase adenine

    SciTech Connect

    Huber, D.; Beikircher, M.; Denifl, S.; Zappa, F.; Matejcik, S.; Bacher, A.; Grill, V.; Maerk, T. D.; Scheier, P.

    2006-08-28

    The dissociative electron attachment to the gas phase nucleobase adenine is studied using two different experiments. A double focusing sector field mass spectrometer is utilized for measurements requiring high mass resolution, high sensitivity, and relative ion yields for all the fragment anions and a hemispherical electron monochromator instrument for high electron energy resolution. The negative ion mass spectra are discussed at two different electron energies of 2 and 6 eV. In contrast to previous gas phase studies a number of new negative ions are discovered in the mass spectra. The ion efficiency curves for the negative ions of adenine are measured for the electron energy range from about 0 to 15 eV with an electron energy resolution of about 100 meV. The total anion yield derived via the summation of all measured fragment anions is compared with the total cross section for negative ion formation measured recently without mass spectrometry. For adenine the shape of the two cross section curves agrees well, taking into account the different electron energy resolutions; however, for thymine some peculiar differences are observed.

  6. The nucleobase adenine as a signalling molecule in the kidney.

    PubMed

    Thimm, D; Schiedel, A C; Peti-Peterdi, J; Kishore, B K; Müller, C E

    2015-04-01

    In 2002, the first receptor activated by the nucleobase adenine was discovered in rats. In the past years, two adenine receptors (AdeRs) in mice and one in Chinese hamsters, all of which belong to the family of G protein-coupled receptors (GPCRs), were cloned and pharmacologically characterized. Based on the nomenclature for other purinergic receptor families (P1 for adenosine receptors and P2 for nucleotide, e.g. ATP, receptors), AdeRs were designated P0 receptors. Pharmacological data indicate the existence of G protein-coupled AdeRs in pigs and humans as well; however, those have not been cloned so far. Current data suggest a role for adenine and AdeRs in renal proximal tubules. Furthermore, AdeRs are suggested to be functional counterplayers of vasopressin in the collecting duct system, thus exerting diuretic effects. We are only at the beginning of understanding the significance of this new class of purinergic receptors, which might become future drug targets.

  7. Fragmentation mechanisms of cytosine, adenine and guanine ionized bases.

    PubMed

    Sadr-Arani, Leila; Mignon, Pierre; Chermette, Henry; Abdoul-Carime, Hassan; Farizon, Bernadette; Farizon, Michel

    2015-05-07

    The different fragmentation channels of cytosine, adenine and guanine have been studied through DFT calculations. The electronic structure of bases, their cations, and the fragments obtained by breaking bonds provides a good understanding of the fragmentation process that can complete the experimental approach. The calculations allow assigning various fragments to the given peaks. The comparison between the energy required for the formation of fragments and the peak intensity in the mass spectrum is used. For cytosine and guanine the elimination of the HNCO molecule is a major route of dissociation, while for adenine multiple loss of HCN or HNC can be followed up to small fragments. For cytosine, this corresponds to the initial bond cleavage of N3-C4/N1-C2, which represents the main dissociation route. For guanine the release of HNCO is obtained through the N1-C2/C5-C6 bond cleavage (reverse order also possible) leading to the largest peak of the spectrum. The corresponding energies of 3.5 and 3.9 eV are typically in the range available in the experiments. The loss of NH3 or HCN is also possible but requires more energy. For adenine, fragmentation consists of multiple loss of the HCN molecule and the main route corresponding to HC8N9 loss is followed by the release of HC2N1.

  8. Biosensor reveals multiple sources for mitochondrial NAD⁺.

    PubMed

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

    2016-06-17

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

  9. Bacterial Renalase: Structure and Kinetics of an Enzyme with 2- and 6-Dihydro-β-NAD(P) Oxidase Activity from Pseudomonas phaseolicola.

    PubMed

    Hoag, Matthew R; Roman, Joseph; Beaupre, Brett A; Silvaggi, Nicholas R; Moran, Graham R

    2015-06-23

    Despite a lack of convincing in vitro evidence and a number of sound refutations, it is widely accepted that renalase is an enzyme unique to animals that catalyzes the oxidative degradation of catecholamines in blood in order to lower vascular tone. Very recently, we identified isomers of β-NAD(P)H as substrates for renalase (Beaupre, B. A. et al. (2015) Biochemistry, 54, 795-806). These molecules carry the hydride equivalent on the 2 or 6 position of the nicotinamide base and presumably arise in nonspecific redox reactions of nicotinamide dinucleotides. Renalase serves to rapidly oxidize these isomers to form β-NAD(P)⁺ and then pass the electrons to dioxygen, forming H₂O₂. We have also shown that these substrate molecules are highly inhibitory to dehydrogenase enzymes and thus have proposed an intracellular metabolic role for this enzyme. Here, we identify a renalase from an organism without a circulatory system. This bacterial form of renalase has the same substrate specificity profile as that of human renalase but, in terms of binding constant (K(d)), shows a marked preference for substrates derived from β-NAD⁺. 2-dihydroNAD(P) substrates reduce the enzyme with rate constants (k(red)) that greatly exceed those for 6-dihydroNAD(P) substrates. Taken together, k(red)/K(d) values indicate a minimum 20-fold preference for 2DHNAD. We also offer the first structures of a renalase in complex with catalytically relevant ligands β-NAD⁺ and β-NADH (the latter being an analogue of the substrate(s)). These structures show potential electrostatic repulsion interactions with the product and a unique binding orientation for the substrate nicotinamide base that is consistent with the identified activity.

  10. SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The sirtuin family of nicotinamide adenine dinucleotide-dependent (NAD) deacetylases plays an important role in aging and metabolic regulation. In yeast, the Sir2 gene and its homolog Hst2 independently mediate the action of caloric restriction on lifespan extension. The mammalian Sir2 ortholog, SIR...

  11. Pleiotropic effects of the sirtuin inhibitor sirtinol involves concentration-dependent modulation of multiple nuclear receptor-mediated pathways in the androgen-responsive prostate cancer cell LNCaP

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sirtinol, a purported specific inhibitor of the nicotinamide adenine dinucleotide (NAD)-dependent type III histone deacetylase (also known as sirtuin), has been used extensively to identify chemopreventive/chemotherapeutic agents that modulate the activity of this group of enzymes. However, the mole...

  12. Metabolic precursors in astrophysical ice analogs: implications for meteorites and comets.

    PubMed

    Smith, Karen E; Gerakines, Perry A; Callahan, Michael P

    2015-07-28

    We report the synthesis of complex organic compounds including nicotinic and quinolinic acid, two members involved in the nicotinamide adenine dinucleotide (NAD) biosynthetic pathway, in irradiated astrophysical ice analogs. If delivered to Earth by meteorites and comets, these compounds may have contributed to the origin and early evolution of life.

  13. A Highly Reactive Imidazolium-Bridged Dinucleotide Intermediate in Nonenzymatic RNA Primer Extension.

    PubMed

    Walton, Travis; Szostak, Jack W

    2016-09-14

    Because of its importance for the origin of life, the nonenzymatic copying of RNA templates has been the subject of intense study for several decades. Previous characterizations of template-directed primer extension using 5'-phosphoryl-2-methylimidazole-activated nucleotides (2-MeImpNs) as substrates have assumed a classical in-line nucleophilic substitution mechanism, in which the 3'-hydroxyl of the primer attacks the phosphate of the incoming monomer, displacing the 2-methylimidazole leaving group. However, we have found that the initial rate of primer extension depends on the pH and concentration at which the activated monomer is maintained prior to the primer extension reaction. These and other results suggest an alternative mechanism, in which two monomers react with each other to form an imidazolium-bridged dinucleotide intermediate, which then binds to the template. Subsequent attack of the 3'-hydroxyl of the primer displaces an activated nucleotide as the leaving group and results in extension of the primer by one nucleotide. Analysis of monomer solutions by NMR indicates formation of the proposed imidazolium-bridged dinucleotide in the expected pH-dependent manner. We have used synthetic methods to prepare material that is enriched in this proposed intermediate and show that it is a highly reactive substrate for primer extension. The formation of an imidazolium-bridged dinucleotide intermediate provides a mechanistic interpretation of previously observed catalysis by an activated nucleotide located downstream from the site of primer extension.

  14. Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins.

    PubMed

    Römling, Ute; Liang, Zhao-Xun; Dow, J Maxwell

    2017-03-01

    Cyclic di-GMP was the first cyclic dinucleotide second messenger described, presaging the discovery of additional cyclic dinucleotide messengers in bacteria and eukaryotes. The GGDEF diguanylate cyclase (DGC) and EAL and HD-GYP phosphodiesterase (PDE) domains conduct the turnover of cyclic di-GMP. These three unrelated domains belong to superfamilies that exhibit significant variations in function, and they include both enzymatically active and inactive members, with a subset involved in synthesis and degradation of other cyclic dinucleotides. Here, we summarize current knowledge of sequence and structural variations that underpin the functional diversification of cyclic di-GMP turnover proteins. Moreover, we highlight that superfamily diversification is not restricted to cyclic di-GMP signaling domains, as particular DHH/DHHA1 domain and HD domain proteins have been shown to act as cyclic di-AMP phosphodiesterases. We conclude with a consideration of the current limitations that such diversity of action places on bioinformatic prediction of the roles of GGDEF, EAL, and HD-GYP domain proteins.

  15. Effects of spinally administered adenine on dorsal horn neuronal responses in a rat model of inflammation.

    PubMed

    Matthews, Elizabeth A; Dickenson, Anthony H

    2004-02-19

    A novel G-protein-coupled receptor with adenine identified as the endogenous ligand has recently been described. In vivo electrophysiological techniques in the rat were used to record the response of dorsal horn neurones in response to transcutaneous electrical stimulation to the hindpaw receptive field. Spinal adenine (1-1000 microg) exerted facilitatory effects on the electrically-evoked neuronal responses, in a mildly dose-related manner. After establishment of carrageenan-induced inflammation to the hindpaw this excitatory effect of adenine was still apparent, yet reduced. C-fibre-evoked responses and other nociceptive related measures were most susceptible to the effects of adenine, whereas non-nociceptive Abeta-fibre evoked activity remained unaffected. Thus, activation of the adenine receptor site, via spinally applied adenine, suggests a pronociceptive role in nociceptive sensory transmission.

  16. 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.

  17. Sulfur and adenine metabolisms are linked, and both modulate sulfite resistance in wine yeast.

    PubMed

    Aranda, Agustín; Jiménez-Martí, Elena; Orozco, Helena; Matallana, Emilia; Del Olmo, Marcellí

    2006-08-09

    Sulfite treatment is the most common way to prevent grape must spoilage in winemaking because the yeast Saccharomyces cerevisiae is particularly resistant to this chemical. In this paper we report that sulfite resistance depends on sulfur and adenine metabolism. The amount of adenine and methionine in a chemically defined growth medium modulates sulfite resistance of wine yeasts. Mutations in the adenine biosynthetic pathway or the presence of adenine in a synthetic minimal culture medium increase sulfite resistance. The presence of methionine has the opposite effect, inducing a higher sensitivity to SO(2). The concentration of methionine, adenine, and sulfite in a synthetic grape must influences the progress of fermentation and at the transcriptional level the expression of genes involved in sulfur (MET16), adenine (ADE4), and acetaldehyde (ALD6) metabolism. Sulfite alters the pattern of expression of all these genes. This fact indicates that the response to this stress is complex and involves several metabolic pathways.

  18. HYDROGEN-BONDED DIMERS OF ADENINE AND URACIL DERIVATIVES.

    PubMed

    HAMLIN, R M; LORD, R C; RICH, A

    1965-06-25

    In concentrated solutions of either 9-ethyladenine or 1-cyclohexyluracil in deuterochloroform, absorption bands in the infrared spectrum demonstrate hydrogen bonding of the adenine and uracil derivatives with themselves. In dilute solutions, there is very little hydrogen bonding. However, when dilute solutions of 9-ethyladenine and 1-cyclohexyluracil are mixed, a series of bands appear which show that these molecules are hydrogen-bonding with each other much more strongly than with themselves. A study of the stoichiometry of this association indicates formation of 1:1 hydrogen-bonded pairs in solution.

  19. Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine.

    PubMed

    Sereda, Michal J

    2010-08-01

    The Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine meeting, held in Tarragona, Spain, included topics covering new findings in the field of purinergic signaling and the development of purine-based drugs. This conference report highlights selected presentations on developments in purinerigic signaling, medicinal chemistry, the therapeutic potential of purine-based drugs, and the role of purines and adenosine receptors in neurodegenerative disorders, sickle cell disease, bone homeostasis, pulmonary fibrosis and pain. Investigational drugs discussed include CF-101 (Can-Fite BioPharma Ltd/NIH/Kwang Dong Pharmaceutical Co Ltd/Seikagaku Corp) and denufosol tetrasodium (Cystic Fibrosis Foundation Therapeutics Inc/Inspire Pharmaceuticals Inc).

  20. Investigation of coordination properties of isolated adenine to copper metal: a systematic spectroscopic and DFT study.

    PubMed

    Prakash, Om; Singh, Sachin Kumar; Singh, Bachcha; Singh, Ranjan K

    2013-08-01

    The coordination properties of copper with adenine have been studied by the analyzing the changes in Fourier Transform Infra-red (FTIR) and Raman spectra of adenine and adenine-copper complex. The geometry of adenine and adenine copper complex were optimized and theoretical Infra-red and Raman spectra of the optimized structures were calculated using Density Functional Theory (DFT). During synthesis of adenine-copper complex specific procedure was adopted to attach the Cu atom with particular N-atom of adenine (N9). The results of Raman and DFT confirmed the attachment. The Raman bands at 625, 330 and 230 cm(-1) of adenine-copper complex contain significant contribution of the vibrational motions of Cu metal coordinated to N9 and Cl atoms. The DFT calculations give additional vibrational modes containing the Cu, N9 and N9* atoms, which are not observed in FTIR and Raman spectra. The Raman, IR and DFT study confirm that Cu metal has good binding affinity to the isolated adenine base.

  1. 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.

  2. 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.

  3. 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

  4. A9145, a new adenine-containing antifungal antibiotic: fermentation.

    PubMed

    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 Co(2+), PO(4) (3-), and CaCO(3), 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 O(2) level was also critical, the optimal concentration being 3 x 10(-2) to 4.5 x 10(-2) mumole of O(2)/ml. Optimization of various fermentation parameters increased antibiotic titers approximately 135-fold in shaken flask fermentors and 225-fold in stirred vessels.

  5. On the deactivation mechanisms of adenine-thymine base pair.

    PubMed

    Gobbo, João Paulo; Saurí, Vicenta; Roca-Sanjuán, Daniel; Serrano-Andrés, Luis; Merchán, Manuela; Borin, Antonio Carlos

    2012-04-05

    In this contribution, the multiconfigurational second-order perturbation theory method based on a complete active space reference wave function (CASSCF/CASPT2) is applied to study all possible single and double proton/hydrogen transfers between the nucleobases in the adenine-thymine (AT) base pair, analyzing the role of excited states with different nature [localized (LE) and charge transfer (CT)], and considering concerted as well as step-wise mechanisms. According to the findings, once the lowest excited states, localized in adenine, are populated during UV irradiation of the Watson-Crick base pair, the proton transfer in the N-O bridge does not require high energy in order to populate a CT state. The latter state will immediately relax toward a crossing with the ground state, which will funnel the system to either the canonical structure or the imino-enol tautomer. The base pair is also capable of repairing itself easily since the imino-enol species is unstable to thermal conversion.

  6. 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.

  7. NAD+/NADH and skeletal muscle mitochondrial adaptations to exercise

    PubMed Central

    White, Amanda T.

    2012-01-01

    The pyridine nucleotides, NAD+ and NADH, are coenzymes that provide oxidoreductive power for the generation of ATP by mitochondria. In skeletal muscle, exercise perturbs the levels of NAD+, NADH, and consequently, the NAD+/NADH ratio, and initial research in this area focused on the contribution of redox control to ATP production. More recently, numerous signaling pathways that are sensitive to perturbations in NAD+(H) have come to the fore, as has an appreciation for the potential importance of compartmentation of NAD+(H) metabolism and its subsequent effects on various signaling pathways. These pathways, which include the sirtuin (SIRT) proteins SIRT1 and SIRT3, the poly(ADP-ribose) polymerase (PARP) proteins PARP1 and PARP2, and COOH-terminal binding protein (CtBP), are of particular interest because they potentially link changes in cellular redox state to both immediate, metabolic-related changes and transcriptional adaptations to exercise. In this review, we discuss what is known, and not known, about the contribution of NAD+(H) metabolism and these aforementioned proteins to mitochondrial adaptations to acute and chronic endurance exercise. PMID:22436696

  8. Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

    PubMed

    Ting, Kai Yiu; Leung, Christina F P; Graeff, Richard M; Lee, Hon Cheung; Hao, Quan; Kotaka, Masayo

    2016-03-01

    Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively.

  9. SIRT1 and NAD as regulators of ageing.

    PubMed

    Rehan, Leopold; Laszki-Szcząchor, Krystyna; Sobieszczańska, Małgorzata; Polak-Jonkisz, Dorota

    2014-06-06

    The recent research on ageing processes in mammals throws new light on the biochemistry of circadian clock. The already known regulatory pathways for biological rhythms and metabolism, combined with newly discovered functions of sirtuins, unveil a perspective for new hypotheses, regarding possible links between ageing and circadian rhythms. The NAD World hypothesis - postulated as a systemic regulatory network for the metabolism and ageing, linked with mammalian, NAD+ dependent Sirtuin 1 - conceptually involves two critical elements. One is the systemic, Nampt-controlled NAD+ (nicotinamide phosphoribosyltransferase) biosynthesis, where Nampt (nicotinamide phosphoribosyltransferase) acts as "propulsion" for metabolism and the other is NAD+ dependent deacetylase (SIRT1) - a regulator responsible for various biological effects, depending on its localisation in organism. In this approach, the role of sirtuins, which are evolutionary conservative, NAD+ dependent histone deacetylases, may be very important for the mammalian metabolic clock. This paper is a review of current research on possible links among SIRT1 (Sirtuin 1), metabolism and ageing with particular consideration of the NAD World hypothesis.

  10. Parp mutations protect against mitochondrial dysfunction and neurodegeneration in a PARKIN model of Parkinson's disease

    PubMed Central

    Lehmann, S; Costa, A C; Celardo, I; Loh, S H Y; Martins, L M

    2016-01-01

    The co-enzyme nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular energy generation in mitochondria as well as for DNA repair mechanisms in the cell nucleus involving NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Mitochondrial function is compromised in animal models of Parkinson's disease (PD) associated with PARKIN mutations. Here, we uncovered alterations in NAD+ salvage metabolism in Drosophila parkin mutants. We show that a dietary supplementation with the NAD+ precursor nicotinamide rescues mitochondrial function and is neuroprotective. Further, by mutating Parp in parkin mutants, we show that this increases levels of NAD+ and its salvage metabolites. This also rescues mitochondrial function and suppresses dopaminergic neurodegeneration. We conclude that strategies to enhance NAD+ levels by administration of dietary precursors or the inhibition of NAD+-dependent enzymes, such as PARP, that compete with mitochondria for NAD+ could be used to delay neuronal death associated with mitochondrial dysfunction. PMID:27031963

  11. NdnR is an NAD-responsive transcriptional repressor of the ndnR operon involved in NAD de novo biosynthesis in Corynebacterium glutamicum.

    PubMed

    Teramoto, Haruhiko; Inui, Masayuki; Yukawa, Hideaki

    2012-04-01

    The Corynebacterium glutamicum ndnR gene, which is chromosomally located in a gene cluster involved in NAD de novo biosynthesis, negatively regulates expression of the cluster genes, i.e. nadA, nadC, nadS and ndnR itself. Although ndnR encodes a member of the recently identified NrtR family of transcriptional regulators, whether or not the NdnR protein directly regulates these NAD biosynthesis genes remains to be verified. Here, two NdnR binding sites in the promoter region of the ndnR-nadA-nadC-nadS operon in C. glutamicum were confirmed by in vitro DNA binding assay and analysis of in vivo expression of the chromosomally integrated ndnR promoter-lacZ reporter fusion. Electrophoretic mobility shift assay revealed that the NdnR protein binds to the 5'-upstream region of ndnR, and that the binding is significantly enhanced by NAD. Mutation in two 21 bp NdnR binding motifs in the ndnR promoter region inhibited the binding of NdnR in vitro. The mutation also enhanced the promoter activity in cells cultured in the presence of nicotinate, which is utilized in NAD biosynthesis, resulting in the loss of the repression in response to an exogenous NAD precursor; this is consistent with the effect of deletion of ndnR reported in our previous study. These results indicate that NAD acts as a co-repressor for the NdnR protein that directly regulates the ndnR operon involved in NAD de novo biosynthesis; the NAD-NdnR regulatory system likely plays an important role in the control of NAD homeostasis in C. glutamicum.

  12. The Maize Ncs2 Abnormal Growth Mutant Has a Chimeric Nad4-Nad7 Mitochondrial Gene and Is Associated with Reduced Complex I Function

    PubMed Central

    Marienfeld, J. R.; Newton, K. J.

    1994-01-01

    The molecular basis of the maternally inherited, heteroplasmic NCS2 mutant of maize was investigated. Analysis of the NCS2 mtDNA showed that it closely resembles the progenitor cmsT mitochondrial genome, except that the mutant genome contains a fused nad4-nad7 gene and is deleted for the small fourth exon of nad4. The rearrangement has occurred at a 16-bp repeat present in the third intron of the nad4 gene and in the second intron of the nad7 gene. Transcripts containing exon 4 of the nad4 gene are greatly reduced in mtRNA preparations from heteroplasmic NCS2 plants; larger transcripts are associated with the first three nad4 exons. Identical 5' ends of the nad4 transcripts have been mapped 396 and 247 bp upstream of the start codon in mtRNAs from both NCS2 and related non-NCS plants. The putative transcription termination signal of nad4 is deleted in mutant DNA, resulting in the production of the unique longer transcripts. The complex transcript pattern associated with nad7 is also altered in the mutant. Both nad4 and nad7 encode subunits of complex I (NADH dehydrogenase) of the mitochondrial electron transfer chain. Oxygen uptake experiments show that the functioning of complex I is specifically reduced in mitochondria isolated from NCS2 mutant plants. PMID:7851780

  13. "Clocks" in the NAD World: NAD as a metabolic oscillator for the regulation of metabolism and aging.

    PubMed

    Imai, Shin-Ichiro

    2010-08-01

    SIR2 (silent information regulator 2) proteins, now called "sirtuins," are an evolutionarily conserved family of NAD-dependent protein deacetylases/ADP-ribosyltransferases. Sirtuins have recently attracted major attention in the field of aging research, and it has been demonstrated that SIR2 and its orthologs regulate aging and longevity in yeast, worms, and flies. In mammals, the SIR2 ortholog SIRT1 coordinates important metabolic responses to nutritional availability in multiple tissues. Most recently, it has been demonstrated that SIRT1 regulates the amplitude and the duration of circadian gene expression through the interaction and the deacetylation of key circadian clock regulators, such as BMAL1 and PER2. More strikingly, we and others have discovered a novel circadian clock feedback loop in which both the rate-limiting enzyme in mammalian NAD biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT), and NAD levels display circadian oscillations and modulate CLOCK:BMAL1-mediated circadian transcriptional regulation through SIRT1, demonstrating a new function of NAD as a "metabolic oscillator." These findings reveal a novel system dynamics of a recently proposed systemic regulatory network regulated by NAMPT-mediated NAD biosynthesis and SIRT1, namely, the NAD World. In the light of this concept, a new connection between physiological rhythmicity, metabolism, and aging will be discussed.

  14. 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.

  15. Renoprotective effects of aliskiren on adenine-induced tubulointerstitial nephropathy: possible underlying mechanisms.

    PubMed

    Hussein, Abdelaziz M; Malek, Hala Abdel; Saad, Mohamed-Ahdy

    2016-08-01

    The present study investigated the possible renoprotective effect of direct renin inhibitor (aliskiren) on renal dysfunctions, as well as its underlying mechanisms in rat model of adenine-induced tubulointerstitial nephropathy. Forty male Sprague-Dawley rats were randomized into 4 groups; normal group, aliskiren group (normal rats received 10 mg/kg aliskiren), adenine group (animals received high-adenine diet for 4 weeks and saline for 12 weeks), and adenine + aliskiren group (animals received adenine for 4 weeks and aliskiren 10 mg/kg for 12 weeks). It was found that adenine caused significant decrease in body mass, Hb, HR, serum Ca(2+), eNOS and nrf2 expression, GSH, and catalase in kidney tissues with significant increase in arterial blood pressure (ABP), serum creatinine, BUN, plasma renin activity (PRA), K(+) and P, urinary albumin excretion (UAE), caspase-3, and MDA (lipid peroxidation marker) in kidney tissues compared to normal group (p < 0.05). Administration of aliskiren caused significant improvement in all studied parameters compared to adenine group (p < 0.05). We concluded that aliskiren has renoprotective effect against adenine-induced nephropathy. This might be due to inhibition of PRA, attenuation of oxidative stress, activation of Nrf2 and eNOS genes, and suppression of caspase-3.

  16. Comparative genomics of NAD(P) biosynthesis and novel antibiotic drug targets.

    PubMed

    Bi, Jicai; Wang, Honghai; Xie, Jianping

    2011-02-01

    NAD(P) is an indispensable cofactor for all organisms and its biosynthetic pathways are proposed as promising novel antibiotics targets against pathogens such as Mycobacterium tuberculosis. Six NAD(P) biosynthetic pathways were reconstructed by comparative genomics: de novo pathway (Asp), de novo pathway (Try), NmR pathway I (RNK-dependent), NmR pathway II (RNK-independent), Niacin salvage, and Niacin recycling. Three enzymes pivotal to the key reactions of NAD(P) biosynthesis are shared by almost all organisms, that is, NMN/NaMN adenylyltransferase (NMN/NaMNAT), NAD synthetase (NADS), and NAD kinase (NADK). They might serve as ideal broad spectrum antibiotic targets. Studies in M. tuberculosis have in part tested such hypothesis. Three regulatory factors NadR, NiaR, and NrtR, which regulate NAD biosynthesis, have been identified. M. tuberculosis NAD(P) metabolism and regulation thereof, potential drug targets and drug development are summarized in this paper.

  17. NAD homeostasis in the bacterial response to DNA/RNA damage.

    PubMed

    Sorci, Leonardo; Ruggieri, Silverio; Raffaelli, Nadia

    2014-11-01

    In mammals, NAD represents a nodal point for metabolic regulation, and its availability is critical to genome stability. Several NAD-consuming enzymes are induced in various stress conditions and the consequent NAD decline is generally accompanied by the activation of NAD biosynthetic pathways to guarantee NAD homeostasis. In the bacterial world a similar scenario has only recently begun to surface. Here we review the current knowledge on the involvement of NAD homeostasis in bacterial stress response mechanisms. In particular, we focus on the participation of both NAD-consuming enzymes (DNA ligase, mono(ADP-ribosyl) transferase, sirtuins, and RNA 2'-phosphotransferase) and NAD biosynthetic enzymes (both de novo, and recycling enzymes) in the response to DNA/RNA damage. As further supporting evidence for such a link, a genomic context analysis is presented showing several conserved associations between NAD homeostasis and stress responsive genes.

  18. Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

    PubMed

    VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

    2015-11-13

    The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

  19. Major and minor groove conformations of DNA trimers modified on guanine or adenine by 4-aminobiphenyl: Adenine adducts favor the minor groove

    SciTech Connect

    Shapiro, R.; Ellis, S.; Hingerty, B.E.

    1995-01-01

    We have studied the conformational effects of 4-aminobiphenyl modification at C-8 of guanine or adenine on double-stranded DNA trimers. We used sequences with the modified purine at the central base pair and all 16 possible neighboring sequences at the outer pairs. Minimized potential energy calculations were carried out using the molecular mechanics program DUPLEX to survey the conformation space of these adducts, using a total of 1280 starting structures both in the modified guanine series and in the modified adenine series. Conformer families in which the bound 4-aminobiphenyl was located in the DNA major groove, and in the minor groove, were located for both adenine and guanine modification. In the modified guanine series, the major and minor groove families were roughly comparable in energy, and the sequence context determined which was more stable in a particular case. In the modified adenine series, however, the minor groove structure was more that 10 kcal/mol more stable than the major groove for all sequences. As a result, minor groove adducts provided most of the global minima in the adenine-modified series. This result may be relevant to a previous mutagenesis study [Lasko et al. (1988) J. Biol. Chem. 263, 15429-15435] in which the hot spot of most frequent occurrence was located at an adenine, in the sequence GAT. 25 refs., 9 figs., 4 tabs.

  20. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides.

    PubMed

    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.

  1. Vectorette PCR isolation of microsatellite repeat sequences using anchored dinucleotide repeat primers.

    PubMed Central

    Lench, N J; Norris, A; Bailey, A; Booth, A; Markham, A F

    1996-01-01

    We have developed a vectorette PCR approach to provide an improved method for isolation of microsatellite repeats. The modified procedure relies on PCR amplification using a vectorette-specific primer in combination with one of a panel of anchored dinucleotide repeat primers. The target DNA to be screened for microsatellite sequences can be from YAC, P1, cosmid, bacteriophage or plasmid clones. We have used this technique to isolate novel, polymorphic microsatellite repeats from clones containing the amelogenin gene (AMGX) located on human chromosome Xp22.3. PMID:8668553

  2. 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.

  3. Electrochemical characterization of redox polymer modified electrode developed for monitoring of adenine.

    PubMed

    Kuralay, Filiz; Erdem, Arzum; Abacı, Serdar; Ozyörük, Haluk

    2013-05-01

    Electrochemical characterization of redox polymer for monitoring of adenine was described in this study using poly(vinylferrocenium) (PVF(+)) modified platinum (Pt) electrode. Scanning electron microscope (SEM) was used for the surface characterization. The electrochemical behaviors of polymer modified and adenine immobilized polymer modified electrodes were investigated by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). In order to obtain more sensitive and improved electrochemical signals, analytical parameters such as the effects of polymeric film thickness, immobilization time of adenine, pH and adenine concentration were examined on the response of the polymer modified electrode. Alternating current (AC) impedance spectroscopy was used for the characterization of polymer modified and adenine immobilized polymer modified electrodes. The effect of possible interferents on the response of the electrode was examined.

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

    PubMed

    Rajendiran, N; Thulasidhasan, J

    2015-06-05

    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.

  5. Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis

    PubMed Central

    Youn, Hyung-Seop; Gyun Kim, Tae; Kim, Mun-Kyoung; Bu Kang, Gil; Youn Kang, Jung; Lee, Jung-Gyu; Yop An, Jun; Ryoung Park, Kyoung; Lee, Youngjin; Jun Im, Young; Hyuck Lee, Jun; Hyun Eom, Soo

    2016-01-01

    Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear. Here, we present the crystal structures of hexameric human QPRT in the apo form and its complexes with reactant or product. We found that the interaction between dimeric subunits was dramatically altered during the reaction process by conformational changes of two flexible loops in the active site at the dimer-dimer interface. In addition, the N-terminal short helix α1 was identified as a critical hexamer stabilizer. The structural features, size distribution, heat aggregation and ITC studies of the full-length enzyme and the enzyme lacking helix α1 strongly suggest that human QPRT acts as a hexamer for cooperative reactant binding via three dimeric subunits and maintaining stability. Based on our comparison of human QPRT structures in the apo and complex forms, we propose a drug design strategy targeting malignant glioma. PMID:26805589

  6. Dissecting systemic control of metabolism and aging in the NAD World: the importance of SIRT1 and NAMPT-mediated NAD biosynthesis.

    PubMed

    Imai, Shin-ichiro

    2011-06-06

    Many countries are facing social and economic problems due to increased elderly demographics. With these demands, it is now critical to understand the fundamental regulatory mechanism for aging and longevity in mammals. Our studies on the mammalian NAD-dependent deacetylase SIRT1 and nicotinamide phosphoribosyltransferase (NAMPT)-mediated systemic NAD biosynthesis led us to propose a comprehensive model for the systemic regulatory network connecting metabolism and aging, termed the "NAD World". In this article, I will discuss the importance of SIRT1 and NAMPT-mediated NAD biosynthesis in the NAD World and the system dynamics of this hierarchical network for the connection between metabolism and aging.

  7. Gender differences in adenine-induced chronic kidney disease and cardiovascular complications in rats.

    PubMed

    Diwan, Vishal; Small, David; Kauter, Kate; Gobe, Glenda C; Brown, Lindsay

    2014-12-01

    Gender contributes to differences in incidence and progression of chronic kidney disease (CKD) and associated cardiovascular disease. To induce kidney damage in male and female Wistar rats (n = 12/group), a 0.25% adenine diet for 16 wk was used. Kidney function (blood urea nitrogen, plasma creatinine, proteinuria) and structure (glomerular damage, tubulointerstitial atrophy, fibrosis, inflammation); cardiovascular function (blood pressure, ventricular stiffness, vascular responses, echocardiography) and structure (cardiac fibrosis); plasma testosterone and estrogen concentrations; and protein expression for oxidative stress [heme oxygenase-1, inflammation (TNF-α), fibrosis (transforming growth factor-β), ERK1/2, and estrogen receptor-α (ER-α)] were compared in males and females. Adenine-fed females had less decline in kidney function than adenine-fed males, although kidney atrophy, inflammation, and fibrosis were similar. Plasma estrogen concentrations increased and plasma testosterone concentrations decreased in adenine-fed males, with smaller changes in females. CKD-associated molecular changes in kidneys were more pronounced in males than females except for expression of ER-α in the kidney, which was completely suppressed in adenine-fed males but unchanged in adenine-fed females. Both genders showed increased blood pressure, ventricular stiffness, and cardiac fibrosis with the adenine diet. Cardiovascular changes with adenine were similar in males and females, except males developed concentric, and females eccentric cardiac hypertrophy. In hearts from adenine-fed male and female rats, expression of ER-α and activation of the ERK1/2 pathway were increased, in part explaining changes in cardiac hypertrophy. In summary, adenine-induced kidney damage may be increased in males due to the suppression of ER-α.

  8. 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.

  9. Identification of a repressor gene involved in the regulation of NAD de novo biosynthesis in Salmonella typhimurium.

    PubMed Central

    Zhu, N; Olivera, B M; Roth, J R

    1988-01-01

    Mutations at the nadI locus affect expression of the first two genes of NAD synthesis, nadA and nadB, which are unlinked. Genetic data imply that the regulatory effects of nadI mutations are not due to indirect consequences of physiological alterations. Two types of mutations map in the nadI region. Common null mutations (nadI) show constitutive high-level expression of the nadB and nadA genes. Rare nadIs mutations cause constitutive low-level expression of nadB and nadA. Some nadIs mutations shut off the expression of the biosynthetic genes sufficiently to cause a nicotinic acid auxotrophy. Spontaneous revertants of auxotrophic nadIs mutants have a NadI- phenotype, including some with deletions of the nadI locus. The nadI locus encodes a repressor protein acting on the unlinked nadA and nadB genes. PMID:3275606

  10. Evolutionary mechanism and biological functions of 8-mers containing CG dinucleotide in yeast.

    PubMed

    Zheng, Yan; Li, Hong; Wang, Yue; Meng, Hu; Zhang, Qiang; Zhao, Xiaoqing

    2017-02-09

    The rules of k-mer non-random usage and the biological functions are worthy of special attention. Firstly, the article studied human 8-mer spectra and found that only the spectra of cytosine-guanine (CG) dinucleotide classification formed independent unimodal distributions when the 8-mers were classified into three subsets under 16 dinucleotide classifications. Secondly, the distribution rules were reproduced by other seven species including yeast, which showed that the evolution phenomenon had species universality. It followed that we proposed two theoretical conjectures: (1) CG1 motifs (8-mers including 1 CG) are the nucleosome-binding motifs. (2) CG2 motifs (8-mers including two or more than two CG) are the modular units of CpG islands. Our conjectures were confirmed in yeast by the following results: a maximum of average area under the receiver operating characteristic (AUC) resulted from CG1 information during nucleosome core sequences, and linker sequences were distinguished by three CG subsets; there was a one-to-one relationship between abundant CG1 signal regions and histone positions; the sequence changing of squeezed nucleosomes was relevant with the strength of CG1 signals; and the AUC value of 0.986 was based on CG2 information when CpG islands and non-CpG islands were distinguished by the three CG subsets.

  11. CG dinucleotide clustering is a species-specific property of the genome.

    PubMed

    Glass, Jacob L; Thompson, Reid F; Khulan, Batbayar; Figueroa, Maria E; Olivier, Emmanuel N; Oakley, Erin J; Van Zant, Gary; Bouhassira, Eric E; Melnick, Ari; Golden, Aaron; Fazzari, Melissa J; Greally, John M

    2007-01-01

    Cytosines at cytosine-guanine (CG) dinucleotides are the near-exclusive target of DNA methyltransferases in mammalian genomes. Spontaneous deamination of methylcytosine to thymine makes methylated cytosines unusually susceptible to mutation and consequent depletion. The loci where CG dinucleotides remain relatively enriched, presumably due to their unmethylated status during the germ cell cycle, have been referred to as CpG islands. Currently, CpG islands are solely defined by base compositional criteria, allowing annotation of any sequenced genome. Using a novel bioinformatic approach, we show that CG clusters can be identified as an inherent property of genomic sequence without imposing a base compositional a priori assumption. We also show that the CG clusters co-localize in the human genome with hypomethylated loci and annotated transcription start sites to a greater extent than annotations produced by prior CpG island definitions. Moreover, this new approach allows CG clusters to be identified in a species-specific manner, revealing a degree of orthologous conservation that is not revealed by current base compositional approaches. Finally, our approach is able to identify methylating genomes (such as Takifugu rubripes) that lack CG clustering entirely, in which it is inappropriate to annotate CpG islands or CG clusters.

  12. Identification of a Soybean Protein That Interacts with GAGA Element Dinucleotide Repeat DNA1

    PubMed Central

    Sangwan, Indu; O'Brian, Mark R.

    2002-01-01

    Dinucleotide repeat DNA with the pattern (GA)n/(TC)n, so-called GAGA elements, control gene expression in animals, and are recognized by a specific regulatory protein. Here, a yeast one-hybrid screen was used to isolate soybean (Glycine max) cDNA encoding a GAGA-binding protein (GBP) that binds to (GA)n/(CT)n DNA. Soybean GBP was dissimilar from the GAGA factor of Drosophila melanogaster. Recombinant GBP protein did not bind to dinucleotide repeat sequences other than (GA)n/(CT)n. GBP bound to the promoter of the heme and chlorophyll synthesis gene Gsa1, which contains a GAGA element. Removal of that GAGA element abrogated binding of GBP to the promoter. Furthermore, insertion of the GAGA element to a nonspecific DNA conferred GBP-binding activity on that DNA. Thus, the GAGA element of the Gsa1 promoter is both necessary and sufficient for GBP binding. Gbp mRNA was expressed in leaves and was induced in symbiotic root nodules elicited by the bacterium Bradyrhizobium japonicum. In addition, Gbp transcripts were much higher in leaves of dark-treated etiolated plantlets than in those exposed to light for 24 h. Homologs of GBP were found in other dicots and in the monocot rice (Oryza sativa), as well. We suggest that interaction between GAGA elements and GBP-like proteins is a regulatory feature in plants. PMID:12177492

  13. The tungsten formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic for enzymes containing molybdopterin dinucleotide.

    PubMed

    Hochheimer, A; Schmitz, R A; Thauer, R K; Hedderich, R

    1995-12-15

    Formylmethanofuran dehydrogenases are molybdenum or tungsten iron-sulfur proteins containing a pterin dinucleotide cofactor. We report here on the primary structures of the four subunits FwdABCD of the tungsten enzyme from Methanobacterium thermoautotrophicum which were determined by cloning and sequencing the encoding genes fwdABCD. FwdB was found to contain sequence motifs characteristic for molybdopterin-dinucleotide-containing enzymes indicating that this subunit harbors the active site. FwdA, FwdC and FwdD showed no significant sequence similarity to proteins in the data bases. Northern blot analysis revealed that the four fwd genes form a transcription unit together with three additional genes designated fwdE, fwdF and fwdG. A 17.8-kDa protein and an 8.6-kDa protein, both containing two [4Fe-4S] cluster binding motifs, were deduced from fwdE and fwdG. The open reading frame fwdF encodes a 38.6-kDa protein containing eight binding motifs for [4Fe-4S] clusters suggesting the gene product to be a novel polyferredoxin. All seven fwd genes were expressed in Escherichia coli yielding proteins of the expected size. The fwd operon was found to be located in a region of the M. thermoautotrophicum genome encoding molybdenum enzymes and proteins involved in molybdopterin biosynthesis.

  14. Base-boronated dinucleotides: synthesis and effect of N7-cyanoborane substitution on the base protons.

    PubMed Central

    Hasan, A; Li, H; Tomasz, J; Shaw, B R

    1996-01-01

    Boron-modified nucleic acids comprise a new set of DNA mimics that have potential biological and therapeutic applications. A series of nine dinucleotides containing N7-cyanoborane-2'-deoxyguanosine ((7b)dG) at the 3', 5' or both positions of the phosphodiester linkage have been synthesized using solution phase phosphoramidite chemistry. Fmoc was used as the 5'-protecting group because of incompatibility of the cyanoborane moiety with 5'-DMT cations generated during the deprotection step. The presence of the cyanoborane group was confirmed on the basis of Fab-MS and 1H NMR spectroscopy. The H-8 proton of (7b)dG in the dinucleotides shifted 0.35-0.80 p.p.m. downfield relative to that of unmodified dG. A comparison of the D20 exchange kinetics of the H-8 proton at 60 degrees C showed that H-8 of (7b)dG is very labile relative to unmodified dG, indicating that the N7-cyanoborane modification increases the acidity of the H-8 proton of (7b)dG. These studies illustrate the feasibility of synthesizing boron-containing oligonucleotides which are modified at the N7-guanine to block Hoogsteen pairing in the DNA major groove. PMID:8668548

  15. Structure-guided reprogramming of human cGAS dinucleotide linkage specificity.

    PubMed

    Kranzusch, Philip J; Lee, Amy S Y; Wilson, Stephen C; Solovykh, Mikhail S; Vance, Russell E; Berger, James M; Doudna, Jennifer A

    2014-08-28

    Cyclic dinucleotides (CDNs) play central roles in bacterial pathogenesis and innate immunity. The mammalian enzyme cGAS synthesizes a unique cyclic dinucleotide (cGAMP) containing a 2'-5' phosphodiester linkage essential for optimal immune stimulation, but the molecular basis for linkage specificity is unknown. Here, we show that the Vibrio cholerae pathogenicity factor DncV is a prokaryotic cGAS-like enzyme whose activity provides a mechanistic rationale for the unique ability of cGAS to produce 2'-5' cGAMP. Three high-resolution crystal structures show that DncV and human cGAS generate CDNs in sequential reactions that proceed in opposing directions. We explain 2' and 3' linkage specificity and test this model by reprogramming the human cGAS active site to produce 3'-5' cGAMP, leading to selective stimulation of alternative STING adaptor alleles in cells. These results demonstrate mechanistic homology between bacterial signaling and mammalian innate immunity and explain how active site configuration controls linkage chemistry for pathway-specific signaling.

  16. Klebsiella pneumoniae 1,3-propanediol:NAD+ oxidoreductase.

    PubMed Central

    Johnson, E A; Lin, E C

    1987-01-01

    Fermentative utilization of glycerol, a more reduced carbohydrate than aldoses and ketoses, requires the disposal of the two extra hydrogen atoms. This is accomplished by sacrificing an equal quantity of glycerol via an auxiliary pathway initiated by glycerol dehydratase. The product, 3-hydroxypropionaldehyde, is then reduced by 1,3-propanediol NAD+:oxidoreductase (1,3-propanediol dehydrogenase; EC 1.1.1.202), resulting in the regeneration of NAD+ from NADH. The pathway for the assimilation of glycerol is initiated by an NAD-linked dehydrogenase. In Klebsiella pneumoniae the two pathways are encoded by the dha regulon which is inducible only anaerobically. In this study 1,3-propanediol:NAD+ oxidoreductase was purified from cells grown anaerobically on glycerol. The enzyme was immunochemically distinct from the NAD-linked glycerol dehydrogenase and was an octamer or hexamer of a polypeptide of 45,000 +/- 3,000 daltons. When tested as a dehydrogenase, only 1,3-propanediol served as a substrate; no activity was detected with ethanol, 1-propanol, 1,2-propanediol, glycerol, or 1,4-butanediol. The enzyme was inhibited by chelators of divalent cations. An enzyme preparation inhibited by alpha,alpha'-dipyridyl was reactivated by the addition of Fe2+ or Mn2+ after removal of the chelator by gel filtration. As for glycerol dehydrogenase, 1,3-propanediol oxidoreductase is apparently inactivated by oxidation during aerobic metabolism, under which condition the enzyme becomes superfluous. Images PMID:3553154

  17. Structural and functional characterization of human NAD kinase.

    PubMed

    Lerner, F; Niere, M; Ludwig, A; Ziegler, M

    2001-10-19

    NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.

  18. An interbacterial NAD(P)+ glycohydrolase toxin requires elongation factor Tu for delivery to target cells

    SciTech Connect

    Whitney, John C.; Quentin, Dennis; Sawai, Shin; LeRoux, Michele; Harding, Brittany N.; Ledvina, Hannah E.; Tran, Bao Q.; Robinson, Howard; Goo, Young Ah; Goodlett, David R.; Raunser, Stefan; Mougous, Joseph D.

    2015-10-08

    Type VI secretion (T6S) influences the composition of microbial communities by catalyzing the delivery of toxins between adjacent bacterial cells. Here, we demonstrate that a T6S integral membrane toxin from Pseudomonas aeruginosa, Tse6, acts on target cells by degrading the universally essential dinucleotides NAD+ and NADP+. Structural analyses of Tse6 show that it resembles mono-ADP-ribosyltransferase proteins, such as diphtheria toxin, with the exception of a unique loop that both excludes proteinaceous ADP-ribose acceptors and contributes to hydrolysis. We find that entry of Tse6 into target cells requires its binding to an essential housekeeping protein, translation elongation factor Tu (EF-Tu). These proteins participate in a larger assembly that additionally directs toxin export and provides chaperone activity. Lastly, visualization of this complex by electron microscopy defines the architecture of a toxin-loaded T6S apparatus and provides mechanistic insight into intercellular membrane protein delivery between bacteria.

  19. Enzymatic assay for calmodulins based on plant NAD kinase activity

    SciTech Connect

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.

    1984-01-01

    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  20. NAD+ metabolism and the control of energy homeostasis - a balancing act between mitochondria and the nucleus

    PubMed Central

    Cantó, Carles; Menzies, Keir; Auwerx, Johan

    2015-01-01

    NAD+ has emerged as a vital cofactor that can rewire metabolism, activate sirtuins and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD+ metabolism revived interest in NAD+ boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD+ metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited. PMID:26118927

  1. 6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) from slow-growing Rhizobia.

    PubMed Central

    Martínez-Drets, G; Gardiol, A; Arias, A

    1977-01-01

    6-Phospho-D-gluconate:NAD+ 2-oxidoreductase (decarboxylating) (NAD+-6PGD) was detected in several slow-growing strains of rhizobia, and no activity involving NADP+ was found in the same extracts. By contrast, fast-growing strains of rhizobia had NADP+-6PGD activity; most of them also had NAD+-6PGD activity. NAD+-6PGD was partially purified from the slow-growing strain Rhizobium japonicum 5006. The reaction was shown to be an oxidative decarboxylation. PMID:16867

  2. NAD(+) Metabolism and the Control of Energy Homeostasis: A Balancing Act between Mitochondria and the Nucleus.

    PubMed

    Cantó, Carles; Menzies, Keir J; Auwerx, Johan

    2015-07-07

    NAD(+) has emerged as a vital cofactor that can rewire metabolism, activate sirtuins, and maintain mitochondrial fitness through mechanisms such as the mitochondrial unfolded protein response. This improved understanding of NAD(+) metabolism revived interest in NAD(+)-boosting strategies to manage a wide spectrum of diseases, ranging from diabetes to cancer. In this review, we summarize how NAD(+) metabolism links energy status with adaptive cellular and organismal responses and how this knowledge can be therapeutically exploited.

  3. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans.

    PubMed

    Trammell, Samuel A J; Schmidt, Mark S; Weidemann, Benjamin J; Redpath, Philip; Jaksch, Frank; Dellinger, Ryan W; Li, Zhonggang; Abel, E Dale; Migaud, Marie E; Brenner, Charles

    2016-10-10

    Nicotinamide riboside (NR) is in wide use as an NAD(+) precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD(+) metabolism in humans. We report that human blood NAD(+) can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD(+) with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD(+) metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD(+), is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD(+) repletion.

  4. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans

    PubMed Central

    Trammell, Samuel A. J.; Schmidt, Mark S.; Weidemann, Benjamin J.; Redpath, Philip; Jaksch, Frank; Dellinger, Ryan W.; Li, Zhonggang; Abel, E. Dale; Migaud, Marie E.; Brenner, Charles

    2016-01-01

    Nicotinamide riboside (NR) is in wide use as an NAD+ precursor vitamin. Here we determine the time and dose-dependent effects of NR on blood NAD+ metabolism in humans. We report that human blood NAD+ can rise as much as 2.7-fold with a single oral dose of NR in a pilot study of one individual, and that oral NR elevates mouse hepatic NAD+ with distinct and superior pharmacokinetics to those of nicotinic acid and nicotinamide. We further show that single doses of 100, 300 and 1,000 mg of NR produce dose-dependent increases in the blood NAD+ metabolome in the first clinical trial of NR pharmacokinetics in humans. We also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en route for the conversion of NR to NAD+, is formed from NR and discover that the rise in NAAD is a highly sensitive biomarker of effective NAD+ repletion. PMID:27721479

  5. N-Alkane oxidation enzymes of a pseudomonad.

    PubMed Central

    Parekh, V R; Traxler, R W; Sobek, J M

    1977-01-01

    A nicotinamide adenine dinucleotide (NAD)-dependent n-alkane dehydrogenase and an NAD phosphate (reduced form)-dependent alkane hydroxylase have been purified from cell-free extracts of Pseudomonas sp. strain 196Aa grown anaerobically on n-alkane. The n-alkane dehydrogenase (fraction R-3), obtained as a single peak from Bio-Gel P-60, showed an overall 135-fold purification and was demonstrated by infrared spectroscopy and gas chromatography to convert n-decane to 1-decene. The alkene hydroxylase activity in the S-3 fraction, purified 167 times from diethylaminoethyl-cellulose, was shown by the same methodology to convert decene to decanol. Commercial ferredoxin has been shown to increase the alkane dehydrogenase activity. An NAD-, flavine adenine dinucleotide-, and iron-dependent alcohol dehydrogenase was demonstrated in the R-3 fraction. A mechanism for the anaerobic conversion of n-alkane to fatty acid has been proposed. PMID:869535

  6. Slow deactivation channels in UV-photoexcited adenine DNA.

    PubMed

    Chen, Xuebo; Fang, Weihai; Wang, Haobin

    2014-03-07

    The molecular mechanism for removing the excess energy in DNA bases is responsible for the high photostability of DNA and is thus the subject of intense theoretical/computational investigation. To understand why the excited state decay of the stacked bases is significantly longer than that of the monomers, we carried out electronic structure calculations on an adenine monomer and an aqueous (dA)5 oligonucleotide employing the CASPT2//CASSCF and CASPT2//CASSCF/AMBER levels of theory. The newly-found bright excited state pair Sstack1((1)ππ*) and Sstack2((1)ππ*) of d(A)5, originated from base stacking, is of intra-base charge transfer nature and occurs in different stacked bases with charge transfer along opposite directions. Two slow deactivation channels of d(A)5 were proposed as a result of the sizable barriers along the relaxation paths starting from the FC point of the Sstack1((1)ππ*) state. The SN1P((1)nπ*) state of d(A)5 serves as an intermediate state in one relaxation channel, to which a nonadiabatic decay from the Sstack1((1)ππ*) state occurs in an energy degeneracy region. A relatively high barrier in this state is found and attributed to the steric hindrance of the DNA environment due to the large NH2 group twisting, which gives a weak and red-shifted fluorescence. Another direct relaxation channel, induced by the C2-H2 bond twisting motion, is found to go through a conical intersection between the Sstack1((1)ππ*) and the ground state. The barrier found here enables fluorescence from the Sstack1((1)ππ*) state and may explain the bright state emission observed in the fluorescence upconversion measurements. The inter-molecular SCT((1)ππ*) state may be involved in the slow relaxation process of the photoexcited adenine oligomers through efficient internal conversion to the intra-base Sstack1((1)ππ*) state.

  7. The NAD/NARB System: Advertising Self-Regulation at Work.

    ERIC Educational Resources Information Center

    Hays, Robert

    Self-regulation, as defined by the National Advertising Division/National Advertising Review Board (NAD/NARB), is a process whereby the advertising industry regulates itself and turns to the federal government only if the system fails. The NAD/NARB system involves a two-step process: complaints are initially handled by the NAD and then are either…

  8. Regulation of SIRT2-dependent α-tubulin deacetylation by cellular NAD levels.

    PubMed

    Skoge, Renate Hvidsten; Dölle, Christian; Ziegler, Mathias

    2014-11-01

    Acetylation of α-tubulin on lysine 40 is one of the major posttranslational modifications of microtubules. The acetylation reaction is catalyzed by alpha-tubulin N-acetyltransferase and the modification can be reversed by either the NAD-independent class II histone deacetylase HDAC6 or the NAD-dependent deacetylase SIRT2. In this study, we assessed to what extent cellular NAD levels are involved in the regulation of the α-tubulin acetylation state. Cells were subjected to different treatments known to influence cellular NAD content. In response to NAD depletion caused by inhibition of NAD synthesis from nicotinamide, α-tubulin was hyperacetylated. Under these conditions, the normal tubulin acetylation state could be restored by providing the cells with alternative NAD precursors. Likewise, decreasing the rate of endogenous NAD consumption using an inhibitor of poly-ADP-ribosylation also stabilized the acetylation of α-tubulin. Conversely, the level of acetylated α-tubulin decreased when NAD synthesis was enhanced by overexpression of an NAD biosynthetic enzyme. Combined, these results show that the tubulin acetylation status is reciprocally regulated by cellular NAD levels. Furthermore, we provide evidence confirming that the NAD-dependent regulation of tubulin acetylation is mediated by SIRT2.

  9. Design of laser pulses for selective vibrational excitation of the N6-H bond of adenine and adenine-thymine base pair using optimal control theory.

    PubMed

    Sharma, Sitansh; Sharma, Purshotam; Singh, Harjinder; Balint-Kurti, Gabriel G

    2009-06-01

    Time dependent quantum dynamics and optimal control theory are used for selective vibrational excitation of the N6-H (amino N-H) bond in free adenine and in the adenine-thymine (A-T) base pair. For the N6-H bond in free adenine we have used a one dimensional model while for the hydrogen bond, N6-H(A)...O4(T), present in the A-T base pair, a two mathematical dimensional model is employed. The conjugate gradient method is used for the optimization of the field dependent cost functional. Optimal laser fields are obtained for selective population transfer in both the model systems, which give virtually 100% excitation probability to preselected vibrational levels. The effect of the optimized laser field on the other hydrogen bond, N1(A)...H-N3(T), present in A-T base pair is also investigated.

  10. 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

  11. Radiolysis of aqueous adenine (vitamin B4) and 8-hydroxyadenine

    NASA Astrophysics Data System (ADS)

    Hartmann, J.; Quint, R. M.; Getoff, N.

    2007-05-01

    The radiolysis of adenine (vitamin B4) was studied in aqueous solution (pH˜7.4) saturated either with argon (operating radicals: 44% e -aq, 46% OH, 10% H) or with air (46% OH, 54% O 2rad - ) and with N 2O (90% OH, 10% H), respectively. The obtained initial Gi-values are: 0.88, 1.16 and 1.45. As main radiolytic product was determined 8-hydroxyadenine (8-HOA), whose yield depends on the OH concentration in the reacting media. Hence, under the same experimental conditions the Gi-values are in media saturated with argon: 0.1, in air: 0.15 and in N 2O: 0.29. In aerated solution also a mixture of aldehydes as well as of carboxylic acids were formed, but they were not identified. 8-HOA is of some biological interest; therefore, its radiolysis was also investigated under the same conditions. The determined Gi(-8HOA)-values were in airfree solution negligible, in aerated solutions: 3.1 and in the presence of N 2O: 4.0. For explanation of the product formation some probable reaction mechanisms were given.

  12. 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.

  13. Extracellular NAD(+): a danger signal hindering regulatory T cells.

    PubMed

    Adriouch, Sahil; Haag, Friedrich; Boyer, Olivier; Seman, Michel; Koch-Nolte, Friedrich

    2012-11-01

    Endogenous danger signals released during cell damage contribute to alert the immune system. Typically, their release results in the activation and maturation of innate immune cells, and the production of pro-inflammatory cytokines. In addition, extracellular NAD(+) stimulates immune responses by hindering regulatory T cells (Tregs), and could, therefore, represent the prototype of a new category of danger signals.

  14. In vitro metabolic engineering for the salvage synthesis of NAD(.).

    PubMed

    Honda, Kohsuke; Hara, Naoya; Cheng, Maria; Nakamura, Anna; Mandai, Komako; Okano, Kenji; Ohtake, Hisao

    2016-05-01

    Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat-treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60°C, the NAD(+) concentration could be kept almost constant for 15h.

  15. 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.

  16. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays.

    PubMed

    Aleshin, Vasily A; Artiukhov, Artem V; Oppermann, Henry; Kazantsev, Alexey V; Lukashev, Nikolay V; Bunik, Victoria I

    2015-08-21

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay.

  17. Mitochondrial Impairment May Increase Cellular NAD(P)H: Resazurin Oxidoreductase Activity, Perturbing the NAD(P)H-Based Viability Assays

    PubMed Central

    Aleshin, Vasily A.; Artiukhov, Artem V.; Oppermann, Henry; Kazantsev, Alexey V.; Lukashev, Nikolay V.; Bunik, Victoria I.

    2015-01-01

    Cellular NAD(P)H-dependent oxidoreductase activity with artificial dyes (NAD(P)H-OR) is an indicator of viability, as the cellular redox state is important for biosynthesis and antioxidant defense. However, high NAD(P)H due to impaired mitochondrial oxidation, known as reductive stress, should increase NAD(P)H-OR yet perturb viability. To better understand this complex behavior, we assayed NAD(P)H-OR with resazurin (Alamar Blue) in glioblastoma cell lines U87 and T98G, treated with inhibitors of central metabolism, oxythiamin, and phosphonate analogs of 2-oxo acids. Targeting the thiamin diphosphate (ThDP)-dependent enzymes, the inhibitors are known to decrease the NAD(P)H production in the pentose phosphate shuttle and/or upon mitochondrial oxidation of 2-oxo acids. Nevertheless, the inhibitors elevated NAD(P)H-OR with resazurin in a time- and concentration-dependent manner, suggesting impaired NAD(P)H oxidation rather than increased viability. In particular, inhibition of the ThDP-dependent enzymes affects metabolism of malate, which mediates mitochondrial oxidation of cytosolic NAD(P)H. We showed that oxythiamin not only inhibited mitochondrial 2-oxo acid dehydrogenases, but also induced cell-specific changes in glutamate and malate dehydrogenases and/or malic enzyme. As a result, inhibition of the 2-oxo acid dehydrogenases compromises mitochondrial metabolism, with the dysregulated electron fluxes leading to increases in cellular NAD(P)H-OR. Perturbed mitochondrial oxidation of NAD(P)H may thus complicate the NAD(P)H-based viability assay. PMID:26308058

  18. Structural and biochemical characterization of linear dinucleotide analogs bound to the c-di-GMP-I aptamer†,‡

    PubMed Central

    Smith, Kathryn D.; Lipchock, Sarah V.; Strobel, Scott A.

    2011-01-01

    The cyclic dinucleotide c-di-GMP regulates lifestyle transitions in many bacteria, such as the change from a free motile state to a biofilm-forming community. Riboswitches that bind this second messenger are important downstream targets in this bacterial signaling pathway. The breakdown of c-di-GMP in the cell is accomplished enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding riboswitches must be able to discriminate between their cognate cyclic ligand and linear dinucleotides in order to be selective biological switches. It has been reported that the cdi-GMP-I riboswitch binds c-di-GMP five orders of magnitude better than the linear pGpG, but the cause of this large energetic difference in binding is unknown. Here we report binding data and crystal structures of several linear c-di-GMP analogs in complex with the c-di-GMP-I riboswitch. These data reveal the parameters for phosphate recognition and the structural basis of linear dinucleotide binding to the riboswitch. Additionally, the pH dependence of binding shows that exclusion of pGpG is not due to the additional negative charge on the ligand. These data reveal principles that, along with published work, will contribute to the design of c-di-GMP analogs with properties desirable for use as chemical tools and potential therapeutics. PMID:22148472

  19. Neutrophil gelatinase-associated lipocalin in a triphasic rat model of adenine-induced kidney injury.

    PubMed

    Gil, Amnon; Brod, Vera; Awad, Hoda; Heyman, Samuel N; Abassi, Zaid; Frajewicki, Victor

    2016-10-01

    The aim of this study is to investigate whether NGAL, given its advantages over traditional biomarkers, can be used to describe the dynamic characteristics of the renal tubulointerstitial insult caused by adenine. Subsequently, it will be possible to assess NGAL as a biomarker of any acute kidney injury, on top of chronic interstitial disease, if NGAL levels are stable through the chronic phase of our adenine model. Study group rats were fed an adenine diet, and control group rats were fed a regular diet only. Blood and urine samples for urea, creatinine and NGAL were drawn from each rat at the beginning of the study and after 1, 3, 4, 5, 6, 7 and 8 weeks. Kidney slices from these rats were stained with Hematoxylin-eosin (HE) and β-actin stainings. Serum urea, creatinine and NGAL levels and urinary NGAL/creatinine ratio in the study group were higher than baseline and than in the control group; these differences were statistically significant in some of the intervals. Tubulointerstitial changes and adenine crystals were evident in the study group rats. In the rats fed adenine, serum urea, creatinine and NGAL levels and urinary NGAL/creatinine ratio followed a triphasic pattern of kidney injury: an acute phase while on the adenine diet, a partial recovery phase after switching to the regular diet and a chronic kidney disease phase after stabilization of renal function. NGAL can serve a biomarker for acute kidney injury and possibly for chronic kidney disease in the tubulointerstitial rat model.

  20. Improved growth and stress tolerance in the Arabidopsis oxt1 mutant triggered by altered adenine metabolism.

    PubMed

    Sukrong, Suchada; Yun, Kil-Young; Stadler, Patrizia; Kumar, Charan; Facciuolo, Tony; Moffatt, Barbara A; Falcone, Deane L

    2012-11-01

    Plants perceive and respond to environmental stresses with complex mechanisms that are often associated with the activation of antioxidant defenses. A genetic screen aimed at isolating oxidative stress-tolerant lines of Arabidopsis thaliana has identified oxt1, a line that exhibits improved tolerance to oxidative stress and elevated temperature but displays no apparent deleterious growth effects under non-stress conditions. Oxt1 harbors a mutation that arises from the altered expression of a gene encoding adenine phosphoribosyltransferase (APT1), an enzyme that converts adenine to adenosine monophosphate (AMP), indicating a link between purine metabolism, whole-plant growth responses, and stress acclimation. The oxt1 mutation results in decreased APT1 expression that leads to reduced enzymatic activity. Correspondingly, oxt1 plants possess elevated levels of adenine. Decreased APT enzyme activity directly correlates with stress resistance in transgenic lines that ectopically express APT1. The metabolic alteration in oxt1 plants also alters the expression of several antioxidant defense genes and the response of these genes to oxidative challenge. Finally, it is shown that manipulation of adenine levels can induce stress tolerance to wild-type plants. Collectively, these results show that alterations in cellular adenine levels can trigger stress tolerance and improve growth, leading to increases in plant biomass. The results also suggest that adenine might play a part in the signals that modulate responses to abiotic stress and plant growth.

  1. 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.

  2. NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion

    PubMed Central

    Sasaki, Yo; Nakagawa, Takashi; Mao, Xianrong; DiAntonio, Aaron; Milbrandt, Jeffrey

    2016-01-01

    Overexpression of the NAD+ biosynthetic enzyme NMNAT1 leads to preservation of injured axons. While increased NAD+ or decreased NMN levels are thought to be critical to this process, the mechanism(s) of this axon protection remain obscure. Using steady-state and flux analysis of NAD+ metabolites in healthy and injured mouse dorsal root ganglion axons, we find that rather than altering NAD+ synthesis, NMNAT1 instead blocks the injury-induced, SARM1-dependent NAD+ consumption that is central to axon degeneration. DOI: http://dx.doi.org/10.7554/eLife.19749.001 PMID:27735788

  3. Molecular Basis of Ligand-Dependent Regulation of NadR, the Transcriptional Repressor of Meningococcal Virulence Factor NadA

    PubMed Central

    Liguori, Alessia; Malito, Enrico; Lo Surdo, Paola; Fagnocchi, Luca; Cantini, Francesca; Haag, Andreas F.; Brier, Sébastien; Pizza, Mariagrazia; Delany, Isabel; Bottomley, Matthew J.

    2016-01-01

    Neisseria adhesin A (NadA) is present on the meningococcal surface and contributes to adhesion to and invasion of human cells. NadA is also one of three recombinant antigens in the recently-approved Bexsero vaccine, which protects against serogroup B meningococcus. The amount of NadA on the bacterial surface is of direct relevance in the constant battle of host-pathogen interactions: it influences the ability of the pathogen to engage human cell surface-exposed receptors and, conversely, the bacterial susceptibility to the antibody-mediated immune response. It is therefore important to understand the mechanisms which regulate nadA expression levels, which are predominantly controlled by the transcriptional regulator NadR (Neisseria adhesin A Regulator) both in vitro and in vivo. NadR binds the nadA promoter and represses gene transcription. In the presence of 4-hydroxyphenylacetate (4-HPA), a catabolite present in human saliva both under physiological conditions and during bacterial infection, the binding of NadR to the nadA promoter is attenuated and nadA expression is induced. NadR also mediates ligand-dependent regulation of many other meningococcal genes, for example the highly-conserved multiple adhesin family (maf) genes, which encode proteins emerging with important roles in host-pathogen interactions, immune evasion and niche adaptation. To gain insights into the regulation of NadR mediated by 4-HPA, we combined structural, biochemical, and mutagenesis studies. In particular, two new crystal structures of ligand-free and ligand-bound NadR revealed (i) the molecular basis of ‘conformational selection’ by which a single molecule of 4-HPA binds and stabilizes dimeric NadR in a conformation unsuitable for DNA-binding, (ii) molecular explanations for the binding specificities of different hydroxyphenylacetate ligands, including 3Cl,4-HPA which is produced during inflammation, (iii) the presence of a leucine residue essential for dimerization and conserved in

  4. 6-Aminonicotinamide-resistant mutants of Salmonella typhimurium.

    PubMed Central

    Hughes, K T; Cookson, B T; Ladika, D; Olivera, B M; Roth, J R

    1983-01-01

    Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD. PMID:6222034

  5. Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

    PubMed

    Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

    2015-01-16

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.

  6. Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism*

    PubMed Central

    Frederick, David W.; Davis, James G.; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A.; Nakamaru-Ogiso, Eiko; Baur, Joseph A.

    2015-01-01

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

  7. Thiaminylated adenine nucleotides. Chemical synthesis, structural characterization and natural occurrence.

    PubMed

    Frédérich, Michel; Delvaux, David; Gigliobianco, Tiziana; Gangolf, Marjorie; Dive, Georges; Mazzucchelli, Gabriel; Elias, Benjamin; De Pauw, Edwin; Angenot, Luc; Wins, Pierre; Bettendorff, Lucien

    2009-06-01

    Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.

  8. 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.

  9. 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

  10. Hairpin stabilized fluorescent silver nanoclusters for quantitative detection of NAD(+) and monitoring NAD(+)/NADH based enzymatic reactions.

    PubMed

    Jain, Priyamvada; Chakma, Babina; Patra, Sanjukta; Goswami, Pranab

    2017-03-01

    A set of 90 mer long ssDNA candidates, with different degrees of cytosine (C-levels) (% and clusters) was analyzed for their function as suitable Ag-nanocluster (AgNC) nucleation scaffolds. The sequence (P4) with highest C-level (42.2%) emerged as the only candidate supporting the nucleation process as evident from its intense fluorescence peak at λ660 nm. Shorter DNA subsets derived from P4 with only stable hairpin structures could support the AgNC formation. The secondary hairpin structures were confirmed by PAGE, and CD studies. The number of base pairs in the stem region also contributes to the stability of the hairpins. A shorter 29 mer sequence (Sub 3) (ΔG = -1.3 kcal/mol) with 3-bp in the stem of a 7-mer loop conferred highly stable AgNC. NAD(+) strongly quenched the fluorescence of Sub 3-AgNC in a concentration dependent manner. Time resolved photoluminescence studies revealed the quenching involves a combined static and dynamic interaction where the binding constant and number of binding sites for NAD(+) were 0.201 L mol(-1) and 3.6, respectively. A dynamic NAD(+) detection range of 50-500 μM with a limit of detection of 22.3 μM was discerned. The NAD(+) mediated quenching of AgNC was not interfered by NADH, NADP(+), monovalent and divalent ions, or serum samples. The method was also used to follow alcohol dehydrogenase and lactate dehydrogenase catalyzed physiological reactions in a turn-on and turn-off assay, respectively. The proposed method with ssDNA-AgNC could therefore be extended to monitor other NAD(+)/NADH based enzyme catalyzed reactions in a turn-on/turn-off approach.

  11. NAD Acts as an Integral Regulator of Multiple Defense Layers1[OPEN

    PubMed Central

    Patrit, Oriane; Tcherkez, Guillaume; Gakière, Bertrand

    2016-01-01

    Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea. Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens. PMID:27621425

  12. Regulation of NAD+ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae.

    PubMed

    Kato, Michiko; Lin, Su-Ju

    2014-11-01

    Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD(+) is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD(+) homeostasis is essential for proper cellular function and aberrant NAD(+) metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD(+) metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD(+) metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD(+) metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD(+) metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD(+) metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD(+)-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD(+) intermediates, and their potential roles in NAD(+) homeostasis. To date, it remains unclear how NAD(+) and NAD(+) intermediates

  13. Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism.

    PubMed

    Gossmann, Toni I; Ziegler, Mathias

    2014-11-01

    NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification.

  14. Chemical probing of adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA

    SciTech Connect

    Rairkar, A.; Rubino, H.M.; Lockard, R.E.

    1988-01-26

    The location of unpaired adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA was determined by chemical probing. Naked /sup 18/S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked /sup 18/S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of /sup 32/P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in /sup 18/S rRNA determined by comparative sequence analysis. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit /sup 18/S rRNA than for E. coli /sup 16/S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian /sup 18/S rRNA, as compared with prokaryotic /sup 16/S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

  15. Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

    PubMed

    Estill, Molly; Kerwin-Iosue, Christine L; Wykoff, Dennis D

    2015-05-01

    In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

  16. Glibenclamide improves kidney and heart structure and function in the adenine-diet model of chronic kidney disease.

    PubMed

    Diwan, Vishal; Gobe, Glenda; Brown, Lindsay

    2014-01-01

    The development of chronic kidney disease (CKD) and associated cardiovascular disease involves free radical damage and inflammation. Addition of adenine to the diet induces inflammation followed by CKD and cardiovascular disease. NOD-like receptor protein-3 (NLRP-3) is pro-inflammatory in the kidney; glibenclamide inhibits production of NLRP-3. Male Wistar rats were fed either control rat food or adenine (0.25%) in this food for 16 weeks. Glibenclamide (10 mg/kg/day) was administered to two groups with and without adenine for the final 8 weeks. Kidney function (blood urea nitrogen/BUN, plasma creatinine/PCr, plasma uric acid, proteinuria), kidney structure (fibrosis, inflammation), cardiovascular parameters (blood pressure, left ventricular stiffness, vascular responses and echocardiography) and protein expression of markers for oxidative stress (HO-1), and inflammation (TNF-α, NLRP-3) were assessed. In adenine-fed rats, glibenclamide decreased BUN (controls: 6±0.6; adenine: 56.6±5.4; adenine+glibenclamide: 19.4±2.7 mmol/L), PCr (controls: 42±2.8; adenine: 268±23; adenine+glibenclamide: 81±10 μmol/L), proteinuria (controls: 150±7.4; adenine: 303±19; adenine+glibenclamide: 220±13 μmol/L) (all p<0.05). Glibenclamide decreased infiltration of chronic inflammatory cells, fibrosis, tubular damage and expression of HO-1, TNF-α and NLRP-3 in the kidney. Glibenclamide did not alter plasma uric acid concentrations (controls: 38±1; adenine: 63±4; adenine+glibenclamide: 69±14 μmol/L). Cardiovascular changes included decreased systolic blood pressure and improved vascular responses although cardiac fibrosis, left ventricular stiffness and hypertrophy were not reduced. Glibenclamide improved kidney structure and function in CKD and decreased some cardiovascular parameters. Inflammatory markers and cell populations were attenuated by glibenclamide in kidneys.

  17. The NAD World: a new systemic regulatory network for metabolism and aging--Sirt1, systemic NAD biosynthesis, and their importance.

    PubMed

    Imai, Shin-Ichiro

    2009-01-01

    For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named "NAD World", for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular Nampt functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.

  18. Evolution of hypervariable microsatellites in apomictic polyploid lineages of Ranunculus carpaticola: directional bias at dinucleotide loci.

    PubMed

    Paun, Ovidiu; Hörandl, Elvira

    2006-09-01

    Microsatellites are widely used in genetic and evolutionary analyses, but their own evolution is far from simple. The mechanisms maintaining the mutational patterns of simple repeats and the typical stable allele-frequency distributions are still poorly understood. Asexual lineages may provide particularly informative models for the indirect study of microsatellite evolution, because their genomes act as complete linkage groups, with mutations being the only source of genetic variation. Here, we study the direction of accumulated dinucleotide microsatellite mutations in wild asexual lineages of hexaploid Ranunculus carpaticola. Whereas the overall number of contractions is not significantly different from that of expansions, the within-locus frequency of contractions, but not of expansions, significantly increases with allele length. Moreover, within-locus polymorphism is positively correlated with allele length, but this relationship is due solely to the influence of contraction mutations. Such asymmetries may explain length constraints generally observed with microsatellites and are consistent with stable, bell-shaped allele-frequency distributions. Although apomictic and allohexaploid, the R. carpaticola lineages show mutational patterns resembling the trends observed in a broad range of organisms, including sexuals and diploids, suggesting that, even if not of germline origin, the mutations in these apomicts may be the consequence of similar mechanisms.

  19. Detection of mercury-TpT dinucleotide binding by Raman spectra: a computational study.

    PubMed

    Benda, Ladislav; Straka, Michal; Sychrovský, Vladimír; Bouř, Petr; Tanaka, Yoshiyuki

    2012-08-16

    The Hg(2+) ion stabilizes the thymine-thymine mismatched base pair and provides new ways of creating various DNA structures. Recently, such T-Hg-T binding was detected by the Raman spectroscopy. In this work, detailed differences in vibrational frequencies and Raman intensity patterns in the free TpT dinucleotide and its metal-mediated complex (TpT·Hg)(2) are interpreted on the basis of quantum chemical modeling. The computations verified specific marker Raman bands indicating the effect of mercury binding to DNA. Although the B3LYP functional well-describes the Raman frequencies, a dispersion correction had to be added for all atoms including mercury to obtain realistic geometry of the (TpT·Hg)(2) dimer. Only then, the DFT complex structure agreed with those obtained with the wave function-based MP2 method. The aqueous solvent modeled as a polarizable continuum had a minor effect on the dispersion interaction, but it stabilized conformations of the sugar and phosphate parts. A generalized definition of internal coordinate force field was introduced to monitor covalent bond mechanical strengthening and weakening upon the Hg(2+) binding. Induced vibrational frequency shifts were rationalized in terms of changes in electronic structure. The simulations thus also provided reliable insight into the complex structure and stability.

  20. 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

    2016-01-22

    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/.

  1. 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/.

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

    PubMed Central

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

    2014-01-01

    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

  3. Sources and implications of NADH/NAD+ redox imbalance in diabetes and its complications

    PubMed Central

    Wu, Jinzi; Jin, Zhen; Zheng, Hong; Yan, Liang-Jun

    2016-01-01

    NAD+ is a fundamental molecule in metabolism and redox signaling. In diabetes and its complications, the balance between NADH and NAD+ can be severely perturbed. On one hand, NADH is overproduced due to influx of hyperglycemia to the glycolytic and Krebs cycle pathways and activation of the polyol pathway. On the other hand, NAD+ can be diminished or depleted by overactivation of poly ADP ribose polymerase that uses NAD+ as its substrate. Moreover, sirtuins, another class of enzymes that also use NAD+ as their substrate for catalyzing protein deacetylation reactions, can also affect cellular content of NAD+. Impairment of NAD+ regeneration enzymes such as lactate dehydrogenase in erythrocytes and complex I in mitochondria can also contribute to NADH accumulation and NAD+ deficiency. The consequence of NADH/NAD+ redox imbalance is initially reductive stress that eventually leads to oxidative stress and oxidative damage to macromolecules, including DNA, lipids, and proteins. Accordingly, redox imbalance-triggered oxidative damage has been thought to be a major factor contributing to the development of diabetes and its complications. Future studies on restoring NADH/NAD+ redox balance could provide further insights into design of novel antidiabetic strategies. PMID:27274295

  4. Sources and implications of NADH/NAD(+) redox imbalance in diabetes and its complications.

    PubMed

    Wu, Jinzi; Jin, Zhen; Zheng, Hong; Yan, Liang-Jun

    2016-01-01

    NAD(+) is a fundamental molecule in metabolism and redox signaling. In diabetes and its complications, the balance between NADH and NAD(+) can be severely perturbed. On one hand, NADH is overproduced due to influx of hyperglycemia to the glycolytic and Krebs cycle pathways and activation of the polyol pathway. On the other hand, NAD(+) can be diminished or depleted by overactivation of poly ADP ribose polymerase that uses NAD(+) as its substrate. Moreover, sirtuins, another class of enzymes that also use NAD(+) as their substrate for catalyzing protein deacetylation reactions, can also affect cellular content of NAD(+). Impairment of NAD(+) regeneration enzymes such as lactate dehydrogenase in erythrocytes and complex I in mitochondria can also contribute to NADH accumulation and NAD(+) deficiency. The consequence of NADH/NAD(+) redox imbalance is initially reductive stress that eventually leads to oxidative stress and oxidative damage to macromolecules, including DNA, lipids, and proteins. Accordingly, redox imbalance-triggered oxidative damage has been thought to be a major factor contributing to the development of diabetes and its complications. Future studies on restoring NADH/NAD(+) redox balance could provide further insights into design of novel antidiabetic strategies.

  5. 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

  6. 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...

  7. The metabolic fate of the products of citrate cleavage. Adenosine triphosphate citrate lyase and nicotinamide–adenine dinucleotide phosphate-linked malate dehydrogenase in foetal and adult liver from ruminants and non-ruminants

    PubMed Central

    Hanson, R. W.; Ballard, F. J.

    1968-01-01

    1. Foetal rat liver slices incorporate the C-3 of aspartate and C-2 of glutamate into fatty acids at rates equal to those observed with adult rat liver slices. Incorporation of either of these labelled carbon atoms into fatty acids would require a functioning citrate-cleavage pathway which consists of the enzymes ATP–citrate lyase, NAD–malate dehydrogenase and NADP–malate dehydrogenase. However, NADP–malate dehydrogenase is present in foetal rat liver at only 5% of the activity detectable in adult rat liver. 2. From these findings and the effect of cofactors on the formation of 14CO2 from [1,5-14C2]citrate in liver supernatant fractions (100000g), it is suggested that NADP–malate dehydrogenase limits the citrate-cleavage sequence. 3. Measurement of the citrate-cleavage pathway by incorporation studies with [3-14C]aspartate and [U-14C]glucose and by determining the activities of ATP–citrate lyase and NADP–malate dehydrogenase have shown that this sequence of reactions is present in the liver of the bovine foetus but not in the adult. However, C-2 of glutamate is not incorporated into fatty acids or non-saponifiable lipid by bovine liver slices. This finding as well as those presented above for the adult and foetal rat liver are interpreted on the basis of a competition between phosphoenolpyruvate carboxykinase and NAD–malate dehydrogenase for oxaloacetate produced by the cleavage of citrate in the cytosol. PMID:4386407

  8. Activation of SIRT3 by the NAD⁺ precursor nicotinamide riboside protects from noise-induced hearing loss.

    PubMed

    Brown, Kevin D; Maqsood, Sadia; Huang, Jing-Yi; Pan, Yong; Harkcom, William; Li, Wei; Sauve, Anthony; Verdin, Eric; Jaffrey, Samie R

    2014-12-02

    Intense noise exposure causes hearing loss by inducing degeneration of spiral ganglia neurites that innervate cochlear hair cells. Nicotinamide adenine dinucleotide (NAD(+)) exhibits axon-protective effects in cultured neurons; however, its ability to block degeneration in vivo has been difficult to establish due to its poor cell permeability and serum instability. Here, we describe a strategy to increase cochlear NAD(+) levels in mice by administering nicotinamide riboside (NR), a recently described NAD(+) precursor. We find that administration of NR, even after noise exposure, prevents noise-induced hearing loss (NIHL) and spiral ganglia neurite degeneration. These effects are mediated by the NAD(+)-dependent mitochondrial sirtuin, SIRT3, since SIRT3-overexpressing mice are resistant to NIHL and SIRT3 deletion abrogates the protective effects of NR and expression of NAD(+) biosynthetic enzymes. These findings reveal that administration of NR activates a NAD(+)-SIRT3 pathway that reduces neurite degeneration caused by noise exposure.

  9. The Circadian NAD+ Metabolism: Impact on Chromatin Remodeling and Aging

    PubMed Central

    Bessho, Yasumasa

    2016-01-01

    Gene expression is known to be a stochastic phenomenon. The stochastic gene expression rate is thought to be altered by topological change of chromosome and/or by chromatin modifications such as acetylation and methylation. Changes in mechanical properties of chromosome/chromatin by soluble factors, mechanical stresses from the environment, or metabolites determine cell fate, regulate cellular functions, or maintain cellular homeostasis. Circadian clock, which drives the expression of thousands of genes with 24-hour rhythmicity, has been known to be indispensable for maintaining cellular functions/homeostasis. During the last decade, it has been demonstrated that chromatin also undergoes modifications with 24-hour rhythmicity and facilitates the fine-tuning of circadian gene expression patterns. In this review, we cover data which suggests that chromatin structure changes in a circadian manner and that NAD+ is the key metabolite for circadian chromatin remodeling. Furthermore, we discuss the relationship among circadian clock, NAD+ metabolism, and aging/age-related diseases. In addition, the interventions of NAD+ metabolism for the prevention and treatment of aging and age-related diseases are also discussed. PMID:28050554

  10. One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

    PubMed

    Oza, Mihir D; Prasad, Kamalesh; Siddhanta, A K

    2012-08-01

    New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications.

  11. 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.

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

    PubMed

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

    2016-01-15

    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.

  13. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

    PubMed

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

    2016-01-05

    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 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-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.

  14. Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

    SciTech Connect

    Fraser, Marie E.; Cherney, Maia M.; Marcato, Paola; Mulvey, George L.; Armstrong, Glen D.; James, Michael N. G.

    2006-07-01

    Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active as an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.

  15. 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.

  16. Electrochemical studies on the oxidation of guanine and adenine at cyclodextrin modified electrodes.

    PubMed

    Abbaspour, Abdolkarim; Noori, Abolhassan

    2008-12-01

    An electrochemical sensor for guanine and adenine using cyclodextrin-modified poly(N-acetylaniline) (PNAANI) on a carbon paste electrode has been developed. The oxidation mechanism of guanine and adenine on the surface of the electrode was investigated by cyclic voltammetry. It was found that the electrode processes are irreversible, pH dependent, and involve several reaction products. The electron transfer process occurs in consecutive steps with the formation of a strongly adsorbed intermediate on the electrode surface. Also, a new method for estimating the apparent formation constants of guanine and adenine with the immobilized cyclodextrins, through the change of surface coverage of studied analytes has been reported. Both guanine and adenine showed linear concentrations in the range of 0.1-10 microM by using differential pulse voltammetry, with an experimental limit of detection down to 0.05 microM. Linear concentration ranges of 2-150 microM for guanine and 6-104 microM for adenine have been found when cyclic voltammetry was used for determination of both analytes.

  17. Alkylation by propylene oxide of deoxyribonucleic acid, adenine, guanosine and deoxyguanylic acid

    PubMed Central

    Lawley, P. D.; Jarman, M.

    1972-01-01

    1. Propylene oxide reacts with DNA in aqueous buffer solution at about neutral pH to yield two principal products, identified as 7-(2-hydroxypropyl)guanine and 3-(2-hydroxypropyl)adenine, which hydrolyse out of the alkylated DNA at neutral pH values at 37°C. 2. These products were obtained in quantity by reactions between propylene oxide and guanosine or adenine respectively. 3. The reactions between propylene oxide and adenine in acetic acid were parallel to those between dimethyl sulphate and adenine in neutral aqueous solution; the alkylated positions in adenine in order of decreasing reactivity were N-3, N-1 and N-9. A method for separating these alkyladenines is described. 4. Deoxyguanylic acid sodium salt was alkylated at N-7 by propylene oxide in neutral aqueous solution. 5. The nature of the side chain in the principal alkylation products was established by mass spectrometry, and the nature of the products is consistent with their formation by the bimolecular reaction mechanism. PMID:5073240

  18. Regulation of active site coupling in glutamine-dependent NAD[superscript +] synthetase

    SciTech Connect

    LaRonde-LeBlanc, Nicole; Resto, Melissa; Gerratana, Barbara

    2009-05-21

    NAD{sup +} is an essential metabolite both as a cofactor in energy metabolism and redox homeostasis and as a regulator of cellular processes. In contrast to humans, Mycobacterium tuberculosis NAD{sup +} biosynthesis is absolutely dependent on the activity of a multifunctional glutamine-dependent NAD{sup +} synthetase, which catalyzes the ATP-dependent formation of NAD{sup +} at the synthetase domain using ammonia derived from L-glutamine in the glutaminase domain. Here we report the kinetics and structural characterization of M. tuberculosis NAD{sup +} synthetase. The kinetics data strongly suggest tightly coupled regulation of the catalytic activities. The structure, the first of a glutamine-dependent NAD{sup +} synthetase, reveals a homooctameric subunit organization suggesting a tight dependence of catalysis on the quaternary structure, a 40-{angstrom} intersubunit ammonia tunnel and structural elements that may be involved in the transfer of information between catalytic sites.

  19. Excited-state lifetime of adenine near the first electronic band origin

    NASA Astrophysics Data System (ADS)

    Kang, Hyuk; Chang, Jinyoung; Lee, Sang Hak; Ahn, Tae Kyu; Kim, Nam Joon; Kim, Seong Keun

    2010-10-01

    The excited-state lifetime of supersonically cooled adenine was measured in the gas phase by femtosecond pump-probe transient ionization as a function of excitation energy between 36 100 and 37 500 cm-1. The excited-state lifetime of adenine is ˜2 ps around the 0-0 band of the L1b ππ ∗ state (36 105 cm-1). The lifetime drops to ˜1 ps when adenine is excited to the L1a ππ ∗ state with the pump energy at 36 800 cm-1 and above. The excited-state lifetimes of L1a and L1b ππ∗ states are differentiated in accordance with previous frequency-resolved and computational studies.

  20. 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.

  1. The basal proton conductance of mitochondria depends on adenine nucleotide translocase content

    PubMed Central

    2005-01-01

    The basal proton conductance of mitochondria causes mild uncoupling and may be an important contributor to metabolic rate. The molecular nature of the proton-conductance pathway is unknown. We show that the proton conductance of muscle mitochondria from mice in which isoform 1 of the adenine nucleotide translocase has been ablated is half that of wild-type controls. Overexpression of the adenine nucleotide translocase encoded by the stress-sensitive B gene in Drosophila mitochondria increases proton conductance, and underexpression decreases it, even when the carrier is fully inhibited using carboxyatractylate. We conclude that half to two-thirds of the basal proton conductance of mitochondria is catalysed by the adenine nucleotide carrier, independently of its ATP/ADP exchange or fatty-acid-dependent proton-leak functions. PMID:16076285

  2. 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

  3. De novo synthesis of adenine nucleotides in different skeletal muscle fiber types

    SciTech Connect

    Tullson, P.C.; John-Alder, H.B.; Hood, D.A.; Terjung, R.L.

    1988-09-01

    Management of adenine nucleotide catabolism differs among skeletal muscle fiber types. This study evaluated whether there are corresponding differences in the rates of de novo synthesis of adenine nucleotide among fiber type sections of skeletal muscle using an isolated perfused rat hindquarter preparation. Label incorporation into adenine nucleotides from the (1-14C)glycine precursor was determined and used to calculate synthesis rates based on the intracellular glycine specific radioactivity. Results show that intracellular glycine is closely related to the direct precursor pool. Rates of de novo synthesis were highest in fast-twitch red muscle (57.0 +/- 4.0, 58.2 +/- 4.4 nmol.h-1.g-1; deep red gastrocnemius and vastus lateralis), relatively high in slow-twitch red muscle (47.0 +/- 3.1; soleus), and low in fast-twitch white muscle (26.1 +/- 2.0 and 21.6 +/- 2.3; superficial white gastrocnemius and vastus lateralis). Rates for four mixed muscles were intermediate, ranging between 32.3 and 37.3. Specific de novo synthesis rates exhibited a strong correlation (r = 0.986) with muscle section citrate synthase activity. Turnover rates (de novo synthesis rate/adenine nucleotide pool size) were highest in high oxidative muscle (0.82-1.06%/h), lowest in low oxidative muscle (0.30-0.35%/h), and intermediate in mixed muscle (0.44-0.55%/h). Our results demonstrate that differences in adenine nucleotide management among fiber types extends to the process of de novo adenine nucleotide synthesis.

  4. The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch.

    PubMed Central

    Schöndorf, T; Hachtel, W

    1995-01-01

    Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate. PMID:7784504

  5. The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch.

    PubMed

    Schöndorf, T; Hachtel, W

    1995-05-01

    Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain. To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase. These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes. The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation. Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low. Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone. Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.

  6. Nutritional energy stimulates NAD+ production to promote tankyrase-mediated PARsylation in insulinoma cells.

    PubMed

    Zhong, Linlin; Yeh, Tsung-Yin J; Hao, Jun; Pourtabatabaei, Nasim; Mahata, Sushil K; Shao, Jianhua; Chessler, Steven D; Chi, Nai-Wen

    2015-01-01

    The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD+ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD+ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD+ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD+ since it is mirrored by the NAD+ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD+ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD+ production to expand NAD+ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD+ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD+ production by ATP suggests that nutritional energy may enhance the functions of other NAD+-driven enzymes including sirtuins.

  7. Efficacy of the acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against feline immunodeficiency virus.

    PubMed

    Hartmann, K; Kuffer, M; Balzarini, J; Naesens, L; Goldberg, M; Erfle, V; Goebel, F D; De Clercq, E; Jindrich, J; Holy, A; Bischofberger, N; Kraft, W

    1998-02-01

    The acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated for their efficacy and side effects in a double-blind placebo-controlled trial using naturally occurring feline immunodeficiency virus (FIV)-infected cats. This natural retrovirus animal model is considered highly relevant for the pathogenesis and chemotherapy of HIV in humans. Both PMEA and FPMPA proved effective in ameliorating the clinical symptoms of FIV-infected cats, as measured by several clinical parameters including the incidence and severity of stomatitis, Karnofsky's score, immunologic parameters such as relative and absolute CD4+ lymphocyte counts, and virologic parameters including proviral DNA levels in peripheral blood mononuclear cells (PBMC) of drug-treated animals. In contrast with PMEA, FPMPA showed no hematologic side effects at a dose that was 2.5-fold higher than PMEA.

  8. 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.

  9. Ricin Activity Assay by Direct Analysis in Real Time Mass Spectrometry Detection of Adenine Release

    DTIC Science & Technology

    2010-02-01

    direct analysis in real time mass spectrometry. The release of adenine from the inhomo- geneous substrate herring sperm DNA by ricin was determined to...chain catalyzes cleavage at adenosine 4324 (in rat RNA) of 28S rRNA to release adenine.10 This action inhibits protein synthesis, leading to cell...death. In addition to RNA, herring sperm DNA (hsDNA) is a substrate for ricin.11 We chose to employ hsDNA for this assay because it is relatively stable

  10. The Secret Life of NAD+: An Old Metabolite Controlling New Metabolic Signaling Pathways

    PubMed Central

    Houtkooper, Riekelt H.; Cantó, Carles; Wanders, Ronald J.; Auwerx, Johan

    2010-01-01

    A century after the identification of a coenzymatic activity for NAD+, NAD+ metabolism has come into the spotlight again due to the potential therapeutic relevance of a set of enzymes whose activity is tightly regulated by the balance between the oxidized and reduced forms of this metabolite. In fact, the actions of NAD+ have been extended from being an oxidoreductase cofactor for single enzymatic activities to acting as substrate for a wide range of proteins. These include NAD+-dependent protein deacetylases, poly(ADP-ribose) polymerases, and transcription factors that affect a large array of cellular functions. Through these effects, NAD+ provides a direct link between the cellular redox status and the control of signaling and transcriptional events. Of particular interest within the metabolic/endocrine arena are the recent results, which indicate that the regulation of these NAD+-dependent pathways may have a major contribution to oxidative metabolism and life span extension. In this review, we will provide an integrated view on: 1) the pathways that control NAD+ production and cycling, as well as its cellular compartmentalization; 2) the signaling and transcriptional pathways controlled by NAD+; and 3) novel data that show how modulation of NAD+-producing and -consuming pathways have a major physiological impact and hold promise for the prevention and treatment of metabolic disease. PMID:20007326

  11. Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

    PubMed

    Shetty, Kartika N; Latha, Vakada Lavanya; Rao, Rameshwaram Nagender; Nadimpalli, Siva Kumar; Suguna, Kaza

    2013-07-01

    Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

  12. The membrane expression of Neisseria meningitidis adhesin A (NadA) increases the proimmune effects of MenB OMVs on human macrophages, compared with NadA- OMVs, without further stimulating their proinflammatory activity on circulating monocytes.

    PubMed

    Tavano, Regina; Franzoso, Susanna; Cecchini, Paola; Cartocci, Elena; Oriente, Francesca; Aricò, Beatrice; Papini, Emanuele

    2009-07-01

    Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA(+) OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we investigated the activity of purified free NadA(Delta351-405) and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines (IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA(+) OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations approximately 10(6) times lower than those required to stimulate cells with free NadA(Delta351-405).

  13. Synthesis, cyclopolymerization and cyclo-copolymerization of 9-(2-diallylaminoethyl)adenine and its hydrochloride salt.

    PubMed

    Bouhadir, Kamal H; Abramian, Lara; Ezzeddine, Alaa; Usher, Karyn; Vladimirov, Nikolay

    2012-11-08

    We report herein the synthesis and characterization of 9-(2-diallylaminoethyl) adenine. We evaluated two different synthetic routes starting with adenine where the optimal route was achieved through coupling of 9-(2-chloroethyl)adenine with diallylamine. The cyclopolymerization and cyclo-copolymerization of 9-(2-diallylaminoethyl)adenine hydrochloride salt resulted in low molecular weight oligomers in low yields. In contrast, 9-(2-diallylaminoethyl)adenine failed to cyclopolymerize, however, it formed a copolymer with SO₂ in relatively good yields. The molecular weights of the cyclopolymers were around 1,700-6,000 g/mol, as estimated by SEC. The cyclo-copolymer was stable up to 226 °C. To the best of our knowledge, this is the first example of a free-radical cyclo-copolymerization of a neutral alkyldiallylamine derivative with SO₂. These polymers represent a novel class of carbocyclic polynucleotides.

  14. Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes.

    PubMed

    Ashihara, Hiroshi; Zheng, Xin-Qiang; Katahira, Riko; Morimoto, Masayuki; Ogita, Shinjiro; Sano, Hiroshi

    2006-05-01

    In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.

  15. NAD⁺ content and its role in mitochondria.

    PubMed

    Li, Wei; Sauve, Anthony A

    2015-01-01

    Nicotinamide adenine dinucleotide (NAD(+)) is a central metabolic coenzyme/cosubstrate involved in cellular energy metabolism and energy production. It can readily be reduced by two electron equivalents and forms the NADH form, which is the minority species to NAD(+) under most physiologic conditions. NAD(+) plays an important role in not only oxidation-reduction reactions in cells but also as a signaling molecule. For example, NAD(+) plays a key role in mitochondrial function via participation in pyruvate dehydrogenase, tricarboxylic acid cycle, and oxidative phosphorylation chemistries. It also serves as a substrate for deacylases SIRT3, SIRT4, and SIRT5, which modify protein posttranslational modifications on lysine within the mitochondrial compartment. Recent work has highlighted the biological significance of dynamic changes to mitochondrial NAD(+). This has increased the need for standardized and effective methods to measure NAD(+) contents in this organelle. To determine NAD(+) concentrations in cells, and specifically in mitochondria, we describe two assays for NAD(+) determinations: An Enzymatic Cycling Assay and Isotope Dilution. The cycling assay contains sample NAD(+), lactate, lactate dehydrogenase, diaphorase, and resazurin. The isotope dilution assay uses synthetic (18)O-NAD(+) as an internal standard, and treated samples are fractionated by HPLC and then NAD(+) concentration determined by the (16)O- and (18)O-NAD(+) peak (664/666) ratio in positive mode MS.

  16. Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis.

    PubMed

    Sorci, Leonardo; Cimadamore, Flavio; Scotti, Stefania; Petrelli, Riccardo; Cappellacci, Loredana; Franchetti, Palmarisa; Orsomando, Giuseppe; Magni, Giulio

    2007-04-24

    Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.

  17. Biochemical Issues in Estimation of Cytosolic Free NAD/NADH Ratio

    PubMed Central

    Xie, Jiansheng; Hu, Xun

    2012-01-01

    Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P. PMID:22570687

  18. NAD+-dependent glutamate dehydrogenase of the edible mushroom Agaricus bisporus: biochemical and molecular characterization.

    PubMed

    Kersten, M A; Müller, Y; Baars, J J; Op den Camp, H J; van der Drift, C; Van Griensven, L J; Visser, J; Schaap, P J

    1999-04-01

    The NAD+-dependent glutamate dehydrogenase (NAD-GDH) of Agaricus bisporus, a key enzyme in nitrogen metabolism, was purified to homogeneity. The apparent molecular mass of the native enzyme is 474 kDa comprising four subunits of 116 kDa. The isoelectric point of the enzyme is about 7.0. Km values for ammonium, 2-oxoglutarate, NADH, glutamate and NAD+ were 6.5, 3.5, 0.06, 37.1 and 0.046 mM, respectively. The enzyme is specific for NAD(H). The gene encoding this enzyme (gdhB) was isolated from an A. bisporus H39 recombinant lambda phage library. The deduced amino acid sequence specifies a 1029-amino acid protein with a deduced molecular mass of 115,463 Da, which displays a significant degree of similarity with NAD-GDH of Saccharomyces cerevisiae and Neurospora crassa. The ORF is interrupted by fifteen introns. Northern analysis combined with enzyme activity measurements suggest that NAD-GDH from A. bisporus is regulated by the nitrogen source. NAD-GDH levels in mycelium grown on glutamate were higher than NAD-GDH levels in mycelium grown on ammonium as a nitrogen source. Combined with the kinetic parameters, these results suggest a catabolic role for NAD-GDH. However, upon addition of ammonium to the culture transcription of the gene is not repressed as strongly as that of the gene encoding NADP-GDH (gdhA). To date, tetrameric NAD-GDHs with large subunits, and their corresponding genes, have only been isolated from a few species. This enzyme represents the first NAD-GDH of basidiomycete origin to be purified and is the first such enzyme from basidiomycetes whose sequence has been determined.

  19. Diminution in adenine nucleotide hydrolysis by platelets and serum from rats submitted to Walker 256 tumour.

    PubMed

    Buffon, Andréia; Ribeiro, Vanessa B; Schanoski, Alessandra S; Sarkis, João J F

    2006-01-01

    Extracellular adenine nucleotide hydrolysis in the circulation is mediated by the action of an NTPDase (CD39, apyrase) and of a 5'-nucleotidase (CD73), presenting as a final product, adenosine. Among other properties described for adenine nucleotides, an anti-cancer activity is suggested, since ATP is considered a cytotoxic molecule in several tumour cell systems. Conversely, some studies demonstrate that adenosine presents a tumour-promoting activity. In this study, we evaluated the pattern of adenine nucleotide hydrolysis by serum and platelets from rats submitted to the Walker 256 tumour model. Extracellular adenine nucleotide hydrolysis by blood serum and platelets obtained from rats at, 6, 10 and 15 days after the subcutaneous Walker 256 tumour inoculation, was evaluated. Our results demonstrate a significant reduction in ATP, ADP and AMP hydrolysis in blood serum at 6, 10 and 15 days after tumour induction. In platelets, a significant reduction in ATP and AMP hydrolysis was observed at 10 and 15 days after tumour induction, while an inhibition of ADP hydrolysis was observed at all times studied. Based on these results, it is possible to suggest a physiologic protection mechanism against the tumoral process in circulation. The inhibition in nucleotide hydrolysis observed probably maintains ATP levels elevated (cytotoxic compound) and, at the same time, reduces the adenosine production (tumour-promoting molecule) in the circulation.

  20. Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

    PubMed Central

    Ali, Badreldin H.; Adham, Sirin A.; Al Za’abi, Mohammed; Waly, Mostafa I.; Yasin, Javed; Nemmar, Abderrahim; Schupp, Nicole

    2015-01-01

    Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered. PMID:25909514

  1. Ameliorative effect of chrysin on adenine-induced chronic kidney disease in rats.

    PubMed

    Ali, Badreldin H; Adham, Sirin A; Al Za'abi, Mohammed; Waly, Mostafa I; Yasin, Javed; Nemmar, Abderrahim; Schupp, Nicole

    2015-01-01

    Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine - induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.

  2. Effect of atracylodes rhizome polysaccharide in rats with adenine-induced chronic renal failure.

    PubMed

    Yang, C; Liu, C; Zhou, Q; Xie, Y C; Qiu, X M; Feng, X

    2015-01-01

    The aim of the study was to elucidate the therapeutic effects of Atracylodes rhizome polysaccharide on adenine-induced chronic renal failure in rats. Fifty male Sprague Dawley rats were selected and randomly divided in to 5 groups (n=10 rats per group): The normal control group, the chronic renal failure pathological control group, the dexamethasone treatment group and two Atracylodes rhizome polysaccharide treatment groups, treated with two different concentrations of the polysaccharide, the Atracylodes rhizome polysaccharide high group and the Atracylodes rhizome polysaccharide low group. All the rats, except those in the normal control group were fed adenine-enriched diets, containing 10 g adenine per kg food for 3 weeks. After being fed with adenine, the dexamethasone treatment group, Atracylodes rhizome polysaccharide high group and Atracylodes rhizome polysaccharide low group rats were administered the drug orally for 2 weeks. On day 35, the kidney coefficient of the rats and the serum levels of creatinine, blood urea nitrogen, total protein and hemalbumin were determined. Subsequent to experimentation on a model of chronic renal failure in rats, the preparation was proven to be able to reduce serum levels of creatinine, blood urea nitrogen and hemalbumin levels (P<0.05) and improve renal function. Atracylodes rhizome polysaccharide had reversed the majority of the indices of chronic renal failure in rats.

  3. The Effect of Adenine Repeats on G-quadruplex/hemin Peroxidase Mimicking DNAzyme Activity.

    PubMed

    Chen, Jielin; Guo, Yuehua; Zhou, Jun; Ju, Huangxian

    2017-03-23

    The catalytic activity of G-quadruplex/hemin is much lower than that of proteinous enzymes, so it is very important to increase its activity. Very recently, flanking sequences, which can be regarded as an external part of G-quadruplexes, were found to enhance the activity of G-quadruplex/hemin DNAzyme. However, little is known about the effect of internal parts, such as loop sequences and linkers, on the activity. In the present study, adenine repeats were incorporated into several designed G-quadruplex structures either in the loops, bulges, or linkers, and the constructed G-quadruplex/hemin DNAzyme exhibit about fivefold improvement in peroxidase-mimicking activity in some cases. The enhancement effect may result from the formation of compound I, protoporphyrin⋅Fe(IV) =O(.+) , accelerated by dA repeats, which was demonstrated by H2 O2 decay kinetics and pH dependency analysis. The novel enhancement methods described here may help in the development of high-activity DNAzymes, illustrated by a dimer G-quadruplex with flanking adenine at one end, a relatively long adenine run in one loop, and another adenine run in the linker.

  4. Macrophage Trafficking as Key Mediator of Adenine-Induced Kidney Injury

    PubMed Central

    Braga, Tárcio Teodoro; Felizardo, Raphael José Ferreira; Andrade-Oliveira, Vinícius; Hiyane, Meire Ioshie; da Silva, João Santana; Câmara, Niels Olsen Saraiva

    2014-01-01

    Macrophages play a special role in the onset of several diseases, including acute and chronic kidney injuries. In this sense, tubule interstitial nephritis (TIN) represents an underestimated insult, which can be triggered by different stimuli and, in the absence of a proper regulation, can lead to fibrosis deposition. Based on this perception, we evaluated the participation of macrophage recruitment in the development of TIN. Initially, we provided adenine-enriched food to WT and searched for macrophage presence and action in the kidney. Also, a group of animals were depleted of macrophages with the clodronate liposome while receiving adenine-enriched diet. We collected blood and renal tissue from these animals and renal function, inflammation, and fibrosis were evaluated. We observed higher expression of chemokines in the kidneys of adenine-fed mice and a substantial protection when macrophages were depleted. Then, we specifically investigated the role of some key chemokines, CCR5 and CCL3, in this TIN experimental model. Interestingly, CCR5 KO and CCL3 KO animals showed less renal dysfunction and a decreased proinflammatory profile. Furthermore, in those animals, there was less profibrotic signaling. In conclusion, we can suggest that macrophage infiltration is important for the onset of renal injury in the adenine-induced TIN. PMID:25132730

  5. The effect of activated charcoal on adenine-induced chronic renal failure in rats.

    PubMed

    Ali, Badreldin H; Alza'abi, Mohamed; Ramkumar, Aishwarya; Al-Lawati, Intisar; Waly, Mostafa I; Beegam, Sumaya; Nemmar, Abderrahim; Brand, Susanne; Schupp, Nicole

    2014-03-01

    Activated charcoal (AC) is a sorbent that has been shown to remove urinary toxins like urea and indoxyl sulfate. Here, the influence of AC on kidney function of rats with experimental chronic renal failure (CRF) is investigated. CRF was induced in rats by feeding adenine (0.75%) for four weeks. As an intervention, AC was added to the feed at concentrations of 10%, 15% or 20%. Adenine treatment impaired kidney function: it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and vanin-1. Furthermore, it raised plasma concentrations of the uremic toxins indoxyl sulfate, phosphate and uric acid. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activity, total antioxidant capacity and reduced glutathione were adversely affected. Most of these changes were significantly ameliorated by dietary administration of AC at a concentration of 20%, while effects induced by lower doses of dietary AC on adenine nephrotoxicity were not statistically significant. The results suggest that charcoal is a useful sorbent agent in dietary adenine-induced CRF in rats and that its usability as a nephroprotective agent in human kidney disease should be studied.

  6. High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation.

    PubMed

    Azzu, Vian; Parker, Nadeene; Brand, Martin D

    2008-07-15

    Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.

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

    PubMed

    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; Pacheco e Silva Filho, Alvaro; Câmara, Niels O S

    2015-06-18

    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.

  8. 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{id