Sample records for adenine dinucleotide detection

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

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


    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.

  2. A Nicotinamide Adenine Dinucleotide Dispersed Multi-walled Carbon Nanotubes Electrode for Direct and Selective Electrochemical Detection of Uric Acid. (United States)

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


    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.

  3. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration. (United States)

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


    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.

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

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


    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.

  5. Fabrication and characterization of Meldola's blue/zinc oxide hybrid electrodes for efficient detection of the reduced form of nicotinamide adenine dinucleotide at low potential

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    Kumar, S. Ashok [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan (China)]. E-mail:; Chen Shenming [Department of Chemical Engineering and Biotechnology, National Taipei University of Technology, No. 1, Section 3, Chung-Hsiao East Road, Taipei 106, Taiwan (China)]. E-mail:


    We report the synthesis and the electrochemical properties of hybrid films made of zinc oxide (ZnO) and Meldola's blue dye (MB) using cyclic voltammetry (CV). MB/ZnO hybrid films were electrochemically deposited onto glassy carbon, gold and indium tin oxide-coated glass (ITO) electrodes at room temperature (25 {+-} 2 deg. C) from the bath solution containing 0.1 M Zn(NO{sub 3}){sub 2}, 0.1 M KNO{sub 3} and 1 x 10{sup -4} M MB. The surface morphology and deposition kinetics of MB/ZnO hybrid films were studied by means of scanning electron microscopy (SEM), atomic force microscopy (AFM) and electrochemical quartz crystal microbalance (EQCM) techniques, respectively. SEM and AFM images of MB/ZnO hybrid films have revealed that the surfaces are well crystallized, porous and micro structured. MB molecules were immobilized and strongly fixed in a transparent inorganic matrix. MB/ZnO hybrid films modified glassy carbon electrode (MB/ZnO/GC) showed one reversible redox couple centered at formal potential (E {sup 0}') -0.12 V (pH 6.9). The surface coverage ({gamma}) of the MB immobilized on ZnO/GC was about 9.86 x 10{sup -12} mol cm{sup -2} and the electron transfer rate constant (ks) was determined to be 38.9 s{sup -1}. The MB/ZnO/GC electrode acted as a sensor and displayed an excellent specific electrocatalytic response to the oxidation of nicotinamide adenine dinucleotide (NADH). The linear response range between 50 and 300 {mu}M NADH concentration at pH 6.9 was observed with a detection limit of 10 {mu}M (S/N = 3). The electrode was stable during the time it was used for the full study (about 1 month) without a notable decrease in current. Indeed, dopamine (DA), ascorbic acid (AA), acetaminophen (AP) and uric acid (UA) did not show any interference during the detection of NADH at this modified electrode.

  6. Role of adenine in thymine-dimer repair by reduced flavin-adenine dinucleotide. (United States)

    Li, Guifeng; Sichula, Vincent; Glusac, Ksenija D


    We present a study of excited-state behavior of reduced flavin cofactors using femtosecond optical transient absorption spectroscopy. The reduced flavin cofactors studied were in two protonation states: flavin-adenine dinucleotide (FADH2 and FADH-) and flavin-mononucleotide (FMNH2 and FMNH-). We find that FMNH- exhibits multiexponential decay dynamics due to the presence of two bent conformers of the isoalloxazine ring. FMNH2 exhibits an additional fast deactivation component that is assigned to an iminol tautomer. Reduced flavin cofactors also exhibit a long-lived component that is attributed to the semiquinone and the hydrated electron that are produced in photoinduced electron transfer to the solvent. The presence of adenine in FADH2 and FADH- further changes the excited-state dynamics due to intramolecular electron transfer from the isoalloxazine to the adenine moiety of cofactors. This electron transfer is more pronounced in FADH2 due to pi-stacking interactions between two moieties. We further studied cyclobutane thymine dimer (TT-dimer) repair via FADH- and FMNH- and found that the repair is much more efficient in the case of FADH-. These results suggest that the adenine moiety plays a significant role in the TT-dimer repair dynamics. Two possible explanations for the adenine mediation are presented: (i) a two-step electron transfer process, with the initial electron transfer occurring from flavin to adenine moiety of FADH-, followed by a second electron transfer from adenine to TT-dimer; (ii) the preconcentration of TT-dimer molecules around the flavin cofactor due to the hydrophobic nature of the adenine moiety.

  7. Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1

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    Lin, C.; Robertson, D.E.; Ahmad, M. [Univ. of Pennsylvania, Philadelphia, PA (United States)] [and others


    The Arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH{center_dot}). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity. 27 refs., 4 figs.


    Institute of Scientific and Technical Information of China (English)

    DING Dalian; JIANG Haiyan; FU Yong; LI Yongqi; Richard Salvi; Shinichi Someya; Masaru Tanokura


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

  9. Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Bystrykh, Leonid V.; Dijkhuizen, Lubbert; Harder, Willem


    Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch

  10. Structural Analysis of a Stereochemical Modification of Flavin Adenine Dinucleotide in Alcohol Oxidase from Methylotrophic Yeasts

    NARCIS (Netherlands)

    Kellogg, Richard M.; Kruizinga, Wim; Bystrykh, Leonid V.; Dijkhuizen, Lubbert; Harder, Wim


    Alcohol oxidase (MOX), a major peroxisomal protein of methanol-utilizing yeasts, contains two different forms of flavin adenine dinucleotide, one of which is identical with natural FAD whereas the other (mFAD) is a stereochemical modification of the natural coenzyme. This modification occurs spontan

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

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


    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.

  12. Determination of the B2 vitamer flavin-adenine dinucleotide in whole blood by high-performance liquid chromatography with fluorometric detection

    NARCIS (Netherlands)

    Speek, A.J.; Schaik, F. van; Schrijver, J.; Schreurs, W.H.P.


    A reliable high-performance liquid chromatographic (HPLC) method is developed for the analysis of FAD in whole blood of humans. The method is able to separate FAD, FMN and Rb from each other and from interfering compounds. A reliable and sensitive detection of FAD has been obtained by selecting the

  13. Kinetic and thermodynamic study of the reaction catalyzed by glucose-6-phosphate dehydrogenase with nicotinamide adenine dinucleotide

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    Martin del Campo, Julia S. [Departamento de Fisica Aplicada, Centro de Investigacion y de Estudios Avanzados - Unidad Merida, Carretera antigua a Progreso Km. 6, A.P. 73 Cordemex, 97310, Merida, Yucatan (Mexico); Patino, Rodrigo, E-mail: [Departamento de Fisica Aplicada, Centro de Investigacion y de Estudios Avanzados - Unidad Merida, Carretera antigua a Progreso Km. 6, A.P. 73 Cordemex, 97310, Merida, Yucatan (Mexico)


    Research highlights: {yields} The reaction catalyzed by one enzyme of the pentose phosphate pathway was studied. {yields} A spectrophotometric method is proposed for kinetic and thermodynamic analysis. {yields} The pH and the temperature influences are reported on physical chemical properties. {yields} Relative concentrations of substrates are also important in the catalytic process. - Abstract: The enzyme glucose-6-phosphate dehydrogenase (G6PD, EC from Leuconostoc mesenteroides has a dual coenzyme specificity with oxidized nicotinamide adenine dinucleotide (NAD{sub ox}) and oxidized nicotinamide adenine dinucleotide phosphate as electron acceptors. The G6PD coenzyme selection is determined by the metabolic cellular prevailing conditions. In this study a kinetic and thermodynamic analysis is presented for the reaction catalyzed by G6PD from L. mesenteroides with NAD{sub ox} as coenzyme in phosphate buffer. For this work, an in situ spectrophotometric technique was employed based on the detection of one product of the reaction. Substrate and coenzyme concentrations as well as temperature and pH effects were evaluated. The apparent equilibrium constant, the Michaelis constant, and the turnover number were determined as a function of each experimental condition. The standard transformed Gibbs energy of reaction was determined from equilibrium constants at different initial conditions. For the product 6-phospho-D-glucono-1,5-lactone, a value of the standard Gibbs energy of formation is proposed, {Delta}{sub f}G{sup o} = -1784 {+-} 5 kJ mol{sup -1}.

  14. Nicotinic acid adenine dinucleotide phosphate-mediated calcium signalling in effector T cells regulates autoimmunity of the central nervous system (United States)

    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.


    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

  15. Expanding antitumor therapeutic windows by targeting cancer-specific nicotinamide adenine dinucleotide phosphate-biogenesis pathways

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    Chakrabarti G


    Full Text Available Gaurab Chakrabarti,1,2,4 David E Gerber,3,4 David A Boothman1,2,4 1Department of Pharmacology, 2Department of Radiation Oncology, 3Division of Hematology and Oncology, 4Harold C Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, TX, USA Abstract: Nicotinamide adenine dinucleotide phosphate (NADPH biogenesis is an essential mechanism by which both normal and cancer cells maintain redox balance. While antitumor approaches to treat cancers through elevated reactive oxygen species (ROS are not new ideas, depleting specific NADPH-biogenesis pathways that control recovery and repair pathways are novel, viable approaches to enhance cancer therapy. However, to elicit efficacious therapies exploiting NADPH-biogenic pathways, it is crucial to understand and specifically define the roles of NADPH-biogenesis pathways used by cancer cells for survival or recovery from cell stress. It is equally important to select NADPH-biogenic pathways that are expendable or not utilized in normal tissue to avoid unwanted toxicity. Here, we address recent literature that demonstrates specific tumor-selective NADPH-biogenesis pathways that can be exploited using agents that target specific cancer cell pathways normally not utilized in normal cells. Defining NADPH-biogenesis profiles of specific cancer-types should enable novel strategies to exploit these therapeutic windows for increased efficacy against recalcitrant neoplastic disease, such as pancreatic cancers. Accomplishing the goal of using ROS as a weapon against cancer cells will also require agents, such as NQO1 bioactivatable drugs, that selectively induce elevated ROS levels in cancer cells, while normal cells are protected. Keywords: reactive oxygen species (ROS, NQO1-bioactivatable drugs, nicotinamide adenine dinucleotide phosphate (NADPH, glutathione (GSH, biogenic pathways, antioxidant

  16. Flavin adenine dinucleotide binding is the crucial step in alcohol oxidase assembly in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Evers, Melchior E.; Titorenko, Vladimir; Harder, Wim; Klei, Ida van der; Veenhuis, Marten


    We have studied the role of flavin adenine dinucleotide (FAD) in the in vivo assembly of peroxisomal alcohol oxidase (AO) in the yeast Hansenula polymorpha. In previous studies, using a riboflavin (Rf) autotrophic mutant, an unequivocal judgement could not be made, since Rf-limitation led to a parti

  17. Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies. (United States)

    Leung, Kevin Ka Ki; Litchfield, David W; Shilton, Brian H


    Quinone reductase 2 (NQO2) is a broadly expressed enzyme implicated in responses to a number of compounds, including protein kinase inhibitors, resveratrol, and antimalarial drugs. NQO2 includes a flavin adenine dinucleotide (FAD) cofactor, but X-ray crystallographic analysis of human NQO2 expressed in Escherichia coli showed that electron density for the isoalloxazine ring of FAD was weak and there was no electron density for the adenine mononucleotide moiety. Reversed-phase high-performance liquid chromatography (HPLC) of the NQO2 preparation indicated that FAD was not present and only 38% of the protomers contained flavin mononucleotide (FMN), explaining the weak electron density for FAD in the crystallographic analysis. A method for purifying NQO2 and reconstituting with FAD such that the final content approaches 100% occupancy with FAD is presented here. The enzyme prepared in this manner has a high specific activity, and there is strong electron density for the FAD cofactor in the crystal structure. Analysis of NQO2 crystal structures present in the Protein Data Bank indicates that many may have sub-stoichiometric cofactor content and/or contain FMN rather than FAD. This method of purification and reconstitution will help to optimize structural and functional studies of NQO2 and possibly other flavoproteins.

  18. Studies of yeast cell oxygenation and energetics by laser fluorometry of reduced nicotinamide adenine dinucleotide (United States)

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


    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 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.

  19. Decrease in nicotinamide adenine dinucleotide dehydrogenase is related to skin pigmentation. (United States)

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


    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.

  20. Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. (United States)

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey


    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.

  1. Magnitude of malate-aspartate reduced nicotinamide adenine dinucleotide shuttle activity in intact respiring tumor cells. (United States)

    Greenhouse, W V; Lehninger, A L


    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.

  2. Interaction of flavin adenine dinucleotide (FAD) with a glassy carbon electrode surface. (United States)

    Wei, Haizhen; Omanovic, Sasha


    The interaction of flavin adenine dinucleotide (FAD) with a glassy carbon electrode (GCE) surface was investigated in terms of the FAD adsorption thermodynamics and kinetics, the subsequent electroreduction mechanism, and the corresponding electron-transfer rate. The kinetics of FAD electroreduction at the GCE was found to be an adsorption-controlled process. A set of electroreduction kinetic parameters was calculated: the true number of electrons involved in the FAD reduction, n=1.76, the apparent transfer coefficient, alpha(app)=0.41, and the apparent heterogeneous electron-transfer rate constant, k(app)=1.4 s(-1). The deviation of the number of exchanged electrons from the theoretical value for the complete reduction of FAD to FADH(2) (n=2) indicates that a small portion of FAD goes to a semiquinone state during the redox process. The FAD adsorption was well described by the Langmuir adsorption isotherm. The large negative apparent Gibbs energy of adsorption (DeltaG(ads)=-39.7 +/-0.4 kJ mol(-1)) indicated a highly spontaneous and strong adsorption of FAD on the GCE. The energetics of the adsorption process was found to be independent of the electrode surface charge in the electrochemical double-layer region. The kinetics of FAD adsorption was modeled using a pseudo-first-order kinetic model.

  3. Thermal stabilization of formaldehyde dehydrogenase by encapsulation in liposomes with nicotinamide adenine dinucleotide. (United States)

    Yoshimoto, Makoto; Yamashita, Takayuki; Kinoshita, Satoshi


    The thermal stability of formaldehyde dehydrogenase (FaDH) from Pseudomonas sp. was examined and controlled by encapsulation in liposomes with β-reduced nicotinamide adenine dinucleotide (NADH). The activity of 4.8 μg/mL free FaDH at pH 8.5 in catalyzing the oxidation of 50mM formaldehyde was highly dependent on temperature so that the activity at 60 °C was 27 times larger than that at 25 °C. Thermal stability of the FaDH activity was examined with and without liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Rapid deactivation of free FaDH was observed at 60 °C because of its dissociation into two subunits. The rate of dissociative deactivation of POPC liposome-encapsulated FaDH was smaller than that of the free enzyme. The liposomal FaDH was however progressively deactivated for the incubation period of 60 min eventually leading to complete loss of its activity. The free FaDH and NADH molecules were revealed to form the thermostable binary complex. The thermal stability of POPC liposome-encapsulated FaDH and NADH system was significantly higher than the liposomal enzyme without cofactor. The above results clearly show that NADH is a key molecule that controls the activity and stability of FaDH in liposomes at high temperatures.

  4. Electrochemical study in both classical cell and microreactors of flavin adenine dinucleotide as a redox mediator for NADH regeneration

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    Tzedakis, Theodore, E-mail: [Laboratoire de Genie Chimique, UMR 5503, Universite Paul Sabatier, 31062 Toulouse cedex 04 (France); Cheikhou, Kane [Ecole Superieure Polytechnique de Dakar BP: 16263 Dakar-Fann (Senegal); Jerome, Roche; Karine, Groenen Serrano; Olivier, Reynes [Laboratoire de Genie Chimique, UMR 5503, Universite Paul Sabatier, 31062 Toulouse cedex 04 (France)


    The electrochemical reduction of flavin adenine dinucleotide (FAD) is studied in a classical electrochemical cell as well as in two types of microreactors: the first one is a one-channel reactor and the other one, a multichannel filter-press reactor. The ultimate goal is to use the reduced form of flavin (FADH{sub 2}), in the presence of formate dehydrogenase (FDH), in order to continuously regenerate the reduced form of nicotinamide adenine dinucleotide (NADH) for chiral syntheses. Various voltammetric and adsorption measurements were carried out for a better understanding of the redox behavior of the FAD as well as its adsorption on gold. Diffusivity and kinetic electrochemical parameters of FAD were determined.

  5. Changes of collagen and nicotinamide adenine dinucleotide in human cancerous and normal prostate tissues studied using native fluorescence spectroscopy with selective excitation wavelength (United States)

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


    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.

  6. Association of nicotinamide adenine dinucleotide phosphate oxidase p22phox gene 549C>T polymorphism with coronary artery disease

    Institute of Scientific and Technical Information of China (English)

    LIU Tong-tao; WANG Li-li; FANG Sheng-xia; JIA Chong-qi


    Background The p22phox is a critical component of the superoxide-generating vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.Several polymorphisms in p22phox gene are studied for their association with cardiovascular diseases.However,no publication is available to assess the relation of 549C>T polymorphism in p22phox gene to coronary artery disease (CAD) risk.This study was to investigate the effect of the p22phox gene 549C>T polymorphism on CAD risk.Methods Hospital-based case-control study was conducted with 297 CAD patients and 343 healthy persons as the control group.Polymerase chain reaction and pyrosequencing using PSQ 96 MA Pyrosequencer (Biotage AB) were used to detect the polymorphisms.Multiple Logistic regression model was used to adjust the potential confounders and to estimate odds ratio (OR) with 95% confidence intervals (Cls).Results The observed genotype frequencies of this polymorphism obeyed the Hardy-Weinberg equilibrium in both cases (P=0.439) and controls (P=0.668).The frequency of mutant genotypes (TT+CT) in cases (41.08%) was higher than that in controls (36.73%) with an OR=-1.20 (95% CI=0.87-1.65).After the adjustment of the potential confounders,there was a significant association of the mutant genotypes with increased risk of CAD (OR=1.57,95% CI=1.01-2.46,P=0.047).Conclusions The mutant genotypes of the p22phox gene 549C>T polymorphism had a significant effect on the increased risk of CAD in this studied population.

  7. Induction of ischemic tolerance in rat liver via reduced nicotinamide adenine dinucleotide phosphate oxidase in Kupffer cells

    Institute of Scientific and Technical Information of China (English)


    AIM: To elucidate the mechanisms of hepatocyte preconditioning by H2O2 to better understand the pathophysiology of ischemic preconditioning.METHODS: The in vitro effect of H2O2 pretreatment was investigated in rat isolated hepatocytes subjected to anoxia/reoxygenation. Cell viability was assessed with propidium iodide fluorometry. In other experiments, rat livers were excised and subjected to warm ischemia/reperfusion in an isolated perfused liver system to determine leakage of liver enzymes. Preconditioning was performed by H2O2 perfusion, or by stopping the perfusion for 10 min followed by 10 min of reperfusion.To inhibit Kupffer cell function or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,gadolinium chloride was injected prior to liver excision, or diphenyleneiodonium, an inhibitor of NADPH oxidase, was added to the perfusate, respectively. Histological detection of o~gen radical formation in Kupffer cells was performed by perfusion with nitro blue tetrazolium.RESULTS: Anoxia/reoxygenation decreased hepatocyte viability compared to the controls. Pretreatment with H2O2 did not improve such hepatocyte injury. In liver perfusion experiments, however, H2O2 preconditioning reduced warm ischemia/reperfusion injury, which was reversed by inhibition of Kupffer cell function or NADPH oxidase. Histological examination revealed that H2O2 preconditioning induced oxygen radical formation in Kupffer cells. NADPH oxidase inhibition also reversed hepatoprotection by ischemic preconditioning.CONCLUSION: H2O2 preconditioning protects hepatocytes against warm ischemia/reperfusion injury via NADPH oxidase in Kupffer cells, and not directly. NADPH oxidase also mediates hepatoprotection by ischemic preconditioning.

  8. Catalytic activity of iron hexacyanoosmate(II) towards hydrogen peroxide and nicotinamide adenine dinucleotide and its use in amperometric biosensors

    Energy Technology Data Exchange (ETDEWEB)

    Kotzian, Petr; Janku, Tereza [Department of Analytical Chemistry, University of Pardubice, Nam. Cs. Legii 565, CZ-532 10 Pardubice (Czech Republic); Kalcher, Kurt [Institute of Chemistry - Analytical Chemistry, Karl-Franzens University, Universitaetsplatz 1, A-8010 Graz (Austria); Vytras, Karel [Department of Analytical Chemistry, University of Pardubice, Nam. Cs. Legii 565, CZ-532 10 Pardubice (Czech Republic)], E-mail:


    Hydrogen peroxide and nicotinamide adenine dinucleotide (NADH) may be determined amperometrically using screen-printed electrodes chemically modified with iron(III) hexacyanoosmate(II) (Osmium purple) in flow injection analysis (FIA). The determination is based on the exploitation of catalytic currents resulting from the oxidation/reduction of the modifier. The performance of the sensor was characterized and optimized by controlling several operational parameters (applied potential, pH and flow rate of the phosphate buffer). Comparison has been made with analogous complexes of ruthenium (Ruthenium purple) and iron (Prussian blue). Taking into account the sensitivity and stability of corresponding sensors, the best results were obtained with the use of Osmium purple. The sensor exhibited a linear increase of the amperometric signal with the concentration of hydrogen peroxide in the range of 0.1-100 mg L{sup -1} with a detection limit (evaluated as 3{sigma}) of 0.024 mg L{sup -1} with a R.S.D. 1.5% for 10 mg L{sup -1} H{sub 2}O{sub 2} under optimized flow rate of 0.4 mL min{sup -1} in 0.1 M phosphate buffer carrier (pH 6) and a working potential of +0.15 V versus Ag/AgCl. Afterwards, a biological recognition element - either glucose oxidase or ethanol dehydrogenase - was incorporated to achieve a sensor facilitating the determination of glucose or ethanol, respectively. The glucose sensor gave linearity between current and concentration in the range from 1 to 250 mg L{sup -1} with a R.S.D. 2.4% for 100 mg L{sup -1} glucose, detection limit 0.02 mg L{sup -1} (3{sigma}) and retained its original activity after 3 weeks when stored at 6 deg. C. Optimal parameters in the determination of ethanol were selected as: applied potential +0.45 V versus Ag/AgCl, flow rate 0.2 mL min{sup -1} in 0.1 M phosphate buffer carrier (pH 7). Different structural designs of the ethanol sensor were tested and linearity obtained was up to 1000 mg L{sup -1} with a maximum R.S.D. of 5

  9. Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay. (United States)

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


    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.

  10. Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase. (United States)

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


    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.

  11. Purification and characterization of the enzymes involved in nicotinamide adenine dinucleotide degradation by Penicillium brevicompactum NRC 829. (United States)

    Ali, Thanaa Hamed; El-Ghonemy, Dina Helmy


    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.

  12. Interaction between thymine dimer and flavin-adenine dinucleotide: a DFT and direct ab initio molecular dynamics study. (United States)

    Tachikawa, Hiroto; Kawabata, Hiroshi


    The interaction between the fully reduced flavin-adenine dinucleotide (FADH (-)) and thymine dimer (T) 2 has been investigated by means of density functional theory (DFT) calculations. The charges of FADH (-) and (T) 2 were calculated to be -0.9 and -0.1, respectively, at the ground state. By photoirradiation, an electron transfer occurred from FADH (-) to (T) 2 at the first excited state. Next, the reaction dynamics of electron capture of (T) 2 have been investigated by means of the direct ab initio molecular dynamics (MD) method (HF/3-21G(d) and B3LYP/6-31G(d) levels) in order to elucidate the mechanism of the repair process of thymine dimer caused by the photoenzyme. The thymine dimer has two C-C single bonds between thymine rings (C 5-C 5' and C 6-C 6' bonds) at the neutral state, which is expressed by (T) 2. After the electron capture of (T) 2, the C 5-C 5' bond was gradually elongated and then it was preferentially broken. The time scale of the C-C bond breaking and formation of the intermediate with a single bond (T) 2 (-) was estimated to be 100-150 fs. The present calculations confirmed that the repair reaction of thymine dimer takes place efficiently via an electron-transfer process from the FADH (-) enzyme.

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


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


    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 le...

  14. Discovery of Nicotinamide Adenine Dinucleotide Binding Proteins in the Escherichia coli Proteome Using a Combined Energetic- and Structural-Bioinformatics-Based Approach. (United States)

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


    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.

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



    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 ...

  16. Spectroscopy and Speciation Studies on the Interactions of Aluminum (III with Ciprofloxacin and β-Nicotinamide Adenine Dinucleotide Phosphate in Aqueous Solutions

    Directory of Open Access Journals (Sweden)

    Xiaodi Yang


    Full Text Available In this study, both experimental and theoretical approaches, including absorption spectra, fluorescence emission spectra, 1H- and 31P-NMR, electrospray ionization mass spectrometry (ESI-MS, pH-potentiometry and theoretical approaches using the BEST & SPE computer programs were applied to study the competitive complexation between ciprofloxacin (CIP and b-nicotinamide adenine dinucleotide phosphate (NADP with aluminum (III in aqueous solutions. Rank annihilation factor analysis (RAFA was used to analyze the absorption and fluorescence emission spectra of the ligands, the binary complexes and the ternary complexes. It is found, at the mM total concentration level and pH = 7.0, the bidentate mononuclear species [Al(CIP]2+ and [Al(NADP] predominate in the aqueous solutions of the Al(III-CIP and Al(III-NADP systems, and the two complexes have similar conditional stability constants. However, the pH-potentiometry results show at the mM total concentration level and pH = 7.0, the ternary species [Al(CIP(HNADP] predominates in the ternary complex system. Comparing predicted NMR spectra with the experimental NMR results, it can be concluded that for the ternary complex, CIP binds to aluminum ion between the 3-carboxylic and 4-carbonyl groups, while the binding site of oxidized coenzyme II is through the oxygen of phosphate, which is linked to adenosine ribose, instead of pyrophosphate. The results also suggested CIP has the potential to be a probe molecular for the detection of NADP and the Al(III-NADP complexes under physiological condition.

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

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


    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.

  18. 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. (United States)

    Rose, Nicholas D; Regan, John M


    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.

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

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


    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.

  20. Two novel mutations in the reduced nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase gene of a patient with generalized type, hereditary methemoglobinemia. (United States)

    Manabe, J; Arya, R; Sumimoto, H; Yubisui, T; Bellingham, A J; Layton, D M; Fukumaki, Y


    Hereditary methemoglobinemia due to reduced nicotinamide adenine dinucleotide (NADH) cytochrome b5 reductase (b5R) deficiency is classified into two types, an erythrocyte (type I) and a generalized (type II). We investigated the b5R gene of a patient with type II from a white United Kingdom (UK) family and found that the patient was a compound heterozygote for two novel mutations. The first mutation was a C-to-A transversion changing codon 42 (TAC: Tyr) to a stop codon in the one allele. From this mutant allele, the product without the catalytic portion of the enzyme is generated. The second one was a missense mutation at codon 95 (CCC-->CAC) in the other allele with the result that Pro changed to His within the flavin adenine dinucleotide (FAD)-binding domain of the enzyme. To characterize effects of this missense mutation on the enzyme function, we compared glutathione S-transferase (GST)-fused b5R with the GST-fused mutant enzyme with the codon 95 missense mutation (P95H) expressed in Escherichia coll. The mutant enzyme showed less catalytic activity, less thermostability, and a greater susceptibility to trypsin than did the normal counterpart. The absorption spectrum of the mutant enzyme in the visual region differed from that of the wild-type. These results suggest that this amino acid substitution influences both secondary structure and catalytic activity of the enzyme. The compound heterozygosity for the nonsense and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient.

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

    KAUST Repository

    Rose, Nicholas D.


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

  2. Relationship between reduced nicotinamide adenine dinucleotide phosphate oxidase subunit p22phox gene polymorphism and obstructive sleep apnea-hypopnea syndrome in the Chinese Han population

    Institute of Scientific and Technical Information of China (English)

    LIU Hui-guo; LIU Kui; ZHOU Yan-ning; XU Yong-jian


    Background Increased production of reactive oxygen species (ROS) is thought to play a major role in the pathogenesis of obstructive sleep apnea-hypopnea syndrome (OSAHS). The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is an important source of ROS. The p22phox subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site. This study aimed to investigate the relationship between NADPH oxidase subunit p22phox gene polymorphism and OSAHS. Methods The genotypes of p22phox polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in 176 unrelated subjects of the Han population in southern region of China (including 107 OSAHS subjects and 69 non-OSAHS subjects), while the plasma concentration of superoxide dismutase (SOD) was detected in the two groups, and p22phox mRNA expression in peripheral blood mononuclear cell (PBMC) was determined with reverse transcription polymerase chain reaction (RT-PCR).Results The phagocyte NADPH oxidase subunit p22phox mRNA expression was significantly increased in the OSAHS group than that in the non-OSAHS group (P<0.01). Compared with the non-OSAHS control group ((85.31±9.23) U/ml), the levels of SOD were lower in patients with OSAHS ((59.65±11.61) U/ml (P<0.01). There were significant differences in genotypes distribution in p22phox polymorphism between the two groups (P=0.02). Compared with the non-OSAHS control group, the OSAHS group had a significantly higher T allele frequency in p22phox polymorphism (P=0.03). There were independent effects of p22phox polymorphism on body mass index (BMI), neck circumference (NC), waist-to-hip ratio (WHR) in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, systolic blood pressure (SBP), diastolic blood pressure (DBP) and apnea-hypopnea index (AHI) (P <0.05), but the carriers of the T allele had lower SOD

  3. A novel twist on molecular interactions between thioredoxin and nicotinamide adenine dinucleotide phosphate-dependent thioredoxin reductase

    DEFF Research Database (Denmark)

    Kirkensgaard, Kristine Groth; Hägglund, Per; Shahpiri, Azar;


    dinucleotide (FAD)-binding domain of HvNTR2 to strongly affect the interaction with Trx. In particular, Trp42 and Met43 play key roles for recognition of the endogenous HvTrxh2. Trx from Arabidopsis thaliana is also efficiently recycled by HvNTR2 but turnover in this case appears to be less dependent...... on these two residues, suggesting a distinct mode for NTR:Trx recognition. Comparison between the HvNTR2:HvTrxh2 model and the crystal structure of the Escherichia coli NTR:Trx complex reveals major differences in interactions involving the FAD- and NADPH-binding domains as supported by our experiments...

  4. Immobilization of flavin adenine dinucleotide (FAD) onto carbon cloth and its application as working electrode in an electroenzymatic bioreactor. (United States)

    Jayabalan, R; Sathishkumar, M; Jeong, E S; Mun, S P; Yun, S E


    A high porosity carbon cloth with immobilized FAD was employed as working electrode in electrochemical NADH-regeneration procedure. Carbon cloth was oxidized with hot acids to create surface carboxyl group and then coupled by adenine amino group of FAD with carbodiimide in the presence of N-hydroxysulfosuccinimide. The bioelectrocatalytic NADH-regeneration was coupled to the conversion of achiral substrate pyruvate into chiral product l-lactate by l-lactate dehydrogenase (l-LDH) within the same reactor. The conversion was completed at 96h in bioreactor with FAD-modified carbon cloth, resulting in about 6mM of l-lactate from 10mM of pyruvate. While with bare carbon cloth, the yield at 120h was around 5mM. Immobilized FAD on the surface of carbon cloth electrode facilitated it to carry electrons from electrode to electron transfer enzymes; thereby NADH-regeneration was accelerated to drive the enzymatic reaction efficiently.

  5. Cyclic dinucleotide detection with riboswitch-G-quadruplex hybrid. (United States)

    Tsuji, Genichiro; Sintim, Herman O


    A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.

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

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


    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.

  7. Inhibition of lactate production in rat brain extracts and synaptosomes by 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate. (United States)

    Cooper, A J; Lai, J C; Coleman, A E; Pulsinelli, W A


    In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.

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

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


    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.

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

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


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

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

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


    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.

  11. Enhancement of anaerobic degradation of azo dye with riboflavin and nicotinamide adenine dinucleotide harvested by osmotic lysis of wasted fermentation yeasts. (United States)

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


    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.

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

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


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

  13. Methods for detection of methyl-CpG dinucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Dunn, John J


    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.

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

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


    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.

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

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


    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.

  16. A Novel Nicotinamide Adenine Dinucleotide Correction Method for Mitochondrial Ca(2+) Measurement with FURA-2-FF in Single Permeabilized Ventricular Myocytes of Rat. (United States)

    Lee, Jeong Hoon; Ha, Jeong Mi; Leem, Chae Hun


    Fura-2 analogs are ratiometric fluoroprobes that are widely used for the quantitative measurement of [Ca(2+)]. 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 [Ca(2+)], as no available ratiometric dyes are excited in the visible range. Thus, NADH interference cannot be avoided during quantitative measurement of [Ca(2+)] 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, [Ca(2+)], and pH/mitochondrial membrane potential; Fura-2-FF for mitochondrial [Ca(2+)] and TMRE for Ψm or carboxy-SNARF-1 for pH were used. With this novel method, we found that the resting mitochondrial [Ca(2+)] concentration was 1.03 µM. This 1 µM cytosolic Ca(2+) could theoretically increase to more than 100 mM in mitochondria. However, the mitochondrial [Ca(2+)] increase was limited to ~30 µM in the presence of 1 µM cytosolic Ca(2+). 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.

  17. Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase. (United States)

    Xun, L; Sandvik, E R


    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 (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), 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 FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.

  18. Characterization of chlorophenol 4-monooxygenase (TftD) and NADH:flavin adenine dinucleotide oxidoreductase (TftC) of Burkholderia cepacia AC1100. (United States)

    Gisi, Michelle R; Xun, Luying


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

  19. G-tensors of the flavin adenine dinucleotide radicals in glucose oxidase: a comparative multifrequency electron paramagnetic resonance and electron-nuclear double resonance study. (United States)

    Okafuji, Asako; Schnegg, Alexander; Schleicher, Erik; Möbius, Klaus; Weber, Stefan


    The flavin adenine dinucleotide (FAD) cofactor of Aspergillus niger glucose oxidase (GO) in its anionic (FAD*-) and neutral (FADH*) radical form was investigated by electron paramagnetic resonance (EPR) at high microwave frequencies (93.9 and 360 GHz) and correspondingly high magnetic fields and by pulsed electron-nuclear double resonance (ENDOR) spectroscopy at 9.7 GHz. Because of the high spectral resolution of the frozen-solution continuous-wave EPR spectrum recorded at 360 GHz, the anisotropy of the g-tensor of FAD*- could be fully resolved. By least-squares fittings of spectral simulations to experimental data, the principal values of g have been established with high precision: gX=2.00429(3), gY=2.00389(3), gZ=2.00216(3) (X, Y, and Z are the principal axes of g) yielding giso=2.00345(3). The gY-component of FAD*- from GO is moderately shifted upon deprotonation of FADH*, rendering the g-tensor of FAD*- slightly more axially symmetric as compared to that of FADH*. In contrast, significantly altered proton hyperfine couplings were observed by ENDOR upon transforming the neutral FADH* radical into the anionic FAD*- radical by pH titration of GO. That the g-principal values of both protonation forms remain largely identical demonstrates the robustness of g against local changes in the electron-spin density distribution of flavins. Thus, in flavins, the g-tensor reflects more global changes in the electronic structure and, therefore, appears to be ideally suited to identify chemically different flavin radicals.

  20. Effect of telmisartan on the expression of adiponectin receptors and nicotinamide adenine dinucleotide phosphate oxidase in the heart and aorta in type 2 diabetic rats

    Directory of Open Access Journals (Sweden)

    Guo Zhixin


    Full Text Available Abstract Background Diabetic cardiovascular disease is associated with decreased adiponectin and increased oxidative stress. This study investigated the effect of telmisartan on the expression of adiponectin receptor 2 (adipoR2 and nicotinamide adenine dinucleotide phosphate (NADPH oxidase subunits in the heart and the expression of adiponectin receptor 1 (adipoR1 in aorta in type 2 diabetic rats. Methods Type 2 diabetes was induced by high-fat and high-sugar diet and intraperitoneal injection of a low dose of streptozotocin (STZ. Heart function, adipoR2, p22phox, NOX4, glucose transporter 4(GLUT4, monocyte chemoattractant protein-1(MCP-1 and connective tissue growth factor (CTGFin the heart, and adipoR1, MCP-1 and nuclear factor kappa B (NF-κB in aorta were analyzed in controls and diabetic rats treated with or without telmisartan (5mg/kg/d by gavage for 12 weeks. Results Heart function, plasma and myocardial adiponectin levels, the expression of myocardial adipoR2 and GLUT4 were significantly decreased in diabetic rats (P Conclusions Our results suggest that telmisartan upregulates the expression of myocardial adiponectin, its receptor 2 and GLUT4. Simultaneously, it downregulates the expression of myocardial p22phox, NOX4, MCP-1, and CTGF, contributing so to the improvement of heart function in diabetic rats. Telmisartan also induces a protective role on the vascular system by upregulating the expression of adipoR1 and downregulating the expression of MCP-1 and NF-κB in the abdominal aorta in diabetic rats.

  1. Ryanodine receptor type I and nicotinic acid adenine dinucleotide phosphate receptors mediate Ca2+ release from insulin-containing vesicles in living pancreatic beta-cells (MIN6). (United States)

    Mitchell, Kathryn J; Lai, F Anthony; Rutter, Guy A


    We have demonstrated recently (Mitchell, K. J., Pinton, P., Varadi, A., Tacchetti, C., Ainscow, E. K., Pozzan, T., Rizzuto, R., and Rutter, G. A. (2001) J. Cell Biol. 155, 41-51) that ryanodine receptors (RyR) are present on insulin-containing secretory vesicles. Here we show that pancreatic islets and derived beta-cell lines express type I and II, but not type III, RyRs. Purified by subcellular fractionation and membrane immuno-isolation, dense core secretory vesicles were found to possess a similar level of type I RyR immunoreactivity as Golgi/endoplasmic reticulum (ER) membranes but substantially less RyR II than the latter. Monitored in cells expressing appropriately targeted aequorins, dantrolene, an inhibitor of RyR I channels, elevated free Ca(2+) concentrations in the secretory vesicle compartment from 40.1 +/- 6.7 to 90.4 +/- 14.8 microm (n = 4, p < 0.01), while having no effect on ER Ca(2+) concentrations. Furthermore, nicotinic acid adenine dinucleotide phosphate (NAADP), a novel Ca(2+)-mobilizing agent, decreased dense core secretory vesicle but not ER free Ca(2+) concentrations in permeabilized MIN6 beta-cells, and flash photolysis of caged NAADP released Ca(2+) from a thapsigargin-insensitive Ca(2+) store in single MIN6 cells. Because dantrolene strongly inhibited glucose-stimulated insulin secretion (from 3.07 +/- 0.51-fold stimulation to no significant glucose effect; n = 3, p < 0.01), we conclude that RyR I-mediated Ca(2+)-induced Ca(2+) release from secretory vesicles, possibly potentiated by NAADP, is essential for the activation of insulin secretion.

  2. Sensitive and selective detection of adenine using fluorescent ZnS nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Meerabai Devi, L; Negi, Devendra P S, E-mail: [Department of Chemistry, North-Eastern Hill University, Permanent Campus, Shillong 793022 (India)


    We have used fluorescent ZnS nanoparticles as a probe for the determination of adenine. A typical 2 x 10{sup -7} M concentration of adenine quenches 39.3% of the ZnS fluorescence. The decrease in ZnS fluorescence as a function of adenine concentration was found to be linear in the concentration range 5 x 10{sup -9}-2 x 10{sup -7} M. The limit of detection (LOD) of adenine by this method is 3 nM. Among the DNA bases, only adenine quenched the fluorescence of ZnS nanoparticles in the submicromolar concentration range, thus adding selectivity to the method. The amino group of adenine was important in determining the quenching efficiency. Steady-state fluorescence experiments suggest that one molecule of adenine is sufficient to quench the emission arising from a cluster of ZnS consisting of about 20 molecules. Time-resolved fluorescence measurements indicate that the adenine molecules block the sites on the surface of ZnS responsible for emission with the longest lifetime component. This method may be applied for the determination of adenine in biological samples since the measurements have been carried out at pH 7.

  3. Detecting horizontally transferred and essential genes based on dinucleotide relative abundance. (United States)

    Baran, Robert H; Ko, Hanseok


    Various methods have been developed to detect horizontal gene transfer in bacteria, based on anomalous nucleotide composition, assuming that compositional features undergo amelioration in the host genome. Evolutionary theory predicts the inevitability of false positives when essential sequences are strongly conserved. Foreign genes could become more detectable on the basis of their higher order compositions if such features ameliorate more rapidly and uniformly than lower order features. This possibility is tested by comparing the heterogeneities of bacterial genomes with respect to strand-independent first- and second-order features, (i) G + C content and (ii) dinucleotide relative abundance, in 1 kb segments. Although statistical analysis confirms that (ii) is less inhomogeneous than (i) in all 12 species examined, extreme anomalies with respect to (ii) in the Escherichia coli K12 genome are typically co-located with essential genes.

  4. 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 (United States)

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


    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

  5. Effects of aqueous extract of Ruta graveolens and its ingredients on cytochrome P450, uridine diphosphate (UDP-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH-quinone oxidoreductase in mice

    Directory of Open Access Journals (Sweden)

    Yune-Fang Ueng


    Full Text Available Ruta graveolens (the common rue has been used for various therapeutic purposes, including relief of rheumatism and treatment of circulatory disorder. To elucidate the effects of rue on main drug-metabolizing enzymes, effects of an aqueous extract of the aerial part of rue and its ingredients on cytochrome P450 (P450/CYP, uridine diphosphate (UDP-glucuronosyltransferase, and reduced nicotinamide adenine dinucleotide (phosphate (NAD(PH:quinone oxidoreductase were studied in C57BL/6JNarl mice. Oral administration of rue extract to males increased hepatic Cyp1a and Cyp2b activities in a dose-dependent manner. Under a 7-day treatment regimen, rue extract (0.5 g/kg induced hepatic Cyp1a and Cyp2b activities and protein levels in males and females. This treatment increased hepatic UDP-glucuronosyltransferase activity only in males. However, NAD(PH:quinone oxidoreductase activity remained unchanged. Based on the contents of rutin and furanocoumarins of mouse dose of rue extract, rutin increased hepatic Cyp1a activity and the mixture of furanocoumarins (Fmix increased Cyp2b activities in males. The mixture of rutin and Fmix increased Cyp1a and Cyp2b activities. These results revealed that rutin and Fmix contributed at least in part to the P450 induction by rue.

  6. Pioglitazone inhibits the expression of nicotinamide adenine dinucleotide phosphate oxidase and p38 mitogen-activated protein kinase in rat mesangial cells

    Institute of Scientific and Technical Information of China (English)

    WANG Shan; YE Shan-dong; SUN Wen-jia; HU Yuan-yuan


    Background Oxidative Stress and p38 mitogen-activated protein kinase (p38MAPK) play a vital role in renal fibrosis.Pioglitazone can protect kidney but the underlying mechanisms are less clear.The purpose of this study was to investigate the effect of pioglitazone on oxidative stress and whether the severity of oxidative stress was associated with the phosphorylation level of p38MAPK.Methods Rat mesangial cells were cultured and randomly assigned to control group,high glucose group and pioglitazone group.After 48-hour exposure,the supernatants and ceils were collected.The protein levels of p22phox,p47phox,phosphorylated p38MAPK,total p38MAPK were measured by Western blotting.The gene expressions of p22phox,p47phox were detected by RT-PCR.The levels of intracellular reactive oxygen species (ROS) were determined by flow cytometry.The levels of superoxide dismutase (SOD) and maleic dialdehyde (MDA) in the supernatant were determined respectively.Results Compared with the control group,the expression levels of p22phox,p47phox,phospho-p38 and ROS significantly increased,activity of SOD decreased in high glucose group,while the level of MDA greatly increased (P <0.01).Pioglitazone significantly suppressed p22phox,p47phox expressions and oxidative stress induced by high glucose.The expressions of p22phox,p47phox,phospho-p38MAPK and ROS generation were markedly reduced after pioglitazone treatment (P <0.05).The activity of SOD in the the supernatant increased (P <0.05),while the level of MDA decreased greatly by pioglitazone (P <0.05).The level of oxidative stress was associated with the phosphorylation level of p38MAPK (P <0.01).Conclusion Pioglitazone can inhibit oxidative stress through suppressing NADPH oxidase expression and p38MAPK phosphorylation.

  7. Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer

    NARCIS (Netherlands)

    Abd-Elsalam, K.; Bahkali, A.H.; Moslem, M.; Wit, de P.J.G.M.; Verreet, J.A.


    Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were develop

  8. 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. (United States)

    Chifotides, Helen T; Dunbar, Kim R


    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.

  9. Quadracyclic adenine

    DEFF Research Database (Denmark)

    Dierckx, Anke; Miannay, Francois-Alexandre; Ben Gaied, Nouha;


    Fluorescent-base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation...... and base-pairing properties of a new environment-sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid-phase synthesis procedures. In DNA qA serves...... as an adenine analogue that preserves the B-form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2-aminopurine, and the recently developed triazole adenine...

  10. Molecular diagnosis of Prader-Willi syndrome: Parent-of-origin dependent methylation sites and non-isotopic detection of (CA){sub n} dinucleotide repeat polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Lerer, I.; Meiner, V.; Pashut-Lavon, I.; Abeliovich, D.


    We describe our experience in the molecular diagnosis of 22 patients suspected of Prader-Willi syndrome (PWS) using a DNA probe PW71 (D15S63) which detects a parent-of-origin specific methylated site in the PWS critical region. The cause of the syndrome was determined as deletion or uniparental disomy according to the segregation of (CA){sub n} dinucleotide repeat polymorphisms of the PWS/AS region and more distal markers of chromosome 15. In 10 patients the clinical diagnosis was confirmed by the segregation of (CA){sub n}, probably due to paternal microdeletion in the PWs critical region which did not include the loci D15S97, D15S113, GABRB3, and GABRA5. This case demonstrates the advantage of the DNA probe PW71 in the diagnosis of PWS. 31 refs., 2 figs., 3 tabs.

  11. Synthesis and Biological Evaluation of Nicotinamide Adenine Dinucleotides Analogues as Inhibitors of CD38%烟酰胺腺嘌呤二核苷酸类CD38抑制剂的合成及生物活性评价

    Institute of Scientific and Technical Information of China (English)

    陈哲; KWONGAnnaKa-Yee; 杨振军; 张亮仁; LEEHonCheung; 张礼和


    CD38 is the main mammalian ADP-ribosyl cyclase and a signaling enzyme responsible for catalyzing the synthesis of Ca2+-messengers and plays a critical role in a wide range of physiological functions. It is of great interest to develop specific and generally applicable inhibitors of CD38. Fluoro-substituted nicotina-mide adenine dinucleotides( NAD) , such as ara-F NMN and ara-F NAD, are catalysis-dependent inhibitors of CD38 and are often used as probes for investigating the function of CD38. For understanding the effect of fluo-ro-substitution on activity in more detail and discovery of active inhibitors of CD38, compounds 2a-2c were synthesized and their inhibition against the hydrolysis activities of CD38 were evaluated. The syntheses were performed by starting from the corresponding fluoro-substituted sugar, then followed by coupling with nicoti-namide, regio-seleclive 5 '-phosphorylation and condensation with adenosine monophosphate, successively. All target compounds were purified by HPLC and characterized by NMR and HRMS. Two compounds showed strong inhibitions, especially 2'-deoxy-2'-fluororibonofuranosyl which gave activity with IC50 of 0. 056μmol/L and was two orders of magnitude higher than positive control ara-F NAD. The results also showed that the activity was greatly affected by the number and the position of fluorine atom on the sugar ring, as well as the configuration of the inhibitors. The detailed biological investigation and structure-activity relationship are underway.%分别以1,3,5-三苯甲酰基-α-D-核糖、3,5-二苯甲酰基-2-脱氧-2,2-氟戊呋喃糖-1-酮和D-木糖为原料,经由烟酰胺核苷及烟酰胺核苷酸中间体,合成了系列糖环经氟原子取代的烟酰胺腺嘌呤二核苷酸(NAD)类CD38抑制剂.基于对CD38的水解抑制能力的考察,评价了所合成氟代NAD类似物的活性.结果表明,糖环上氟原子取代的数目和位置对抑制剂活性的影响十分明显,烟酰胺核苷的端

  12. Preparation of a sol-gel-derived carbon nanotube ceramic electrode by microwave irradiation and its application for the determination of adenine and guanine

    Energy Technology Data Exchange (ETDEWEB)

    Abbaspour, Abdolkarim, E-mail: [Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Fars 71456-85464 (Iran, Islamic Republic of); Ghaffarinejad, Ali [Department of Chemistry, College of Sciences, Shiraz University, Shiraz, Fars 71456-85464 (Iran, Islamic Republic of)


    In this study, microwave irradiation was used for the fast preparation (min) of a sol-gel-derived carbon nanotube ceramic electrode (MW-CNCE). For confirmation of the preparation of the ceramic by MW irradiation, Fourier transform infrared, X-ray diffraction spectra and scanning electron microscopy images of the produced ceramic were compared with those of conventional ceramic (which is produced by drying the ceramic in air for 48 h). The electrochemical behavior of MW-CNCE in nicotinamide adenine dinucleotide, L-cysteine, adenine and guanine was compared with that of a conventional sol-gel-derived carbon nanotube ceramic electrode (CNCE). In all systems, similar peak potentials and lower background currents were obtained with respect to CNCE. Finally, the MW-CNCE was used for the simultaneous determination of adenine and guanine using differential pulse voltammetry. The linear ranges of 0.1-10 and 0.1-20 muM were obtained for adenine and guanine, respectively. These results are comparable with some modified electrodes that have recently been reported for the determination of adenine and guanine, with the advantage that the proposed electrode did not contain modifier. In addition, the proposed electrode was successfully used for the oxidation of adenine and guanine in DNA, and the detection limit for this measurement was 0.05 mug mL{sup -1} DNA.

  13. DNA damage induced by the environmental carcinogen butadiene: identification of a diepoxybutane-adenine adduct and its detection by 32P-postlabelling. (United States)

    Leuratti, C; Jones, N J; Marafante, E; Kostiainen, R; Peltonen, K; Waters, R


    To date only a few studies have been undertaken on DNA adducts formed by epoxybutene (EB) and diepoxybutane (DEB), the two active metabolites of 1,3-butadiene. Our interests have focused on further investigating DNA alkylation by the two epoxides, especially in relation to the development of a method for human biomonitoring. Here, following the reaction of deoxyadenosine monophosphate and poly(dA-dT)(dA-dT) with DEB and subsequent HPLC, we have identified an adenine adduct. MS analyses indicate the structure of an adenine adducted by DEB at the N6 position. A HPLC/32P-postlabelling method was developed for its measurement in DNA samples and the adduct was detected in calf thymus DNA and DNA from Chinese hamster ovary cells exposed to DEB. The 100% labelling efficiency during postlabelling, the amount of the adduct and its elution before the normal nucleotides during HPLC suggest it could be a suitable indicator of BUT exposure.

  14. 脊髓烟酰胺腺嘌呤二核苷酸磷酸氧化酶在大鼠糖尿病神经痛维持中的作用%Role of nicotinamide adenine dinucleotide phosphate oxidase in spinal cord in maintenance of diabetic neuropathic pain in rats

    Institute of Scientific and Technical Information of China (English)

    黄晓雷; 李晓芸; 吴铭广; 文亚杰; 齐晓非; 胡薇; 李元涛


    目的 评价脊髓烟酰胺腺嘌呤二核苷酸磷酸氧化酶(NADPH氧化酶)在大鼠糖尿病神经痛维持中的作用.方法 清洁级雄性SD大鼠,2月龄,体重180 ~ 220 g,采用腹腔注射1%链脲佐菌素(STZ)的方法制备糖尿病模型,注射STZ后72 h血糖>16.7 mmol/L的大鼠作为糖尿病大鼠.采用随机数字表法,将20只糖尿病大鼠分为2组(n=10):糖尿病神经痛组(DN组)和NADPH氧化酶特异性抑制剂香荚兰乙酮组(A组),另取10只大鼠为正常对照组(C组).A组于注射STZ后28 d腹腔注射香荚兰乙酮5 mg/kg,1次/d,连续7d.分别于注射STZ前(T1)、注射STZ后7、14、21、28、35 d(T2-6)时测定机械缩足反应阈(PWT).于T6时PWT测定结束后,处死大鼠,取L4,5节段脊髓组织,检测NADPH氧化酶亚基gp91phox和p47phox的表达、MDA含量及SOD活性.结果 与C组比较,DN组和A组T3-5时PWT降低,脊髓gp91phpx和p47phox的表达上调,MDA含量升高,SOD活性降低(P<0.05);与DN组比较,A组T6时PWT升高,脊髓gp91phox和p47phox的表达下调,MDA含量降低,SOD活性升高(P<0.05).结论 脊髓NADPH氧化酶参与了大鼠糖尿病神经痛的维持.%Objective To evaluate the role of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) in the spinal cord in the maintenance of diabetic neuropathic pain in rats.Methods Pathogenfree male Sprague-Dawley rats,aged 2 months,weighing 180-220 g,were used in the study.Diabetes mellitus was induced by intraperitoneal streptozotocin (STZ) 60mg/kg and confirmed by blood glucose > 16.7 mmol/L at 72 h after STZ injection.Twenty diabetic rats were randomly allocated to diabetic neuropathic pain group (DN group,n =10) and apocynin (specific NADPH oxidase inhibitor) group (A group,n =10).Another 10 agematched normal rats served as control group (C group,n =10).Twenty-eight days after STZ injection,apyconin 5 mg/kg was injected intraperitoneally once a day for 7 consecutive days in A group.Paw withdrawal threshold to yon

  15. Automated genotyping of dinucleotide repeat markers

    Energy Technology Data Exchange (ETDEWEB)

    Perlin, M.W.; Hoffman, E.P. [Carnegie Mellon Univ., Pittsburgh, PA (United States)]|[Univ. of Pittsburgh, PA (United States)


    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.

  16. 尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4源性活性氧过量产生抑制胚胎干细胞向心肌细胞的分化%Excessive production of nicotinamide adenine dinucleotide phosphate oxidase 4-dependent reactive oxygen species suppresses cardiomyocyte differentiation from embrvonic stem cells

    Institute of Scientific and Technical Information of China (English)

    张小勇; 国汉邦; 黎健


    二核苷酸磷酸氧化酶4过表达诱导的细胞凋亡.结果:①不同水平的活性氧对心肌细胞分化具有不同的效应.在分化后4 d用较低浓度(1~100 nmol/L)的过氧化氢处理胚小体2 h可明显促进心肌细胞分化(P<0.01),而较高浓度(> 1 μmol/L)的过氧化氢则显示出抑制作用(P<0.01).②尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4程小鼠胚胎干细胞中的表达水平最高,尼克酰胺腺嘌呤二核苷酸磷酸氧化酶3虽然也在胚胎干细胞中表达,但表达水平低,丽尼克酰胺腺嘌呤二核苷酸磷酸氧化酶1、2在胚胎干细胞中不表达.RT-PCR检测结果显示,与单纯转染pcDNA3.1细胞相比,转染pcDNA3.1-NOX4质粒的CGR8细胞中尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达.③四唑氮蓝实验检测结果显示,高水平表达的尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4引起过量活性氧产生(P<0.05).与未转染质粒的细胞相比,尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达抑制了心肌细胞的分化(P<0.01). Western Blot结果显示高水平尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4导致胚小体内MLC2v蛋白水平降低.④p21和p53可能参与了NADPH氧化酶4诱导的凋亡过程.转染尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4的p53-/-ES细胞R72D27并未发生凋亡,高水平的Bcl-2可以抑制尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4过表达诱导的细胞凋亡.结论:尼克酰胺腺嘌呤二核苷酸磷酸氧化酶4在心肌细胞分化中起关键作用,p53和p21以及Bcl-2可能参与了凋亡信号通路的调控.%BACKGROUND: Reactive oxygen species (ROS), including superoxide anion (O2) and hydrogen peroxide (H2O2), have been recognized as specific second messengers in signaling cascades involved in the growth and differentiation of cells.The generation of excessive ROS initiates cardiomyocyte apoptosis. This paper is aimed to corroborate the hypothesis that excessive amounts of nicotinamide adenine

  17. Creation of bioorthogonal redox systems depending on nicotinamide flucytosine dinucleotide. (United States)

    Ji, Debin; Wang, Lei; Hou, Shuhua; Liu, Wujun; Wang, Jinxia; Wang, Qian; Zhao, Zongbao K


    Many enzymes catalyzing biological redox chemistry depend on the omnipresent cofactor, nicotinamide adenine dinucleotide (NAD). NAD is also involved in various nonredox processes. It remains challenging to disconnect one particular NAD-dependent reaction from all others. Here we present a bioorthogonal system that catalyzes the oxidative decarboxylation of l-malate with a dedicated abiotic cofactor, nicotinamide flucytosine dinucleotide (NFCD). By screening the multisite saturated mutagenesis libraries of the NAD-dependent malic enzyme (ME), we identified the mutant ME-L310R/Q401C, which showed excellent activity with NFCD, yet marginal activity with NAD. We found that another synthetic cofactor, nicotinamide cytosine dinucleotide (NCD), also displayed similar activity with the ME mutants. Inspired by these observations, we mutated d-lactate dehydrogenase (DLDH) and malate dehydrogenase (MDH) to DLDH-V152R and MDH-L6R, respectively, and both mutants showed fully active with NFCD. When coupled with DLDH-V152R, ME-L310R/Q401C required only a catalytic amount of NFCD to convert l-malate. Our results opened the window to engineer bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.

  18. Effects of glucagon-like peptide-1 on the expression of nicotinamide adenine dinucleotide phosphate oxidase subunits in the heart of type 1 diabetic rats%胰高血糖素样肽1对1型糖尿病大鼠心肌烟酰胺腺嘌呤二核苷酸氧化酶亚单位表达的影响

    Institute of Scientific and Technical Information of China (English)

    俞媛贤; 郭志新; 齐伟; 杜时晶; 刘晋津; 吴杰萍


    Objective To explore the effect of glucagon-like peptide-1 on the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox and Nox4 in the heart tissue of type 1 diabetic rats.Methods Forty-two male Sprague-Dawley(SD) rats were randomly divided into normal control group (group A,n=7) and diabetic model group (n =35) with the random number table.Type 1 diabetic model was established by intraperitoneal injection of streptozotocin.Twenty-nine successfully-induced diabetic rats were randomly divided into diabetic (group DM,n =10),diabetic treated with low-dose of GLP-1 (group DL,n =10) and diabetic treated with high-dose of GLP-1 (group DH,n =9) with the random number table method.The rats in group DL were given exenatide in dose of 1 μg/kg twice daily by subcutaneous injection.The rats in group DH were given exenatide in dose of 5 μg/kg twice daily by subcutaneous injection.All rats were sacrificed after exenatide treatment for eight weeks.The mRNA expression of myocardial p22phox and Nox4 in the rats of four groups was detected by real-time fluorescence quantitative polymerase chain reaction(PCR),and the protein expression of myocardial copper zinc-superoxide dismutase (Cu-Zn-SOD) was detected by immunohistochemical staining.Statistical analysis among groups was performed by using one way ANOVA.Results Compared with group NC,the mRNA expression of myocardial p22phox and Nox4 and the myocardial Cu-Zn-SOD protein expression increased significantly in group DM(t =5.77,5.36,59.91,all P <0.05).After exenatide treatment for 8 weeks,the mRNA expression of myocardial p22phox and Nox4 and the myocardial Cu-Zn-SOD protein expression decreased significantly in group DL and DH (t =16.86,7.66 and 16.11,7.59 and 56.00,47.05,and all P < 0.05).Compared with group DL,the mRNA expression of myocardial p22phox and Nox4 decreased significantly in group DH (t =10.14,8.67,both P < 0.05).There was no significant difference in the expression of

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

    Directory of Open Access Journals (Sweden)

    Sunil Ojha


    Full Text Available 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

  20. New Approach to Detecting Phenoxyl Free Radicals Generated in Enzyme Reaction by Stopped-flow Spectrophotometry

    Institute of Scientific and Technical Information of China (English)

    Jing XU; Lan Hua ZHAO; Xin Guo WU; Hong Mei WANG; Ru Xiu CAI


    A highly sensitive stopped-flow spectrophotometry kinetic method was proposed forquantification phenoxyl radicals based on their accelerating effect on the oxidation of nicotinamide adenine dinucleotide (NADH). Phenoxyl radicals generated from as low as 1×10-8 mol/L 2,4-DCP can be readily detected with the proposed method and the detecting limit was 2.5×10-9mol/L.

  1. 小分子干扰RNA抑制高氧暴露下人肺腺癌A549细胞中的硫氧还蛋白-2对还原型烟酰胺腺嘌呤二核苷酸脱氢酶亚单位1、细胞色素C氧化酶工表达的影响%Suppressed expression of thioredoxin-2 by small interference RNA in A549 cells exposed to hyperoxia reduced expression of nicotinamide-adenine dinucleotide dehydrogenase subunit 1 and cytochrome C oxidase Ⅰ

    Institute of Scientific and Technical Information of China (English)

    蔡成; 常立文; 李文斌; 陈燕; 单瑞艳; 刘伟; 潘睿


    Objective To explore the effects of expression of thioredoxin-2(Trx-2) suppressed by small interference RNA(SiRNA) in A549 cells exposed to hyperoxia on expression of nicotinamide adenine dinucleotide(NADH) dehydrogenase subunit 1(ND1)and cytochrome C oxidase Ⅰ(COX Ⅰ). Methods A549 cells were gained by serial subcultivation in vitro and transfered with synthetic Trx-2 sequence-specific SiRNA and then were randomly divided into air group without interference,hyperoxia group without interference,air group after interference,and hyperoxia group after interference.After exposure to oxygen or room air for 12,24 and 48 h,expressions of Trx-2,ND1 and COX Ⅰ mRNA of these cells were detected by reverse transcription-polymerase chain reaction (RT-PCR),and Trx-2 protein was detected by Western blot. Results (1)Sequence-specific SiRNA targeting Trx-2 could significantly down-regulate its expression in A549 cells.(2)Trx-2 mRNA levds in hyperoxia group without interference at 24 h was higher than those in air group without interference(0.7799±0.1249 VS 0.4424±Ⅰ.1140,P<0.05).Th-2 mRNA levels in hyperoxia group after ireedcrence at 24 h and 48 h were 0.2774±0.0174 and 0.2587±0.0069,lower than those in air group after interference and hyperoxia group without interference (P<0.05).(3)ND1 mRNA levels in hyperoxia group without interference at 24 h was 0.6609±0.0368,lower than those in air group without interference(0.8898±0.1049)(P<0.05).ND1 mRNA levels in hyperoxia group after interference at 12 h was 0.8848±0.0135,higher than those in air group after imederence(P<0.05).ND1 mRNA levels in hypemxia group after interference at 48 h was 0.3808±0.0937,lower than those in air group after imerference and hyperoxia group without interference(P<0.05).(4)COXI mRNA levels in hypemxia group without inteference at 12,24 and 48 h were 1.7313±0.4331,2.1929±0.6722 and 2.0754±0.2584,higher than those in air group witheUt interference,respectively (P<0.05). Conclusions ND1 and

  2. Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides. (United States)

    Videla, L A; Salim-Hanna, M; Lissi, E A


    The study of the interaction of alkylperoxyl radicals generated by the aerobic thermolysis of 2,2'-azobis(2-amidinopropane) (AAP) with yeast alcohol dehydrogenase (YADH) revealed a high reactivity of the enzyme, with an average of about 20 radicals per added YADH tetramer being needed to elicit its total inactivation. NAD+ enhanced YADH inactivation at NAD+/YADH molar ratios from 0.25 to 1, decreasing the rate of the process when added in excess to the enzyme concentration. At NADH/YADH molar ratios greater than 1, NADH exhibited a protective effect characterized by a poorly defined induction time and lower inactivation rates, which progressively increased during the reaction period. These changes occurred concomitantly with the oxidation of NADH into NAD+, which might counteract the protective effect of NADH. Under similar conditions, NADP+ did not modify AAP-induced YADH inactivation, while NADPH exhibited a modest protection at NADPH/YADH molar ratios greater than 1. It is concluded that YADH inactivation by alkylperoxyl radicals is strongly dependent on the redox state of the NADH-NAD+ couple, as the rates of the process at different time intervals inversely correlate with the respective NADH/NAD+ ratios.

  3. Folding dynamics of flavin adenine dinucleotide (FAD) inside non-aqueous and aqueous reverse micelles (United States)

    Sengupta, Abhigyan; Gavvala, Krishna; Koninti, Raj Kumar; Chaudhuri, Haribandhu; Hazra, Partha


    Present Letter describes dynamics of FAD in non-aqueous and aqueous reverse micelles (RMs). FAD in non-aqueous reverse micelles (containing MeOH, glycerol, formamide or DMF) shows a prominent rise of quantum yield with increasing solvent loading, whereas in water RM FAD shows a rise in quantum yield only up to w0 = 10 and afterwards drops sharply up to bulk. A relative difference in polarity of dry AOT to solvent loaded RMs can be attributed to the opposite trend of changes in emission. This Letter of FAD in different RMs and its preferential orientation might be useful for understanding FAD dynamics inside flavoproteins.

  4. Quantum-chemical study of interactions of trans-resveratrol with guanine-thymine dinucleotide and DNA-nucleobases. (United States)

    Mikulski, Damian; Szeląg, Małgorzata; Molski, Marcin


    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.

  5. Synthesis and Chiral Separation of Dinucleotide(TpAZT) Phosphoramidates

    Institute of Scientific and Technical Information of China (English)

    Chang Xue LIN; Hua FU; Guang Zhong TU; Yu Fen ZHAO


    Dinucleotide (TpAZT) phosphoramidates were synthesized by Todd reaction of dinucleoside H-phosphonates and amino acid methyl esters, and their diastereomers (Rp and Sp) were separated by crystallization, and the results showed that natural and cheap methyl esters of alanine and phenylalanine can be used for large-scale synthesis of dinucleotide analogs.

  6. Detecting Industrial Chemicals in Water With Microbial Fuel Cells and Artificial Neural Networks (United States)


    Transfer FM First Moment mA milli-Ampere MFC Microbial Fuel Cell NADH Nicotinamide adenine dinucleotide PA Peak Area PH...transfer of electrons across the inner membrane. Nicotinamide adenine dinucleotide (NADH) derived from the oxidation of organic matter is taken up by...filters (Mahle et al., 2003). It is a weak cholinesterase inhibitor and is an irritant of the skin , eyes, mucous membranes and upper respiratory tract

  7. Analysis of dinucleotide signatures in HIV-1 subtype B genomes

    Indian Academy of Sciences (India)

    Aridaman Pandit; Jyothirmayi Vadlamudi; Somdatta Sinha


    Dinucleotide usage is known to vary in the genomes of organisms. The dinucleotide usage profiles or genome signatures are similar for sequence samples taken from the same genome, but are different for taxonomically distant species. This concept of genome signatures has been used to study several organisms including viruses, to elucidate the signatures of evolutionary processes at the genome level. Genome signatures assume greater importance in the case of host–pathogen interactions, where molecular interactions between the two species take place continuously, and can influence their genomic composition. In this study, analyses of whole genome sequences of the HIV-1 subtype B, a retrovirus that caused global pandemic of AIDS, have been carried out to analyse the variation in genome signatures of the virus from 1983 to 2007.We show statistically significant temporal variations in some dinucleotide patterns highlighting the selective evolution of the dinucleotide profiles of HIV-1 subtype B, possibly a consequence of host specific selection.

  8. Excited State Pathways Leading to Formation of Adenine Dimers. (United States)

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


    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).

  9. Identification of prophages in bacterial genomes by dinucleotide relative abundance difference.

    Directory of Open Access Journals (Sweden)

    K V Srividhya

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

  10. Novel non-specific DNA adenine methyltransferases (United States)

    Drozdz, Marek; Piekarowicz, Andrzej; Bujnicki, Janusz M.; Radlinska, Monika


    The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N6-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N6-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N6-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation. PMID:22102579

  11. Bound anionic states of adenine

    Energy Technology Data Exchange (ETDEWEB)

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


    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Nakano, Koji [Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)], E-mail:; Ohkubo, Kimihiko; Taira, Hiroaki [Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Takagi, Makoto [Fukuoka Women' s University, 1-1-1, Kasumigaoka, Higashi-ku, Fukuoka 813-8529 (Japan); Imato, Toshihiko [Department of Applied Chemistry, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan)


    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{sup -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{sup -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.

  13. Alternative Splicing and Differential Expression of Two Transcripts of Nicotine Adenine Dinucleotide Phosphate Oxidase B Gene from Zea mays

    Institute of Scientific and Technical Information of China (English)

    Fan Lin; Yun Zhang; Ming-Yi Jiang


    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.Altemative splicing of ZmrbohB has generated two transcript isoforms, ZmrbohB-α and -β. Spliced transcript ZmrbohB-β 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-α. The transcripts of ZmrbohB-α accumulated markedly when the maize seedlings were subjected to various abiotic stimuli, such as wounding, cold (4℃), heat (40℃), 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.

  14. The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption. (United States)

    Cheng, X; Hookway, E S; Kashima, T; Oppermann, U; Galione, A; Athanasou, N A


    Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20 mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

  15. Syntheses of nicotinamide riboside and derivatives: effective agents for increasing nicotinamide adenine dinucleotide concentrations in mammalian cells. (United States)

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


    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.

  16. On the origin of multiexponential fluorescence decays from 2-aminopurine-labeled dinucleotides. (United States)

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


    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.

  17. Water-soluble, electroactive, and photoluminescent quaterthiophene-dinucleotide conjugates. (United States)

    Alesi, Silvia; Brancolini, Giorgia; Melucci, Manuela; Capobianco, Massimo Luigi; Venturini, Alessandro; Camaioni, Nadia; Barbarella, Giovanna


    Quaterthiophene-dinucleotide conjugates 5'TA3'-t4-3'AT5', 5'AA3'-t4-3'AA5', and 5'TT3'-t4-3'TT5' (TA: thymidine-adenosine, AA: adenosine-adenosine, TT: thymidine-thymidine) were synthesized and analyzed by a combination of spectroscopy and microscopy, electrical characterization, and theoretical calculations. Circular dichroism (CD) experiments demonstrated a transfer of chirality from the dinucleotides to quaterthiophene at high ionic strength and in cast films. The films were photoluminescent and electroactive. CD and photoluminescence spectra and current density/voltage plots (measured under dynamic vacuum) displayed significant variation on changing the dinucleotide scaffold. Molecular mechanics and molecular dynamics calculations indicated that the conformation and packing modes of the conjugates are the result of a balance between intra- and intermolecular nucleobase-thiophene stacking interactions and intramolecular hydrogen bonding between the nucleobases.

  18. A fiber-optic sorbitol biosensor based on NADH fluorescence detection toward rapid diagnosis of diabetic complications. (United States)

    Gessei, Tomoko; Arakawa, Takahiro; Kudo, Hiroyuki; Mitsubayashi, Kohji


    Accumulation of sorbitol in the tissue is known to cause microvascular diabetic complications. In this paper, a fiber-optic biosensor for sorbitol which is used as a biomarker of diabetic complications was developed and tested. The biosensor used a sorbitol dehydrogenase from microorganisms of the genus Flavimonas with high substrate specificity and detected the fluorescence of reduced nicotinamide adenine dinucleotide (NADH) by the enzymatic reaction. An ultraviolet light emitting diode (UV-LED) was used as the excitation light source of NADH. The fluorescence of NADH was detected using a spectrometer or a photomultiplier tube (PMT). The UV-LED and the photodetector were coupled using a Y-shaped optical fiber. In the experiment, an optical fiber probe with a sorbitol dehydrogenase immobilized membrane was placed in a cuvette filled with a phosphate buffer containing the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). The changes in NADH fluorescence intensity were measured after adding a standard sorbitol solution. According to the experimental assessment, the calibration range of the sorbitol biosensor systems using a spectrometer and a PMT was 5.0-1000 μmol L(-1) and 1.0-1000 μmol L(-1), respectively. The sorbitol biosensor system using the sorbitol dehydrogenase from microorganisms of the genus Flavimonas has high selectivity and sensitivity compared with that from sheep liver. The sorbitol biosensor allows for point-of-care testing applications or daily health care tests for diabetes patients.

  19. Dinucleotide controlled null models for comparative RNA gene prediction

    Directory of Open Access Journals (Sweden)

    Gesell Tanja


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

  20. Design and synthesis of novel adenine fluorescence probe based on Eu(III) complexes with dtpa-bis(guanine) ligand. (United States)

    Tian, Fengyun; Jiang, Xiaoqing; Dou, Xuekai; Wu, Qiong; Wang, Jun; Song, Youtao


    A novel adenine (Ad) fluorescence probe (Eu(III)-dtpa-bis(guanine)) was designed and synthesized by improving experimental method based on the Eu(III) complex and dtpa-bis(guanine) ligand. The dtpa-bis(guanine) ligand was first synthesized by the acylation action between dtpaa and guanine (Gu), and the corresponding Eu(III) complex was successfully prepared through heat-refluxing method with dtpa-bis(guanine) ligand. As a novel fluorescence probe, the Eu(III)-dtpa-bis(guanine) complex can detect adenine (Ad) with characteristics of strong targeting, high specificity and high recognition ability. The detection mechanism of the adenine (Ad) using this probe in buffer solution was studied by ultraviolet-visible (UV-vis) and fluorescence spectroscopy. When the Eu(III)-dtpa-bis(guanine) was introduced to the adenine (Ad) solution, the fluorescence emission intensity was significantly enhanced. However, adding other bases such as guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) with similar composition and structure to that of adenine (Ad) to the Eu(III)-dtpa-bis(guanine) solution, the fluorescence emission intensities are nearly invariable. Meanwhile, the interference of guanine (Gu), xanthine (Xa), hypoxanthine (Hy) and uric acid (Ur) on the detection of the adenine using Eu(III)-dtpa-bis(guanine) probe was also studied. It was found that presence of these bases does not affect the detection of adenine (Ad). A linear response of fluorescence emission intensities of Eu(III)-dtpa-bis(guanine) at 570nm as a function of adenine (Ad) concentration in the range of 0.00-5.00×10(-5)molL(-1) was observed. The detection limit is about 4.70×10(-7)molL(-1).

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

    Kokina, Agnese; Kibilds, Juris; Liepins, Janis


    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. Conformational control of benzophenone-sensitized charge transfer in dinucleotides


    Merz, Thomas; Wenninger, Matthias; Weinberger, Michael; Riedle, Eberhard; Wagenknecht, Hans-Achim; Schuetz, Martin


    Charge transfer in DNA cannot be understood without addressing the complex conformational flexibility, which occurs on a wide range of timescales. In order to reduce this complexity four dinucleotide models 1X consisting of benzophenone linked by a phosphodiester to one of the natural nucleosides X = A, G, T, C were studied in water and methanol. The theoretical work focuses on the dynamics and electronic structure of 1G. Predominant conformations in the two solvents were obtained by molecula...

  3. Optical biopsy - a new armamentarium to detect disease using light (United States)

    Pu, Yang; Alfano, Robert R.


    Optical spectroscopy has been considered a promising method for cancer detection for past thirty years because of its advantages over the conventional diagnostic methods of no tissue removal, minimal invasiveness, rapid diagnoses, less time consumption and reproducibility since the first use in 1984. It offers a new armamentarium. Human tissue is mainly composed of extracellular matrix of collagen fiber, proteins, fat, water, and epithelial cells with key molecules in different structures. Tissues contain a number of key fingerprint native endogenous fluorophore molecules, such as tryptophan, collagen, elastin, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD) and porphyrins. It is well known that abnormalities in metabolic activity precede the onset of a lot of main diseases: carcinoma, diabetes mellitus, atherosclerosis, Alzheimer, and Parkinson's disease, etc. Optical spectroscopy may help in detecting various disorders. Conceivably the biochemical or morphologic changes that cause the spectra variations would appear earlier than the histological aberration. Therefore, "optical biopsy" holds a great promise as clinical tool for diagnosing early stage of carcinomas and other deceases by combining with available photonic technology (e.g. optical fibers, photon detectors, spectrographs spectroscopic ratiometer, fiber-optic endomicroscope and nasopharyngoscope) for in vivo use. This paper focuses on various methods available to detect spectroscopic changes in tissues, for example to distinguish cancerous prostate tissues and/or cells from normal prostate tissues and/or cells. The methods to be described are fluorescence, stokes shift, scattering, Raman, and time-resolved spectroscopy will be reviewed. The underlying physical and biological basis for these optical approaches will be discussed with examples. The idea is to present some of the salient works to show the usefulness and methods of Optical Biopsy for cancer detection and

  4. Electrochemical studies on the oxidation of guanine and adenine at cyclodextrin modified electrodes. (United States)

    Abbaspour, Abdolkarim; Noori, Abolhassan


    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.

  5. The catalase activity of diiron adenine deaminase

    Energy Technology Data Exchange (ETDEWEB)

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


    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. The overlapping host responses to bacterial cyclic dinucleotides. (United States)

    Abdul-Sater, Ali A; Grajkowski, Andrzej; Erdjument-Bromage, Hediye; Plumlee, Courtney; Levi, Assaf; Schreiber, Michael T; Lee, Carolyn; Shuman, Howard; Beaucage, Serge L; Schindler, Christian


    Macrophages respond to infection with Legionella pneumophila by the induction of inflammatory mediators, including type I Interferons (IFN-Is). To explore whether the bacterial second messenger cyclic 3'-5' diguanylate (c-diGMP) activates some of these mediators, macrophages were infected with L. pneumophila strains in which the levels of bacterial c-diGMP had been altered. Intriguingly, there was a positive correlation between c-diGMP levels and IFN-I expression. Subsequent studies with synthetic derivatives of c-diGMP, and newly described cyclic 3'-5' diadenylate (c-diAMP), determined that these molecules activate overlapping inflammatory responses in human and murine macrophages. Moreover, UV crosslinking studies determined that both dinucleotides physically associate with a shared set of host proteins. Fractionation of macrophage extracts on a biotin-c-diGMP affinity matrix led to the identification of a set of candidate host binding proteins. These studies suggest that mammalian macrophages can sense and mount a specific inflammatory response to bacterial dinucleotides.

  7. Influence of Magnetic Microparticles Isolation on Adenine Homonucleotides Structure

    Directory of Open Access Journals (Sweden)

    Monika Kremplova


    Full Text Available The electroactivity of purine and pyrimidine bases is the most important property of nucleic acids that is very useful for determining oligonucleotides using square wave voltammetry. This study was focused on the electrochemical behavior of adenine-containing oligonucleotides before and after their isolation using paramagnetic particles. Two peaks were detected—peak A related to the reduction of adenine base and another peak B involved in the interactions between individual adenine strands and contributes to the formation of various spatial structures. The influence of the number of adenine bases in the strand in the isolation process using paramagnetic particles was investigated too.

  8. CpG dinucleotide frequencies reveal the role of host methylation capabilities in parvovirus evolution. (United States)

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


    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.

  9. Detection of ATP and NADH: A Bioluminescent Experience. (United States)

    Selig, Ted C.; And Others


    Described is a bioluminescent assay for adenosine triphosphate (ATP) and reduced nicotineamide-adenine dinucleotide (NADH) that meets the requirements of an undergraduate biochemistry laboratory course. The 3-hour experiment provides students with experience in bioluminescence and analytical biochemistry yet requires limited instrumentation,…

  10. Surface-enhanced Raman spectroscopy (SERS) for identifying traces of adenine in different mineral and rock samples (United States)

    Lafuente, B.; Navarro, R.; Sansano, A.; Rull, F.


    The aim of this study is to analyze the potentials of SERS as a technique for in-situ identification of life traces in Mars surface explorations using the Raman instrument (RLS), payload of the ESA Mars mission Exomars. This preliminary study focused on detection of adenine on a variety of rocks soils samples using macro-SERS detection.

  11. Radiation and thermal stabilities of adenine nucleotides. (United States)

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


    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.

  12. Dinucleotide composition in animal RNA viruses is shaped more by virus family than host species. (United States)

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


    Viruses use the cellular machinery of their hosts for replication. It has therefore been proposed that the nucleotide and dinucleotide composition of viruses should match that of their host species. If 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.

  13. CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes for electrochemical determination of guanine and adenine

    Energy Technology Data Exchange (ETDEWEB)

    Wei Yan [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Huang Qinan [Department of Chemistry, Wannan Medical College, Wuhu 241002 (China); Li Maoguo [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Huang Xingjiu [Institute of Intelligent Machines, Chinese Academy of Sciences, Hefei 230031 (China); Fang Bin, E-mail: [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China); Wang Lun, E-mail: [College of Chemistry and Materials Sciences, Anhui Normal University, Wuhu 241000 (China)


    Sub-10 nm CeO{sub 2} nanoparticles decorated multi-walled carbon nanotubes has been constructed for electrochemial determination of guanine and adenine. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) were used to characterize the nanoparticles CeO{sub 2}/MWCNTs. Electrochemical impedance spectroscopy (EIS) was used to characterize the electrode modifying process. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to study the electrocatalytic activity toward the electrochemical oxidation of guanine and adenine. The detection limit (S/N = 3) for adenine and guanine was found to be 20 and 10 nM, respectively. The obtained sensitivity toward guanine and adenine was 1.26 and 1.13 {mu}A/{mu}M in the linear concentration range 5-50 {mu}M and 5-35 {mu}M, respectively. These results demonstrate that the carbon nanotubes could provide huge locations and facilitate the adsorptive accumulation of the guanine and adenine, and the CeO{sub 2} nanoparticles are promising substrates for the development of high-performance electrocatalysts for biosensing.

  14. Simultaneous Determination of Adenine and Guanine Using Cadmium Selenide Quantum Dots-Graphene Oxide Nanocomposite Modified Electrode. (United States)

    Kalaivani, Arumugam; Narayanan, Sangilimuthu Sriman


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

  15. Discrepancy variation of dinucleotide microsatellite repeats in eukaryotic genomes. (United States)

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


    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.

  16. The tendency to recreate ancestral CG dinucleotides in the human genome

    Directory of Open Access Journals (Sweden)

    Li Mingkun


    Full Text Available Abstract Background The CG dinucleotides are known to be deficient in the human genome, due to a high mutation rate from 5-methylated CG to TG and its complementary pair CA. Meanwhile, many cellular functions rely on these CG dinucleotides, such as gene expression controlled by cytosine methylation status. Thus, CG dinucleotides that provide essential functional substrates should be retained in genomes. How these two conflicting processes regarding the fate of CG dinucleotides - i.e., high mutation rate destroying CG dinucleotides, vs. functional processes that require their preservation remains an unsolved question. Results By analyzing the mutation and frequency spectrum of newly derived alleles in the human genome, a tendency towards generating more CGs was observed, which was mainly contributed by an excess number of mutations from CA/TG to CG. Simultaneously, we found a fixation preference for CGs derived from TG/CA rather than CGs generated by other dinucleotides. These tendencies were observed both in intergenic and genic regions. An analysis of Integrated Extended Haplotype Homozygosity provided no evidence of selection for newly derived CGs. Conclusions Ancestral CG dinucleotides that were subsequently lost by mutation tend to be recreated in the human genome, as indicated by a biased mutation and fixation pattern favoring new CGs that derived from TG/CA.

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

    Directory of Open Access Journals (Sweden)

    Mohita Upadhyay

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

  18. What is adenine doing in photolyase? (United States)

    Acocella, Angela; Jones, Garth A; Zerbetto, Francesco


    The short answer to the title question is that it acts as an electrostatic bouncer that shoves the charge flow from flavin toward the DNA lesion that photolyase repairs. This explanation is provided by an explicit time-dependent quantum mechanical approach, which is used to investigate the electron transfer process that triggers the repair mechanism. The transfer occurs from the flavin photolyase cofactor to the cyclobutane ring of DNA, previously formed by light-induced cycloaddition of adjacent pyrimidine bases. The electron wave function dynamics accurately accounts for the previously proposed mechanism of transfer via the terminal methyl group of the flavin moiety present in the catalytic electron-donor cofactor, FADH(-), which also contains adenine. This latter moiety, which has often been assumed to be present mainly for structural reasons, instantaneously modifies the interaction between acceptor and donor by a variation of the electrostatic interactions so that the presence of its local atomic charges is necessary to trigger the transfer. In principle, knowledge of the details of the electron transfer dynamics and of the important role of polarization effects can be exploited to improve the efficiency of the repair mechanism in artificial systems.

  19. Conformational control of benzophenone-sensitized charge transfer in dinucleotides. (United States)

    Merz, Thomas; Wenninger, Matthias; Weinberger, Michael; Riedle, Eberhard; Wagenknecht, Hans-Achim; Schütz, Martin


    Charge transfer in DNA cannot be understood without addressing the complex conformational flexibility, which occurs on a wide range of timescales. In order to reduce this complexity four dinucleotide models 1X consisting of benzophenone linked by a phosphodiester to one of the natural nucleosides X = A, G, T, C were studied in water and methanol. The theoretical work focuses on the dynamics and electronic structure of 1G. Predominant conformations in the two solvents were obtained by molecular dynamics simulations. 1G in MeOH adopts mainly an open geometry with a distance of 12–16 Å between the two aromatic parts. In H2O the two parts of 1G form primarily a stacked conformation yielding a distance of 5–6 Å. The low-lying excited states were investigated by electronic structure theory in a QM/MM environment for representative snapshots of the trajectories. Photo-induced intramolecular charge transfer in the S1 state occurs exclusively in the stacked conformation. Ultrafast transient absorption spectroscopy with 1X reveals fast charge transfer from S1 in both solvents with varying yields. Significant charge transfer from the T1 state is only found for the nucleobases with the lowest oxidation potential: in H2O, charge transfer occurs with 3.2 × 10(9) s(-1) for 1A and 6.0 × 10(9) s(-1) for 1G. The reorganization energy remains nearly unchanged going from MeOH to the more polar H2O. The electronic coupling is rather low even for the stacked conformation with H(AB) = 3 meV and explains the moderate charge transfer rates. The solvent controls the conformational distribution and therefore gates the charge transfer due to differences in distance and stacking.

  20. Role of p73 Dinucleotide Polymorphism in Prostate Cancer and p73 Protein Isoform Balance

    Directory of Open Access Journals (Sweden)

    L. Michael Carastro


    Full Text Available Background. Molecular markers for prostate cancer (PCa risks are currently lacking. Here we address the potential association of a dinucleotide polymorphism (DNP in exon 2 of the p73 gene with PCa risk/progression and discern any disruption of p73 protein isoforms levels in cells harboring a p73 DNP allele. Methods. We investigated the association between p73 DNP genotype and PCa risk/aggressiveness and survival by fitting logistic regression models in 1,292 incident cases and 682 controls. Results. Although we detected no association between p73 DNP and PCa risk, a significant inverse relationship between p73 DNP and PCa aggressiveness (AT/AT + GC/AT versus GC/GC, OR = 0.55, 95%Cl = 0.31–0.99 was detected. Also, p73 DNP is marginally associated with overall death (dominant model, HR = 0.76, 95%Cl = 0.57–1.00, P=0.053 as well as PCa specific death (HR = 0.69, 95%Cl = 0.45–1.06, P=0.09. Western blot analyses for p73 protein isoforms indicate that cells heterozygous for the p73 DNP have lower levels of ∆Np73 relative to TAp73 (P<0.001. Conclusions. Our findings are consistent with an association between p73 DNP and low risk for PCa aggressiveness by increasing the expressed TAp73/∆Np73 protein isoform ratio.

  1. Presynaptic signalling mediated by mono- and dinucleotides in the central nervous system. (United States)

    Miras-Portugal, M T; Díaz-Hernández, M; Gomez-Villafuerte, R; Gualix, J; Castro, E; Pintor, J


    Synaptosomal preparations from rat midbrain exhibit specific responses to both ATP and Ap(5)A, which elicit a Ca(2+) entrance to the presynaptic terminals. Studies of isolated single terminals showed that not all the terminals contain ionotropic receptors for nucleotides, in fact only 46% of them do. Of these, 12% responded only to the dinucleotide Ap(5)A, and 20% to the mononucleotide ATP. At the presynaptic level, diinosine pentaphosphate, Ip(5)I, is a good tool to specifically block dinucleotide responses, which are inhibited at low nM concentration, versus the high microM concentrations required to block ATP responses. There is evidence for a presynaptic control of mononucleotide and dinucleotide responses, mediated by metabotropic and ionotropic receptors. Stimulation of adenosine A1 receptors increases the affinity of dinucleotide receptors by five orders of magnitude, from 30 microM to 680 pM for control and in the presence of A1 agonist, respectively.

  2. The activity of uridine diphosphate-D-glucose: Nicotinamide-adenine dinucleotide oxidoreductase in cambial tissue and differentiating xylem isolated from sycamore trees. (United States)

    Rubery, P H


    The activity of UDPGlc: NAD oxidoreductase is measured in enzyme preparations obtained from sycamore cambium and xylem tissue. The activity of this enzyme is greater in xylem than in cambium whether expressed on a specific activity basis or on a per-cell basis. It is suggested that, in developing xylem, direct oxidation of UDPGlc may contribute significantly to the biosynthesis of polysaccharide precursors.

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

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


    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

  4. Enhanced Reduced Nicotinamide Adenine Dinucleotide electrocatalysis onto multi-walled carbon nanotubes-decorated gold nanoparticles and their use in hybrid biofuel cell (United States)

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


    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.

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

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


    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.

  6. AB154. Testosterone improves erectile function through regulation of nicotinamide adenine dinucleotide phosphate-oxidase and cyclooxygenase-2 expression in castrated rats (United States)

    Li, Rui; Wang, Tao; Yang, Jun; Zhang, Yan; Niu, Yonghua; Wang, Shaogang; Ye, Zhangqun; Rao, Ke; Liu, Jihong


    Objective Testosterone significantly improves hypogonadal-related erectile dysfunction (ED). However, the molecular mechanisms are poorly understood. The purpose of this study was to explore the effect and mechanism of testosterone in castrated rats. Methods Forty male Sprague-Dawley rats were randomized to 4 groups (control, sham-operated, castration and castration-with-testosterone-replacement). After 2 months, reactive oxygen species (ROS) production was measured by dihydroethidium (DHE) staining. Erectile function was tested by recording intracavernosal pressure (ICP) and mean arterial blood pressure (MAP). Protein expression levels were examined by Western blot. Results Castration reduced erectile function, and testosterone restored it. The concentrations of testosterone, cyclic guanosine mono-phosphate (cGMP) and cyclic adenosine monophosphate (cAMP) were lower in castrated rats than in controls, and testosterone restored these decreases (each P<0.05). The expression levels of cyclooxygenase-2 (COX-2), prostacyclin synthase (PTGIS or PGIS), endothelial nitric oxide synthase (eNOS) and phospho-eNOS were reduced in castrated rats compared with controls. The expression levels were significantly elevated in rats treated with testosterone (each P<0.05). The expression levels of p40phox and p67phox were increased in castrated rats, and testosterone significantly reduced these increases (each P<0.05). ROS production was markedly enhanced in castrated rats, and testosterone administration reversed this effect (P<0.05). Conclusions Testosterone can ameliorate ED after castration by reducing ROS production and increasing activity of the eNOS/cGMP and COX-2/PTGIS/cAMP signaling pathways.

  7. Wiring of Glucose Oxidizing Flavin Adenine Dinucleotide-Dependent Enzymes by Methylene Blue-Modified Third Generation Poly(amidoamine) Dendrimers Attached to Spectroscopic Graphite Electrodes

    DEFF Research Database (Denmark)

    Castaing, Victor; Álvarez-Martos, Isabel; Ferapontova, Elena


    ordered multiple redox centers, represent an advanced alternative to the existing approaches. Here we show that methylene blue (MB)-labeled G3 PAMAM dendrimers covalently attached to the high-surface area spectroscopic graphite (Gr) electrodes form stable and spatially resolved electronic wires...

  8. White spot syndrome virus VP12 interacts with adenine nucleotide translocase of Litopenaeus vannamei. (United States)

    Ma, Fang-fang; Chou, Zhi-guang; Liu, Qing-hui; Guan, Guangkuo; Li, Chen; Huang, Jie


    White spot syndrome virus VP12 contains cell attachment motif RGD which is considered to be critical for host cell binding. Until now, the function of this protein remains undefined. In this study, we explored the interaction of VP12 with host cells. A new shrimp protein (adenine nucleotide translocase of Litopenaeus vannamei, LvANT) is selected by far-western overlay assay. Tissue distribution of adenine nucleotide translocase mRNA showed that it was commonly spread in all the tissues detected. Cellular localization of LvANT in shrimp hemocytes showed that it was primarily located in the cytoplasm of hemocytes and colocalized with mitochondria. ELISA and far-western blot assay confirmed that VP12 interacted with LvANT. In vivo neutralization assay showed that anti-LvANT antibody can significantly reduce the mortality of shrimp challenged by WSSV at 48h post-treatment. Our results collectively showed that VP12 is involved in host cell binding via interaction with adenine nucleotide translocase.

  9. A new microplatform based on titanium dioxide nanofibers/graphene oxide nanosheets nanocomposite modified screen printed carbon electrode for electrochemical determination of adenine in the presence of guanine. (United States)

    Arvand, Majid; Ghodsi, Navid; Zanjanchi, Mohammad Ali


    The current techniques for determining adenine have several shortcomings such as high cost, high time consumption, tedious pretreatment steps and the requirements for highly skilled personnel often restrict their use in routine analytical practice. This paper describes the development and utilization of a new nanocomposite consisting of titanium dioxide nanofibers (TNFs) and graphene oxide nanosheets (GONs) for screen printed carbon electrode (SPCE) modification. The synthesized GONs and TNFs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR). The modified electrode (TNFs/GONs/SPCE) was used for electrochemical characterization of adenine. The TNFs/GONs/SPCE exhibited an increase in peak current and the electron transfer kinetics and decrease in the overpotential for the oxidation reaction of adenine. Using differential pulse voltammetry (DPV), the prepared sensor showed good sensitivity for determining adenine in two ranges from 0.1-1 and 1-10 μM, with a detection limit (DL) of 1.71 nM. Electrochemical studies suggested that the TNFs/GONs/SPCE provided a synergistic augmentation on the voltammetric behavior of electrochemical oxidation of adenine, which was indicated by the improvement of anodic peak current and a decrease in anodic peak potential. The amount of adenine in pBudCE4.1 plasmid was determined via the proposed sensor and the result was in good compatibility with the sequence data of pBudCE4.1 plasmid.

  10. Characterization of new G protein-coupled adenine receptors in mouse and hamster. (United States)

    Thimm, Dominik; Knospe, Melanie; Abdelrahman, Aliaa; Moutinho, Miguel; Alsdorf, Bernt B A; von Kügelgen, Ivar; Schiedel, Anke C; Müller, Christa E


    The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation "P0-receptors" has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [(3)H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure-activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins.

  11. Effects of Low-Molecular-Weight-Chitosan on the Adenine- Induced Chronic Renal Failure Ratsin vitro andin vivo

    Institute of Scientific and Technical Information of China (English)

    ZHI Xuan; HAN Baoqin; SUI Xianxian; HU Rui; LIU Wanshun


    Theeffects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigatedin vivoand in vitro. Chitosan were hydrolyzed using chitosanase at pH 6–7 and 37℃ for 24h to obtain LMWC.In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For theinvivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the controlgroup, indicating that the rat chronic renal failure model worked successfully. The re-sults after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100mgkg−1d−1.

  12. Simultaneous determination of adenine,uridine and adenosine in cordyceps sinensis and its substitutes by LC/ESI-MS

    Institute of Scientific and Technical Information of China (English)

    黄兰芳; 吴名剑; 孙贤军; 郭方遒; 梁逸曾; 李晓如


    A simple, sensitive and reproducible high performance liquid chromatography-mass spectrometry coupled with electrospray ionization method for simultaneous separation and determination of adenine, adenosine and uridine was developed. The analytical column is a 2.0 mm× 150 mm Shimadzu VP-ODS column and volume fraction of the mobile phase is 86.5 %water, 12.0%methanol and 1.5%formic acid. 2-chloroadenosine was used as internal standard. Selective ion monitoring mode and selective ion monitoring ions at ratio of mass to electric charge of 136 for adenine, 268 for adenosine and 267 for uridine were chosen for quantitative analysis of the three active components. The results show that the regression equations and linear range are Y=0. 062X+0. 005 and 2.0 - 140.0μg · mL 1for adenine, Y=0. 049X+0. 004 and 4. 0 - 115.0 μg · mL-1 for uridine, Y=0. 154X+0. 014 and 1.0 - 125.0 μg · mL 1 for adenosine. The limits of detection are 0.6 μg · mL 1 for adenine, 1.0μg · mL-1 for uri dine and 0.2 μg · mL 1 for adenosine.The recoveries of the three constituents are from 96.6% to 103.2%.

  13. Human nucleosomes: special role of CG dinucleotides and Alu-nucleosomes

    Directory of Open Access Journals (Sweden)

    Trifonov Edward N


    Full Text Available Abstract Background The periodical occurrence of dinucleotides with a period of 10.4 bases now is undeniably a hallmark of nucleosome positioning. Whereas many eukaryotic genomes contain visible and even strong signals for periodic distribution of dinucleotides, the human genome is rather featureless in this respect. The exact sequence features in the human genome that govern the nucleosome positioning remain largely unknown. Results When analyzing the human genome sequence with the positional autocorrelation method, we found that only the dinucleotide CG shows the 10.4 base periodicity, which is indicative of the presence of nucleosomes. There is a high occurrence of CG dinucleotides that are either 31 (10.4 × 3 or 62 (10.4 × 6 base pairs apart from one another - a sequence bias known to be characteristic of Alu-sequences. In a similar analysis with repetitive sequences removed, peaks of repeating CG motifs can be seen at positions 10, 21 and 31, the nearest integers of multiples of 10.4. Conclusions Although the CG dinucleotides are dominant, other elements of the standard nucleosome positioning pattern are present in the human genome as well. The positional autocorrelation analysis of the human genome demonstrates that the CG dinucleotide is, indeed, one visible element of the human nucleosome positioning pattern, which appears both in Alu sequences and in sequences without repeats. The dominant role that CG dinucleotides play in organizing human chromatin is to indicate the involvement of human nucleosomes in tuning the regulation of gene expression and chromatin structure, which is very likely due to cytosine-methylation/-demethylation in CG dinucleotides contained in the human nucleosomes. This is further confirmed by the positions of CG-periodical nucleosomes on Alu sequences. Alu repeats appear as monomers, dimers and trimers, harboring two to six nucleosomes in a run. Considering the exceptional role CG dinucleotides play in the

  14. Differences in dinucleotide frequencies of thermophilic genes encoding water soluble and membrane proteins

    Institute of Scientific and Technical Information of China (English)

    Hiroshi NAKASHIMA; Yuka KURODA


    The occurrence frequencies of the dinucleotides of genes of three thermophilic and three mesophilic species from both archaea and eubacteria were investigated in this study. The genes encoding water soluble proteins were rich in the dinucleotides of purine dimers, whereas the genes encoding membrane proteins were rich in pyrimidine dimers. The dinucleotides of purine dimers are the counterparts of pyrimidine dimers in a double-stranded DNA. The purine/pyrimidine dimers were favored in the thermophiles but not in the mesophiles, based on comparisons of observed and expected frequencies. This finding is in agreement with our previous study which showed that purine/pyrimidine dimers are positive factors that increase the thermal stability of DNA. The dinucleotides AA, AG, and GA are components of the codons of charged residues of Glu, Asp, Lys, and Arg, and the dinucleotides TT, CT, and TC are components of the codons of hydrophobic residues of Leu, He, and Phe. This is consistent with the suitabilities of the different amino acid residues for water soluble and membrane proteins. Our analysis provides a picture of how thermophilic species produce water soluble and membrane proteins with distinctive characters: the genes encoding water soluble proteins use DNA sequences rich in purine dimers, and the genes encoding membrane proteins use DNA sequences rich in pyrimidine dimers on the opposite strand.

  15. Typing dinucleotide repeat loci using microplate array diagonal gel electrophoresis: proof of principle. (United States)

    Rodríguez, Santiago; Chen, Xiao-He; Day, Ian N M


    Polymorphic dinucleotide repeat loci ('microsatellite markers') are found in varying abundance throughout the genomes of most organisms. They have been extensively used for genetic studies, but conventional techniques used for their genotyping require sophisticated equipment. Microplate array diagonal gel electrophoresis (MADGE) has previously been extended to economical high-throughput genotyping of trinucleotide and tetranucleotide microsatellite amplicons. However, the capability of this technique to resolve the alleles of dinucleotide repeat loci has not been explored previously. Here we show that a modified microsatellite-MADGE approach can provide sufficient resolution for dinucleotide repeat typing. This enables economical and convenient set up for analysis of single markers in many samples in parallel, suitable, for example, for population association studies.

  16. Biofabrication of chitosan-silver composite SERS substrates enabling quantification of adenine by a spectroscopic shift

    Energy Technology Data Exchange (ETDEWEB)

    Luo, X L; Bentley, W E [Institute for Bioscience and Biotechnology Research (IBBR), University of Maryland, College Park, MD 20742 (United States); Buckhout-White, S; Rubloff, G W, E-mail: [Department of Materials Science and Engineering, University of Maryland, College Park, MD 20742 (United States)


    Surface-enhanced Raman scattering (SERS) has grown dramatically as an analytical tool for the sensitive and selective detection of molecules adsorbed on nano-roughened noble metal structures. Quantification with SERS based on signal intensity remains challenging due to the complicated fabrication process to obtain well-dispersed nanoparticles and well-ordered substrates. We report a new biofabrication strategy of SERS substrates that enable quantification through a newly discovered spectroscopic shift resulting from the chitosan-analyte interactions in solution. We demonstrate this phenomenon by the quantification of adenine, which is an essential part of the nucleic acid structure and a key component in pathways which generate signal molecules for bacterial communications. The SERS substrates were fabricated simply by sequential electrodeposition of chitosan on patterned gold electrodes and electroplating of a silver nitrate solution through the chitosan scaffold to form a chitosan-silver nanoparticle composite. Active SERS signals of adenine solutions were obtained in real time from the chitosan-silver composite substrates with a significant concentration-dependent spectroscopic shift. The Lorentzian curve fitting of the dominant peaks suggests the presence of two separate peaks with a concentration-dependent area percentage of the separated peaks. The chitosan-mediated composite SERS substrates can be easily biofabricated on predefined electrodes within microfluidic channels for real-time detection in microsystems.

  17. Study on the oxidation form of adenine in phosphate buffer solution. (United States)

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


    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.

  18. RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies. (United States)

    Tulloch, Fiona; Atkinson, Nicky J; Evans, David J; Ryan, Martin D; Simmonds, Peter


    Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.

  19. Adenine nucleotide concentrations in patients with erythrocyte autoantibodies.


    Strong, V F; Sokol, R J; Rodgers, S A; Hewitt, S.


    Erythrocyte adenine nucleotide concentrations were measured in 154 patients with erythrocyte autoantibodies and 811 normal subjects using a luciferin-luciferase bioluminescent assay. The patients were initially divided into haemolysing and non-haemolysing groups. Red cell adenosine triphosphate (ATP) concentrations were significantly raised in the 96 patients with active haemolysis compared with the normal subjects and with the 58 patients in the non-haemolysing group. Although the patients c...

  20. The family of N9-adenine: New entry for adenine-benzamide conjugates linked via versatile spacers

    Indian Academy of Sciences (India)

    Prabhpreet Singh


    We have prepared 4-nitrobenzamide-adenine conjugates (8, 13 and 14) linked with versatile spacer such as triethylene glycol (TEG), aminocaproic acid and ethyl chains which were eventually reduced to obtain the corresponding 4-aminobenzamide-adenine conjugates (1-3) in good yields. These conjugates bear a nucleobase for DNA recognition or self-assembly through base-pair complementarity, a biocompatible linker for interfacing with biological system, and a p-aminobenzamide moiety for pharmacological applications. The use of hydrophilic or lipophilic linkers may tune the dispersibility of these conjugates in different solvents, as well as impart different properties. In the preliminary experiments the versatility and application of these linkers has been tested for functionalization of SWCNTs.

  1. Simultaneous determination of adenine and guanine in ruminant bacterial pellets by ion-pair HPLC. (United States)

    García del Moral, Pilar; Arín, María Jesús; Resines, José Antonio; Díez, María Teresa


    An ion-pair reversed-phase high-performance liquid chromatography with gradient elution and UV detection was used to measure adenine (A) and guanine (G) in lyophilized bacterial pellets from ruminants using allopurinol as internal standard. The separation was performed on a Symmetry C18 column and the detection was monitored at 280 nm. Calibration curves were found to be linear in the concentration range from 5 to 50 mg/l with correlation coefficients (r2)>0.999. Mean recoveries of A and G standards added to bacterial samples were 102.2 and 98.2, respectively. The method proposed yielded sharp, well-resolved peaks within 25 min and was successfully applied for the determination of A and G in bacterial pellets.

  2. Application of the VPp1 bacteriophage combined with a coupled enzyme system in the rapid detection of Vibrio parahaemolyticus. (United States)

    Peng, Yong; Jin, Yanqiu; Lin, Hong; Wang, Jingxue; Khan, Muhammad Naseem


    For rapid and quantitative detection of Vibrio parahaemolyticus, a method combining the specific lysis of bacteriophages with a bacterial luciferase-flavin mononucleotide:nicotinamide adenine dinucleotide oxidoreductase bioluminescent system in vitro was developed. A V. parahaemolyticus detection system was established by optimizing three main influencing factors: bacteriophage titer, volume ratio of the bacteriophage to its host bacterium, and lysis time. A standard curve between the number of bacteria and the luminescence intensity of the coupled enzyme system was studied and revealed a good linear relationship. More than 10(7)colony-forming units (cfu)·ml(-1) bacteria in pure culture and >10(8) cfu·ml(-1) bacteria in oyster samples were readily detected without pre-enrichment. Furthermore, >10(0) cfu·ml(-1) bacteria in oyster samples were readily detected after 4h of enrichment culture. Because of its rapid detection, high specificity, and simplicity in operation, this method is an effective tool for detecting living bacteria in food and environmental samples.

  3. Carbon Nanomaterials Based Electrochemical Sensors/Biosensors for the Sensitive Detection of Pharmaceutical and Biological Compounds

    Directory of Open Access Journals (Sweden)

    Bal-Ram Adhikari


    Full Text Available Electrochemical sensors and biosensors have attracted considerable attention for the sensitive detection of a variety of biological and pharmaceutical compounds. Since the discovery of carbon-based nanomaterials, including carbon nanotubes, C60 and graphene, they have garnered tremendous interest for their potential in the design of high-performance electrochemical sensor platforms due to their exceptional thermal, mechanical, electronic, and catalytic properties. Carbon nanomaterial-based electrochemical sensors have been employed for the detection of various analytes with rapid electron transfer kinetics. This feature article focuses on the recent design and use of carbon nanomaterials, primarily single-walled carbon nanotubes (SWCNTs, reduced graphene oxide (rGO, SWCNTs-rGO, Au nanoparticle-rGO nanocomposites, and buckypaper as sensing materials for the electrochemical detection of some representative biological and pharmaceutical compounds such as methylglyoxal, acetaminophen, valacyclovir, β-nicotinamide adenine dinucleotide hydrate (NADH, and glucose. Furthermore, the electrochemical performance of SWCNTs, rGO, and SWCNT-rGO for the detection of acetaminophen and valacyclovir was comparatively studied, revealing that SWCNT-rGO nanocomposites possess excellent electrocatalytic activity in comparison to individual SWCNT and rGO platforms. The sensitive, reliable and rapid analysis of critical disease biomarkers and globally emerging pharmaceutical compounds at carbon nanomaterials based electrochemical sensor platforms may enable an extensive range of applications in preemptive medical diagnostics.

  4. Cellular localization of adenine receptors in the rat kidney and their functional significance in the inner medullary collecting duct. (United States)

    Kishore, Bellamkonda K; Zhang, Yue; Gevorgyan, Haykanush; Kohan, Donald E; Schiedel, Anke C; Müller, Christa E; Peti-Peterdi, János


    The Gi-coupled adenine receptor (AdeR) binds adenine with high affinity and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in the renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex and outer medulla compared with the inner medulla. Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid COOH-terminal sequence of rat AdeR, which we generated, detected two bands between ∼30 and 40 kDa (molecular mass of native protein: 37 kDa) in the cortex, outer medulla, and inner medulla. These bands were ablated by preadsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature, including the glomerular arterioles, and less intense labeling in the cells of the collecting duct system. Confocal immunofluorescence imaging colocalized AdeR with aquaporin-2 protein to the apical plasma membrane in the collecting duct. Functionally, adenine (10 μM) significantly decreased (P < 0.01) 1-deamino-8-d-arginine vasopressin (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 μM, P < 0.01), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in the renal vasculature and collecting ducts and its functional relevance. This study may open a new avenue for the exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease.

  5. Self-organization, optical, and electrical properties of alpha-quinquethiophene-dinucleotide conjugates. (United States)

    Alesi, Silvia; Brancolini, Giorgia; Viola, Ilenia; Capobianco, Massimo Luigi; Venturini, Alessandro; Camaioni, Nadia; Gigli, Giuseppe; Melucci, Manuela; Barbarella, Giovanna


    The synthesis and properties of (5')TA(3')-t5 (8a) and (5')CG(3')-t5 (8b) conjugates, in which the self-complementary dinucleotides TA and CG are covalently bound to the central ring of alpha-quinquethiophene (t5), are described. According to molecular mechanics calculations, the preferred conformation of both 8a and 8b is that with the dinucleotide folded over the planar t5 backbone, with the nucleobases facing t5 at stacking distance. The calculations show that the aggregation process of 8a and 8b is driven by a mix of nucleobase-thiophene interactions, hydrogen bonding between nucleobases (non Watson-Crick (W&C) in TA, and W&C in CG), van der Waals, and electrostatic interactions. While 8b is scarcely soluble in any solvents, 8a is soluble in water, indicating that the aggregates of the former are more stable than those of the latter. Microfluidic-induced self-assembly studies of 8a showed the formation of lamellar, spherulitic, and dendritic supramolecular structures, depending on the concentration and solvent evaporation time. The self-assembled structures displayed micrometer dimensions in the xy plane of the substrate and nanometer dimensions in the z direction. Spatially resolved confocal microscopy and spectroscopy showed that the aggregates were characterized by intense fluorescence emission. Cast films of 8a from water solutions showed chirality transfer from the dinucleotide to t5. The hole mobility of the cast films of 8a was estimated using a two-electrode device under high vacuum and found to be up to two orders of magnitude greater than those previously measured for dinucleotide-quarterthiophene conjugates under the same experimental conditions.

  6. Examination of tyrosine/adenine stacking interactions in protein complexes. (United States)

    Copeland, Kari L; Pellock, Samuel J; Cox, James R; Cafiero, Mauricio L; Tschumper, Gregory S


    The π-stacking interactions between tyrosine amino acid side chains and adenine-bearing ligands are examined. Crystalline protein structures from the protein data bank (PDB) exhibiting face-to-face tyrosine/adenine arrangements were used to construct 20 unique 4-methylphenol/N9-methyladenine (p-cresol/9MeA) model systems. Full geometry optimization of the 20 crystal structures with the M06-2X density functional theory method identified 11 unique low-energy conformations. CCSD(T) complete basis set (CBS) limit interaction energies were estimated for all of the structures to determine the magnitude of the interaction between the two ring systems. CCSD(T) computations with double-ζ basis sets (e.g., 6-31G*(0.25) and aug-cc-pVDZ) indicate that the MP2 method overbinds by as much as 3.07 kcal mol(-1) for the crystal structures and 3.90 kcal mol(-1) for the optimized structures. In the 20 crystal structures, the estimated CCSD(T) CBS limit interaction energy ranges from -4.00 to -6.83 kcal mol(-1), with an average interaction energy of -5.47 kcal mol(-1), values remarkably similar to the corresponding data for phenylalanine/adenine stacking interactions. Geometry optimization significantly increases the interaction energies of the p-cresol/9MeA model systems. The average estimated CCSD(T) CBS limit interaction energy of the 11 optimized structures is 3.23 kcal mol(-1) larger than that for the 20 crystal structures.

  7. Few-layer graphene sheets with embedded gold nanoparticles for electrochemical analysis of adenine

    Directory of Open Access Journals (Sweden)

    Biris AR


    Full Text Available Alexandru R Biris,1 Stela Pruneanu,1 Florina Pogacean,1 Mihaela D Lazar,1 Gheorghe Borodi,1 Stefania Ardelean,1 Enkeleda Dervishi,2 Fumiya Watanabe,2 Alexandru S Biris2 1National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania; 2Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR, USA Abstract: This work describes the synthesis of few-layer graphene sheets embedded with various amounts of gold nanoparticles (Gr-Au-x over an Aux/MgO catalytic system (where x = 1, 2, or 3 wt%. The sheet-like morphology of the Gr-Au-x nanostructures was confirmed by transmission electron microscopy and high resolution transmission electron microscopy, which also demonstrated that the number of layers within the sheets varied from two to seven. The sample with the highest percentage of gold nanoparticles embedded within the graphitic layers (Gr-Au-3 showed the highest degree of crystallinity. This distinct feature, along with the large number of edge-planes seen in high resolution transmission electron microscopic images, has a crucial effect on the electrocatalytic properties of this material. The reaction yields (40%–50% and the final purity (96%–98% of the Gr-Au-x composites were obtained by thermogravimetric analysis. The Gr-Au-x composites were used to modify platinum substrates and subsequently to detect adenine, one of the DNA bases. For the bare electrode, no oxidation signal was recorded. In contrast, all of the modified electrodes showed a strong electrocatalytic effect, and a clear peak for adenine oxidation was recorded at approximately +1.05 V. The highest increase in the electrochemical signal was obtained using a platinum/Gr-Au-3-modified electrode. In addition, this modified electrode had an exchange current density (I0, obtained from the Tafel plot one order of magnitude higher than that of the bare platinum electrode, which also confirmed that

  8. Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo (United States)

    Zhi, Xuan; Han, Baoqin; Sui, Xianxian; Hu, Rui; Liu, Wanshun


    The effects of low-molecular-weight-chitosan (LMWC) on chronic renal failure (CRF) rats induced by adenine were investigated in vivo and in vitro. Chitosan were hydrolyzed using chitosanase at pH 6-7 and 37° for 24 h to obtain LMWC. In vitro, the effect of LMWC on the proliferation of renal tubular epithelial cells (RTEC) showed that it had no cytotoxic effect and could promote cell growth. For the in vivo experiment, chronic renal failure rats induced by adenine were randomly divided into control group, Niaoduqing group, and high-, medium- and low-dose LMWC groups. For each group, we detected serum creatinine (SCR), blood urea nitrogen (BUN), and total superoxide dismutase (T-SOD), glutathione oxidase (GSH-Px) activities of renal tissue, and obtained the ratio of kidney weight/body weight, pathological changes of kidney. The levels of serum SCR, BUN were higher in the adenine-induced rats than those in the control group, indicating that the rat chronic renal failure model worked successfully. The results after treatment showed that LMWC could reduce the SCR and BUN levels and enhance the activities/levels of T-SOD and GSH-PX in kidney compared to control group. Histopathological examination revealed that adenine-induced renal alterations were restored by LMWC at three tested dosages, especially at the low dosage of 100 mg kg-1 d-1.

  9. Absorption spectroscopy of adenine, 9-methyladenine, and 2-aminopurine in helium nanodroplets

    NARCIS (Netherlands)

    S. Smolarek; A.M. Rijs; W.J. Buma; M. Drabbels


    High-resolution absorption spectra of adenine, 9-methyladenine and 2-aminopurine in helium nanodroplets have been recorded. In contrast to molecular beam experiments, large variations in linewidths are observed for adenine and 9-methyladenine. At the same time, the spectrum of 2-aminopurine remains

  10. Absorption by DNA single strands of adenine isolated in vacuo: The role of multiple chromophores

    DEFF Research Database (Denmark)

    Nielsen, L.M.; Pedersen, S.O.; Kirketerp, M.-B.S.;


    strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (~3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar...

  11. Suppression of feline immunodeficiency virus infection in vivo by 9-(2-phosphonomethoxyethyl)adenine

    NARCIS (Netherlands)

    Horzinek, M.C.; Egberink, H.F.; Borst, M.; Niphuis, H.; Balzarini, J.; Neu, H.; Schellekens, H.; Clercq, H. de; Koolen, M.J.M.


    The acyclic purine nucleoside analogue 9-(2-phosphonomethoxyethyl)adenine [PMEA; formerly referred to as 9-(2-phosphonylmethoxyethyl)adenine] is a potent and selective inhibitor of human immunodeficiency virus replication in vitro and of Moloney murine sarcoma virus-induced tumor formation in mice.

  12. Design, synthesis and biological evaluation of dinucleotide mRNA cap analog containing propargyl moiety. (United States)

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


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

  13. Gender differences in adenine-induced chronic kidney disease and cardiovascular complications in rats. (United States)

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


    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.

  14. Investigation of fluorescence methods for rapid detection of municipal wastewater impact on drinking water sources (United States)

    Peleato, Nicolas M.; Legge, Raymond L.; Andrews, Robert C.


    Fluorescence spectroscopy as a means to detect low levels of treated wastewater impact on two source waters was investigated using effluents from five wastewater facilities. To identify how best to interpret the fluorescence excitation-emission matrices (EEMs) for detecting the presence of wastewater, several feature selection and classification methods were compared. An expert supervised regional integration approach was used based on previously identified features which distinguish biologically processed organic matter including protein-like fluorescence and the ratio of protein to humic-like fluorescence. Use of nicotinamide adenine dinucleotide-like (NADH) fluorescence was found to result in higher linear correlations for low levels of wastewater presence. Parallel factors analysis (PARAFAC) was also applied to contrast an unsupervised multiway approach to identify underlying fluorescing components. A humic-like component attributed to reduced semiquinone-like structures was found to best correlate with wastewater presence. These fluorescent features were used to classify, by volume, low (0.1-0.5%), medium (1-2%), and high (5-15%) levels by applying support vector machines (SVMs) and logistic regression. The ability of SVMs to utilize high-dimensional input data without prior feature selection was demonstrated through their performance when considering full unprocessed EEMs (66.7% accuracy). The observed high classification accuracies are encouraging when considering implementation of fluorescence spectroscopy as a water quality monitoring tool. Furthermore, the use of SVMs for classification of fluorescence data presents itself as a promising novel approach by directly utilizing the high-dimensional EEMs.

  15. Detection of antibiotic resistant Avibacterium paragallinarum from broiler chickens in Bangladesh

    Directory of Open Access Journals (Sweden)

    Mst. Mousumi Khatun


    Full Text Available Objective: An attempt was undertaken for the detection and characterization of Avibacterium paragallinarum from clinically sick broiler chickens during field outbreaks. Materials and methods: Nasal and ocular discharges (n=6, tracheal swab (n=6, tracheal washing (n=4 and infraorbital sinus exudates (n=4 were collected aseptically from broiler chickens (n=10. To isolate A. paragallinarum, the clinical samples were cultured onto blood agar and chocolate agar enriched with Nicotinamide Adenine Dinucleotide (NAD and feeder organism (Staphylococcus aureus. Identification of A. paragallinarum was performed by Gram staining reaction, sugar fermentation profiles using five basic sugars (Dextrose, Maltose, Sucrose, Lactose and Mannitol and biochemical tests (Indole, Voges Proskauer and Methyl red tests. Antibiogram of the bacterial isolates of infected chicken was performed against five antibiotics namely Ciprofloxacin, Azithromycin, Gentamicin, Ampicillin and Cefalexin using disk diffusion method. Results: Results of colonial morphology, Gram staining reaction, sugar fermentation and biochemical tests confirmed one isolate as A. paragallinarum. The overall prevalence of IC in broiler chicken was 10% (1 of 10. This isolate was found to be sensitive to Ciprofloxacin, Azithroycin and Gentamicin and resistant to Ampicillin and Cefalexin. Conclusion: This is the first report of detection of A. paragallinarum from broiler chicken in Bangladesh. [J Adv Vet Anim Res 2016; 3(2.000: 173-177

  16. An electrochemical biosensor based on DNA tetrahedron/graphene composite film for highly sensitive detection of NADH. (United States)

    Li, Zonglin; Su, Wenqiong; Liu, Shuopeng; Ding, Xianting


    Dihydronicotinamide adenine dinucleotide (NADH) is a major biomarker correlated with lethal diseases such as cancers and bacterial infection. Herein, we report a graphene-DNA tetrahedron-gold nanoparticle modified gold disk electrode for highly sensitive NADH detection. By assembling the DNA tetrahedron/graphene composite film on the gold disk electrode surface which prior harnessed electrochemical deposition of gold nanoparticles to enhance the effective surface area, the oxidation potential of NADH was substantially decreased to 0.28V (vs. Ag/AgCl) and surface fouling effects were successfully eliminated. Furthermore, the lower detection limit of NADH by the presented platform was reduced down to 1fM, with an upper limit of 10pM. Both the regeneration and selectivity of composite film-modified electrode are investigated and proved to be robust. The novel sensor developed here could serve as a highly sensitive probe for NADH detection, which would further benefit the field of NADH related disease diagnostics.

  17. Structural Flexibility and Conformation Features of Cyclic Dinucleotides in Aqueous Solutions. (United States)

    Che, Xing; Zhang, Jun; Zhu, Yanyu; Yang, Lijiang; Quan, Hui; Gao, Yi Qin


    Cyclic dinucleotides are able to trigger the innate immune system by activating STING. It was found that the binding affinity of asymmetric 2'3'-cGAMP to symmetric dimer of STING is 3 orders of magnitude higher than that of the symmetric 3'3'-cyclic dinucleotides. Such a phenomenon has not been understood yet. Here we show that the subtle changes in phosphodiester linkage of CDNs lead to their distinct structural properties which correspond to the varied binding affinities. 2'-5' and/or 3'-5' linked CDNs adopt specific while different types of ribose puckers and backbone conformations. That ribose conformations and base types have different propensities for anti or syn glycosidic conformations further affects the overall flexibility of CDNs. The counterbalance between backbone ring tension and electrostatic repulsion, both affected by the ring size, also contributes to the different flexibility of CDNs. Our calculations reveal that the free energy cost for 2'3'-cGAMP to adopt the STING-bound structure is smaller than that for 3'3'-cGAMP and cyclic-di-GMP. These findings may serve as a reference for design of CDN-analogues as vaccine adjuvants. Moreover, the cyclization pattern of CDNs closely related to their physiological roles suggests the importance of understanding structural properties in the study of protein-ligand interactions.

  18. File list: Oth.Lar.50.Adenine_N6-methylation.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Lar.50.Adenine_N6-methylation.AllCell ce10 TFs and others Adenine N6-methylatio...n Larvae ...

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  17. No evidence that mRNAs have lower folding free energies than random sequences with the same dinucleotide distribution

    DEFF Research Database (Denmark)

    Workman, Christopher; Krogh, Anders Stærmose


    , dinucleotide content is important when assessing the significance of predicted free energy as the physical stability of RNA secondary structure is known to depend on dinucleotide base stacking energies. Even known RNA secondary structures, like tRNAs, can be shown to have predicted free energies......This work investigates whether mRNA has a lower estimated folding free energy than random sequences. The free energy estimates are calculated by the mfold program for prediction of RNA secondary structures. For a set of 46 mRNAs it is shown that the predicted free energy is not significantly...... different from random sequences with the same dinucleotide distribution. For random sequences with the same mononucleotide distribution it has previously been shown that the native mRNA sequences have a lower predicted free energy, which indicates a more stable structure than random sequences. However...

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

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


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

  19. Ozone therapy could attenuate tubulointerstitial injury in adenine-induced CKD rats by mediating Nrf2 and NF-κB

    Directory of Open Access Journals (Sweden)

    Gang Yu


    Full Text Available Objective(s: This study aims to determine the effects of ozone therapy on restoring impaired Nrf2 activation to ameliorate chronic tubulointerstitial injury in rats with adenine-induced CKD. Materials and Methods: Sprague–Dawley rats were fed with 0.75% adenine-containing diet to induce CKD and chronic tubulointerstitial injury. Ozone therapy was administered by rectal insufflation. After 4 weeks, serum and kidney samples were collected and analyzed. Renal function and systemic electrolyte level were detected. Pathological changes in kidney were assessed by hematoxylin–eosin staining and Masson trichrome staining. Nrf2 activation was detected by immunohistochemistry and Western blot analyses. The levels of SOD, CAT, GSH, PCO, and MDA were detected in the kidney. Immunohistochemistry, Western blot, and real-time PCR analyses were performed to evaluate the activation of the nuclear factor kappa B (NF-κB P65 pathway and inflammation infiltration in the tubulointerstitium of the rats. Results: Ozone therapy improved severe renal insufficiency and tubulointerstitial morphology injury as well as restored Nrf2 activation and inhibited the NF-κB pathway in rats with adenine-induced CKD. Ozone therapy also up-regulated anti-oxidation enzymes (SOD, CAT, and GSH and down-regulated oxidation products (PCO and MDA, as well as inflammatory cytokines (IL-1β, IL-6, TNF-α, and ICAM-1 in the kidney. Conclusion:These findings indicated that ozone therapy could attenuate tubulointerstitial injury in rats with adenine-induced CKD by mediating Nrf2 and NF-κB.

  20. Ozone therapy could attenuate tubulointerstitial injury in adenine-induced CKD rats by mediating Nrf2 and NF-κB (United States)

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


    Objective(s): This study aims to determine the effects of ozone therapy on restoring impaired Nrf2 activation to ameliorate chronic tubulointerstitial injury in rats with adenine-induced CKD. Materials and Methods: Sprague–Dawley rats were fed with 0.75% adenine-containing diet to induce CKD and chronic tubulointerstitial injury. Ozone therapy was administered by rectal insufflation. After 4 weeks, serum and kidney samples were collected and analyzed. Renal function and systemic electrolyte level were detected. Pathological changes in kidney were assessed by hematoxylin–eosin staining and Masson trichrome staining. Nrf2 activation was detected by immunohistochemistry and Western blot analyses. The levels of SOD, CAT, GSH, PCO, and MDA were detected in the kidney. Immunohistochemistry, Western blot, and real-time PCR analyses were performed to evaluate the activation of the nuclear factor kappa B (NF-κB) P65 pathway and inflammation infiltration in the tubulointerstitium of the rats. Results: Ozone therapy improved severe renal insufficiency and tubulointerstitial morphology injury as well as restored Nrf2 activation and inhibited the NF-κB pathway in rats with adenine-induced CKD. Ozone therapy also up-regulated anti-oxidation enzymes (SOD, CAT, and GSH) and down-regulated oxidation products (PCO and MDA), as well as inflammatory cytokines (IL-1β, IL-6, TNF-α, and ICAM-1) in the kidney. Conclusion: These findings indicated that ozone therapy could attenuate tubulointerstitial injury in rats with adenine-induced CKD by mediating Nrf2 and NF-κB. PMID:27872711

  1. Highly sensitive and stable Ag@SiO2 nanocubes for label-free SERS-photoluminescence detection of biomolecules (United States)

    Nguyen, Minh-Kha; Su, Wei-Nien; Chen, Ching-Hsiang; Rick, John; Hwang, Bing-Joe


    Surface-enhanced Raman scattering (SERS) and fluorescence microscopy are a widely used biological and chemical characterization techniques. However, the peak overlapping in multiplexed experiments and rapid photobleaching of fluorescent organic dyes is still the limitations. When compared to Ag nanocubes (NCs), higher SERS sensitivities can be obtained with thin shelled silica Ag@SiO2 NCs, in contrast metal-enhanced photoluminescence (MEPL) is only found with NCs that have thicker silica shells. A 'dual functionality' represented by the simultaneous strengthening of SERS and MEPL signals can be achieved by mixing Ag@SiO2 NCs, with a silica shell thickness of 1.5 nm and 4.4 nm. This approach allows both the Ag@SiO2 NCs SERS and MEPL sensitivities to be maintained at 90% after 12 weeks of storage. Based on the distinguished detection of creatinine and flavin adenine dinucleotide in the mixture, the integration of SERS and MEPL together on a stable single plasmonic nanoparticle platform offers an opportunity to enhance both biomarker detection sensitivity and specificity.

  2. Estrogen receptor alpha dinucleotide repeat polymorphism in Japanese patients with autoimmune thyroid diseases

    Directory of Open Access Journals (Sweden)

    Tozaki Teruaki


    Full Text Available Abstract Background The autoimmune thyroid diseases (AITDs, comprising Graves' disease (GD and Hashimoto's thyroiditis (HT, appear to develop as a result of complex interactions between predisposing genes and environmental triggers. Susceptibility to AITDs is conferred by genes in the human leukocyte antigen (HLA and genes unlinked to HLA, including the CTLA-4 gene. Recently, an association to some estrogen receptor (ERα genotypes with breast cancer, hypertension, osteoporosis, generalized osteoarthritis, and some autoimmune diseases such as rheumatoid arthritis has been reported. We have analyzed a dinucleotide (TAn repeat polymorphism lying upstream of the human ERα gene in patients with AITDs and in normal subjects. Results Seventeen different alleles were found in 130 patients with GD, 93 patients with HT, and 190 control subjects. There was no significant difference in the distributions of ERα alleles between patients and controls. Conclusions The present results do not support an association between the ERα gene and AITD in the Japanese population.

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

    Energy Technology Data Exchange (ETDEWEB)

    Casimir, C.M.; Bu-Ghanim, H.N.; Rowe, P.; Segal, A.W. (University College London (England)); Rodaway, A.R.F.; Bentley, D.L. (Imperial Cancer Research Fund Lab., London (England))


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

  4. Design of laser pulses for selective vibrational excitation of the N6-H bond of adenine and adenine-thymine base pair using optimal control theory. (United States)

    Sharma, Sitansh; Sharma, Purshotam; Singh, Harjinder; Balint-Kurti, Gabriel G


    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.

  5. One-step fabrication of integrated disposable biosensor based on ADH/NAD+/meldola's blue/graphitized mesoporous carbons/chitosan nanobiocomposite for ethanol detection. (United States)

    Hua, Erhui; Wang, Li; Jing, Xiaoying; Chen, Changtao; Xie, Guoming


    A novel strategy to simplify the dehydrogenase-based electrochemical biosensor fabrication through one-step drop-coating nanobiocomposite on a screen printed electrode (SPE) was developed. The nanobiocomposite was prepared by successively adding graphitized mesoporous carbons (GMCs), meldola's blue (MDB), alcohol dehydrogenase (ADH) and cofactor nicotinamide adenine dinucleotide (NAD(+)) in chitosan (CS) solution. MDB/GMCs/CS film was prepared. Cyclic voltammetry measurements demonstrated that MDB was strongly adsorbed on GMCs. After optimizing the concentration of MDB and the working potential, the MDB/GMCs/CS film presented a fast amperometric response (5s), excellent sensitivity (10.36 nA μM(-1)), wide linear range (10-410 μM) toward NADH and without any other interference signals (such as AA, UA, DA, H2O2 and metal ions). Furthermore, concentrations of ADH and NAD(+) in nanobiocomposite and the detection conditions (temperature and pH) were also optimized. The constructed disposable ethanol biosensor showed an excellent linear response ranged from 0.5 to 15 mM with high sensitivity (67.28 nA mM(-1)) and a low limit of detection (80 μM) and a remarkable long-term stability (40 days). The intra-batch and inter-batch variation coefficients were both less than 5% (n=5). The ethanol recovery test demonstrated that the proposed biosensor offered a remarkable and accurate method for ethanol detection in the real blood samples.

  6. Design of an instrument for real-time detection of bioaerosols using simultaneous measurement of particle aerodynamic size and intrinsic fluorescence. (United States)

    Hairston, P P; Ho, J; Quant, F R


    A prototype instrument has been constructed to measure individual airborne particles based on their aerodynamic size and their intrinsic fluorescence at selected excitation and emission wavelength bands. The instrument combines features of an aerodynamic particle sizing device with capabilities similar to those of a liquid flow cytometer. The goal of the instrument is to provide real-time data indicative of particle characteristics, and it is especially targeted to respond to bioaerosols from 0.5 to 10 micrometers (aerodynamic diameter) with intrinsic fluorescence exited at a wavelength of 325 nm and emitting from 420 to 580 nm. This size range covers individual airborne bacteria and bacteria clusters, and the fluorescence sensitivity is selected for biological molecules commonly found in cellular systems, for example, reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] and riboflavin. Initial tests with nebulised Bacillus subtilis var. niger (BG, ATCC 9372) spores have shown that, for both individual spores and spore clumps, a low level of fluorescence is detected from 17% of the particles. This detection percentage is on the same order as previous experiments that have measured viability of about 12% for mechanically dispersed BG spores (Ho and Fisher (1993) Defense Research Establishment Suffield Memorandum 1421) and suggests a need for further investigation into the possible relationship between the detected fluorescence and viability of bacterial spores.

  7. Interference Reduction in Glucose Detection by Redox Potential Tuning: New Glucose Meter Development. (United States)

    Cho, Seong Je; Cho, Chul-Ho; Kim, Kwang Bok; Lee, Min-Hyoung; Kim, Jae Hong; Lee, Suho; Cho, Jaegeol; Jung, Suntae; Kim, Dong-Min; Shim, Yoon-Bo


    A new glucose meter was developed employing a novel disposable glucose sensor strip comprising a nicotinamide adenine dinucleotide-glucose dehydrogenase (NAD-GDH) and a mixture of Fe compounds as a mediator. An iron complex, 5-(2,5-di(thiophen-2-yl)-1H-pyrrol-1-yl)-1,10-phenanthroline iron(III) chloride (Fe-PhenTPy), was synthesized as a new mediator for the NAD-GDH system. Due to the high oxidation potential of the mediator, the detection potential was tuned to be more closely fitted toward the enzyme reaction potential, less than 400 mV (vs. Ag/AgCl), by mixing with an additional iron mediator. The impedance spectrometry for the enzyme sensor containing the mixed mediators showed an enhanced charge transfer property. In addition, a new cartridge-type glucose meter was manufactured using effective aligned-electrodes, which showed an enhanced response compared with conventional electrode alignment. The proposed glucose sensor resulted in a wide dynamic range in the concentration range of 30 - 500 mg dL(-1) with a reduced interference effect and a good sensitivity of 0.57 μA mM(-1).

  8. Effects of hypobaric hypoxia on adenine nucleotide pools, adenine nucleotide transporter activity and protein expression in rat liver

    Institute of Scientific and Technical Information of China (English)

    Cong-Yang Li; Jun-Ze Liu; Li-Ping Wu; Bing Li; Li-Fen Chen


    AIM: To explore the effect of hypobaric hypoxia on mitochondrial energy metabolism in rat liver.METHODS: Adult male Wistar rats were exposed to a hypobaric chamber simulating 5000 m high altitude for 23 h every day for 0 (HO), 1 (H1), 5 (HS), 15 (H15) and 30 d (H30) respectively. Rats were sacrificed by decapitation and liver was removed. Liver mitochondria were isolated by differential centrifugation program. The size of adenine nucleotide pool (ATP, ADP, and AMP) in tissue and mitochondria was separated and measured by high performance liquid chromatography (HPLC). The adenine nucleotide transporter (ANT) activity was determined by isotopic technique. The ANT total protein level was determined by Western blot. RESULTS: Compared with HO group, intra-mitochondrial ATP content decreased in all hypoxia groups. However,the H5 group reached the lowest point (70.6%) (P< 0.01)when compared to the control group. Intra-mitochondrial ADP and AMP level showed similar change in all hypoxia groups and were significantly lower than that in HO group. In addition, extra-mitochondrial ATP and ADP content decreased significantly in all hypoxia groups.Furthermore, extra-mitochondrial AMP in groups H5, H15and H30 was significantly lower than that in HO group,whereas H1 group had no marked change compared to the control situation. The activity of ANT in hypoxia groups decreased significantly, which was the lowest in H5 group (55.7%) (P<0.01) when compared to HO group. ANT activity in H30 group was higher than in H15 group, but still lower than that in HO group. ANT protein level in H5, H15, H30 groups, compared with HO group decreased significantly, which in H5 group was the lowest, being 27.1% of that in HO group (P<0.01). ANT protein level in H30 group was higher than in H15 group,but still lower than in HO group.CONCLUSION: Hypobaric hypoxia decreases the mitochondrial ATP content in rat liver, while mitochondrial ATP level recovers during long-term hypoxia exposure.The lower

  9. Suppression of feline immunodeficiency virus infection in vivo by 9-(2-phosphonomethoxyethyl)adenine


    Horzinek, M.C.; Egberink, H F; Borst, M.; Niphuis, H; Balzarini, J; Neu, H.; Schellekens, H.; De Clercq, H; Koolen, M.J.M.


    The acyclic purine nucleoside analogue 9-(2-phosphonomethoxyethyl)adenine [PMEA; formerly referred to as 9-(2-phosphonylmethoxyethyl)adenine] is a potent and selective inhibitor of human immunodeficiency virus replication in vitro and of Moloney murine sarcoma virus-induced tumor formation in mice. In the latter system PMEA has stronger antiretroviral potency and selectivity than 3'-azido-3'-thymidine (AZT). We have now investigated the effect of the drug in cats infected with the feline immu...

  10. Improved Growth and Stress Tolerance in the Arabidopsis oxt1 Mutant Triggered by Altered Adenine Metabolism

    Institute of Scientific and Technical Information of China (English)

    Suchada Sukrong; Kil-Young Yun; Patrizia Stadler; Charan Kumar; Tony Facciuolo; Barbara A.Moffatt; Deane L.Falcone


    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.

  11. Benchmark Thermochemistry for Biologically Relevant Adenine and Cytosine. A Combined Experimental and Theoretical Study. (United States)

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


    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.

  12. Sequence-dependent folding landscapes of adenine riboswitch aptamers (United States)

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

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

  13. Drug: D04959 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D04959 Mixture, Drug Liver extract - flavin adenine dinucleotide; Liver extract - f...lavin adenine dinucleotide sodium; Adelavin (TN) Liver extract, (Flavin adenine dinucleotide [DR:D00005] | F...erapeutics 3262 Hepatic agents D04959 Liver extract - flavin adenine dinucleotide PubChem: 17398233 ...

  14. Biofuel cell based self-powered sensing platform for L-cysteine detection. (United States)

    Hou, Chuantao; Fan, Shuqin; Lang, Qiaolin; Liu, Aihua


    L-cysteine (L-Cys) detection is of great importance because of its crucial roles in physiological and clinical diagnoses. In this study, a glucose/O2 biofuel cell (BFC) was assembled by using flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH)-based bioanode and laccase-based biocathode. Interestingly, the open circuit potential (OCP) of the BFC could be inhibited by Cu(2+) and subsequently activated by L-Cys, by which a BFC-based self-powered sensing platform for the detection of L-Cys was proposed. The FAD-GDH activity can be inhibited by Cu(2+) and, in turn, subsequent reversible activation by L-Cys because of the binding preference of L-Cys toward Cu(2+) by forming the Cu-S bond. The preferential interaction between L-Cys and Cu(2+) facilitated Cu(2+) to remove from the surface of the bioanode, and thus, the OCP of the system could be turned on. Under optimized conditions, the OCP of the BFC was systematically increased upon the addition of the L-Cys. The OCP increment (ΔOCP) was linear with the concentration of L-Cys within 20 nM to 3 μM. The proposed sensor exhibited lower detection limit of 10 nM L-Cys (S/N = 3), which is significantly lower than those values for other methods reported so far. Other amino acids and glutathione did not affect L-Cys detection. Therefore, this developed approach is sensitive, facile, cost-effective, and environmental-friendly, and could be very promising for the reliable clinically detecting of L-Cys. This work would trigger the interest of developing BFCs based self-powered sensors for practical applications.

  15. Predicting DNA Methylation State of CpG Dinucleotide Using Genome Topological Features and Deep Networks. (United States)

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


    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

  16. 聚硫堇/石墨烯复合材料修饰电极对NADH的电催化氧化研究%Grapheme functionalized with poly thionine for electrochemical determination of nicotinamide adenine dinucleotide

    Institute of Scientific and Technical Information of China (English)

    张天祎; 赵曼竹; 魏倾鹤; 齐斌


    采用循环伏安法将硫堇在石墨烯修饰的玻碳电极表面聚合,得到了一种新的聚硫堇/石墨烯修饰电极,此电极兼备了石墨烯和聚硫堇的特性.实验表明:该修饰电极能有效降低NADH的过电位;对NADH的检测范围为2.4×10-6~4.89×10-3 mol·L-1;检出限为6.826×10-7 mol·L-1;对尿酸和抗坏血酸的干扰有很好的消除作用;此电极稳定性、重现性较好,有很高的实际应用价值.

  17. Binding of adenine to Stx2, the protein toxin from Escherichia coli O157:H7

    Energy Technology Data Exchange (ETDEWEB)

    Fraser, Marie E., E-mail: [Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4 (Canada); Cherney, Maia M. [Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton AB T6G 2H7 (Canada); Marcato, Paola [Department of Medical Microbiology and Immunology, University of Alberta, Edmonton AB T6G 2H7 (Canada); Mulvey, George L.; Armstrong, Glen D. [Department of Microbiology and Infectious Diseases, University of Calgary, 3330 Hospital Drive NW, Calgary AB T2N 4N1 (Canada); James, Michael N. G. [Group in Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton AB T6G 2H7 (Canada); Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary AB T2N 1N4 (Canada)


    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.

  18. Excited-state lifetime of adenine near the first electronic band origin. (United States)

    Kang, Hyuk; Chang, Jinyoung; Lee, Sang Hak; Ahn, Tae Kyu; Kim, Nam Joon; Kim, Seong Keun


    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 500cm(-1). The excited-state lifetime of adenine is ∼2ps around the 0-0 band of the (1)L(b) ππ(∗) state (36 105cm(-1)). The lifetime drops to ∼1ps when adenine is excited to the (1)L(a) ππ(∗) state with the pump energy at 36 800cm(-1) and above. The excited-state lifetimes of (1)L(a) and (1)L(b) ππ(∗) states are differentiated in accordance with previous frequency-resolved and computational studies.

  19. The Innate Immune DNA Sensor cGAS Produces a Noncanonical Cyclic Dinucleotide that Activates Human STING

    Directory of Open Access Journals (Sweden)

    Elie J. Diner


    Full Text Available The presence of foreign DNA in the cytosol of mammalian cells elicits a potent antiviral interferon response. Recently, cytosolic DNA was proposed to induce the synthesis of cyclic GMP-AMP (cGAMP upon binding to an enzyme called cGAMP synthase (cGAS. cGAMP activates an interferon response by binding to a downstream receptor called STING. Here, we identify natural variants of human STING (hSTING that are poorly responsive to cGAMP yet, unexpectedly, are normally responsive to DNA and cGAS signaling. We explain this paradox by demonstrating that the cGAS product is actually a noncanonical cyclic dinucleotide, cyclic [G(2′-5′pA(3′-5′p], which contains a single 2′-5′ phosphodiester bond. Cyclic [G(2′-5′pA(3′-5′p] potently activates diverse hSTING receptors and, therefore, may be a useful adjuvant or immunotherapeutic. Our results indicate that hSTING variants have evolved to distinguish conventional (3′-5′ cyclic dinucleotides, known to be produced mainly by bacteria, from the noncanonical cyclic dinucleotide produced by mammalian cGAS.

  20. Efficacy of the acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) against feline immunodeficiency virus. (United States)

    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


    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.

  1. High negative ion production yield in 30 keV F{sup 2+} + adenine (C{sub 5}H{sub 5}N{sub 5}) collisions

    Energy Technology Data Exchange (ETDEWEB)

    Li, B; Ma, X; Zhu, X L; Zhang, S F; Liu, H P; Feng, W T; Qian, D B; Zhang, D C [Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Chen, L; Bredy, R; Montagne, G; Bernard, J; Martin, S [Universite de Lyon, F-69622, Lyon (France) and Universite Lyon 1, Villeurbanne; CNRS, UMR5579, LASIM (France)], E-mail:


    In collisions between slow F{sup 2+} ions (30 keV) and molecular targets, adenine, scattered particle production yields have been measured directly by simultaneous detection of neutrals, positive and negative ions. The relative cross-section for a negative ion formation channel was measured to be 1%. Despite a slight decrease compared to a larger target, the fullerene C{sub 60}, the measured negative ion formation cross section is still at least one order of magnitude larger than the yield in ion-atom interactions.

  2. Synthesis of 9-[1-(1 -hydroxyethyl)-3-(phosphonomethoxy)propyl]adenine and prodrug as possible antiviral agents. (United States)

    Ghosh, Ajit; El-Kattan, Yahya; Wu, Minwan; Lin, Tsu-Hsing; Vadlakonda, Satish; Kotian, Pravin L; Babu, Yarlagadda S; Chand, Pooran


    The appropriately protected C-1'-hydroxyethyl-3-hydroxypropyl-N9-adenine nucleoside was prepared from 1-pivaloyloxy-5-tert-butyldiphenylsilyloxy-3-pentanol and adenine through the Mitsunobu reaction. One of the terminal hydroxyls was converted to the phosphonomethoxy derivative and the prodrug.

  3. Defining and Detecting Complex Peak Relationships in Mass Spectral Data: The Mz.unity Algorithm. (United States)

    Mahieu, Nathaniel G; Spalding, Jonathan L; Gelman, Susan J; Patti, Gary J


    Analysis of a single analyte by mass spectrometry can result in the detection of more than 100 degenerate peaks. These degenerate peaks complicate spectral interpretation and are challenging to annotate. In mass spectrometry-based metabolomics, this degeneracy leads to inflated false discovery rates, data sets containing an order of magnitude more features than analytes, and an inefficient use of resources during data analysis. Although software has been introduced to annotate spectral degeneracy, current approaches are unable to represent several important classes of peak relationships. These include heterodimers and higher complex adducts, distal fragments, relationships between peaks in different polarities, and complex adducts between features and background peaks. Here we outline sources of peak degeneracy in mass spectra that are not annotated by current approaches and introduce a software package called mz.unity to detect these relationships in accurate mass data. Using mz.unity, we find that data sets contain many more complex relationships than we anticipated. Examples include the adduct of glutamate and nicotinamide adenine dinucleotide (NAD), fragments of NAD detected in the same or opposite polarities, and the adduct of glutamate and a background peak. Further, the complex relationships we identify show that several assumptions commonly made when interpreting mass spectral degeneracy do not hold in general. These contributions provide new tools and insight to aid in the annotation of complex spectral relationships and provide a foundation for improved data set identification. Mz.unity is an R package and is freely available at as well as our laboratory Web site .

  4. Degradation of Adenine on the Martian Surface in the Presence of Perchlorates and Ionizing Radiation: A Reflectron Time-of-flight Mass Spectrometric Study (United States)

    Góbi, Sándor; Bergantini, Alexandre; Kaiser, Ralf I.


    The aim of the present work is to unravel the radiolytic decomposition of adenine (C5H5N5) under conditions relevant to the Martian surface. Being the fundamental building block of (deoxy)ribonucleic acids, the possibility of survival of this biomolecule on the Martian surface is of primary importance to the astrobiology community. Here, neat adenine and adenine–magnesium perchlorate mixtures were prepared and irradiated with energetic electrons that simulate the secondary electrons originating from the interaction of the galactic cosmic rays with the Martian surface. Perchlorates were added to the samples since they are abundant—and therefore relevant oxidizers on the surface of Mars—and they have been previously shown to facilitate the radiolysis of organics such as glycine. The degradation of the samples were monitored in situ via Fourier transformation infrared spectroscopy and the electron ionization quadruple mass spectrometric method; temperature-programmed desorption profiles were then collected by means of the state-of-the-art single photon photoionization reflectron time-of-flight mass spectrometry (PI-ReTOF-MS), allowing for the detection of the species subliming from the sample. The results showed that perchlorates do increase the destruction rate of adenine by opening alternative reaction channels, including the concurrent radiolysis/oxidation of the sample. This new pathway provides a plethora of different radiolysis products that were identified for the first time. These are carbon dioxide (CO2), isocyanic acid (HNCO), isocyanate (OCN‑), carbon monoxide (CO), and nitrogen monoxide (NO); an oxidation product containing carbonyl groups (R1R2–C=O) with a constrained five-membered cyclic structure could also be observed. Cyanamide (H2N–C≡N) was detected in both irradiated samples as well.

  5. SERS, XPS, and DFT Study of Adenine Adsorption on Silver and Gold Surfaces. (United States)

    Pagliai, Marco; Caporali, Stefano; Muniz-Miranda, Maurizio; Pratesi, Giovanni; Schettino, Vincenzo


    The adsorption of adenine on silver and gold surfaces has been investigated combining density functional theory calculations with surface-enhanced Raman scattering and angle-resolved X-ray photoelectron spectroscopy measurements, obtaining useful insight into the orientation and interaction of the nucleobase with the metal surfaces.

  6. Effect of AST-120 on Endothelial Dysfunction in Adenine-Induced Uremic Rats

    Directory of Open Access Journals (Sweden)

    Yuko Inami


    Full Text Available Aim. Chronic kidney disease (CKD represents endothelial dysfunction. Monocyte adhesion is recognized as the initial step of arteriosclerosis. Indoxyl sulfate (IS is considered to be a risk factor for arteriosclerosis in CKD. Oral adsorbent AST-120 retards deterioration of renal function, reducing accumulation of IS. In the present study, we determined the monocyte adhesion in the adenine-induced uremic rats in vivo and effects of AST-120 on the adhesion molecules. Methods. Twenty-four rats were divided into control, control+AST-120, adenine, and adenine+AST-120 groups. The number of monocytes adherent to the endothelium of thoracic aorta by imaging the entire endothelial surface and the mRNA expressions of adhesion and atherosclerosis-related molecules were examined on day 49. The mRNA expressions of ICAM-1 and VCAM-1 in human umbilical vein endothelial cells were also examined. Results. Adenine increased the number of adherent monocytes, and AST-120 suppressed the increase. The monocyte adhesion was related to serum creatinine and IS in sera. Overexpression of VCAM-1 and TGF-β1 mRNA in the arterial walls was observed in uremic rats. IS induced increase of the ICAM-1 and VCAM-1 mRNA expressions in vitro. Conclusion. It appears that uremic condition introduces the monocyte adhesion to arterial wall and AST-120 might inhibit increasing of the monocyte adherence with CKD progression.

  7. Gas-phase spectroscopy of protonated adenine, adenosine 5′-monophosphate and monohydrated ions

    DEFF Research Database (Denmark)

    Pedersen, S.O.; Støchkel, K.; Byskov, C.S.


    Microsolvation of chromophore ions commonly has large effects on their electronic structure and as a result on their optical absorption spectra. Here spectroscopy of protonated adenine (AdeH+) and its complex with one water molecule isolated in vacuo was done using a home-built mass spectrometer...

  8. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  9. The effect of activated charcoal on adenine-induced chronic renal failure in rats. (United States)

    Ali, Badreldin H; Alza'abi, Mohamed; Ramkumar, Aishwarya; Al-Lawati, Intisar; Waly, Mostafa I; Beegam, Sumaya; Nemmar, Abderrahim; Brand, Susanne; Schupp, Nicole


    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.

  10. Effect of atracylodes rhizome polysaccharide in rats with adenine-induced chronic renal failure. (United States)

    Yang, C; Liu, C; Zhou, Q; Xie, Y C; Qiu, X M; Feng, X


    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 (Prenal function. Atracylodes rhizome polysaccharide had reversed the majority of the indices of chronic renal failure in rats.

  11. Probing electronic coupling between adenine bases in RNA strands from synchrotron radiation circular dichroism experiments

    DEFF Research Database (Denmark)

    Nielsen, Lisbeth Munksgård; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted


    Circular dichroism spectra (176–330 nm) of RNA adenine oligomers, (rA)n (n = 1–10, 12, 15, and 20), reveal electronic coupling between two bases in short strands. The number of interacting bases in long strands is more and larger than that reported previously for the corresponding DNA strands....

  12. Structure of STING bound to cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the immune system. (United States)

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


    STING (stimulator of interferon genes) is an innate immune sensor of cyclic dinucleotides that regulates the induction of type I interferons. STING's C-terminal domain forms a V-shaped dimer and binds a cyclic diguanylate monophosphate (c-di-GMP) at the dimer interface by both direct and solvent-mediated hydrogen bonds. Guanines of c-di-GMP stack against the phenolic rings of a conserved tyrosine, and mutations at the c-di-GMP binding surface reduce nucleotide binding and affect signaling.

  13. Watson-Crick Base Pairing, Electronic and Photophysical Properties of Triazole Modified Adenine Analogues: A Computational Study

    KAUST Repository

    Das, Shubhajit


    We employ first-principles Density Functional Theory (DFT) and time-dependent DFT (TDDFT) to elucidate structural, electronic and optical properties of a few recently reported triazole adenine nucleobase analogues. The results are compared against the findings obtained for both natural adenine nucleobase and available experimental data. The optical absorption of these adenine analogues are calculated both in gas-phase and in solvent (methanol) using Polarized Continuum Model (PCM). We find that all the analogues show a red-shifted absorption profile as compared to adenine. Our simulated emission spectra in solvent compare fairly well with experimentally observed results. We investigate base paring ability of these adenine analogues with thymine. The calculations on the intrinsic stability of these base pairs ascertain that all the adenine analogues form the hydrogen bonded Watson-Crick base pair with similar H-bonding energy as obtained for natural adenine-thymine base pair. In our study, we provide a microscopic origin of the low-energy absorption and emission peaks, observed experimentally.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  15. The influence of pH on the structure of adenine monolayers adsorbed at Au(110)/electrolyte interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Bowfield, A.; Smith, C.I.; Mansley, C.P.; Weightman, P. [Department of Physics, Oliver Lodge Laboratory, University of Liverpool, L69 7ZE (United Kingdom)


    The pH of the solution is shown to significantly effect the reflection anisotropy spectroscopy (RAS) profiles of adenine adsorbed at Au(110)/electrolyte interfaces. At pH 12.8 the net adsorption is very weak due the formation of negative adenine ions in solution. The sensitivity of the RAS profiles to the pH of the solution is probably due to a change in the geometry of the adsorbed molecules caused by a disruption of the base stacking configuration that is adopted when adenine is adsorbed from solutions at pH 7.1. (Abstract Copyright [2010], Wiley Periodicals, Inc.)

  16. Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside

    Institute of Scientific and Technical Information of China (English)

    Xiao-kun WEI; Qing-bao DING; Lu ZHANG; Yong-li GUO; Lin OU; Chang-lu WANG


    Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells ofEnterobacter aerogenes DCJO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

  17. Induction of nucleoside phosphorylase in Enterobacter aerogenes and enzymatic synthesis of adenine arabinoside. (United States)

    Wei, Xiao-Kun; Ding, Qing-Bao; Zhang, Lu; Guo, Yong-Li; Ou, Lin; Wang, Chang-Lu


    Nucleoside phosphorylases (NPases) were found to be induced in Enterobacter aerogenes DGO-04, and cytidine and cytidine 5'-monophosphate (CMP) were the best inducers. Five mmol/L to fifteen mmol/L cytidine or CMP could distinctly increase the activities of purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase) and thymidine phosphorylase (TPase) when they were added into medium from 0 to 8 h. In the process of enzymatic synthesis of adenine arabinoside from adenine and uracil arabinoside with wet cells of Enterobacter aerogenes DGO-04 induced by cytidine or CMP, the reaction time could be shortened from 36 to 6 h. After enzymatic reaction the activity of NPase in the cells induced remained higher than that in the cells uninduced.

  18. Two Adenine Nucleotide Translocase Paralogues Involved in Cell Proliferation and Spermatogenesis in the Silkworm Bombyx mori


    Ryohei Sugahara; Akiya Jouraku; Takayo Nakakura; Takahiro Kusakabe; Takenori Yamamoto; Yasuo Shinohara; Hideto Miyoshi; Takahiro Shiotsuki


    Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for m...

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

    Energy Technology Data Exchange (ETDEWEB)

    Baei, Mohammad T. [Department of Chemistry, Islamic Azad University, Azadshahr Branch, Azadshahr, Golestan (Iran, Islamic Republic of); Taghartapeh, Mohammad Ramezani [Young Researchers and Elite Club, Islamic Azad University, Gorgan Branch, Gorgan (Iran, Islamic Republic of); Lemeski, E. Tazikeh [Department of Chemistry, Islamic Azad University, Gorgan Branch, Gorgan (Iran, Islamic Republic of); Soltani, Alireza, E-mail: [Young Researchers and Elite Club, Islamic Azad University, Gorgan Branch, Gorgan (Iran, Islamic Republic of)


    Density-functional theory calculations are used to investigate the interaction of Al{sub 12}N{sub 12} and B{sub 12}N{sub 12} clusters with the adenine (A), uracil (U), and cytosine (C) molecules. The current calculations demonstrate that these hybrid adsorbent materials are able to adsorb the adenine, uracil, and cytosine molecules through exothermic processes. Our theoretical results reveal improvement in the adsorption of adenine, uracil, and cytosine on Al{sub 12}N{sub 12} and B{sub 12}N{sub 12}. It is observed that B{sub 12}N{sub 12} is highly sensitive to adenine, uracil, and cytosine compared with Al{sub 12}N{sub 12} to serve as a biochemical sensor.

  20. Spectroscopic assessment of argon gas discharge induced radiolysis of aqueous adenine and thymine

    Energy Technology Data Exchange (ETDEWEB)

    Su Xi [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Shushanhu Road 350, Hefei 230031 (China); Huang Qing, E-mail: [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Shushanhu Road 350, Hefei 230031 (China); Dang Bingrong [Institute of Modern Physics, Chinese Academy of Sciences, 509 Nanchang Road, Lanzhou 730000 (China); Wang Xiangqin; Yu Zengliang [Key Laboratory of Ion Beam Bio-engineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, P.O. Box 1138, Shushanhu Road 350, Hefei 230031 (China)


    Ionizing radiation influences life profoundly for it can modify genetic materials. It is a long-standing task to investigate the interaction between energetic particles and DNA together with its components such as nucleotides, nucleosides and bases so as to predict and assess the potential biological effects. In this study, argon gas discharge was employed to produce energetic ions and electrons. The gas discharge caused the radiolysis of aqueous bases and the involved reactions were analyzed by means of spectroscopic tools including UV-vis absorption, fluorescence and Fourier transformation infrared (FTIR) spectroscopy, also assisted by liquid chromatography/mass spectrometry (LC/MS). It was found that the discharge resulted in the adenine-derived lesions such as 4,6-diamino-5-formamidopyrimidine, 8-OH-Ade and 2-OH-Ade in the radiolysis of aqueous adenine, as well as the thymine-derived lesions such as thymine glycol, 5-hydroxy-6-hydrothymine and/or 6-hydroxy-5-hydrothymine, 5-hydroxymethyluracil and 5-formyluracil in the radiolysis of aqueous thymine. The study of radio-sensitivity showed that adenine was more resistant to the discharge. The mechanisms of the involved reactions were studied in detail, confirming that the hydroxyl radical played a dominant role. - Highlights: > Effective new way to study radiolysis of bases via a home-made argon discharge apparatus. > Quantitative analysis of base radiolysis employing spectroscopic tools combined with HPLC/MS. > Discovery of different radiolysis effect compared with other forms of ionizing radiations.

  1. L-Arginine Intake Effect on Adenine Nucleotide Metabolism in Rat Parenchymal and Reproductive Tissues

    Directory of Open Access Journals (Sweden)

    G. Kocic


    Full Text Available L-arginine is conditionally essetcial amino acid, required for normal cell growth, protein synthesis, ammonia detoxification, tissue growth and general performance, proposed in the treatment of men sterility and prevention of male impotence. The aim of the present paper was to estimate the activity of the enzymes of adenine nucleotide metabolism: 5′-nucleotidase (5′-NU, adenosine deaminase (ADA, AMP deaminase, and xanthine oxidase (XO, during dietary intake of L-arginine for a period of four weeks of male Wistar rats. Adenosine concentration in tissues is maintained by the relative activities of the adenosine-producing enzyme, 5′-NU and the adenosine-degrading enzyme-ADA adenosine deaminase. Dietary L-arginine intake directed adenine nucleotide metabolism in liver, kidney, and testis tissue toward the activation of adenosine production, by increased 5′-NU activity and decreased ADA activity. Stimulation of adenosine accumulation could be of importance in mediating arginine antiatherosclerotic, vasoactive, immunomodulatory, and antioxidant effects. Assuming that the XO activity reflects the rate of purine catabolism in the cell, while the activity of AMP deaminase is of importance in ATP regeneration, reduced activity of XO, together with the increased AMP-deaminase activity, may suggest that adenine nucleotides are presumably directed to the ATP regenerating process during dietary L-arginine intake.

  2. Prolonged Pulmonary Exposure to Diesel Exhaust Particles Exacerbates Renal Oxidative Stress, Inflammation and DNA Damage in Mice with Adenine-Induced Chronic Renal Failure

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    Abderrahim Nemmar


    Full Text Available Background/Aims: Epidemiological evidence indicates that patients with chronic kidney diseases have increased susceptibility to adverse outcomes related to long-term exposure to particulate air pollution. However, mechanisms underlying these effects are not fully understood. Methods: Presently, we assessed the effect of prolonged exposure to diesel exhaust particles (DEP on chronic renal failure induced by adenine (0.25% w/w in feed for 4 weeks, which is known to involve inflammation and oxidative stress. DEP (0.5m/kg was intratracheally (i.t. instilled every 4th day for 4 weeks (7 i.t. instillation. Four days following the last exposure to either DEP or saline (control, various renal endpoints were measured. Results: While body weight was decreased, kidney weight increased in DEP+adenine versus saline+adenine or DEP. Water intake, urine volume, relative kidney weight were significantly increased in adenine+DEP versus DEP and adenine+saline versus saline. Plasma creatinine and urea increased and creatinine clearance decreased in adenine+DEP versus DEP and adenine+saline versus saline. Tumor necrosis factor α, lipid peroxidation and reactive oxygen species were significantly increased in adenine+DEP compared with either DEP or adenine+saline. The antioxidant calase was significantly decreased in adenine+DEP compared with either adenine+saline or DEP. Notably, renal DNA damage was significantly potentiated in adenine+DEP compared with either adenine+saline or DEP. Similarly, systolic blood pressure was increased in adenine+DEP versus adenine+saline or DEP, and in DEP versus saline. Histological evaluation revealed more collagen deposition, higher number of necrotic cell counts and dilated tubules, cast formation and collapsing glomeruli in adenine+DEP versus adenine+saline or DEP. Conclusion: Prolonged pulmonary exposure to diesel exhaust particles worsen renal oxidative stress, inflammation and DNA damage in mice with adenine-induced chronic

  3. Isolation and characterisation of a dinucleotide microsatellite set for a parentage and biodiversity study in domestic guinea pig (Cavia porcellus

    Directory of Open Access Journals (Sweden)

    Diana Aviles


    Full Text Available The domestic guinea pig is a valuable genetic resource because it is part of local folklore and food tradition in many South American countries. The economic importance of the guinea pig is due to its high feed efficiency and the quality of animal protein produced. For these reasons, our study is aimed to design a complete dinucleotide microsatellite marker set following international recommendation to assess the genetic diversity and genealogy management of guinea pigs. We selected a total of 20 microsatellites, looking for laboratory efficiency and good statistical parameters. The set was tested in 100 unrelated individuals of guinea pigs from Ecuador, Peru, Colombia, Bolivia and Spain. Our results show a high degree of polymorphisms with a total of 216 alleles and a mean number of 10.80±3.49 for markers with a combined exclusion probability of 0.99.

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

    Energy Technology Data Exchange (ETDEWEB)

    Minaev, B. F., E-mail:, E-mail: [Bohdan Khmelnitsky National University, 18031 Cherkasy (Ukraine); Tomsk State University, 634050 Tomsk (Russian Federation); Shafranyosh, M. I.; Svida, Yu. Yu; Sukhoviya, M. I.; Shafranyosh, I. I. [Uzhgorod National University, 88000 Uzhgorod (Ukraine); Baryshnikov, G. V.; Minaeva, V. A. [Bohdan Khmelnitsky National University, 18031 Cherkasy (Ukraine)


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

  5. Dynamic simulation and metabolome analysis of long-term erythrocyte storage in adenine-guanosine solution.

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    Taiko Nishino

    Full Text Available Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1 adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2 adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3 guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with in silico analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.

  6. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor. (United States)

    Wigand, R


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

  7. A novel missense adenine nucleotide translocator-1 gene mutation in a Greek adPEO family. (United States)

    Napoli, L; Bordoni, A; Zeviani, M; Hadjigeorgiou, G M; Sciacco, M; Tiranti, V; Terentiou, A; Moggio, M; Papadimitriou, A; Scarlato, G; Comi, G P


    Autosomal dominant progressive external ophthalmoplegia (adPEO) is caused by mutations in at least three different genes: ANT1 (chromosome 4q34-35), TWINKLE, and POLG. The ANT1 gene encodes the adenine nucleotide translocator-1 (ANT1). We identified a heterozygous T293C mutation of the ANT1 gene in a Greek family with adPEO. The resulting leucine to proline substitution likely modifies the secondary structure of the ANT1 protein. ANT1 gene mutations may account for adPEO in families with different ethnic backgrounds.

  8. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2'-Amino-α-l-LNA Adenine Monomers

    DEFF Research Database (Denmark)

    Andersen, Nicolai K; Anderson, Brooke A; Wengel, Jesper


    The development of conformationally restricted nucleotide building blocks continues to attract considerable interest because of their successful use within antisense, antigene, and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-l-LNA are two interesting examples...... (ONs) modified with 2'-amino-α-l-LNA adenine monomers W-Z. The synthesis of the target phosphoramidites 1-4 is initiated from pentafuranose 5, which upon Vorbrüggen glycosylation, O2'-deacylation, O2'-activation and C2'-azide introduction yields nucleoside 8. A one-pot tandem Staudinger...

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  10. Comparative study between transcriptionally- and translationally-acting adenine riboswitches reveals key differences in riboswitch regulatory mechanisms.

    Directory of Open Access Journals (Sweden)

    Jean-François Lemay


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

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

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


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

  12. Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors (United States)

    El-Gendy, Dalia M.; Ghany, Nabil A. Abdel; El Sherbini, E. E. Foad; Allam, Nageh K.


    A simple method is demonstrated to prepare spongy adenine-functionalized graphene (SFG) as interconnected, porous 3-dimensional (3D) network crinkly sheets. Such 3D network structure provides better contact at the electrode/electrolyte interface and facilitates the charge transfer kinetics. The fabricated SFG was characterized by X-ray diffraction (XRD), FTIR, scanning electron microscopy (FESEM), Raman spectroscopy, thermogravimetric analysis (TGA), UV−vis absorption spectroscopy, and transmission electron microscopy (TEM). The synthesized materials have been evaluated as supercapacitor materials in 0.5 M H2SO4 using cyclic voltammetry (CV) at different potential scan rates, and galvanostatic charge/discharge tests at different current densities. The SFG electrodes showed a maximum specific capacitance of 333 F/g at scan rate of 1 mV/s and exhibited excellent cycling retention of 102% after 1000 cycles at 200 mV/s. The energy density was 64.42 Wh/kg with a power density of 599.8 W/kg at 1.0 A/g. Those figures of merit are much higher than those reported for graphene-based materials tested under similar conditions. The observed high performance can be related to the synergistic effects of the spongy structure and the adenine functionalization. PMID:28216668

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

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


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

  14. DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus. (United States)

    Jeon, Taeck J


    The expression of amoeba sams genes is switched from sams1 to sams2 when amoebae are infected with Legionella jeonii. To elucidate the mechanism for the inactivation of host sams1 gene by endosymbiotic bacteria, methylation states of the sams1 gene of D and xD amoebae was compared in this study. The sams1 gene of amoebae was methylated at an internal adenine residue of GATC site in symbiont-bearing xD amoebae but not in symbiont-free D amoebae, suggesting that the modification might have caused the inactivation of sams1 in xD amoebae. The sams1 gene of xD amoebae was inactivated at the transcriptional level. Analysis of DNA showed that adenine residues in L. jeonii sams were also methylated, implying that L. jeonii bacteria belong to a Dam methylase-positive strain. In addition, both SAM and Met appeared to act as negative regulators for the expression of sams1 whereas the expression of sams2 was not affected in amoebae.

  15. Thymine- and Adenine-Functionalized Polystyrene Form Self-Assembled Structures through Multiple Complementary Hydrogen Bonds

    Directory of Open Access Journals (Sweden)

    Yu-Shian Wu


    Full Text Available In this study, we investigated the self-assembly of two homopolymers of the same molecular weight, but containing complementary nucleobases. After employing nitroxide-mediated radical polymerization to synthesize poly(vinylbenzyl chloride, we converted the polymer into poly(vinylbenzyl azide through a reaction with NaN3 and then performed click chemistry with propargyl thymine and propargyl adenine to yield the homopolymers, poly(vinylbenzyl triazolylmethyl methylthymine (PVBT and poly(vinylbenzyl triazolylmethyl methyladenine (PVBA, respectively. This PVBT/PVBA blend system exhibited a single glass transition temperature over the entire range of compositions, indicative of a miscible phase arising from the formation of multiple strong complementary hydrogen bonds between the thymine and adenine groups of PVBT and PVBA, respectively; Fourier transform infrared and 1H nuclear magnetic resonance spectroscopy confirmed the presence of these noncovalent interactions. In addition, dynamic rheology, dynamic light scattering and transmission electron microscopy provided evidence for the formation of supramolecular network structures in these binary PVBT/PVBA blend systems.

  16. Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors (United States)

    El-Gendy, Dalia M.; Ghany, Nabil A. Abdel; El Sherbini, E. E. Foad; Allam, Nageh K.


    A simple method is demonstrated to prepare spongy adenine-functionalized graphene (SFG) as interconnected, porous 3-dimensional (3D) network crinkly sheets. Such 3D network structure provides better contact at the electrode/electrolyte interface and facilitates the charge transfer kinetics. The fabricated SFG was characterized by X-ray diffraction (XRD), FTIR, scanning electron microscopy (FESEM), Raman spectroscopy, thermogravimetric analysis (TGA), UV‑vis absorption spectroscopy, and transmission electron microscopy (TEM). The synthesized materials have been evaluated as supercapacitor materials in 0.5 M H2SO4 using cyclic voltammetry (CV) at different potential scan rates, and galvanostatic charge/discharge tests at different current densities. The SFG electrodes showed a maximum specific capacitance of 333 F/g at scan rate of 1 mV/s and exhibited excellent cycling retention of 102% after 1000 cycles at 200 mV/s. The energy density was 64.42 Wh/kg with a power density of 599.8 W/kg at 1.0 A/g. Those figures of merit are much higher than those reported for graphene-based materials tested under similar conditions. The observed high performance can be related to the synergistic effects of the spongy structure and the adenine functionalization.

  17. Microwave-assisted stereospecific synthesis of novel tetrahydropyran adenine isonucleosides and crystal structures determination (United States)

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


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

  18. High-NaCl diet impairs dynamic renal blood flow autoregulation in rats with adenine-induced chronic renal failure

    DEFF Research Database (Denmark)

    Saeed, Aso; DiBona, Gerald F; Grimberg, Elisabeth


    This study examined the effects of 2 wk of high-NaCl diet on kidney function and dynamic renal blood flow autoregulation (RBFA) in rats with adenine-induced chronic renal failure (ACRF). Male Sprague-Dawley rats received either chow containing adenine or were pair-fed an identical diet without...... increase the susceptibility to hypertensive end-organ injury and progressive renal failure by facilitating pressure transmission to the microvasculature....... adenine (controls). After 10 wk, rats were randomized to either remain on the same diet (0.6% NaCl) or to be switched to high 4% NaCl chow. Two weeks after randomization, renal clearance experiments were performed under isoflurane anesthesia and dynamic RBFA, baroreflex sensitivity (BRS), systolic...

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

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


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

  20. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation. (United States)

    Zeinyeh, Waël; Mahiout, Zahia; Radix, Sylvie; Lomberget, Thierry; Dumoulin, Axel; Barret, Roland; Grenot, Catherine; Rocheblave, Luc; Matera, Eva-Laure; Dumontet, Charles; Walchshofer, Nadia


    Bivalent ligands were designed on the basis of the described close proximity of the ATP-site and the putative steroid-binding site of P-glycoprotein (ABCB1). The syntheses of 19 progesterone-adenine hybrids are described. Their abilities to inhibit P-glycoprotein-mediated daunorubicin efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein were evaluated versus progesterone. The hybrid with a hexamethylene linker chain showed the best inhibitory potency. The efficiency of these progesterone-adenine hybrids depends on two main factors: (i) the nature of the linker and (ii) its attachment point on the steroid skeleton.

  1. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function (United States)

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.


    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  2. Development of di-nucleotide microsatellite markers and construction of genetic linkage map in mango (Mangifera indica L.

    Directory of Open Access Journals (Sweden)

    Chataporn Chunwongse


    Full Text Available Forty-two di-nucleotide microsatellite, or simple-sequence repeat (SSR, markers were developed using CA and CTenriched genomic libraries of Mangifera indica L. Six cultivated mangoes and two wild species were tested for primer amplifications. Most loci could amplify M. caloneura Kruz and M. foetida. The average number of alleles per locus was 4.4. The average expected heterozygosity and the maximum polymorphism information content value were 0.57 and 0.53, respectively. The SSRs developed in this study together with 65 SSRs and 145 restriction fragment length polymorphism (RFLP markers reported previously were used in the genetic linkage analysis. A partial genetic linkage map was constructed based on 31 F1 progenies from a cross between ‘Alphonso’ and ‘Palmer’. The map spanned a distance of 529.9 centiMorgan (cM and consisted of 9 microsatellite markers (6 from this study and 67 RFLP markers. The new SSR markers and the present map will be useful for mango genetic studies and breeding applications in the future.

  3. Dissection of thousands of cell type-specific enhancers identifies dinucleotide repeat motifs as general enhancer features. (United States)

    Yáñez-Cuna, J Omar; Arnold, Cosmas D; Stampfel, Gerald; Boryń, Lukasz M; Gerlach, Daniel; Rath, Martina; Stark, Alexander


    Gene expression is determined by genomic elements called enhancers, which contain short motifs bound by different transcription factors (TFs). However, how enhancer sequences and TF motifs relate to enhancer activity is unknown, and general sequence requirements for enhancers or comprehensive sets of important enhancer sequence elements have remained elusive. Here, we computationally dissect thousands of functional enhancer sequences from three different Drosophila cell lines. We find that the enhancers display distinct cis-regulatory sequence signatures, which are predictive of the enhancers' cell type-specific or broad activities. These signatures contain transcription factor motifs and a novel class of enhancer sequence elements, dinucleotide repeat motifs (DRMs). DRMs are highly enriched in enhancers, particularly in enhancers that are broadly active across different cell types. We experimentally validate the importance of the identified TF motifs and DRMs for enhancer function and show that they can be sufficient to create an active enhancer de novo from a nonfunctional sequence. The function of DRMs as a novel class of general enhancer features that are also enriched in human regulatory regions might explain their implication in several diseases and provides important insights into gene regulation.

  4. On the existence of the H3 tautomer of adenine in aqueous solution. Rationalizations based on hybrid quantum mechanics/molecular mechanics predictions

    DEFF Research Database (Denmark)

    Aidas, Kestutis; Mikkelsen, Kurt V; Kongsted, Jacob


    The (15)N NMR spectrum of adenine in aqueous solution has been modeled using high-level combined density functional theory/molecular mechanics techniques coupled to a dynamical averaging scheme. The explicit consideration of the three lowest-energy tautomers of adenine-H9, H7 and H3-allows for a ...

  5. Chronic kidney disease induced by adenine: a suitable model of growth retardation in uremia. (United States)

    Claramunt, Débora; Gil-Peña, Helena; Fuente, Rocío; García-López, Enrique; Loredo, Vanessa; Hernández-Frías, Olaya; Ordoñez, Flor A; Rodríguez-Suárez, Julián; Santos, Fernando


    Growth retardation is a major manifestation of chronic kidney disease (CKD) in pediatric patients. The involvement of the various pathogenic factors is difficult to evaluate in clinical studies. Here, we present an experimental model of adenine-induced CKD for the study of growth failure. Three groups (n = 10) of weaning female rats were studied: normal diet (control), 0.5% adenine diet (AD), and normal diet pair fed with AD (PF). After 21 days, serum urea nitrogen, creatinine, parathyroid hormone (PTH), weight and length gains, femur osseous front advance as an index of longitudinal growth rate, growth plate histomorphometry, chondrocyte proliferative activity, bone structure, aorta calcifications, and kidney histology were analyzed. Results are means ± SE. AD rats developed renal failure (serum urea nitrogen: 70 ± 6 mg/dl and creatinine: 0.6 ± 0.1 mg/dl) and secondary hyperparathyroidism (PTH: 480 ± 31 pg/ml). Growth retardation of AD rats was demonstrated by lower weight (AD rats: 63.3 ± 4.8 g, control rats: 112.6 ± 4.7 g, and PF rats: 60.0 ± 3.8 g) and length (AD rats: 7.2 ± 0.2 cm, control rats: 11.1 ± 0.3 cm, and PF rats: 8.1 ± 0.3 cm) gains as well as lower osseous front advances (AD rats: 141 ± 13 μm/day, control rats: 293 ± 16 μm/day, and PF rats: 251 ± 10 μm/day). The processes of chondrocyte maturation and proliferation were impaired in AD rats, as shown by lower growth plate terminal chondrocyte height (21.7 ± 2.3 vs. 26.2 ± 1.9 and 23.9 ± 1.3 μm in control and PF rats) and proliferative activity index (AD rats: 30 ± 2%, control rats: 38 ± 2%, and PF rats: 42 ± 3%). The bone primary spongiosa structure of AD rats was markedly disorganized. In conclusion, adenine-induced CKD in young rats is associated with growth retardation and disturbed endochondral ossification. This animal protocol may be a useful new experimental model to study growth in CKD.

  6. Hydroxyl radical reactions with adenine: reactant complexes, transition states, and product complexes. (United States)

    Cheng, Qianyi; Gu, Jiande; Compaan, Katherine R; Schaefer, Henry F


    In order to address problems such as aging, cell death, and cancer, it is important to understand the mechanisms behind reactions causing DNA damage. One specific reaction implicated in DNA oxidative damage is hydroxyl free-radical attack on adenine (A) and other nucleic acid bases. The adenine reaction has been studied experimentally, but there are few theoretical results. In the present study, adenine dehydrogenation at various sites, and the potential-energy surfaces for these reactions, are investigated theoretically. Four reactant complexes [A···OH]* have been found, with binding energies relative to A+OH* of 32.8, 11.4, 10.7, and 10.1 kcal mol(-1). These four reactant complexes lead to six transition states, which in turn lie +4.3, -5.4, (-3.7 and +0.8), and (-2.3 and +0.8) kcal mol(-1) below A+OH*, respectively. Thus the lowest lying [A···OH]* complex faces the highest local barrier to formation of the product (A-H)*+H(2)O. Between the transition states and the products lie six product complexes. Adopting the same order as the reactant complexes, the product complexes [(A-H)···H(2)O]* lie at -10.9, -22.4, (-24.2 and -18.7), and (-20.5 and -17.5) kcal mol(-1), respectively, again relative to separated A+OH*. All six A+OH* → (A-H)*+H(2)O pathways are exothermic, by -0.3, -14.7, (-17.4 and -7.8), and (-13.7 and -7.8) kcal mol(-1), respectively. The transition state for dehydrogenation at N(6) lies at the lowest energy (-5.4 kcal mol(-1) relative to A+OH*), and thus reaction is likely to occur at this site. This theoretical prediction dovetails with the observed high reactivity of OH radicals with the NH(2) group of aromatic amines. However, the high barrier (37.1 kcal mol(-1)) for reaction at the C(8) site makes C(8) dehydrogenation unlikely. This last result is consistent with experimental observation of the imidazole ring opening upon OH radical addition to C(8). In addition, TD-DFT computed electronic transitions of the N(6) product around 420 nm

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

    Energy Technology Data Exchange (ETDEWEB)

    Nenov, Artur, E-mail:; Giussani, Angelo; Segarra-Martí, Javier; Jaiswal, Vishal K. [Dipartimento di Chimica “G. Ciamician,” Università di Bologna, Via Selmi 2, IT-40126 Bologna (Italy); Rivalta, Ivan [Université de Lyon, CNRS, Institut de Chimie de Lyon, École Normale Supérieure de Lyon, 46 Allée d’Italie, F-69364 Lyon Cedex 07 (France); Cerullo, Giulio [Dipartimento di Fisica, Politecnico di Milano, IFN-CNR, Piazza Leonardo Da Vinci 32, IT-20133 Milano (Italy); Mukamel, Shaul [Department of Chemistry, University of California, Irvine, California 92697-2025 (United States); Garavelli, Marco, E-mail:, E-mail: [Dipartimento di Chimica “G. Ciamician,” Università di Bologna, Via Selmi 2, IT-40126 Bologna (Italy); Université de Lyon, CNRS, Institut de Chimie de Lyon, École Normale Supérieure de Lyon, 46 Allée d’Italie, F-69364 Lyon Cedex 07 (France)


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

  8. A distinct sequence in the adenine nucleotide translocase from Artemia franciscana embryos is associated with insensitivity to bongkrekate and atypical effects of adenine nucleotides on Ca2+ uptake and sequestration. (United States)

    Konràd, Csaba; Kiss, Gergely; Töröcsik, Beata; Lábár, János L; Gerencser, Akos A; Mándi, Miklós; Adam-Vizi, Vera; Chinopoulos, Christos


    Mitochondria isolated from embryos of the crustacean Artemia franciscana lack the Ca(2+)-induced permeability transition pore. Although the composition of the pore described in mammalian mitochondria is unknown, the impacts of several effectors of the adenine nucleotide translocase (ANT) on pore opening are firmly established. Notably, ADP, ATP and bongkrekate delay, whereas carboxyatractyloside hastens, Ca(2+)-induced pore opening. Here, we report that adenine nucleotides decreased, whereas carboxyatractyloside increased, Ca(2+) uptake capacity in mitochondria isolated from Artemia embryos. Bongkrekate had no effect on either Ca(2+) uptake or ADP-ATP exchange rate. Transmission electron microscopy imaging of Ca(2+)-loaded Artemia mitochondria showed needle-like formations of electron-dense material in the absence of adenine nucleotides, and dot-like formations in the presence of adenine nucleotides or Mg(2+). Energy-filtered transmission electron microscopy showed the material to be rich in calcium and phosphorus. Sequencing of the Artemia mRNA coding for ANT revealed that it transcribes a protein with a stretch of amino acids in the 198-225 region with 48-56% similarity to those from other species, including the deletion of three amino acids in positions 211, 212 and 219. Mitochondria isolated from the liver of Xenopus laevis, in which the ANT shows similarity to that in Artemia except for the 198-225 amino acid region, demonstrated a Ca(2+)-induced bongkrekate-sensitive permeability transition pore, allowing the suggestion that this region of ANT may contain the binding site for bongkrekate.

  9. Prebiotic Synthesis of Adenine and Amino Acids Under Europa-like Conditions (United States)

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


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

  10. Role of adenine nucleotide translocator 1 in mtDNA maintenance. (United States)

    Kaukonen, J; Juselius, J K; Tiranti, V; Kyttälä, A; Zeviani, M; Comi, G P; Keränen, S; Peltonen, L; Suomalainen, A


    Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.

  11. Adenine phosphoribosyltransferase deficiency: an underdiagnosed cause of lithiasis and renal failure. (United States)

    Marra, Giuseppina; Vercelloni, Paolo Gilles; Edefonti, Alberto; Manzoni, Gianantonio; Pavesi, Maria Angela; Fogazzi, Giovanni Battista; Garigali, Giuseppe; Mockel, Lionel; Picot, Irene Ceballos


    We describe an infant affected by adenine phosphoribosyltransferase (APRT) deficiency diagnosed at 18 months of age with a de novo mutation that has not been previously reported. APRT deficiency is a rare defect of uric acid catabolism that leads to the accumulation of 2,8 dihydroxyadenine (2,8-DHA), a highly insoluble substance excreted by the kidneys that may precipitate in urine and form stones. The child suffered from renal colic due to a stone found in the peno-scrotal junction of the bulbar urethra. Stone spectrophotometric analysis allowed us to diagnose the disease and start kidney-saving therapy in order to avoid irreversible chronic kidney damage. APRT deficiency should always be considered in the differential diagnosis of pediatric urolithiasis.

  12. 3-Methyl-2-butenal: an enzymatic degradation product of the cytokinin, N-6-(delta-2 isopentenyl)adenine. (United States)

    Brownlee, B G; Hall, R H; Whitty, C D


    An enzyme preparation from immature corn kernels catalyzed cleavage of N-6-(delta-2-isopentenyl)adenine to give the aldehyde, 3-methyl-2-butenal, as the major side-chain derived product. This product, in the form of the semicarbazone, was identical with an authentic product by several criteria: chromatographic behavior, mass and ultraviolet spectra.

  13. Kinetics and thermodynamics of the reaction between the •OH radical and adenine – a theoretical investigation

    DEFF Research Database (Denmark)

    Milhøj, Birgitte Olai; Sauer, Stephan P. A.


    The accessibility of all possible reaction paths for the reaction between the nucleobase adenine and the •OH radical is investigated through quantum chemical calculations of barrier heights and rate constants at the wB97X-D/6-311++G(2df,2pd) level with Eckart tunneling corrections. First the comp...

  14. Adenine adsorption on Au(1 1 1) and Au(1 0 0) electrodes: Characterisation, surface reconstruction effects and thermodynamic study

    Energy Technology Data Exchange (ETDEWEB)

    Prado, Cesar [Department of Physical Chemistry, University of Sevilla, c/ Prof. Garcia Gonzalez n 2, Sevilla 41012 (Spain); Prieto, Francisco [Department of Physical Chemistry, University of Sevilla, c/ Prof. Garcia Gonzalez n 2, Sevilla 41012 (Spain); Rueda, Manuela [Department of Physical Chemistry, University of Sevilla, c/ Prof. Garcia Gonzalez n 2, Sevilla 41012 (Spain)]. E-mail:; Feliu, Juan [Department of Physical Chemistry, University of Alicante, Apart 99, Alicante E-03080 (Spain); Aldaz, Antonio [Department of Physical Chemistry, University of Alicante, Apart 99, Alicante E-03080 (Spain)


    Adsorption of adenine on Au(1 1 1) and Au(1 0 0) electrodes is studied by cyclic voltammetry, impedance and chronoamperometric measurements in 0.1 M and 0.01 M KClO{sub 4} and in 0.5 M NaF solutions. The experiments performed with flame-annealed electrodes at different contact potentials, scan potential limits and scan rates, suggest different adsorption behaviour on the unreconstructed and reconstructed surface domains. This is confirmed by comparing the results obtained with electrochemically annealed unreconstructed and with flame-annealed reconstructed surfaces. In both cases the initial electrode surface state is characterised by the E {sub pzc} values. The adsorption on reconstructed surfaces takes place at more positive potentials than on the unreconstructed surfaces and induces the lifting of the reconstruction. The thermodynamic analysis is performed on the chronoamperometric data for adenine desorption on well characterised unreconstructed Au(1 1 1) surfaces. To this end a new methodology of the chronoamperometric experiments is introduced. Quantitative thermodynamic adsorption parameters such as surface tension, Gibbs surface excess, Gibbs energy of adsorption, potential versus Gibbs excess slope and electrosorption valency are determined. Weak chemisorption of adenine is inferred with a molecular orientation independent on the coverage and on the electrode potential. It is proposed that adsorbed adenine molecules adopt a tilted orientation at the surface to facilitate the coordination to the gold atoms.

  15. Regulation of Salmonella enterica pathogenicity island 1 by DNA adenine methylation. (United States)

    López-Garrido, Javier; Casadesús, Josep


    DNA adenine methylase (Dam(-)) mutants of Salmonella enterica are attenuated in the mouse model and present multiple virulence-related defects. Impaired interaction of Salmonella Dam(-) mutants with the intestinal epithelium has been tentatively correlated with reduced secretion of pathogenicity island 1 (SPI-1) effectors. In this study, we show that S. enterica Dam(-) mutants contain lowered levels of the SPI-1 transcriptional regulators HilA, HilC, HilD, and InvF. Epistasis analysis indicates that Dam-dependent regulation of SPI-1 requires HilD, while HilA, HilC, and InvF are dispensable. A transcriptional hilDlac fusion is expressed at similar levels in Dam(+) and Dam(-) hosts. However, lower levels of hilD mRNA are found in a Dam(-) background, thus providing unsuspected evidence that Dam methylation might exert post-transcriptional regulation of hilD expression. This hypothesis is supported by the following lines of evidence: (i) lowered levels of hilD mRNA are found in Salmonella Dam(-) mutants when hilD is transcribed from a heterologous promoter; (ii) increased hilD mRNA turnover is observed in Dam(-) mutants; (iii) lack of the Hfq RNA chaperone enhances hilD mRNA instability in Dam(-) mutants; and (iv) lack of the RNA degradosome components polynucleotide phosphorylase and ribonuclease E suppresses hilD mRNA instability in a Dam(-) background. Our report of Dam-dependent control of hilD mRNA stability suggests that DNA adenine methylation plays hitherto unknown roles in post-transcriptional control of gene expression.

  16. Excretory Function of Intestinal Tract Enhanced in Kidney Impaired Rats Caused by Adenine (United States)

    Yun, Yu; Gao, Tao; Li, Yue; Gao, Zhiyi; Duan, Jinlian; Yin, Hua


    The main aim of the study was to prove the compensative effect of intestine for renal function. Rat kidney was impaired by intragastrically administrating adenine (400 mg per day for 5 days). Intestinal tract was harvested and equally divided into 20 segments except cecum. Kidneys were harvested and histologically examined with hematoxylin-eosin staining kits. Uric acid, urea (BUN), and creatinine in serum were determined with assay kits, and BUN and creatinine in every intestinal segment were also determined. The results showed that adenine was able to increase uric acid level in serum from 20.98 ± 6.98 μg/mL to 40.77 ± 7.52 μg/mL and cause renal function damage with BUN (from 3.87 ± 0.62 mM to 12.33 ± 3.27 mM) and creatinine (from 51.48 ± 6.98 μM to 118.25 ± 28.63 μM) increasing in serum and with abnormally micromorphological changes in kidney. The amount of BUN and creatinine distributed in intestinal tract was positively correlated with those in blood. In impaired renal function rats, the amount of BUN (from 4.26 ± 0.21 μMole to 10.72 ± 0.55 μMole) and creatinine (from 681.4 ± 23.3 nMole to 928.7 ± 21.3 nMole) distributed in intestinal tract significantly increased. All the results proved that intestinal tract had excretory function compensative for renal function. PMID:27975080

  17. Lack of association between estrogen receptor β dinucleotide repeat polymorphism and autoimmune thyroid diseases in Japanese patients

    Directory of Open Access Journals (Sweden)

    Tomita Motowo


    Full Text Available Abstract Background The autoimmune thyroid diseases (AITDs, such as Graves' disease (GD and Hashimoto's thyroiditis (HT, appear to develop as a result of complex interactions between predisposing genes and environmental triggers. Susceptibility to AITDs is conferred by genes in the human leukocyte antigen (HLA and genes unlinked to HLA, including the CTLA-4 gene. Recently, estrogen receptor (ER β, located at human chromosome 14q23-24.1, was identifed. We analyzed a dinucleotide (CAn repeat polymorphism located in the flanking region of ERβ gene in patients with AITDs and in normal subjects. High heterozygosity makes this polymorphism a useful marker in the genetic study of disorders affecting female endocrine systems. We also correlated a ERβ gene microsatellite polymorphism with bone mineral density (BMD in the distal radius and biochemical markers of bone turnover in patients with GD in remission. Results Fourteen different alleles were found in 133 patients with GD, 114 patients with HT, and 179 controls subjects. The various alleles were designated as allele*1 through allele*14 according to the number of the repeats, from 18 to 30. There was no significant difference in the distributions of ERβ alleles between patient groups and controls. Although recent study demonstrated a significant relation between a allele*9 in the ERβ gene and BMD in postmenopausal Japanese women, there were no statistically significant interaction between this allele and BMD in the distal radius, nor biochemical markers in patients with GD in remission. Conclusions The present results do not support an association between the ERβ microsatellite marker and AITD in the Japanese population. We also suggest that the ERβ microsatellite polymorphism has at most a minor pathogenic importance in predicting the risk of osteoporosis as a complication of GD.

  18. Lack of hepcidin ameliorates anemia and improves growth in an adenine-induced mouse model of chronic kidney disease. (United States)

    Akchurin, Oleh; Sureshbabu, Angara; Doty, Steve B; Zhu, Yuan-Shan; Patino, Edwin; Cunningham-Rundles, Susanna; Choi, Mary E; Boskey, Adele; Rivella, Stefano


    Growth delay is common in children with chronic kidney disease (CKD), often associated with poor quality of life. The role of anemia in uremic growth delay is poorly understood. Here we describe an induction of uremic growth retardation by a 0.2% adenine diet in wild-type (WT) and hepcidin gene (Hamp) knockout (KO) mice, compared with their respective littermates fed a regular diet. Experiments were started at weaning (3 wk). After 8 wk, blood was collected and mice were euthanized. Adenine-fed WT mice developed CKD (blood urea nitrogen 82.8 ± 11.6 mg/dl and creatinine 0.57 ± 0.07 mg/dl) and were 2.1 cm shorter compared with WT controls. WT adenine-fed mice were anemic and had low serum iron, elevated Hamp, and elevated IL6 and TNF-α. WT adenine-fed mice had advanced mineral bone disease (serum phosphorus 16.9 ± 3.1 mg/dl and FGF23 204.0 ± 115.0 ng/ml) with loss of cortical and trabecular bone volume seen on microcomputed tomography. Hamp disruption rescued the anemia phenotype resulting in improved growth rate in mice with CKD, thus providing direct experimental evidence of the relationship between Hamp pathway and growth impairment in CKD. Hamp disruption ameliorated CKD-induced growth hormone-insulin-like growth factor 1 axis derangements and growth plate alterations. Disruption of Hamp did not mitigate the development of uremia, inflammation, and mineral and bone disease in this model. Taken together, these results indicate that an adenine diet can be successfully used to study growth in mice with CKD. Hepcidin appears to be related to pathways of growth retardation in CKD suggesting that investigation of hepcidin-lowering therapies in juvenile CKD is warranted.

  19. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana. (United States)

    Yoshida, Keisuke; Hisabori, Toru


    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance.

  20. Reference: 529 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available nine dinucleotide (NaAD) from nicotinate mononucleotide (NaMN) in the Preiss-Handler-dependent pathway, and ...of nicotinamide adenine dinucleotide (NAD) from nicotiamide mononucleotide (NMN) in the Pre...iss-Handler-independent pathway. Prominent AtNMNAT expression was detected in the male gametophyte. More...over, AtNMNAT expression was spatio-temporally regulated during microspore development and pollen tube... growth. Disruption of the AtNMNAT gene (atnmnat mutant) was characterized by a decre

  1. Efficacy of Adenine in the Treatment of Leukopenia and Neutropenia Associated with an Overdose of Antipsychotics or Discontinuation of Lithium Carbonate Administration: Three Case Studies (United States)

    Tomita, Takashi; Goto, Hidekazu; Sumiya, Kenji; Yoshida, Tadashi; Tanaka, Katsuya; Kohda, Yukinao


    Because adenine is effective for managing cases of radiation-induced and drug-induced leukopenia, it may be effective in cases of antipsychotic-induced leukopenia and neutropenia. Here, we report our experience with patients with leukopenia and neutropenia caused by an antipsychotic overdose or discontinuation of lithium carbonate, in whom adenine administration ameliorated the white blood cell and neutrophil counts. The progress of patients suggests that adenine is effective in cases of leukopenia and neutropenia associated with lithium carbonate discontinuation and an antipsychotic overdose. PMID:27776394

  2. New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus. (United States)

    Bowman, Lisa; Zeden, Merve S; Schuster, Christopher F; Kaever, Volkhard; Gründling, Angelika


    Nucleotide signaling networks are key to facilitate alterations in gene expression, protein function, and enzyme activity in response to diverse stimuli. Cyclic di-adenosine monophosphate (c-di-AMP) is an important secondary messenger molecule produced by the human pathogen Staphylococcus aureus and is involved in regulating a number of physiological processes including potassium transport. S. aureus must ensure tight control over its cellular levels as both high levels of the dinucleotide and its absence result in a number of detrimental phenotypes. Here we show that in addition to the membrane-bound Asp-His-His and Asp-His-His-associated (DHH/DHHA1) domain-containing phosphodiesterase (PDE) GdpP, S. aureus produces a second cytoplasmic DHH/DHHA1 PDE Pde2. Although capable of hydrolyzing c-di-AMP, Pde2 preferentially converts linear 5'-phosphadenylyl-adenosine (pApA) to AMP. Using a pde2 mutant strain, pApA was detected for the first time in S. aureus, leading us to speculate that this dinucleotide may have a regulatory role under certain conditions. Moreover, pApA is involved in a feedback inhibition loop that limits GdpP-dependent c-di-AMP hydrolysis. Another protein linked to the regulation of c-di-AMP levels in bacteria is the predicted regulator protein YbbR. Here, it is shown that a ybbR mutant S. aureus strain has increased acid sensitivity that can be bypassed by the acquisition of mutations in a number of genes, including the gene coding for the diadenylate cyclase DacA. We further show that c-di-AMP levels are slightly elevated in the ybbR suppressor strains tested as compared with the wild-type strain. With this, we not only identified a new role for YbbR in acid stress resistance in S. aureus but also provide further insight into how c-di-AMP levels impact acid tolerance in this organism.

  3. Adenine nucleotide effect on intraocular pressure: Involvement of the parasympathetic nervous system. (United States)

    Peral, Assumpta; Gallar, Juana; Pintor, Jesús


    Nucleotides are present in the aqueous humor possibly exerting physiological effects on intraocular pressure (IOP). To determine the effect of nucleotides such as ATP and its related derivatives on IOP, New Zealand white rabbits were used. IOP was measured in rabbits treated topically either with saline (control) or with a single dose (10 microg/microL) of adenine nucleotides (ATP, 2-meS-ATP, ATP-gamma-S, alpha,beta-meADP, alpha,beta-meATP and beta,gamma-meATP). Those nucleotides reducing IOP (alpha,beta-meATP and beta,gamma-meATP) were then tested in concentrations ranging from 1 to 100 microg/microL to obtain the IC(50) value. Several antagonists for the P2 and adenosine A1 receptors (all at 10 microg/microL) were assayed 30 min before the application of the hypotensive nucleotide beta,gamma-meATP. To see whether the nucleotide was acting directly on the structures involved in aqueous humor dynamics or on the autonomic nerves controlling IOP, animal denervation and sympathetic (yohimbine and ICI-118,551 at 10 microg/microL) and parasympathetic (atropine and hexametonium at 10 microg/microL) receptors' antagonists were used 30 min before the instillation of beta,gamma-meATP. alpha,beta-meATP and beta,gamma-meATP decreased IOP to 60% of control value (basal IOP=23.2+/-1.3 mmHg), with IC(50) of 1.59+/-0.21 microg/microLand 0.56+/-0.62 microg/microL, which corresponds to 3mM and 1mM respectively. Denervation completely abolished the effect of beta,gamma-meATP. Sympathetic antagonists did not modify the hypotensive effect of beta,gamma-meATP, but parasympathetic antagonists were able to abolish it. Among the series of adenine nucleotide tested, alpha,beta-meATP and beta,gamma-meATP presented hypotensive actions on IOP. beta,gamma-meATP seems to stimulate cholinergic terminals being its final effect the IOP reduction. Therefore, these two nucleotides are interesting pharmacological tools for those pathologies related with high intraocular pressure.

  4. Investigations on the performance of poly(o-anisidine)/graphene nanocomposites for the electrochemical detection of NADH

    Energy Technology Data Exchange (ETDEWEB)

    Sangamithirai, D. [Department of Nuclear Physics, University of Madras, Guindy Campus, Chennai 600025 (India); Narayanan, V. [Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600025 (India); Muthuraaman, B. [Department of Energy, University of Madras, Guindy Campus, Chennai 600025 (India); Stephen, A., E-mail: [Department of Nuclear Physics, University of Madras, Guindy Campus, Chennai 600025 (India)


    The electrocatalytic oxidation of dihydronicotinamide adenine dinucleotide (NADH) based on poly(o-anisidine)/graphene (POA/GR) nanocomposites modified glassy carbon electrode (GCE) was explored for the first time. POA/GR nanocomposites were synthesized via chemical oxidative polymerization method. X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), high resolution transmission electron microscopy (HRTEM), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and UV–Vis spectroscopy results demonstrate that nanocomposites are successfully synthesized. An intriguing composite structure was observed using different ratios of o-anisidine monomer and graphene. The electrical properties and electrochemical properties of these nanocomposites are investigated by impedance spectroscopy technique and cyclic voltammetric (CV) method, respectively. The synthesized nanocomposites were used to modify glassy carbon electrode (GCE), and the modified electrodes were found to exhibit electrocatalytic activity for oxidation of NADH at low potential range of + 0.045 V in a neutral environment. The fabricated sensor based on POA/GR31-modified GCE exhibited enhanced current response with very high sensitivity of 47.1 μA μM{sup −1} for the detection of NADH. The developed POA/GR-modified GCE exhibited excellent reproducibility, stability, and selectivity for the determination of NADH. The practical analytical utility of the proposed method was demonstrated by NADH spiked ascorbic acid (AA) and the results confirmed that the proposed method is suitable for the determination of NADH in the presence of AA. This can open up new opportunities for simple and selective detection of NADH and provide a promising platform for biosensor designs. - Highlights: • NADH sensor was prepared by poly(o-anisidine)/graphene nanocomposite modified GCE. • FESEM technique revealed the growth of POA nanofibers on the surface of graphene. • Good

  5. 基于 WST-8细胞活性实验的表面增强拉曼光谱研究%WST-8 Assay for Living Cell Viability Detection by Surface-enhanced Raman Spectroscopy†

    Institute of Scientific and Technical Information of China (English)

    杨津; 刘卓; 王晓慧; 隋会敏; 毛竹; 赵冰


    采用常规的用于测定活细胞个数的试剂2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐(WST-8四唑盐)建立了一种快速测定细胞活性的表面增强拉曼散射光谱(SERS)新方法.考察了pH 值及还原剂烟酰胺腺嘌噙二核苷酸(NADH)用量对 WST-8甲瓒信号的影响.结果表明,与传统的 WST-8比色法相比, SERS 法具有更高的检测灵敏度和选择性,对 WST-8甲瓒的检测限可达到10μmol/ L.%Surface-enhanced Raman scattering(SERS) technique was used to develop and optimize a novel and quantitative 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt(WST-8) assay for living cell viability. The influences of pH and dosage of reductant nicoti-namide adenine dinucleotide(NADH) on WST-8 formazan signal were examined. SERS signals of formazan with high resolution and great diversity between WST-8 and formazan were observed in neutral conditions. Meanwhile, the signals of formazan(560, 1351, 1598, 1616 cm-1 ) could be found clearly when the concen-tration of NADH was four times that of WST-8. Compared with the traditional method, this assay appears higher sensitivity and selectivity for WST-8 formazan. Moreover, the detection limit of formazan is as low as 10μmol/ L.

  6. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process. (United States)

    Yang, C. C.; Oro, J.


    Study of the formation of purines, pyrimidines, and other bases from CO, H2, and NH3 under conditions similar to those used in the Fischer-Tropsch process. It is found that industrial nickel/iron alloy catalyzes the synthesis of adenine, guanine, cytosine, and other nitrogenous compounds from mixtures of CO, H2, and NH3 at temperatures of about 600 C. Sufficient sample was accumulated to isolate as solid products adenine, guanine, and cytosine, which were identified by infrared spectrophotometry. In the absence of nickel/iron catalyst, at 650 C, or in the presence of this catalyst, at 450 C, no purines or pyrimidines were synthesized. These results confirm and extend some of the work reported by Kayatsu et al. (1968).

  7. The contribution of adenines in the catalytic core of 10-23 DNAzyme improved by the 6-amino group modifications. (United States)

    Zhu, Junfei; Li, Zhiwen; Wang, Qi; Liu, Yang; He, Junlin


    In the catalytic core of 10-23 DNAzyme, its five adenine residues are moderate conservative, but with highly conserved functional groups like 6-amino group and 7-nitrogen atom. It is this critical conservation that these two groups could be modified for better contribution. With 2'-deoxyadenosine analogues, several functional groups were introduced at the 6-amino group of the five adenine residues. 3-Aminopropyl substituent at 6-amino group of A15 resulted in a five-fold increase of kobs. More efficient DNAzymes are expected by delicate design of the linkage and the external functional groups for this 6-amino group of A15. With this modification approach, other functional groups or residues could be optimized for 10-23 DNAzyme.

  8. Design and Facile Synthesis of New Dinucleotide Cap Analog Containing Both 2' and 3'-OH Modification on M⁷Guanosine Moiety. (United States)

    Kore, Anilkumar R; Bugarin, Alejandro; Shanmugasundaram, Muthian


    The first example of the synthesis of new dinucleotide cap analog containing 2('),3(')-diacetyl group on m(7)guanosine moiety is described. The desired modified cap analog, m(7,2)(')(,3)(')(-diacetyl)G[5(')]ppp[5(')]G has been obtained by the coupling reaction of triethylamine salt of m(7,2)(')(,3)(')(-diacetyl)GDP with ImGMP in presence of ZnCl2 as a catalyst in 62% yield with high purity. The structure of new cap analog has been confirmed by (1)H and (31)P NMR and mass data.

  9. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Directory of Open Access Journals (Sweden)

    Gaëlle Demarre


    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  10. Poly-adenine-based programmable engineering of gold nanoparticles for highly regulated spherical DNAzymes. (United States)

    Zhu, Dan; Pei, Hao; Chao, Jie; Su, Shao; Aldalbahi, Ali; Rahaman, Mostafizur; Wang, Lihua; Wang, Lianhui; Huang, Wei; Fan, Chunhai; Zuo, Xiaolei


    Enzyme complexes are assembled at the two-dimensional lipid membrane or prearranged on three-dimensional scaffolding proteins to regulate their catalytic activity in cells. Inspired by nature, we have developed gold nanoparticle-based spherical DNAzymes (SNAzymes) with programmably engineered activities by exploiting poly-adenine (polyA)-Au interactions. In a SNAzyme, AuNPs serve as the metal core, which is decorated with a functional shell of DNAzymes. Conventional thiolated DNAzyme-based assembly leads to disordered structures with suppressed activity. In contrast, by using an anchoring block of polyA tails, we find that the activity of SNAzymes can be programmably regulated. By using a polyA30 tail, SNAzymes demonstrated remarkably enhanced binding affinity compared to the thiolated DNAzyme-based assembly (∼75-fold) or individual DNAzymes in the solution phase (∼10-fold). More significantly, this increased affinity is directly translated to the sensitivity improvement in the SNAzyme-based lead sensor. Hence, this design of SNAzymes may provide new opportunities for developing biosensors and bioimaging probes for theranostic applications.

  11. Laser pulse trains for controlling excited state dynamics of adenine in water. (United States)

    Petersen, Jens; Wohlgemuth, Matthias; Sellner, Bernhard; Bonačić-Koutecký, Vlasta; Lischka, Hans; Mitrić, Roland


    We investigate theoretically the control of the ultrafast excited state dynamics of adenine in water by laser pulse trains, with the aim to extend the excited state lifetime and to suppress nonradiative relaxation processes. For this purpose, we introduce the combination of our field-induced surface hopping method (FISH) with the quantum mechanical-molecular mechanical (QM/MM) technique for simulating the laser-driven dynamics in the condensed phase under explicit inclusion of the solvent environment. Moreover, we employ parametric pulse shaping in the frequency domain in order to design simplified laser pulse trains allowing to establish a direct link between the pulse parameters and the controlled dynamics. We construct pulse trains which achieve a high excitation efficiency and at the same time keep a high excited state population for a significantly extended time period compared to the uncontrolled dynamics. The control mechanism involves a sequential cycling of the population between the lowest and higher excited states, thereby utilizing the properties of the corresponding potential energy surfaces to avoid conical intersections and thus to suppress the nonradiative decay to the ground state. Our findings provide a means to increase the fluorescence yield of molecules with an intrinsically very short excited state lifetime, which can lead to novel applications of shaped laser fields in the context of biosensing.

  12. Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox. (United States)

    Magrini, V; Salmi, D; Thomas, D; Herbert, S K; Hartzell, P L; Youderian, P


    Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.

  13. Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD. (United States)

    Mishima, Eikan; Fukuda, Shinji; Shima, Hisato; Hirayama, Akiyoshi; Akiyama, Yasutoshi; Takeuchi, Yoichi; Fukuda, Noriko N; Suzuki, Takehiro; Suzuki, Chitose; Yuri, Akinori; Kikuchi, Koichi; Tomioka, Yoshihisa; Ito, Sadayoshi; Soga, Tomoyoshi; Abe, Takaaki


    The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

  14. The experimental and theoretical gas phase acidities of adenine, guanine, cytosine, uracil, thymine and halouracils (United States)

    Chen, Edward C. M.; Herder, Charles; Chen, Edward S.


    The gas phase acidities GPA (Δ H (298) for deprotonation) of the most stable tautomers of adenine, guanine, cytosine, uracil and thymine are evaluated. New GPA are obtained from electron impact spectra and acid dissociation constants measured in dimethylsulfoxide for A, U and 5-FU. The average experimental GPA are: [N1 sbnd H] C 340(2); T 333(2); U 333(2); 5-FU 329(4); [N9 sbnd H] A 333(1); G 332(4); all in kcal/mol. Only cytosine is a weaker acid than HCl in the gas phase. The most acidic hydrogens in the nucleotides are replaced by the sugar in DNA and RNA. The experimental N3 sbnd H GPA are G 334(4); U 347(2), T 347(4), while the predicted N3 sbnd H 5-FU GPA is 343 kcal/mol. The NH sbnd H GPA are: C 346(4); A 352(2); G 336(4) (all in kcal/mol). These are supported by semi-empirical multiconfiguration configuration interaction calculations. The predicted C8 sbnd H acidities of G and A and the C6 sbnd H of T are about the same, 360(2) kcal/mol. The remaining CH acidities are 370-380 kcal/mol. The 5-halouracils are predicted to be more acidic than HCl.

  15. Ischemic preconditioning protects post-ischemic renal function in anesthetized dogs: role of adenosine and adenine nucleotides

    Institute of Scientific and Technical Information of China (English)

    Fan-zhu LI; Shoji KIMURA; Akira NISHIYAMA; Matlubur RAHMAN; Guo-xing ZHANG; Youichi ABE


    Aim: To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury.Methods: Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902),respectively.The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1-3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology.Results: One 10-min IPC, adenosine A1 receptor antagonist (KW3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP).Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia.All of these recovery times were dependent on the cycles of 10-min IPC.The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively.Conclusion: A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively.Renal IPC can protect against ischemiareperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function.

  16. IR Vibrational spectra of H-bonded complexes of adenine, 2-aminopurine and 2-aminopurine+ with cytosine and thymine: Quantum-chemical study (United States)

    Brovarets', O. O.; Hovorun, D. M.


    Using theoretical study on the B3LYP/6-311++G(d,p) level of theory, we have compared vibrational spectra of 2-aminopurine (as neutral or protonated at N1 atom species) with adenine and H-bonded complexes of 2-aminopurine (as neutral or protoned at N1 atom species) · cytosine or 2-aminopurine · thymine with adenine · cytosine and adenine · thymine base pairs. The nature of the base pairing between adenine, 2-aminopurine, 2-aminopurine+ and cytosine or thymine have been investigated by means of quantum-mechanical calculations. We have investigated the effect of the hydrogen bond formation on the vibrational spectra of the investigated base pairs. The main differences in the vibrational spectra as for bases so for base pairs have been observed in the high-frequency region.

  17. High-NaCl diet impairs dynamic renal blood flow autoregulation in rats with adenine-induced chronic renal failure. (United States)

    Saeed, Aso; DiBona, Gerald F; Grimberg, Elisabeth; Nguy, Lisa; Mikkelsen, Minne Line Nedergaard; Marcussen, Niels; Guron, Gregor


    This study examined the effects of 2 wk of high-NaCl diet on kidney function and dynamic renal blood flow autoregulation (RBFA) in rats with adenine-induced chronic renal failure (ACRF). Male Sprague-Dawley rats received either chow containing adenine or were pair-fed an identical diet without adenine (controls). After 10 wk, rats were randomized to either remain on the same diet (0.6% NaCl) or to be switched to high 4% NaCl chow. Two weeks after randomization, renal clearance experiments were performed under isoflurane anesthesia and dynamic RBFA, baroreflex sensitivity (BRS), systolic arterial pressure variability (SAPV), and heart rate variability were assessed by spectral analytical techniques. Rats with ACRF showed marked reductions in glomerular filtration rate and renal blood flow (RBF), whereas mean arterial pressure and SAPV were significantly elevated. In addition, spontaneous BRS was reduced by ∼50% in ACRF animals. High-NaCl diet significantly increased transfer function fractional gain values between arterial pressure and RBF in the frequency range of the myogenic response (0.06-0.09 Hz) only in ACRF animals (0.3 ± 4.0 vs. -4.4 ± 3.8 dB; P renal failure by facilitating pressure transmission to the microvasculature.

  18. A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3',3'-cGAMP. (United States)

    Roembke, Benjamin T; Zhou, Jie; Zheng, Yue; Sayre, David; Lizardo, Allan; Bernard, Laurentee; Sintim, Herman O


    Cyclic dinucleotides have emerged as second messengers that regulate diverse processes in bacteria, as well as regulating the production of type I interferons in metazoans. Fluorescent sensors for these important second messengers are highly sought-after for high-throughput inhibitor discovery, yet most sensors reported to date are not amenable for high-throughput screening purposes. Herein, we demonstrate that a new analog, 3',3'-cG(d2AP)MP, which is a 2-aminopurine (2AP)-containing cyclic dinucleotide, self-associates in the presence of Mn(2+) with an association constant of 120,000 M(-1). 3'3'-cG(d2AP)MP can also form a heterodimer with cGAMP, activator of immune regulator, STING, or the bacterial biofilm regulator, c-di-GMP in the presence of Mn(II). Upon dimer formation, the fluorescence of 3',3'-cG(d2AP)MP is quenched and this provides a convenient method to monitor the enzymatic processing of both DGC and PDE enzymes, opening up several opportunities for the discovery of inhibitors of nucleotide signaling.

  19. An ensemble of B-DNA dinucleotide geometries lead to characteristic nucleosomal DNA structure and provide plasticity required for gene expression

    Directory of Open Access Journals (Sweden)

    Bansal Manju


    Full Text Available Abstract Background A nucleosome is the fundamental repeating unit of the eukaryotic chromosome. It has been shown that the positioning of a majority of nucleosomes is primarily controlled by factors other than the intrinsic preference of the DNA sequence. One of the key questions in this context is the role, if any, that can be played by the variability of nucleosomal DNA structure. Results In this study, we have addressed this question by analysing the variability at the dinucleotide and trinucleotide as well as longer length scales in a dataset of nucleosome X-ray crystal structures. We observe that the nucleosome structure displays remarkable local level structural versatility within the B-DNA family. The nucleosomal DNA also incorporates a large number of kinks. Conclusions Based on our results, we propose that the local and global level versatility of B-DNA structure may be a significant factor modulating the formation of nucleosomes in the vicinity of high-plasticity genes, and in varying the probability of binding by regulatory proteins. Hence, these factors should be incorporated in the prediction algorithms and there may not be a unique 'template' for predicting putative nucleosome sequences. In addition, the multimodal distribution of dinucleotide parameters for some steps and the presence of a large number of kinks in the nucleosomal DNA structure indicate that the linear elastic model, used by several algorithms to predict the energetic cost of nucleosome formation, may lead to incorrect results.

  20. Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study. (United States)

    Allnér, Olof; Nilsson, Lennart; Villa, Alessandra


    Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.

  1. Two adenine nucleotide translocase paralogues involved in cell proliferation and spermatogenesis in the silkworm Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Ryohei Sugahara

    Full Text Available Mitochondrial adenine nucleotide translocase (ANT specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4 and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4 is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1 and the testis-specific paralogue (BmANTI2. The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1, but not those of other insect species (or PxANTI2, restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4.

  2. Two adenine nucleotide translocase paralogues involved in cell proliferation and spermatogenesis in the silkworm Bombyx mori. (United States)

    Sugahara, Ryohei; Jouraku, Akiya; Nakakura, Takayo; Kusakabe, Takahiro; Yamamoto, Takenori; Shinohara, Yasuo; Miyoshi, Hideto; Shiotsuki, Takahiro


    Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4.

  3. Adenine Nucleotide Translocase 4 Is Expressed Within Embryonic Ovaries and Dispensable During Oogenesis (United States)

    Lim, Chae Ho; Brower, Jeffrey V.; Resnick, James L.; Oh, S. Paul


    Adenine nucleotide translocase (Ant) facilitates the exchange of adenosine triphosphate across the mitochondrial inner membrane and plays a critical role for bioenergetics in eukaryotes. Mice have 3 Ant paralogs, Ant1 (Slc25a4), Ant2 (Slc25a5), and Ant4 (Slc25a31), which are expressed in a tissue-dependent manner. We previously identified that Ant4 was expressed exclusively in testicular germ cells in adult mice and essential for spermatogenesis and subsequently male fertility. Further investigation into the process of spermatogenesis revealed that Ant4 was particularly highly expressed during meiotic prophase I and indispensable for normal progression of leptotene spermatocytes to the stages thereafter. In contrast, the expression and roles of Ant4 in female germ cells have not previously been elucidated. Here, we demonstrate that the Ant4 gene is expressed during embryonic ovarian development during which meiotic prophase I occurs. We confirmed embryonic ovary-specific Ant4 expression using a bacterial artificial chromosome transgene. In contrast to male, however, Ant4 null female mice were fertile although the litter size was slightly decreased. They showed apparently normal ovarian development which was morphologically indistinguishable from the control animals. These data indicate that Ant4 is a meiosis-specific gene expressed during both male and female gametogenesis however indispensable only during spermatogenesis and not oogenesis. The differential effects of Ant4 depletion within the processes of male and female gametogenesis may be explained by meiosis-specific inactivation of the X-linked Ant2 gene in male, a somatic paralog of the Ant4 gene. PMID:25031318

  4. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function. (United States)

    Reynafarje, B; Lehninger, A L


    This paper describes the properties and a possible biological role of a transport process across the inner membrane of rat liver mitochondria resulting in the exchange of ATP(4-) (out) for ADP(3-) (in) + 0.5 phosphate(2-) (in). This transmembrane exchange reaction, designated as the ATP-ADP-phosphate exchange, is specific for the ligands shown, electroneutral, insensitive to N-ethylmaleimide or mersalyl, inhibited by atractyloside, and appears to occur only in the direction as written. It is thus distinct from the well-known phosphate-hydroxide and phosphate-dicarboxylate exchange systems, which are inhibited by mersalyl, and from the ATP-ADP exchanger, which does not transport phosphate. During ATP hydrolysis by mitochondria, half of the phosphate formed from ATP passes from the matrix to the medium by the mersalyl-insensitive ATP-ADP-phosphate exchange and the other half by the well-known mersalyl-sensitive phosphate-hydroxide exchange. These and other considerations have led to a hypothesis for the pathway and stoichiometry of ATP-dependent reverse electron transport, characterized by a requirement of 1.33 molecules of ATP per pair of electrons reversed and by the utilization of a different membrane transport pathway for phosphate and adenine nucleotides than is taken in forward electron flow and oxidative phosphorylation. The possible occurrence of independent pathways for ATP-forming forward electron flow and ATP-consuming reverse electron flow is consonant with the fact that the opposing degradative and synthetic pathways in the central routes of cell metabolism generally have different pathways that are independently regulated.

  5. Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate. (United States)

    Li, Quansong; Giussani, Angelo; Segarra-Martí, Javier; Nenov, Artur; Rivalta, Ivan; Voityuk, Alexander A; Mukamel, Shaul; Roca-Sanjuán, Daniel; Garavelli, Marco; Blancafort, Lluís


    The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D-UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter-base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long-lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the (1) Lb , S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D-UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm(-1) in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D-UV spectroscopy to disentangle the photophysics of multichromophoric systems.

  6. Preliminary evidence for an association of a dinucleotide repeat polymorphism at the MAOA gene with early onset alcoholism/substance abuse

    Energy Technology Data Exchange (ETDEWEB)

    Vanyukov, M.M.; Moss, H.B.; Tarter, R.E. [Univ. of Pittsburg, PA (United States)] [and others


    An association between the liability to early onset alcoholism/substance abuse and a recently discovered dinucleotide repeat length polymorphism at the MAOA gene (MAOCA-1) was examined using polymerase chain reaction (PCR). A significant correlation between the presence/absence of the disorder and the length of the MAOCA-1 repeat was found in males, but not females, with {open_quotes}long{close_quotes} alleles (repeat length above 115 bp) associated with both increased risk for the disorder and lower age of onset of substance abuse. These preliminary data suggest that further exploration of the relationship between the MAOA gene and behavioral traits in an expanded sample is warranted. 22 refs., 1 fig., 3 tabs.

  7. Adsorption of adenine and thymine on zeolites: FT-IR and EPR spectroscopy and X-ray diffractometry and SEM studies. (United States)

    Baú, João Paulo T; Carneiro, Cristine E A; de Souza Junior, Ivan G; de Souza, Cláudio M D; da Costa, Antonio C S; di Mauro, Eduardo; Zaia, Cássia T B V; Coronas, Joaquin; Casado, Clara; de Santana, Henrique; Zaia, Dimas A M


    The interactions of adenine and thymine with and adsorption on zeolites were studied using different techniques. There were two main findings. First, as shown by X-ray diffractometry, thymine increased the decomposition of the zeolites (Y, ZSM-5) while adenine prevented it. Second, zeolite Y adsorbed almost the same amount of adenine and thymine, thus both nucleic acid bases could be protected from hydrolysis and UV radiation and could be available for molecular evolution. The X-ray diffractometry and SEM showed that artificial seawater almost dissolved zeolite A. The adsorption of adenine on ZSM-5 zeolite was higher than that of thymine (Student-Newman-Keuls test-SNK pzeolite, when compared to other zeolites (SNK pzeolites was not statistically different (SNK p>0.05). The adsorption of adenine and thymine on zeolites did not depend on pore size or Si/Al ratio and it was not explained only by electrostatic forces; rather van der Waals interactions should also be considered.

  8. Evaluation of Porin Interaction with Adenine Nucleotide Translocase and Cyclophilin-D Proteins after Brain Ischemia and Reperfusion

    Directory of Open Access Journals (Sweden)

    Mohammad Ali Atlasi


    Full Text Available Objective (s Porin is a mitochondrial outer membrane channel, which usually functions as the pathway for the movement of various substances in and out of the mitochondria and is considered to be a component of the permeability transition (PT pore complex that plays a role in the PT. We addressed the hypothesis that porin interacts with other mitochondrial proteins after ischemic injury.Materials and MethodsFor this purpose, we used in vivo 4-vessel occlusion model of rat brain and porin purification method by hydroxyapatite column. After SDS gel electrophoresis and silver nitrate staining, Western blotting was done for porin, adenine nucleotide translocase and cyclophilin-D proteins.Results Porin was purified from mitochondrial mixture in ischemic brain and control groups. Investigation of interaction of adenine nucleotide transposes (ANT and cyclophilin-D with porin by Western blotting showed no proteins co-purified with porin from injured tissues.Conclusion The present study implies that there may not be interaction between porin, and ANT or cyclophilin-D, and if there is any, it is not maintained during the purification procedure.

  9. Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice

    Energy Technology Data Exchange (ETDEWEB)

    Engle, S.J.; Chen, J.; Tischfield, J.A. [Indiana Univ., School of Medicine, Indianapolis, IN (United States)] [and others


    Adenine phosphoribosyltransferase (APRT: EC, a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine kinase gene at the 3{prime} end of the homology as a negative selection marker. The vector was introduced into D3 ES cells by electroporation and the cells were selected for G418 and ganciclovir (GANC) resistance. G418-GANC resistant clones were screened by Southern blot. One of several correctly targeted clones was expanded and used for blastocyst microinjection to produce chimeric mice. Chimeric animals were bred and agouti progeny heterozygous for the targeted allele were obtained. Heterozygous animals have been bred to produce APRT-deficient animals. Matings are currently underway to determine the phenotype of APRT/HPRT-deficient animals.

  10. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence.

    Directory of Open Access Journals (Sweden)

    Allison E James

    Full Text Available DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam. To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

  11. Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence. (United States)

    James, Allison E; Rogovskyy, Artem S; Crowley, Michael A; Bankhead, Troy


    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence.

  12. Control of dinucleoside polyphosphates by the FHIT-homologous HNT2 gene, adenine biosynthesis and heat shock in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Bieganowski Pawel


    Full Text Available Abstract Background The FHIT gene is lost early in the development of many tumors. Fhit possesses intrinsic ApppA hydrolase activity though ApppA cleavage is not required for tumor suppression. Because a mutant form of Fhit that is functional in tumor suppression and defective in catalysis binds ApppA well, it was hypothesized that Fhit-substrate complexes are the active, signaling form of Fhit. Which substrates are most important for Fhit signaling remain unknown. Results Here we demonstrate that dinucleoside polyphosphate levels increase 500-fold to hundreds of micromolar in strains devoid of the Saccharomyces cerevisiae homolog of Fhit, Hnt2. Accumulation of dinucleoside polyphosphates is reversed by re-expression of Hnt2 and is active site-dependent. Dinucleoside polyphosphate levels depend on an intact adenine biosynthetic pathway and time in liquid culture, and are induced by heat shock to greater than 0.1 millimolar even in Hnt2+ cells. Conclusions The data indicate that Hnt2 hydrolyzes both ApppN and AppppN in vivo and that, in heat-shocked, adenine prototrophic yeast strains, dinucleoside polyphosphates accumulate to levels in which they may saturate Hnt2.

  13. Demographic changes and marker properties affect detection of human population differentiation

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    Sanichwankul Kittipong


    Full Text Available Abstract Background Differentiating genetically between populations is valuable for admixture and population stratification detection and in understanding population history. This is easy to achieve for major continental populations, but not for closely related populations. It has been claimed that a large marker panel is necessary to reliably distinguish populations within a continent. We investigated whether empirical genetic differentiation could be accomplished efficiently among three Asian populations (Hmong, Thai, and Chinese using a small set of highly variable markers (15 tetranucleotide and 17 dinucleotide repeats. Results Hmong could be differentiated from Thai and Chinese based on multi-locus genotypes, but Thai and Chinese were indistinguishable from each other. We found significant evidence for a recent population bottleneck followed by expansion in the Hmong that was not present in the Thai or Chinese. Tetranucleotide repeats were less useful than dinucleotide repeat markers in distinguishing between major continental populations (Asian, European, and African while both successfully distinguished Hmong from Thai and Chinese. Conclusion Demographic history contributes significantly to robust detection of intracontinental population structure. Populations having experienced a rapid size reduction may be reliably distinguished as a result of a genetic drift -driven redistribution of population allele frequencies. Tetranucleotide markers, which differ from dinucleotide markers in mutation mechanism and rate, are similar in information content to dinucleotide markers in this situation. These factors should be considered when identifying populations suitable for gene mapping studies and when interpreting interpopulation relationships based on microsatellite markers.

  14. Toxicity associated with high dosage 9-[(2R,5R-2,5-dihydro-5-phosphonomethoxy)-2-furanyl]adenine therapy off attempts to abort early FIV infection. (United States)

    Hartmann, K; Ferk, G; North, T W; Pedersen, N C


    9-[(2R,5R-2,5-dihydro-5-phosphonomethoxy)-2-furanyl]adenine, or D4API, was tested in the feline immunodeficiency virus (FIV) infection model and found to be significantly more inhibitory in vitro than its parent compound 9-phosphonylmethoxethyl adenine (PMEA). Cytotoxicity was less than for PMEA or azidothymidine (AZT) for culture periods of 7 days, but more toxic after 10 days. D4API was rapidly absorbed by cats following subcutaneous inoculation, with a plasma half-life of less than 1 h after intravenous inoculation and between 2 and 3 h after subcutaneous injection. Peripheral blood mononuclear cells collected from cats given a single dose of D4API were refractory, however, to FIV infection in vitro for up to 24 h. Given its prolonged intracellular phase and high selectivity index, high dose D4API therapy was tested for its ability to abort an acute (i.e. 2 week) FIV infection. A divided daily dose of D4API, which was one-fourth the toxic dose and 125 times the concentration that would totally inhibit virus replication in vitro, completely abrogated the anticipated viremia and antibody responses. Unfortunately, a majority of treated/uninfected and treated/infected test cats died acutely of drug toxicity after 47 days of treatment. Toxicity in vivo mirrored what was observed in vitro, being precipitous and cumulative in nature. Toxic signs included widespread hepatic and lymphoid necrosis. A surviving treated/FIV infected cat remained healthy to day 175 when the study was terminated; antibodies appeared 2 months later than in untreated/infected cats and virus was only detectable at low levels on day 175. In contrast, untreated/infected cats were viremic and antibody positive from 3 to 4 weeks post-infection onwards. Therefore, it was possible to alter, but not abort, an early FIV infection with prolonged, high-dose D4API treatment.

  15. Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation. (United States)

    Stephansen, Anne B; King, Sarah B; Yokoi, Yuki; Minoshima, Yusuke; Li, Wei-Li; Kunin, Alice; Takayanagi, Toshiyuki; Neumark, Daniel M


    Dipole bound (DB) and valence bound (VB) anions of binary iodide-adenine complexes have been studied using one-color and time-resolved photoelectron imaging at excitation energies near the vertical detachment energy. The experiments are complemented by quantum chemical calculations. One-color spectra show evidence for two adenine tautomers, the canonical, biologically relevant A9 tautomer and the A3 tautomer. In the UV-pump/IR-probe time-resolved experiments, transient adenine anions can be formed by electron transfer from the iodide. These experiments show signals from both DB and VB states of adenine anions formed on femto- and picosecond time scales, respectively. Analysis of the spectra and comparison with calculations suggest that while both the A9 and A3 tautomers contribute to the DB signal, only the DB state of the A3 tautomer undergoes a transition to the VB anion. The VB anion of A9 is higher in energy than both the DB anion and the neutral, and the VB anion is therefore not accessible through the DB state. Experimental evidence of the metastable A9 VB anion is instead observed as a shape resonance in the one-color photoelectron spectra, as a result of UV absorption by A9 and subsequent electron transfer from iodide into the empty π-orbital. In contrast, the iodide-A3 complex constitutes an excellent example of how DB states can act as doorway state for VB anion formation when the VB state is energetically available.

  16. A concise and simple synthesis of 1-hydroxy-phenethylamine derivatives: Formal synthesis of naturally occurring norephedrine, virolin and 3-hydroxy-2-phosphonylmethoxypropyl adenine

    Indian Academy of Sciences (India)

    S Saha; P Chakraborty; S C Roy


    A concise and simple synthesis of 1-hydroxy-phenethylamine derivatives has been achieved following classical organic transformations using commercially available chiral pools. The said derivatives were explored for the synthesis of naturally occurring bio-active small molecules. Formal synthesis of norephedrine, virolin and 3-hydroxy-2-phosphonylmethoxypropyl adenine has been demonstrated.

  17. Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions

    NARCIS (Netherlands)

    Vaartjes, W.J.; Breejen, J.N. den; Geelen, M.J.H.; Bergh, S.G. van den


    1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite d

  18. Metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl-CoA effects - Implications for a mechanism linking obesity and type 2 diabetes

    NARCIS (Netherlands)

    Ciapaite, Jolita; Bakker, Stephan J. L.; Diamant, Michaela; van Eikenhorst, Gerco; Heine, Robert J.; Westerhoff, Hans V.; Krab, Klaas


    Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitoc

  19. Nitric oxide interacts with oxygen free radicals to evoke the release of adenosine and adenine nucleotides from rat hippocampal slices. (United States)

    Broad, R M; Fallahi, N; Fredholm, B B


    The present study examined some possible mechanisms underlying the previously demonstrated release of adenosine by nitric oxide (NO) donors. Perfusion with the NO-donor S-nitroso-N-acetyl penicillamine (SNAP; 300 microM) led to a significant increase in the release of [3H]purines from both unstimulated and electrically stimulated hippocampal slices prelabeled with [3H]adenine. The NO-donor also evoked the release of endogenous ATP and ADP from unstimulated slices and, when combined with electrical stimulation, the release of ATP, AMP and adenosine. The SNAP-induced [3H]purine release was calcium-dependent, but not affected by the glutamate receptor antagonists MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]-cyclohepten-5,10-imine;100 nM) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; 10 microM). Zaprinast (5 microM), an inhibitor of the cyclic GMP-dependent phosphodiesterase and 8-Br-cyclic GMP (0.01-1 mM) failed to evoke the release of purines, whereas generation of oxygen free radicals by xanthine plus xanthine oxidase did evoke purine release. Coperfusion of SNAP with the free radical scavengers superoxide dismutase (SOD; 60 microg/ml) and catalase (50 microg/ml) reduced or eliminated the ability of the NO-donor to enhance [3H]purine release, but the poly (ADP-ribosyl) synthetase (PARS) inhibitor benzamide (500 microM) did not affect it. These data indicate that NO interacts with superoxide, likely forming peroxynitrite, which subsequently acts to release adenosine and adenine nucleotides from hippocampal tissue.

  20. Mismatch base pairing of the mutagen 8-oxoguanine and its derivatives with adenine: A theoretical search for possible antimutagenic agents (United States)

    Singh, A. K.; Mishra, P. C.

    Molecular geometries of 8-oxoguanine (8OG), those of its substituted derivatives with the substitutions CH2, CF2, CO, CNH, O, and S in place of the N7H7 group, adenine (A), and the base pairs of 8OG and its substituted derivatives with adenine were optimized using the RHF/6-31+G* and B3LYP/6-31+G* methods in gas phase. All the molecules and their hydrogen-bonded complexes were solvated in aqueous media employing the polarized continuum model (PCM) of the self-consistent reaction field (SCRF) theory using the RHF/6-31+G* and B3LYP/6-31+G* methods. The optimized geometrical parameters of the 8OG-A base pair at the RHF/6-31+G* and B3LYP/6-31+G* levels of theory agree satisfactorily with those of an oligonucleotide containing the base pair found from X-ray crystallography. The pattern of hydrogen bonding in the CF2- and O-substituted 8OG-A base pair is of Watson-Crick type and that in the unsubstituted and CH2-, CNH-, and S-substituted base pairs is of Hoogsteen type. In the CO-substituted base pair, the hydrogen bonding pattern is of neither Watson-Crick nor Hoogsteen type. The CF2-substitution appears to introduce steric hindrance for stacking of DNA bases. On the basis of these results, it appears that among all the substituted 8OG molecules considered here, the O-substituted derivative may be useful as an antimutagenic drug. It is, however, subject to experimental verification. Content:text/plain; charset="UTF-8"

  1. Expansion of GA Dinucleotide Repeats Increases the Density of CLAMP Binding Sites on the X-Chromosome to Promote Drosophila Dosage Compensation.

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    Guray Kuzu


    Full Text Available Dosage compensation is an essential process that equalizes transcript levels of X-linked genes between sexes by forming a domain of coordinated gene expression. Throughout the evolution of Diptera, many different X-chromosomes acquired the ability to be dosage compensated. Once each newly evolved X-chromosome is targeted for dosage compensation in XY males, its active genes are upregulated two-fold to equalize gene expression with XX females. In Drosophila melanogaster, the CLAMP zinc finger protein links the dosage compensation complex to the X-chromosome. However, the mechanism for X-chromosome identification has remained unknown. Here, we combine biochemical, genomic and evolutionary approaches to reveal that expansion of GA-dinucleotide repeats likely accumulated on the X-chromosome over evolutionary time to increase the density of CLAMP binding sites, thereby driving the evolution of dosage compensation. Overall, we present new insight into how subtle changes in genomic architecture, such as expansions of a simple sequence repeat, promote the evolution of coordinated gene expression.

  2. The rates of G:C[yields]T:A and G:C[yields]C:G transversions at CpG dinucleotides in the human factor IX gene

    Energy Technology Data Exchange (ETDEWEB)

    Ketterling, R.P.; Vielhaber, E.; Sommer, S.S. (Mayo Clinic/Foundation, Rochester, MN (United States))


    The authors have identified eight independent transversions at CpG in 290 consecutive families with hemophilia B. These eight transversions account for 16.3% of all independent transversions in the sample, yet the expected frequency of CpG transversions at random in the factor IX gene is only 2.6% (P<0.1). The aggregate data suggest that the two types of CpG transversions (G:C[yields]T:A and G:C[yields]C:G) possess similar mutation rates (24.8 [times] 10[sup [minus]10] and 20.6 [times] 10[sup [minus]10], respectively), which are about fivefold greater than the comparable rates for transversions at non-CpG dinucleotides. The enhancement of transversions at CpG suggest that the model by which mutations occur at CpG may need to be reevaluated. The relationship, if any, between deamination of 5-methyl cytosine and enhancement of transversions at CpG remains to be defined. 28 refs., 2 tabs.

  3. iROS-gPseKNC: Predicting replication origin sites in DNA by incorporating dinucleotide position-specific propensity into general pseudo nucleotide composition (United States)

    Xiao, Xuan; Ye, Han-Xiao; Liu, Zi; Jia, Jian-Hua; Chou, Kuo-Chen


    DNA replication, occurring in all living organisms and being the basis for biological inheritance, is the process of producing two identical replicas from one original DNA molecule. To in-depth understand such an important biological process and use it for developing new strategy against genetics diseases, the knowledge of duplication origin sites in DNA is indispensible. With the explosive growth of DNA sequences emerging in the postgenomic age, it is highly desired to develop high throughput tools to identify these regions purely based on the sequence information alone. In this paper, by incorporating the dinucleotide position-specific propensity information into the general pseudo nucleotide composition and using the random forest classifier, a new predictor called iROS-gPseKNC was proposed. Rigorously cross–validations have indicated that the proposed predictor is significantly better than the best existing method in sensitivity, specificity, overall accuracy, and stability. Furthermore, a user-friendly web-server for iROS-gPseKNC has been established at, by which users can easily get their desired results without the need to bother the complicated mathematics, which were presented just for the integrity of the methodology itself. PMID:27147572

  4. Effect of base stacking on the acid-base properties of the adenine cation radical [A*+] in solution: ESR and DFT studies. (United States)

    Adhikary, Amitava; Kumar, Anil; Khanduri, Deepti; Sevilla, Michael D


    In this study, the acid-base properties of the adenine cation radical are investigated by means of experiment and theory. Adenine cation radical (A*(+)) is produced by one-electron oxidation of dAdo and of the stacked DNA-oligomer (dA)6 by Cl2*(-) in aqueous glass (7.5 M LiCl in H2O and in D2O) and investigated by ESR spectroscopy. Theoretical calculations and deuterium substitution at C8-H and N6-H in dAdo aid in our assignments of structure. We find the pKa value of A*(+) in this system to be ca. 8 at 150 K in seeming contradiction to the accepted value of or = 160 K, complete deprotonation of A*(+) occurs in dAdo in these glassy systems even at pH ca. 3. A*(+) found in (dA)6 at 150 K also deprotonates on thermal annealing. The stability of A*(+) at 150 K in these systems is attributed to charge delocalization between stacked bases. Theoretical calculations at various levels (DFT B3LYP/6-31G*, MPWB95, and HF-MP2) predict binding energies for the adenine stacked dimer cation radical of 12 to 16 kcal/mol. Further DFT B3LYP/6-31G* calculations predict that, in aqueous solution, monomeric A*(+) should deprotonate spontaneously (a predicted pKa of ca. -0.3 for A*(+)). However, the charge resonance stabilized dimer AA*(+) is predicted to result in a significant barrier to deprotonation and a calculated pKa of ca. 7 for the AA*(+) dimer which is 7 pH units higher than the monomer. These theoretical and experimental results suggest that A*(+) isolated in solution and A*(+) in adenine stacks have highly differing acid-base properties resulting from the stabilization induced by hole delocalization within adenine stacks.

  5. Combining pseudo dinucleotide composition with the Z curve method to improve the accuracy of predicting DNA elements: a case study in recombination spots. (United States)

    Dong, Chuan; Yuan, Ya-Zhou; Zhang, Fa-Zhan; Hua, Hong-Li; Ye, Yuan-Nong; Labena, Abraham Alemayehu; Lin, Hao; Chen, Wei; Guo, Feng-Biao


    Pseudo dinucleotide composition (PseDNC) and Z curve showed excellent performance in the classification issues of nucleotide sequences in bioinformatics. Inspired by the principle of Z curve theory, we improved PseDNC to give the phase-specific PseDNC (psPseDNC). In this study, we used the prediction of recombination spots as a case to illustrate the capability of psPseDNC and also PseDNC fused with Z curve theory based on a novel machine learning method named large margin distribution machine (LDM). We verified that combining the two widely used approaches could generate better performance compared to only using PseDNC with a support vector machine based (SVM-based) model. The best Mathew's correlation coefficient (MCC) achieved by our LDM-based model was 0.7037 through the rigorous jackknife test and improved by ∼6.6%, ∼3.2%, and ∼2.4% compared with three previous studies. Similarly, the accuracy was improved by 3.2% compared with our previous iRSpot-PseDNC web server through an independent data test. These results demonstrate that the joint use of PseDNC and Z curve enhances performance and can extract more information from a biological sequence. To facilitate research in this area, we constructed a user-friendly web server for predicting hot/cold spots, HcsPredictor, which can be freely accessed from . In summary, we provided a united algorithm by integrating Z curve with PseDNC. We hope this united algorithm could be extended to other classification issues in DNA elements.

  6. The electrochemical reduction of the purines guanine and adenine at platinum electrodes in several room temperature ionic liquids

    Energy Technology Data Exchange (ETDEWEB)

    Zanoni, Maria Valnice Boldrin, E-mail: [Department of Analytical Chemistry, Institute of Chemistry, University of Sao Paulo State, Araraquara, R. Prof. Francisco Degni, CP 355, 14801-970, SP (Brazil); Rogers, Emma I. [Department of Chemistry, Physical and Theoretical Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QZ (United Kingdom); Hardacre, Christopher, E-mail: [School of Chemistry and Chemical Engineering/QUILL, Queen' s University Belfast, Belfast, Northern Ireland BT9 5AG (United Kingdom); Compton, Richard G., E-mail: [Department of Chemistry, Physical and Theoretical Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QZ (United Kingdom)


    The reduction of guanine was studied by microelectrode voltammetry in the room temperature ionic liquids (RTILs) N-hexyltriethylammonium bis (trifluoromethanesulfonyl) imide [N{sub 6,2,2,2}][N(Tf){sub 2}], 1-butyl-3-methylimidazolium hexafluorosphosphate [C{sub 4}mim][PF{sub 6}], N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide [C{sub 4}mpyrr][N(Tf){sub 2}], 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C{sub 4}mim][N(Tf){sub 2}], N-butyl-N-methyl-pyrrolidinium dicyanamide [C{sub 4}mpyrr][N(NC){sub 2}] and tris(P-hexyl)-tetradecylphosphonium trifluorotris(pentafluoroethyl)phosphate [P{sub 14,6,6,6}][FAP] on a platinum microelectrode. In [N{sub 6,2,2,2}][NTf{sub 2}] and [P{sub 14,6,6,6}][FAP], but not in the other ionic liquids studied, guanine reduction involves a one-electron, diffusion-controlled process at very negative potential to produce an unstable radical anion, which is thought to undergo a dimerization reaction, probably after proton abstraction from the cation of the ionic liquid. The rate of this subsequent reaction depends on the nature of the ionic liquid, and it is faster in the ionic liquid [P{sub 14,6,6,6}][FAP], in which the formation of the resulting dimer can be voltammetrically monitored at less negative potentials than required for the reduction of the parent molecule. Adenine showed similar behaviour to guanine but the pyrimidines thymine and cytosine did not; thymine was not reduced at potentials less negative than required for solvent (RTIL) decomposition while only a poorly defined wave was seen for cytosine. The possibility for proton abstraction from the cation in [N{sub 6,2,2,2}][NTf{sub 2}] and [P{sub 14,6,6,6}][FAP] is noted and this is thought to aid the electrochemical dimerization process. The resulting rapid reaction is thought to shift the reduction potentials for guanine and adenine to lower values than observed in RTILs where the scope for proton abstraction is not present. Such shifts are

  7. Mechanisms of Mitochondrial Defects in Gulf War Syndrome (United States)


    FADH2: flavin adenine dinucleotide, reduced; O2: oxygen; NADH: Nicotinamide Adenine Dinucleotide, reduced; NAD: Nicotinamide Adenine Dinucleotide...criteria, an appointment is made for clinical examination by the P.I., blood draw, and skin biopsy. Modified criteria for chronic fatigue syndrome and...muscle and we have now established reference ranges in uncultured skin cells, fibroblasts, and EBV transformed cell lines. Sample testing is

  8. DPT tautomerization of the long A∙A Watson-Crick base pair formed by the amino and imino tautomers of adenine: combined QM and QTAIM investigation. (United States)

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M


    Combining quantum-mechanical (QM) calculations with quantum theory of atoms in molecules (QTAIM) and using the methodology of sweeps of the energetic, electron-topological, geometric and polar parameters, which describe the course of the tautomerization along the intrinsic reaction coordinate (IRC), we showed for the first time that the biologically important A∙A base pair (Cs symmetry) formed by the amino and imino tautomers of adenine (A) tautomerizes via asynchronous concerted double proton transfer (DPT) through a transition state (TS), which is the A(+)∙A(-) zwitterion with the separated charge, with Cs symmetry. The nine key points, which can be considered as electron-topological "fingerprints" of the asynchronous concerted A∙A ↔A ∙A tautomerization process via the DPT, were detected and completely investigated along the IRC of the A∙A*↔A*∙A tautomerization. Based on the sweeps of the H-bond energies, it was found that intermolecular antiparallel N6Н⋯N6 (7.01 kcal mol(-1)) and N1H⋯N1 (6.88 kcal mol(-1)) H-bonds are significantly cooperative and mutually reinforce each other. It was shown for the first time that the A∙A ↔A ∙A tautomerization is assisted by the third C2H⋯HC2 dihydrogen bond (DHB), which, in contrast to the two others N6H⋯N6 and N1H⋯N1 H-bonds, exists within the IRC range from -2.92 to 2.92 Å. The DHB cooperatively strengthens, reaching its maximum energy 0.42 kcal mol(-1) at IRC = -0.52 Å and minimum energy 0.25 kcal mol(-1) at IRC = -2.92 Å, and is accompanied by strengthening of the two other aforementioned classical H-bonds. We established that the C2H⋯HC2 DHB completely satisfies the electron-topological criteria for H-bonding, in particular Bader's and all eight "two-molecule" Koch and Popelier's criteria. The positive value of the Grunenberg's compliance constant (5.203 Å/mdyn) at the TSA∙A ↔A ∙A proves that the C2H⋯HC2 DHB is a stabilizing interaction. NBO analysis predicts transfer

  9. Theoretical Investigation on the Substituent Effect of Halogen Atoms at the C8 Position of Adenine: Relative Stability, Vibrational Frequencies, and Raman Spectra of Tautomers. (United States)

    Chen, Yan-Li; Wu, De-Yin; Tian, Zhong-Qun


    We have theoretically investigated the substituent effect of adenine at the C8 position with a substituent X = H, F, Cl, and Br by using the density functional theory (DFT) at the B3LYP/6-311+G(d, p) level. The aim is to study the substituent effect of halogen atoms on the relative stability, vibrational frequencies, and solvation effect of tautomers. Our calculated results show that for substituted adenine molecules the N9H8X tautomer to be the most stable structure in gas phase at the present theoretical level. Here N9H8X denotes the hydrogen atom binds to the N9 position of imidazole ring and X denotes H, F, Cl, and Br atoms. The influence of the induced attraction of the fluorine substituent is significantly larger than chlorine and bromine ones. The halogen substituent effect has a significant influence on changes of vibrational frequencies and Raman intensities.

  10. Structure of dimeric, recombinant Sulfolobus solfataricus phosphoribosyl diphosphate synthase: a bent dimer defining the adenine specificity of the substrate ATP. (United States)

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


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

  11. Exercise effects on activities of Na(+),K(+)-ATPase, acetylcholinesterase and adenine nucleotides hydrolysis in ovariectomized rats. (United States)

    Ben, Juliana; Soares, Flávia Mahatma Schneider; Cechetti, Fernanda; Vuaden, Fernanda Cenci; Bonan, Carla Denise; Netto, Carlos Alexandre; Wyse, Angela Terezinha de Souza


    Hormone deficiency following ovariectomy causes activation of Na(+),K(+)-ATPase and acetylcholinesterase (AChE) that has been related to cognitive deficits in experimental animals. Considering that physical exercise presents neuroprotector effects, we decide to investigate whether exercise training would affect enzyme activation in hippocampus and cerebral cortex, as well as adenosine nucleotide hydrolysis in synaptosomes from cerebral cortex of ovariectomized rats. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries), exercise, ovariectomized (Ovx) and Ovx plus exercise. Thirty days after surgery, animals were submitted to one month of exercise training, three times per week. After, rats were euthanized, blood serum was collected and hippocampus and cerebral cortex were dissected. Data demonstrated that exercise reversed the activation of Na(+),K(+)-ATPase and AChE activities both in hippocampus and cerebral cortex of ovariectomized rats. Ovariectomy decreased AMP hydrolysis in cerebral cortex and did not alter adenine nucleotides hydrolysis in blood serum. Exercise per se decreased ADP and AMP hydrolysis in cerebral cortex. On the other hand, AMP hydrolysis in blood serum was increased by exercise in ovariectomized adult rats. Present data support that physical exercise might have beneficial effects and constitute a therapeutic alternative to hormone replacement therapy for estrogen deprivation.

  12. Yeast species utilizing uric acid, adenine, n-alkylamines or diamines as sole source of carbon and energy. (United States)

    Middelhoven, W J; De Kievit, H; Biesbroek, A L


    Yeast strains utilizing uric acid, adenine, monoamines or diamines as sole source of carbon and energy were isolated from several soil samples by the enrichment culture method. The most common species was Trichosporon cutaneum. Strains of Candida catenulata, C. famata, C. parapsilosis, C. rugosa, Cryptococcus laurentii, Stephanoascus ciferrii and Tr. adeninovorans were also isolated. All strains utilizing uric acid as sole carbon source utilized some primary n-alkyl-l-amines, hydroxyamines or diamines as well. The ascomycetous yeast strains showing these characteristics all belonged to species known to assimilate hydrocarbons. Type strains of hydrocarbon-positive yeast species which were not found in the enrichment cultures generally assimilated putrescine, some type strains also butylamine or pentylamine, but none assimilated uric acid. Methanol-positive species were not isolated. Type strains of methanol-positive and of hydrocarbon-negative species did not assimilate uric acid, butylamine or putrescine. Assimilation of putrescine as sole source of carbon and energy may be a valuable diagnostic criterion in yeast taxonomy.

  13. Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis. (United States)

    Šmídková, Markéta; Dvoráková, Alexandra; Tloust'ová, Eva; Česnek, Michal; Janeba, Zlatko; Mertlíková-Kaiserová, Helena


    Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

  14. Study of the chemical evolution and spectral signatures of some interstellar precursor molecules of adenine, glycine alanine

    CERN Document Server

    Majumdar, Liton; Chakrabarti, Sandip K; Chakrabarti, Sonali; 10.1016/j.newast.2012.09.002


    We carry out a quantum chemical calculation to obtain the infrared and electronic absorption spectra of several complex molecules of the interstellar medium (ISM). These molecules are the precursors of adenine, glycine & alanine. They could be produced in the gas phase as well as in the ice phase. We carried out a hydro-chemical simulation to predict the abundances of these species in the gas as well as in the ice phase. Gas and grains are assumed to be interacting through the accretion of various species from the gas phase on to the grain surface and desorption (thermal evaporation and photo-evaporation) from the grain surface to the gas phase. Depending on the physical properties of the cloud, the calculated abundances varies. The influence of ice on vibrational frequencies of different pre-biotic molecules was obtained using Polarizable Continuum Model (PCM) model with the integral equation formalism variant (IEFPCM) as default SCRF method with a dielectric constant of 78.5. Time dependent density func...

  15. Multi-pathway excited state relaxation of adenine oligomers in aqueous solution: a joint theoretical and experimental study. (United States)

    Banyasz, Akos; Gustavsson, Thomas; Onidas, Delphine; Changenet-Barret, Pascale; Markovitsi, Dimitra; Improta, Roberto


    The singlet excited states of adenine oligomers, model systems widely used for the understanding of the interaction of ultraviolet radiation with DNA, are investigated by fluorescence spectroscopy and time-dependent (TD) DFT calculations. Fluorescence decays, fluorescence anisotropy decays, and time-resolved fluorescence spectra are recorded from the femtosecond to the nanosecond timescales for single strand (dA)20 in aqueous solution. These experimental observations and, in particular, the comparison of the fluorescence behavior upon UVC and UVA excitation allow the identification of various types of electronic transitions with different energy and polarization. Calculations performed for up to five stacked 9-methyladenines, taking into account the solvent, show that different excited states are responsible for the absorption in the UVC and UVA spectral domains. Independently of the number of bases, bright excitons may evolve toward two types of excited dimers having π-π* or charge-transfer character, each one distinguished by its own geometry and spectroscopic signature. According to the picture arising from the joint experimental and theoretical investigation, UVC-induced fluorescence contains contribution from 1) exciton states with a different degree of localization, decaying within a few ps, 2) "neutral" excited dimers decaying on the sub-nanosecond timescale, being the dominant species, and 3) charge-transfer states decaying on the nanosecond timescale. The majority of the photons emitted upon UVA excitation are related to charge-transfer states.

  16. Continuing Exposure to Low-Dose Nonylphenol Aggravates Adenine-Induced Chronic Renal Dysfunction and Role of Rosuvastatin Therapy

    Directory of Open Access Journals (Sweden)

    Yen Chia-Hung


    Full Text Available Abstract Background Nonylphenol (NP, an environmental organic compound, has been demonstrated to enhance reactive-oxygen species (ROS synthesis. Chronic exposure to low-dose adenine (AD has been reported to induce chronic kidney disease (CKD. Methods In this study, we tested the hypothesis that chronic exposure to NP will aggravate AD-induced CKD through increasing generations of inflammation, ROS, and apoptosis that could be attenuated by rosuvastatin. Fifty male Wistar rats were equally divided into group 1 (control, group 2 (AD in fodder at a concentration of 0.25%, group 3 (NP: 2 mg/kg/day, group 4 (combined AD & NP, and group 5 (AD-NP + rosuvastatin: 20 mg/kg/day. Treatment was continued for 24 weeks for all animals before being sacrificed. Results By the end of 24 weeks, serum blood urea nitrogen (BUN and creatinine levels were increased in group 4 than in groups 1–3, but significantly reduced in group 5 as compared with group 4 (all p  Conclusion NP worsened AD-induced CKD that could be reversed by rosuvastatin therapy.

  17. The tumour metabolism inhibitors GSAO and PENAO react with cysteines 57 and 257 of mitochondrial adenine nucleotide translocase

    Directory of Open Access Journals (Sweden)

    Park Danielle


    Full Text Available Abstract Background GSAO (4-(N-(S-glutathionylacetylamino phenylarsonous acid and PENAO (4-(N-(S-penicillaminylacetylamino phenylarsonous acid are tumour metabolism inhibitors that target adenine nucleotide translocase (ANT of the inner-mitochondrial membrane. Both compounds are currently being trialled in patients with solid tumours. The trivalent arsenical moiety of GSAO and PENAO reacts with two matrix facing cysteine residues of ANT, inactivating the transporter. This leads to proliferation arrest and death of tumour and tumour-supporting cells. Results The two reactive ANT cysteine residues have been identified in this study by expressing cysteine mutants of human ANT1 in Saccharomyces cerevisiae and measuring interaction with the arsenical moiety of GSAO and PENAO. The arsenic atom of both compounds cross-links cysteine residues 57 and 257 of human ANT1. Conclusions The sulphur atoms of these two cysteines are 20 Å apart in the crystal structures of ANT and the optimal spacing of cysteine thiolates for reaction with As (III is 3-4 Å. This implies that a significant conformational change in ANT is required for the organoarsenicals to react with cysteines 57 and 257. This conformational change may relate to the selectivity of the compounds for proliferating cells.

  18. An extended version of Boyd's force field method applicable to heteroatomic molecules. Part 1. Adenine and uracil (United States)

    Espinosa-Müller, A. W.; Bravo, A. N.

    The force field method developed by Boyd is extended to include molecules containing atoms other than C and H (e.g., N, O, P, S, Cl, Br,…). A new set of force field parameters is determined in order to redefine the potential energy functions that govern the dynamics of the internal (valence coordinates) degrees of freedom of a molecule. It is shown that the minimum of the partial potential energy surface is significantly affected by electrostatic intramolecular interactions. In this regard the non-bonded interactions appears to be less important than the dipole-dipole type interactions for a given interatomic distance when heteroatoms are present in the molecular framework. The reliability of the extended method as regards minimized structure, vibrational spectra and thermodynamic properties has been checked for more than 20 polyatomic molecules. From the correlation between calculated and experimental properties it is concluded that the method has good potential for further applications on polyatomic molecules with increasing size and topological compexities such as adenine and uracil.

  19. UV-Induced Adenine Radicals Induced in DNA A-Tracts: Spectral and Dynamical Characterization. (United States)

    Banyasz, Akos; Ketola, Tiia-Maaria; Muñoz-Losa, Aurora; Rishi, Sunny; Adhikary, Amitava; Sevilla, Michael D; Martinez-Fernandez, Lara; Improta, Roberto; Markovitsi, Dimitra


    Adenyl radicals generated in DNA single and double strands, (dA)20 and (dA)20·(dT)20, by one- and two-photon ionization by 266 nm laser pulses decay at 600 nm with half-times of 1.0 ± 0.1 and 4 ± 1 ms, respectively. Though ionization initially forms the cation radical, the radicals detected for (dA)20 are quantitatively identified as N6-deprotonated adenyl radicals by their absorption spectrum, which is computed quantum mechanically employing TD-DFT. Theoretical calculations show that deprotonation of the cation radical induces only weak spectral changes, in line with the spectra of the adenyl radical cation and the deprotonated radical trapped in low temperature glasses.

  20. Can an excess electron localize on a purine moiety in the adenine-thymine Watson-Crick base pair? A computational study (United States)

    Mazurkiewicz, Kamil; Harańczyk, Maciej; Gutowski, Maciej; Rak, Janusz

    The electron affinity and the propensity to electron-induced proton transfer (PT) of hydrogen-bonded complexes between the Watson-Crick adenine-thymine pair (AT) and simple organic acid (HX), attached to adenine in the Hoogsteen-type configuration, were studied at the B3LYP/6-31+G** level. Although the carboxyl group is deprotonated at physiological pH, its neutral form, COOH, resembles the peptide bond or the amide fragment in the side chain of asparagine (Asn) or glutamine (Gln). Thus, these complexes mimic the interaction between the DNA environment (e.g., proteins) and nucleobase pairs incorporated in the biopolymer. Electron attachment is thermodynamically feasible and adiabatic electron affinities range from 0.41 to 1.28 eV, while the vertical detachment energies of the resulting anions span the range of 0.39-2.88 eV. Low-energy activation barriers separate the anionic minima: aHX(AT) from the more stable single-PT anionic geometry, aHX(AT)-SPT, and aHX(AT)-SPT from the double-PT anionic geometry, aHX(AT)-DPT. Interaction between the adenine of the Watson-Crick AT base pair with an acidic proton donor probably counterbalances the larger EA of isolated thymine, as SOMO is almost evenly delocalized over both types of nucleic bases in the aHX(AT) anions. Moreover, as a result of PT the excess electron localizes entirely on adenine. Thus, in DNA interacting with its physiological environment, damage induced by low-energy electrons could begin, contrary to the current view, with the formation of purine anions, which are not formed in isolated DNA because of the greater stability of anionic pyrimidines.0

  1. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model (United States)

    Li, Chunhua; Lv, Dashuai; Zhang, Lei; Yang, Feng; Wang, Cunxin; Su, Jiguo; Zhang, Yang


    Riboswitches are noncoding mRNA segments that can regulate the gene expression via altering their structures in response to specific metabolite binding. We proposed a coarse-grained Gaussian network model (GNM) to examine the unfolding and folding dynamics of adenosine deaminase (add) A-riboswitch upon the adenine dissociation, in which the RNA is modeled by a nucleotide chain with interaction networks formed by connecting adjoining atomic contacts. It was shown that the adenine binding is critical to the folding of the add A-riboswitch while the removal of the ligand can result in drastic increase of the thermodynamic fluctuations especially in the junction regions between helix domains. Under the assumption that the native contacts with the highest thermodynamic fluctuations break first, the iterative GNM simulations showed that the unfolding process of the adenine-free add A-riboswitch starts with the denature of the terminal helix stem, followed by the loops and junctions involving ligand binding pocket, and then the central helix domains. Despite the simplified coarse-grained modeling, the unfolding dynamics and pathways are shown in close agreement with the results from atomic-level MD simulations and the NMR and single-molecule force spectroscopy experiments. Overall, the study demonstrates a new avenue to investigate the binding and folding dynamics of add A-riboswitch molecule which can be readily extended for other RNA molecules.

  2. A high-resolution map of genes, microsatellite markers, and new dinucleotide repeats from UBE1 to the GATA locus in the region Xp11.23

    Energy Technology Data Exchange (ETDEWEB)

    Kwan, Sau-Ping; Hagemann, T.L. [Rush Medical School, Chicago, IL (United States); Rosen, F.S. [Harvard Medical School, Boston, MA (United States)] [and others


    Several new genes and markers have recently been identified on the proximal short arm of the human X chromosome in the area of Xp11.23. We had previously generated at YAC contig in this region extending from UBE1 to the OATL1 locus. In this report two polymorphic dinucleotide repeats, DXS6949 and DXS6950, were isolated and characterized from the OATL1 locus. A panel of YAC deletion derivatives from the distal portion of the contig was used in conjunction with the rest of the YAC map to position the new microsatellites and order other markers localizing to this interval. The marker order was determined to be DXS1367-ZNF81-DXS6849-ZNF21-DXS6616-DXS6950-DXS6949. In the proximal region below OATL1, we have isolated a pair of YACs from the GATA locus, B1026 and C01160. Mapping within these YACs indicates the orientation of DXS1126 and DXS1240, while a cosmid near the OATL1 region reveals the overlap between the YAC contigs from the two loci. This cosmid contains the gene responsible for Wiskott-Aldrich syndrome (WAS) and localizes the disease gene between OATL1 and GATA. These data enable the expansion of the present physical map of the X chromosome from UBE1 to the GATA locus, covering a large portion of the Xp11.23 region. Genetic crossovers in Xp11.23 support the marker orientation and the position of WAS, contrary to previous reports. With the integration of both physical and genetic maps we have predicted the following marker order: Xpter-UBE1-SYN1/ARAF1/TIMP1/DXS1367-ZNF81-DXS-6849-ZNF21-DXSy6616-(OATL1, DXS6950-DXS6949)-WAS-(GATA,DXS1126)-DXS12410-Xcen. This orientation identifies DXS6949 and DXS1126 as the nearest flanking polymorphic markers for WAS and provides useful anchor positions for the analysis of other disease genes that have been localized to this area including three different retinal defects and X-linked nephrolithiasis. 39 refs., 3 figs., 1 tab.

  3. A reagentless enzymatic amperometric biosensor using vertically aligned carbon nanofibers (VACNF)

    Energy Technology Data Exchange (ETDEWEB)

    Weeks, Martha L [University of Tennessee, Knoxville (UTK); Rahman, Touhidur [ORNL; Frymier, Paul Dexter [ORNL; Islam, Syed K [University of Tennessee, Knoxville (UTK); McKnight, Timothy E [ORNL


    A reagentless amperometric enzymatic biosensor is constructed on a carbon substrate for detection of ethanol. Yeast alcohol dehydrogenase (YADH), an oxidoreductase, and its cofactor nicotinamide adenine dinucleotide (NAD+) are immobilized by adsorption and covalent attachment to the carbon substrate. Carbon nanofibers grown by plasma enhanced chemical vapor deposition (PECVD) are chosen as the electrode material due to their excellent structural and electrical properties. Electrochemical techniques are employed to test the functionality and performance of the biosensor using reduced form of nicotinamide adenine dinucleotide (NADH) which also determines the oxidation peak potential of NADH. Subsequently, amperometric measurements are conducted for detection of ethanol to determine the electrical current response due to the increase in analyte concentration. The detection range, storage stability, reusability, and response time of the biosensor are also examined.

  4. Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells. (United States)

    Onetti, C G; Lara, J; García, E


    Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.

  5. Hypothesis on skeletal muscle aging : mitochondrial adenine nucleotide translocator decreases reactive oxygen species production while preserving coupling efficiency

    Directory of Open Access Journals (Sweden)

    Philippe eDIOLEZ


    Full Text Available Mitochondrial membrane potential is the major regulator of mitochondrial functions, including coupling efficiency and production of reactive oxygen species (ROS. Both functions are crucial for cell bioenergetics. We previously presented evidences for a specific modulation of adenine nucleotide translocase (ANT appearing during aging that results in a decrease in membrane potential - and therefore ROS production – but surprisingly increases coupling efficiency under conditions of low ATP turnover. Careful study of the bioenergetic parameters (oxidation and phosphorylation rates, membrane potential of isolated mitochondria from skeletal muscles (gastrocnemius of aged and young rats revealed a remodeling at the level of the phosphorylation system, in the absence of alteration of the inner mitochondrial membrane (uncoupling or respiratory chain complexes regulation. We further observed a decrease in mitochondrial affinity for ADP in aged isolated mitochondria, and higher sensitivity of ANT to its specific inhibitor atractyloside. This age-induced modification of ANT results in an increase in the ADP concentration required to sustain the same ATP turnover as compared to young muscle, and therefore in a lower membrane potential under phosphorylating - in vivo - conditions. Thus, for equivalent ATP turnover (cellular ATP demand, coupling efficiency is even higher in aged muscle mitochondria, due to the down-regulation of inner membrane proton leak caused by the decrease in membrane potential. In the framework of the radical theory of aging, these modifications in ANT function may be the result of oxidative damage caused by intra mitochondrial ROS and may appear like a virtuous circle where ROS induce a mechanism that reduces their production, without causing uncoupling, and even leading in improved efficiency. Because of the importance of ROS as therapeutic targets, this new mechanism deserves further studies.

  6. A QM/QTAIM microstructural analysis of the tautomerisationviathe DPT of the hypoxanthine·adenine nucleobase pair (United States)

    Brovarets', Ol'ha O.; Zhurakivsky, Roman O.; Hovorun, Dmytro M.


    We provide a pathway for the tautomerisation of the biologically important hypoxanthine.adenine (Hyp.Ade) nucleobase pair (Cs) formed by the keto tautomer of the Hyp and the amino tautomer of the Ade into the Hyp*.Ade* base pair (Cs) formed by the enol tautomer of the Hyp and the imino tautomer of the Ade by applying quantum-mechanical calculations and Bader's Quantum Theory of Atoms in Molecules analysis. It was found out that the dipole active Hyp.Ade↔Hyp*.Ade* tautomerisation occurs via the asynchronous concerted double proton transfer (DPT) through the TSHyp.Ade↔Hyp*.Ade* (Cs). Based on the sweeps of the energies of the intermolecular H-bonds along the intrinsic reaction coordinate, it was established that the N6H...O6 H-bond (5.40) is cooperative with the N1H...N1 H-bond (6.99) in the Hyp.Ade base pair, as well as the O6H...N6 H-bond (11.50) is cooperative with the N1H...N1 H-bond (7.28 kcal.mol-1) in the Hyp*.Ade* base pair, mutually strengthening each other. The Hyp*.Ade* base pair possesses an extremely short lifetime 2.68.10-14 s, which is predetermined by the negative value of the Gibbs free energy of the reverse barrier of its tautomerisation, and all of the six low-frequency intermolecular vibrations cannot develop during this period of time. Consequently, the Hyp.Ade→Hyp*.Ade* DPT tautomerisation cannot serve as a source of the rare tautomers of the bases.

  7. Hydration properties of natural and synthetic DNA sequences with methylated adenine or cytosine bases in the R.DpnI target and BDNF promoter studied by molecular dynamics simulations (United States)

    Shanak, Siba; Helms, Volkhard


    Adenine and cytosine methylation are two important epigenetic modifications of DNA sequences at the levels of the genome and transcriptome. To characterize the differential roles of methylating adenine or cytosine with respect to their hydration properties, we performed conventional MD simulations and free energy perturbation calculations for two particular DNA sequences, namely the brain-derived neurotrophic factor (BDNF) promoter and the R.DpnI-bound DNA that are known to undergo methylation of C5-methyl cytosine and N6-methyl adenine, respectively. We found that a single methylated cytosine has a clearly favorable hydration free energy over cytosine since the attached methyl group has a slightly polar character. In contrast, capping the strongly polar N6 of adenine with a methyl group gives a slightly unfavorable contribution to its free energy of solvation. Performing the same demethylation in the context of a DNA double-strand gave quite similar results for the more solvent-accessible cytosine but much more unfavorable results for the rather buried adenine. Interestingly, the same demethylation reactions are far more unfavorable when performed in the context of the opposite (BDNF or R.DpnI target) sequence. This suggests a natural preference for methylation in a specific sequence context. In addition, free energy calculations for demethylating adenine or cytosine in the context of B-DNA vs. Z-DNA suggest that the conformational B-Z transition of DNA transition is rather a property of cytosine methylated sequences but is not preferable for the adenine-methylated sequences investigated here.

  8. New Dihydro OO'Bis(Salicylidene 2,2' Aminobenzothiazolyl Borate Complexes: Kinetic and Voltammetric Studies of Dimethyltin Copper Complex with Guanine, Adenine, and Calf Thymus DNA

    Directory of Open Access Journals (Sweden)


    Full Text Available The newly synthesized ligand, dihydro OO'bis(salicylidene 2,2' aminobenzothiazolyl borate (2, was derived from the reaction of Schiff base of 2-aminobenzothiazole and salicylaldehyde with KBH 4 . Cu II (3 and Zn II (4 complexes of (2 were synthesized and further metallated with dimethyltindichloride to yield heterobimetallic complexes (5 and (6. All complexes have been thoroughly characterized by elemental analysis, and IR, NMR, EPR, and UV-Vis spectroscopy and conductance measurements. The spectroscopic data support square planar environment around the Cu II atom, while the Sn IV atom acquires pentacoordinate geometry. The interaction of complex (5 with guanine, adenine, and calf thymus DNA was studied by spectrophotometric, electrochemical, and kinetic methods. The absorption spectra of complex (5 exhibit a remarkable "hyperchromic effect" in the presence of guanine and calf thymus DNA. Indicative of strong binding of the complex to calf thymus DNA preferentially binds through N 7 position of guanine base, while the adenine shows binding to a lesser extent. The kinetic data were obtained from the rate constants, k obs , values under pseudo-first-order conditions. Cyclic voltammetry was employed to study the interaction of complex (5 with guanine, adenine, and calf thymus DNA. The CV of complex (5 in the absence and in the presence of guanine and calf thymus DNA altered drastically, with a positive shift in formal peak potential E pa and E pc values and a significant increase in peak current. The positive shift in formal potentials with increase in peak current favours strong interaction of complex (5 with calf thymus DNA. The net shift in E 1/2 has been used to estimate the ratio of equilibrium constants for the binding of Cu(II and Cu(I complexes to calf thymus DNA.

  9. New Dihydro OO'Bis(Salicylidene) 2,2' Aminobenzothiazolyl Borate Complexes: Kinetic and Voltammetric Studies of Dimethyltin Copper Complex with Guanine, Adenine, and Calf Thymus DNA. (United States)

    Arjmand, Farukh; Mohani, Bhawana; Parveen, Shamima


    The newly synthesized ligand, dihydro OO'bis(salicylidene) 2,2' aminobenzothiazolyl borate (2), was derived from the reaction of Schiff base of 2-aminobenzothiazole and salicylaldehyde with KBH(4). Cu(II) (3) and Zn(II) (4) complexes of (2) were synthesized and further metallated with dimethyltindichloride to yield heterobimetallic complexes (5) and (6). All complexes have been thoroughly characterized by elemental analysis, and IR, NMR, EPR, and UV-Vis spectroscopy and conductance measurements. The spectroscopic data support square planar environment around the Cu(II) atom, while the Sn(IV) atom acquires pentacoordinate geometry. The interaction of complex (5) with guanine, adenine, and calf thymus DNA was studied by spectrophotometric, electrochemical, and kinetic methods. The absorption spectra of complex (5) exhibit a remarkable "hyperchromic effect" in the presence of guanine and calf thymus DNA. Indicative of strong binding of the complex to calf thymus DNA preferentially binds through N(7) position of guanine base, while the adenine shows binding to a lesser extent. The kinetic data were obtained from the rate constants, k(obs), values under pseudo-first-order conditions. Cyclic voltammetry was employed to study the interaction of complex (5) with guanine, adenine, and calf thymus DNA. The CV of complex (5) in the absence and in the presence of guanine and calf thymus DNA altered drastically, with a positive shift in formal peak potential E(pa) and E(pc) values and a significant increase in peak current. The positive shift in formal potentials with increase in peak current favours strong interaction of complex (5) with calf thymus DNA. The net shift in E(1/2) has been used to estimate the ratio of equilibrium constants for the binding of Cu(II) and Cu(I) complexes to calf thymus DNA.

  10. XRD and optical microscopic studies of Co(III) complexes containing 5-cyano-6-(4-pyridyl)-2-thiouracil, thymine and adenine bases

    Indian Academy of Sciences (India)

    Lallan Mishra; Brajesh Pathak; R K Mandal


    Multifunctional ligand 5-cyano-6-(-4-pyridyl)-2-thiouracil (L) was prepared and allowed to react with trans [Co(en)2Cl2]+Cl– resulting into [Co(en)2LCl]2+.2Cl– which upon further reaction with equimolar ratio of ligand [L] gave the complex [Co(en)2L2]3+.3Cl–. These metal complexes were then separately reacted with thymine and adenine bases. Complexes thus prepared after characterization by their elemental analysis, FAB mass and spectral (IR, 1HNMR, UV-visible) data were studied for their powder X-ray diffraction and optical microscopic characteristics.

  11. Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers. (United States)

    Holland, Joseph G; Malin, Jessica N; Jordan, David S; Morales, Esmeralda; Geiger, Franz M


    This article reports nonlinear optical measurements that quantify, for the first time directly and without labels, how many Mg(2+) cations are bound to DNA 21-mers covalently linked to fused silica/water interfaces maintained at pH 7 and 10 mM NaCl, and what the thermodynamics are of these interactions. The overall interaction of Mg(2+) with adenine, thymine, guanine, and cytosine is found to involve -10.0 ± 0.3, -11.2 ± 0.3, -14.0 ± 0.4, and -14.9 ± 0.4 kJ/mol, and nonspecific interactions with the phosphate and sugar backbone are found to contribute -21.0 ± 0.6 kJ/mol for each Mg(2+) ion bound. The specific and nonspecific contributions to the interaction energy of Mg(2+) with oligonucleotide single strands is found to be additive, which suggests that within the uncertainty of these surface-specific experiments, the Mg(2+) ions are evenly distributed over the oligomers and not isolated to the most strongly binding nucleobase. The nucleobases adenine and thymine are found to bind only three Mg(2+) ions per 21-mer oligonucleotide, while the bases cytosine and guanine are found to bind eleven Mg(2+) ions per 21-mer oligonucleotide.

  12. Specificities and pH profiles of adenine and hypoxanthine-guanine-xanthine phosphoribosyltransferases (nucleotide synthases) of the thermoacidophile archaeon Sulfolobus solfataricus

    DEFF Research Database (Denmark)

    Hansen, Michael Riis; Jensen, Kristine Steen; Rasmussen, Mads Skytte;


    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransfe......Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine...... phosphoribosyltransferase (PRTase), the protein product turned out to be a PRTase highly specific for adenine and we suggest that the reading frame should be renamed apT. The other reading frame SSO2424 (gpT-2) proved to be a true 6-oxopurine PRTase active with hypoxanthine, xanthine and guanine as substrates, and we...... suggest that the gene should be renamed gpT. Both enzymes exhibited unusual profiles of activity versus pH. The adenine PRTase showed the highest activity at pH 7.5-8.5, but had a distinct peak of activity also at pH 4.5. The 6-oxo PRTase showed maximal activity with hypoxanthine and guanine around pH 4...

  13. Acidity and complex formation studies of 3-(adenine-9-yl)-propionic and 3-(thymine-1-yl)-propionic acids in ethanol-water media (United States)

    Hammud, Hassan H.; El Shazly, Shawky; Sonji, Ghassan; Sonji, Nada; Bouhadir, Kamal H.


    The ligands 3-(adenine-9-yl)propionic acid (AA) and 3-(thymine-1-yl)propionic acid (TA) were prepared by N9-alkylation of adenine and N1-alkylation of thymine with ethylacrylate in presence of a base catalyst, followed by acid hydrolysis of the formed ethyl esters to give the corresponding propionic acid derivatives. The products were characterized by spectral methods (FTIR, 1H NMR and 13C NMR), which confirm their structures. The dissociation constants of ligands, were potentiometrically determined in 0.3 M KCl at 20-50 °C temperature range. The work was extended to study complexation behavior of AA and TA with various biologically important divalent metal ions (Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Mn2+ and Pb2+) in 50% v/v water-ethanol medium at four different temperatures, keeping ionic strength constant (0.3 M KCl). The order of the stability constants of the formed complexes decreases in the sequence Cu2+ > Pb2+ > Zn2+ > Ni2+ > Co2+ > Mn2+ > Cd2+ for both ligands. The effect of temperature was also studied and the corresponding thermodynamic functions (ΔG, ΔH, ΔS) were derived and discussed. The formation of metal complexes has been found to be spontaneous, and the stability constants were dependant markedly on the basicity of the ligands.

  14. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen


    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2, was subjected to crystallographic, kinetic and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5......-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product AMP or the inhibitor ADP in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its...... undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor, ADP binds like the product AMP with both the α- and β-phosphates occupying the 5’-phosphoribosyl binding site. The enzyme shows activity over a wide p...

  15. Drug: D07633 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D07633 Mixture, Drug Chondroitin sulfate sodium - flavin adenine dinucleotide sodium mixt; sulfate sodium - FAD sodium mixt; Mucofadin (TN); Mucotear (TN) Chondroitin sulfate sodi...rgans 13 Agents affecting sensory organs 131 Ophthalmic agents 1319 Others D07633 Chondroitin sulfate sodium - flavin adenine dinucleotide sodium mixt PubChem: 96024455 ...

  16. Liquid Chromatography-Mass Spectrometry Interface for Detection of Extraterrestrial Organics (United States)

    Southard, Adrian E.; Getty, Stephanie A.; Balvin, Manuel; Cook, Jamie E.; Espiritu, Ana Mellina; Kotecki, Carl; Towner, Deborah W.; Dworkin, J. P.; Glavin, Daniel P.; Mahaffy, Paul R.; Ferrance, J.


    The OASIS (Organics Analyzer for Sampling Icy surfaces) microchip enables electrospray or thermospray of analyte for subsequent analysis by the OASIS time-of-flight mass spectrometer. Electrospray of buffer solution containing the nucleobase adenine was performed using the microchip and detected by a commercial time-of-flight mass spectrometer. Future testing of thermospray and electrospray capability will be performed using a test fixture and vacuum chamber developed especially for optimization of ion spray at atmosphere and in low pressure environments.

  17. Model-based analysis of clinical fluorescence spectroscopy for in vivo detection of cervical intraepithelial dysplasia (United States)

    Chang, Sung K.; Marín, Nena; Follen, Michelle; Richards-Kortum, Rebecca R.


    We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.

  18. A preliminary study on adenine-limited control during the ribavirin fermentation%利巴韦林发酵过程中腺嘌呤限量供应的初步研究

    Institute of Scientific and Technical Information of China (English)

    刘莉; 王法松; 李燕军; 谢希贤; 陈宁


    Bacillus amyloliquefaciens TA208-LM is a guanosine producer with adenine auxotrophy.The adenine concentration in the fermentation broth is a critical factor because too little adenine will affect the growth of bacteria,while too much will restrain the synthesis of keyenzymes.Firstly the type of adenine donor and its appropriate concentration were optimized.The results indicated that Springer 2506 was the best adenine donor,and 15 g/L was optimal for ribavirin accumulation.However,the concentration of adenine released from this dose of Springer 2506 was too high at the end of fermentation.Therefore,the initial quantity of Springer 2506 was reduced and extra adenine was added.The results indicated that the activity of PRPP amidotransferase and the titer of ribavirin were both highest when the optimal initial adenine concentration was 47.95 mg/L and accordingly the adenine in the fermentation liquid was maintained at 40 ~ 50 mg/L.This data indicated that controlling the initial addition of yeast extract and further feeding appropriate quantity of yeast extract was likely a feasible way to control adenine concentration in the fermentation.Springer 2506 fed-batch fermentation in 7.5 L fermentation tank was studied.When 7.5 g/L Springer 2506 was used as substrate and another 7.5 g/L Springer 2506 was fed into the fermentation broth,the ribavirin production increased by 11.29% compared to one-time addition of 15 g/L Springer 2506.The study on limited control of adenine would provide some useful guidance for industrial production of ribavirin.%解淀粉芽孢杆菌(Bacillus amyloliquefaciens)TA208-LM是腺嘌呤缺陷型突变株,腺嘌呤浓度过低会影响菌体生长,过高又会反馈阻遏关键酶的合成.首先对腺嘌呤供体的种类和用量进行了研究,摇瓶发酵结果表明Springer 2506是最佳的腺嘌呤供体,用量在15 g/L时最有利于利巴韦林积累,但发酵后期腺嘌呤浓度过高.因此在降低Springer 2506的初始浓度的基

  19. Detection of non-ΔGT NCF-1 mutations in chronic granulomatous disease

    DEFF Research Database (Denmark)

    Jakobsen, Marianne Antonius; Pedersen, Svend Stenvang; Barington, Torben


    to the functional NCF-1 has complicated the identification of other mutations in the gene. The aim of this study was to find a general technique for detection of non-GT deletion mutations in the coding region of NCF-1. RESULTS: A technique involving GeneScan analysis followed by amplification of cDNA with intact...... dinucleotide repeat was set up and used to identify a novel mutation in exon 7 of NCF-1 in a patient with autosomal recessive CGD, explaining the disease by changing a UGG codon to a premature UGA STOP codon. CONCLUSION: The method is generally applicable for the detection of NCF-1 mutations in patients...

  20. Investigation of Rho Signaling Pathways in 3-D Collagen Matrices with Multidimensional Microscopy and Visualization Techniques (United States)


    Rueden (lead software developer at LOCI) using Image J, VisBio, and SlimPlotter. The goal is to create an efficient and accurate method to translate...MPLSM imaging of en- dogenous signals from collagen and fluorophores such as nicotinamide adenine dinucleotide NADH or flavin ad- enine ...phores imaged with MPLSM are tryptophan, nicotinamide ad- enine dinucleotide NADH and flavin adenine dinucleotide FAD, as well as endogenous SHG

  1. Selective inhibitory effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine and 2'-nor-cyclic GMP on adenovirus replication in vitro. (United States)

    Baba, M; Mori, S; Shigeta, S; De Clercq, E


    The inhibitory effects of 20 selected antiviral compounds on the replication of adenoviruses (types 1 to 8) in vitro were investigated. While 18 compounds were ineffective, 2 compounds, namely (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA] and 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide (2'-nor-cyclic GMP), were highly effective against all adenovirus types assayed in human embryonic fibroblast cultures. Their 50% inhibitory doses were 1.1 microgram/ml for (S)-HPMPA and 4.1 micrograms/ml for 2'-nor-cyclic GMP. They were nontoxic for the host cells at the effective antiviral doses.

  2. Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells. (United States)

    Park, Hyung Seo; Betzenhauser, Matthew J; Zhang, Yu; Yule, David I


    Secretagogue-stimulated intracellular Ca(2+) signals are fundamentally important for initiating the secretion of the fluid and ion component of saliva from parotid acinar cells. The Ca(2+) signals have characteristic spatial and temporal characteristics, which are defined by the specific properties of Ca(2+) release mediated by inositol 1,4,5-trisphosphate receptors (InsP(3)R). In this study we have investigated the role of adenine nucleotides in modulating Ca(2+) release in mouse parotid acinar cells. In permeabilized cells, the Ca(2+) release rate induced by submaximal [InsP(3)] was increased by 5 mM ATP. Enhanced Ca(2+) release was not observed at saturating [InsP(3)]. The EC(50) for the augmented Ca(2+) release was ∼8 μM ATP. The effect was mimicked by nonhydrolysable ATP analogs. ADP and AMP also potentiated Ca(2+) release but were less potent than ATP. In acini isolated from InsP(3)R-2-null transgenic animals, the rate of Ca(2+) release was decreased under all conditions but now enhanced by ATP at all [InsP(3)]. In addition the EC(50) for ATP potentiation increased to ∼500 μM. These characteristics are consistent with the properties of the InsP(3)R-2 dominating the overall features of InsP(3)R-induced Ca(2+) release despite the expression of all isoforms. Finally, Ca(2+) signals were measured in intact parotid lobules by multiphoton microscopy. Consistent with the release data, carbachol-stimulated Ca(2+) signals were reduced in lobules exposed to experimental hypoxia compared with control lobules only at submaximal concentrations. Adenine nucleotide modulation of InsP(3)R in parotid acinar cells likely contributes to the properties of Ca(2+) signals in physiological and pathological conditions.

  3. Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis (United States)

    Seib, Kate L.; Jen, Freda E.-C.; Tan, Aimee; Scott, Adeana L.; Kumar, Ritesh; Power, Peter M.; Chen, Li-Tzu; Wu, Hsing-Ju; Wang, Andrew H.-J.; Hill, Dorothea M. C.; Luyten, Yvette A.; Morgan, Richard D.; Roberts, Richard J.; Maiden, Martin C. J.; Boitano, Matthew; Clark, Tyson A.; Korlach, Jonas; Rao, Desirazu N.; Jennings, Michael P.


    Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N6-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N6-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGYm6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-ACm6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CCm6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGYm6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGCm6AGG-3′ sites, to 100% at 5′-ACGTm6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching. PMID:25845594

  4. Data supporting the involvement of the adenine nucleotide translocase conformation in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria

    Directory of Open Access Journals (Sweden)

    Sergey M. Korotkov


    Full Text Available There we made available information about the effects of the adenine nucleotide translocase (ANT ‘c’ conformation fixers (phenylarsine oxide (PAO, tert-butylhydroperoxide (tBHP, and carboxyatractyloside as well as thiol reagent (4,4′-diisothiocyanostilbene-2,2′-disulfonate (DIDS on isolated rat liver mitochondria. We observed a decrease in A540 (mitochondrial swelling and respiratory control rates (RCRADP [state 3/state 4] and RCRDNP [2,4-dinitrophenol-uncoupled state/basal state or state 4], as well as an increase in Ca2+-induced safranin fluorescence (F485/590, arbitrary units, showed a dissipation in the inner membrane potential (ΔΨmito, in experiments with energized rat liver mitochondria, injected into the buffer containing 25–75 mM TlNO3, 125 mM KNO3, and 100 µM Ca2+. The fixers and DIDS, in comparison to Ca2+ alone, greatly increased A540 decline and the rate of Ca2+-induced ΔΨmito dissipation. These reagents also markedly decreased RCRADP and RCRDNP. The MPTP inhibitors (ADP, cyclosporin A, bongkrekic acid, and N-ethylmaleimide fixing the ANT in ‘m’ conformation significantly hindered the above-mentioned effects of the fixers and DIDS. A more complete scientific analysis of these findings may be obtained from the manuscript “To involvement the conformation of the adenine nucleotide translocase in opening the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria” (Korotkov et al., 2016 [1].

  5. An important role for adenine, cholera toxin, hydrocortisone and triiodothyronine in the proliferation, self-renewal and differentiation of limbal stem cells in vitro. (United States)

    Yu, Min; Bojic, Sanja; Figueiredo, Gustavo S; Rooney, Paul; de Havilland, Julian; Dickinson, Anne; Figueiredo, Francisco C; Lako, Majlinda


    The cornea is a self-renewing tissue located at the front of the eye. Its transparency is essential for allowing light to focus onto the retina for visual perception. The continuous renewal of corneal epithelium is supported by limbal stem cells (LSCs) which are located in the border region between conjunctiva and cornea known as the limbus. Ex vivo expansion of LSCs has been successfully applied in the last two decades to treat patients with limbal stem cell deficiency (LSCD). Various methods have been used for their expansion, yet the most widely used culture media contains a number of ingredients derived from animal sources which may compromise the safety profile of human LSC transplantation. In this study we sought to understand the role of these components namely adenine, cholera toxin, hydrocortisone and triiodothyronine with the aim of re-defining a safe and GMP compatible minimal media for the ex vivo expansion of LSCs on human amniotic membrane. Our data suggest that all four components play a critical role in maintaining LSC proliferation and promoting LSC self-renewal. However removal of adenine and triiodothyronine had a more profound impact and led to LSC differentiation and loss of viability respectively, suggesting their essential role for ex vivo expansion of LSCs. Replacement of each of the components with GMP-grade reagents resulted in equal growth to non-GMP grade media, however an enhanced differentiation of LSCs was observed, suggesting that additional combinations of GMP grade reagents need to be tested to achieve similar or better level of LSC maintenance in the same manner as the traditional LSC media.

  6. 二核苷酸STR基因座D6S261的多态性检测%Genetic Polymorphisms of the Dinucleotide STR Locus D6S261

    Institute of Scientific and Technical Information of China (English)

    杨鹏; 刘亚楠; 聂燕钗; 周怀谷; 赵子琴


    Objective To investigate the application of dinucleotide STR locus in paternity testing. Methods Dinucleotide STR locus D6S261 was selected and the paternity testing blood samples were amplified using 200 random blood samples, 16 family samples and 193 paternity test samples. Data of the PCR products were collected by 3130XL Genetic Analyzer and the genetic parameters of population were calculated by PowerStats v12. Results Fifteen alleles and 50 genotypes were found and H, DP, PE and PIC were 0.850, 0.953, 0.695, and 0.820, respectively. The typing results of both family samples and paternity test samples were accord with the law of inheritance, which no mutation was discovered. Conclusion The genetic polymorphisms of D6S261 show good characteristics with low mutation rate and high stability. It can be an effective method to solve the indetermination caused by mutation in paternity testing if the stutter bands can be decreased.%目的 研究二核苷酸STR基因座在亲子鉴定中的应用.方法 选取二核苷酸STR基因座D6S261,采取200份随机血样,家系样本16份,亲子鉴定193份血样进行扩增,采用3130XL遗传分析仪收集数据,PowerStats v12计算群体遗传学参数.结果 获取15种等位基因及50种基因型,H为0.850、DP为0.953、PE为0.695、PIC为0.820.家系样本和亲子鉴定样本分型结果均符合遗传定律,未发现突变.结论 D6S261具有良好的遗传多态性,具有突变率低、稳定性好的特点,若进一步降低影子带(stutter bands)干扰,可作为解决亲权鉴定中基因突变难题的有效手段.

  7. Gender and chronological age affect erythrocyte membrane oxidative indices in citrate phosphate dextrose adenine-formula 1 (CPDA-1) blood bank storage condition. (United States)

    Erman, Hayriye; Aksu, Uğur; Belce, Ahmet; Atukeren, Pınar; Uzun, Duygu; Cebe, Tamer; Kansu, Ahmet D; Gelişgen, Remisa; Uslu, Ezel; Aydın, Seval; Çakatay, Ufuk


    It is well known that in vitro storage lesions lead to membrane dysfunction and decreased number of functional erythrocytes. As erythrocytes get older, in storage media as well as in peripheral circulation, they undergo a variety of biochemical changes. In our study, the erythrocytes with different age groups in citrate phosphate dextrose adenine-formula 1 (CPDA-1) storage solution were used in order to investigate the possible effect of gender factor on oxidative damage. Oxidative damage biomarkers in erythrocyte membranes such as ferric reducing antioxidant power, pro-oxidant-antioxidant balance, protein-bound advance glycation end products, and sialic acid were analyzed. Current study reveals that change in membrane redox status during blood-bank storage condition also depends on both gender depended homeostatic factors and the presence of CPDA-1. During the storage period in CPDA-1, erythrocytes from the male donors are mostly affected by free radical-mediated oxidative stress but erythrocytes obtained from females are severely affected by glyoxidative stress.

  8. Identification of a compound heterozygote for adenine phosphoribosyltransferase deficiency (APRT*J/APART*Q0) leading to 2,8-dihydroxyadenine urolithiasis. (United States)

    Kamatani, N; Kuroshima, S; Yamanaka, H; Nakashe, S; Take, H; Hakoda, M


    Homozygous deficiency of a purine salvage enzyme, adenine phosphoribosyltransferase (APRT), causes urolithiasis and renal failure. There are two known types of homozygous APRT deficiencies; type I patients completely lack APRT activity while type II patients only partially lack such activity. All type II patients possess at least one APRT*J allele with a substitution from ATG (Met) to ACG (Thr) at codon 136. Type I patients are considered to possess two alleles (APRT*Q0) both of which code for complete deficiencies. Thus, some patients with type II APRT deficiencies may have a genotype of APRT*J/APRT*Q0. As no individuals with such a genotype have previously been identified, we performed extensive analysis on four members of a family by (1) the T-cell method for the identification of a homozygote, (2) the B-cell method for the identification of heterozygotes, and (3) oligonucleotide hybridization after in vitro amplification of a part of genomic APRT sequence for the identification of APRT*J and non-APRT*J alleles. We report here the first evidence that 2,8-dihydroxyadenine urolithiasis developed in a boy aged 2 years with a genotype of APRT*J/APRT*Q0.

  9. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis. (United States)

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, Tarsis F; Pávek, Petr; Trejtnar, František; Watts, Val J; Janeba, Zlatko


    Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

  10. Nitrogen Substituted Polycyclic Aromatic Hydrocarbon As Capable Interstellar Infrared Spectrum Source Considering Astronomical Chemical Evolution Step To Biological Organic Purine And Adenine

    CERN Document Server

    Ota, Norio


    In order to find out capable chemical evolution step from astronomically created organic in interstellar space to biological organic on the earth, infrared spectrum of nitrogen substituted carbon pentagon-hexagon coupled polycyclic aromatic hydrocarbon was analyzed by the density functional theory. Ionization was modeled from neutral to tri-cation. Among one nitrogen and two nitrogen substituted NPAH, we could find good examples showing similar IR behavior with astronomically well observed one as like C8H6N1, C7H5N2, and C7H5N2. We can imagine that such ionized NPAH may be created in interstellar space by attacks of high energy nitrogen and photon. Whereas, in case of three and four nitrogen substituted cases as like C6H4N3 and C5H3N4, there were no candidate showing similar behavior with observed one. Also, IR of typical biological organic with four and five nitrogen substituted one as like purine and adenine resulted no good similarity with observed one. By such theoretical comparison, one capable story of ...

  11. Quantum-classical effective-modes dynamics of the pipi* --> npi* decay in 9H-adenine. A quadratic vibronic coupling model. (United States)

    Picconi, David; Ferrer, Francisco José Avila; Improta, Roberto; Lami, Alessandro; Santoro, Fabrizio


    We present mixed quantum-classical simulation of the internal conversion between the lowest energy pipi* (S(La)) and npi* (S(n)) excited electronic states in adenine in the gas phase, adopting a quadratic vibronic model (QVC), parametrized with the help of PBE0 density functional calculations. Our approach is based on a hierarchical representation of the QVC Hamiltonian and a subsequent treatment of the most relevant coordinates at accurate time-dependent quantum level and of the other 'bath' modes at classical level. We predict an ultrafast transfer (-30 fs) of approximately 75% of the initial population excited on S(La) to S(n). Within an adiabatic picture, on the same timescale the wave packet concentrates almost completely on the lowest S1 state, where however it shows a very broad distribution with different characteristics (due to the different 'diabatic' character). It is shown that the proposed methodology offers a practicable route to describe the quantum dynamics of internal conversion processes in large semi-rigid systems.

  12. {sup 19}F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Sochor, F. [Johann Wolfgang Goethe-University Frankfurt, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany); Silvers, R. [Massachusetts Institute of Technology, Department of Chemistry, Francis Bitter Magnet Laboratory (United States); Müller, D.; Richter, C.; Fürtig, B., E-mail:; Schwalbe, H., E-mail: [Johann Wolfgang Goethe-University Frankfurt, Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ) (Germany)


    In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus {sup 19}F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5′-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the {sup 19}F isotope. The thermal stability of the {sup 19}F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a {sup 1}H,{sup 15}N-HSQC allow the identification of Watson–Crick base paired uridine signals and the {sup 19}F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of {sup 19}F-labeling even for sizeable RNAs in the range of 70 nucleotides.

  13. Structure-guided mutational analysis of gene regulation by the Bacillus subtilis pbuE adenine-responsive riboswitch in a cellular context. (United States)

    Marcano-Velázquez, Joan G; Batey, Robert T


    Riboswitches are a broadly distributed form of RNA-based gene regulation in Bacteria and, more rarely, Archaea and Eukarya. Most often found in the 5'-leader sequence of bacterial mRNAs, they are generally composed of two functional domains: a receptor (aptamer) domain that binds an effector molecule and a regulatory domain (or expression platform) that instructs the expression machinery. One of the most studied riboswitches is the Bacillus subtilis adenine-responsive pbuE riboswitch, which regulates gene expression at the transcriptional level, up-regulating expression in response to increased intracellular effector concentrations. In this work, we analyzed sequence and structural elements that contribute to efficient ligand-dependent regulatory activity in a co-transcriptional and cellular context. Unexpectedly, we found that the P1 helix, which acts as the antitermination element of the switch in this RNA, supported ligand-dependent activation of a reporter gene over a broad spectrum of lengths from 3 to 10 bp. This same trend was also observed using a minimal in vitro single-turnover transcription assay, revealing that this behavior is intrinsic to the RNA sequence. We also found that the sequences at the distal tip of the terminator not directly involved in alternative secondary structure formation are highly important for efficient regulation. These data strongly support a model in which the switch is highly localized to the P1 helix adjacent to the ligand-binding pocket that likely presents a local kinetic block to invasion of the aptamer by the terminator.

  14. Detecting Chemically Modified DNA Bases Using Surface Enhanced Raman Spectroscopy. (United States)

    Barhoumi, Aoune; Halas, Naomi J


    Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics.

  15. TGF-β1 induction of the adenine nucleotide translocator 1 in astrocytes occurs through Smads and Sp1 transcription factors

    Directory of Open Access Journals (Sweden)

    Wallace Douglas C


    Full Text Available Abstract Background The adenine nucleotide translocator 1 (Ant1 is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant1 is upregulated by transforming growth factor-beta (TGF-β in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-β1 regulates Ant1 gene expression in cultured primary rodent astrocytes. Results Transcription reporter analysis verified that TGF-β1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-β1 responsive element of the Ant1 promoter was also identified. Electrophoretic mobility shift assays demonstrated that astrocyte nuclear proteins bind to this response element and TGF-β1 treatment recruits additional nuclear protein binding to this element. Antibody supershift and promoter deletion analyses demonstrated that Sp1 consensus binding sites in the RE are important for TGF-β1 regulation of Ant1 in astrocytes. Additionally, we demonstrate that Smad 2, 3 and 4 transcription factors are expressed in injured cerebral cortex and in primary astrocyte cultures. TGF-β1 activated Smad transcription factors also contribute to Ant1 regulation since transcription reporter assays in the presence of dominant negative (DN-Smads 3 and 4 significantly reduced induction of Ant1 by TGF-β1. Conclusion The specific regulation of Ant1 by TGF-β1 in astrocytes involves a cooperative interaction of both Smad and Sp1 binding elements located immediately upstream of the transcriptional start site. The first report of expression of Smads 2, 3 and 4 in astrocytes provided here is consistent with a regulation of Ant1 gene expression by these transcription factors in reactive astrocytes. Given the similarity in TGF-β1 regulation of Ant1 with other genes that are thought to promote neuronal survival, this interaction may

  16. Is the DPT tautomerization of the long A·G Watson-Crick DNA base mispair a source of the adenine and guanine mutagenic tautomers? A QM and QTAIM response to the biologically important question. (United States)

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M


    Herein, we first address the question posed in the title by establishing the tautomerization trajectory via the double proton transfer of the adenine·guanine (A·G) DNA base mispair formed by the canonical tautomers of the A and G bases into the A*·G* DNA base mispair, involving mutagenic tautomers, with the use of the quantum-mechanical calculations and quantum theory of atoms in molecules (QTAIM). It was detected that the A·G ↔ A*·G* tautomerization proceeds through the asynchronous concerted mechanism. It was revealed that the A·G base mispair is stabilized by the N6H···O6 (5.68) and N1H···N1 (6.51) hydrogen bonds (H-bonds) and the N2H···HC2 dihydrogen bond (DH-bond) (0.68 kcal·mol(-1) ), whereas the A*·G* base mispair-by the O6H···N6 (10.88), N1H···N1 (7.01) and C2H···N2 H-bonds (0.42 kcal·mol(-1) ). The N2H···HC2 DH-bond smoothly and without bifurcation transforms into the C2H···N2 H-bond at the IRC = -10.07 Bohr in the course of the A·G ↔ A*·G* tautomerization. Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···O6 H-bond is anticooperative to the two others-N1H···N1 and N2H···HC2 in the A·G base mispair, while the latters are significantly cooperative, mutually strengthening each other. In opposite, all three O6H···N6, N1H···N1, and C2H···N2 H-bonds are cooperative in the A*·G* base mispair. All in all, we established the dynamical instability of the А*·G* base mispair with a short lifetime (4.83·10(-14) s), enabling it not to be deemed feasible source of the A* and G* mutagenic tautomers of the DNA bases. The small lifetime of the А*·G* base mispair is predetermined by the negative value of the Gibbs free energy for the A*·G* → A·G transition. Moreover, all of the six low-frequency intermolecular vibrations cannot develop during this lifetime that additionally confirms the aforementioned results. Thus, the A*·G* base mispair cannot be

  17. Spermidine and resveratrol induce autophagy by distinct pathways converging on the acetylproteome

    DEFF Research Database (Denmark)

    Morselli, Eugenia; Mariño, Guillermo; Bennetzen, Martin V


    Autophagy protects organelles, cells, and organisms against several stress conditions. Induction of autophagy by resveratrol requires the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1). In this paper, we show that the acetylase inhibitor spermidine stimulates autophagy ...

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

    NARCIS (Netherlands)

    van de Weijer, T.; Phielix, E.; Bilet, L.; Williams, E.G.; Ropelle, E.R.; Bierwagen, A.; Livingstone, R.; Nowotny, P.; Sparks, L.M.; Paglialunga, S.A.; Szendroedi, J.; Havekes, B.; Moullan, N.; Pirinen, E.; Hwang, J.H.; Schrauwen-Hinderling, V.B.; Hesselink, M.K.; Auwerx, J.; Roden, M.; Schrauwen, P.


    Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide : NAD+ : precursors to increase oxidative phosphorylation and improve metabolic health, but human data is lacking. Here, we hypothesized that the nicotinic acid derivative Acipimox, a NAD+ precursor, would directly

  19. Decreased visfatin after exercise training correlates with improved glucose tolerance

    DEFF Research Database (Denmark)

    Haus, Jacob M; Solomon, Thomas; Marchetti, Christine M;


    Nampt/pre-B-cell colony-enhancing factor/visfatin (visfatin) release from adipocytes has recently been suggested to be nutrient responsive and linked to systemic nicotinamide adenine dinucleotide biosynthesis and regulation of pancreatic beta-cell function....

  20. Primaquine (United States)

    ... carry oxygen to the tissues in the body),nicotinamide adenine dinucleotide (NADH) deficiency (a genetic condition), glucose- ... these symptoms, call your doctor immediately: tiredness pale skin shortness of breath fast heartbeat yellowing of the ...

  1. The structures of pyruvate oxidase from Aerococcus viridans with cofactors and with a reaction intermediate reveal the flexibility of the active-site tunnel for catalysis


    Juan, Ella Czarina Magat; Hoque, Md Mominul; Hossain, Md Tofazzal; Yamamoto, Tamotsu; Imamura, Shigeyuki; Suzuki, Kaoru; Sekiguchi, Takeshi; Takénaka, Akio


    The crystal structures of pyruvate oxidase from A. viridans in complex with flavin adenine dinucleotide, thiamine diphosphate and the reaction intermediate 2-acetyl-thiamine diphosphate reveal details of substrate recognition and catalysis.

  2. Studies of Hepatic Lesion on Hyperuricemia Rat-model Induced by Adenine and Guanine%腺嘌呤、鸟嘌呤致高尿酸血症大鼠肝脏损害的研究

    Institute of Scientific and Technical Information of China (English)

    宋燕郡; 于维森; 殷凡; 宋扬


    Objective:To observe adenine and guanine effect liver function of hyperuricemia-model rat, and the changes under ultra microstructure.Method:The 36 male Wistar rats were randomly divided into 6 groups (A-control group,B-model group,C-adenine group,D-adenine starch paste group,E-adenine and guanine group,F-guanine group),each group of 6 cases.Groups B, C, D, E, F continuously given Yeast Extract Solution 15 g/(kg·d) to fill the stomach,7 days induced hyperuricemia-model rats.After the success of the building, group B for starch paste to fill the stomach.Group C:20 mg/(kg·d) adenine starch paste mixture.Group D:10 mg/(kg·d) adenine starch paste mixture.Group E:10 mg/(kg·d) adenine mixed 10 mg/(kg·d) guanine starch suspension.Group F:20 mg/(kg·d) guanine starch paste mixture to fill the stomach.For 14 days,the determination serum ALT,AST,UA and liver tissue electron microscope observation of liver injury of each group rats.Result:(1)Ultra microstructure observation the lysosome of group C increased obviously,scattered around the nucleus,and had dark grain material.Group D lysosome amount increased slightly,increased lipid drops, enter the lysosome.Group E the amount of lysosome increasing, increased lipid droplets, appear dark granular material.Group F bile duct in a small dark granular material.(2)ALT,AST of C-F groups were compared with group B,the differences were statistically significant(P cause ALT and AST levels significantly in hyperuricemia-model rats, the effect is higher than the same dose of guanine.Adenine has obvious effect to rat blood uric acid levels.%目的:观察腺嘌呤、鸟嘌呤作用高尿酸血症大鼠肝脏时,肝脏功能变化情况及透射电镜下肝脏超微结构的变化。方法:选用实验用雄性Wistar大鼠36只,随机分为A组(对照组)、B组(造模对照组)、C组(腺嘌呤组)、D组(腺嘌呤淀粉糊组)、E组(腺嘌呤、鸟嘌呤组)、F组(鸟嘌呤组),每组6只。B、C

  3. The Postconditioning Effects Of Diazoxide in the Brain Following Hemorrhagic Shock and Cerebral Hypoperfusion (United States)


    mmHg millimeter of mercury mPTP mitochondrial permeability transition pore Na sodium NAD+ nicotinamide adenine dinucleotide ion NADH... nicotinamide adenine dinucleotide NaHCO3 sodium bicarbonate NBD nucleotide binding domain nm nanometer xix     NS normal saline O2 oxygen PBS...saline-locked and secured to the tail using 3-0 silk thread. Three 18 gauge needles were then placed through small folds of skin on the right upper

  4. In vitro Toxicity and Inflammatory Response Induced by Copper Nanoparticles in Rat Alveolar Macrophages (United States)


    ingestion (via the gastrointestinal tract) or skin absorption routes of exposure (Tsuji, et al., 2006:43). More research is needed to determine if...nanoparticles can penetrate the skin (Tsuji, et al., 2006:44), because little information exists as to whether nanoparticles can be absorbed through...below). The conversion in viable cells is done by nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (reduced

  5. Botanical Extracts as Medical Countermeasures for Radiation Induced DNA Damage (United States)


    date June 2013 Listed medicinal ingredients - Grape seed extract standardized to 85 % polyphenols - Grape skin extract standardized to 15...Control wells contained dH2O or ethanol (concentration dependent on test substance solvent), NADPH (beta- nicotinamide adenine dinucleotide phosphate...statistical number of repeats NaCl sodium chloride NADPH beta- nicotinamide adenine dinucleotide phosphate nm nanometer (10-9) PBS phosphate buffered

  6. Detection of neuroinflammation through the retina by means of Raman spectroscopy and multivariate analysis (United States)

    Marro, Monica; Taubes, Alice; Villoslada, Pablo; Petrov, Dmitri


    Retinal nervous tissue sustains a substantial damage during the autoimmune inflammatory processes characteristic for Multiple Sclerosis (MS). The damage can be characterized non-surgically by Raman Spectroscopy, a non-invasive optical imaging technology. We used non-resonant near-infrared Raman spectrosocopy to create a spectral library of eight pivotal biomolecules known to be involved in neuroinflammation: Nicotinamide Adenine Dinucliotide (NADH), Flavin Adenine Nucleotide (FAD), Lactate, Cytochrome C, Glutamate, N-Acetyl- Aspartate (NAA), Phosphotidylcholine, with Advanced Glycolization End Products (AGEs) analyzed as a reference. Principal Component Analysis (PCA) of 50 spectra taken of murine retinal tissue culture undergoing an inflammatory response and healthy controls was used in order to characterize the molecular makeup of the inflammation. The loading plots revealed a heavy influence of peaks related to Glutamate, NADH, and Phosphotidylcholine to inflammation-related spectral changes. Partial Least Squares - Discriminant analysis (PLS-DA) was performed to create a multivariate classifier for the spectral diagnosis of neuroinflammed tissue and yielded a diagnostic sensitivity of 100% and specificity of 100%. We demonstrate then the effectiveness of combining Raman spectroscopy with PCA and PLS-DA statistical techniques to detect and monitor neuroinflamation in retina. With this technique Glutamate, NAA and NADH are detected in retina tissue as signs for neuroinflammation.

  7. 基于SERS光谱的银溶胶溶液中腺嘌呤的定量分析%Quantitative Analysis with Adenine in Silver Colloidal Sol by Surface-enhanced Raman Scattering Spectroscopic Technique

    Institute of Scientific and Technical Information of China (English)

    冯小平; 曹晓卫; 汪佳俐; 李玲; 张志学


    Surface-enhanced Raman scattering (SERS) spectroscopic technique was used to perform quantitative analyses with adenine, an important basic group from nucleic acid, by using silver colloidal sol as the SERS substrate. Sodium polyacrylate was added into the silver colloidal sol in order to improve its stability. In addition, during different SERS measurements, pyridine was used an internal standard to eliminate the impact of the variations in laser power, optical alignment and focusing on the SERS signal intensity of the an-alyte. It reveals that a good linear relationship is presented between the concentration of adenine and the characteristic SERS signal intensity ratio of the analyte adenine (733 cm-1) to the internal standard pyridine (1003 cm-1) in the range of 1× 10-4~1× 10-3mol ·L-1.%碱基是构成核酸的物质基础.碱基的含量分析对于生物体的生理活动及新陈代谢过程研究具有重要意义.本文利用表面增强拉曼散射(SERS)光谱技术研究了腺嘌呤在相对稳定的银溶胶溶液中的SERS光谱信号强度与浓度的定量关系.研究表明添加聚合物作为稳定剂的银溶胶具有良好的稳定性.进一步研究表明,在1×10-4~1×10-2mol·L-1的浓度范围内,以吡啶作为内标,分析物腺嘌呤与内标物特征谱峰强度之比与腺嘌呤的浓度之间呈现良好的线性关系.

  8. Netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex. Antibiotic binding at adenine . thymine base pairs in the minor groove of the self-complementary octanucleotide duplex. (United States)

    Patel, D J


    The structure of the netropsin . dG-dG-dA-dA-dT-dT-dC-dC complex (one antibiotic molecule/self-complementary octanucleodide duplex) and its dynamics as a function of temperature have been monitored by the nuclear magnetic resonances of the Watson-Crick protons, the nonexchangeable base and sugar protons and the backbone phosphates. The antibiotic forms a complex with the nucleic acid duplex at the dA . dT-containing tetranucleotide segment dA-dA-dT-dT, with slow migration amongst potential binding sites at low temperature. The downfield shifts in the exchangeable protons of netropsin on complex formation demonstrate the contributions of hydrogen-bonding interactions between the antibiotic and the nucleic acid to the stability of the complex. Complex formation results in changes in the glycosidic torsion angles of both thymidine residues and one deoxyadenosine residue as monitored by chemical shift changes in the thymine C-6 and adenine C-8 protons. The close proximity of the pyrrole rings of the antibiotic and the base-pair edges in the minor groove is manifested in the downfield shifts (0.3--0.5 ppm) of the pyrrole C-3 protons of netropsin and one adenine C-2 proton and one thymine N-3 base-pair proton on complex formation. The internucleotide phosphates of the octanucleotide undergo 31P chemical shift changes on addition of netropsin and these may reflect, in part, contributions from electrostatic interactions between the charged ends of the antibiotic and the backbone phosphates of the nucleic acid.

  9. A quick look at biochemistry : Carbohydrate metabolism

    NARCIS (Netherlands)

    Dashty, Monireh


    In mammals, there are different metabolic pathways in cells that break down fuel molecules to transfer their energy into high energy compounds such as adenosine-5'-triphosphate (ATP), guanosine-5'-triphosphate (GTP), reduced nicotinamide adenine dinucleotide (NADH2), reduced flavin adenine dinucleot

  10. Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models (United States)


    have been using Fluorescence Lifetime Imaging Microscopy (FLIM) (7) to examine Nicotinamide adenine dinucleotide (NADH) and Flavin optical imaging window into the skin of these mice above the tumor. We will then collect SHG from collagen and FLIM data for NADH using an MPM

  11. The NADPH-dependent O-.2-generating oxidase from human neutrophils. (United States)

    Gabig, T G


    A subcellular particulate fraction from normal neutrophils that was enriched in NADPH-dependent O-.2-generating activity (Gabig, T. G., Schervish, E. W., and Santinga, J. T. (1982) J. Biol. Chem. 257, 4114-4119) has been further characterized. This preparation contained 0.25 +/- 0.02 nmol of flavin adenine dinucleotide/mg of protein and 0.28 +/- 0.01 nmol of cytochrome b/mg of protein. Measurable amounts of riboflavin or flavin mononucleotide were not present. The flavoprotein was completely resolved from the cytochrome b by selective bile salt extraction of the particulate oxidase fraction. The identical subcellular particulate fraction was studied in the neutrophils from two male patients with chronic granulomatous disease. The neutrophil oxidase fraction from one of the chronic granulomatous disease patients had a cytochrome b component that was spectrally abnormal, but a normal content of flavin adenine dinucleotide. The fraction from this patient's neutrophils corresponding to the resolved flavoprotein from normal cells had fluorescence excitation and emission spectra that were identical to the normal flavoprotein. The neutrophil oxidase fraction from the second chronic granulomatous disease patient had a quantitatively and spectrally normal cytochrome b but less than 8% of the normal amount of flavin adenine dinucleotide. The fraction from the latter patient's neutrophils corresponding to the resolved flavoprotein from normal cells had no detectable flavoprotein by fluorescence excitation and emission spectroscopy. It is postulated that these two patients represent distinct mutants in two separate components of the neutrophil NADPH-dependent O-.2-generating oxidase system, flavoprotein and cytochrome b.

  12. Detection of N6-methyladenine in GATC sequences of Selenomonas ruminantium. (United States)

    Pristas, P; Molnarova, V; Javorsky, P


    The presence of N6-methyladenine in GATC sequences in DNA of Selenomonas ruminantium was investigated using sensitive methylation discriminating isochizomeric restriction enzymes analysis. Methylated adenine was detected in 8 out of 18 tested strains belonging to the subsp. lactilytica of S. ruminantium. No corresponding restriction activity was detected in three tested strains. No GATC methylation was detected in 3 analysed S. ruminantium subsp. ruminantium strains. Sustainable progress was achieved in the molecular biology of ruminal microorganisms in the last decade. Many different genes acting in the cell wall degradation were cloned and characterized. As practically all cloning experiments were done in Escherichia coli cells, there is a lack of data about regulation of gene(s) expression in the natural hosts. However, much better understanding of molecular genetics of ruminal bacteria is required for improving rumen functions by genetic modifications of rumen bacteria. DNA methylation is main mechanism of the control of gene expression in eukaryotes. In prokaryotes, DNA methylation influences wide variety of important cellular functions as accessibility of DNA to digestion by restriction endonucleases, control of replication initiation, transposition, phage DNA packaging, including positive and negative regulation of gene expression. Most of the DNA methyltransferases; enzymes which facilitate methylation; identified in prokaryotes are part of restriction modification systems (WILSON and MURRAY 1991. Another class of methyltransferase are independent methylases like Dam and Dcm in Escherichia coli. Dam methylase recognizes the sequence GATC and methylates adenine at N6 position (BARRAS and MARINUS 1989). The methylation is the only documented case of prokaryotic methylation involved in the regulation of cellular process (NOYER-WIEDNER and TRAUTNER 1993). Screening of large number of bacteria have detected the presence of Dam methylation in cyanobacteria as

  13. Alteration of marker enzymes in adenine-induced impaired testis and the interference of total flavonoids of herba epimedii in rats%腺嘌呤致大鼠睾丸损伤中标志酶的变化及淫羊藿总黄酮的干预作用

    Institute of Scientific and Technical Information of China (English)

    张作涛; 谢高宇; 陈凯; 齐敏友; 刘浩然


    treatment from the 15th day,and then sacrificed after 30 days. The mass of testis, epididymis, seminal vesicles and rats were weighted,and the organ indexes were calculated. Serum testosterone,follicle-stimulating hormone(FSH) ,luteinizing hormone (LH) and testicular lactate dehydrogenase (LDH) , acid phosphatase ( ACP) , gamma-glutamyl transpeptidase(γ-GT),superoxide dismutase (SOD) ,glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) content were detected. Results Compared with the normal control group,the gonadal organ indexes, serum testosterone value and testicular LDH,ACP,γ-GT activity decreased, while MDA content increased in model group,with significantly difference(P<0. 05). The rats organ indexes, serum testosterone levels and specific enzyme activity were increased in TFE group when compared to those in model group(P<0. 05). Conclusion TFE may improve the activity of marker enzymes in adenine-induced impaired testis through promoting the testosterone release and anti-oxidation.

  14. Nafion-CNT coated carbon-fiber microelectrodes for enhanced detection of adenosine. (United States)

    Ross, Ashley E; Venton, B Jill


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

  15. Detection of regional DNA methylation using DNA-graphene affinity interactions. (United States)

    Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A


    We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues.

  16. Electrocatalytic oxidation behavior of NADH at Pt/Fe{sub 3}O{sub 4}/reduced-graphene oxide nanohybrids modified glassy carbon electrode and its determination

    Energy Technology Data Exchange (ETDEWEB)

    Roushani, Mahmoud, E-mail: [Department of Chemistry, Faculty of Sciences, Ilam University, Ilam, 69315516 (Iran, Islamic Republic of); Hoseini, S. Jafar [Department of Chemistry, Faculty of Sciences, Yasouj University, Yasouj, 7591874831 (Iran, Islamic Republic of); Azadpour, Mitra [Department of Chemistry, Faculty of Sciences, Ilam University, Ilam, 69315516 (Iran, Islamic Republic of); Heidari, Vahid; Bahrami, Mehrangiz; Maddahfar, Mahnaz [Department of Chemistry, Faculty of Sciences, Yasouj University, Yasouj, 7591874831 (Iran, Islamic Republic of)


    We have developed Pt/Fe{sub 3}O{sub 4}/reduced-graphene oxide nanohybrids modified glassy carbon (Pt/Fe{sub 3}O{sub 4}/RGO/GC) electrode as a novel system for the preparation of electrochemical sensing platform. Characterization of as-made composite was determined using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscopy (AFM) and energy-dispersive analysis of X-ray (EDAX) where the Pt, Fe, Si, O and C elements were observed. The Pt/Fe{sub 3}O{sub 4}/RGO/GC electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Due to the synergistic effect between Pt, Fe{sub 3}O{sub 4} and RGO, the nanohybrid exhibited excellent performance toward dihydronicotinamide adenine dinucleotide (NADH) oxidation in 0.1 M phosphate buffer solution, pH 7.0, with a low detection limit of 5 nM. - Highlights: • Preparation of a novel electrochemical sensing platform system • Excellent performance of Pt/Fe{sub 3}O{sub 4}/reduced-graphene oxide nanohybrids • Dihydronicotinamide adenine dinucleotide oxidation with a low detection limit of 5 nM.

  17. A bridged nucleic acid, 2',4'-BNA COC: synthesis of fully modified oligonucleotides bearing thymine, 5-methylcytosine, adenine and guanine 2',4'-BNA COC monomers and RNA-selective nucleic-acid recognition. (United States)

    Mitsuoka, Yasunori; Kodama, Tetsuya; Ohnishi, Ryo; Hari, Yoshiyuki; Imanishi, Takeshi; Obika, Satoshi


    Recently, we synthesized pyrimidine derivatives of the 2'-O,4'-C-methylenoxymethylene-bridged nucleic-acid (2',4'-BNA(COC)) monomer, the sugar conformation of which is restricted in N-type conformation by a seven-membered bridged structure. Oligonucleotides (BNA(COC)) containing this monomer show high affinity with complementary single-stranded RNA and significant resistance to nuclease degradation. Here, BNA(COC) consisting of 2',4'-BNA(COC) monomers bearing all four bases, namely thymine, 5-methylcytosine, adenine and guanine was efficiently synthesized and properties of duplexes containing the 2',4'-BNA(COC) monomers were investigated by UV melting experiments and circular dichroism (CD) spectroscopy. The UV melting curve analyses showed that the BNA(COC)/BNA(COC) duplex possessed excellent thermal stability and that the BNA(COC) increased thermal stability with a complementary RNA strand. On the other hand, BNA(COC)/DNA heteroduplexes showed almost the same thermal stability as RNA/DNA heteroduplexes. Furthermore, mismatched sequence studies showed that BNA(COC) generally improved the sequence selectivity with Watson-Crick base-pairing compared to the corresponding natural DNA and RNA. A CD spectroscopic analysis indicated that the BNA(COC) formed duplexes with complementary DNA and RNA in a manner similar to natural RNA.

  18. Efeito da sacarose, cinetina, isopentenil adenina e zeatina no desenvolvimento de embriões de Heliconia rostrata in vitro Effect of sucrose, kinetin, isopentenyl adenine and zeatin on the development of embryos of Heliconia rostrata in vitro

    Directory of Open Access Journals (Sweden)

    Antonio Carlos Torres


    Full Text Available Embriões provenientes de frutos maturos de Heliconia rostrata Ruiz & Pavon foram excisados e inoculados em meio de cultura contendo os sais básicos MS, vitaminas e sacarose. A adição de sacarose foi essencial para o desenvolvimento dos embriões. Em meio desprovido de sacarose os embriões morreram em cultura. Concentrações de 1%, 2% e 3% (p/v de sacarose favoreceram o desenvolvimento dos embriões. Concentrações de 6%, 9% e 12% (p/v de sacarose inibiram o crescimento dos embriões. A adição de cinetina, isopentenil adenina e zeatina não favoreceram o crescimento e o desenvolvimento dos embriões.Embryos from mature fruits of Heliconia rostrata were excised and cultured in basal medium containing MS salts, vitamins and sucrose. Sucrose was essential for embryo development. In meclium without sucrose the embryos died in culture. Sucrose concentrations of 1%, 2% and 3% (w/v were beneficial for embryo development. Sucrose concentrations of 6%, 9% and 12% (w/v inhibited embryo growth. The addition of kinetin, isopentenyl adenine and zeatin did not improve embryo growth and development.

  19. Supernova detection

    Energy Technology Data Exchange (ETDEWEB)

    Nakahata, Masayuki [Kamioka Observatory, Institute for Cosmic Ray research, University of Tokyo, Higashi-Mozumi, Kamioka-cho, Hida-shi, Gifu, Japan, 506-1205 (Japan)], E-mail:


    The detection of supernova neutrinos is reviewed, focusing on the current status of experiments to detect supernova burst neutrinos and supernova relic neutrinos. The capabilities of each detector currently operating and in development are assessed and the likely neutrino yield for a future supernova is estimated. It is expected that much more information will be obtained if a supernova burst were to occur in our Galaxy than was obtained for supernova SN1987A. The detection of supernova relic neutrinos is considered and it is concluded that a large volume detector with a neutron tagging technique is necessary.

  20. Statistical methods for detecting periodic fragments in DNA sequence data

    Directory of Open Access Journals (Sweden)

    Ying Hua


    period detection in the presence of eroded periodicity. The autocorrelation method was identified as poorly suited for use with the blockwise bootstrap. Application of our methods to the genomes of two model organisms revealed a striking proportion of the yeast and mouse genomes are spanned by NPS. Despite their markedly different sizes, roughly equivalent proportions (19-21% of the genomes lie within period-10 spans of the NPS dinucleotides {AA, TT, TA}. The biological significance of these regions remains to be demonstrated. To facilitate this, the genomic coordinates are available as Additional files 1, 2, and 3 in a format suitable for visualisation as tracks on popular genome browsers. Reviewers This article was reviewed by Prof Tomas Radivoyevitch, Dr Vsevolod Makeev (nominated by Dr Mikhail Gelfand, and Dr Rob D Knight.

  1. Multiphoton fluorescence lifetime imaging of metabolic status in mesenchymal stem cell during adipogenic differentiation (United States)

    Meleshina, A. V.; Dudenkova, V. V.; Shirmanova, M. V.; Bystrova, A. S.; Zagaynova, E. V.


    Non-invasive imaging of cell metabolism is a valuable approach to assess the efficacy of stem cell therapy and understand the tissue development. In this study we analyzed metabolic trajectory of the mesenchymal stem cells (MCSs) during differentiation into adipocytes by measuring fluorescence lifetimes of free and bound forms of the reduced nicotinamide adenine dinucleotide (NAD(P)H) and flavine adenine dinucleotide (FAD). Undifferentiated MSCs and MSCs on the 5, 12, 19, 26 days of differentiation were imaged on a Zeiss 710 microscope with fluorescence lifetime imaging (FLIM) system B&H (Germany). Fluorescence of NAD(P)H and FAD was excited at 750 nm and 900 nm, respectively, by a femtosecond Ti:sapphire laser and detected in a range 455-500 nm and 500-550 nm, correspondingly. We observed the changes in the NAD(P)H and FAD fluorescence lifetimes and their relative contributions in the differentiated adipocytes compare to undifferentiated MSCs. Increase of fluorescence lifetimes of the free and bound forms of NAD(P)H and the contribution of protein-bound NAD(P)H was registered, that can be associated with a metabolic switch from glycolysis to oxidative phosphorylation and/or synthesis of lipids in adipogenically differentiated MSCs. We also found that the contribution of protein-bound FAD decreased during differentiation. After carrying out appropriate biochemical measurements, the observed changes in cellular metabolism can potentially serve to monitor stem cell differentiation by FLIM.

  2. Analysis of stereochemistry and biosynthesis of epicatechin in tea plants by chiral phase high performance liquid chromatography. (United States)

    Qian, Yumei; Zhao, Xianqian; Zhao, Lei; Cui, Lilan; Liu, Li; Jiang, Xiaolan; Liu, Yajun; Gao, Liping; Xia, Tao


    Tea (Camellia sinensis) is rich in flavan-3-ols (catechins), especially epicatechin (EC), which is the predominant extension unit of polymeric proanthocyanidins (PAs). However, studies assessing EC's stereochemistry are scarce. Here, a high performance liquid chromatography column using amylose tris-(3, 5-dimethylphenylcarbamate) immobilized on silica-gel as chiral stationary phases (CSPs) was applied to explore its stereochemistry and biosynthetic pathway in tea plants. The results revealed (-)-epicatechin [(-)-EC] was the predominant di-hyroxy-non-galloylated-catechins, while (+)-epicatechin [(+)-EC] was not detected. Interestingly, (-)-EC was the only product obtained from cyanidin using the partially purified native C. sinensis anthocyanidin reductase (CsANR) in the presence of reduction nicotinamide adenine dinucleotide phosphate (NADPH); meanwhile, (+)-EC was the main product using recombinant CsANR in the same conditions. In addition, (-)-EC could be obtained from (+)-catechin [(+)-C] using recombinant CsANR, which displayed C3-epimerase activity in the presence of oxidation nicotinamide adenine dinucleotide phosphate (NADP(+)). But the partially purified native CsANR did not possess this function. Finally, (-)-EC could result from the de-gallate acid reaction of epicatechin gallate (ECG) catalyzed by a novel partially purified native galloylated catechins hydrolase (GCH) from tea leaves. In summary, (-)-EC is likely the product of native protein from the tea plants, and (+)-EC is only produced in a reaction catalyzed by recombinant CsANR in vitro.

  3. Biodegradation of pendimethalin by Bacillus subtilis Y3. (United States)

    Ni, Haiyan; Yao, Li; Li, Na; Cao, Qin; Dai, Chen; Zhang, Jun; He, Qin; He, Jian


    A bacterium strain Y3, capable of efficiently degrading pendimethalin, was isolated from activated sludge and identified as Bacillus subtilis according to its phenotypic features and 16S rRNA phylogenetic analysis. This strain could grow on pendimethalin as a sole carbon source and degrade 99.5% of 100mg/L pendimethalin within 2.5days in batch liquid culture, demonstrating a greater efficiency than any other reported strains. Three metabolic products, 6-aminopendimethalin, 5-amino-2-methyl-3-nitroso-4-(pentan-3-ylamino) benzoic acid, and 8-amino-2-ethyl-5-(hydroxymethyl)-1,2-dihydroquinoxaline-6-carboxylic acid, were identified by HPLC-MS/MS, and a new microbial degradation pathway was proposed. A nitroreductase catalyzing nitroreduction of pendimethalin to 6-aminopendimethalin was detected in the cell lysate of strain Y3. The cofactor was nicotinamide adenine dinucleotide phosphate (NADPH) or more preferably nicotinamide adenine dinucleotide (NADH). The optimal temperature and pH for the nitroreductase were 30°C and 7.5, respectively. Hg(2+), Ni(2+), Pb(2+), Co(2+), Mn(2+) Cu(2+), Ag(+), and EDTA severely inhibited the nitroreductase activity, whereas Fe(2+), Mg(2+), and Ca(2+) enhanced it. This study provides an efficient pendimethalin-degrading microorganism and broadens the knowledge of the microbial degradation pathway of pendimethalin.

  4. Self-assembled CNTs/CdS/dehydrogenase hybrid-based amperometric biosensor triggered by photovoltaic effect. (United States)

    Tang, Longhua; Zhu, Yihua; Yang, Xiaoling; Sun, Jinjie; Li, Chunzhong


    A novel multi-components hybrid material, self-assembled quantum dots (CdS) and glutamate dehydrogenase (GDH) onto multiwall carbon nanotubes (CNTs), was designed for amperometric biosensing system. The zeta-potential and transmission electron microscopy (TEM) analyses confirmed the uniform growth of the CdS/GDH onto carboxyl-functionalized CNTs. Compared with the single CdS, the resulting hybrid material showed more efficient generation of photocurrent upon illumination. The incident light excites CdS and generates charge carriers, and then CNTs facilitates the charge transfer. For dehydrogenase-based biosensor, normally, the cofactor of beta-nicotinamide adenine dinucleotide (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate (NADP(+)) is necessary. Furthermore, we found the photovoltaic effect of CNTs/CdS/GDH can trigger the dehydrogenase enzymatic reaction in the absence of the NAD(+) or NADP(+) cofactors. The electrochemical experiment results also demonstrate that the cofactor-independent dehydrogenase biosensing system had series attractive characteristics, such as a good sensitivity (11.9 nA/microM), lower detection limit (up to 50 nM), an acceptable reproducibility and stability. These studies aid in understanding the combination of the semiconductor nanohybrids (CNTs/QDs, etc.) and biomolecules (enzymes, etc.), which has potential for the applications in biosensor, biofuel cell, biomedical and other bioelectronics field.

  5. Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy (United States)

    Schweitzer, Dietrich; Deutsch, Lydia; Klemm, Matthias; Jentsch, Susanne; Hammer, Martin; Peters, Sven; Haueisen, Jens; Müller, Ulrich A.; Dawczynski, Jens


    The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included in the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τi, amplitudes αi, and relative contributions Qi were statistically compared between corresponding groups in two spectral channels (490diabetic patients and age-matched controls (p450 ps, and the shift of τ3 from ˜3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine dinucleotide at the fundus. AGE also accumulated in the crystalline lens.

  6. PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae. (United States)

    Sauer, Elizabeta; Merdanovic, Melisa; Mortimer, Anne Price; Bringmann, Gerhard; Reidl, Joachim


    The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.

  7. Arteriosclerosis Detection (United States)


    The Veterans Administration Hospital used computer image-processing techniques to detect arteriosclerosis. Originally developed by Goddard Space Flight Center engineers to enhance spacecraft pictures, this device replaced the previous testing for this disease which was extremely painful and time consuming. With this instrument, computer detected edges are shown along with an estimate of location of pre-arteriosclerosis vessel wall. The difference between the two represents the relative amount of disease in the blood vessel. Instrumentation will be expanded again in 1976 to analyze the coronary arteries and the blood vessels of the retina.

  8. Does the tautomeric status of the adenine bases change upon the dissociation of the A*·A(syn) Topal-Fresco DNA mismatch? A combined QM and QTAIM atomistic insight. (United States)

    Brovarets', Ol'ha O; Zhurakivsky, Roman O; Hovorun, Dmytro M


    We have scrupulously explored the tautomerisation mechanism via the double proton transfer of the A*·A(syn) Topal-Fresco base mispair (C(s) symmetry), formed by the imino and amino tautomers of the adenine DNA base in the anti- and syn-conformations, respectively, bridging quantum-mechanical calculations with Bader's quantum theory of atoms in molecules. It was found that the A*·A(syn) ↔ A·A*(syn) tautomerisation is the asynchronous concerted process. It was established that the A*·A(syn) DNA mismatch is stabilized by the N6H···N6 (6.35) and N1H···N7 (6.17) hydrogen (H) bonds, whereas the A·A*(syn) base mispair (Cs) by the N6H···N6 (8.82) and N7H···N1 (9.78) H-bonds and the C8H···HC2 HH-bond (0.30 kcal mol(-1)). Using the sweeps of the energies of the intermolecular H-bonds, it was observed that the N6H···N6 and N1H···N7/N7H···N1 H-bonds are anti-cooperative and mutually weaken each other in the A*·A(syn) and A·A*(syn) mispairs. It was revealed that the A·A*(syn) DNA mismatch is a dynamically unstable structure with a short lifetime of 1.12 × 10(-13) s and any of its 6 low-frequency intermolecular vibrations can develop during this period of time. This observation makes it impossible to change the tautomeric status of the A bases upon the dissociation of the A*·A(syn) base mispair into the monomers during DNA replication.

  9. Carboxyatractyloside effects on brown-fat mitochondria imply that the adenine nucleotide translocator isoforms ANT1 and ANT2 may be responsible for basal and fatty-acid-induced uncoupling respectively. (United States)

    Shabalina, Irina G; Kramarova, Tatiana V; Nedergaard, Jan; Cannon, Barbara


    In brown-fat mitochondria, fatty acids induce thermogenic uncoupling through activation of UCP1 (uncoupling protein 1). However, even in brown-fat mitochondria from UCP1-/- mice, fatty-acid-induced uncoupling exists. In the present investigation, we used the inhibitor CAtr (carboxyatractyloside) to examine the involvement of the ANT (adenine nucleotide translocator) in the mediation of this UCP1-independent fatty-acid-induced uncoupling in brown-fat mitochondria. We found that the contribution of ANT to fatty-acid-induced uncoupling in UCP1-/- brown-fat mitochondria was minimal (whereas it was responsible for nearly half the fatty-acid-induced uncoupling in liver mitochondria). As compared with liver mitochondria, brown-fat mitochondria exhibit a relatively high (UCP1-independent) basal respiration ('proton leak'). Unexpectedly, a large fraction of this high basal respiration was sensitive to CAtr, whereas in liver mitochondria, basal respiration was CAtr-insensitive. Total ANT protein levels were similar in brown-fat mitochondria from wild-type mice and in liver mitochondria, but the level was increased in brown-fat mitochondria from UCP1-/- mice. However, in liver, only Ant2 mRNA was found, whereas in brown adipose tissue, Ant1 and Ant2 mRNA levels were equal. The data are therefore compatible with a tentative model in which the ANT2 isoform mediates fatty-acid-induced uncoupling, whereas the ANT1 isoform may mediate a significant part of the high basal proton leak in brown-fat mitochondria.

  10. Fingerprint detection

    Energy Technology Data Exchange (ETDEWEB)

    Saunders, G.C.


    This patent describes a method for detection and visualization of latent fingerprints. It comprises contacting a substrate containing a latent print thereon the a colloidal metal composition at a pH from about 2.5 to about 4.0 for time sufficient to allow reaction of the colloidal metal composition with the latent print; and, preserving or recording the observable print.

  11. Detection device (United States)

    Smith, J.E.


    The present invention is directed to a detection device comprising: (1) an entrance chamber; (2) a central chamber; and (3) an exit chamber. The central chamber includes an ionizing gas, anode, and means for connecting the anode with an external power supply and pulse counter.

  12. Animal Detectives (United States)

    Mulvey, Bridget; Warnock, Carly


    During a two-week inquiry-based 5E learning cycle unit, children made observations and inferences to guide their explorations of animal traits and habitats (Bybee 2014). The children became "animal detectives" by studying a live-feed webcam and digital images of wolves in their natural habitat, reading books and online sources about…

  13. Detection of transposable elements by their compositional bias

    Directory of Open Access Journals (Sweden)

    Anxolabéhère Dominique


    Full Text Available Abstract Background Transposable elements (TE are mobile genetic entities present in nearly all genomes. Previous work has shown that TEs tend to have a different nucleotide composition than the host genes, either considering codon usage bias or dinucleotide frequencies. We show here how these compositional differences can be used as a tool for detection and analysis of TE sequences. Results We compared the composition of TE sequences and host gene sequences using probabilistic models of nucleotide sequences. We used hidden Markov models (HMM, which take into account the base composition of the sequences (occurrences of words n nucleotides long, with n ranging here from 1 to 4 and the heterogeneity between coding and non-coding parts of sequences. We analyzed three sets of sequences containing class I TEs, class II TEs and genes respectively in three species: Drosophila melanogaster, Cænorhabditis elegans and Arabidopsis thaliana. Each of these sets had a distinct, homogeneous composition, enabling us to distinguish between the two classes of TE and the genes. However the particular base composition of the TEs differed in the three species studied. Conclusions This approach can be used to detect and annotate TEs in genomic sequences and complements the current homology-based TE detection methods. Furthermore, the HMM method is able to identify the parts of a sequence in which the nucleotide composition resembles that of a coding region of a TE. This is useful for the detailed annotation of TE sequences, which may contain an ancient, highly diverged coding region that is no longer fully functional.

  14. Detection of Macromolecular Fractions in HCN Polymers Using Electrophoretic and Ultrafiltration Techniques. (United States)

    Marín-Yaseli, Margarita R; Cid, Cristina; Yagüe, Ana I; Ruiz-Bermejo, Marta


    Elucidating the origin of life involves synthetic as well as analytical challenges. Herein, for the first time, we describe the use of gel electrophoresis and ultrafiltration to fractionate HCN polymers. Since the first prebiotic synthesis of adenine by Oró, HCN polymers have gained much interest in studies on the origins of life due to the identification of biomonomers and related compounds within them. Here, we demonstrate that macromolecular fractions with electrophoretic mobility can also be detected within HCN polymers. The migration of polymers under the influence of an electric field depends not only on their sizes (one-dimensional electrophoresis) but also their different isoelectric points (two-dimensional electrophoresis, 2-DE). The same behaviour was observed for several macromolecular fractions detected in HCN polymers. Macromolecular fractions with apparent molecular weights as high as 250 kDa were detected by tricine-SDS gel electrophoresis. Cationic macromolecular fractions with apparent molecular weights as high as 140 kDa were also detected by 2-DE. The HCN polymers synthesized were fractionated by ultrafiltration. As a result, the molecular weight distributions of the macromolecular fractions detected in the HCN polymers directly depended on the synthetic conditions used to produce these polymers. The implications of these results for prebiotic chemistry will be discussed.

  15. Simultaneous immobilization of dehydrogenases on polyvinylidene difluoride resin after separation by non-denaturing two-dimensional electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Shimazaki, Youji [Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Bunkyo-cho 2-5, Matsuyama City 790-8577 (Japan)], E-mail:; Kadota, Mariko [Faculty of Science, Ehime University, Matsuyama (Japan)


    We detected mouse liver malate, sorbitol and aldehyde dehydrogenases by negative staining, analysis of malate and sorbitol dehydrogenase activities using each substrate, and electron transfers including nicotinamide adenine dinucleotide (NAD) and nitroblue tetrazolium in non-denaturing two-dimensional electrophoresis (2-DE) gel. Dehydrogenases were also identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) after 2-DE separation and protein detection by negative staining. Spots of dehydrogenases separated by 2-DE were excised, and simultaneously transferred and immobilized on polyvinylidene difuoride (PVDF) resin by electrophoresis. The dehydrogenase activities remained intact after immobilization. In conclusion, resin-immobilized dehydrogenases can be simultaneously obtained after separation by non-denaturing 2-DE, detection by negative staining and transferring to resins.

  16. On-chip microfluidic systems for determination of L-glutamate based on enzymatic recycling of substrate

    DEFF Research Database (Denmark)

    Laiwattanapaisal, W.; Yakovleva, J.; Bengtsson, Martin


    Two microfluidic systems have been developed for specific analysis of L-glutamate in food based on substrate recycling fluorescence detection. L-glutamate dehydrogenase and a novel enzyme, D-phenylglycine aminotransferase, were covalently immobilized on (i) the surface of silicon microchips....... The reaction was accompanied by reduction of nicotinamide adenine dinucleotide (NAD(+)) to NADH, which was monitored by fluorescence detection (epsilon(ex)=340 nm, epsilon(em)=460 nm). First, the microchip-based system, L-glutamate was detected within a range of 3.1-50.0 mM. Second, to be automatically......). In the case of SIA, the beads were introduced and removed from the microchip automatically. The immobilized beads could be stored in a 20% glycerol and 0.5 mM ethylenediaminetetraacetic acid solution maintained at a pH of 7.0 using a phosphate buffer for at least 15 days with 72% of the activity remaining...

  17. Edge Detection, (United States)


    PROJECT. T ASK0 Artificial Inteligence Laboratory AREA It WORK UNIT NUMBERS V 545 Technology Square ( Cambridge, HA 02139 I I* CONTOOL1LIN@4OFFICE NAME...ARD-A1t62 62 EDGE DETECTION(U) NASSACNUSETTS INST OF TECH CAMBRIDGE 1/1 ARTIFICIAL INTELLIGENCE LAB E C HILDRETH SEP 85 AI-M-8 N99SI4-8S-C-6595...used to carry out this analysis. cce~iO a N) ’.~" D LI’BL. P p ------------ Sj. t i MASSACHUSETTS INSTITUTE OF TECHNOLOGY i ARTIFICIAL INTELLIGENCE

  18. Second-Generation Fluorescent Quadracyclic Adenine Analogues

    DEFF Research Database (Denmark)

    Dumat, Blaise; Bood, Mattias; Wranne, Moa S.;


    Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putativ...

  19. DNA adenine methyltransferase (Dam) controls the expression of the cytotoxic enterotoxin (act) gene of Aeromonas hydrophila via tRNA modifying enzyme-glucose-inhibited division protein (GidA). (United States)

    Erova, Tatiana E; Kosykh, Valeri G; Sha, Jian; Chopra, Ashok K


    Aeromonas hydrophila is both a human and animal pathogen, and the cytotoxic enterotoxin (Act) is a crucial virulence factor of this bacterium because of its associated hemolytic, cytotoxic, and enterotoxic activities. Previously, to define the role of some regulatory genes in modulating Act production, we showed that deletion of a glucose-inhibited division gene (gidA) encoding tRNA methylase reduced Act levels, while overproduction of DNA adenine methyltransferase (Dam) led to a concomitant increase in Act-associated biological activities of a diarrheal isolate SSU of A. hydrophila. Importantly, there are multiple GATC binding sites for Dam within an upstream sequence of the gidA gene and one such target site in the act gene upstream region. We showed the dam gene to be essential for the viability of A. hydrophila SSU, and, therefore, to better understand the interaction of the encoding genes, Dam and GidA, in act gene regulation, we constructed a gidA in-frame deletion mutant of Escherichia coli GM28 (dam(+)) and GM33 (∆dam) strains. We then tested the expressional activity of the act and gidA genes by using a promoterless pGlow-TOPO vector containing a reporter green fluorescent protein (GFP). Our data indicated that in GidA(+) strains of E. coli, constitutive methylation of the GATC site(s) by Dam negatively regulated act and gidA gene expression as measured by GFP production. However, in the ∆gidA strains, irrespective of the presence or absence of constitutively active Dam, we did not observe any alteration in the expression of the act gene signifying the role of GidA in positively regulating Act production. To determine the exact mechanism of how Dam and GidA influence Act, a real-time quantitative PCR (RT-qPCR) assay was performed. The analysis indicated an increase in gidA and act gene expression in the A. hydrophila Dam-overproducing strain, and these data matched with Act production in the E. coli GM28 strain. Thus, the extent of DNA methylation

  20. 益生菌发酵驼乳对慢性肾功能衰竭的治疗作用%The rapeutic Action of Probiotic Fermented Camel Milk on Chronic Renal Failure Rats Caused by Adenine

    Institute of Scientific and Technical Information of China (English)

    李建美; 潘蕾; 张敏; 王娟; 刘薇; 郭春燕; 李擎; 吉日木图


    Purpose: To compare the different therapeutic Failure(CRF) of rats. Method: CRF rat models induced by fermentation camel milk. And then evaluated the therapeutic action of probiotics felTnented camel milk on Chronic Renal adenine were received intragastric administration with probiotic effect of three kinds of fermented camel milk by measure the diet and water intake, weight, kidney index, serum creatinine(Scr), Urea Nitrogen(BUN), nitric oxide(NO), superoxide dismutase(SOD), serum total protein(STP), calcium(Ca), phosphorus(P), urine volume in 24 hours, urine protein(UP) and the Renal Biopsy. Result: The result showed that fermentation camel milk can reduce the serum level of Set and BUN, slow down UP, ameliorate the balance of calcium and phosphorus, enhanced the level of SOD and STP and alleviated pathological changes of the kidney tissue. Conclusion: Different kinds of probiotic fermented camel milk are effective in treating CRF of rats and the effect of camel milk fermented by L.casei Zhang is superior to camel milk fermented by other probiotics.%目的:比较不同菌种发酵的驼乳制品对腺嘌呤所致大鼠慢性。肾功能衰竭(CRF)的缓解作用。方法:采用腺嘌呤复制CRF大鼠模型,以不同发酵剂发酵的驼乳作为受试物进行灌胃干预。通过检测大鼠的饮食饮水情况、排尿量、尿液和血清常规指标及肾脏病理组织学变化,评估各发酵驼乳对CRF大鼠的治疗效果。结果:发酵驼乳均可改善肾功能衰竭大鼠的一般生理状况,可降低大鼠血肌酐(Scr)、尿素氦(BUN)水平,减缓尿蛋白(UP),调节Ca、P的含量,提高过氧化物歧化酶(SOD)、血清总蛋白(STP)水平,具有保护肾功能的作用,其中LIcaseiZhang发酵的驼乳对CRF大鼠的改善效果最佳。

  1. Short-hairpin RNA-induced suppression of adenine nucleotide translocase-2 in breast cancer cells restores their susceptibility to TRAIL-induced apoptosis by activating JNK and modulating TRAIL receptor expression

    Directory of Open Access Journals (Sweden)

    Kim Chul-Woo


    Full Text Available Abstract Background Tumor necrosis factor (TNF-related apoptosis-inducing ligand (TRAIL; apo2 ligand induces apoptosis in cancer cells but has little effect on normal cells. However, many cancer cell types are resistant to TRAIL-induced apoptosis, limiting the clinical utility of TRAIL as an anti-cancer agent. We previously reported that the suppression of adenine nucleotide translocase-2 (ANT2 by short-hairpin RNA (shRNA induces apoptosis of breast cancer cells, which frequently express high levels of ANT2. In the present study, we examined the effect of RNA shRNA-induced suppression of ANT2 on the resistance of breast cancer cells to TRAIL-induced apoptosis in vitro and in vivo. Results ANT2 shRNA treatment sensitized MCF7, T47 D, and BT474 cells to TRAIL-induced apoptosis by up-regulating the expression of TRAIL death receptors 4 and 5 (DR4 and DR5 and down-regulating the TRAIL decoy receptor 2 (DcR2. In MCF7 cells, ANT2 knockdown activated the stress kinase c-Jun N-terminal kinase (JNK, subsequently stabilizing and increasing the transcriptional activity of p53 by phosphorylating it at Thr81; it also enhanced the expression and activity of DNA methyltransferase 1 (DNMT1. ANT2 shRNA-induced overexpression of DR4/DR5 and TRAIL sensitization were blocked by a p53 inhibitor, suggesting that p53 activation plays an important role in the transcriptional up-regulation of DR4/DR5. However, ANT2 knockdown also up-regulated DR4/DR5 in the p53-mutant cell lines BT474 and T47 D. In MCF7 cells, ANT2 shRNA treatment led to DcR2 promoter methylation and concomitant down-regulation of DcR2 expression, consistent with the observed activation of DNMT1. Treatment of the cells with a demethylating agent or JNK inhibitor prevented the ANT2 shRNA-induced down-regulation of DcR2 and activation of both p53 and DNMT1. In in vivo experiments using nude mice, ANT2 shRNA caused TRAIL-resistant MCF7 xenografts to undergo TRAIL-induced cell death, up-regulated DR4/DR5

  2. Utilizing Gold Nanoparticle Probes to Visually Detect DNA Methylation (United States)

    Chen, Kui; Zhang, Mingyi; Chang, Ya-Nan; Xia, Lin; Gu, Weihong; Qin, Yanxia; Li, Juan; Cui, Suxia; Xing, Gengmei


    The surface plasmon resonance (SPR) effect endows gold nanoparticles (GNPs) with the ability to visualize biomolecules. In the present study, we designed and constructed a GNP probe to allow the semi-quantitative analysis of methylated tumor suppressor genes in cultured cells. To construct the probe, the GNP surfaces were coated with single-stranded DNA (ssDNA) by forming Au-S bonds. The ssDNA contains a thiolated 5'-end, a regulatory domain of 12 adenine nucleotides, and a functional domain with absolute pairing with methylated p16 sequence (Met- p16). The probe, paired with Met- p16, clearly changed the color of aggregating GNPs probe in 5 mol/L NaCl solution. Utilizing the probe, p16 gene methylation in HCT116 cells was semi-quantified. Further, the methylation of E-cadherin, p15, and p16 gene in Caco2, HepG2, and HCT116 cell lines were detected by the corresponding probes, constructed with three domains. This simple and cost-effective method was useful for the diagnosis of DNA methylation-related diseases.

  3. 金匮肾气丸对腺嘌呤致不育大鼠精子质量与肾脏功能的影响%Effect of Jinkui Shenqi Pill on Sperm Quality and Renal Function in Rats Adenine-induced Infertility

    Institute of Scientific and Technical Information of China (English)

    马亮; 刘华剑; 南亚昀; 刘镘利; 王南丁; 陈艳秋; 王宗仁; 马静


    Objective:To observe the influence of Jinkui Shenqi Pill on sperm quality and renal function in rats with adenine-induced infertility. Method: Thirty adult male SD rats were randomly divided into Jinkui Shenqi Pill group, model group and control group. Sperm qualities of experimental rats were detected by automatic sperm quality analyzer, and contents of testosterone(T), estradiol( E2 ), luteinizing hormone(LH) and follicle-stimulating hormone (FSH) were measured by radioimmunoassay. Contents of BUN and Scr were ested by automatic biochemical analyzer. Renal tissue was observed by optical microscope. Testis was observed by transmission electron microscopy. Result: Jinkui Shenqi Pill could significantly improve symptoms of Shen-yang deficiency in adenineinduced infertility rats, and it could improve the sperm quality, including sperm density, sperm motility and sperm activity. It could increase content of T obviously, lower contents of E2, LH and FSH, lower contents of BUN and Scr. And it can better the damaged kidney tissue in model rats. Conclusion: Jinkui Shenqi pill can improve sperm quality significantly and improve function of kidney. And the mechanism of improving sperm quality may be related to the increase in content of T by stimulating the interstitial cells and lowering level of E2.%目的:观察金匮肾气丸对腺嘌呤致雄性不育大鼠精子质量及肾功能的影响.方法:30只成年雄性SD大鼠随机分为空白组、模型组、金匮肾气丸组.应用自动精子质量分析仪检测实验大鼠的精子质量(精子密度、活率及活动度);应用放射免疫法测定血清睾酮(T)、雌二醇(E2)、促黄体激素(LH)和促卵泡激素(FSH)含量;自动生化分析仪分析血清尿素氮(BUN)、肌酐(Scr)含量;HE染色、光镜观察肾脏组织变化,透射电镜观察实验大鼠睾丸中精子鞭毛密度.结果:金匮肾气丸能明显增加大鼠精子密度、活率及活动度,提高大鼠血清T含量,降低E2,LH,FSH含

  4. Smoke detection

    Energy Technology Data Exchange (ETDEWEB)

    Warmack, Robert J. Bruce; Wolf, Dennis A.; Frank, Steven Shane


    Various apparatus and methods for smoke detection are disclosed. In one embodiment, a method of training a classifier for a smoke detector comprises inputting sensor data from a plurality of tests into a processor. The sensor data is processed to generate derived signal data corresponding to the test data for respective tests. The derived signal data is assigned into categories comprising at least one fire group and at least one non-fire group. Linear discriminant analysis (LDA) training is performed by the processor. The derived signal data and the assigned categories for the derived signal data are inputs to the LDA training. The output of the LDA training is stored in a computer readable medium, such as in a smoke detector that uses LDA to determine, based on the training, whether present conditions indicate the existence of a fire.

  5. Detection of E-cyanomethanimine towards Sagittarius B2(N) in the Green Bank Telescope PRIMOS Survey

    CERN Document Server

    Zaleski, Daniel P; Steber, Amanda L; Muckle, Matt T; Loomis, Ryan A; Corby, Joanna F; Martinez,, Oscar; Crabtree, Kyle N; Jewell, Philip R; Hollis, Jan M; Lovas, Frank J; Vasquez, David; Nyiramahirwe, Jolie; Sciortino, Nicole; Johnson, Kennedy; McCarthy, Michael C; Remijan, Anthony J; Pate, Brooks H


    The detection E-cyanomethanimine (E-HNCHCN) towards Sagittarius B2(N) is made by comparing the publicly available Green Bank Telescope (GBT) PRIMOS survey spectra (Hollis et al.) to laboratory rotational spectra from a reaction product screening experiment. The experiment uses broadband molecular rotational spectroscopy to monitor the reaction products produced in an electric discharge source using a gas mixture of NH3 and CH3CN. Several transition frequency coincidences between the reaction product screening spectra and previously unassigned interstellar rotational transitions in the PRIMOS survey have been assigned to E cyanomethanimine. A total of 8 molecular rotational transitions of this molecule between 9 and 50 GHz are observed with the GBT. E-cyanomethanimine, often called the HCN dimer, is an important molecule in prebiotic chemistry because it is a chemical intermediate in proposed synthetic routes of adenine, one of the two purine nucleobases found in DNA and RNA. New analyses of the rotational spe...

  6. A Novel Conductive Poly(3,4-ethylenedioxythiophene-BSA Film for the Construction of a Durable HRP Biosensor Modified with NanoAu Particles

    Directory of Open Access Journals (Sweden)

    Fangcheng Xu


    Full Text Available In this study, we have investigated the contribution of bovine serum albumin (BSA to the durability of the electrochemically synthesized poly(3,4-ethylenedioxythiophene (PEDOT film on a platinum (Pt electrode. The electrode was capable to effectively adsorb the nano Au particles (AuNPs to form a uniform layout, which was then able to immobilize the horseradish peroxidase (HRP to construct a functional HRP/AuNPs/PEDOT(BSA/Pt biosensor. Cyclic voltammetry was employed to evaluate the performance of the biosensor through the measurement of hydrogen peroxide. Our results revealed a satisfied linear correlation between the cathodic current and the concentration of H2O2. Furthermore, the addition of oxidized form of nicotinamide adenine dinucleotide, or NAD+, as the electron transfer mediator in the detection solution could dramatically enhance the sensitivity of detection by about 35.5%. The main advantages of the current biosensor are its durability, sensitivity, reliability, and biocompatibility.

  7. A hyperspectral fluorescence lifetime probe for skin cancer diagnosis (United States)

    De Beule, P. A. A.; Dunsby, C.; Galletly, N. P.; Stamp, G. W.; Chu, A. C.; Anand, U.; Anand, P.; Benham, C. D.; Naylor, A.; French, P. M. W.


    The autofluorescence of biological tissue can be exploited for the detection and diagnosis of disease but, to date, its complex nature and relatively weak signal levels have impeded its widespread application in biology and medicine. We present here a portable instrument designed for the in situ simultaneous measurement of autofluorescence emission spectra and temporal decay profiles, permitting the analysis of complex fluorescence signals. This hyperspectral fluorescence lifetime probe utilizes two ultrafast lasers operating at 355 and 440nm that can excite autofluorescence from many different biomolecules present in skin tissue including keratin, collagen, nicotinamide adenine dinucleotide (phosphate), and flavins. The instrument incorporates an optical fiber probe to provide sample illumination and fluorescence collection over a millimeter-sized area. We present a description of the system, including spectral and temporal characterizations, and report the preliminary application of this instrument to a study of recently resected (skin lesions, illustrating its potential for skin cancer detection and diagnosis.

  8. Reagentless phosphate ion sensor system for environmental monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, M.; Kurata, H.; Inoue, Y.; Shin, H. [Kyushu Institute of Technology, Fukuoka (Japan). Faculty of computer Science and Systems; Kubo, I. [Soka University, Tokyo (Japan). Faculty of Engineering; Nakamura, H.; Ikebukuro, K.; Karube, I. [The University of Tokyo, Tokyo (Japan). Research Center for Advanced Science and Technology


    Phosphate ion sensor system using an electrochemical detector was developed by the use of recombinant pyruvate oxidase (PyOD) from Lactobacillus plantarum, which needs no addition of thiamine pyrophosphate and flavin adenine dinucleotide for reaction. This system could detect 2 nM hydrogen peroxide. Response time for phosphate ion was 80 s and total measurement time for one sample was 3 min. Citrate buffer solution (pH 6.3) was most suitable for the measurement and optimum flow rate was 0.6 ml/min. Under these optimum conditions minimum detection limit of phosphate ion was 15 nM, which was enough for the determination of phosphate ion in dam-lake. 6 refs., 5 figs., 1 tab.

  9. Hypothesis: intracellular acidification contributes to infertility in varicocele. (United States)

    Ghabili, Kamyar; Shoja, Mohammadali M; Agutter, Paul S; Agarwal, Ashok


    We suggest that varicocele leads to male factor infertility by a mechanism involving underperfusion of the testis, a shortfall in glucose supply to the tissue, decreased flux through the pentose phosphate pathway, lowering of the reduced nicotinamide-adenine dinucleotide phosphate/oxidized nicotinamide-adenine dinucleotide phosphate ratio and the supply of glutathione to the antioxidant systems, increased levels of reactive oxygen species, peroxidation of spermatozoon membrane lipids, and the consequent generation of acidic degradation products and sequestering of spermine. Acidification of the seminal plasma impairs sperm motility and also inhibits most antioxidant enzymes, exacerbating the accumulation of reactive oxygen species and the resultant lowering of pH.

  10. On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Andersen, Ann Zahle; Poulsen, Allan K; Olsen, Lars Folke


    We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found that the ......We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found...

  11. Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*


    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A.


    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino ...

  12. Resolution of overlapping skin auto-fluorescence for development of non-invasive applications (United States)

    Su, Yu-Zheng; Lin, Li-Wu; Chen, Chuen-Yau; Hung, Min-Wei; Huang, Kuo-Cheng


    Skin auto-fluorescence spectra can provide useful biological information, but the obtained spectrum is overlapped and is difficult to distinguish each contributed component. We applied the genetic algorithm to decompose the overlapping spectrum. First, we simulate the overlapping spectral to confirm our feasible algorithm. The skin auto-fluorescence spectra were obtained from the normal human skin with 337 nm excitation light source. The nicotinamide adenine dinucleotid (NADH) and flavin adenine dinucleotide (FAD) are accurately decomposed and demonstrated. The developed algorithm can be widely applied to achieve qualitative and quantitative analysis for overlapping spectra.

  13. Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, L.S.; Hoffman, E.P. (Univ. of Pittsburgh Schoool of Medicine, Pittsburgh, PA (United States)); Tarleton, J. (Self Memorial Hospital, Greenwood, SC (United States)); Popovich, B. (Children' s Hosptial and Health Center, San Diego, CA (United States)); Seltzer, W.K. (Univ. of Colorado Health Sciences Center, Denver, CO (United States))


    The authors have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)[sub n] repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. The authors present the successful application of this protocol in families who proved refractory to more traditional analyses. 22 refs., 3 figs.

  14. Development of extraction protocols for life detection biosensor-based instruments (United States)

    Fornaro, Teresa; Brucato, John Robert; Pucci, Amaranta; Branciamore, Sergio


    Extraction experiments were performed to evaluate suitable sample-extraction and processing protocols that will be used by bioanalytical instruments like Life Marker Chip (LMC). These instruments will focus on the detection of molecules associated with life that will be extracted from the Martian soil. LMC is an antibody microarray biosensor instrument with optical readout, which uses fluorescently labeled antibodies, to detect and quantify the presence of polar and non-polar biomolecules, extracted from the Martian soil. The success of the LMC biomarkers detection experiment is dependent on the sample extraction protocol. The currently recommended extraction procedure, optimized for aliphatic biomarkers, consists in sonicating the samples using as solvent system 20:80 methanol:water with 1.5 mg mL-1 tween 80, which respects all the tight requirements associated with LMC biosensor. Examining this procedure for a particular class of biomarkers, the nucleobases adenine, cytosine, uracil and hypoxanthine adsorbed onto magnesium oxide (MgO), we observed low extraction efficiency, confirming that the selected solvent system is not able to extract with high efficiency aromatic biomarkers.

  15. Tuning plasmonic and chemical enhancement for SERS detection on graphene-based Au hybrids (United States)

    Liang, Xiu; Liang, Benliang; Pan, Zhenghui; Lang, Xiufeng; Zhang, Yuegang; Wang, Guangsheng; Yin, Penggang; Guo, Lin


    Various graphene-based Au nanocomposites have been developed as surface-enhanced Raman scattering (SERS) substrates recently. However, efficient use of SERS has been impeded by the difficulty of tuning SERS enhancement effects induced from chemical and plasmonic enhancement by different preparation methods of graphene. Herein, we developed graphene-based Au hybrids through physical sputtering gold NPs on monolayer graphene prepared by chemical vapor deposition (CVD) as a CVD-G/Au hybrid, as well as graphene oxide-gold (GO/Au) and reduced-graphene oxide (rGO/Au) hybrids prepared using the chemical in situ crystallization growth method. Plasmonic and chemical enhancements were tuned effectively by simple methods in these as-prepared graphene-based Au systems. SERS performances of CVD-G/Au, rGO/Au and GO/Au showed a gradually monotonic increasing tendency of enhancement factors (EFs) for adsorbed Rhodamine 6G (R6G) molecules, which show clear dependence on chemical bonds between graphene and Au, indicating that the chemical enhancement can be steadily controlled by chemical groups in a graphene-based Au hybrid system. Most notably, we demonstrate that the optimized GO/Au was able to detect biomolecules of adenine, which displayed high sensitivity with a detection limit of 10-7 M as well as good reproducibility and uniformity.Various graphene-based Au nanocomposites have been developed as surface-enhanced Raman scattering (SERS) substrates recently. However, efficient use of SERS has been impeded by the difficulty of tuning SERS enhancement effects induced from chemical and plasmonic enhancement by different preparation methods of graphene. Herein, we developed graphene-based Au hybrids through physical sputtering gold NPs on monolayer graphene prepared by chemical vapor deposition (CVD) as a CVD-G/Au hybrid, as well as graphene oxide-gold (GO/Au) and reduced-graphene oxide (rGO/Au) hybrids prepared using the chemical in situ crystallization growth method. Plasmonic

  16. 石榴皮鞣质对腺嘌呤性慢性肾衰大鼠保护作用的研究%Protective Effect of Tannin in Punica granatum L.on Chronic Renal Failure in Rats Induced by Adenine

    Institute of Scientific and Technical Information of China (English)

    周本宏; 郭志磊; 王慧媛; 冯琪


    目的 观察石榴皮鞣质对腺嘌呤性慢性肾功能衰竭(CRF)大鼠的影响.方法 用250 mg·kg~(-1)腺嘌呤溶液连续灌胃21 d使Wistar大鼠产生类似慢性肾袁的症状.21d后,治疗组大鼠每天用石榴皮鞣质(40,20,10mg·kg~(-1).d~(-1))灌胃,阳性组用20%尿毒清混悬液按2.1 g·kg~(-1)·灌胃.60 d后收集尿液,处死大鼠,取血、留取肾组织,测定血Ca~(2+)、P~(3+)及血肌酐(Scr)、尿素氮(BUN),血脂和24h尿蛋白定量、尿Ca~(2+)、P~(3+),进行统计学处理,并进行形态学观察.结果 石榴皮鞣质对慢性肾衰竭大鼠的血Ca~(2+)、P~(3+)及Scr、BUN和24 h尿蛋白定量、尿Ca~(2+)、P~(3+)有不同程度的改善.结论 石榴皮鞣质对腺嘌呤导致的大鼠慢性肾衰竭有一定的保护作用.%OBJECTIVE To observe the effeets of tannin in Punica granatum L.on chronic renal failure(CRF)in rats induced by adenine.METHODS The Wistar rats were administered intragastrically with adenine(200 mg·kg~(-1)·d~(-1))for 21 d to induce the symptoms similar to chronic renal failure.Then,the rats were administered intragastrically with tannin(40,20 and 10 blood urea nitrogen(BUN),calcium ion(Ca~(2+)),phosphorus ion(P~(3+)),blood lipids and urine protein of 24 h were determined and analyzed.Meanwhile morphology was observed.RESULTS Serum levels of Scr,BUN and P~(3+) were decreased but Ca~(2+) level was increased by tannin in Punica granatum L.CONCLUSION Tannin in Punica granatum L can improve the renal function injuried by metabolic products in adenine-modeled CRF rats.

  17. Detection of neuronal tissue in meat using tissue specific DNA modifications

    Directory of Open Access Journals (Sweden)

    Harris N.


    Full Text Available A method has been developed to differentiate between non-muscle tissues such as liver, kidney and heart and that of muscle in meat samples using tissue specific DNA detection. Only muscle tissue is considered meat from the point of view of labelling (Food Labelling [Amendment] (England Regulations 2003 and Quantitative Ingredient Declaration (QUID, and also certain parts of the carcass are prohibited to be used in raw meat products (Meat Products [England] Regulations 2003. Included in the prohibited offal are brain and spinal cord. The described methodology has therefore been developed primarily to enforce labelling rules but also to contribute to the enforcement of BSE legislation on the detection of Central Nervous System (CNS tissue. The latter requires the removal of Specified Risk Material (SRM, such as bovine and ovine brain and spinal cord, from the food chain. Current methodologies for detection of CNS tissue include histological examination, analysis of cholesterol content and immunodetection. These can potentially be time consuming, less applicable to processed samples and may not be readily adapted to high throughput sample analysis. The objective of this work was therefore to develop a DNAbased detection assay that exploits the sensitivity and specificity of PCR and is potentially applicable to more highly processed food samples. For neuronal tissue, the DNA target selected was the promoter for Glial Fibrillary Acidic Protein (GFAP, a gene whose expression is restricted to astroglial cells within CNS tissue. The promoter fragments from both cattle and sheep have been isolated and key differences in the methylation patterns of certain CpG dinucleotides in the sequences from bovine and sheep brain and spinal cord and the corresponding skeletal muscle identified. These have been used to design a PCR assay exploiting Methylation Specific PCR (MSP to specifically amplify the neuronal tissue derived sequence and therefore identify the

  18. Silver nanoparticles embedded in amine-functionalized silicate sol-gel network assembly for sensing cysteine, adenosine and NADH

    Energy Technology Data Exchange (ETDEWEB)

    Maduraiveeran, Govindhan; Ramaraj, Ramasamy, E-mail: [Madurai Kamaraj University, Centre for Photoelectrochemistry, School of Chemistry (India)


    Silver nanoparticles embedded in amine-functionalized silicate sol-gel network were synthesized and used for sensing biomolecules such as cysteine, adenosine, and {beta}-nicotinamide adenine dinucleotide (NADH). The sensing of these biomolecules by the assembly of silver nanoparticles was triggered by the optical response of the surface plasmon resonance (SPR) of the silver nanoparticles. The optical sensor exhibited the lowest detection limit (LOD) of 5, 20, and 5 {mu}M for cysteine, adenosine, and NADH, respectively. The sensing of biomolecules in the micromolar range by using the amine-functionalized silicate sol-gel embedded silver nanoparticles was studied in the presence of interference molecules like uridine, glycine, guanine, and guanosine. Thus, the present approach might open up a new avenue for the development of silver nanoparticles-based optical sensor devices for biomolecules.

  19. Network analysis of biochemical logic for noise reduction and stability: a system of three coupled enzymatic and gates. (United States)

    Privman, Vladimir; Arugula, Mary A; Halámek, Jan; Pita, Marcos; Katz, Evgeny


    We develop an approach aimed at optimizing the parameters of a network of biochemical logic gates for reduction of the "analog" noise buildup. Experiments for three coupled enzymatic AND gates are reported, illustrating our procedure. Specifically, starch, one of the controlled network inputs, is converted to maltose by beta-amylase. With the use of phosphate (another controlled input), maltose phosphorylase then produces glucose. Finally, nicotinamide adenine dinucleotide (NAD(+)), the third controlled input, is reduced under the action of glucose dehydrogenase to yield the optically detected signal. Network functioning is analyzed by varying selective inputs and fitting standardized few-parameters "response-surface" functions assumed for each gate. This allows a certain probe of the individual gate quality, but primarily yields information on the relative contribution of the gates to noise amplification. The derived information is then used to modify our experimental system to put it in a regime of a less noisy operation.

  20. Evaluating Edge Detection through Boundary Detection

    Directory of Open Access Journals (Sweden)

    Wang Song


    Full Text Available Edge detection has been widely used in computer vision and image processing. However, the performance evaluation of the edge-detection results is still a challenging problem. A major dilemma in edge-detection evaluation is the difficulty to balance the objectivity and generality: a general-purpose edge-detection evaluation independent of specific applications is usually not well defined, while an evaluation on a specific application has weak generality. Aiming at addressing this dilemma, this paper presents new evaluation methodology and a framework in which edge detection is evaluated through boundary detection, that is, the likelihood of retrieving the full object boundaries from this edge-detection output. Such a likelihood, we believe, reflects the performance of edge detection in many applications since boundary detection is the direct and natural goal of edge detection. In this framework, we use the newly developed ratio-contour algorithm to group the detected edges into closed boundaries. We also collect a large data set ( of real images with unambiguous ground-truth boundaries for evaluation. Five edge detectors (Sobel, LoG, Canny, Rothwell, and Edison are evaluated in this paper and we find that the current edge-detection performance still has scope for improvement by choosing appropriate detectors and detector parameters.

  1. Using CdTe/ZnSe core/shell quantum dots to detect DNA and damage to DNA (United States)

    Moulick, Amitava; Milosavljevic, Vedran; Vlachova, Jana; Podgajny, Robert; Hynek, David; Kopel, Pavel; Adam, Vojtech


    CdTe/ZnSe core/shell quantum dot (QD), one of the strongest and most highly luminescent nanoparticles, was directly synthesized in an aqueous medium to study its individual interactions with important nucleobases (adenine, guanine, cytosine, and thymine) in detail. The results obtained from the optical analyses indicated that the interactions of the QDs with different nucleobases were different, which reflected in different fluorescent emission maxima and intensities. The difference in the interaction was found due to the different chemical behavior and different sizes of the formed nanoconjugates. An electrochemical study also confirmed that the purines and pyrimidines show different interactions with the core/shell QDs. Based on these phenomena, a novel QD-based method is developed to detect the presence of the DNA, damage to DNA, and mutation. The QDs were successfully applied very easily to detect any change in the sequence (mutation) of DNA. The QDs also showed their ability to detect DNAs directly from the extracts of human cancer (PC3) and normal (PNT1A) cells (detection limit of 500 pM of DNA), which indicates the possibilities to use this easy assay technique to confirm the presence of living organisms in extreme environments. PMID:28243089

  2. Chitosan-coated anisotropic silver nanoparticles as a SERS substrate for single-molecule detection (United States)

    Potara, Monica; Baia, Monica; Farcau, Cosmin; Astilean, Simion


    Surface-enhanced Raman spectroscopy (SERS) is a technique that has become widely used for identifying and providing structural information about molecular species in low concentration. There is an ongoing interest in finding optimum particle size, shape and spatial distribution for optimizing the SERS substrates and pushing the sensitivity toward the single-molecule detection limit. This work reports the design of a novel, biocompatible SERS substrate based on small clusters of anisotropic silver nanoparticles embedded in a film of chitosan biopolymer. The SERS efficiency of the biocompatible film is assessed by employing Raman imaging and spectroscopy of adenine, a significant biological molecule. By combining atomic force microscopy with SERS imaging we find that the chitosan matrix enables the formation of small clusters of silver nanoparticles, with junctions and gaps that greatly enhance the Raman intensities of the adsorbed molecules. The study demonstrates that chitosan-coated anisotropic silver nanoparticle clusters are sensitive enough to be implemented as effective plasmonic substrates for SERS detection of nonresonant analytes at the single-molecule level.

  3. Detection of Factor XI Deficiency (FXID and Complex Vertebral Malformation (CVM in Bali Cattle

    Directory of Open Access Journals (Sweden)

    S. W. Siswanti


    Full Text Available Factor XI Deficiency (FXID is caused by imperfect insertion of poly adenine which is resulted in introduction of premature stop codon in FXI gene. Substitution of guanine into thymine in SLC35A3 gene caused Complex Vertebral Malformation (CVM. The research was aimed to detect the presence or absence of a genetic defect mainly CVM using SLC35A3 gene and FXID using FXI gene in Indonesian Bali cattle. The presence of this genetic defect may have a significant economic impact on the breeding program. The research of genetic defect was done mostly in dairy cattle, but there was no report for screening of genetic defect in Bali cattle. In this study, 303 fresh blood samples and 22 semen samples which were collected from Indonesian Bali cattle breeding center (BPTU HMT Denpasar, BPT HMT Serading West Nusa Tenggara and district Barru South Sulawesi and artificial insemination centre (BBIB Singosari and BIBD Baturiti were used for screening of FXID and CVM. The amplicons of FXI gene were obtained by using PCR and that for SLC35A3 gene were obtained by using PCR-RFLP method with PstI restriction enzyme. These PCR products were analyzed by using 2% agarose gels electrophoresis. All genotypes were confirmed by DNA sequencing to determine an allele mutant. The allele mutant was not found in all of the samples. The result of this study showed that CVM and FXID were not detected in Bali cattle from Indonesian Bali cattle breeding and artificial insemination centres.

  4. Multicolor excitation two-photon microscopy: in vivo imaging of cells and tissues (United States)

    Li, Dong; Zheng, Wei; Qu, Jianan Y.


    Two-photon microscopy based on endogenous fluorescence provides non-invasive imaging of living biological system. Reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), keratin, collagen and elastin are the endogenous fluorophores widely used as the contrast agents for imaging metabolism and morphology of living cells and tissue. The fluorescence of tryptophan, a kind of essential amino acid, conveys the information on cellular protein content, structure and microenvironment. However, it can't be effectively excited by the commonly used Ti:sapphire femtosecond laser. Because each endogenous fluorophore provides limited information, it is desirable to simultaneously excite fluorescence from as many fluorophores as possible to obtain accurate biochemical and morphological information on biomedical samples. In this study, we demonstrate that the supercontinuum generation from a photonic crystal fiber (PCF) excited by an ultrafast source can be used to excite multiple endogenous nonlinear optical signals simultaneously. By employing the spectral lifetime detection capability, this technology provides a unique approach to sense the fine structure, protein distribution and cellular metabolism of cells and tissues in vivo. In particular, with application of acetic acid, a safe contrast agent used for detection cervical cancer for many years, the tryptophan signals reveal cellular morphology and even cell-cell junctions clearly. Moreover, it was found that the pH value dependent lifetime of tryptophan fluorescence could provide the qualitative information on the gradient of pH value in epithelial tissue. Finally, we will demonstrate the potential of our multi-color TPEF microscopy to investigate the early development of cancer in epithelial tissue.

  5. Novel Flurometric Tool to Assess Mitochondrial Redox State of Isolated Perfused Rat Lungs After Exposure to Hyperoxia (United States)

    Audi, Said H.; Staniszewski, Kevin S.; Haworth, Steven T.; Jacobs, Elizabeth R.; Ranji, Mahsa; Zablocki, Clement J.


    Recently, we demonstrated the utility of optical fluorometry to detect a change in the redox status of mitochondrial autofluorescent coenzymes nicotinamide adenine dinucleotide (NADH) and oxidized form of flavin adenine dinucleotide \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}$({\\rm FADH}_{2})$\\end{document} (FAD), as a measure of mitochondrial function in isolated perfused rat lungs (IPL). The objective of this paper was to utilize optical fluorometry to evaluate the effect of rat exposure to hyperoxia (\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{upgreek} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} }{}${>}{95\\%}~{\\rm O}_{2}$\\end{document} for 48 h) on lung tissue mitochondrial redox status of NADH and FAD in a nondestructive manner in IPL. Surface NADH and FAD signals were measured before and after lung perfusion with perfusate containing rotenone (ROT, complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor), and/or pentachlorophenol (PCP, uncoupler). ROT- or KCN-induced increase in NADH signal is considered a measure of complex I activity, and KCN-induced decrease in FAD signal is considered a measure of complex II activity. The results show that hyperoxia decreased complex I and II activities by 63% and 55%, respectively, when compared to lungs of rats exposed to room air (normoxic rats). Mitochondrial complex I and II activities in lung homogenates were also lower (77% and 63%, respectively) for hyperoxic than for normoxic lungs. These results suggest that the mitochondrial matrix is more reduced in hyperoxic lungs than in normoxic lungs, and demonstrate the ability of optical fluorometry to detect a change

  6. Raman Spectroscopy of the Interferon-Induced 2’,5’-Oligoadenylates (United States)


    generation of the Raman spectrum of triethyl ammonium ion ••••••••••••••••••••••••••••••• 41 12. structures of purine, adenine, purine riboside , adenosine...ribose 5 1-phosphate, AMP, and ATP........ 48 13. Raman spectra of adenine and purine •••••••.••••••••• 49 14. Raman spectra of purine riboside and... nicotinamide adenine dinucleotide; TFAB, triethyl anunonium bicarbonate; TFA, triethyl amm::mium. ion; CD circular _dichroism; NMR, nuclear magnetic

  7. Enhanced energy transfer in respiratory-deficient endothelial cells probed by microscopic fluorescence excitation spectroscopy (United States)

    Schneckenburger, Herbert; Gschwend, Michael H.; Bauer, Manfred; Strauss, Wolfgang S. L.; Steiner, Rudolf W.


    Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.

  8. Adenine translocase 1gene transfection induces apoptosis of vascular smooth muscle cells in rats with carotid balloon injury%转染ANT1基因诱导颈动脉球囊损伤大鼠血管平滑肌细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    杨易; 李燕; 王霄; 刘洁; 卢巍; 宋耀明


    Objective To study the effect of adenine nucleotide translocase 1 (ANT1) gene over-expression on apoptosis of vascular smooth muscle cells ( VSMC ) in a rat carotid balloon injury model induced by adenovirus plasmid. Methods Seventy-two SD rats were randomly divided into normal group, non-transfection group, Ad-GFP transfection group and Ad-ANTl transfection group (18 in each group). ANT1 gene was trans-fected into rat carotid arteries with Ad-ANTl adenovirus after a rat carotid balloon injury model was established. The arteries were harvested on days 7, 14 and 28 after operation. Expression of ANT-1, BAX, and Bcl-2 in arteries was detected by RT-PCR, Western blotting, and immunohistochemistry, respectively, with HE staining. Apoptosis of VSMC in tunica intima and tunica media of the model was assayed with TUNEL staining. Results The ANT1 gene was significantly expressed in rat carotid arteries after Ad-ANTl transfection and reached its peak level on day 14, which was significantly higher in Ad-ANTl transfection group than in Ad-GFP transfection group and balloon injury group (P 0. 05). The apoptosis rate of VSMC in tunica intima and tunica media of the model was significantly higher in Ad-ANTl transfection group than in the other 3 groups (P <0. 05). The tunica intima/ tunica media area ratio in the model was lower in Ad-ANTl transfection group than in Ad-GFP transfection group and balloon injury group on days 14 and 28 after transfection(P<0. 05). Conclusion Adenovirus plasmid-induced over-expression of ANT1 gene induces apoptosis of VSMC in tunica intima/ tunica media of the model by up-regulating the expression of BAX.%目的 通过腺病毒载体在体转染大鼠颈总动脉球囊损伤模型,研究过表达腺嘌呤核苷酸转位酶-1( adeninenucleotide translocase 1,ANT1)对血管平滑肌细胞凋亡的影响.方法 将72只雄性SD大鼠随机分为正常组、单纯损伤组、Ad-GFP转染组、Ad-ANT1转染组,每组18只.用携带ANT1

  9. Thiamin and riboflavin vitamers in human milk: effects of lipid-based nutrient supplementation and stage of lactation on vitamer secretion and contributions to total vitamin content (United States)

    While thiamin and riboflavin in breast milk have been analyzed for over 50 years, less attention has been given to the different forms of each vitamin. Thiamin-monophosphate (TMP) and free thiamin contribute to total thiamin content; flavin adenine-dinucleotide (FAD) and free riboflavin are the main...

  10. Efficient regeneration of NADPH in a 3-enzyme cascade reaction by in situ generation of glucose 6-phosphate from glucose and pyrophosphate

    NARCIS (Netherlands)

    Hartog, A.F.; van Herk, T.; Wever, R.


    We report here a promising method to regenerate NADPH (nicotinamide adenine dinucleotide phosphate) using the intermediate formation of glucose 6-phosphate (G6P) from glucose and pyrophosphate (PPi) catalyzed by the acid phosphatase from Shigella flexneri (PhoN-Sf). The G6P formed is used in turn by

  11. SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction (United States)

    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...

  12. Metabolic precursors in astrophysical ice analogs: implications for meteorites and comets. (United States)

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


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

    NARCIS (Netherlands)

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


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

  14. Innate Immunity Dysregulation in Myelodysplastic Syndromes (United States)


    IL, interleukin; ITAM, immunoreceptor tyrosine-based activating motif; MDS, myelodysplastic syndromes; NADPH, nicotinamide adenine dinucleotide...differentiation, including macrophage, skin cells and neurons.19,2on In this study, we have shown that knock down of JMJ03 in BM C034 + cells of lower

  15. Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1

    NARCIS (Netherlands)

    Sucharitakul, J.; Wongnate, T.; Montersino, S.; Berkel, van W.J.H.; Chaiyen, P.


    3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB

  16. Riboflavin carrier protein-targeted fluorescent USPIO for the assessment of vascular metabolism in tumors

    NARCIS (Netherlands)

    Jayapaul, J.; Arns, S.; Lederle, W.; Lammers, T.G.G.M.; Comba, P.; Gätjens, J.; Kiessling, F.


    Abstract Riboflavin (Rf) and its metabolic analogs flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are essential for normal cellular growth and function. Their intracellular transport is regulated by the riboflavin carrier protein (RCP), which has been shown to be over-expressed b

  17. Biosensors Based on Ultrathin Film Composite Membranes (United States)


    adenine dinucleotide (FAD) cofactor associated with the enzyme in its reduced state ( FADH 2) (17). FADH2 is reo:idized by the 3 oxidized form of the...this type. This is because 02 can also oxidize FADH ,. Thus, if the 02 concentration in the analyte solution changes during an analysis, the response

  18. Reversible resolution of flavin and pterin cofactors of His-tagged Escherichia coli DNA photolyse.

    NARCIS (Netherlands)

    Xu, L.; Zhang, D.; Mu, W.; Berkel, van W.J.H.; Luo, Z.


    Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein-chrom

  19. Detection of Terahertz Radiation

    DEFF Research Database (Denmark)


    The present invention relates to a system for detecting terahertz radiation, a camera device, and a method for detecting terahertz radiation.......The present invention relates to a system for detecting terahertz radiation, a camera device, and a method for detecting terahertz radiation....

  20. On Edge Detection, (United States)


    T. 0. "On boundary detection." A. I. Memo 183, MIT, 1980. Hildreth, E. C. "Implementation of a theory of edge detection ." A. /. Memo 579, MIT, 1980...Detection." IEEE Trans. PAMI, 6, 678-680, 1983. Marr, 0. C. and Hildreth, E. C, " Theory of edge detection ." Proc. R. Soc. Lond. B, 207, 187-217, 1980. Marr

  1. Outlier detection using autoencoders

    CERN Document Server

    Lyudchik, Olga


    Outlier detection is a crucial part of any data analysis applications. The goal of outlier detection is to separate a core of regular observations from some polluting ones, called “outliers”. We propose an outlier detection method using deep autoencoder. In our research the invented method was applied to detect outlier points in the MNIST dataset of handwriting digits. The experimental results show that the proposed method has a potential to be used for anomaly detection.

  2. Metabolomics analysis of metabolic effects of nicotinamide phosphoribosyltransferase (NAMPT) inhibition on human cancer cells. (United States)

    Tolstikov, Vladimir; Nikolayev, Alexander; Dong, Sucai; Zhao, Genshi; Kuo, Ming-Shang


    Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide-consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry-based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level.

  3. Optical metabolic imaging of treatment response in human head and neck squamous cell carcinoma.

    Directory of Open Access Journals (Sweden)

    Amy T Shah

    Full Text Available Optical metabolic imaging measures fluorescence intensity and lifetimes from metabolic cofactors nicotinamide adenine dinucleotide (NADH and flavin adenine dinucleotide (FAD. These molecular level measurements provide unique biomarkers for early cellular responses to cancer treatments. Head and neck squamous cell carcinoma (HNSCC is an attractive target for optical imaging because of easy access to the site using fiber optic probes. Two HNSCC cell lines, SCC25 and SCC61, were treated with Cetuximab (anti-EGFR antibody, BGT226 (PI3K/mTOR inhibitor, or cisplatin (chemotherapy for 24 hours. Results show increased redox ratio, NADH α1 (contribution from free NADH, and FAD α1 (contribution from protein-bound FAD for malignant cells compared with the nonmalignant cell line OKF6 (p<0.05. In SCC25 and SCC61 cells, the redox ratio is unaffected by cetuximab treatment and decreases with BGT226 and cisplatin treatment (p<0.05, and these results agree with standard measurements of proliferation rates after treatment. For SCC25, NADH α1 is reduced with BGT226 and cisplatin treatment. For SCC61, NADH α1 is reduced with cetuximab, BGT226, and cisplatin treatment. Trends in NADH α1 are statistically similar to changes in standard measurements of glycolytic rates after treatment. FAD α1 is reduced with cisplatin treatment (p<0.05. These shifts in optical endpoints reflect early metabolic changes induced by drug treatment. Overall, these results indicate that optical metabolic imaging has potential to detect early response to cancer treatment in HNSCC, enabling optimal treatment regimens and improved patient outcomes.

  4. Metabolomics analysis of metabolic effects of nicotinamide phosphoribosyltransferase (NAMPT inhibition on human cancer cells.

    Directory of Open Access Journals (Sweden)

    Vladimir Tolstikov

    Full Text Available Nicotinamide phosphoribosyltransferase (NAMPT plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide-consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry-based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer and HCT-116 (colorectal cancer cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA, and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level.

  5. Raman spectroscopic study of acute oxidative stress induced changes in mice skeletal muscles (United States)

    Sriramoju, Vidyasagar; Alimova, Alexandra; Chakraverty, Rahul; Katz, A.; Gayen, S. K.; Larsson, L.; Savage, H. E.; Alfano, R. R.


    The oxidative stress due to free radicals is implicated in the pathogenesis of tissue damage in diseases such as muscular dystrophy, Alzheimer dementia, diabetes mellitus, and mitochrondrial myopathies. In this study, the acute oxidative stress induced changes in nicotinamide adenine dinucleotides in mouse skeletal muscles are studied in vitro using Raman spectroscopy. Mammalian skeletal muscles are rich in nicotinamide adenine dinucleotides in both reduced (NADH) and oxidized (NAD) states, as they are sites of aerobic and anaerobic respiration. The relative levels of NAD and NADH are altered in certain physiological and pathological conditions of skeletal muscles. In this study, near infrared Raman spectroscopy is used to identify the molecular fingerprints of NAD and NADH in five-week-old mice biceps femoris muscles. A Raman vibrational mode of NADH is identified in fresh skeletal muscle samples suspended in buffered normal saline. In the same samples, when treated with 1% H IIO II for 5 minutes and 15 minutes, the Raman spectrum shows molecular fingerprints specific to NAD and the disappearance of NADH vibrational bands. The NAD bands after 15 minutes were more intense than after 5 minutes. Since NADH fluoresces and NAD does not, fluorescence spectroscopy is used to confirm the results of the Raman measurements. Fluorescence spectra exhibit an emission peak at 460 nm, corresponding to NADH emission wavelength in fresh muscle samples; while the H IIO II treated muscle samples do not exhibit NADH fluorescence. Raman spectroscopy may be used to develop a minimally invasive, in vivo optical biopsy method to measure the relative NAD and NADH levels in muscle tissues. This may help to detect diseases of muscle, including mitochondrial myopathies and muscular dystrophies.

  6. In vivo diagnosis of colonic precancer and cancer using near-infrared autofluorescence spectroscopy and biochemical modeling (United States)

    Shao, Xiaozhuo; Zheng, Wei; Huang, Zhiwei


    The aim of this study is to evaluate the biochemical foundation and clinical capability of an image-guided near-infrared (NIR) autofluorescence (AF) spectroscopy technique for in vivo diagnosis of colonic malignancies during clinical colonoscopy. A novel endoscopic fiber-optic AF system was utilized for in vivo NIR AF measurements at 785 nm excitation. A total of 263 in vivo NIR AF spectra of colonic tissues were measured from 100 patients, in which 164 spectra were from benign tissue (116 normal and 48 hyperplastic polyps), 34 spectra were from precancer (adenomatous polyps), and 65 spectra were from cancer. The non-negativity constrained least squares minimization biochemical modeling was explored to estimate the biochemical compositions of colonic tissue using nine basis reference spectra from the representative biochemicals (i.e., collagen I, elastin, β-nicotinamide adenine dinucleotide, flavin adenine dinucleotide, L-tryptophan, hematoporphyrin, 4-pyridoxic acid, pyridoxal 5'-phosphate, and water) associated with structural or cellular metabolic progression in colonic precancer and cancer. High-quality in vivo NIR AF spectra in the spectral range of 810 to 1000 nm were acquired from colonic benign, precancerous, and cancerous mucosa under white-light reflectance endoscopic imaging guidance. Partial least squares discriminant analysis, together with the leave-one tissue site-out, cross validation on in vivo NIR AF spectra yields diagnostic sensitivities of 85.4%, 76.5%, and 84.6%, and specificities of 89.9%, 93.4%, and 91.4%, respectively, for classification of benign, precancer, and cancer in the colon. This work demonstrates that image-guided NIR AF spectroscopy in conjunction with biochemical modeling has promising potential for improving in vivo detection and diagnosis of colonic precancer and cancer during clinical colonoscopic screening.

  7. Time-gated FLIM microscope for corneal metabolic imaging (United States)

    Silva, Susana F.; Batista, Ana; Domingues, José Paulo; Quadrado, Maria João.; Morgado, António Miguel


    Detecting corneal cells metabolic alterations may prove a valuable tool in the early diagnosis of corneal diseases. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are autofluorescent metabolic co-factors that allow the assessment of metabolic changes through non-invasive optical methods. These co-factors exhibit double-exponential fluorescence decays, with well-separated short and lifetime components, which are related to their protein-bound and free-states. Corneal metabolism can be assessed by measuring the relative contributions of these two components. For that purpose, we have developed a wide-field time-gated fluorescence lifetime microscope based on structured illumination and one-photon excitation to record FAD lifetime images from corneas. NADH imaging was not considered as its UV excitation peak is regarded as not safe for in vivo measurements. The microscope relies on a pulsed blue diode laser (λ=443 nm) as excitation source, an ultra-high speed gated image intensifier coupled to a CCD camera to acquire fluorescence signals and a Digital Micromirror Device (DMD) to implement the Structured Illumination technique. The system has a lateral resolution better than 2.4 μm, a field of view of 160 per 120 μm and an optical sectioning of 6.91 +/- 0.45 μm when used with a 40x, 0.75 NA, Water Immersion Objective. With this setup we were able to measure FAD contributions from ex-vivo chicken corneas collected from a local slaughterhouse..

  8. Synchronous in-field application of life-detection techniques in planetary analog missions (United States)

    Amador, Elena S.; Cable, Morgan L.; Chaudry, Nosheen; Cullen, Thomas; Gentry, Diana; Jacobsen, Malene B.; Murukesan, Gayathri; Schwieterman, Edward W.; Stevens, Adam H.; Stockton, Amanda; Yin, Chang; Cullen, David C.; Geppert, Wolf


    Field expeditions that simulate the operations of robotic planetary exploration missions at analog sites on Earth can help establish best practices and are therefore a positive contribution to the planetary exploration community. There are many sites in Iceland that possess heritage as planetary exploration analog locations and whose environmental extremes make them suitable for simulating scientific sampling and robotic operations. We conducted a planetary exploration analog mission at two recent lava fields in Iceland, Fimmvörðuháls (2010) and Eldfell (1973), using a specially developed field laboratory. We tested the utility of in-field site sampling down selection and tiered analysis operational capabilities with three life detection and characterization techniques: fluorescence microscopy (FM), adenine-triphosphate (ATP) bioluminescence assay, and quantitative polymerase chain reaction (qPCR) assay. The study made use of multiple cycles of sample collection at multiple distance scales and field laboratory analysis using the synchronous life-detection techniques to heuristically develop the continuing sampling and analysis strategy during the expedition. Here we report the operational lessons learned and provide brief summaries of scientific data. The full scientific data report will follow separately. We found that rapid in-field analysis to determine subsequent sampling decisions is operationally feasible, and that the chosen life detection and characterization techniques are suitable for a terrestrial life-detection field mission. In-field analysis enables the rapid obtainment of scientific data and thus facilitates the collection of the most scientifically relevant samples within a single field expedition, without the need for sample relocation to external laboratories. The operational lessons learned in this study could be applied to future terrestrial field expeditions employing other analytical techniques and to future robotic planetary exploration

  9. Revolution in Detection Affairs

    Energy Technology Data Exchange (ETDEWEB)

    Stern W.


    The detection of nuclear or radioactive materials for homeland or national security purposes is inherently difficult. This is one reason detection efforts must be seen as just one part of an overall nuclear defense strategy which includes, inter alia, material security, detection, interdiction, consequence management and recovery. Nevertheless, one could argue that there has been a revolution in detection affairs in the past several decades as the innovative application of new technology has changed the character and conduct of detection operations. This revolution will likely be most effectively reinforced in the coming decades with the networking of detectors and innovative application of anomaly detection algorithms.

  10. Colorimetric DNA detection of transgenic plants using gold nanoparticles functionalized with L-shaped DNA probes (United States)

    Nourisaeid, Elham; Mousavi, Amir; Arpanaei, Ayyoob


    In this study, a DNA colorimetric detection system based on gold nanoparticles functionalized with L-shaped DNA probes was prepared and evaluated. We investigated the hybridization efficiency of the L-shaped probes and studied the effect of nanoparticle size and the L-shaped DNA probe length on the performance of the as-prepared system. Probes were attached to the surface of gold nanoparticles using an adenine sequence. An optimal sequence of 35S rRNA gene promoter from the cauliflower mosaic virus, which is frequently used in the development of transgenic plants, and the two complementary ends of this gene were employed as model target strands and probe molecules, respectively. The spectrophotometric properties of the as-prepared systems indicated that the large NPs show better changes in the absorption spectrum and consequently present a better performance. The results of this study revealed that the probe/Au-NPs prepared using a vertical spacer containing 5 thymine oligonucleotides exhibited a stronger spectrophotometric response in comparison to that of larger probes. These results in general indicate the suitable performance of the L-shaped DNA probe-functionalized Au-NPs, and in particular emphasize the important role of the gold nanoparticle size and length of the DNA probes in enhancing the performance of such a system.

  11. Interior intrusion detection systems

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, J.R.; Matter, J.C. (Sandia National Labs., Albuquerque, NM (United States)); Dry, B. (BE, Inc., Barnwell, SC (United States))


    The purpose of this NUREG is to present technical information that should be useful to NRC licensees in designing interior intrusion detection systems. Interior intrusion sensors are discussed according to their primary application: boundary-penetration detection, volumetric detection, and point protection. Information necessary for implementation of an effective interior intrusion detection system is presented, including principles of operation, performance characteristics and guidelines for design, procurement, installation, testing, and maintenance. A glossary of sensor data terms is included. 36 figs., 6 tabs.

  12. Synthesis of salicylaldehyde-adenine schiff base and its application for the spectrophotometric determination of copper(Ⅱ)%水杨醛缩腺嘌呤席夫碱的合成及其光度法测定铜(Ⅱ)的研究

    Institute of Scientific and Technical Information of China (English)

    洪涛; 龙巍然; 陶晋飞; 刘鹏; 曹秋娥


    以水杨醛和腺嘌呤为原料,合成了一个新的席夫碱(水杨醛缩腺嘌呤席夫碱,SASB),并对其结构进行了鉴定.以该席夫碱为显色剂,在优化了反应条件后,建立了一个测定痕量Cu2+的新光度分析方法.方法在400 nm处的表现摩尔吸光系数为7.68×104 L-mol-1·cm-1,线性范围为0.05~0.60μg/mL.常见离子都不干扰测定,加入2 mL 10.0 g/L NH4F溶液作为掩蔽剂时,Al3+和Fe3+的允许量可分别提高到75倍和25倍.将方法用于铝合金中铜的测定,结果同原子吸收光谱法的测定值一致,相对标准偏差在2.5%以内.%A new schiff base (salicylaldehyde-adenine schiff base. SASB) has been synthesized by the condensation reaction between salicylaldehyde and adenine. And its structure has been identified. With schiff base as color reagents, a new spectrophotometric method for the determination of trace Cu2+ has been established after the conditions of the reaction between SASB and Cu2+ was optimized. The linear range and apparent molar absorptivity at 400nm was 0. 05-0. 60 μg/mL and 7. 68 ×104 L · mol-1 · cm-1, respectively. The method, which was free from the interference of much common ions, could be used for the determination of copper in the presence of 75 times of Al3+ and 25 times of Fe3+ after 2 mL of 10. 0 g/L NH4F was added as screening agent. The results for the determination of trace copper in aluminium alloys obtained by this method with the standard deviation of less than 2. 5% was in agreement with that obtained by AAS.

  13. Enzyme detection by microfluidics

    DEFF Research Database (Denmark)


    Microfluidic-implemented methods of detecting an enzyme, in particular a DNA-modifying enzyme, are provided, as well as methods for detecting a cell, or a microorganism expressing said enzyme. The enzyme is detected by providing a nucleic acid substrate, which is specifically targeted...... by that enzyme...

  14. Autonomous Forest Fire Detection

    NARCIS (Netherlands)

    Breejen, E. den; Breuers, M.; Cremer, F.; Kemp, R.A.W.; Roos, M.; Schutte, K.; Vries, J.S. de


    Forest fire detection is a very important issue in the pre-suppression process. Timely detection allows the suppression units to reach the fire in its initial stages and this will reduce the suppression costs considerably. The autonomous forest fire detection principle is based on temporal contrast

  15. Development of a Fluorescent Multiplex Assay for Detection of MSI-High Tumors

    Directory of Open Access Journals (Sweden)

    Jeffery W. Bacher


    Full Text Available Determining whether a tumor exhibits microsatellite instability (MSI is useful in identifying patients with hereditary non-polyposis colorectal cancer and sporadic gastrointestinal cancers with defective DNA mismatch repair (MMR. The assessment of MSI status aids in establishing a clinical prognosis and may be predictive of tumor response to chemotherapy. A reference panel of five markers was suggested for MSI analysis by a National Cancer Institute (NCI workshop in 1997 that has helped to standardize testing. But this panel of markers has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumors with MMR deficiencies compared to other types of markers that are currently available. This study demonstrates that mononucleotides are the most sensitive and specific markers for detection of tumors with defects in MMR and identifies an optimal panel of markers for detection of MSI-H tumors. A set of 266 mono-, di-, tetra- and penta-nucleotide repeat microsatellite markers were used to screen for MSI in colorectal tumors. The best markers for detection of MSI-H tumors were selected for a MSI Multiplex System, which included five mononucleotide markers: BAT-25, BAT-26, NR-21, NR-24 and MONO-27. In addition, two pentanucleotide markers were added to identify sample mix-ups and/or contamination. We classified 153 colorectal tumors using the new MSI Multiplex System and compared the results to those obtained with a panel of 10 microsatellite markers combined with immunohistochemical (IHC analysis. We observed 99% concordance between the two methods with nearly 100% accuracy in detection of MSI-H tumors. Approximately 5% of the MSI-H tumors had normal levels of four MMR proteins and as a result would have been misclassified based solely on IHC analysis, emphasizing the importance of performing MSI testing. The new MSI Multiplex System offers several distinct advantages over other methods

  16. Can Gravitons Be Detected?

    CERN Document Server

    Rothman, T; Boughn, Stephen; Rothman, Tony


    Freeman Dyson has questioned whether any conceivable experiment in the real universe can detect a single graviton. If not, is it meaningful to talk about gravitons as physical entities? We attempt to answer Dyson's question and find it is possible concoct an idealized thought experiment capable of detecting one graviton; however, when anything remotely resembling realistic physics is taken into account, detection becomes impossible, indicating that Dyson's conjecture is very likely true. We also point out several mistakes in the literature dealing with graviton detection and production.

  17. Explicit Content Image Detection

    Directory of Open Access Journals (Sweden)

    Jorge Alberto Marcial Basilio


    Full Text Available This paper proposes a system gives for explicit content image detection based on Computer VisionAlgorithms, pattern recognition and FTK software Explicit Image Detection. In the first stage, HSV colormodel is used for the input images for the purpose of discriminating elements that are not human skinimages. Then the image is filtered using skin detection. The output image only contains the areas of whichit is composed. The results show a comparison between the proposed system and the company softwareAccess Data called Forensic Toolkit 3.1 Explicit Image Detection isperformed.

  18. Explicit Content Image Detection

    Directory of Open Access Journals (Sweden)

    Jorge Alberto Marcial Basilio


    Full Text Available This paper proposes a system gives for explicit content image detection based on Computer Vision Algorithms, pattern recognition and FTK software Explicit Image Detection. In the first stage, HSV color model is used for the input images for the purpose of discriminating elements that are not human skin images. Then the image is filtered using skin detection. The output image only contains the areas of which it is composed. The results show a comparison between the proposed system and the company software Access Data called Forensic Toolkit 3.1 Explicit Image Detection isperformed.

  19. Lean blowoff detection sensor (United States)

    Thornton, Jimmy; Straub, Douglas L.; Chorpening, Benjamin T.; Huckaby, David


    Apparatus and method for detecting incipient lean blowoff conditions in a lean premixed combustion nozzle of a gas turbine. A sensor near the flame detects the concentration of hydrocarbon ions and/or electrons produced by combustion and the concentration monitored as a function of time are used to indicate incipient lean blowoff conditions.

  20. Array for detecting microbes

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Gary L.; DeSantis, Todd D.


    The present embodiments relate to an array system for detecting and identifying biomolecules and organisms. More specifically, the present embodiments relate to an array system comprising a microarray configured to simultaneously detect a plurality of organisms in a sample at a high confidence level.

  1. Detecting Illicit Nuclear Materials

    Energy Technology Data Exchange (ETDEWEB)

    Kouzes, Richard T.


    The threat that weapons of mass destruction might enter the United States has led to a number of efforts for the detection and interdiction of nuclear, radiological, chemical, and biological weapons at our borders. There have been multiple deployments of instrumentation to detect radiation signatures to interdict radiological material, including weapons and weapons material worldwide.

  2. Fraud detection tools

    Directory of Open Access Journals (Sweden)

    Katerina Hawlova


    Full Text Available This article aims to introduce to readers the topic of fraud management – detection of fraudulent behaviour. The article is divided into two parts. The first part presents what is meant by fraud and fraudulent behaviour. In the second part a case study dealing with fraudulent behaviour detection in the procurement area is introduced.

  3. Semiconductor radiation detection systems

    CERN Document Server


    Covers research in semiconductor detector and integrated circuit design in the context of medical imaging using ionizing radiation. This book explores other applications of semiconductor radiation detection systems in security applications such as luggage scanning, dirty bomb detection and border control.

  4. Using CdTe/ZnSe core/shell quantum dots to detect DNA and damage to DNA

    Directory of Open Access Journals (Sweden)

    Moulick A


    Full Text Available Amitava Moulick,1,2 Vedran Milosavljevic,1,2 Jana Vlachova,1,2 Robert Podgajny,3 David Hynek,1,2 Pavel Kopel,1,2 Vojtech Adam1,2 1Department of Chemistry and Biochemistry, Mendel University, 2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic; 3Faculty of Chemistry, Jagiellonian University, Krakow, Poland Abstract: CdTe/ZnSe core/shell quantum dot (QD, one of the strongest and most highly luminescent nanoparticles, was directly synthesized in an aqueous medium to study its individual interactions with important nucleobases (adenine, guanine, cytosine, and thymine in detail. The results obtained from the optical analyses indicated that the interactions of the QDs with different nucleobases were different, which reflected in different fluorescent emission maxima and intensities. The difference in the interaction was found due to the different chemical behavior and different sizes of the formed nanoconjugates. An electrochemical study also confirmed that the purines and pyrimidines show different interactions with the core/shell QDs. Based on these phenomena, a novel QD-based method is developed to detect the presence of the DNA, damage to DNA, and mutation. The QDs were successfully applied very easily to detect any change in the sequence (mutation of DNA. The QDs also showed their ability to detect DNAs directly from the extracts of human cancer (PC3 and normal (PNT1A cells (detection limit of 500 pM of DNA, which indicates the possibilities to use this easy assay technique to confirm the presence of living organisms in extreme environments. Keywords: nanoparticles, nucleobases, biosensor, fluorescence, mutation

  5. Analogue MIMO Detection

    Directory of Open Access Journals (Sweden)

    McNamara Darren


    Full Text Available In this contribution we propose an analogue receiver that can perform turbo detection in MIMO systems. We present the case for a receiver that is built from nonlinear analogue devices, which perform detection in a "free-flow" network (no notion of iterations. This contribution can be viewed as an extension of analogue turbo decoder concepts to include MIMO detection. These first analogue implementations report reductions of few orders of magnitude in the number of required transistors and in consumed energy, and the same order of improvement in processing speed. It is anticipated that such analogue MIMO decoder could bring about the same advantages, when compared to traditional digital implementations.

  6. Light Detection And Ranging (United States)

    U.S. Geological Survey, Department of the Interior — LiDAR (Light Detection and Ranging) discrete-return point cloud data are available in the American Society for Photogrammetry and Remote Sensing (ASPRS) LAS format....

  7. Anomaly Detection in Sequences (United States)

    National Aeronautics and Space Administration — We present a set of novel algorithms which we call sequenceMiner, that detect and characterize anomalies in large sets of high-dimensional symbol sequences that...

  8. Lead Poison Detection (United States)


    With NASA contracts, Whittaker Corporations Space Science division has developed an electro-optical instrument to mass screen for lead poisoning. Device is portable and detects protoporphyrin in whole blood. Free corpuscular porphyrins occur as an early effect of lead ingestion. Also detects lead in urine used to confirm blood tests. Test is inexpensive and can be applied by relatively unskilled personnel. Similar Whittaker fluorometry device called "drug screen" can measure morphine and quinine in urine much faster and cheaper than other methods.

  9. Sensor for metal detection

    KAUST Repository

    Kodzius, Rimantas


    NOVELTY - The sensor has a microfluidic flow channel that is provided with an inlet port, an outlet port, and a detection chamber. The detection chamber is provided with a group of sensing electrodes (4) having a working electrode (8), a counter electrode (9), and a reference electrode (10). A flow sensor is configured to measure flow in the channel. A temperature sensor (6) is configured to measure temperature in the channel (3). An electrical connection is configured to connect the sensor to a sensing device. USE - Sensor for detecting metal such as toxic metal in sample such as clinical sample such as stool, saliva, sputum, bronchial lavage, urine, vaginal swab, nasal swab, biopsy, tissue, tears, breath, blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, pleural fluid, pericardial fluid, joint fluid, and amniotic fluid, water sample, food sample, air sample, and soil sample (all claimed). ADVANTAGE - The sensor for use with the portable analytical instrument is configured for detection of metalsin samples. The sensor can provide the excellent solution for on-site metal detection, including heavy metal detection. The sensors can provide significant advantages in higher throughput, lower cost, at the same time being less labor intensive and less dependent on individual skills. The disposable design of the sensor, the enhanced reliability and repeatability of measurements can be obtained. The sensors can be widely applied in various industries. DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the following: (1) a system for detecting metal in sample; and (2) a method for using sensor for detecting metal in sample. DESCRIPTION OF DRAWING(S) - The drawing shows a schematic view of the sensor prototype. Channel (3) Sensing electrodes (4) Temperature sensor (6) Working electrode (8) Counter electrode (9) Reference electrode (10)

  10. Object Duplicate Detection


    Vajda, Péter


    With the technological evolution of digital acquisition and storage technologies, millions of images and video sequences are captured every day and shared in online services. One way of exploring this huge volume of images and videos is through searching a particular object depicted in images or videos by making use of object duplicate detection. Therefore, need of research on object duplicate detection is validated by several image and video retrieva...

  11. Dengue disease outbreak detection. (United States)

    Dayama, Pankaj; Sampath, Kameshwaran


    Early detection of outbreak of a disease may help in timely and effective public health interventions. Our motivation for this work is to assist EHD planning officer to analyze the incidence data and based on it declare whether there is an outbreak or not. In this paper, we develop ensemble of multiple techniques for detecting dengue disease outbreak. These techniques are applied to dengue incidence data from Singapore and results are summarized.

  12. Emotion Detection from Text

    CERN Document Server

    Shivhare, Shiv Naresh


    Emotion can be expressed in many ways that can be seen such as facial expression and gestures, speech and by written text. Emotion Detection in text documents is essentially a content - based classification problem involving concepts from the domains of Natural Language Processing as well as Machine Learning. In this paper emotion recognition based on textual data and the techniques used in emotion detection are discussed.

  13. Imprinting mutations in Angelman syndrome detected by Southern blotting using a probe containing exon {alpha} of SNRPN

    Energy Technology Data Exchange (ETDEWEB)

    Beuten, J.; Sutcliffe, J.S.; Nakao, M. [Baylor College of Medicine, Houston, TX (United States)] [and others


    Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are associated with paternal and maternal deficiencies respectively, of gene expression within human chromosome 15q11-q13, and are caused by deletion, uniparental disomy (UPD), or other mutations. The SNRPN gene maps in this region, is paternally expressed, and is a candidate gene for PWS. Southern blotting using methylation-sensitive enzymes and a genomic DNA probe from the CpG island containing exon {alpha} of the SNRPN gene reveals methylation specific for the maternal allele. In cases of the usual deletions or UPD, the probe detects absence of an unmethylated allele in PWS and absence of a methylated allele in AS. We have analyzed 21 nondeletion/nonUPD AS patients with this probe and found evidence for an imprinting mutation (absence of a methylated allele) in 3 patients. Southern blotting with methylation-sensitive enzymes using the exon {alpha} probe, like use of the PW71 probe, should detect abnormalities in all known PWS cases and in 3 of the 4 forms of AS: deletion, UPD and imprinting mutations. This analysis provides a valuable diagnostic approach for PWS and AS. In efforts to localize the imprinting mutations in AS, one patient was found with failure to inherit a dinucleotide repeat polymorphism near probe 189-1 (D15S13). Analysis of this locus in AS families and CEPH families demonstrates a polymorphism that impairs amplification and a different polymorphism involving absence of hybridization to the 189-1 probe. The functional significance, if any, of deletion of the 189-1 region is unclear.

  14. DNA-templated silver nanoclusters based label-free fluorescent molecular beacon for the detection of adenosine deaminase. (United States)

    Zhang, Kai; Wang, Ke; Xie, Minhao; Zhu, Xue; Xu, Lan; Yang, Runlin; Huang, Biao; Zhu, Xiaoli


    A general and reliable fluorescent molecular beacon is proposed in this work utilizing DNA-templated silver nanoclusters (AgNCs). The fluorescent molecular beacon has been employed for sensitive determination of the concentration of adenosine deaminase (ADA) and its inhibition. A well-designed oligonucleotide containing three functional regions (an aptamer region for adenosine assembly, a sequence complementary to the region of the adenosine aptamer, and an inserted six bases cytosine-loop) is adopted as the core element in the strategy. The enzymatic reaction of adenosine catalyzed by ADA plays a key role as well in the regulation of the synthesis of the DNA-templated AgNCs, i.e. the signal indicator. The intensity of the fluorescence signal may thereby determine the concentration of the enzyme and its inhibitor. The detection limit of the ADA can be lowered to 0.05 UL(-1). Also, 100 fM of a known inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride (EHNA) is enough to present distinguishable fluorescence emission. Moreover, since the fluorescent signal indicator is not required to be bound with the oligonucleotide, this fluorescent molecular beacon may integrate the advantages of both the label-free and signal-on strategies.


    Energy Technology Data Exchange (ETDEWEB)

    Zaleski, Daniel P.; Seifert, Nathan A.; Steber, Amanda L.; Muckle, Matt T.; Loomis, Ryan A.; Vasquez, David; Nyiramahirwe, Jolie; Sciortino, Nicole; Johnson, Kennedy; Pate, Brooks H. [Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22904 (United States); Corby, Joanna F. [Department of Astronomy, University of Virginia, McCormick Road, Charlottesville, VA 22904 (United States); Martinez, Oscar Jr.; Crabtree, Kyle N.; McCarthy, Michael C. [Harvard-Smithsonian Center for Astrophysics, 60 Garden Street, Cambridge, MA 02138 (United States); Jewell, Philip R.; Remijan, Anthony J. [National Radio Astronomy Observatory, 520 Edgemont Road, Charlottesville, VA 22903-2475 (United States); Hollis, Jan M. [NASA Goddard Space Flight Center, Greenbelt, MD 20771 (United States); Lovas, Frank J., E-mail:, E-mail:, E-mail: [National Institute of Standards and Technology, Gaithersburg, MD 20899 (United States)


    The detection of E-cyanomethanimine (E-HNCHCN) toward Sagittarius B2(N) is made by comparing the publicly available Green Bank Telescope (GBT) PRIMOS survey spectra to laboratory rotational spectra from a reaction product screening experiment. The experiment uses broadband molecular rotational spectroscopy to monitor the reaction products produced in an electric discharge source using a gas mixture of NH{sub 3} and CH{sub 3}CN. Several transition frequency coincidences between the reaction product screening spectra and previously unassigned interstellar rotational transitions in the PRIMOS survey have been assigned to E-cyanomethanimine. A total of eight molecular rotational transitions of this molecule between 9 and 50 GHz are observed with the GBT. E-cyanomethanimine, often called the HCN dimer, is an important molecule in prebiotic chemistry because it is a chemical intermediate in proposed synthetic routes of adenine, one of the two purine nucleobases found in DNA and RNA. New analyses of the rotational spectra of both E-cyanomethanimine and Z-cyanomethanimine that incorporate previous millimeter-wave measurements are also reported.

  16. Methods of Endotoxin Detection. (United States)

    Su, Wenqiong; Ding, Xianting


    Endotoxin, present in the outer membrane of all gram-negative bacteria, can pose serious risks to human health, from irreversible shock to death. Therefore, it is essential to develop sensitive, accurate, and rapid methods for its detection. The rabbit pyrogen test is the first standard technique for endotoxin detection and, nowadays, has been replaced by the Limulus Amoebocyte Lysate test, which is the most popular detection technique for endotoxin. With in-depth understanding of endotoxin, biosensors based on endotoxin-sensing components are promising alternatives to pursue in developing low-cost, easy-operation, and fast-response endotoxin detection techniques. This article summarizes the recent advances of endotoxin detection methods with a particular emphasis on optical and electrochemical biosensors based on various sensing elements ranging from nature biomolecules to artificial materials. As the research and technological revolution continues, the highly integrated and miniaturized commercial devices for sensitively and reliably detecting endotoxin will provide a wide range of applications in people's daily life.

  17. Proactive malware detection (United States)

    Gloster, Jonathan; Diep, Michael; Dredden, David; Mix, Matthew; Olsen, Mark; Price, Brian; Steil, Betty


    Small-to-medium sized businesses lack resources to deploy and manage high-end advanced solutions to deter sophisticated threats from well-funded adversaries, but evidence shows that these types of businesses are becoming key targets. As malicious code and network attacks become more sophisticated, classic signature-based virus and malware detection methods are less effective. To augment the current malware methods of detection, we developed a proactive approach to detect emerging malware threats using open source tools and intelligence to discover patterns and behaviors of malicious attacks and adversaries. Technical and analytical skills are combined to track adversarial behavior, methods and techniques. We established a controlled (separated domain) network to identify, monitor, and track malware behavior to increase understanding of the methods and techniques used by cyber adversaries. We created a suite of tools that observe the network and system performance looking for anomalies that may be caused by malware. The toolset collects information from open-source tools and provides meaningful indicators that the system was under or has been attacked. When malware is discovered, we analyzed and reverse engineered it to determine how it could be detected and prevented. Results have shown that with minimum resources, cost effective capabilities can be developed to detect abnormal behavior that may indicate malicious software.

  18. Forensic audio watermark detection (United States)

    Steinebach, Martin; Zmudzinski, Sascha; Petrautzki, Dirk


    Digital audio watermarking detection is often computational complex and requires at least as much audio information as required to embed a complete watermark. In some applications, especially real-time monitoring, this is an important drawback. The reason for this is the usage of sync sequences at the beginning of the watermark, allowing a decision about the presence only if at least the sync has been found and retrieved. We propose an alternative method for detecting the presence of a watermark. Based on the knowledge of the secret key used for embedding, we create a mark for all potential marking stages and then use a sliding window to test a given audio file on the presence of statistical characteristics caused by embedding. In this way we can detect a watermark in less than 1 second of audio.

  19. Neurology and detective writing. (United States)

    Kempster, Peter A; Lees, Andrew J


    When searching for clues to reach a diagnosis, neurologists often empathise with the detective who is trying to solve a case. The premise of this article is that detective stories have been part of the fabric of neurology ever since the time that it evolved into a discrete medical speciality. We will examine how this form of narrative has found expression in detective mystery fiction and popular science publications created by 20th century neurologist physician-writers. We will also investigate the power of the neurologist's alter ego, Sherlock Holmes: his relationship to founders of clinical neuroscience such as Jean-Martin Charcot, William Gowers and Sigmund Freud, and his influences on neurological practice and its literary traditions.

  20. Changing change detection

    DEFF Research Database (Denmark)

    Kyllingsbæk, Søren; Bundesen, Claus


    The change detection paradigm is a popular way of measuring visual short-term memory capacity. Using the paradigm, researchers have found evidence for a capacity of about four independent visual objects, confirming classic estimates that were based on the number of items that could be reported....... Here, we determine the reliability of capacity measures found by change detection. We derive theoretical predictions of the variance of the capacity estimates and show how they depend on the number of items to be remembered and the guessing strategy of the observer. We compare the theoretically derived...... variance to the variance estimated over repeated blocks of trials with the same observer and find close correspondence between predicted and observed variances. Also, we propose a new version of the two-alternative choice change detection paradigm, in which the choice is unforced. This new paradigm reduces...

  1. Detection of solar events (United States)

    Fischbach, Ephraim; Jenkins, Jere


    A flux detection apparatus can include a radioactive sample having a decay rate capable of changing in response to interaction with a first particle or a field, and a detector associated with the radioactive sample. The detector is responsive to a second particle or radiation formed by decay of the radioactive sample. The rate of decay of the radioactive sample can be correlated to flux of the first particle or the field. Detection of the first particle or the field can provide an early warning for an impending solar event.

  2. Detecting Patterns of Anomalies (United States)


    detect anomalies in the dataset is used in [Leung and Leckie, 2005] and [Eskin et al., 2002]. One-class SVMs [Li et al., 2003, Heller et al., 2003] and...IEE Proceedings F, 140(2): 107–113, 1993. J.D.F. Habbema, J. Hermans , and K. Vandenbroek. A stepwise discriminant analysis pro- gram using density...Technometrics, 29(4):409–412, 1987. K.A. Heller , K.M. Svore, A. Keromytis, and S.J. Stolfo. One class support vector machines for detecting anomalous

  3. Handbook for Language Detectives

    DEFF Research Database (Denmark)

    Bryanne, Ulla; Bruntt, Karen Scheel Lassen

    Handbook for Language Detectives gives a thorough presentation of English grammar and discusses how to teach grammar. The book unveils to the readers, who will be working as grammar detectives, the fascinating world of language. It does not only deal with "traditional grammar" but also discusses...... be done. The book serves a double purpose: - English grammar and language usage at bachelor level from a functional linguistic point of view. - How to teach English grammar within a communicative approach. The book is mainly intended for Danish student teachers of English, but anyone else interested...

  4. Readers as research detectives

    Directory of Open Access Journals (Sweden)

    Gøtzsche Peter C


    Full Text Available Abstract Flaws in research papers are common but it may require arduous detective work to unravel them. Checklists are helpful, but many inconsistencies will only be revealed through repeated cross-checks of every little detail, just like in a crime case. As a major deterrent for dishonesty, raw data from all trials should be posted on a public website. This would also make it much easier to detect errors and flaws in publications, and it would allow many research projects to be performed without collecting new data. The prevailing culture of secrecy and ownership to data is not in the best interests of patients.

  5. Readers as research detectives

    DEFF Research Database (Denmark)

    Gøtzsche, Peter C


    Flaws in research papers are common but it may require arduous detective work to unravel them. Checklists are helpful, but many inconsistencies will only be revealed through repeated cross-checks of every little detail, just like in a crime case. As a major deterrent for dishonesty, raw data from...... all trials should be posted on a public website. This would also make it much easier to detect errors and flaws in publications, and it would allow many research projects to be performed without collecting new data. The prevailing culture of secrecy and ownership to data is not in the best interests...

  6. Pretentiously detecting power cancellation

    CERN Document Server

    Jung, Junehyuk


    Granville and Soundararajan have recently introduced the notion of pretentiousness in the study of multiplicative functions of modulus bounded by 1, essentially the idea that two functions which are similar in a precise sense should exhibit similar behavior. It turns out, somewhat surprisingly, that this does not directly extend to detecting power cancellation - there are multiplicative functions which exhibit as much cancellation as possible in their partial sums that, modified slightly, give rise to functions which exhibit almost as little as possible. We develop two new notions of pretentiousness under which power cancellation can be detected, one of which applies to a much broader class of multiplicative functions.

  7. Sequential Detection of Digital Watermarking

    Institute of Scientific and Technical Information of China (English)

    LI Li; YU Yu-lian; WANG Pei


    The paper analyzed a new watermarking detection paradigm including double detection thresholds based on sequential hypothesis testing. A joint design of watermarking encoding and detection was proposed. The paradigm had good immunity to noisy signal attacks and high detection probability. Many experiments proved that the above algorithm can detect watermarks about 66% faster than popular detectors, which could have significant impact on many applications such as video watermarking detection and watermark-searching in a large database of digital contents.

  8. Immunity Based Worm Detection System

    Institute of Scientific and Technical Information of China (English)

    HONG Zheng; WU Li-fa; WANG Yuan-yuan


    Current worm detection methods are unable to detect multi-vector polymorphic worms effectively.Based on negative selection mechanism of the immune system,a local network worm detection system that detects worms was proposed.Normal network service requests were represented by self-strings,and the detection system used self-strings to monitor the network for anomaly.According to the properties of worm propagation,a control center correlated the anomalies detected in the form of binary trees to ensure the accuracy of worm detection.Experiments show the system to be effective in detecting the traditional as well as multi-vector polymorphic worms.

  9. Detecting Android Malware Using Clone Detection

    Institute of Scientific and Technical Information of China (English)

    陈健; Manar H. Alalfi; Member; ACM; IEEE; Thomas R. Dean; 邹颖


    Android is currently one of the most popular smartphone operating systems. However, Android has the largest share of global mobile malware and significant public attention has been brought to the security issues of Android. In this paper, we investigate the use of a clone detector to identify known Android malware. We collect a set of Android applications known to contain malware and a set of benign applications. We extract the Java source code from the binary code of the applications and use NiCad, a near-miss clone detector, to find the classes of clones in a small subset of the malicious applications. We then use these clone classes as a signature to find similar source files in the rest of the malicious applications. The benign collection is used as a control group. In our evaluation, we successfully decompile more than 1 000 malicious apps in 19 malware families. Our results show that using a small portion of malicious applications as a training set can detect 95% of previously known malware with very low false positives and high accuracy at 96.88%. Our method can effectively and reliably pinpoint malicious applications that belong to certain malware families.

  10. Detection of colorectal neoplasia

    DEFF Research Database (Denmark)

    Wilhelmsen, Michael; Christensen, Ib J; Rasmussen, Louise


    Serological biomarkers may be an option for early detection of colorectal cancer (CRC). The present study assessed 8 cancer-associated protein biomarkers in plasma from subjects undergoing first time ever colonoscopy due to symptoms attributable to colorectal neoplasia. Plasma AFP, CA19-9, CEA, hs...

  11. Evolutionary Theories of Detection

    Energy Technology Data Exchange (ETDEWEB)

    Fitch, J P


    Current, mid-term and long range technologies for detection of pathogens and toxins are briefly described in the context of performance metrics and operational scenarios. Predictive (evolutionary) and speculative (revolutionary) assessments are given with trade-offs identified, where possible, among competing performance goals.

  12. Electrically detected ferromagnetic resonance

    NARCIS (Netherlands)

    Goennenwein, S.T.B.; Schink, S.W.; Brandlmaier, A.; Boger, A.; Opel, M.; Gross, R.; Keizer, R.S.; Klapwijk, T.M.; Gupta, A.; Huebl, H.; Bihler, C.; Brandt, M.S.


    We study the magnetoresistance properties of thin ferromagnetic CrO2 and Fe3O4 films under microwave irradiation. Both the sheet resistance ρ and the Hall voltage VHall characteristically change when a ferromagnetic resonance (FMR) occurs in the film. The electrically detected ferromagnetic resonanc

  13. Automatic detection of laughter

    NARCIS (Netherlands)

    Truong, K.P.; Leeuwen, D.A. van


    In the context of detecting ‘paralinguistic events’ with the aim to make classification of the speaker’s emotional state possible, a detector was developed for one of the most obvious ‘paralinguistic events’, namely laughter. Gaussian Mixture Models were trained with Perceptual Linear Prediction fea

  14. Soybean allergen detection methods

    DEFF Research Database (Denmark)

    Pedersen, Mona H; Holzhauser, Thomas; Bisson, Caroline


    Soybean containing products are widely consumed, thus reliable methods for detection of soy in foods are needed in order to make appropriate risk assessment studies to adequately protect soy allergic patients. Six methods were compared using eight food products with a declared content of soy...

  15. Change Detection Tools

    NARCIS (Netherlands)

    Dekker, R.J.; Kuenzer, C.; Lehner, M.; Reinartz, P.; Niemeyer, I.; Nussbaum, S.; Lacroix, V.; Sequeira, V.; Stringa, E.; Schöpfer, E.


    In this chapter a wide range of change detection tools is addressed. They are grouped into methods suitable for optical and multispectral data, synthetic aperture radar (SAR) images, and 3D data. Optical and multispectral methods include unsupervised approaches, supervised and knowledge-based approa

  16. Detection of counterfeit currency (United States)

    Burns, D.A.


    A method is disclosed of detecting counterfeit currency by contacting the currency to be tested with near infrared beams in the spectrum below 1,250 nanometers, measuring reflectance of the near infrared beams and comparing the reflectance values with those from genuine currency. 18 figs.

  17. Global Disease Detectives

    Centers for Disease Control (CDC) Podcasts


    This podcast documents U.S. efforts to prevent, detect, and control emerging infectious diseases, such as SARS and pandemic influenza.  Created: 9/21/2010 by CDC Center for Global Health.   Date Released: 9/21/2010.

  18. Electrical load detection aparatus

    DEFF Research Database (Denmark)


    A load detection technique for a load comprising multiple frequency-dependant sub-loads comprises measuring a representation of the impedance characteristic of the load; providing stored representations of a multiplicity of impedance characteristics of the load; each one of the stored representat...

  19. Portable raman explosives detection

    Energy Technology Data Exchange (ETDEWEB)

    Moore, David Steven [Los Alamos National Laboratory; Scharff, Robert J [Los Alamos National Laboratory


    Recent advances in portable Raman instruments have dramatically increased their application to emergency response and forensics, as well as homeland defense. This paper reviews the relevant attributes and disadvantages of portable Raman spectroscopy, both essentially and instrumentally, to the task of explosives detection in the field.

  20. Bayesian microsaccade detection (United States)

    Mihali, Andra; van Opheusden, Bas; Ma, Wei Ji


    Microsaccades are high-velocity fixational eye movements, with special roles in perception and cognition. The default microsaccade detection method is to determine when the smoothed eye velocity exceeds a threshold. We have developed a new method, Bayesian microsaccade detection (BMD), which performs inference based on a simple statistical model of eye positions. In this model, a hidden state variable changes between drift and microsaccade states at random times. The eye position is a biased random walk with different velocity distributions for each state. BMD generates samples from the posterior probability distribution over the eye state time series given the eye position time series. Applied to simulated data, BMD recovers the “true” microsaccades with fewer errors than alternative algorithms, especially at high noise. Applied to EyeLink eye tracker data, BMD detects almost all the microsaccades detected by the default method, but also apparent microsaccades embedded in high noise—although these can also be interpreted as false positives. Next we apply the algorithms to data collected with a Dual Purkinje Image eye tracker, whose higher precision justifies defining the inferred microsaccades as ground truth. When we add artificial measurement noise, the inferences of all algorithms degrade; however, at noise levels comparable to EyeLink data, BMD recovers the “true” microsaccades with 54% fewer errors than the default algorithm. Though unsuitable for online detection, BMD has other advantages: It returns probabilities rather than binary judgments, and it can be straightforwardly adapted as the generative model is refined. We make our algorithm available as a software package. PMID:28114483