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Sample records for adenine dinucleotide biosynthesis

  1. Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

    PubMed Central

    Nelson, R E; Selitrennikoff, C P; Siegel, R W

    1975-01-01

    A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase. Images PMID:165174

  2. Nicotinamide adenine dinucleotide-dependent and nicotinamide adenine dinucleotide-independent lactate dehydrogenases in homofermentative and heterofermentative lactic acid bacteria.

    PubMed

    Doelle, H W

    1971-12-01

    Three homofermentative (Lactobacillus plantarum B38, L. plantarum B33, Pediococcus pentosaceus B30) and three heterofermentative (Leuconostoc mesenteroides 39, L. oenos B70, Lactobacillus brevis) lactic acid bacteria were examined for the presence or absence of nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent d- and l-lactate dehydrogenases. Two of the six strains investigated, P. pentosaceus and L. oenos, did not exhibit an NAD-independent enzyme activity capable of reducing dichlorophenol indophenol. The pH optima of the lactic dehydrogenases were determined. The NAD-dependent enzymes from homofermentative strains exhibited optima at pH 7.8 to 8.8, whereas values from 9.0 to 10.0 were noted for these enzymes from heterofermentative organisms. The optima for the NAD-independent enzymes were between 5.8 and 6.6. The apparent Michaelis-Menten constants determined for both NAD and the substrates demonstrated the existence of a greater affinity for d- than l-lactic acid. A comparison of the specific NAD-dependent and NAD-independent lactate dehydrogenase activities revealed a direct correlation of the d/l ratios of these activities with the type of lactic acid produced during the growth of the organism.

  3. Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Isolated Corn Mitochondria 1

    PubMed Central

    Koeppe, D. E.; Miller, Raymond J.

    1972-01-01

    Isolated corn (Zea mays L.) mitochondria were found to oxidize reduced nicotinamide adenine dinucleotide phosphate in a KCl reaction medium. This oxidation was dependent on the presence of calcium or phosphate or both. Strontium and manganese substituted for calcium, but magnesium or barium did not. The oxidation of NADPH produced contraction of mitochondria swollen in KCl. Further evidence that the oxidation of NADPH was coupled was observed in respiratory control and adenosine diphosphate-oxygen ratios that were comparable to those reported for reduced nicotinamide adenine dinucleotide. The pathways of electron flow from NADH and NADPH were compared through the addition of electron transport inhibitors. The only difference between the two dinucleotides was that amytal was found to inhibit almost totally the state 3 oxidation of NADPH, but had little effect on the state 3 oxidation of NADH. The hypothetical pathways for electron flow from NADPH are discussed, as are the possible sites of calcium and phosphate stimulation. PMID:16657960

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

    PubMed

    Barry, S; O'Carra, P

    1973-12-01

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

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

    PubMed Central

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

    2010-01-01

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

  6. Two-dimensional infrared spectroscopy of azido-nicotinamide adenine dinucleotide in water

    NASA Astrophysics Data System (ADS)

    Dutta, Samrat; Rock, William; Cook, Richard J.; Kohen, Amnon; Cheatum, Christopher M.

    2011-08-01

    Mid-IR active analogs of enzyme cofactors have the potential to be important spectroscopic reporters of enzyme active site dynamics. Azido-nicotinamide adenine dinucleotide (NAD+), which has been recently synthesized in our laboratory, is a mid-IR active analog of NAD+, a ubiquitous redox cofactor in biology. In this study, we measure the frequency-frequency time correlation function for the antisymmetric stretching vibration of the azido group of azido-NAD+ in water. Our results are consistent with previous studies of pseudohalides in water. We conclude that azido-NAD+ is sensitive to local environmental fluctuations, which, in water, are dominated by hydrogen-bond dynamics of the water molecules around the probe. Our results demonstrate the potential of azido-NAD+ as a vibrational probe and illustrate the potential of substituted NAD+-analogs as reporters of local structural dynamics that could be used for studies of protein dynamics in NAD-dependent enzymes.

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

    PubMed

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

    2016-05-15

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

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

    PubMed Central

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

    2004-01-01

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

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

    SciTech Connect

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

    1987-05-05

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

  10. Flavin adenine dinucleotide content of quinone reductase 2: analysis and optimization for structure-function studies.

    PubMed

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

    2012-01-01

    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.

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

    PubMed

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

    2012-03-01

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

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

    PubMed

    Greenhouse, W V; Lehninger, A L

    1977-11-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1991-03-01

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

  15. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes.

    PubMed Central

    Eng, J; Lynch, R M; Balaban, R S

    1989-01-01

    Nicotinamide adenine dinucleotide (NADH) plays a critical role in oxidative phosphorylation as the primary source of reducing equivalents to the respiratory chain. Using a modified fluorescence microscope, we have obtained spectra and images of the blue autofluorescence from single rat cardiac myocytes. The optical setup permitted rapid acquisition of fluorescence emission spectra (390-595 nm) or intensified digital video images of individual myocytes. The spectra showed a broad fluorescence centered at 447 +/- 0.2 nm, consistent with mitochondrial NADH. Addition of cyanide resulted in a 100 +/- 10% increase in fluorescence, while the uncoupler FCCP resulted in a 82 +/- 4% decrease. These two transitions were consistent with mitochondrial NADH and implied that the myocytes were 44 +/- 6% reduced under the resting control conditions. Intracellular fluorescent structures were observed that correlated with the distribution of a mitochondrial selective fluorescent probe (DASPMI), the mitochondrial distribution seen in published electron micrographs, and a metabolic digital subtraction image of the cyanide fluorescence transition. These data are consistent with the notion that the blue autofluorescence of rat cardiac myocytes originates from mitochondrial NADH. Images FIGURE 9 FIGURE 10 FIGURE 2 FIGURE 3 FIGURE 8 FIGURE 11 PMID:2720061

  16. Nicotinamide adenine dinucleotide: An essential factor in preserving hearing in cisplatin-induced ototoxicity.

    PubMed

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

    2015-08-01

    Ototoxicity is an important issue in patients receiving cisplatin chemotherapy. Numerous studies have demonstrated that several mechanisms, including oxidative stress, DNA damage, and inflammatory responses, are closely associated with cisplatin-induced ototoxicity. Although much attention has been directed at identifying ways to protect the inner ear from cisplatin-induced damage, the precise underlying mechanisms have not yet been elucidated. The cofactor nicotinamide adenine dinucleotide (NAD(+)) has emerged as an important regulator of cellular energy metabolism and homeostasis. NAD(+) acts as a cofactor for various enzymes including sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), and therefore, maintaining adequate NAD(+) levels has therapeutic benefits because of its effect on NAD(+)-dependent enzymes. Recent studies demonstrated that disturbance in intracellular NAD(+) levels is critically involved in cisplatin-induced cochlear damage associated with oxidative stress, DNA damage, and inflammatory responses. In this review, we describe the importance of NAD(+) in cisplatin-induced ototoxicity and discuss potential strategies for the prevention or treatment of cisplatin-induced ototoxicity with a particular focus on NAD(+)-dependent cellular pathways. PMID:25891352

  17. Kinetic properties of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release.

    PubMed

    Genazzani, A A; Mezna, M; Summerhill, R J; Galione, A; Michelangeli, F

    1997-03-21

    Three endogenous molecules have now been shown to release Ca2+ in the sea urchin egg: inositol trisphosphate (InsP3), cyclic adenosine 5'-diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), a derivative of NADP. While the mechanism through which the first two molecules are able to release Ca2+ is established and well characterized with InsP3 and cADPR-activating InsP3 and ryanodine receptors, respectively, the newly described NAADP has been shown to release Ca2+ via an entirely different mechanism. The most striking feature of this novel Ca2+ release mechanism is its inactivation, since subthreshold concentrations of NAADP are able to fully and irreversibly desensitize the channel. In the present study we have investigated the fast kinetics of activation and inactivation of NAADP-induced Ca2+ release. NAADP was found to release Ca2+ in a biphasic manner, and such release was preceded by a pronounced latent period, which was inversely dependent on concentration. Moreover, the kinetic features of NAADP-induced Ca2+ release were not altered by pretreatment with low concentrations of NAADP, although the extent of Ca2+ release was greatly affected. Our data suggest that the inactivation of NAADP-induced Ca2+ release is an all-or-none phenomenon, and while some receptors have been fully inactivated, those that remain sensitive to NAADP do so without any change in kinetic features. PMID:9065423

  18. Reduced nicotinamide adenine dinucleotide-activated phosphoenolpyruvate carboxylase in Pseudomonas MA: potential regulation between carbon assimilation and energy production.

    PubMed Central

    Newaz, S S; Hersh, L B

    1975-01-01

    Comparison of enzyme activities in crude extracts of methylamine-grown Pseudomonas MA (ATCC 23319) to those in succinate-grown cells indicates the involvement of an acetyl coenzyme A-independent phosphoenolpyruvate carboxylase in one-carbon metabolism. The purified phosphoenolpyruvate carboxylase is activated specifically by reduced nicotinamide adenine dinucleotide (KA = 0.2 mM). The regulatory properties of this enzyme suggests that phosphoenolpyruvate serves as a focal point for both carbon assimilation and energy metabolism. PMID:171253

  19. RESPIRATORY PATHWAYS IN THE MYCOPLASMA. II. PATHWAY OF ELECTRON TRANSPORT DURING OXIDATION OF REDUCED NICOTINAMIDE ADENINE DINUCLEOTIDE BY MYCOPLASMA HOMINIS.

    PubMed

    VANDEMARK, P J; SMITH, P F

    1964-07-01

    VanDemark, P. J. (University of South Dakota, Vermillion), and P. F. Smith. Respiratory pathways in the Mycoplasma. II. Pathway of electron transport during oxidation of reduced nicotinamide adenine dinucleotide by Mycoplasma hominis. J. Bacteriol. 88:122-129. 1964.-Unlike the flavin-terminated respiratory pathway of the fermentative Mycoplasma, the respiratory chain of the nonfermentative M. hominis strain 07 appears to be more complex, involving quinones and cytochromes in addition to flavins. In addition to reduction by reduced nicotine adenine dinucleotide (NADH) and reduced nicotine adenine dinucleotide phosphate, nonpyridine nucleotide-linked reduction of the respiratory chain of this organism occurred with succinate, lactate, and short-chained acyl coenzyme A derivatives as electron donors. Enzymes catalyzing the oxidation of NADH included an NADH oxidase, a diaphorase, a quinone reductase, and a cytochrome c reductase. The oxidation of NADH was sensitive to a variety of inhibitors, including 10(-4)m Atabrine, 10(-3)m sodium amytal, 10(-5)mp-chloromercuribenzoate, 10(-4)m antimycin A, and 10(-4)m potassium cyanide. The oxidase was resolved by the addition of 5% trichloroacetic acid and reactivated by the addition of flavin adenine dinucleotide but not flavin mononucleotide. The M. hominis sonic extract contained an NADH-coenzyme Q reductase. The oxidation of NADH was stimulated by the addition of either menadione or vitamin K(2) (C(35)). The oxidase was inactivated by extraction with ether or irradiation at 360 mmu. The ether-inactivated enzyme was partially reactivated by the addition of "lipid" extract of the enzyme and coenzyme Q(6). Difference spectra of the cell extracts revealed the presence of "b" and "a" type cytochromes. These cell extracts were found to contain a cyanide-and azide-sensitive cytochrome oxidase and catalase. PMID:14197876

  20. Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice

    PubMed Central

    Gariani, Karim; Menzies, Keir J.; Ryu, Dongryeol; Wegner, Casey J.; Wang, Xu; Ropelle, Eduardo R.; Moullan, Norman; Zhang, Hongbo; Perino, Alessia; Lemos, Vera; Kim, Bohkyung; Park, Young‐Ki; Piersigilli, Alessandra; Pham, Tho X.; Yang, Yue; Ku, Chai Siah; Koo, Sung I.; Fomitchova, Anna; Cantó, Carlos; Schoonjans, Kristina; Sauve, Anthony A.

    2015-01-01

    With no approved pharmacological treatment, nonalcoholic fatty liver disease (NAFLD) is now the most common cause of chronic liver disease in Western countries and its worldwide prevalence continues to increase along with the growing obesity epidemic. Here, we show that a high‐fat high‐sucrose (HFHS) diet, eliciting chronic hepatosteatosis resembling human fatty liver, lowers hepatic nicotinamide adenine dinucleotide (NAD+) levels driving reductions in hepatic mitochondrial content, function, and adenosine triphosphate (ATP) levels, in conjunction with robust increases in hepatic weight, lipid content, and peroxidation in C57BL/6J mice. To assess the effect of NAD+ repletion on the development of steatosis in mice, nicotinamide riboside, a precursor of NAD+ biosynthesis, was added to the HFHS diet, either as a preventive strategy or as a therapeutic intervention. We demonstrate that NR prevents and reverts NAFLD by inducing a sirtuin (SIRT)1‐ and SIRT3‐dependent mitochondrial unfolded protein response, triggering an adaptive mitohormetic pathway to increase hepatic β‐oxidation and mitochondrial complex content and activity. The cell‐autonomous beneficial component of NR treatment was revealed in liver‐specific Sirt1 knockout mice (Sirt1hep−/−), whereas apolipoprotein E‐deficient mice (Apoe −/−) challenged with a high‐fat high‐cholesterol diet affirmed the use of NR in other independent models of NAFLD. Conclusion: Our data warrant the future evaluation of NAD+ boosting strategies to manage the development or progression of NAFLD. (Hepatology 2016;63:1190–1204) PMID:26404765

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

    PubMed

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

    2013-10-01

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

  2. Photoaffinity Labeling of High Affinity Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-Binding Proteins in Sea Urchin Egg*♦

    PubMed Central

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

    2012-01-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Recent studies have identified two-pore channels (TPCs) as endolysosomal channels that are regulated by NAADP; however, the nature of the NAADP receptor binding site is unknown. To further study NAADP binding sites, we have synthesized and characterized [32P-5-azido]nicotinic acid adenine dinucleotide phosphate ([32P-5N3]NAADP) as a photoaffinity probe. Photolysis of sea urchin egg homogenates preincubated with [32P-5N3]NAADP resulted in specific labeling of 45-, 40-, and 30-kDa proteins, which was prevented by inclusion of nanomolar concentrations of unlabeled NAADP or 5N3-NAADP, but not by micromolar concentrations of structurally related nucleotides such as NAD, nicotinic acid adenine dinucleotide, nicotinamide mononucleotide, nicotinic acid, or nicotinamide. [32P-5N3]NAADP binding was saturable and displayed high affinity (Kd ∼10 nm) in both binding and photolabeling experiments. [32P-5N3]NAADP photolabeling was irreversible in a high K+ buffer, a hallmark feature of NAADP binding in the egg system. The proteins photolabeled by [32P-5N3]NAADP have molecular masses smaller than the sea urchin TPCs, and antibodies to TPCs do not detect any immunoreactivity that comigrates with either the 45-kDa or the 40-kDa photolabeled proteins. Interestingly, antibodies to TPC1 and TPC3 were able to immunoprecipitate a small fraction of the 45- and 40-kDa photolabeled proteins, suggesting that these proteins associate with TPCs. These data suggest that high affinity NAADP binding sites are distinct from TPCs. PMID:22117077

  3. Reduction of nicotinamide adenine dinucleotide by pyruvate:lipoate oxidoreductase in anaerobic, dark-grown Rhodospirillum rubrum mutant C.

    PubMed Central

    Gorrell, T E; Uffen, R L

    1978-01-01

    Cell extracts from fermentatively grown Rhodospirillum rubrum reduced about 80 nmol of nicotinamide adenine dinucleotide (NAD) per mg of protein per min under anaerobic conditions with sodium pyruvate. The reaction was specific for pyruvate and NAD; NAD phosphate was not reduced. Results indicated that pyruvate-linked NAD reduction occurred via pyruvate:lipoate oxidoreductase. The reaction required catalytic amounts of both coenzyme A and thiamine pyrophosphate. Addition of sodium arsenite inhibited enzyme activity by 90%. Pyruvate:lipoate oxidoreductase was the only system detected in anaerobic, dark-grown R. rubrum cell extracts which operated to produce reduced NAD. The low activity of the enzyme system suggested that it was not quantitatively important in ATP formation. PMID:207677

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

    PubMed

    Meneely, Kathleen M; Lamb, Audrey L

    2007-10-23

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1995-04-01

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

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

    PubMed

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

    2001-11-01

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

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

    PubMed

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

    1976-06-01

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

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

    PubMed

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

    2015-02-15

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

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

    PubMed

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

    2012-01-01

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

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

    PubMed

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

    2012-01-01

    A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration. PMID:22091984

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

    PubMed Central

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

    2016-01-01

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

  13. Molecular beacon based bioassay for highly sensitive and selective detection of nicotinamide adenine dinucleotide and the activity of alanine aminotransferase.

    PubMed

    Tang, Zhiwen; Liu, Pei; Ma, Changbei; Yang, Xiaohai; Wang, Kemin; Tan, Weihong; Lv, Xiaoyuan

    2011-04-01

    We have developed a new approach to detect nicotinamide adenine dinucleotide (NAD(+)) with high specificity and sensitivity using molecular beacons (MBs) and employed it in the investigation of NAD(+) related biological processes, such as calorie restriction and alanine aminotransferase (ALT) activation. The E. coli DNA ligase would catalyze the ligation of two short oligonucleotides that complement with an MB only in the presence of NAD(+), resulting in the opening of the MB and the restoration of fluorescent signal. Thanks to the high sensitivity of the MB probe and the fidelity of E. coli DNA ligase toward its substrates, this approach can detect 0.3 nM NAD(+) with high selectivity against other NAD(+) analogs. This novel assay can also provide a convenient and robust way to analyze NAD(+) in biological samples such as cell lysate. As NAD(+) plays an essential role in many biochemical processes, this method can be used to investigate NAD(+) related life processes. For instance, the effect of calorie restriction on the intracellular NAD(+) level in MCF7 cells has been studied using this new assay. Moreover, this approach was also successfully used to analyze the activity of ALT. Therefore, this novel NAD(+) assay holds wide applicability as an analytical tool in biochemical and biomedical research.

  14. Electrochemical detection of nicotinamide adenine dinucleotide based on molecular beacon-like DNA and E. coli DNA ligase.

    PubMed

    He, Xiaoxiao; Ni, Xiaoqi; Wang, Yonghong; Wang, Kemin; Jian, Lixin

    2011-01-15

    An electrochemical method for nicotinamide adenine dinucleotide (NAD(+)) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5'-SH and 3'-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD(+), E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD(+)-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD(+) range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD(+) from its analogues.

  15. Unique kinetics of nicotinic acid-adenine dinucleotide phosphate (NAADP) binding enhance the sensitivity of NAADP receptors for their ligand.

    PubMed Central

    Patel, S; Churchill, G C; Galione, A

    2000-01-01

    Nicotinic acid-adenine dinucleotide phosphate (NAADP) is a novel and potent Ca(2+)-mobilizing agent in sea urchin eggs and other cell types. Little is known, however, concerning the properties of the putative intracellular NAADP receptor. In the present study we have characterized NAADP binding sites in sea urchin egg homogenates. [(32)P]NAADP bound to a single class of high-affinity sites that were reversibly inhibited by NaCl but insensitive to pH and Ca(2+). Binding of [(32)P]NAADP was lost in preparations that did not mobilize Ca(2+) in response to NAADP, indicating that [(32)P]NAADP probably binds to a receptor mediating Ca(2+) mobilization. Addition of excess unlabelled NAADP, at various times after initiation of [(32)P]NAADP binding, did not result in displacement of bound [(32)P]NAADP. These data show that NAADP becomes irreversibly bound to its receptor immediately upon association. Accordingly, incubation of homogenates with low concentrations of NAADP resulted in maximal labelling of NAADP binding sites. This unique property renders NAADP receptors exquisitely sensitive to their ligand, thereby allowing detection of minute changes in NAADP levels. PMID:11104679

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

    PubMed

    Goran, Jacob M; Stevenson, Keith J

    2013-11-01

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

  17. EPR and potentiometric studies of copper(II) binding to nicotinamide adenine dinucleotide (NAD+) in water solution.

    PubMed

    Hoffmann, Stanisław K; Goslar, Janina; Lijewski, Stefan; Basiński, Kamil; Gąsowska, Anna; Łomozik, Lechosław

    2012-06-01

    Coordination of Cu(II) by nicotinamide adenine dinucleotide (NAD(+)) molecule has been studied in water solutions of various pH by potentiometry and electron paramagnetic resonance (EPR) and electron spin echo (ESE) spectroscopy. Potentiometric results indicate Cu(II) coordination by protonated NAD(+) at low pH and by deprotonated NAD(+) at high pH. At medium pH value (around pH=7) NAD(+) is not able to coordinate Cu(II) ions effectively and mainly the Cu(H(2)O)(6) complexes exist in the studied solution. This has been confirmed by EPR results. Electronic structure of Cu(II)-NAD complex and coordination sites is determined from EPR and ESE measurements in frozen solutions (at 77K and 6K). EPR spectra exclude coordination with nitrogen atoms. Detailed analysis of EPR parameters (g(||)=2.420, g(perpendicular)==2.080, A(||)=-131×10(-4)cm(-1) and A(perpendicular)=8×10(-4)cm(-1)) performed in terms of molecular orbital (MO) theory shows that Cu(II)NAD complex has elongated axial octahedral symmetry with a relatively strong delocalization of unpaired electron density on in-plane and axial ligands. The distortion of octahedron is analyzed using A(||) vs. g(||) diagram for various CuO(x) complexes. Electron spin echo decay modulation excludes the coordination by oxygen atoms of phosphate groups. We postulate a coordination of Cu(II) by two hydroxyl oxygen atoms of two ribose moieties of the NAD molecules and four solvated water molecules both at low and high pH values with larger elongation of the octahedron at higher pH.

  18. Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: effects of acute oxidative stress.

    PubMed

    Masihzadeh, Omid; Ammar, David A; Lei, Tim C; Gibson, Emily A; Kahook, Malik Y

    2011-09-01

    The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H(2)O(2)) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H(2)O(2) on the biochemistry of the TM cells and the initial response to oxidative stress. In this report, we have used two-photon excitation autofluorescence (2PAF) to monitor changes to TM cell nicotinamide adenine dinucleotide (NADPH). 2PAF allows non-destructive, real-time analysis of concentration of intracellular NADPH. Coupled to reduced glutathione, NADPH, is a major component in the anti-oxidant defense of TM cells. Cultured human TM cells were monitored for over 30 min in control and H(2)O(2)-containing solutions. Peroxide caused both a dose- and time-dependent decrease in NADPH signal. NADPH fluorescence in control and in 4 mM H(2)O(2) solutions showed little attenuation of NADPH signal (4% and 9% respectively). TM cell NADPH fluorescence showed a linear decrease with exposure to 20 mM H(2)O(2) (-29%) and 100 mM H(2)O(2) (37%) after a 30 min exposure. Exposure of TM cells to 500 mM H(2)O(2) caused an exponential decrease in NADPH fluorescence to a final attenuation of 46% of starting intensity. Analysis of individual TM cells indicates that cells with higher initial NADPH fluorescence are more refractive to the apparent loss of viability caused by H(2)O(2) than weakly fluorescing TM cells. We conclude that 2PAF of intracellular NADPH is a valuable tool for studying TM cell metabolism in response to oxidative insult. PMID:21354135

  19. ß-nicotinamide adenine dinucleotide is an enteric inhibitory neurotransmitter in human and non-human primate colons

    PubMed Central

    Hwang, Sung Jin; Durnin, Leonie; Dwyer, Laura; Rhee, Poong-Lyul; Ward, Sean M.; Koh, Sang Don; Sanders, Kenton M.; Mutafova-Yambolieva, Violeta N.

    2010-01-01

    Background & Aims An important component of enteric inhibitory neurotransmission is mediated by a purine neurotransmitter, such as adenosine 5’-triphosphate (ATP), binding to P2Y1 receptors and activating small conductance K+ channels. In murine colon ß-nicotinamide adenine dinucleotide (ß-NAD) is released with ATP and mimics the pharmacology of inhibitory neurotransmission better than ATP. Here ß-NAD and ATP were compared as possible inhibitory neurotransmitters in human and monkey colons. Methods A small-volume superfusion assay and HPLC with fluorescence detection were used to evaluate spontaneous and nerve-evoked overflow of ß-NAD, ATP and metabolites. Postjunctional responses to nerve stimulation, ß-NAD and ATP were compared using intracellular membrane potential and force measurements. Effects of ß-NAD on smooth muscle cells (SMCs) were recorded by patch clamp. P2Y receptor transcripts and proteins were assayed by RT-PCR. Results In contrast to ATP, overflow of ß-NAD evoked by electrical field stimulation correlated with stimulation frequency and was diminished by neurotoxins, tetrodotoxin and ω-conotoxin GVIA. Inhibitory junction potentials and responses to exogenous ß-NAD, but not ATP, were blocked by P2Y receptor antagonists suramin, PPADS, MRS2179 and MRS2500. ß-NAD activated non-selective cation currents in SMCs, but failed to activate outward currents. Conclusions ß-NAD meets the criteria for a neurotransmitter better than ATP in human and monkey colons and therefore may contribute to neural regulation of colonic motility. SMCs are unlikely targets for inhibitory purine neurotransmitters because dominant responses of SMCs were activation of net inward, rather than outward, current. PMID:20875415

  20. Morphological features, distribution and compartmental organization of the nicotinamide adenine dinucleotide phosphate reduced-diaphorase interneurons in the human striatum.

    PubMed

    Bernácer, Javier; Prensa, Lucía; Giménez-Amaya, José Manuel

    2005-08-29

    Striatal nicotinamide adenine dinucleotide phosphate reduced-diaphorase (NADPH-d)-positive (+) cells are one of the major classes of striatal interneurons. The present study analyzes their somatodendritic morphology, distribution pattern, and compartmental organization in the caudate nucleus (CN) and putamen (Put) of nine normal human brains. The following striatal territories are examined: 1) the precommissural head of the CN; 2) the postcommissural head of the CN; 3) the body of the CN; 4) the gyrus of the CN; 5) the tail of the CN; 6) the precommissural Put; and 7) the postcommissural Put. Three morphologically distinct types of NADPH-d+ neurons were found in each of these territories. The two most common NADPH-d+ neurons displayed an ovoid or triangular perikaryon from which several thick primary dendrites emerged, although much less numerous, bipolar-shaped NADPH-d+ cells were also observed. The highest density of NADPH-d+ neurons was found in the gyrus of the CN, followed by the body of the CN, tail of the CN, postcommissural head of the CN, postcommissural Put, precommissural head of the CN, and precommissural Put. The matrix was the striatal compartment with the densest NADPH-d+ neuronal population. Some of these cells also occurred in the center and peripheral regions of the striosomes located in the head of the CN and in the Put. In the body and gyrus of the CN, the striosomes were largely devoid of these striatal interneurons. Knowledge of the density and distribution of these interneurons should advance our understanding of the organization of the normal human striatum and help to evaluate the effects of neurodegenerative processes on cell density.

  1. Real-time measurements of nicotinamide adenine dinucleotide in live human trabecular meshwork cells: Effects of acute oxidative stress✩

    PubMed Central

    Masihzadeh, Omid; Ammar, David A.; Lei, Tim C.; Gibson, Emily A.; Kahook, Malik Y.

    2016-01-01

    The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H2O2) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H2O2 on the biochemistry of the TM cells and the initial response to oxidative stress. In this report, we have used two-photon excitation autofluorescence (2PAF) to monitor changes to TM cell nicotinamide adenine dinucleotide (NADPH). 2PAF allows non-destructive, real-time analysis of concentration of intracellular NADPH. Coupled to reduced glutathione, NADPH, is a major component in the anti-oxidant defense of TM cells. Cultured human TM cells were monitored for over 30 min in control and H2O2-containing solutions. Peroxide caused both a dose- and time-dependent decrease in NADPH signal. NADPH fluorescence in control and in 4 mM H2O2 solutions showed little attenuation of NADPH signal (4% and 9% respectively). TM cell NADPH fluorescence showed a linear decrease with exposure to 20 mM H2O2 (−29%) and 100 mM H2O2 (37%) after a 30 min exposure. Exposure of TM cells to 500 mM H2O2 caused an exponential decrease in NADPH fluorescence to a final attenuation of 46% of starting intensity. Analysis of individual TM cells indicates that cells with higher initial NADPH fluorescence are more refractive to the apparent loss of viability caused by H2O2 than weakly fluorescing TM cells. We conclude that 2PAF of intracellular NADPH is a valuable tool for studying TM cell metabolism in response to oxidative insult. PMID:21354135

  2. Evidence for the Degradation of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutamate Dehydrogenase of Candida utilis During Rapid Enzyme Inactivation

    PubMed Central

    Hemmings, Brian A.

    1978-01-01

    The nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADP-GDH) from the food yeast Candida utilis was found to be rapidly inactivated when cultures were starved of a carbon source. The addition of glutamate or alanine to the starvation medium stimulated the rate of inactivation. Loss of enzyme activity was irreversible since the reappearance of enzyme activity, following the addition of glucose to carbon-starved cultures, was blocked by cycloheximide. A specific rabbit antibody was prepared against the NADP-GDH from C. utilis and used to quantitate the enzyme during inactivation promoted by carbon starvation. The amount of precipitable antigenic material paralleled the rapid decrease of enzyme activity observed after transition of cells from NH4+-glucose to glutamate medium. No additional small-molecular-weight protein was precipitated by the antibody as a result of the inactivation, suggesting that the enzyme is considerably altered during the primary steps of the inactivation process. Analysis by immunoprecipitation of the reappearance of enzyme activity after enzyme inactivation showed that increase of NADP-GDH activity was almost totally due to de novo synthesis, ruling out the possibility that enzyme activity modulation is achieved by reversible covalent modification. Enzyme degradation was also measured during steady-state growth and other changes in nitrogen and carbon status of the culture media. In all instances so far estimated, the enzyme was found to be very stable and not normally subject to high rates of degradation. Therefore, the possibility that inactivation was caused by a change in the ratio of synthesis to degradation can be excluded. Images PMID:24041

  3. A functional role for nicotinic acid adenine dinucleotide phosphate in oxytocin-mediated contraction of uterine smooth muscle from rat.

    PubMed

    Aley, Parvinder K; Noh, Hyun J; Gao, Xin; Tica, Andrei A; Brailoiu, Eugen; Churchill, Grant C

    2010-06-01

    Conventionally, G protein-coupled receptors are thought to increase calcium via inositol 1,4,5-trisphosphate (InsP(3)). More recent evidence shows that an alternative second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), also has a role to play, causing researchers to question established calcium releasing pathways. With the recent development, by our group, of cell-permeant NAADP (NAADP-aceteoxymethyl ester) and a selective NAADP receptor antagonist (Ned-19; 1-(3-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid),the ability to investigate this signaling pathway has improved. Therefore, we investigated a role for NAADP in oxytocin-mediated responses in the rat uterus. Oxytocin- and NAADP-mediated effects were investigated by using contractile measurements of whole uterine strips from rat in organ baths. Responses were correlated to calcium release in cultured rat uterine smooth muscle cells measured by fluorescence microscopy. Inhibition of both oxytocin-induced contraction and calcium release by the traditional NAADP-signaling disrupter bafilomycin and the NAADP receptor antagonist Ned-19 clearly demonstrated a role for NAADP in oxytocin-induced signaling. A cell-permeant form of NAADP was able to produce both uterine contractions and calcium release. This response was unaffected by depletion of sarcoplasmic reticulum stores with thapsigargin, but was abolished by both bafilomycin and Ned-19. Crucially, oxytocin stimulated an increase in NAADP in rat uterine tissue. The present study demonstrates directly that NAADP signaling plays a role in rat uterine contractions. Moreover, investigation of this signaling pathway highlights yet another component of oxytocin-mediated signaling, stressing the need to consider the action of new components as they are discovered, even in signaling pathways that are thought to be well established.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-07-30

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

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

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

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

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

    PubMed

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

    2014-02-20

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

  8. Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I.

    PubMed

    Lorenz, Nicole; Olsovská, Jana; Sulc, Miroslav; Tudzynski, Paul

    2010-03-01

    Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I. PMID:20118373

  9. Regulation of Enzymes Involved in the Conversion of Tryptophan to Nicotinamide Adenine Dinucleotide in a Colorless Strain of Xanthomonas pruni1

    PubMed Central

    Brown, Albert T.; Wagner, Conrad

    1970-01-01

    A colorless strain of Xanthomonas pruni was isolated which is capable of converting tryptophan to nicotinamide adenine dinucleotide (NAD). The enzymes responsible for the conversion of tryptophan to quinolinic acid were shown to be present. Nicotinic acid-requiring mutants were isolated, and it was found that the growth of these mutants can be supported by various intermediates on the pathway from tryptophan to NAD. The first three enzymes on this pathway are induced coordinately by l-tryptophan. Gratuitous inducers of these enzymes include d-tryptophan, α-methyl-dl-tryptophan, and 4-methyl-dl-tryptophan; formyl-l-kynurenine and l-kynurenine were not effective as inducers. These data suggest that at least the first three enzymes in the pathway from tryptophan to NAD are under common regulatory control. PMID:4313053

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

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

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

    PubMed

    Ido, Yasuo

    2016-07-01

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

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

    PubMed

    Ido, Yasuo

    2016-07-01

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

  13. Influence of surface adsorption on the interfacial electron transfer of flavin adenine dinucleotide and glucose oxidase at carbon nanotube and nitrogen-doped carbon nanotube electrodes.

    PubMed

    Goran, Jacob M; Mantilla, Sandra M; Stevenson, Keith J

    2013-02-01

    The adsorption of flavin adenine dinucleotide (FAD) and glucose oxidase (GOx) onto carbon nanotube (CNT) and nitrogen-doped CNT (N-CNT) electrodes was investigated and found to obey Langmuir adsorption isotherm characteristics. The amount adsorbed and adsorption maximum are dependent on exposure time, the concentration of adsorbate, and the ionic strength of the solution. The formal potentials measured for FAD and GOx are identical, indicating that the observed electroactivity is from FAD, the redox reaction center of GOx. When glucose is added to GOx adsorbed onto CNT/N-CNT electrodes, direct electron transfer (DET) from enzyme-active FAD is not observed. However, efficient mediated electron transfer (MET) occurs if an appropriate electron mediator is placed in solution, or the natural electron mediator oxygen is used, indicating that GOx is adsorbed and active on CNT/N-CNT electrodes. The observed surface-confined redox reaction at both CNT and N-CNT electrodes is from FAD that either specifically adsorbs from solution or adsorbs from the holoprotein subsequently inactivating the enzyme. The splitting of cathodic and anodic peak potentials as a function of scan rate provides a way to measure the heterogeneous electron-transfer rate constant (k(s)) using Laviron's method. However, the measured k(s) was found to be under ohmic control, not under the kinetic control of an electron-transfer reaction, suggesting that k(s) for FAD on CNTs is faster than the measured value of 7.6 s(-1).

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

    PubMed

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

    1984-09-01

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

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

    PubMed

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

    2016-03-15

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

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

    PubMed

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

    2012-11-08

    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.

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

    PubMed

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

    2015-08-28

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2014-03-25

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

  20. Ordered carbohydrate-derived porous carbons immobilized gold nanoparticles as a new electrode material for electrocatalytical oxidation and determination of nicotinamide adenine dinucleotide.

    PubMed

    Hosseini, Hadi; Behbahani, Mohammad; Mahyari, Mojtaba; Kazerooni, Hanif; Bagheri, Akbar; Shaabani, Ahmad

    2014-09-15

    The ordered carbohydrate-derived porous carbons (OC-DPCs) were first functionalized with thiol groups (-SH) and then immobilized with gold nanoparticles (AuNPs). The Au-SH-OC-DPCs were characterized by CHN analysis, transmission electron microscopy (TEM), fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and x-ray diffraction (XRD). The Au-SH-OC-DPCs were applied for the fabrication of a new electrochemical sensor. The electrocatalytic capabilities of the new sensor were tested by the oxidation of nicotinamide adenine dinucleotide (NADH) in a 0.1 M Robinson buffer solution (pH 7.0) using cyclic voltammetry (CV), linear sweep voltammetry (LSV), and differential pulse voltammetry (DPV). The Au-SH-OC-DPCs showed a good voltammetric performance in the electrochemical detection of NADH with a low detection limit (1.0 nM), high sensitivity (4.934 μA/μM), and wide linear concentration range (5.0 nM-10 µM).

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

    PubMed

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

    2015-08-28

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

  2. Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic β-cells and adipocytes.

    PubMed

    Park, Kwang-Hyun; Kim, Byung-Ju; Shawl, Asif Iqbal; Han, Myung-Kwan; Lee, Hon Cheung; Kim, Uh-Hyun

    2013-12-01

    Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger for mobilizing Ca(2+) from intracellular stores in various cell types. Extracellular application of NAADP has been shown to elicit intracellular Ca(2+) signals, indicating that it is readily transported into cells. However, little is known about the functional role of this NAADP uptake system. Here, we show that NAADP is effectively transported into selected cell types involved in glucose homeostasis, such as adipocytes and pancreatic β-cells, but not the acinar cells, in a high glucose-dependent manner. NAADP uptake was inhibitable by Ned-19, a NAADP mimic; dipyridamole, a nucleoside inhibitor; or NaN3, a metabolic inhibitor or under Ca(2+)-free conditions. Furthermore, NAADP was found to be released from pancreatic islets upon stimulation by high glucose. Consistently, administration of NAADP to type 2 diabetic mice improved glucose tolerance. We propose that NAADP is functioning as an autocrine/paracrine hormone important in glucose homeostasis. NAADP is thus a potential antidiabetic agent with therapeutic relevance.

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

    NASA Astrophysics Data System (ADS)

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

    2011-03-01

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

  4. Differential regulation of nicotinic acid-adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP.

    PubMed Central

    Wilson, H L; Galione, A

    1998-01-01

    The sea urchin egg has been used as a system to study calcium-release mechanisms induced by inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR), and more recently, nicotinic acid-adenine dinucleotide phosphate (NAADP). In order that cADPR and NAADP may be established as endogenous messengers for calcium release, the existence of intracellular enzymes capable of metabolizing these molecules must be demonstrated. In addition, intracellular levels of cADPR and NAADP should be under the control of extracellular stimuli. It has been shown that cGMP stimulates the synthesis of cADPR in the sea urchin egg. The present study shows that the sea urchin egg is capable of synthesizing and degrading NAADP. cADPR and NAADP synthetic activities appear to be separate, with different cellular localizations, pH and temperature optima. We suggest that in the sea urchin egg, cADPR and NAADP production may be differentially regulated by receptor-coupled second messengers, with cADPR production being regulated by cGMP and NAADP production modulated by cAMP. PMID:9560312

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

    PubMed Central

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

    1998-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

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

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

    PubMed

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

    2003-03-28

    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.

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

    PubMed

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

    2015-12-01

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2015-07-01

    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.

  11. Dietary adenine controls adult lifespan via adenosine nucleotide biosynthesis and AMPK, and regulates the longevity benefit of caloric restriction

    PubMed Central

    Stenesen, Drew; Suh, Jae Myoung; Seo, Jin; Yu, Kweon; Lee, Kyu-Sun; Kim, Jong-Seok; Min, Kyung-Jin; Graff, Jonathan M.

    2012-01-01

    SUMMARY A common thread among conserved lifespan regulators lies within intertwined roles in metabolism and energy homeostasis. We show that heterozygous mutations of adenosine monophosphate (AMP) biosynthetic enzymes extend Drosophila lifespan. The lifespan benefit of these mutations depends upon increased AMP to adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to ATP ratios and adenosine monophosphate-activated protein kinase (AMPK). Transgenic expression of AMPK in adult fat body or adult muscle, key metabolic tissues, extended lifespan, while AMPK RNAi reduced lifespan. Supplementing adenine, a substrate for AMP biosynthesis, to the diet of long-lived AMP biosynthesis mutants reversed lifespan extension. Remarkably, this simple change in diet also blocked the pro-longevity effects of dietary restriction. These data establish AMP biosynthesis, adenosine nucleotide ratios, and AMPK as determinants of adult lifespan, provide a mechanistic link between cellular anabolism and energy sensing pathways, and indicate that dietary adenine manipulations might alter metabolism to influence animal lifespan. PMID:23312286

  12. 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

    PubMed Central

    2012-01-01

    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 (CTGF)in 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 <0.05). The expression of myocardial p22phox, NOX4, MCP-1, and CTGF was significantly increased in diabetic rats (P <0.05). The expression of adipoR1 was decreased and the expression of MCP-1 and NF-κB was increased in the abdominal aorta in diabetic rats (P <0.05). Telmisartan treatment significantly attenuated these changes in diabetic rats (P <0.05). 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. PMID:22873349

  13. 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.

    PubMed

    Kumar, S Ashok; Chen, Shen-Ming

    2007-05-29

    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 degrees C) from the bath solution containing 0.1 M Zn(NO3)2, 0.1 M KNO3 and 1x10(-4) MMB. 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 (E0') -0.12 V (pH 6.9). The surface coverage (gamma) of the MB immobilized on ZnO/GC was about 9.86x10(-12) mol cm(-2) and the electron transfer rate constant (ks) was determined to be 38.9 s(-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 microM NADH concentration at pH 6.9 was observed with a detection limit of 10 microM (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.

  14. The regulation of respiration of guinea pig taenia coli in high-K medium: the role of nicotinamide-adenine dinucleotide, adenosine diphosphate and Ca++.

    PubMed

    Tsuda, S; Urakawa, N; Saito, Y; Fukami, J

    1975-10-01

    In an attempt to elucidate the regulation mechanism of respiration in the smooth muscle cell, we investigated the roles of nicotinamide-adenine dinucleotide (NAD), adenosine diphosphate (ADP) and Ca++ in the muscle respiration using the tissues and subcellular fractions from guinea pig taenia coli. The tension in the strips of taenia coli increased with a concomitant increase in O2 consumption in high-K medium (40 mM K) containing 2.5 mM Ca. 10(-3) M amytal and 10(-5)M ouabain decreased the high-K induced tension and O2 consumption of the muscle. 10(-4)M 2,4-dinitrophenol (DNP) relieved the decreased respiration induced by ouabain, but not that with amytal. From these data it is suggested that NADH-linked respiration plays an important role in the respiration of the muscle. Ca++ in concentrations ranging from 0.5 to 2.5 mM in the high-K medium resulted in an increase in tension and in O2 concumption progressively. In spectrophotometric observations of subcellular fractions of the taenia coli, ADP increased in absorbance change at 340 m mu. Such occurred in mitochondrial fractions and was initiated by the addition of NADH. Therefore it is deduced that the increase in ADP level of the cytoplasm is primarily due to a contraction triggered by Ca++ thus stimulating respiration. On the other hand, at 0.1 mM of Ca++ concentration, the muscle strip increased O2 consumption without tension development in high-K medium. In the spectrophotometric observations, Ca++ and Sr++ increased the absorbance change in the homogenate and in the mitochondrial fraction. Hence, it seems that one part of the Ca++ entering into the smooth muscle treated with the high-K increased O2 consumption in mitochondia independent of an increase in muscle tension. From these results it is concluded that NADH-linked respiration plays an important role in the smooth muscle respiration in high-K medium and that ADP and Ca++ also play a role in regulating respiration. PMID:176493

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2005-10-21

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

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

    PubMed

    Rubery, P H

    1972-06-01

    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.

  18. Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-α-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway

    PubMed Central

    CAO, YANJUN; LIU, FENG; HUANG, ZHUANGZHUANG; ZHANG, YANMIN

    2014-01-01

    The Chinese medicinal formula Guanxin Shutong capsule (GXSTC) has been used for almost 10 years as a clinical treatment for chest pain, depression, palpitation and cardiovascular diseases. The aim of this study was to investigate the effects of GXSTC drug-containing serum on tumor necrosis factor-α (TNF-α)-stimulated endothelial cells. Cell viability was measured by MTT assay, and nitric oxide (NO) levels and NO synthase (NOS) activity were measured as standards of endothelial dysfunction. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were evaluated using commercial kits. In addition, the protein expression of endothelial NOS (eNOS), AKT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits was examined to evaluate the effect of GXSTC drug-containing serum on ECV304 cells. GXSTC significantly reversed the decrease in NO production induced by TNF-α (5 ng/ml) in ECV304 cells. The expression of NADPH oxidase subunits was increased by TNF-α treatment, but markedly inhibited by treatment with GXSTC in TNF-α-stimulated cells. In summary, GXSTC increased the production of NO in ECV304 cells and exerted a protective effect on ECV304 cells stimulated with TNF-α by upregulating the mRNA and protein expression of eNOS. This was accompanied by increased SOD activity and reduced MDA levels. These results suggested that GXSTC protects the endothelium via the NO pathway and exhibits antioxidant effects. PMID:25120637

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

    PubMed Central

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

    2016-01-01

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

  20. Role of the C-terminal extension stacked on the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide prosthetic group in ferredoxin-NADP(+) oxidoreductase from Bacillus subtilis.

    PubMed

    Seo, Daisuke; Asano, Tomoya; Komori, Hirofumi; Sakurai, Takeshi

    2014-08-01

    Ferredoxin-NADP(+) oxidoreductase [EC 1.18.1.2] from Bacillus subtilis (BsFNR) is homologous to the bacterial NADPH-thioredoxin reductase, but possesses a unique C-terminal extension that covers the re-face of the isoalloxazine ring moiety of the flavin adenine dinucleotide (FAD) prosthetic group. In this report, we utilize BsFNR mutants depleted of their C-terminal residues to examine the importance of the C-terminal extension in reactions with NADPH and ferredoxin (Fd) from B. subtilis by spectroscopic and steady-state reaction analyses. The depletions of residues Y313 to K332 (whole C-terminal extension region) and S325 to K332 (His324 intact) resulted in significant increases in the catalytic efficiency with NADPH in diaphorase assay with ferricyanide, whereas Km values for ferricyanide were increased. In the cytochrome c reduction assay in the presence of B. subtilis ferredoxin, the S325-K332 depleted mutant displayed a significant decrease in the turnover rate with an Fd concentration range of 1-10 μM. The Y313-K332 depleted mutant demonstrated an increase in the rate of the direct reduction of horse heart cytochrome c in the absence of Fd. These data indicated that depletion of the C-terminal extension plays an important role in the reaction of BsFNR with ferredoxin.

  1. Nicotinamide Adenine Dinucleotide Based Therapeutics, Update.

    PubMed

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

    2015-01-01

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

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

    PubMed

    Chifotides, Helen T; Dunbar, Kim R

    2007-10-17

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

  3. Is Nicotinamide Adenine Dinucleotide Phosphate an Obligatory Intermediate in Photosynthesis?

    PubMed Central

    Ben-Amotz, Ami; Avron, Mordhay

    1972-01-01

    The site of action of the inhibitors disalicylidenepropanediamine and pyrophosphate was more closely defined as acting on ferredoxin. Three inhibitors which act on the electron transport path between ferredoxin and NADP: disalicylidenepropanediamine, pyrophosphate, and phosphoadenosinediphosphate ribose, had no effect on photosynthesis in cell free preparations of Dunaliela parva at concentrations which completely inhibited the enzymic activity on which each inhibitor acts. The addition of disalicylidenepropanediamine to dark-grown Euglena gracilis cells prevented the light-induced formation of NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, but not of photosynthesis, chlorophyll synthesis, or NAD-dependent glyceraldehyde-3-phosphate dehydrogenase. The above results are interpreted as indicating that, at least under some conditions, a reduced product of photosystem I preceding ferredoxin in the electron transport path can serve as the reductant of CO2 in photosynthesis. PMID:16657933

  4. Binding of nicotinamide–adenine dinucleotides to diphtheria toxin

    PubMed Central

    Montanaro, L.; Sperti, Simonetta

    1967-01-01

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

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

    PubMed

    Fang, Pengfei; Guo, Min

    2015-05-01

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

  6. Search for interstellar adenine

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  7. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

    PubMed

    Yoshimoto, Naoko; Onuma, Misato; Mizuno, Shinya; Sugino, Yuka; Nakabayashi, Ryo; Imai, Shinsuke; Tsuneyoshi, Tadamitsu; Sumi, Shin-ichiro; Saito, Kazuki

    2015-09-01

    S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.

  8. Evolution probabilities and phylogenetic distance of dinucleotides.

    PubMed

    Michel, Christian J

    2007-11-21

    We develop here an analytical evolution model based on a dinucleotide mutation matrix 16 x 16 with six substitution parameters associated with the three types of substitutions in the two dinucleotide sites. It generalizes the previous models based on the nucleotide mutation matrices 4 x 4. It determines at some time t the exact occurrence probabilities of dinucleotides mutating randomly according to these six substitution parameters. Furthermore, several properties and two applications of this model allow to derive 16 evolutionary analytical solutions of dinucleotides and also a dinucleotide phylogenetic distance. Finally, based on this mathematical model, the SED (Stochastic Evolution of Dinucleotides) web server has been developed for deriving evolutionary analytical solutions of dinucleotides.

  9. Photodissociation of dinucleotide ions in a storage ring

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

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

  10. Mutations in the 3β-Hydroxysterol Δ24-Reductase Gene Cause Desmosterolosis, an Autosomal Recessive Disorder of Cholesterol Biosynthesis

    PubMed Central

    Waterham, Hans R.; Koster, Janet; Romeijn, Gerrit Jan; Hennekam, Raoul C.M.; Vreken, Peter; Andersson, Hans C.; FitzPatrick, David R.; Kelley, Richard. I.; Wanders, Ronald J. A.

    2001-01-01

    Desmosterolosis is a rare autosomal recessive disorder characterized by multiple congenital anomalies. Patients with desmosterolosis have elevated levels of the cholesterol precursor desmosterol, in plasma, tissue, and cultured cells; this abnormality suggests a deficiency of the enzyme 3β-hydroxysterol Δ24-reductase (DHCR24), which, in cholesterol biosynthesis, catalyzes the reduction of the Δ24 double bond of sterol intermediates. We identified the human DHCR24 cDNA, by the similarity between the encoded protein and a recently characterized plant enzyme—DWF1/DIM, from Arabidopsis thaliana—catalyzing a different but partially similar reaction in steroid/sterol biosynthesis in plants. Heterologous expression, in the yeast Saccharomyces cerevisiae, of the DHCR24 cDNA, followed by enzyme-activity measurements, confirmed that it encodes DHCR24. The encoded DHCR24 protein has a calculated molecular weight of 60.1 kD, contains a potential N-terminal secretory-signal sequence as well as at least one putative transmembrane helix, and is a member of a recently defined family of flavin adenine dinucleotide (FAD)–dependent oxidoreductases. Conversion of desmosterol to cholesterol by DHCR24 in vitro is strictly dependent on reduced nicotinamide adenine dinucleotide phosphate and is increased twofold by the addition of FAD to the assay. The corresponding gene, DHCR24, was identified by database searching, spans ∼46.4 kb, is localized to chromosome 1p31.1-p33, and comprises nine exons and eight introns. Sequence analysis of DHCR24 in two patients with desmosterolosis revealed four different missense mutations, which were shown, by functional expression, in yeast, of the patient alleles, to be disease causing. Our data demonstrate that desmosterolosis is a cholesterol-biosynthesis disorder caused by mutations in DHCR24. PMID:11519011

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

    SciTech Connect

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

    2006-10-27

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

  12. Hydrogen isotope fractionation during lipid biosynthesis by Haloarcula marismortui

    NASA Astrophysics Data System (ADS)

    Dirghangi, Sitindra S.; Pagani, Mark

    2013-10-01

    We studied the controls on the fractionation of hydrogen isotopes during lipid biosynthesis by Haloarcula marismortui, a halophilic archaea, in pure culture experiments by varying organic substrate, the hydrogen isotope composition (D/H) of water, temperature, and salinity. Cultures were grown on three substrates: succinate, pyruvate and glycerol with known hydrogen isotope compositions, and in water with different hydrogen isotopic compositions. All culture series grown on a particular substrate show strong correlations between δDarchaeol and δDwater. However, correlations are distinctly different for cultures grown on different substrates. Our results indicate that the metabolic pathway of substrate exerts a fundamental influence on the δD value of lipids, likely by influencing the D/H composition of NADPH (nicotinamide adenine dinucleotide phosphate), the reducing agent that contributes hydrogen to carbon atoms during lipid biosynthesis. Temperature and salinity have smaller, but similar effects on δDlipid, primarily due to the way temperature and salinity influence growth rate, as well as temperature effects on the activity of enzymes.

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

    PubMed

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

    2015-08-01

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

  14. Vertical Ionization Energies of Adenine and 9-Methyl Adenine

    NASA Astrophysics Data System (ADS)

    Dolgounitcheva, O.; Zakrzewski, V. G.; Ortiz, J. V.

    2009-07-01

    Vertical ionization energies of 9-H adenine and 9-methyl adenine have been calculated with the following, ab initio, electron propagator methods: the outer valence Green's function (OVGF), partial third-order theory (P3), and the third-order algebraic diagrammatic construction, or ADC(3). Basis set effects have been systematically examined. All methods predict near degeneracy in the π2-n1 and π3-n2 pairs of cationic, adenine final states and larger splittings of the corresponding, cationic states of 9-methyl adenine. P3 results for adenine predict the following order of the first six final states: π1, n1, π2, n2, π3, n3. Coupled-cluster calculations on the first three cationic states of adenine confirm these predictions. OVGF and ADC(3) calculations reverse the order of the second and third states and of the fourth and fifth states. All results confirm previous interpretations of experiments in which the second and third spectral bands correspond to the aforementioned pairs of final states and disagree with a recent reassignment based on time-resolved photoelectron spectra. Lower ionization energies and larger splittings in the methylated molecule are interpreted in terms of phase relationships in the Dyson orbitals. ADC(3) results confirm the qualitative validity of the one-electron approximation for the first six final states of both molecules and disclose its inadequacies for higher ionization energies.

  15. Inactivation of yeast alcohol dehydrogenase by alkylperoxyl radicals. Characteristics and influence of nicotinamide-adenine dinucleotides.

    PubMed

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

    1992-10-01

    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.

  16. The responses of isolated plant mitochondria to external nicotinamide adenine dinucleotide.

    PubMed

    Soole, K L; Dry, I B; Wiskich, J T

    1986-06-01

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

  17. The activity of liver alcohol dehydrogenase with nicotinamide–adenine dinucleotide phosphate as coenzyme

    PubMed Central

    Dalziel, K.; Dickinson, F. M.

    1965-01-01

    1. The separation of nucleotide impurities from commercial NADP preparations by chromatography is described. All the preparations studied contained 0·1–0·2% of NAD. 2. The activity of pure crystalline liver alcohol dehydrogenase with NADP as coenzyme has been confirmed. Initial-rate data are reported for the reaction at pH 6·0 and 7·0 with ethanol and acetaldehyde as substrates. With NADP and NADPH2 of high purity, the maximal specific rates were similar to those obtained with NAD and NADH2, but the Michaelis constants for the former coenzymes were much greater than those for the latter. 3. The oxidation of ethanol by NADP is greatly inhibited by NADH2, and this accounts for low values of certain initial-rate parameters obtained with commercial NADP preparations containing NAD. The kinetics of the inhibition are consistent with competitive inhibition in a compulsory-order mechanism. 4. Initial-rate data with NAD and NADPH2 do not conform to the requirements of the mechanism proposed by Theorell & Chance (1951), in contrast with results previously obtained with NAD and NADH2. The possibility that the deviations are due to competing nucleotide impurity in the oxidized coenzyme cannot be excluded. The data show that the enzyme reacts more slowly with, and has a smaller affinity for, NADP and NADPH2 than NAD and NADH2. 5. Phosphate behaves as a competitive inhibitor towards NADP. PMID:14340079

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

    PubMed

    Ternes, Chad M; Schönknecht, Gerald

    2014-09-01

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

  19. Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

    PubMed Central

    Ternes, Chad M.; Schönknecht, Gerald

    2014-01-01

    NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

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

    PubMed Central

    Tischfield, Jay A.; Ruddle, Frank H.

    1974-01-01

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

  1. Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2′-phosphate

    PubMed Central

    Tóth, Balázs; Iordanov, Iordan

    2015-01-01

    Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid–adenine-dinucleotide (NAAD), and NAAD-2′-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified–specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2′-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest. PMID:25918360

  2. Protein Modification by Adenine Propenal

    PubMed Central

    2015-01-01

    Base propenals are products of the reaction of DNA with oxidants such as peroxynitrite and bleomycin. The most reactive base propenal, adenine propenal, is mutagenic in Escherichia coli and reacts with DNA to form covalent adducts; however, the reaction of adenine propenal with protein has not yet been investigated. A survey of the reaction of adenine propenal with amino acids revealed that lysine and cysteine form adducts, whereas histidine and arginine do not. Nε-Oxopropenyllysine, a lysine–lysine cross-link, and S-oxopropenyl cysteine are the major products. Comprehensive profiling of the reaction of adenine propenal with human serum albumin and the DNA repair protein, XPA, revealed that the only stable adduct is Nε-oxopropenyllysine. The most reactive sites for modification in human albumin are K190 and K351. Three sites of modification of XPA are in the DNA-binding domain, and two sites are subject to regulatory acetylation. Modification by adenine propenal dramatically reduces XPA’s ability to bind to a DNA substrate. PMID:25211669

  3. NAD+-dependent deacetylase Hst1p controls biosynthesis and cellular NAD+ levels in Saccharomyces cerevisiae.

    PubMed

    Bedalov, Antonio; Hirao, Maki; Posakony, Jeffrey; Nelson, Melisa; Simon, Julian A

    2003-10-01

    Nicotine adenine dinucleotide (NAD(+)) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD(+)-dependent protein deacetylases. In the latter two processes, NAD(+) is consumed and converted to ADP-ribose and nicotinamide. NAD(+) levels can be maintained by regeneration of NAD(+) from nicotinamide via a salvage pathway or by de novo synthesis of NAD(+) from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD(+)-dependent deacetylase Hst1p as a sensor of NAD(+) levels and regulator of NAD(+) biosynthesis. Using transcript arrays, we show that low NAD(+) states specifically induce the de novo NAD(+) biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD(+)-dependent deacetylase activity of Hst1p represses de novo NAD(+) biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD(+) biosynthesis genes. The removal of HST1-mediated repression of the NAD(+) de novo biosynthesis pathway leads to increased cellular NAD(+) levels. Transcript array analysis shows that reduction in cellular NAD(+) levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD(+)-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD(+) in comparison to other NAD(+)-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD(+) sensor that monitors and regulates cellular NAD(+) levels. PMID:12972620

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

    PubMed

    Mikulski, Damian; Szeląg, Małgorzata; Molski, Marcin

    2011-12-01

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

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

    PubMed

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

    2015-02-01

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

  6. The Biosynthesis of the Thiazole Moiety of Thiamin in the Archaeon Halobacterium salinarum.

    PubMed

    Hayashi, Maria; Kijima, Yukie; Tazuya-Murayama, Keiko; Yamada, Kazuko

    2015-01-01

    The biosynthetic pathways of the thiazole moiety of thiamin were studied in the archaeon Halobacterium salinarum. Thiamin is generated by the union of 4-amino-5-hydroxymethyl-2-methylpyrimidine (pyrimidine) and 5-(2-hydroxyethyl)-4-methylthiazole (thiazole). The biosynthesis of thiazole is different in facultative anaerobes, aerobes and eukaryotes. In eukaryotes, the C-4, -4', -5, -5' and -5" of the thiazole is biosynthesized from nicotinamide adenine dinucleotide (NAD), with cysteine as S donor and the C-2 and N atoms of glycine. In facultative anaerobic bacteria, such as Escherichia coli, the precursors of the thiazole are the N and C-2 atoms from tyrosine and C-4, -4', -5, -5' and -5" from 1-deoxy-D-xylurose-5-phosphate, again with cysteine as S donor. In aerobic bacteria, such as Bacillus subtilis, L-tyrosine is replaced by glycine. In Archaea, known as the third domain of life, the biosynthetic pathway of thiamin has not yet been elucidated. In the present study in the archaeon H. salinarum, it was shown that both the N and C-2 from glycine are incorporated into the thiazole, rather than the N atom coming from L-tyrosine. These results show that thiazole biosynthesis in H. salinarum more closely resembles the biosynthetic pathway found in eukaryotes.

  7. Automated genotyping of dinucleotide repeat markers

    SciTech Connect

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

    1994-09-01

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

  8. Genetic linkage in Pseudomonas aeruginosa of algT and nadB: mutation in nadB does not affect NAD biosynthesis or alginate production.

    PubMed

    DeVries, C A; Hassett, D J; Flynn, J L; Ohman, D E

    1995-04-14

    The 68-min region of the chromosome of Pseudomonas aeruginosa (Pa) contains the gene algT, encoding a putative alternate sigma factor similar to sigma E in Escherichia coli, that is required for the expression of several genes in the alginate biosynthetic regulon. Sequences immediately upstream from algT were found to contain a divergently expressed open reading frame encoding a 60-kDa protein with 64 and 36% identity to the nadB gene products of E. coli and Bacillus subtilis, respectively. The nadB gene encodes L-aspartate oxidase and has been shown in several bacteria to be essential for de novo nicotinamide-adenine dinucleotide (NAD) biosynthesis. Pa nadB complemented the growth requirement for nicotinic acid in a nadB mutant strain of E. coli, suggesting that this gene encodes a functional homologue of L-aspartate oxidase. A nadB::Tn501 mutant was constructed by gene replacement in the alginate-producing strain, Pa FRD. This NadB- mutant still produced alginate and appeared normal with respect to the regulation of alginate synthesis. Interestingly, the NadB- mutant did not have an auxotrophic phenotype for nicotinic acid, indicating that this nadB was not essential for NAD biosynthesis in Pa. These results suggest the possibility that Pa has an alternate mechanism for de novo NAD biosynthesis.

  9. Nicotinamide mononucleotide synthetase is the key enzyme for an alternative route of NAD biosynthesis in Francisella tularensis

    PubMed Central

    Sorci, Leonardo; Martynowski, Dariusz; Rodionov, Dmitry A.; Eyobo, Yvonne; Zogaj, Xhavit; Klose, Karl E.; Nikolaev, Evgeni V.; Magni, Giulio; Zhang, Hong; Osterman, Andrei L.

    2009-01-01

    Enzymes involved in the last 2 steps of nicotinamide adenine dinucleotide (NAD) cofactor biosynthesis, which catalyze the adenylylation of the nicotinic acid mononucleotide (NaMN) precursor to nicotinic acid dinucleotide (NaAD) followed by its amidation to NAD, constitute promising drug targets for the development of new antibiotics. These enzymes, NaMN adenylyltransferase (gene nadD) and NAD synthetase (gene nadE), respectively, are indispensable and conserved in nearly all bacterial pathogens. However, a comparative genome analysis of Francisella tularensis allowed us to predict the existence of an alternative route of NAD synthesis in this category A priority pathogen, the causative agent of tularaemia. In this route, the amidation of NaMN to nicotinamide mononucleotide (NMN) occurs before the adenylylation reaction, which converts this alternative intermediate to the NAD cofactor. The first step is catalyzed by NMN synthetase, which was identified and characterized in this study. A crystal structure of this enzyme, a divergent member of the NadE family, was solved at 1.9-Å resolution in complex with reaction products, providing a rationale for its unusual substrate preference for NaMN over NaAD. The second step is performed by NMN adenylyltransferase of the NadM family. Here, we report validation of the predicted route (NaMN → NMN → NAD) in F. tularensis including mathematical modeling, in vitro reconstitution, and in vivo metabolite analysis in comparison with a canonical route (NaMN → NaAD → NAD) of NAD biosynthesis as represented by another deadly bacterial pathogen, Bacillus anthracis. PMID:19204287

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2015-01-01

    Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the rate-limiting step in the salvage pathway for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and thereby regulates the deacetylase activity of sirtuins. Here we show accommodative regulation of myocardial NAD+ by monocyte-derived extracellular Nampt (eNampt), which is essential for hemodynamic compensation to pressure overload. Although intracellular Nampt (iNampt) expression was decreased in pressure-overloaded hearts, myocardial NAD+ concentration and Sirt1 activity were preserved. In contrast, iNampt was up-regulated in spleen and monocytes, and circulating eNampt protein and nicotinamide mononucleotide (NMN), a key precursor of NAD+, were significantly increased. Pharmacological inhibition of Nampt by FK866 or depletion of monocytes/macrophages by clodronate liposomes disrupted the homeostatic mechanism of myocardial NAD+ levels and NAD+-dependent Sirt1 activity, leading to susceptibility to cardiomyocyte apoptosis and cardiac decompensation in pressure-overloaded mice. These biochemical and hemodynamic defects were prevented by systemic administration of NMN. Our studies uncover a crucial role of monocyte-derived eNampt in myocardial adaptation to pressure overload, and highlight a potential intervention controlling myocardial NAD+ against heart failure. PMID:26522369

  13. Bound anionic states of adenine

    SciTech Connect

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

    2007-03-20

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

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

    PubMed Central

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

    1970-01-01

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

  15. DiProDB: a database for dinucleotide properties.

    PubMed

    Friedel, Maik; Nikolajewa, Swetlana; Sühnel, Jürgen; Wilhelm, Thomas

    2009-01-01

    DiProDB (http://diprodb.fli-leibniz.de) is a database of conformational and thermodynamic dinucleotide properties. It includes datasets both for DNA and RNA, as well as for single and double strands. The data have been shown to be important for understanding different aspects of nucleic acid structure and function, and they can also be used for encoding nucleic acid sequences. The database is intended to facilitate further applications of dinucleotide properties. A number of property datasets is highly correlated. Therefore, the database comes with a correlation analysis facility. Authors having determined new sets of dinucleotide property values are invited to submit these data to DiProDB.

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

    NASA Technical Reports Server (NTRS)

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

    1978-01-01

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

  17. An investigation of the nicotinamide-adenine dinucleotide-induced 'tightening' of the structure of glyceraldehyde 3-phosphate dehydrogenase.

    PubMed Central

    Osborne, H H; Hollaway, M R

    1976-01-01

    An investigation was made of the effect of NAD+ analogues on subunit interactions in yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenases by using the subunit exchange (hybridization) method described previously [e.g. see Osborne & Hollaway (1975) Biochem. J. 151, 37-45]. The ligands ATP, ITP, ADP, AMP, cyclic AMP and ADP-ribose like NADH, all caused an apparent weakening of intramolecular subunit interactions, whereas NAD+ caused an apparent increase in the stability of the tetrameric enzyme molecules. A mixture of NMN and AMP, although it did not simulate completely the NAD+-induced 'tightening' of the enzyme structure, did result in a more than 20-fold decrease in the rate of subunit exchange compared with that in the presence of AMP alone. These results show that occupancy of the NMN subsite of the enzyme NAD+-binding site is insufficient in itself to give the marked tightening of the enzyme structure induced by NAD+. The 'tightening' effect is specific in that it seems to require a phosphodiester link between NMN and ADP-ribose. These effects are discussed in terms of the detailed X-ray structure of the lobster holoenzyme [Buehner et al. (1974) J. Mol. Biol. 90, 25-49]. Images PLATE 1 PLATE 2 PMID:183744

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

    PubMed Central

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

    1969-01-01

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

  19. Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

    PubMed Central

    Brinkworth, R I; Masters, C J; Winzor, D J

    1975-01-01

    Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase. PMID:175784

  20. Nicotinamide Adenine Dinucleotide Phosphate Oxidase (NOX) in Experimental Liver Fibrosis: GKT137831 as a Novel Potential Therapeutic Agent

    PubMed Central

    Aoyama, Tomonori; Paik, Yong-Han; Watanabe, Sumio; Laleu, Benoît; Gaggini, Francesca; Fioraso-Cartier, Laetitia; Molango, Sophie; Heitz, Freddy; Merlot, Cédric; Szyndralewiez, Cédric; Page, Patrick; Brenner, David A.

    2012-01-01

    Background & Aims NADPH oxidase (NOX) generates reactive oxygen species (ROS) in hepatic stellate cells (HSCs) during liver fibrosis. In response to fibrogenic agonists, such as angiotensin II (Ang II), the NOX1 components form an active complex including Rac1. Superoxide dismutase 1 (SOD1) interacts with the NOX-Rac1 complex to stimulate NOX activity. NOX4 is also induced in activated HSCs/myofibroblast by increased gene expression. Here, we investigate the role of an enhanced activity SOD1 G37R mutation (SODmu) and the effects of GKT137831, a dual NOX1/4 inhibitor, on HSCs and liver fibrosis. Methods To induce liver fibrosis, wild-type (WT) and SOD1mu mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL). Then, to address the role of NOX-SOD1-mediated ROS production in HSC activation and liver fibrosis, mice were treated with a NOX1/4 inhibitor. Fibrosis and ROS generation was assessed by histology and measurement of TBARS and NOX related genes. Primary cultured HSCs isolated from WT, SODmu, and NOX1 knock-out (KO) mice were assessed for ROS production, Rac1 activity, and NOX gene expression. Results Liver fibrosis was increased in SOD1mu mice, and ROS production and Rac1 activity were increased in SOD1mu HSCs. The NOX1/4 inhibitor GKT137831 attenuated liver fibrosis and ROS production in both SOD1mu and WT mice as well as mRNA expression of fibrotic and NOX genes. Treatment with GKT137831 suppressed ROS production and NOX and fibrotic gene expression, but not Rac1 activity, in SOD1mut and WT HSCs. Both Ang II and TGFb upregulated NOX4, but AngII required NOX1. Conclusions SOD1mu induces excessive NOX1 activation through Rac1 in HSCs, causing enhanced NOX4 upregulation, ROS generation, and liver fibrosis. Treatment targeting NOX1/4 may be a new therapy for liver fibrosis. PMID:22806357

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

    SciTech Connect

    Tich, N.R.

    1989-01-01

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

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

    PubMed

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

    2007-12-27

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

  3. Accumulation of DNA damage and reduced levels of nicotine adenine dinucleotide in the brains of Atm-deficient mice.

    PubMed

    Stern, Nora; Hochman, Ayala; Zemach, Naty; Weizman, Nir; Hammel, Ilan; Shiloh, Yosef; Rotman, Galit; Barzilai, Ari

    2002-01-01

    Ataxia-telangiectasia (A-T) is a human genetic disorder caused by mutational inactivation of the ATM gene. A-T patients display a pleiotropic phenotype, in which a major neurological feature is progressive ataxia due to degeneration of cerebellar Purkinje and granule neurons. Disruption of the mouse Atm locus creates a murine model of A-T that exhibits most of the clinical and cellular features of the human disease, but the neurological phenotype is barely expressed. We present evidence for the accumulation of DNA strand breaks in the brains of Atm(-/-), supporting the notion that ATM plays a major role in maintaining genomic stability. We also show a perturbation of the steady state levels of pyridine nucleotides. There is a significant decrease in both the reduced and the oxidized forms of NAD and in the total levels of NADP(T) and NADP(+) in the brains of Atm(-/-) mice. The changes in NAD(T), NADH, NAD(+), NADP(T), and NADP(+) were progressive and observed primarily in the cerebellum of 4-month-old Atm(-/-) mice. Higher rates of mitochondrial respiration were also recorded in 4-month-old Atm(-/-) cerebella. Taken together, our findings support the hypothesis that absence of functional ATM results in continuous stress, which may be an important cause of the degeneration of cerebellar neurons in A-T. PMID:11679583

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

    PubMed Central

    Heath, H.; Fiddick, Rosemary

    1965-01-01

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

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

    PubMed Central

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

    1971-01-01

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

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

    SciTech Connect

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

    2009-02-18

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

  7. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

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

  8. The catalase activity of diiron adenine deaminase.

    PubMed

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

    2011-12-01

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

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

    PubMed

    Kokina, Agnese; Kibilds, Juris; Liepins, Janis

    2014-08-01

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

  10. Biosynthesis and metabolism of retinoic acid: roles of CRBP and CRABP in retinoic acid: roles of CRBP and CRABP in retinoic acid homeostasis.

    PubMed

    Napoli, J L

    1993-02-01

    The enzymes that constitute the pathway of retinoic acid biosynthesis and metabolism may recognize retinoid binding proteins as effectors and substrates. Apocellular retinol-binding protein (CRBP) stimulates a bile-salt independent membrane-bound retinyl ester hydrolase resulting in the hydrolysis of endogenous retinyl esters and the formation of holoCRBP. HoloCRBP delivers retinol to a microsomal nicotin-amide-adenine dinucleotide phosphate-dependent dehydrogenase, protects it from artifactual oxidation and denies enzymes that cannot recognize the binding protein access to retinol. The retinal synthesized may be transferred from the microsomes to the cytosol by CRBP. A cytosolic retinal dehydrogenase has been purified that produces retinoic acid from retinal generated by microsomes in the presence of CRBP and from the complex CRBP-retinal itself. Thus, CRBP(type I) seems to channel retinoids through the reactions of retinoic acid synthesis via a series of protein-protein interactions. Cellular retinoic acid-binding protein (type I) facilitates retinoic acid metabolism by sequestering it and by acting as a low Km substrate, thereby also modulating the steady-state concentrations of retinoic acid. PMID:8381481

  11. The catalase activity of diiron adenine deaminase

    SciTech Connect

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

    2011-12-01

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

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

    PubMed

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

    2016-03-24

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

  13. Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.).

    PubMed

    Katahira, Riko; Ashihara, Hiroshi

    2009-12-01

    As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --> nicotinamide mononucleotide --> nicotinamide riboside --> nicotinic acid riboside --> nicotinic acid mononucleotide --> nicotinic acid adenine dinucleotide --> NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide. PMID:19820966

  14. Profiles of the biosynthesis and metabolism of pyridine nucleotides in potatoes (Solanum tuberosum L.).

    PubMed

    Katahira, Riko; Ashihara, Hiroshi

    2009-12-01

    As part of a research program on nucleotide metabolism in potato tubers (Solanum tuberosum L.), profiles of pyridine (nicotinamide) metabolism were examined based on the in situ metabolic fate of radio-labelled precursors and the in vitro activities of enzymes. In potato tubers, [(3)H]quinolinic acid, which is an intermediate of de novo pyridine nucleotide synthesis, and [(14)C]nicotinamide, a catabolite of NAD, were utilised for pyridine nucleotide synthesis. The in situ tracer experiments and in vitro enzyme assays suggest the operation of multiple pyridine nucleotide cycles. In addition to the previously proposed cycle consisting of seven metabolites, we found a new cycle that includes newly discovered nicotinamide riboside deaminase which is also functional in potato tubers. This cycle bypasses nicotinamide and nicotinic acid; it is NAD --> nicotinamide mononucleotide --> nicotinamide riboside --> nicotinic acid riboside --> nicotinic acid mononucleotide --> nicotinic acid adenine dinucleotide --> NAD. Degradation of the pyridine ring was extremely low in potato tubers. Nicotinic acid glucoside is formed from nicotinic acid in potato tubers. Comparative studies of [carboxyl-(14)C]nicotinic acid metabolism indicate that nicotinic acid is converted to nicotinic acid glucoside in all organs of potato plants. Trigonelline synthesis from [carboxyl-(14)C]nicotinic acid was also found. Conversion was greater in green parts of plants, such as leaves and stem, than in underground parts of potato plants. Nicotinic acid utilised for the biosynthesis of these conjugates seems to be derived not only from the pyridine nucleotide cycle, but also from the de novo synthesis of nicotinic acid mononucleotide.

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

    PubMed

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

    2016-03-24

    The energetic burden of continuously concentrating solutes against gradients along the tubule may render the kidney especially vulnerable to ischaemia. Acute kidney injury (AKI) affects 3% of all hospitalized patients. Here we show that the mitochondrial biogenesis regulator, PGC1α, is a pivotal determinant of renal recovery from injury by regulating nicotinamide adenine dinucleotide (NAD) biosynthesis. Following renal ischaemia, Pgc1α(-/-) (also known as Ppargc1a(-/-)) mice develop local deficiency of the NAD precursor niacinamide (NAM, also known as nicotinamide), marked fat accumulation, and failure to re-establish normal function. Notably, exogenous NAM improves local NAD levels, fat accumulation, and renal function in post-ischaemic Pgc1α(-/-) mice. Inducible tubular transgenic mice (iNephPGC1α) recapitulate the effects of NAM supplementation, including more local NAD and less fat accumulation with better renal function after ischaemia. PGC1α coordinately upregulates the enzymes that synthesize NAD de novo from amino acids whereas PGC1α deficiency or AKI attenuates the de novo pathway. NAM enhances NAD via the enzyme NAMPT and augments production of the fat breakdown product β-hydroxybutyrate, leading to increased production of prostaglandin PGE2 (ref. 5), a secreted autacoid that maintains renal function. NAM treatment reverses established ischaemic AKI and also prevented AKI in an unrelated toxic model. Inhibition of β-hydroxybutyrate signalling or prostaglandin production similarly abolishes PGC1α-dependent renoprotection. Given the importance of mitochondrial health in ageing and the function of metabolically active organs, the results implicate NAM and NAD as key effectors for achieving PGC1α-dependent stress resistance. PMID:26982719

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

    PubMed Central

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

    2008-01-01

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

  17. Cyclic Dinucleotide-Controlled Regulatory Pathways in Streptomyces Species

    PubMed Central

    2015-01-01

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

  18. Association of bone mineral density with a dinucleotide repeat polymorphism at the calcitonin (CT) locus.

    PubMed

    Miyao, M; Hosoi, T; Emi, M; Nakajima, T; Inoue, S; Hoshino, S; Shiraki, M; Orimo, H; Ouchi, Y

    2000-01-01

    Calcitonin (CT), a calcium-regulating hormone, lowers the calcium level in serum by inhibiting bone resorption. Because CT may play a role in the pathogenesis of osteoporosis, genetic variations in or adjacent to the CT gene may be associated with variations in bone mineral density (BMD). The present study examined the correlation between a dinucleotide (cytosine-adenine; CA) repeat polymorphism at the CT locus and BMD in 311 Japanese postmenopausal women (mean age, 64.1 years). Seven alleles were present in this population; each allele contained 10, 11, 16, 17, 18, 19, or 20 CA repeats. Thus, we designated the respective genotypes A10, A11, A16, A17, A18, A19, and A20. The A10 and A17 alleles were the predominant alleles in the population studied. Z scores (a parameter representing deviation from the age-specific weight-adjusted average BMD) were compared between individuals that possessed one or two alleles of each genotype and those that did not possess the allele. Subjects who possessed one or two A10 alleles had lower BMD Z scores than those who did not (lumbar 2-4 BMD Z score; -0.148 +/- 1.23 vs 0.182 +/- 1.54; P = 0.04). No significant relationships were observed between allelic status and background data or biochemical parameters. The significant association observed between BMD and genetic variations at the CT locus implies that polymorphism at this locus may be a useful marker for the genetic study of osteoporosis. PMID:11185743

  19. Auxin Biosynthesis

    PubMed Central

    Zhao, Yunde

    2014-01-01

    lndole-3-acetic acid (IAA), the most important natural auxin in plants, is mainly synthesized from the amino acid tryptophan (Trp). Recent genetic and biochemical studies in Arabidopsis have unambiguously established the first complete Trp-dependent auxin biosynthesis pathway. The first chemical step of auxin biosynthesis is the removal of the amino group from Trp by the TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of transaminases to generate indole-3-pyruvate (IPA). IPA then undergoes oxidative decarboxylation catalyzed by the YUCCA (YUC) family of flavin monooxygenases to produce IAA. This two-step auxin biosynthesis pathway is highly conserved throughout the plant kingdom and is essential for almost all of the major developmental processes. The successful elucidation of a complete auxin biosynthesis pathway provides the necessary tools for effectively modulating auxin concentrations in plants with temporal and spatial precision. The progress in auxin biosynthesis also lays a foundation for understanding polar auxin transport and for dissecting auxin signaling mechanisms during plant development. PMID:24955076

  20. Methods for detection of methyl-CpG dinucleotides

    SciTech Connect

    Dunn, John J

    2013-11-26

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

  1. Methods for detection of methyl-CpG dinucleotides

    SciTech Connect

    Dunn, John J.

    2013-01-29

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

  2. Methods for detection of methyl-CpG dinucleotides

    SciTech Connect

    Dunn, John J.

    2012-09-11

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

  3. Adenine nucleotide transporters in organelles: novel genes and functions.

    PubMed

    Traba, Javier; Satrústegui, Jorgina; del Arco, Araceli

    2011-04-01

    In eukaryotes, cellular energy in the form of ATP is produced in the cytosol via glycolysis or in the mitochondria via oxidative phosphorylation and, in photosynthetic organisms, in the chloroplast via photophosphorylation. Transport of adenine nucleotides among cell compartments is essential and is performed mainly by members of the mitochondrial carrier family, among which the ADP/ATP carriers are the best known. This work reviews the carriers that transport adenine nucleotides into the organelles of eukaryotic cells together with their possible functions. We focus on novel mechanisms of adenine nucleotide transport, including mitochondrial carriers found in organelles such as peroxisomes, plastids, or endoplasmic reticulum and also mitochondrial carriers found in the mitochondrial remnants of many eukaryotic parasites of interest. The extensive repertoire of adenine nucleotide carriers highlights an amazing variety of new possible functions of adenine nucleotide transport across eukaryotic organelles.

  4. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

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

    1995-03-01

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

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

    PubMed

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

    2013-12-01

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

  6. Hepatitis C virus RNA: dinucleotide frequencies and cleavage by RNase L.

    PubMed

    Washenberger, Christopher L; Han, Jian-Qiu; Kechris, Katherina J; Jha, Babal Kant; Silverman, Robert H; Barton, David J

    2007-12-01

    Ribonuclease L (RNase L) is an antiviral endoribonuclease that cleaves hepatitis C virus (HCV) RNA at single-stranded UA and UU dinucleotides throughout the open reading frame (ORF). To determine whether RNase L exerts evolutionary pressure on HCV we examined the frequencies of UA and UU dinucleotides in 162 RNA sequences from the Los Alamos National Labs HCV Database (http://hcv.lanl.gov). Considering the base composition of the HCV ORFs, both UA and UU dinucleotides were less frequent than predicted in each of 162 HCV RNAs. UA dinucleotides were significantly less frequent than predicted at each of the three codon positions while UU dinucleotides were less frequent than predicted predominantly at the wobble position of codons. UA and UU dinucleotides were among the least abundant dinucleotides in HCV RNA ORFs. Furthermore, HCV genotype 1 RNAs have a lower frequency of UA and UU dinucleotides than genotype 2 and 3 RNAs, perhaps contributing to increased resistance of HCV genotype 1 infections to interferon therapy. In vitro, RNase L cleaved both HCV genotype 1 and 2 RNAs efficiently. Thus, RNase L can cleave HCV RNAs efficiently and variably reduced frequencies of UA and UU dinucleotides in HCV RNA ORFs are consistent with the selective pressure of RNase L.

  7. Role of pipecolic acid in the biosynthesis of lysine in Rhodotorula glutinis.

    PubMed

    Kinzel, J J; Bhattacharjee, J K

    1979-05-01

    The role of pipecolic acid in the biosynthesis of lysine was investigated in Rhodotorula glutinis, an aerobic red yeast. Supplementation of pipecolic acid in the minimal medium supported the growth of mutants lys2, lys3, and lys5; alpha-aminoadipic acid supported the growth of lys5; but neither alpha-aminoadipic acid nor pipecolic acid supported the growth of mutants MNNG42 and MNNG37. During the growth of the appropriate mutants, pipecolic acid was removed from the growth medium and the intracellular pool. In tracer experiments, radioactivity from [(14)C]pipecolic acid was selectively incorporated into the cellular lysine of lys5 and the wild-type strain. l-Pipecolic acid-dependent enzyme activity did not require any cofactor and was inhibited by mercuric chloride and potassium cyanide. This activity was present in the wild-type strain and all of the mutants tested and was repressed in mutant lys5 when grown in the presence of higher concentration of lysine. The reaction product of pipecolic acid was converted to saccharopine by lys5 enzyme in the presence of glutamate and reduced nicotin-amide adenine dinucleotide phosphate. Mutant MNNG37 lacked the saccharopine dehydrogenase activity, indicating that this step is involved in the conversion of alpha-aminoadipic acid and pipecolic acid to lysine. Mutants MNNG37 and MNNG42 accumulated a p-dimethylaminobenzaldehyde-reacting product in the culture supernatant and in the intracellular pool. Chromatographic properties of the p-dimethylaminobenzaldehyde adduct and that of the pipecolic acid-dependent reaction product were similar. The reaction product and the accumulation product were characterized on the basis of mass and absorption spectra as alpha-aminoadipic-semialdehyde, which in solution remains in equilibrium with Delta(1)-piperideine-6-carboxylic acid. Since alpha-aminoadipic-semialdehyde is a known intermediate of the alpha-aminoadipic acid pathway for the biosynthesis of lysine, it is concluded that pipecolic

  8. Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Shwing a Covalent Flavin-Aspartate Bond

    SciTech Connect

    Podzelinska, K.; Latimer, R; Bhattacharya, A; Vining, L; Zechel, D; Jia, Z

    2010-01-01

    Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 {angstrom} resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4{alpha})-OOH intermediate. Strikingly, the 8{alpha} carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

  9. What is adenine doing in photolyase?

    PubMed

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

    2010-03-25

    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.

  10. Ultraviolet Photostability of Adenine on Gold and Silicon Surfaces

    NASA Astrophysics Data System (ADS)

    Mateo-Martí, Eva; Pradier, Claire-Marie; Martín-Gago, Jose-Angel

    2009-08-01

    The adenine molecule is a DNA nucleobase, an essential component of genetic material. Because of the important role of DNA nucleobases in terrestrial biochemistry, we have studied the molecular adsorption, orientation, and chemical binding of adenine on metallic and semiconducting surfaces, such as gold and silicon, respectively, and their stability toward ultraviolet radiation by X-ray photoelectron spectroscopy (XPS) and reflection absorption infrared spectroscopy (RAIRS) techniques. We have exposed the adenine surface system to UV radiation (200-400 nm) under a high-vacuum environment (10-7 mbar) to study the photostability and photochemistry of adenine on different surfaces. After 10 or 24 hours of exposure under interplanetary space conditions, UV radiation induces desorption and partial dissociation of the molecule, which is dependant on the nature of the surface. The electronic excitations, induced in the material by UV absorption, play a major role in the photodestruction of the absorbed molecules on the solid surfaces.

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

    SciTech Connect

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

    2015-11-07

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

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2015-11-15

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2005-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    PubMed

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

    2014-10-28

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

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

    PubMed

    Südi, J

    1974-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2002-01-01

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

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

    PubMed

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

    2014-04-01

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

  1. The biosynthesis of the molybdenum cofactors.

    PubMed

    Mendel, Ralf R; Leimkühler, Silke

    2015-03-01

    The biosynthesis of the molybdenum cofactors (Moco) is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified to date. In all molybdoenzymes except nitrogenase, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into three general steps, with a fourth one present only in bacteria and archaea: (1) formation of the cyclic pyranopterin monophosphate, (2) formation of MPT, (3) insertion of molybdenum into molybdopterin to form Moco, and (4) additional modification of Moco in bacteria with the attachment of a nucleotide to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.

  2. Possible prebiotic catalysts formed from adenine and aldehyde

    NASA Astrophysics Data System (ADS)

    Vergne, J.; Dumas, L.; Décout, J.-L.; Maurel, M.-C.

    2000-09-01

    Careful examination of the present metabolism and in vitro selection of various catalytic RNAs strongly support the "RNA World" hypothesis of the origin of life. However, in this scenario, the difficult prebiotic synthesis of ribose and consequently of nucleotides remain a major problem. In order to overcome this problem and obtain nucleoside analogs, we are investigating reactions of the nucleic acid base, adenine 1, with different aldehydes under presumably prebiotic conditions. In the reaction of adenine and pyruvaldehyde 2 in water, we report here the formation in high yield of two isomeric products. These compounds possessing alcohols functions as nucleosides result from condensation of two molecules of pyruvaldehyde on the 6-amino group of one adenine molecule. Their catalytic activities in the model hydrolysis of p-nitrophenylesters appeared interesting in the search of prebiotic catalysts.

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

    PubMed

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

    2004-02-12

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

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

    PubMed Central

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

    2013-01-01

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

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

    SciTech Connect

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

    2014-03-01

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

  6. Thymine, adenine and lipoamino acid based gene delivery systems.

    PubMed

    Skwarczynski, Mariusz; Ziora, Zyta M; Coles, Daniel J; Lin, I-Chun; Toth, Istvan

    2010-05-14

    A novel class of thymine, adenine and lipoamino acid based non-viral carriers for gene delivery has been developed. Their ability to bind to DNA by hydrogen bonding was confirmed by NMR diffusion, isothermal titration calorimetry and transmission electron microscopy experiments.

  7. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

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

    2016-10-01

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

  8. PolyAdenine cryogels for fast and effective RNA purification.

    PubMed

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

    2016-10-01

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

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

    SciTech Connect

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

    2011-03-22

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

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

    SciTech Connect

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

    2011-12-31

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

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

    PubMed Central

    Siddharthan, Rahul

    2010-01-01

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

  12. Copper-Adenine Complex Catalyst for O2 Production from

    NASA Astrophysics Data System (ADS)

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

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

  13. Cyclic (di)nucleotides: the common language shared by microbe and host.

    PubMed

    Gao, Juyi; Tao, Jianli; Liang, Weili; Jiang, Zhengfan

    2016-04-01

    Fluency in a common language allows individuals to convey information and carry out complex activities that otherwise would be difficult or even impossible without the benefit of shared communication. Cyclic (di)nucleotides have recently been recognized as such an accessible language understood by both microbe and the host, ever since remarkable progresses have revealed the molecular details of these nucleotide second messengers used in cellular communication systems. Though undergoing separate evolutionary pathways in prokaryotes and eukaryotes, cyclic (di)nucleotides enable microbes to influence host cells immediately and fiercely by modulating a variety of cellular activities. Here we highlight recent insights in cyclic (di)nucleotides and focus on the balancing of these indispensable signaling molecules by synthases and phosphodiesterases.

  14. Typing dinucleotide repeat loci using microplate array diagonal gel electrophoresis: proof of principle.

    PubMed

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

    2004-04-01

    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.

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

    PubMed

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

    2014-11-19

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

  16. High resolution dissociative electron attachment to gas phase adenine

    SciTech Connect

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

    2006-08-28

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

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

    PubMed

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

    2015-05-01

    The different fragmentation channels of cytosine, adenine and guanine have been studied through DFT calculations. The electronic structure of bases, their cations, and the fragments obtained by breaking bonds provides a good understanding of the fragmentation process that can complete the experimental approach. The calculations allow assigning various fragments to the given peaks. The comparison between the energy required for the formation of fragments and the peak intensity in the mass spectrum is used. For cytosine and guanine the elimination of the HNCO molecule is a major route of dissociation, while for adenine multiple loss of HCN or HNC can be followed up to small fragments. For cytosine, this corresponds to the initial bond cleavage of N3-C4/N1-C2, which represents the main dissociation route. For guanine the release of HNCO is obtained through the N1-C2/C5-C6 bond cleavage (reverse order also possible) leading to the largest peak of the spectrum. The corresponding energies of 3.5 and 3.9 eV are typically in the range available in the experiments. The loss of NH3 or HCN is also possible but requires more energy. For adenine, fragmentation consists of multiple loss of the HCN molecule and the main route corresponding to HC8N9 loss is followed by the release of HC2N1. PMID:25869111

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

    PubMed

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

    2015-03-01

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

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

    NASA Astrophysics Data System (ADS)

    Słowikowska, Joanna Maria; Woźniak, Krzysztof

    1996-01-01

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

  20. Examination of tyrosine/adenine stacking interactions in protein complexes.

    PubMed

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

    2013-11-14

    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.

  1. A comparison of adenine and some derivatives on pig isolated tracheal muscle.

    PubMed Central

    Bach-Dieterle, Y.; Holden, W. E.; Junod, A. F.

    1983-01-01

    We studied the muscle relaxation induced by adenine and several adenine derivatives in strips of tracheal smooth muscle from pigs; in addition their metabolism by the tissue was examined. Adenine relaxed tissue which was contracted by carbachol, histamine, or KCl. Adenine's potency was similar to that of adenosine and ATP (threshold about 4 X 10(-5)M). In tissues with carbachol-induced tone, the adenine effect differed from adenosine and ATP by being slower in onset and in 'washout' time. Furthermore, neither dipyridamole nor theophylline modified the response to adenine. The relationship was examined between pharmacological effects and the metabolism of [3H]-adenosine and [3H]-adenine. Both substrates were taken up by the tissue and converted to nucleotides, but relaxation correlated with nucleotide accumulation only in the case of [3H]-adenine. We conclude that the site and mechanism of adenine-induced relaxation is different from that of adenosine and ATP in porcine tracheal muscle. PMID:6571222

  2. Immune-Stimulatory Dinucleotide at the 5′-End of Oligodeoxynucleotides Is Critical for TLR9-Mediated Immune Responses

    PubMed Central

    2013-01-01

    Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2′-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5′-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses. We designed and synthesized ODNs with two immune-stimulatory dinucleotides, one placed toward the 5′-end region and the other toward the 3′-end region, incorporated 2′-O-methylribonucleotides selectively preceding the 5′- or 3′-immune-stimulatory dinucleotide or both, and studied TLR9-mediated immune responses of these compounds in cell-based assays and in vivo in mice. These studies showed that an immune-stimulatory dinucleotide located closer to the 5′-end is critical for and dictates TLR9-mediated immune responses. These studies provide insights for the use of ODNs when employed as TLR9 agonists and antagonists or antisense agents. PMID:24900663

  3. Modelling proton tunnelling in the adenine-thymine base pair.

    PubMed

    Godbeer, A D; Al-Khalili, J S; Stevenson, P D

    2015-05-21

    The energies of the canonical (standard, amino-keto) and tautomeric (non-standard, imino-enol) charge-neutral forms of the adenine-thymine base pair (A-T and A*-T*, respectively) are calculated using density functional theory. The reaction pathway is then computed using a transition state search to provide the asymmetric double-well potential minima along with the barrier height and shape, which are combined to create the potential energy surface using a polynomial fit. The influence of quantum tunnelling on proton transfer within a base pair H-bond (modelled as the DFT deduced double-well potential) is then investigated by solving the time-dependent master equation for the density matrix. The effect on a quantum system by its surrounding water molecules is explored via the inclusion of a dissipative Lindblad term in the master equation, in which the environment is modelled as a heat bath of harmonic oscillators. It is found that quantum tunnelling, due to transitions to higher energy eigenstates with significant amplitudes in the shallow (tautomeric) side of the potential, is unlikely to be a significant mechanism for the creation of adenine-thymine tautomers within DNA, with thermally assisted coupling of the environment only able to boost the tunnelling probability to a maximum of 2 × 10(-9). This is barely increased for different choices of the starting wave function or when the geometry of the potential energy surface is varied.

  4. Nonselective enrichment for yeast adenine mutants by flow cytometry

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

  5. Analysis of stereochemistry and biosynthesis of epicatechin in tea plants by chiral phase high performance liquid chromatography.

    PubMed

    Qian, Yumei; Zhao, Xianqian; Zhao, Lei; Cui, Lilan; Liu, Li; Jiang, Xiaolan; Liu, Yajun; Gao, Liping; Xia, Tao

    2015-12-01

    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.

  6. Adenine nucleotides as allosteric effectors of pea seed glutamine synthetase.

    PubMed

    Knight, T J; Langston-Unkefer, P J

    1988-08-15

    The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked

  7. Trypanosome Glycosylphosphatidylinositol Biosynthesis

    PubMed Central

    Kinoshita, Taroh

    2009-01-01

    Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis. PMID:19724691

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

    SciTech Connect

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

    1995-01-01

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

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

    PubMed

    Rajendiran, N; Thulasidhasan, J

    2015-06-01

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

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

    NASA Astrophysics Data System (ADS)

    Rajendiran, N.; Thulasidhasan, J.

    2015-06-01

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

  11. Association of poly(N-isopropylacrylamide) containing nucleobase multiple hydrogen bonding of adenine for DNA recognition

    NASA Astrophysics Data System (ADS)

    Yang, Hsiu-Wen; Chen, Jem-Kun; Cheng, Chih-Chia; Kuo, Shiao-Wei

    2013-04-01

    In this study we used the poly(N-isopropylacrylamide) (PNIPAAm) as a medium to generate PNIPAAm-adenine supramolecular complexes. A nucleobase-like hydrogen bonding (NLHB) between PNIPAAm and adenine was found that changed the morphology, crystalline structure, and temperature responsiveness of PNIPAAm microgels relatively to the adenine concentrations. With increasing the adenine concentration, the PNIPAAm-adenine supramolecular complexes gradually altered their morphologies from microgel particles to thin film structures and suppressed the thermodynamical coil-to-globule transition of PNIPAAm because of the NLHB existed between the PNIPAAm amide and ester groups and the adenine amide groups (Cdbnd O⋯Hsbnd N and Nsbnd H⋯Nsbnd R), verified by FTIR spectral analysis. NLHB was also diverse and extensive upon increasing the temperature; therefore, the thermoresponsive behavior of the complexes was altered with the NLBH intensity, evaluated by the inter-association equilibrium constant (Ka) above and below their LCST. Therefore, PNIPAAm can be as a medium to recognize adenine in various concentrations, which could potentially be applied in DNA recognition.

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

    PubMed

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

    2016-06-01

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

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

    PubMed

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

    2016-06-01

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

  14. Isolation and characterization of dinucleotide microsatellite loci in the Asian elephant (Elephas maximus).

    PubMed

    Kongrit, C; Siripunkaw, C; Brockelman, W Y; Akkarapatumwong, V; Wright, T F; Eggert, L S

    2008-01-01

    The endangered Asian elephant is found today primarily in protected areas. We characterized 18 dinucleotide microsatellite loci in this species. Allelic diversity ranged from three to eight per locus, and observed heterozygosity ranged from 0.200 to 0.842 in a wild population. All loci were in Hardy-Weinberg equilibrium, but linkage disequilibrium was detected between two loci in the wild, but not in the zoo elephants. These loci will be useful for the population-level studies of this species.

  15. N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

    PubMed

    Wion, Didier; Casadesús, Josep

    2006-03-01

    N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.

  16. Adenine Phosphoribosyltransferase in Plant Tissues: Some Effects of Kinetin on Enzymic Activity 1

    PubMed Central

    Nicholls, P. B.; Murray, A. W.

    1968-01-01

    Adenine phosphoribosyltransferase activity was measured in extracts of soybean (Glycine max var. Acme) callus and of senescing barley leaves (Hordeum distichon c.v. Prior). The enzyme from soybean callus had Michaelis constants for adenine and 5-phosphoribosyl pyrophosphate of 1.5 and 7.5 μm respectively and was inhibited by AMP and stimulated by ATP. The presence of kinetin was found to considerably increase the activity of adenine phosphoribosyltransferase in extracts of soybean callus and senescing barley leaves. PMID:16656820

  17. Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes

    NASA Astrophysics Data System (ADS)

    Carlson, Jacob C.; Li, Shengying; Gunatilleke, Shamila S.; Anzai, Yojiro; Burr, Douglas A.; Podust, Larissa M.; Sherman, David H.

    2011-08-01

    Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

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

    PubMed Central

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

    2015-01-01

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

  19. Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence.

    PubMed

    Davies, Bryan W; Bogard, Ryan W; Young, Travis S; Mekalanos, John J

    2012-04-13

    The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.

  20. Separation and characterization of oxaliplatin dinucleotides from DNA using HPLC-ESI ion trap mass spectrometry.

    PubMed

    Mowaka, Shereen; Linscheid, Michael

    2008-11-01

    Oxaliplatin is a third-generation platinum complex, and has a broad spectrum of antitumor activity. Such platinum complexes with the DACH carrier ligand have recently received increasing attention since they show efficacy against cisplatin-resistant cell lines. As the foremost indication of antitumor activity of platinum drugs is the formation of adducts with genomic DNA, calf thymus DNA-oxaliplatin adducts were the major target in this study. Calf thymus DNA was incubated with oxaliplatin, resulting in the formation of a large number of platinum-DNA adducts. Treated DNA was digested into the dinucleotides with a combination of enzymes, namely, benzonase, alkaline phosphatase, and nuclease S1. Using a high-performance liquid chromatography, we carried out the separation of individual platinum-DNA adducts which were concurrently identified using electrospray ionization ion trap mass spectrometry (MS). Both 1,2-intrastrand and 1,2-interstrand cross-linked adducts were found; however, those of the intrastrand nature have a considerably higher abundance than those of the interstrand cross-links. Among them, d(GpG)-oxaliplatin was the most abundant bifuctional adduct. To a lesser extent, a few monofunctional adducts were detected as well. MS(n) experiments served to ascertain the detailed structures of oxaliplatin adducts of dinucleoside monophosphates and of dinucleotides.

  1. Base-boronated dinucleotides: synthesis and effect of N7-cyanoborane substitution on the base protons.

    PubMed Central

    Hasan, A; Li, H; Tomasz, J; Shaw, B R

    1996-01-01

    Boron-modified nucleic acids comprise a new set of DNA mimics that have potential biological and therapeutic applications. A series of nine dinucleotides containing N7-cyanoborane-2'-deoxyguanosine ((7b)dG) at the 3', 5' or both positions of the phosphodiester linkage have been synthesized using solution phase phosphoramidite chemistry. Fmoc was used as the 5'-protecting group because of incompatibility of the cyanoborane moiety with 5'-DMT cations generated during the deprotection step. The presence of the cyanoborane group was confirmed on the basis of Fab-MS and 1H NMR spectroscopy. The H-8 proton of (7b)dG in the dinucleotides shifted 0.35-0.80 p.p.m. downfield relative to that of unmodified dG. A comparison of the D20 exchange kinetics of the H-8 proton at 60 degrees C showed that H-8 of (7b)dG is very labile relative to unmodified dG, indicating that the N7-cyanoborane modification increases the acidity of the H-8 proton of (7b)dG. These studies illustrate the feasibility of synthesizing boron-containing oligonucleotides which are modified at the N7-guanine to block Hoogsteen pairing in the DNA major groove. PMID:8668548

  2. Structural Flexibility and Conformation Features of Cyclic Dinucleotides in Aqueous Solutions.

    PubMed

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

    2016-03-17

    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.

  3. A cluster of highly polymorphic dinucleotide repeats in intron 17b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

    PubMed Central

    Zielenski, J; Markiewicz, D; Rininsland, F; Rommens, J; Tsui, L C

    1991-01-01

    A cluster of highly polymorphic dinucleotide repeats has been detected in intron 17b of the CFTR gene, 200 bp downstream from the preceding exon. At least 24 alleles, with sizes ranging from 7 to 56 units of a TA repeat, have been identified in a panel of 92 unrelated carriers of cystic fibrosis (CF). The common ones are those with 7, 30, and 31 dinucleotide units, with frequencies of .22, .19, and .12, respectively, among the non-CF chromosomes. Mendelian, codominant segregation of the alleles has been demonstrated in family studies, as expected. A less polymorphic dinucleotide (CA repeat) cluster has also been detected in a region 167 bp downstream from the TA repeat. The length of the CA repeat cluster varies from 11 to 17 dinucleotide units, and it appears to have an inverse relationship to that of the TA repeats. These dinucleotide repeats should be useful in genetic linkage studies, in counseling for CF families with unknown mutations, and in tracing the origins of the various mutant CF alleles. Images Figure 2 Figure 3 PMID:1720926

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

    SciTech Connect

    Simchi, Hamidreza; Esmaeilzadeh, Mahdi Mazidabadi, Hossein

    2014-01-28

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

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

    PubMed

    Detke, Siegfried; Elsabrouty, Rania

    2008-01-01

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

  6. The expanding roles of c-di-GMP in the biosynthesis of exopolysaccharides and secondary metabolites.

    PubMed

    Liang, Zhao-Xun

    2015-05-01

    The cyclic dinucleotide c-di-GMP has emerged in the last decade as a prevalent intracellular messenger that orchestrates the transition between the motile and sessile lifestyles of many bacterial species. The motile-to-sessile transition is often associated with the formation of extracellular matrix-encased biofilm, an organized community of bacterial cells that often contributes to antibiotic resistance and host-pathogen interaction. It is increasingly clear that c-di-GMP controls motility, biofilm formation and bacterial pathogenicity partially through regulating the production of exopolysaccharides (EPS) and small-molecule secondary metabolites. This review summarizes our current understanding of the regulation of EPS biosynthesis by c-di-GMP in a diversity of bacterial species and highlights the emerging role of c-di-GMP in the biosynthesis of small-molecule secondary metabolites.

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

    PubMed

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

    2015-09-17

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

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

    PubMed

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

    2015-09-17

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

  9. Phosphonate analogues of dinucleotides as substrates for DNA-dependent RNA polymerase from Escherichia coli in primed abortive initiation reaction.

    PubMed

    Cvekl, A; Horská, K; Sebesta, K; Rosenberg, I; Holý, A

    1989-02-01

    Dinucleotides (3'-5')-ApU and UpA and their 3'-O-phosphonylmethyl and 5'-O-phosphonylmethyl analogues were studied as substrates in the primed abortive synthesis catalysed by Escherichia coli DNA-dependent RNA polymerase on poly[d(A-T)] template. All phosphonate analogues of dinucleotides containing the anomalous sugar-phosphate backbone are substrates for the holoenzyme as verified by RNase A and RNase T2 digestion of the trinucleotide analogues obtained. The finding that phosphonate dinucleotides act as primers for transcription indicates that steric requirements at the initiation site are not as specific as previously supposed. Analysis of kinetic constants of ordered bibi reaction Kia, KmA, KmB and Vmax suggests that the instability of short RNA-DNA hybrids contributes to the abortive release of trinucleotides formed.

  10. Labeling of mitochondrial adenine nucleotides of bovine sperm

    SciTech Connect

    Cheetham, J.; Lardy, H.A.

    1986-05-01

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

  11. Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

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

    2010-01-01

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

  12. Detecting horizontally transferred and essential genes based on dinucleotide relative abundance.

    PubMed

    Baran, Robert H; Ko, Hanseok

    2008-10-01

    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.

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

    PubMed Central

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

    2002-01-01

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

  14. Dissection of the PHO pathway in Schizosaccharomyces pombe using epistasis and the alternate repressor adenine.

    PubMed

    Estill, Molly; Kerwin-Iosue, Christine L; Wykoff, Dennis D

    2015-05-01

    In Saccharomyces cerevisiae, intracellular phosphate levels are maintained by the PHO pathway, activation of which is assayed by increased phosphatase activity. The PHO pathway of Schizosaccharomyces pombe upregulates phosphatase activity (encoded by pho1 (+)) during low extracellular phosphate levels, but the underlying mechanism is poorly understood. We utilized an alternate repressor of pho1 (+) expression (adenine supplementation) along with epistasis analysis to develop a model of how S. pombe PHO pathway components interact. Analyzing Pho1 activity in S. pombe PHO pathway deletion mutants during adenine starvation, we observed most mutants with a phosphatase defect in phosphate starvation also had a defect in adenine starvation. Pho7, a transcription factor in the PHO pathway, is necessary for an adenine starvation-mediated increase in Pho1 activity. Comparing adenine starvation to phosphate starvation, there are differences in the degree to which individual mutants regulate the two responses. Through epistasis studies, we identified two positive regulatory arms and one repressive arm of the PHO pathway. PKA activation is a positive regulator of Pho1 activity under both environmental conditions and is critical for transducing adenine concentrations in the cell. The synthesis of IP7 also appears critical for the induction of Pho1 activity during adenine starvation, but IP7 is not critical during phosphate starvation, which differs from S. cerevisiae. Finally, Csk1 is critical for repression of pho1 (+) expression during phosphate starvation. We believe all of these regulatory arms converge to increase transcription of pho1 (+) and some of the regulation acts through pho7 (+).

  15. BIOSYNTHESIS OF YEAST CAROTENOIDS

    PubMed Central

    Simpson, Kenneth L.; Nakayama, T. O. M.; Chichester, C. O.

    1964-01-01

    Simpson, Kenneth L. (University of California, Davis), T. O. M. Nakayama, and C. O. Chichester. Biosynthesis of yeast carotenoids. J. Bacteriol. 88:1688–1694. 1964.—The biosynthesis of carotenoids was followed in Rhodotorula glutinis and in a new strain, 62-506. The treatment of the growing cultures by methylheptenone, or ionone, vapors permitted observations of the intermediates in the biosynthetic pathway. On the basis of concentration changes and accumulation in blocked pathways, the sequence of carotenoid formation is postulated as phytoene, phytofluene, ζ-carotene, neurosporene, β-zeacarotene, γ-carotene, torulin, a C40 aldehyde, and torularhodin. Torulin and torularhodin were established as the main carotenoids of 62-506. PMID:14240958

  16. Biosynthesis of pulcherriminic acid

    PubMed Central

    MacDonald, J. C.

    1965-01-01

    1. Candida pulcherrima was grown on a complex medium to which various compounds had been added to determine their effect on the biosynthesis of pulcherriminic acid. Most of the pulcherriminic acid synthesized by C. pulcherrima PRL2019 was derived from the l-[1-14C]leucine added to the medium. 2. The cyclic dipeptide of l-leucine (cyclo-l-leucyl-l-leucyl) was shown, by trapping experiments involving cycloleucyl-leucyl isomers, to be synthesized by strain PRL2019. Cyclo-l-leucyl-l-leucyl was derived from l-leucine and was converted into pulcherriminic acid. Cyclo-l-leucyl-l-leucyl was a precursor of pulcherriminic acid in strain PRL2007 also. 3. The results supported the hypothesis that pulcherriminic acid is derived from l-leucine and that cyclo-l-leucyl-l-leucyl is an intermediate in the biosynthesis. PMID:5837792

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

    PubMed Central

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

    1997-01-01

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

  18. Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

    PubMed

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

    2016-01-01

    A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  1. Temperature-dependent self-assembly of adenine derivative on HOPG.

    PubMed

    Mu, Zhongcheng; Rubner, Oliver; Bamler, Markus; Blömker, Tobias; Kehr, Gerald; Erker, Gerhard; Heuer, Andreas; Fuchs, Harald; Chi, Lifeng

    2013-08-27

    Temperature-dependent self-assembly formed by the adsorption of the nucleobase adenine derivative on a graphite surface were investigated by in situ scanning tunneling microscopy (STM). The high-resolution STM images reveal two types of structures, α phase and β phase, which are mainly driven by either hydrogen bonding or aromatic π-π interactions between adenine bases, respectively, as well as the interactions of alkyl chains. α-Phase structures can be transformed into β-phase structures by increasing temperature. The reverse is true for decreasing temperature. This reflects structural stabilities resulting from the different interactions. Density functional theory (DFT) calculations were performed to characterize possible arrangements of adjacent adenine moieties systematically in terms of binding energies and structural properties. Via a systematic search algorithm, all possible network structures were determined on a microscopic level. In this way, it is possible to rationalize the structural parameters as found in the STM images.

  2. Deep-UV surface-enhanced resonance Raman scattering of adenine on aluminum nanoparticle arrays.

    PubMed

    Jha, Shankar K; Ahmed, Zeeshan; Agio, Mario; Ekinci, Yasin; Löffler, Jörg F

    2012-02-01

    We report the ultrasensitive detection of adenine using deep-UV surface-enhanced resonance Raman scattering on aluminum nanostructures. Well-defined Al nanoparticle arrays fabricated over large areas using extreme-UV interference lithography exhibited sharp and tunable plasmon resonances in the UV and deep-UV wavelength ranges. Theoretical modeling based on the finite-difference time-domain method was used to understand the near-field and far-field optical properties of the nanoparticle arrays. Raman measurements were performed on adenine molecules coated uniformly on the Al nanoparticle arrays at a laser excitation wavelength of 257.2 nm. With this technique, less than 10 amol of label-free adenine molecules could be detected reproducibly in real time. Zeptomole (~30,000 molecules) detection sensitivity was readily achieved proving that deep-UV surface-enhanced resonance Raman scattering is an extremely sensitive tool for the detection of biomolecules.

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

    PubMed

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

    2015-08-01

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

  4. Counterregulation of nuclear 3,5,3'-triiodo-L-thyronine (T3) binding by oxidized and reduced-nicotinamide adenine dinucleotide phosphates in the presence of cytosolic T3-binding protein in vitro

    SciTech Connect

    Hashizume, K.; Miyamoto, T.; Yamauchi, K.; Ichikawa, K.; Kobayashi, M.; Ohtsuka, H.; Sakurai, A.; Suzuki, S.; Yamada, T.

    1989-04-01

    The role of cytosolic T3-binding protein (CTBP) in the regulation of nuclear T3 binding was studied in vitro. Nuclear (125I)T3 binding was observed in the presence of 1.0 mM dithiothreitol (DTT). When the nuclei prepared from rat kidney were incubated with inactive form of CTBP which was also prepared from rat kidney, (125I)T3 binding to nuclei was not affected. When the nuclei were incubated with inactive form of CTBP in the presence of NADP, (125I)T3 binding to nuclei was increased, whereas binding was diminished when nuclei were incubated with CTBP in the presence of NADPH. The inactive form of CTBP was activated by NADPH. NADP also activated CTBP in the presence of DTT. Both active forms of CTBP were again inactivated by extraction with charcoal, and these inactive forms were reactivated by NADPH or by NADP and DTT, but not by NADP alone. Although the nuclei treated with 0.3 M NaCl lost the binding activity for (125I)T3 in the absence of NADP, the nuclei retained the binding activity for (125I)T3 in the presence of NADP and the inactive form of CTBP. Treatment of the nuclei with 0.5 M NaCl lost the binding activity for (125I)T3 not only in the absence but also in the presence of NADP and CTBP. These results suggested that NADP and NADPH play roles as counterregulatory factors for nuclear T3 binding in the presence of CTBP. Further, it was speculated that binding sites for the T3-CTBP complex, which is generated in the presence of NADP and DTT, are present in nuclei, and that binding sites for the complex are different from nuclear T3 receptors.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. DNA with adenine tracts contains poly(dA).poly(dT) conformational features in solution.

    PubMed

    Brahms, S; Brahms, J G

    1990-03-25

    The conformation of DNA's with adenine-thymine tracts exhibiting retardation in electrophoretic migration and considered as curved were investigated in solution by CD and RAMAN spectroscopy. The following curved multimers with adenine tracts but of different flanking sequences d(CA5TGCC)n, d(TCTCTA6TATATA5)n, d(GA4T4C)n yield CD spectroscopic features indicating a non-B structure of the dA.dT tract with similarities to polyd(A).polyd(T). We suggest that adenine-thymine bases in these multimers contain some of the distinctive conformational features of poly(A).polyd(T) probably with large propeller twist found by NMR (Behling and Kearns, 1987) and by X-ray diffraction on oligonucleotides containing a tract of adenines (Nelson et al. 1987, Coll et al; 1987; DiGabriele et al. 1989). Some elements of distinctive CD features of the contiguous adenines run are also observed in the straight multi-9-mer d(CA5GCC)n which lacks in-phase relation to the helical repeat. Despite the presence of the TpA step in the straight multimer d(GT4A4)n, the altered dA.dT conformation is not completely destroyed. Interruption of adenine tract by a guanine in d(CAAGAATGCC)n leads to a B-like conformation and to a normal electrophoretic mobility. The Raman spectra reveal a rearrangement of the sugar-phosphate backbone of dA.dT tract in the multimer d(CA5TGCC)n with respect to that of polydA.polydT. This is reflected in the presence of an unique Raman band associated to C2'-endo sugar with a predominant contribution of C1'-exo puckering which is exhibited by the multimer whereas two distinct Raman bands characterize poly(dA).poly(dT) backbone conformation.

  7. Solution structure of a five-adenine bulge loop within a DNA duplex.

    PubMed

    Dornberger, U; Hillisch, A; Gollmick, F A; Fritzsche, H; Diekmann, S

    1999-09-28

    The three-dimensional solution structure of a DNA molecule of the sequence 5'-d(GCATCGAAAAAGCTACG)-3' paired with 5'-d(CGTAGCCGATGC)-3' containing a five-adenine bulge loop (dA(5)-bulge) between two double helical stems was determined by 2D (1)H and (31)P NMR, infrared, and Raman spectroscopy. The DNA in both stems adopt a classical B-form double helical structure with Watson-Crick base pairing and C2'-endo sugar conformation. In addition, the two dG/dC base pairs framing the dA(5)-bulge loop are formed and are stable at least up to 30 degrees C. The five adenine bases of the bulge loop are localized at intrahelical positions within the double helical stems. Stacking on the double helical stem is continued for the first four 5'-adenines in the bulge loop. The total rise (the height) of these four stacked adenines roughly equals the diameter of the double helical stem. The stacking interactions are broken between the last of these four 5'-adenines and the fifth loop adenine at the 3'-end. This 3'-adenine partially stacks on the other stem. The angle between the base planes of the two nonstacking adenines (A10 and A11) in the bulge loop reflects the kinking angle of the global DNA structure. The neighboring cytosines opposite the dA(5)-bulge (being parts of the bulge flanking base pairs) do not stack on one another. This disruption of stacking is characterized by a partial shearing of these bases, such that certain sequential NOEs for this base step are preserved. In the base step opposite the loop, an extraordinary hydrogen bond is observed between the phosphate backbone of the 5'-dC and the amino proton of the 3'-dC in about two-thirds of the conformers. This hydrogen bond probably contributes to stabilizing the global DNA structure. The dA(5)-bulge induces a local kink into the DNA molecule of about 73 degrees (+/-11 degrees ). This kinking angle and the mutual orientation of the two double helical stems agree well with results from fluorescence resonance energy

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

    NASA Astrophysics Data System (ADS)

    Wang, Shiliang; Hu, Anguang

    2016-06-01

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

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

    PubMed

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

    2014-04-18

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  12. Structural and Functional Analysis of DDX41: a bispecific immune receptor for DNA and cyclic dinucleotide

    PubMed Central

    Omura, Hiroki; Oikawa, Daisuke; Nakane, Takanori; Kato, Megumi; Ishii, Ryohei; Ishitani, Ryuichiro; Tokunaga, Fuminori; Nureki, Osamu

    2016-01-01

    In the innate immune system, pattern recognition receptors (PRRs) specifically recognize ligands derived from bacteria or viruses, to trigger the responsible downstream pathways. DEAD box protein 41 (DDX41) is an intracellular PRR that triggers the downstream pathway involving the adapter STING, the kinase TBK1, and the transcription factor IRF3, to activate the type I interferon response. DDX41 is unique in that it recognizes two different ligands; i.e., double-stranded DNA (dsDNA) and cyclic dinucleotides (CDN), via its DEAD domain. However, the structural basis for the ligand recognition by the DDX41 DEAD domain has remained elusive. Here, we report two crystal structures of the DDX41 DEAD domain in apo forms, at 1.5 and 2.2 Å resolutions. A comparison of the two crystal structures revealed the flexibility in the ATP binding site, suggesting its formation upon ATP binding. Structure-guided functional analyses in vitro and in vivo demonstrated the overlapped binding surface for dsDNA and CDN, which is distinct from the ATP-binding site. We propose that the structural rearrangement of the ATP binding site is crucial for the release of ADP, enabling the fast turnover of DDX41 for the dsDNA/CDN-induced STING activation pathway. PMID:27721487

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

    PubMed Central

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

    2014-01-01

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

  14. bis-Molybdopterin guanine dinucleotide is required for persistence of Mycobacterium tuberculosis in guinea pigs.

    PubMed

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

    2015-02-01

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

  15. Measurement of liver adenine nucleotides and S-adenosyl amino acids by one-step high-performance liquid chromatography.

    PubMed

    Gourdeau, H; Lavoie, R; Grose, J H; Bélanger, L

    1986-10-01

    A reverse-phase isocratic HPLC method is described for direct simultaneous assay of ATP, ADP, AMP, S-adenosylmethionine, S-adenosylhomocysteine, S-adenosylethionine, and other adenine derivatives in liver microbiopsies. The procedure was tested in conditions which alter the hepatic content of adenine nucleotides and sulfur-adenosyl amino acids in humans, rats, and guinea pigs.

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

    SciTech Connect

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

    1989-05-16

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

  17. Controlling two-phase self-assembly of an adenine derivative on HOPG via kinetic effects.

    PubMed

    Wang, Can; Jana, Pritam Kumar; Zhang, Haiming; Mu, Zhongcheng; Kehr, Gerald; Blömker, Tobias; Erker, Gerhard; Fuchs, Harald; Heuer, Andreas; Chi, Lifeng

    2014-08-21

    Large-area self-assembled structures of a nucleobase adenine derivative were successfully realized through vacuum deposition. STM images reveal two types of structures, which could be regulated by substrate temperature and the evaporation rate, indicating the relevance of kinetic effects. The results are supported by computer simulations.

  18. The effect of activated charcoal on adenine-induced chronic renal failure in rats.

    PubMed

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

    2014-03-01

    Activated charcoal (AC) is a sorbent that has been shown to remove urinary toxins like urea and indoxyl sulfate. Here, the influence of AC on kidney function of rats with experimental chronic renal failure (CRF) is investigated. CRF was induced in rats by feeding adenine (0.75%) for four weeks. As an intervention, AC was added to the feed at concentrations of 10%, 15% or 20%. Adenine treatment impaired kidney function: it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and vanin-1. Furthermore, it raised plasma concentrations of the uremic toxins indoxyl sulfate, phosphate and uric acid. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activity, total antioxidant capacity and reduced glutathione were adversely affected. Most of these changes were significantly ameliorated by dietary administration of AC at a concentration of 20%, while effects induced by lower doses of dietary AC on adenine nephrotoxicity were not statistically significant. The results suggest that charcoal is a useful sorbent agent in dietary adenine-induced CRF in rats and that its usability as a nephroprotective agent in human kidney disease should be studied.

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

    PubMed Central

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

    1978-01-01

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

  20. Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

    PubMed Central

    Ali, Badreldin H.; Adham, Sirin A.; Al Za’abi, Mohammed; Waly, Mostafa I.; Yasin, Javed; Nemmar, Abderrahim; Schupp, Nicole

    2015-01-01

    Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered. PMID:25909514

  1. Macrophage Trafficking as Key Mediator of Adenine-Induced Kidney Injury

    PubMed Central

    Braga, Tárcio Teodoro; Felizardo, Raphael José Ferreira; Andrade-Oliveira, Vinícius; Hiyane, Meire Ioshie; da Silva, João Santana; Câmara, Niels Olsen Saraiva

    2014-01-01

    Macrophages play a special role in the onset of several diseases, including acute and chronic kidney injuries. In this sense, tubule interstitial nephritis (TIN) represents an underestimated insult, which can be triggered by different stimuli and, in the absence of a proper regulation, can lead to fibrosis deposition. Based on this perception, we evaluated the participation of macrophage recruitment in the development of TIN. Initially, we provided adenine-enriched food to WT and searched for macrophage presence and action in the kidney. Also, a group of animals were depleted of macrophages with the clodronate liposome while receiving adenine-enriched diet. We collected blood and renal tissue from these animals and renal function, inflammation, and fibrosis were evaluated. We observed higher expression of chemokines in the kidneys of adenine-fed mice and a substantial protection when macrophages were depleted. Then, we specifically investigated the role of some key chemokines, CCR5 and CCL3, in this TIN experimental model. Interestingly, CCR5 KO and CCL3 KO animals showed less renal dysfunction and a decreased proinflammatory profile. Furthermore, in those animals, there was less profibrotic signaling. In conclusion, we can suggest that macrophage infiltration is important for the onset of renal injury in the adenine-induced TIN. PMID:25132730

  2. Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.

    PubMed

    Immormino, Robert M; Kang, Yanlong; Chiosis, Gabriela; Gewirth, Daniel T

    2006-08-10

    Hsp90 chaperones play a critical role in modulating the activity of many cell signaling proteins and are an attractive target for anti-cancer therapeutics. We report here the structures of the water soluble 8-aryl-sulfanyl adenine class Hsp90 inhibitors, 1 (PU-H71) and 2 (PU-H64), in complex with the N-terminal domain of human Hsp90alpha. The conformation of 1 when bound to Hsp90 differs from previously reported 8-aryl adenine Hsp90 inhibitors including 3 (PU24FCl). While the binding mode for 3 places the 2'-halide of the 8-aryl group on top of the adenine ring, for 1 and 2, we show that the 2'-halide is rotated approximately 180 degrees away. This difference explains the opposing trends in Hsp90 inhibitory activity for the 2'-halo derivatives of the 3',4',5'-trimethoxy series where Cl > Br > I compared to the 4',5'-methylenedioxy series where I > Br > Cl. We also present quantum chemical calculations of 2 and its analogues that illuminate their basis for Hsp90 inhibition. The calculated conformation of 2 agreed well with the crystallographically observed conformations of 1 and 2. The predictive nature of the calculations has allowed the exploration of additional derivatives based on the 8-aryl adenine scaffold.

  3. SERS, XPS, and DFT Study of Adenine Adsorption on Silver and Gold Surfaces.

    PubMed

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

    2012-01-19

    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.

  4. Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics.

    PubMed

    Stern, Noa; Major, Dan Thomas; Gottlieb, Hugo Emilio; Weizman, Daniel; Sayer, Alon Haim; Blum, Eliav; Fischer, Bilha

    2012-08-01

    Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.

  5. Cyclic dinucleotides bind the C-linker of HCN4 to control channel cAMP responsiveness.

    PubMed

    Lolicato, Marco; Bucchi, Annalisa; Arrigoni, Cristina; Zucca, Stefano; Nardini, Marco; Schroeder, Indra; Simmons, Katie; Aquila, Marco; DiFrancesco, Dario; Bolognesi, Martino; Schwede, Frank; Kashin, Dmitry; Fishwick, Colin W G; Johnson, A Peter; Thiel, Gerhard; Moroni, Anna

    2014-06-01

    cAMP mediates autonomic regulation of heart rate by means of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which underlie the pacemaker current If. cAMP binding to the C-terminal cyclic nucleotide binding domain enhances HCN open probability through a conformational change that reaches the pore via the C-linker. Using structural and functional analysis, we identified a binding pocket in the C-linker of HCN4. Cyclic dinucleotides, an emerging class of second messengers in mammals, bind the C-linker pocket (CLP) and antagonize cAMP regulation of the channel. Accordingly, cyclic dinucleotides prevent cAMP regulation of If in sinoatrial node myocytes, reducing heart rate by 30%. Occupancy of the CLP hence constitutes an efficient mechanism to hinder β-adrenergic stimulation on If. Our results highlight the regulative role of the C-linker and identify a potential drug target in HCN4. Furthermore, these data extend the signaling scope of cyclic dinucleotides in mammals beyond their first reported role in innate immune system. PMID:24776929

  6. Methionine Biosynthesis in Lemna

    PubMed Central

    Thompson, Gregory A.; Datko, Anne H.; Mudd, S. Harvey; Giovanelli, John

    1982-01-01

    Regulation of enzymes of methionine biosynthesis was investigated by measuring the specific activities of O-phosphohomoserine-dependent cystathionine γ-synthase, O-phosphohomoserine sulfhydrylase, and O-acetylserine sulfhydrylase in Lemna paucicostata Hegelm. 6746 grown under various conditions. For cystathionine γ-synthase, it was observed that (a) adding external methionine (2 μm) decreased specific activity to 15% of control, (b) blocking methionine synthesis with 0.05 μml-aminoethoxyvinylglycine or with 36 μm lysine plus 4 μm threonine (Datko, Mudd 1981 Plant Physiol 69: 1070-1076) caused a 2- to 3-fold increase in specific activity, and (c) blocking methionine synthesis and adding external methionine led to the decreased specific activity characteristic of methionine addition alone. Activity in extracts from control cultures was unaffected by addition of methionine, lysine, threonine, lysine plus threonine, S-adenosylmethionine, or S-methylmethionine sulfonium to the assay mixture. Parallel studies of O-phosphohomoserine sulfhydrylase and O-acetylserine sulfhydrylase showed that O-phosphohomoserine sulfhydrylase activity responded to growth conditions identically to cystathionine γ-synthase activity, whereas O-acetylserine sulfhydrylase activity remained unaffected. Lemna extracts did not catalyze lanthionine formation from O-acetylserine and cysteine. Estimates of kinetic constants for the three enzyme activities indicate that O-acetylserine sulfhydrylase has much higher activity and affinity for sulfide than O-phosphohomoserine sulfhydrylase. The results suggest that (a) methionine, or one of its products, regulates the amount of active cystathionine γ-synthase in Lemna, (b) O-phosphohomoserine sulfhydrylase and cystathionine γ-synthase are probably activities of one enzyme that has low specificity for its sulfur-containing substrate, and (c) O-acetylserine sulfhydrylase is a separate enzyme. The relatively high activity and affinity for sulfide of

  7. Biosynthesis of methanopterin

    SciTech Connect

    White, R.H. )

    1990-06-05

    The biosynthetic pathway for the generation of the methylated pterin in methanopterins was determined for the methanogenic bacteria Methanococcus volta and Methanobacterium formicicum. Extracts of M. volta were found to readily cleave L-7,8-dihydroneopterin to 7,8-dihydro-6-(hydroxymethyl)pterin, which was confirmed to be a precursor of the pterin portion of the methanopterin. (methylene{sup 2}H)-6-(hydroxymethyl)pterin was incorporated into methanopterin by growing cells of M. volta to an extent of 30%. Both the C-11 and C-12 methyl groups of methanopterin originate from (methyl-{sup 2}H{sub 3})methionine. Cells grown in the presence of (methylene-{sup 2}H)-6-(hydroxymethyl)pterin, (ethyl-{sup 2}H{sub 4})-6-(1 (RS)-hydroxyethyl)pterin, (methyl-{sup 2}H{sub 3})-6-(hydroxymethyl)-7-methylpterin, (ethyl-{sup 2}H{sub 4}, methyl-{sup 2}H{sub 3})-6-(1 (RS)-hydroxyethyl)-7-methylpterin, and (1-ethyl-{sup 3}H)-6-(1 (RS)-hydroxyethyl)-7-methylpterin showed that only the non-7-methylated pterins were incorporated into methanopterin. Cells extracts of M. formicicum readily condensed synthetic (methylene-{sup 3}H)-7,8-H{sub 2}-6-(hydroxymethyl)pterin-PP with methaniline to generate demethylated methanopterin, which is then methylated to methanopterin by the cell extract in the presence of S-adenosylmethionine. These observations indicate that the pterin portion of methanopterin is biosynthetically derived from 7,8-H{sub 2}-6-(hydroxymethyl)pterin, which is coupled to methaniline by a pathway analogous to the biosynthesis of folic acid. This pathway for the biosynthesis of methanopterin represents the first example of the modification of the specificity of a coenzyme through a methylation reaction.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    2011-04-01

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

  10. Agonists of Toll-like receptor 9 containing synthetic dinucleotide motifs.

    PubMed

    Yu, Dong; Putta, Mallikarjuna R; Bhagat, Lakshmi; Li, Yukui; Zhu, Fugang; Wang, Daqing; Tang, Jimmy X; Kandimalla, Ekambar R; Agrawal, Sudhir

    2007-12-13

    Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends. Agonists of TLR9 containing cytosine or guanine modification showed activity in HEK293 cells expressing TLR9, mouse spleen, and human cell-based assays and in vivo in mice. The results presented here provide insight into which specific chemical modifications at C or G of the CpG motif are recognized by TLR9 and the ability to modulate immune responses substituting natural C or G in immune modulatory oligonucleotides. PMID:17988082

  11. Rationale, progress and development of vaccines utilizing STING-activating cyclic dinucleotide adjuvants

    PubMed Central

    Kanne, David B.; Leong, Meredith L.

    2013-01-01

    A principal barrier to the development of effective vaccines is the availability of adjuvants and formulations that can elicit both effector and long-lived memory CD4 and CD8 T cells. Cellular immunity is the presumptive immune correlate of protection against intracellular pathogens: a group composed of bacteria, viruses and protozoans that is responsible for a staggering level of morbidity and mortality on a global scale. T-cell immunity is also correlated with clinical benefit in cancer, and the development of therapeutic strategies to harness the immune system to treat diverse malignancies is currently undergoing a renaissance. Cyclic dinucleotides (CDNs) are ubiquitous small molecule second messengers synthesized by bacteria that regulate diverse processes and are a relatively new class of adjuvants that have been shown to increase vaccine potency. CDNs activate innate immunity by directly binding the endoplasmic reticulum-resident receptor STING (stimulator of interferon genes), activating a signaling pathway that induces the expression of interferon-β (IFN-β) and also nuclear factor-κB (NF-κB) dependent inflammatory cytokines. The STING signaling pathway has emerged as a central Toll-like receptor (TLR) independent mediator of host innate defense in response to sensing cytosolic nucleic acids, either through direct binding of CDNs secreted by bacteria, or, as shown recently, through binding of a structurally distinct CDN produced by a host cell receptor in response to binding cytosolic double-stranded (ds)DNA. Although this relatively new class of adjuvants has to date only been evaluated in mice, newly available CDN-STING cocrystal structures will likely intensify efforts in this field towards further development and evaluation in human trials both in preventive vaccine and immunotherapy settings. PMID:24757520

  12. Diverse inhibitors of aflatoxin biosynthesis.

    PubMed

    Holmes, Robert A; Boston, Rebecca S; Payne, Gary A

    2008-03-01

    Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

  13. Serine biosynthesis and transport defects.

    PubMed

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. PMID:27161889

  14. Possibility of 2,4,5-triamino-6-hydroxypyrimidine as an intermediate in the pathway of riboflavin biosynthesis.

    PubMed

    Nakajima, K; Yamada, Y; Mitsuda, H

    1985-01-01

    It was studied with resting cells of a high flavinogenic mold, Eremothecium ashbyii, whether or not 2,4,5-triamino-6-hydroxypyrimidine (THP) is an intermediate in the early pathway of riboflavin biosynthesis. A small amounts of THP strongly inhibited riboflavin formation in the resting cells, but the inhibition was effectively reversed by the added purines, except for adenine. Radioactive tracer experiments showed that the incorporation of the radioactivity from [2-14C]THP into riboflavin was negligible. The results obtained strongly suggest that THP is not an intermediate but a rigid inhibitor for riboflavin formation, and thus there is non salvage pathway of THP for the pathway of riboflavin biosynthesis in resting cells of E. ashbyii. PMID:4041122

  15. Stereoselectivity in Polyphenol Biosynthesis

    NASA Technical Reports Server (NTRS)

    Lewis, Norman G.; Davin, Laurence B.

    1992-01-01

    Stereoselectivity plays an important role in the late stages of phenyl-propanoid metabolism, affording lignins, lignans, and neolignans. Stereoselectivity is manifested during monolignol (glucoside) synthesis, e.g., where the geometry (E or Z) of the pendant double bond affects the specificity of UDPG:coniferyl alcohol glucosyltransferases in different species. Such findings are viewed to have important ramifications in monolignol transport and storage processes, with roles for both E- and Z-monolignols and their glucosides in lignin/lignan biosynthesis being envisaged. Stereoselectivity is also of great importance in enantiose-lective enzymatic processes affording optically active lignans. Thus, cell-free extracts from Forsythia species were demonstrated to synthesize the enantiomerically pure lignans, (-)-secoisolariciresinol, and (-)-pinoresinol, when NAD(P)H, H2O2 and E-coniferyl alcohol were added. Progress toward elucidating the enzymatic steps involved in such highly stereoselective processes is discussed. Also described are preliminary studies aimed at developing methodologies to determine the subcellular location of late-stage phenylpropanoid metabolites (e.g., coniferyl alcohol) and key enzymes thereof, in intact tissue or cells. This knowledge is essential if questions regarding lignin and lignan tissue specificity and regulation of these processes are to be deciphered.

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

    PubMed

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

    2013-04-01

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

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

    PubMed

    Karolak, Aleksandra; van der Vaart, Arjan

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

  20. First prebiotic generation of a ribonucleotide from adenine, D-ribose and trimetaphosphate.

    PubMed

    Baccolini, Graziano; Boga, Carla; Micheletti, Gabriele

    2011-03-28

    Adenosine monophosphate isomers are obtained by self-assembling of adenine, D-ribose and trimetaphosphate in aqueous solution in good yields. This generation of a ribonucleotide from its three molecular components occurs in a one-pot reaction at room temperature for about 30-40 days and with high chemio-, regio-, and stereo-selectivity. Similar results are obtained with guanine. A mechanism is also proposed. PMID:21305098

  1. Protection of Chinese herbs against adenine-induced chronic renal failure in rats.

    PubMed

    Tong, Yanqing; Han, Bing; Guo, Hongyang; Liu, Yanru

    2010-01-01

    The aim of the study is to evaluate the efficacy of Chinese herbs (Angelica sinensis, Ligusticum wallichii, Salvia miltiorrhiza, Rhizoma dioscoreae, Rhodiola crenilata, Astragalus membranaceus and Angelica sinensis) on adenine-induced chronic renal failure in rats. 30 age-matched male Wistar rats were divided into three groups. Rats in group A (n = 10), B (n = 10) and C (n = 10) were fed a standard laboratory chow and allowed tap water ad libitum. In group B and C, renal failure was induced by the administration of a diet containing 0.75% adenine for 28 days which began at day 0. Rats in group C were given Chinese herbs (40 ml/kg with drug concentration 1.75 g/ml) beginning at day 0. Urine albumin, blood urea nitrogen (BUN) and creatinine were determined at days 0, 14 and 28. At day 28, the animals were killed and their kidneys removed for light microscope evaluation. Body weight in Group B decreased more significantly than that in Group C (p = 0.032) at day 28. The rats in group B demonstrated more severe proteinuria and higher Serum creatinine and BUN levels than group C at day 14 and day 28 (P < 0.05, 0.01). All rats given adenine developed marked structural renal damage involving the tubule and interstitium. The values were much less severe in group C than those in group B. In adenine-induced chronic renal failure rats, the protective effects of these Chinese herbs were of a significant nature. Our results do support the notion that these Chinese herbs are useful in deferring the advance of chronic renal failure. We recommend Chinese herbs as a beneficial treatment for pre-end stage chronic renal failure.

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

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

    1984-09-01

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

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

    PubMed Central

    Türkel, Naciye

    2015-01-01

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

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

    PubMed Central

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

    2007-01-01

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

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

    PubMed

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

    2005-05-01

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

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

    PubMed

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

    2015-12-08

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

  8. Adenine Synthesis in a Model Prebiotic Reaction: Connecting Origin of Life Chemistry with Biology

    PubMed Central

    2011-01-01

    Many high school laboratory experiments demonstrate concepts related to biological evolution, but few exist that allow students to investigate life’s chemical origins. This series of laboratory experiments has been developed to allow students to explore and appreciate the deep connection that exists between prebiotic chemistry, chemical evolution, and contemporary biological systems. In the first experiment of the series, students synthesize adenine, one of the purine nucleobases of DNA and RNA, from plausibly prebiotic precursor molecules. Students compare their product to authentic standards using thin-layer chromatography. The second and third experiments of the series allow students to extract DNA from a familiar organism, the strawberry, and hydrolyze it, releasing adenine, which they can then compare to the previously chemically-synthesized adenine. A fourth, optional experiment is included where the technique of thin-layer chromatography is introduced and chromatographic skills are developed for use in the other three experiments that comprise this series. Concepts relating to organic and analytical chemistry, as well as biochemistry and DNA structure, are incorporated throughout, allowing this series of laboratory experiments to be easily inserted into existing laboratory courses and to reinforce concepts already included in any high school chemistry or biology curriculum. PMID:22075932

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

    PubMed

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

    2015-01-01

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

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

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

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

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

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

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

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

    PubMed

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

    2012-06-24

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

    NASA Technical Reports Server (NTRS)

    Kawamura, K.; Ferris, J. P.

    1994-01-01

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

  14. Adenine photodimerization in deoxyadenylate sequences: elucidation of the mechanism through structural studies of a major d(ApA) photoproduct.

    PubMed Central

    Kumar, S; Joshi, P C; Sharma, N D; Bose, S N; Jeremy, R; Davies, H; Takeda, N; McCloskey, J A

    1991-01-01

    The mechanism of the photodimerization of adjacent adenine bases on the same strand of DNA has been elucidated by determining the structure of one of the two major photoproducts that are formed by UV irradiation of the deoxydinucleoside monophosphate d(ApA). The photoproduct, denoted d(ApA)*, corresponds to a species of adenine photodimer first described by Pörschke (Pörschke, D. (1973) J.Am.Chem.Soc. 95, 8440-8446). From a detailed examination of its chemical and spectroscopic properties, including comparisons with the model compound N-cyano-N1-(1-methylimidazol-5-yl)formamidine, it is deduced that d(ApA)* contains a deoxyadenosine unit covalently linked through its C(8) position to C(4) of an imidazole N(1) deoxyribonucleoside moiety bearing an N-cyanoformamidino substituent at C(5). On treatment with acid, d(ApA)* is degraded with high specificity to 8-(5-amino-imidazol-4-yl)adenine whose identity has been confirmed by independent chemical synthesis. It is concluded that the primary event in adenine photodimerization entails photoaddition of the N(7)-C(8) double bond of the 5'-adenine across the C(6) and C(5) positions of the 3'-adenine. The azetidine species thus generated acts as a common precursor to both types of d(ApA) photoproduct which are formed from it by competing modes of azetidine ring fission. PMID:2057348

  15. Movement and Metabolism of Kinetin-14C and of Adenine-14C in Coleus Petiole Segments of Increasing Age 1

    PubMed Central

    Veen, Henk; Jacobs, William P.

    1969-01-01

    To see if polar movement was typical of growth-regulators other than auxins, the movement of adenine-8-14C and of kinetin-8-14C was studied in segments cut from petioles of increasing age. No polarity was found. In time-course experiments lasting 24 hr, kinetin showed a progressive increase of radioactivity in receiver blocks, while adenine showed a maximum at 8 hr with a decline thereafter. More kinetin moved through older segments than through younger ones. There was no difference in net loss as far as the position of the donor block is concerned. However, the loss of radioactivity from adenine donor blocks was much higher than the loss of radioactivity from kinetin donor blocks. The radioactivity in receiver blocks after 24 hr treatment with kinetin-14C was still with kinetin, judging by location on chromatograms. By the same criterion, adenine and a smaller amount of some other compound were in receiver blocks after a 6 hr transport with adenine-14C in the donors. By contrast, more zones of radioactivity were extracted from petiole segments to which kinetin or adenine had been added. For both purine derivatives the original compound represented no more than 20% of the total radioactivity extracted from the tissue after a transport period of 24 hr. PMID:16657203

  16. UU/UA dinucleotide frequency reduction in coding regions results in increased mRNA stability and protein expression.

    PubMed

    Al-Saif, Maher; Khabar, Khalid S A

    2012-05-01

    UU and UA dinucleotides are rare in mammalian genes and may offer natural selection against endoribonuclease-mediated mRNA decay. This study hypothesized that reducing UU and UA (UW) dinucleotides in the mRNA-coding sequence, including the codons and the dicodon boundaries, may promote resistance to mRNA decay, thereby increasing protein production. Indeed, protein expression from UW-reduced coding regions of enhanced green fluorescent protein (EGFP), luciferase, interferon-α, and hepatitis B surface antigen (HBsAg) was higher when compared to the wild-type protein expression. The steady-state level of UW-reduced EGFP mRNA was higher and the mRNA half-life was also longer. Ectopic expression of the endoribonuclease, RNase L, did not reduce the wild type or UW-reduced mRNA. A mutant form of the mRNA decay-promoting protein, tristetraprolin (TTP/ZFP36), which has a point mutation in the zinc-finger domain (C124R), was used. The wild-type EGFP mRNA but not the UW-reduced mRNA responded to the dominant negative action of the C124R ZFP36/TTP mutant. The results indicate the efficacy of the described rational approach to formulate a general scheme for boosting recombinant protein production in mammalian cells.

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

    PubMed

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

    2006-05-29

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

  18. Auxin biosynthesis and storage forms

    PubMed Central

    Strader, Lucia C.

    2013-01-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development. PMID:23580748

  19. Auxin biosynthesis and storage forms.

    PubMed

    Korasick, David A; Enders, Tara A; Strader, Lucia C

    2013-06-01

    The plant hormone auxin drives plant growth and morphogenesis. The levels and distribution of the active auxin indole-3-acetic acid (IAA) are tightly controlled through synthesis, inactivation, and transport. Many auxin precursors and modified auxin forms, used to regulate auxin homeostasis, have been identified; however, very little is known about the integration of multiple auxin biosynthesis and inactivation pathways. This review discusses the many ways auxin levels are regulated through biosynthesis, storage forms, and inactivation, and the potential roles modified auxins play in regulating the bioactive pool of auxin to affect plant growth and development.

  20. Alternate biosynthesis of valerenadiene and related sesquiterpenes.

    PubMed

    Paknikar, Shashikumar K; Kadam, Shahuraj H; Ehrlich, April L; Bates, Robert B

    2013-09-01

    It is proposed that the biosynthesis of the sesquiterpene valerenadiene, a key intermediate in the biosynthesis of a sedative in valerian, involves cyclopropane and not cyclobutane intermediates and includes as a key step a cyclopropylcarbinylcation-cyclopropylcarbinylcation rearrangement analogous to the one observed in the conversion of presqualene to squalene in triterpene and steroid biosynthesis. Similar mechanisms are proposed for the biosynthesis of the related sesquiterpenes pacifigorgiol, tamariscene and (+)-pacifigorgia-1,10-diene. PMID:24273843

  1. Phermone biosynthesis activation in fire ants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN), was identified to stimulate sex pheromone biosynthesis in a moth. Since then, the physiological role, target site and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in mot...

  2. The Evolution of Aflatoxin Biosynthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biosynthesis of aflatoxin (AF) involves over 20 enzymatic reactions in a complex polyketide pathway that converts acetate and malonate to the intermediates sterigmatocystin (ST) and O-methylsterigmatocysin (OMST), the respective penultimate and ultimate precursors of AF. Although ST, OMST, and ...

  3. Transcriptional control of flavonoid biosynthesis

    PubMed Central

    Li, Shutian

    2014-01-01

    Flavonoids are plant secondary polyphenolic metabolites and fulfil many vital biological functions, offering a valuable metabolic and genetic model for studying transcriptional control of gene expression. Arabidopsis thaliana mainly accumulates 3 types of flavonoids, including flavonols, anthocyanins, and proanthocyanidins (PAs). Flavonoid biosynthesis involves a multitude of well-characterized enzymatic and regulatory proteins. Three R2R3-MYB proteins (MYB11, MYB12, and MYB111) control flavonol biosynthesis via activating the early biosynthetic steps, whereas the production of anthocyanins and PAs requires the MYB-bHLH-WD40 (MBW) complex to activate the late biosynthetic genes. Additional regulators of flavonoid biosynthesis have recently come to light, which interact with R2R3-MYBs or bHLHs to organize or disrupt the formation of the MBW complex, leading to enhanced or compromised flavonoid production. This mini-review gives an overview of how these novel players modulate flavonoid metabolism and thus plant developmental processes and further proposes a fine-tuning mechanism to complete the complex regulatory network controlling flavonoid biosynthesis. PMID:24393776

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    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 × 10-15 and 3.2 × 10-15 cm2, respectively. The total cross section for formation of the negative ions is 6.1 × 10-18 and 7.6 × 10-18 cm2 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. Fragmentation of the adenine and guanine molecules induced by electron collisions

    SciTech Connect

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

    2014-05-07

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

  6. NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy.

    PubMed

    Okabe, Cristiene; Borges, Raquel Lerner; de Almeida, Danilo Candido; Fanelli, Camilla; Barlette, Grasiela Pedreira; Machado, Flavia Gomes; Arias, Simone Costa Alarcon; Malheiros, Denise Maria Avancini Costa; Camara, Niels Olsen Saraiva; Zatz, Roberto; Fujihara, Clarice Kazue

    2013-07-15

    Adenine overload promotes intratubular crystal precipitation and interstitial nephritis. We showed recently that these abnormalities are strongly attenuated in mice knockout for Toll-like receptors-2, -4, MyD88, ASC, or caspase-1. We now investigated whether NF-κB activation also plays a pathogenic role in this model. Adult male Munich-Wistar rats were distributed among three groups: C (n = 17), receiving standard chow; ADE (n = 17), given adenine in the chow at 0.7% for 1 wk and 0.5% for 2 wk; and ADE + pyrrolidine dithiocarbamate (PDTC; n = 14), receiving adenine as above and the NF-κB inhibitor PDTC (120 mg·kg⁻¹·day⁻¹ in the drinking water). After 3 wk, widespread crystal deposition was seen in tubular lumina and in the renal interstitium, along with granuloma formation, collagen accumulation, intense tubulointerstitial proliferation, and increased interstitial expression of inflammatory mediators. Part of the crystals were segregated from tubular lumina by a newly formed cell layer and, at more advanced stages, appeared to be extruded to the interstitium. p65 nuclear translocation and IKK-α increased abundance indicated activation of the NF-κB system. PDTC treatment prevented p65 migration and normalized IKK-α, limited crystal shift to the interstitium, and strongly attenuated interstitial fibrosis/inflammation. These findings indicate that the complex inflammatory phenomena associated with this model depend, at least in part, on NF-κB activation, and suggest that the NF-κB system may become a therapeutic target in the treatment of chronic kidney disease.

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

    SciTech Connect

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

    2009-09-02

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

  8. (-)-Menthol biosynthesis and molecular genetics.

    PubMed

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L; Wildung, Mark R

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint (Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general "allylic oxidation-conjugate reduction" scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1R, 3R, 4S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil. PMID:16292524

  9. (-)-Menthol biosynthesis and molecular genetics

    NASA Astrophysics Data System (ADS)

    Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

    2005-12-01

    (-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

  10. Purine biosynthesis-deficient Burkholderia mutants are incapable of symbiotic accommodation in the stinkbug.

    PubMed

    Kim, Jiyeun Kate; Jang, Ho Am; Won, Yeo Jin; Kikuchi, Yoshitomo; Han, Sang Heum; Kim, Chan-Hee; Nikoh, Naruo; Fukatsu, Takema; Lee, Bok Luel

    2014-03-01

    The Riptortus-Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect-bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host's symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host's symbiotic organ at early instar stages and reduced the host's fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host's symbiotic organ and enhancing the host's fitness values.

  11. The structure, stability, H-bonding pattern, and electrostatic potential of adenine tetrads

    NASA Astrophysics Data System (ADS)

    Gu, Jiande; Leszczynski, Jerzy

    2001-03-01

    Two conformations of the adenine tetrad were investigated at the HF and B3LYP/6-311G(d,p) levels of theory. Both conformations are predicted to be stable only in the nonplanar form. They adopt the bowl type structure. Since the planar form offers better geometry for stacking with the adjacent G-tetrad, both planar forms are expected to be important in the formation of the tetraplexes. Based on electrostatic potential map the positive electrostatic potential in the central area of both conformations is expected to reinforce the stacking between the A-tetrads and the G-tetrads in the tetraplexes.

  12. Strong coupling between adenine nucleobases in DNA single strands revealed by circular dichroism using synchrotron radiation

    NASA Astrophysics Data System (ADS)

    Kadhane, Umesh; Holm, Anne I. S.; Hoffmann, Søren Vrønning; Nielsen, Steen Brøndsted

    2008-02-01

    Circular dichroism (CD) experiments on DNA single strands (dAn) at the ASTRID synchrotron radiation facility reveal that eight adenine (A) bases electronically couple upon 190nm excitation. After n=8 , the CD signal increases linearly with n with a slope equal to the sum of the coupling terms. Nearest neighbor interactions account for only 24% of the CD signal whereas electronic communication is limited to nearest neighbors for two other exciton bands observed at 218 and 251nm (i.e., dimer excited states). Electronic coupling between bases in DNA is important for nonradiative deexcitation of electronically excited states since the hazardous energy is spread over a larger spatial region.

  13. Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

    PubMed Central

    Garin, Intza; Edghill, Emma L.; Akerman, Ildem; Rubio-Cabezas, Oscar; Rica, Itxaso; Locke, Jonathan M.; Maestro, Miguel Angel; Alshaikh, Adnan; Bundak, Ruveyde; del Castillo, Gabriel; Deeb, Asma; Deiss, Dorothee; Fernandez, Juan M.; Godbole, Koumudi; Hussain, Khalid; O’Connell, Michele; Klupa, Thomasz; Kolouskova, Stanislava; Mohsin, Fauzia; Perlman, Kusiel; Sumnik, Zdenek; Rial, Jose M.; Ugarte, Estibaliz; Vasanthi, Thiruvengadam; Johnstone, Karen; Flanagan, Sarah E.; Martínez, Rosa; Castaño, Carlos; Patch, Ann-Marie; Fernández-Rebollo, Eduardo; Raile, Klemens; Morgan, Noel; Harries, Lorna W.; Castaño, Luis; Ellard, Sian; Ferrer, Jorge; de Nanclares, Guiomar Perez; Hattersley, Andrew T.

    2010-01-01

    Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man. PMID:20133622

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

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

    PubMed

    Kalaivani, Arumugam; Narayanan, Sangilimuthu Sriman

    2015-06-01

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

  16. The isolation and characterisation of a new type of dimeric adenine photoproduct in UV-irradiated deoxyadenylates.

    PubMed Central

    Kumar, S; Sharma, N D; Davies, R J; Phillipson, D W; McCloskey, J A

    1987-01-01

    A new type of dimeric adenine photoproduct has been isolated from d(ApA) irradiated at 254 nm in neutral aqueous solution. It is formed in comparable amounts to another, quite distinct, adenine photoproduct first described by Pörschke (J. Am. Chem. Soc. (1973), 95, 8440-8446). Results from high resolution mass spectrometry and 1H NMR indicate that the new photoproduct comprises a mixture of two stereoisomers whose formation involves covalent coupling of the adenine bases in d(ApA) and concomitant incorporation of the elements of one molecule of water. The photoproduct is degraded specifically by acid to 4,6-diamino-5-guanidinopyrimidine (DGPY) whose identity has been confirmed by independent chemical synthesis. Formation of the new photoproduct in UV-irradiated d(pA)2 and poly(dA), but not poly(rA), has been demonstrated by assaying their acid hydrolysates for the presence of DGPY. The properties of the photoproduct are consistent with it being generated by the hydrolytic fission of an azetidine photoadduct in which the N(7) and C(8) atoms of the 5'-adenine in d(ApA) are linked respectively to the C(6) and C(5) positions of the 3'-adenine. PMID:3822822

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

    PubMed

    Yin, Yuling; Watanabe, Shin; Ashihara, Hiroshi

    2012-01-01

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

  18. Accurate crystal molecular dynamics simulations using particle-mesh-Ewald: RNA dinucleotides — ApU and GpC

    NASA Astrophysics Data System (ADS)

    Lee, Hsing; Darden, Thomas; Pedersen, Lee

    1995-09-01

    Long molecular dynamics (MD) simulations for two crystal RNA dinucleotides ApU (2.0 ns) and GpC (1.5 ns) were performed, starting from the crystallographic positions of all heavy atoms in the crystals. By employing the particle-mesh-Ewald algorithm [Darden et al., J. Chem. Phys. 98 (1993) 10089] to accommodate the long-range Coulomb interactions, highly accurate MD structures were obtained for both crystals. The instantaneous root-mean-square positional deviations of the heavy atoms equilibrate at approximately 0.4 Å for both systems, while the experimental and calculated temperature factors are comparable in size. These results describe the first successful crystal MD simulation of RNA molecules.

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

    PubMed

    Bochow, S; Elliman, J; Owens, L

    2012-11-01

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

  20. 3D Magnetically Ordered Open Supramolecular Architectures Based on Ferrimagnetic Cu/Adenine/Hydroxide Heptameric Wheels.

    PubMed

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

    2016-08-01

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

  1. Differentiation alters the unstable expression of adenine phosphoribosyltransferase in mouse teratocarcinoma cells.

    PubMed

    Turker, M S; Tischfield, J A; Rabinovitch, P; Stambrook, P J; Trill, J J; Smith, A C; Ogburn, C E; Martin, G M

    1986-01-01

    Three multipotent mouse teratocarcinoma stem lines, all exhibiting unstable expression for the purine salvage enzyme adenine phosphoribosyltransferase (APRT) were used for the isolation of differentiated cell lines from neoplasms developed in syngeneic mice. Two of the stem cell lines (DAP1B and DAP1C) exhibited homozygous deficiencies for APRT expression while the third stem cell line (E140) exhibited a heterozygous deficiency (Turker, M.S., Smith, A.C., and Martin, G.M.; Somat. Cell Mol. Genet.; 10:55-69; 1984). A total of 16 morphologically differentiated cell lines were established from these neoplasms; most were no longer tumorigenic. Differentiated cell lines derived from the E140-induced tumors segregated homozygous deficient mutants in a single step, consistent with their retention of the heterozygous deficient state. Differentiated homozygous deficient cell lines gave rise to phenotypic revertants at very high frequencies (10(-1) to 10(-2)). The majority of these putative revertants, however, yielded cell-free extracts with little or no detectable APRT activity. These putative revertants were capable of adenine salvage and were therefore termed APRT pseudorevertants. Since the APRT pseudorevertant phenotype was only observed in the differentiated progeny of the APRT deficient stem cell lines, we conclude that this change in the nature of the revertant phenotype was a consequence of cellular differentiation.

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

    NASA Technical Reports Server (NTRS)

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

    2002-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

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

    PubMed

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

    2011-08-11

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

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

    SciTech Connect

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

    2007-03-28

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

  8. 3D Magnetically Ordered Open Supramolecular Architectures Based on Ferrimagnetic Cu/Adenine/Hydroxide Heptameric Wheels.

    PubMed

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

    2016-08-01

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

  9. A van der Waals density functional study of adenine on graphene: Single molecular adsorption and overlayer binding

    SciTech Connect

    Berland, Kristian; Cooper, Valentino R; Langreth, David C.; Schroder, Prof. Elsebeth; Chakarova-Kack, Svetla

    2011-01-01

    The adsorption of an adenine molecule on graphene is studied using a first-principles van der Waals functional (vdW-DF) [Dion et al., Phys. Rev. Lett. 92, 246401 (2004)]. The cohesive energy of an ordered adenine overlayer is also estimated. For the adsorption of a single molecule, we determine the optimal binding configuration and adsorption energy by translating and rotating the molecule. The adsorption energy for a single molecule of adenine is found to be 711 meV, which is close to the calculated adsorption energy of the similar-sized naphthalene. Based on the single molecular binding configuration, we estimate the cohesive energy of a two-dimensional ordered overlayer. We find a significantly stronger binding energy for the ordered overlayer than for single-molecule adsorption.

  10. Gibberellin biosynthesis in Gibberlla fujikuroi

    SciTech Connect

    Johnson, S.W.; Coolbaugh, R.C. )

    1989-04-01

    Gibberellins (GAs) are a group of plant growth hormones which were first isolated from the fungus Gibberella fujikuori. We have examined the biosynthesis of GAs in this fungus in liquid cultures using HPLC followed by GC-MS. Furthermore we have used cell-free enzyme extracts with {sup 14}C-labeled intermediates to examine the regulation of specific parts of the biosynthetic pathway. GA{sub 3} is the predominant GA in well aerated cultures. GA{sub 4} and GA{sub 7}, intermediates in GA{sub 3} biosynthesis, accumulate in cultures with low levels of dissolved oxygen, but are not detectable in more aerated cultures. Light stimulates GA production in G. fujikuroi cultures grown from young stock. Cell-free enzyme studies indicate that light has no effect on incorporation of mevalonic acid into kaurene, but does significantly stimulate the oxidation of kaurenoic acid.

  11. Lignification: Flexibility, Biosynthesis and Regulation.

    PubMed

    Zhao, Qiao

    2016-08-01

    Lignin is a complex phenolic polymer that is deposited in the secondary cell wall of all vascular plants. The evolution of lignin is considered to be a critical event during vascular plant development, because lignin provides mechanical strength, rigidity, and hydrophobicity to secondary cell walls to allow plants to grow tall and transport water and nutrients over a long distance. In recent years, great research efforts have been made to genetically alter lignin biosynthesis to improve biomass degradability for the production of second-generation biofuels. This global focus on lignin research has significantly advanced our understanding of the lignification process. Based on these advances, here I provide an overview of lignin composition, the biosynthesis pathway and its regulation. PMID:27131502

  12. Phosphatidylcholine biosynthesis and lipoprotein metabolism.

    PubMed

    Cole, Laura K; Vance, Jean E; Vance, Dennis E

    2012-05-01

    Phosphatidylcholine (PC) is the major phospholipid component of all plasma lipoprotein classes. PC is the only phospholipid which is currently known to be required for lipoprotein assembly and secretion. Impaired hepatic PC biosynthesis significantly reduces the levels of circulating very low density lipoproteins (VLDLs) and high density lipoproteins (HDLs). The reduction in plasma VLDLs is due in part to impaired hepatic secretion of VLDLs. Less PC within the hepatic secretory pathway results in nascent VLDL particles with reduced levels of PC. These particles are recognized as being defective and are degraded within the secretory system by an incompletely defined process that occurs in a post-endoplasmic reticulum compartment, consistent with degradation directed by the low-density lipoprotein receptor and/or autophagy. Moreover, VLDL particles are taken up more readily from the circulation when the PC content of the VLDLs is reduced, likely due to a preference of cell surface receptors and/or enzymes for lipoproteins that contain less PC. Impaired PC biosynthesis also reduces plasma HDLs by inhibiting hepatic HDL formation and by increasing HDL uptake from the circulation. These effects are mediated by elevated expression of ATP-binding cassette transporter A1 and hepatic scavenger receptor class B type 1, respectively. Hepatic PC availability has recently been linked to the progression of liver and heart disease. These findings demonstrate that hepatic PC biosynthesis can regulate the amount of circulating lipoproteins and suggest that hepatic PC biosynthesis may represent an important pharmaceutical target. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

  13. Biosynthesis of Fungal Indole Alkaloids

    PubMed Central

    Xu, Wei; Gavia, Diego J.; Tang, Yi

    2014-01-01

    This review provides a summary of recent research advances in elucidating the biosynthesis of fungal indole alkaloids. Different strategies used to incorporate and derivatize the indole/indoline moieties in various families of fungal indole alkaloids will be discussed, including tryptophan-containing nonribosomal peptides and polyketide-nonribosomal peptide hybrids; and alkaloids derived from other indole building blocks. This review also includes discussion regarding the downstream modifications that generate chemical and structural diversity among indole alkaloids. PMID:25180619

  14. DFT Studies of the Extent of Hole Delocalization in One-electron Oxidized Adenine and Guanine base Stacks

    PubMed Central

    Kumar, Anil

    2011-01-01

    This study investigates the extent of hole delocalization in one-electron oxidized adenine (A)- and guanine (G)-stacks and shows that new IR vibrational bands are predicted that are characteristic of hole delocalization within A-stacks. The geometries of A-stack (Ai; i = 2 – 8) and G-stack (GG and GGG) in their neutral and one-electron oxidized states were optimized with the bases in a B-DNA conformation using the M06-2X/6-31G* method. The highest occupied molecular orbital (HOMO) is localized on a single adenine in A-stacks and on a single guanine in GG and GGG stacks; located at the 5′-site of the stack. On one-electron oxidation (removal of an electron from the HOMO of the neutral A- and G-stacks) a “hole” is created. Mulliken charge analysis shows that these “holes” are delocalized over 2 – 3 adenine bases in the A-stack. The calculated spin density distribution of (Ai)•+ (i = 2 – 8), also, showed delocalization of the hole predominantly on two adenine bases with some delocalization on a neighboring base. For GG and GGG radical cations, the hole was found to be localized on a single G in the stack. The calculated HFCCs of GG and GGG are in good agreement with the experiment. Further, from the vibrational frequency analysis, it was found that IR spectra of neutral and the corresponding one-electron oxidized adenine stacks are quite different. The IR spectra of (A2)•+ has intense IR peaks between 900 – 1500 cm−1 which are not present in the neutral A2 stack. The presence of (A2)•+ in the adenine stack has a characteristic intense peak at ~1100 cm−1. Thus IR and Raman spectroscopy has potential for monitoring the extent of hole delocalization in A stacks. PMID:21417208

  15. Pyrimidine Biosynthesis in Lactobacillus leichmannii

    PubMed Central

    Hutson, Judith Y.; Downing, Mancourt

    1968-01-01

    Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented. PMID:5686000

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  18. REVERSAL BY ADENINE OF THE ETHIONINE-INDUCED LIPID ACCUMULATION IN THE ENDOPLASMIC RETICULUM OF THE RAT LIVER

    PubMed Central

    Baglio, Corrado M.; Farber, Emmanuel

    1965-01-01

    Within 3.5 to 4 hours after thionine administration, numerous small osmiophilic bodies, liposomes, appear in the endoplasmic reticulum of the liver cells. By fusion, the liposomes lead to the formation of larger collections of fat, giant liposomes. Adenine administration to ethionine-treated rats removes the liposomes from the hepatocytes and causes the transitory appearance of osmiophilic droplets in the sinusoidal space of Disse. The characteristic disaggregation of hepatic polysomes seen in the liver after ethionine administration is corrected by the injection of adenine. PMID:5885431

  19. Progesterone-adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: design, synthesis, characterization and biological evaluation.

    PubMed

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

    2012-10-01

    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.

  20. Dysregulation of Plasmalogen Homeostasis Impairs Cholesterol Biosynthesis.

    PubMed

    Honsho, Masanori; Abe, Yuichi; Fujiki, Yukio

    2015-11-27

    Plasmalogen biosynthesis is regulated by modulating fatty acyl-CoA reductase 1 stability in a manner dependent on cellular plasmalogen level. However, physiological significance of the regulation of plasmalogen biosynthesis remains unknown. Here we show that elevation of the cellular plasmalogen level reduces cholesterol biosynthesis without affecting the isoprenylation of proteins such as Rab and Pex19p. Analysis of intermediate metabolites in cholesterol biosynthesis suggests that the first oxidative step in cholesterol biosynthesis catalyzed by squalene monooxygenase (SQLE), an important regulator downstream HMG-CoA reductase in cholesterol synthesis, is reduced by degradation of SQLE upon elevation of cellular plasmalogen level. By contrast, the defect of plasmalogen synthesis causes elevation of SQLE expression, resulting in the reduction of 2,3-epoxysqualene required for cholesterol synthesis, hence implying a novel physiological consequence of the regulation of plasmalogen biosynthesis.

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

    SciTech Connect

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

    2015-06-07

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-06-01

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

  3. Hydroxyl radical reactions with adenine: reactant complexes, transition states, and product complexes.

    PubMed

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

    2010-10-18

    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

  4. Chronic kidney disease induced by adenine: a suitable model of growth retardation in uremia.

    PubMed

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

    2015-07-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Zhang, Guiqing; Xie, Shijie

    2016-07-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2000-01-01

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

  7. Vacuum-ultraviolet circular dichroism reveals DNA duplex formation between short strands of adenine and thymine.

    PubMed

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

    2012-11-21

    Absorbance spectroscopy is used extensively to tell when two DNA single strands come together and form a double strand. Here we show that circular dichroism in the vacuum ultraviolet region provides an even stronger indication for duplex formation in the case of short strands of adenine and thymine (4 to 16 bases in each strand). Indeed, our results show that a strong positive CD band appears at 179 nm when double strands are formed. Melting experiments were done in aqueous solution with and without added Na(+) counter ions. With additional salt present a huge increase in the 179 nm CD band was observed when lowering the temperature. A 179 nm CD marker band for duplex formation can be used to measure the kinetics for the association of two single strands. Such experiments rely on large changes at one particular wavelength since it is too time-consuming to record a full-wavelength spectrum.

  8. Metal-adeninate vertices for the construction of an exceptionally porous metal-organic framework.

    PubMed

    An, Jihyun; Farha, Omar K; Hupp, Joseph T; Pohl, Ehmke; Yeh, Joanne I; Rosi, Nathaniel L

    2012-01-03

    Metal-organic frameworks comprising metal-carboxylate cluster vertices and long, branched organic linkers are the most porous materials known, and therefore have attracted tremendous attention for many applications, including gas storage, separations, catalysis and drug delivery. To increase metal-organic framework porosity, the size and complexity of linkers has increased. Here we present a promising alternative strategy for constructing mesoporous metal-organic frameworks that addresses the size of the vertex rather than the length of the organic linker. This approach uses large metal-biomolecule clusters, in particular zinc-adeninate building units, as vertices to construct bio-MOF-100, an exclusively mesoporous metal-organic framework. Bio-MOF-100 exhibits a high surface area (4,300 m(2) g(-1)), one of the lowest crystal densities (0.302 g cm(-3)) and the largest metal-organic framework pore volume reported to date (4.3 cm(3) g(-1)).

  9. The isolation and characterization of the Escherichia coli DNA adenine methylase (dam) gene.

    PubMed Central

    Brooks, J E; Blumenthal, R M; Gingeras, T R

    1983-01-01

    The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms. PMID:6300769

  10. Synthesis and enzymatic incorporation of α-L-threofuranosyl adenine triphosphate (tATP).

    PubMed

    Zhang, Su; Chaput, John C

    2013-03-01

    Threose nucleic acid (TNA) is an artificial genetic polymer in which the natural ribose sugar found in RNA has been replaced with an unnatural threose sugar. TNA can be synthesized enzymatically using Therminator DNA polymerase to copy DNA templates into TNA. Here, we expand the substrate repertoire of Therminator DNA polymerase to include threofuranosyl adenine 3'-triphsophate (tATP). We chemically synthesized tATP by two different methods from the 2'-O-acetyl derivative. Enzyme-mediated polymerization reveals that tATP functions as an efficient substrate for Therminator DNA polymerase, indicating that tATP can replace the diaminopurine analogue (tDTP) in TNA transcription reactions. PMID:23352269

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

    PubMed Central

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

    2009-01-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2003-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

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

    2015-01-01

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

  15. Intriguing radical-radical interactions among double-electron oxidized adenine-thymine base pairs

    NASA Astrophysics Data System (ADS)

    Wang, Mei; Zhao, Jing; Zhang, Laibin; Su, Xiyu; Su, Hanlei; Bu, Yuxiang

    2015-01-01

    We present a theoretical investigation of the structural and electronic properties of double-electron oxidized adenine-thymine base pair as well as its deprotonated Watson-Crick derivatives. Double-electron oxidation can destabilize the AT unit, leading to a barrier-hindered metastable A+T+ state with a dissociation channel featuring negative dissociation energy. This unusual energetic phenomenon originates from the competition of electrostatic repulsion and attractively hydrogen-bonding interaction co-existing between Arad + and Trad +. The associated double-proton-transfer process is also explored, suggesting a possible two-step mechanism. Magnetic coupling interactions of various diradical structures are controlled by both intra- and inter-molecular interactions.

  16. Sites of Adsorption of Adenine, Uracil, and Their Corresponding Derivatives on Sodium Montmorillonite

    NASA Astrophysics Data System (ADS)

    Perezgasga, L.; Serrato-Díaz, A.; Negrón-Mendoza, A.; Gal'N, L. De Pablo; Mosqueira, F. G.

    2005-04-01

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

  17. Similarities between UDP-Glucose and Adenine Nucleotide Release in Yeast

    PubMed Central

    Esther, Charles R.; Sesma, Juliana I.; Dohlman, Henrik G.; Ault, Addison D.; Clas, Marién L.; Lazarowski, Eduardo R.; Boucher, Richard C.

    2008-01-01

    Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Δ was reduced by deletion of the YEA4 UDP-N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation. PMID:18693752

  18. Acceleration of adventitious shoots by interaction between exogenous hormone and adenine sulphate in Althaea officinalis L.

    PubMed

    Naz, Ruphi; Anis, M

    2012-11-01

    In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog's (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2 ± 0.58 buds in central vein explants. Addition of different growth regulators (cytokinins-6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins-indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4 ± 0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.

  19. Regulation of Salmonella enterica pathogenicity island 1 by DNA adenine methylation.

    PubMed

    López-Garrido, Javier; Casadesús, Josep

    2010-03-01

    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.

  20. Herpes simplex type 1 defective interfering particles do not affect the antiviral activity of acyclovir, foscarnet and adenine arabinoside.

    PubMed

    Harmenberg, J G; Svensson, L T

    1988-03-01

    The concentration of defective interfering particles (DI-particles) of herpes simplex type 1 virus was analysed by electron microscopy and plaque titration. Fifteen consecutive passages of undiluted virus in green monkey kidney cells were followed. No relationship was found between the concentration of DI-particles and the activity of antiviral substances such as acyclovir, foscarnet and adenine arabinoside.

  1. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration. PMID:19560175

  2. Evolution of rosmarinic acid biosynthesis.

    PubMed

    Petersen, Maike; Abdullah, Yana; Benner, Johannes; Eberle, David; Gehlen, Katja; Hücherig, Stephanie; Janiak, Verena; Kim, Kyung Hee; Sander, Marion; Weitzel, Corinna; Wolters, Stefan

    2009-01-01

    Rosmarinic acid and chlorogenic acid are caffeic acid esters widely found in the plant kingdom and presumably accumulated as defense compounds. In a survey, more than 240 plant species have been screened for the presence of rosmarinic and chlorogenic acids. Several rosmarinic acid-containing species have been detected. The rosmarinic acid accumulation in species of the Marantaceae has not been known before. Rosmarinic acid is found in hornworts, in the fern family Blechnaceae and in species of several orders of mono- and dicotyledonous angiosperms. The biosyntheses of caffeoylshikimate, chlorogenic acid and rosmarinic acid use 4-coumaroyl-CoA from the general phenylpropanoid pathway as hydroxycinnamoyl donor. The hydroxycinnamoyl acceptor substrate comes from the shikimate pathway: shikimic acid, quinic acid and hydroxyphenyllactic acid derived from l-tyrosine. Similar steps are involved in the biosyntheses of rosmarinic, chlorogenic and caffeoylshikimic acids: the transfer of the 4-coumaroyl moiety to an acceptor molecule by a hydroxycinnamoyltransferase from the BAHD acyltransferase family and the meta-hydroxylation of the 4-coumaroyl moiety in the ester by a cytochrome P450 monooxygenase from the CYP98A family. The hydroxycinnamoyltransferases as well as the meta-hydroxylases show high sequence similarities and thus seem to be closely related. The hydroxycinnamoyltransferase and CYP98A14 from Coleus blumei (Lamiaceae) are nevertheless specific for substrates involved in RA biosynthesis showing an evolutionary diversification in phenolic ester metabolism. Our current view is that only a few enzymes had to be "invented" for rosmarinic acid biosynthesis probably on the basis of genes needed for the formation of chlorogenic and caffeoylshikimic acid while further biosynthetic steps might have been recruited from phenylpropanoid metabolism, tocopherol/plastoquinone biosynthesis and photorespiration.

  3. Tetrahydrobiopterin biosynthesis, regeneration and functions.

    PubMed Central

    Thöny, B; Auerbach, G; Blau, N

    2000-01-01

    Tetrahydrobiopterin (BH(4)) cofactor is essential for various processes, and is present in probably every cell or tissue of higher organisms. BH(4) is required for various enzyme activities, and for less defined functions at the cellular level. The pathway for the de novo biosynthesis of BH(4) from GTP involves GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase and sepiapterin reductase. Cofactor regeneration requires pterin-4a-carbinolamine dehydratase and dihydropteridine reductase. Based on gene cloning, recombinant expression, mutagenesis studies, structural analysis of crystals and NMR studies, reaction mechanisms for the biosynthetic and recycling enzymes were proposed. With regard to the regulation of cofactor biosynthesis, the major controlling point is GTP cyclohydrolase I, the expression of which may be under the control of cytokine induction. In the liver at least, activity is inhibited by BH(4), but stimulated by phenylalanine through the GTP cyclohydrolase I feedback regulatory protein. The enzymes that depend on BH(4) are the phenylalanine, tyrosine and tryptophan hydroxylases, the latter two being the rate-limiting enzymes for catecholamine and 5-hydroxytryptamine (serotonin) biosynthesis, all NO synthase isoforms and the glyceryl-ether mono-oxygenase. On a cellular level, BH(4) has been found to be a growth or proliferation factor for Crithidia fasciculata, haemopoietic cells and various mammalian cell lines. In the nervous system, BH(4) is a self-protecting factor for NO, or a general neuroprotecting factor via the NO synthase pathway, and has neurotransmitter-releasing function. With regard to human disease, BH(4) deficiency due to autosomal recessive mutations in all enzymes (except sepiapterin reductase) have been described as a cause of hyperphenylalaninaemia. Furthermore, several neurological diseases, including Dopa-responsive dystonia, but also Alzheimer's disease, Parkinson's disease, autism and depression, have been suggested to be

  4. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

    Mitochondrial metabolism is important for sustaining cellular growth and maintenance; however, the regulatory mechanisms underlying individual processes in plant mitochondria remain largely uncharacterized. Previous redox-proteomics studies have suggested that mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid (TCA) cycle and redox shuttling, is under thiol-based redox regulation as a target candidate of thioredoxin (Trx). In addition, the adenine nucleotide status may be another factor controlling mitochondrial metabolism, as respiratory ATP production in mitochondria is believed to be influenced by several environmental stimuli. Using biochemical and reverse-genetic approaches, we addressed the redox- and adenine nucleotide-dependent regulation of mMDH in Arabidopsis thaliana. Recombinant mMDH protein formed intramolecular disulfide bonds under oxidative conditions, but these bonds did not have a considerable effect on mMDH activity. Mitochondria-localized o-type Trx (Trx-o) did not facilitate re-reduction of oxidized mMDH. Determination of the in vivo redox state revealed that mMDH was stably present in the reduced form even in Trx-o-deficient plants. Accordingly, we concluded that mMDH is not in the class of redox-regulated enzymes. By contrast, mMDH activity was lowered by adenine nucleotides (AMP, ADP, and ATP). Each adenine nucleotide suppressed mMDH activity with different potencies and ATP exerted the largest inhibitory effect with a significantly lower K(I). Correspondingly, mMDH activity was inhibited by the increase in ATP/ADP ratio within the physiological range. These results suggest that mMDH activity is finely controlled in response to variations in mitochondrial adenine nucleotide balance. PMID:26946085

  5. Adenine nucleotide-dependent and redox-independent control of mitochondrial malate dehydrogenase activity in Arabidopsis thaliana.

    PubMed

    Yoshida, Keisuke; Hisabori, Toru

    2016-06-01

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

  6. Characterization of an unbalanced de novo rearrangement, initially by dinucleotide repeat polymorphism typing and subsequent confirmation by FISH

    SciTech Connect

    Zhao, J.; Gordon, P.L.; Wilroy, R.S.

    1994-09-01

    Unbalanced de novo rearrangements not amenable to characterization by conventional cytogenetics can be elucidated using molecular techniques. By microsatellite polymorphism typing, we initially determined the origin and composition of an unbalanced de novo translocation (46,XX,15q+) in a child with multiple congenital anomalies. Microsatellite polymorphism for D5S208 (localized to 5p15) and polymerase chain reaction (PCR) analysis showed that the extra segment originated from the short arm of chromosome 5. Amplification of patient`s DNA with primers for dinucleotide repeats D5S350 and D5S118 showed that most of the short arm of chromosome 5 was present in three copies. The PCR findings were then reconfirmed by a battery of fluorescence in situ hybridizations (FISH). FISH analyses were further helpful in accurately defining the precise trisomic region as well as elucidating the dicentric nature of 46,XX,-15,+der dic (15)t(5;15)(q11.1;p12) de novo translocation. This work demonstrates the benefit of microsatellite polymorphism typing for the identification and characterization of de novo unbalanced rearrangements and apparent tandem duplications in which conventional cytogenetic techniques are usually uninformative. Such study design in cytogenetically equivocal cases could undoubtedly aid in patient management, family counseling and benefit gene mapping studies through accurate genotype-phenotype correlation.

  7. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    PubMed Central

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

    2014-01-01

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

  8. Fatty acid biosynthesis in actinomycetes

    PubMed Central

    Gago, Gabriela; Diacovich, Lautaro; Arabolaza, Ana; Tsai, Shiou-Chuan; Gramajo, Hugo

    2011-01-01

    All organisms that produce fatty acids do so via a repeated cycle of reactions. In mammals and other animals, these reactions are catalyzed by a type I fatty acid synthase (FAS), a large multifunctional protein to which the growing chain is covalently attached. In contrast, most bacteria (and plants) contain a type II system in which each reaction is catalyzed by a discrete protein. The pathway of fatty acid biosynthesis in Escherichia coli is well established and has provided a foundation for elucidating the type II FAS pathways in other bacteria (White et al., 2005). However, fatty acid biosynthesis is more diverse in the phylum Actinobacteria: Mycobacterium, possess both FAS systems while Streptomyces species have only the multi-enzyme FAS II system and Corynebacterium species exclusively FAS I. In this review we present an overview of the genome organization, biochemical properties and physiological relevance of the two FAS systems in the three genera of actinomycetes mentioned above. We also address in detail the biochemical and structural properties of the acyl-CoA carboxylases (ACCases) that catalyzes the first committed step of fatty acid synthesis in actinomycetes, and discuss the molecular bases of their substrate specificity and the structure-based identification of new ACCase inhibitors with anti-mycobacterial properties. PMID:21204864

  9. Oleic acid biosynthesis in cyanobacteria

    SciTech Connect

    VanDusen, W.J.; Jaworski, J.G.

    1986-05-01

    The biosynthesis of fatty acids in cyanobacteria is very similar to the well characterized system found in green plants. However, the initial desaturation of stearic acid in cyanobacteria appears to represent a significant departure from plant systems in which stearoyl-ACP is the exclusive substrate for desaturation. In Anabaena variabilis, the substrate appears to be monoglucosyldiacylglycerol, a lipid not found in plants. The authors examined five different cyanobacteria to determine if the pathway in A. variabilis was generally present in other cyanobacteria. The cyanobacteria studied were A. variabilis, Chlorogloeopsis sp., Schizothrix calcicola, Anacystis marina, and Anacystis nidulans. Each were grown in liquid culture, harvested, and examined for stearoyl-ACP desaturase activity or incubated with /sup 14/CO/sub 2/. None of the cyanobacteria contained any stearoyl-ACP desaturase activity in whole homogenates or 105,000g supernatants. All were capable of incorporating /sup 14/CO/sub 2/ into monoglucosyldiacylglycerol and results from incubations of 20 min, 1 hr, 1 hr + 10 hr chase were consistent with monoglucosyldiacylglycerol serving as precursor for monogalctosyldiacylglycerol. Thus, initial evidence is consistent with oleic acid biosynthesis occurring by desaturation of stearoyl-monoglucosyldiacylglycerol in all cyanobacteria.

  10. Lipoarabinomannans: from structure to biosynthesis.

    PubMed

    Nigou, Jérôme; Gilleron, Martine; Puzo, Germain

    2003-01-01

    Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens and the molecular basis of its virulence remains poorly understood. Here, we review our current knowledge about the structure and biosynthesis of the mycobacterial cell-wall lipoglycans, lipoarabinomannans (LAM). LAM are ubiquitous of mycobacteria and appear as the most potent non-peptidic molecules to modulate the host immune response. Nevertheless, LAM structure differs according to the mycobacterial species and three types of LAM have been described: mannose-capped LAM (ManLAM), phospho-myo-inositol-capped LAM (PILAM) and non-capped LAM (AraLAM). The type of capping is a major structural feature determining the ability of LAM to modulate the immune response. ManLAM, found in slow-growing mycobacteria, such as M. tuberculosis, have been demonstrated to be powerful anti-inflammatory molecules and emerge as key virulence factors that may be relevant drug targets. LAM-like molecules are not only confined to mycobacteria but are also present in actinomycetes (including the genera Rhodococcus, Corynebacterium or Gordonia). This offers the possibility of comparative studies that should help in deciphering the structure-function relationships and biosynthesis of these complex molecules in the future.

  11. Cellulose biosynthesis inhibitors - a multifunctional toolbox.

    PubMed

    Tateno, Mizuki; Brabham, Chad; DeBolt, Seth

    2016-01-01

    In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis.

  12. Cellulose biosynthesis inhibitors - a multifunctional toolbox.

    PubMed

    Tateno, Mizuki; Brabham, Chad; DeBolt, Seth

    2016-01-01

    In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis. PMID:26590309

  13. Current aspects of auxin biosynthesis in plants.

    PubMed

    Kasahara, Hiroyuki

    2015-01-01

    Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.

  14. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity.

    PubMed

    Li, Wang; Li, Yong; Liu, Zhuoliang; Lin, Bin; Yi, Haibo; Xu, Feng; Nie, Zhou; Yao, Shouzhuo

    2016-09-01

    G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3' end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3' adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid-base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions. PMID:27422869

  15. Insight into G-quadruplex-hemin DNAzyme/RNAzyme: adjacent adenine as the intramolecular species for remarkable enhancement of enzymatic activity

    PubMed Central

    Li, Wang; Li, Yong; Liu, Zhuoliang; Lin, Bin; Yi, Haibo; Xu, Feng; Nie, Zhou; Yao, Shouzhuo

    2016-01-01

    G-quadruplex (G4) with stacked G-tetrads structure is able to bind hemin (iron (III)-protoporphyrin IX) to form a unique type of DNAzyme/RNAzyme with peroxidase-mimicking activity, which has been widely employed in multidisciplinary fields. However, its further applications are hampered by its relatively weak activity compared with protein enzymes. Herein, we report a unique intramolecular enhancement effect of the adjacent adenine (EnEAA) at 3′ end of G4 core sequences that significantly improves the activity of G4 DNAzymes. Through detailed investigations of the EnEAA, the added 3′ adenine was proved to accelerate the compound I formation in catalytic cycle and thus improve the G4 DNAzyme activity. EnEAA was found to be highly dependent on the unprotonated state of the N1 of adenine, substantiating that adenine might function as a general acid–base catalyst. Further adenine analogs analysis supported that both N1 and exocyclic 6-amino groups in adenine played key role in the catalysis. Moreover, we proved that EnEAA was generally applicable for various parallel G-quadruplex structures and even G4 RNAzyme. Our studies implied that adenine might act analogously as the distal histidine in protein peroxidases, which shed light on the fundamental understanding and rational design of G4 DNAzyme/RNAzyme catalysts with enhanced functions. PMID:27422869

  16. Topographic heterogeneity in cholesterol biosynthesis.

    PubMed

    Lange, Y; Muraski, M F

    1988-07-01

    We have examined the membrane topography of cholesterol biosynthesis in cultured human fibroblasts. We fed the cells with radioacetate and then interrupted the biosynthetic pathway so as to trap labeled intermediates in their subcellular locations. We analyzed homogenates of human fibroblasts labeled biosynthetically from radioacetate by centrifugation to equilibrium on sucrose gradients. The following two methods were used to interrupt cholesterol biosynthesis: incubation at 10 degrees C and treatment with 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, a specific inhibitor of oxidosqualene cyclase. Incubation at 10 degrees C caused the accumulation of radiolanosterol at the expense of cholesterol. The lanosterol appeared predominantly at an unusually buoyant density (20% (w/w) sucrose; d = 1.08 g/cm3) as well as at the density normally labeled at 37 degrees C (30% sucrose; d = 1.13 g/cm3). 4,4,10 beta-Trimethyl-trans-decal-3 beta-ol treatment caused the accumulation of labeled squalene and squalene 2,3-oxide. Reversal of the block permitted the label to progress rapidly as a wave into lanosterol and ultimately into cholesterol. The profiles of the three precursors did not coincide, suggesting that they were mostly in different membranes. Squalene was uniquely confined to a density of 1.18 g/cm3 (40% sucrose) while squalene 2,3-oxide appeared in peaks of density 1.08 g/cm3 and 1.13 g/cm3 (20% and 30% sucrose). Lanosterol was in a peak of density 1.13 g/cm3. Pulse-chase experiments showed that lanosterol synthesized in the membranes at 20% sucrose moved rapidly to the membranes at 30% sucrose where it was converted to cholesterol. The density gradient profiles of the following organelle markers also were monitored: plasma membrane, cholesterol mass; Golgi apparatus, galactosyltransferase; endoplasmic reticulum, RNA, 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cytochrome c reductase; peroxisomes, catalase. None of these markers appeared at the buoyant density

  17. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine.

    PubMed

    Janowsky, Aaron; Tosh, Dilip K; Eshleman, Amy J; Jacobson, Kenneth A

    2016-04-01

    Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [(125)I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [(3)H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N(6)-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [(125)I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4'-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter

  18. Poly-adenine-based programmable engineering of gold nanoparticles for highly regulated spherical DNAzymes

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  19. Rigid Adenine Nucleoside Derivatives as Novel Modulators of the Human Sodium Symporters for Dopamine and Norepinephrine

    PubMed Central

    Tosh, Dilip K.; Eshleman, Amy J.; Jacobson, Kenneth A.

    2016-01-01

    Thirty-two congeneric rigid adenine nucleoside derivatives containing a North (N)-methanocarba ribose substitution and a 2-arylethynyl group either enhanced (up to 760% of control) or inhibited [125I] methyl (1R,2S,3S)-3-(4-iodophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate (RTI-55) binding at the human dopamine (DA) transporter (DAT) and inhibited DA uptake. Several nucleosides also enhanced [3H]mazindol [(±)-5-(4-chlorophenyl)-3,5-dihydro-2H-imidazo[2,1-a]isoindol-5-ol] binding to the DAT. The combination of binding enhancement and functional inhibition suggests possible allosteric interaction with the tropanes. The structure-activity relationship of this novel class of DAT ligands was explored: small N6-substition (methyl or ethyl) was favored, while the N1 of the adenine ring was essential. Effective terminal aryl groups include thien-2-yl (compounds 9 and 16), with EC50 values of 35.1 and 9.1 nM, respectively, in [125I]RTI-55 binding enhancement, and 3,4-difluorophenyl as in the most potent DA uptake inhibitor (compound 6) with an IC50 value of 92 nM (3-fold more potent than cocaine), but not nitrogen heterocycles. Several compounds inhibited or enhanced binding at the norepinephrine transporter (NET) and serotonin transporter (SERT) and inhibited function in the micromolar range; truncation at the 4′-position in compound 23 allowed for weak inhibition of the SERT. We have not yet eliminated adenosine receptor affinity from this class of DAT modulators, but we identified modifications that remove DAT inhibition as an off-target effect of potent adenosine receptor agonists. Thus, we have identified a new class of allosteric DAT ligands, rigidified adenosine derivatives, and explored their initial structural requirements. They display a very atypical pharmacological profile, i.e., either enhancement by increasing affinity or inhibition of radioligand binding at the DAT, and in some cases the NET and SERT, and inhibition of neurotransmitter uptake

  20. 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

    PubMed Central

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

    2016-01-01

    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

  1. Steroid biosynthesis in adipose tissue.

    PubMed

    Li, Jiehan; Papadopoulos, Vassilios; Vihma, Veera

    2015-11-01

    Tissue-specific expression of steroidogenic enzymes allows the modulation of active steroid levels in a local manner. Thus, the measurement of local steroid concentrations, rather than the circulating levels, has been recognized as a more accurate indicator of the steroid action within a specific tissue. Adipose tissue, one of the largest endocrine tissues in the human body, has been established as an important site for steroid storage and metabolism. Locally produced steroids, through the enzymatic conversion from steroid precursors delivered to adipose tissue, have been proven to either functionally regulate adipose tissue metabolism, or quantitatively contribute to the whole body's steroid levels. Most recently, it has been suggested that adipose tissue may contain the steroidogenic machinery necessary for the initiation of steroid biosynthesis de novo from cholesterol. This review summarizes the evidence indicating the presence of the entire steroidogenic apparatus in adipose tissue and discusses the potential roles of local steroid products in modulating adipose tissue activity and other metabolic parameters.

  2. Acylphloroglucinol Biosynthesis in Strawberry Fruit.

    PubMed

    Song, Chuankui; Ring, Ludwig; Hoffmann, Thomas; Huang, Fong-Chin; Slovin, Janet; Schwab, Wilfried

    2015-11-01

    Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry (Fragaria × ananassa), we discovered four acylphloroglucinol (APG)-glucosides as native Fragaria spp. fruit metabolites whose levels were differently regulated in the transgenic fruits. The biosynthesis of the APG aglycones was investigated by examination of the enzymatic properties of three recombinant Fragaria vesca chalcone synthase (FvCHS) proteins. CHS is involved in anthocyanin biosynthesis during ripening. The F. vesca enzymes readily catalyzed the condensation of two intermediates in branched-chain amino acid metabolism, isovaleryl-Coenzyme A (CoA) and isobutyryl-CoA, with three molecules of malonyl-CoA to form phlorisovalerophenone and phlorisobutyrophenone, respectively, and formed naringenin chalcone when 4-coumaroyl-CoA was used as starter molecule. Isovaleryl-CoA was the preferred starter substrate of FvCHS2-1. Suppression of CHS activity in both transient and stable CHS-silenced fruit resulted in a substantial decrease of APG glucosides and anthocyanins and enhanced levels of volatiles derived from branched-chain amino acids. The proposed APG pathway was confirmed by feeding isotopically labeled amino acids. Thus, Fragaria spp. plants have the capacity to synthesize pharmaceutically important APGs using dual functional CHS/(phloriso)valerophenone synthases that are expressed during fruit ripening. Duplication and adaptive evolution of CHS is the most probable scenario and might be generally applicable to other plants. The results highlight that important promiscuous gene function may be missed when annotation relies solely on in silico analysis. PMID:26169681

  3. Acylphloroglucinol Biosynthesis in Strawberry Fruit.

    PubMed

    Song, Chuankui; Ring, Ludwig; Hoffmann, Thomas; Huang, Fong-Chin; Slovin, Janet; Schwab, Wilfried

    2015-11-01

    Phenolics have health-promoting properties and are a major group of metabolites in fruit crops. Through reverse genetic analysis of the functions of four ripening-related genes in the octoploid strawberry (Fragaria × ananassa), we discovered four acylphloroglucinol (APG)-glucosides as native Fragaria spp. fruit metabolites whose levels were differently regulated in the transgenic fruits. The biosynthesis of the APG aglycones was investigated by examination of the enzymatic properties of three recombinant Fragaria vesca chalcone synthase (FvCHS) proteins. CHS is involved in anthocyanin biosynthesis during ripening. The F. vesca enzymes readily catalyzed the condensation of two intermediates in branched-chain amino acid metabolism, isovaleryl-Coenzyme A (CoA) and isobutyryl-CoA, with three molecules of malonyl-CoA to form phlorisovalerophenone and phlorisobutyrophenone, respectively, and formed naringenin chalcone when 4-coumaroyl-CoA was used as starter molecule. Isovaleryl-CoA was the preferred starter substrate of FvCHS2-1. Suppression of CHS activity in both transient and stable CHS-silenced fruit resulted in a substantial decrease of APG glucosides and anthocyanins and enhanced levels of volatiles derived from branched-chain amino acids. The proposed APG pathway was confirmed by feeding isotopically labeled amino acids. Thus, Fragaria spp. plants have the capacity to synthesize pharmaceutically important APGs using dual functional CHS/(phloriso)valerophenone synthases that are expressed during fruit ripening. Duplication and adaptive evolution of CHS is the most probable scenario and might be generally applicable to other plants. The results highlight that important promiscuous gene function may be missed when annotation relies solely on in silico analysis.

  4. A comparison of genetic map distance and linkage disequilibrium between 15 polymorphic dinucleotide repeat loci in two populations

    SciTech Connect

    Urbanek, M.; Goldman, D.; Long, J.C.

    1994-09-01

    Linkage disequilibrium has recently been used to map the diastrophic dysplasia gene in a Finnish sample. One advantage of this method is that the large pedigrees required by some other methods are unnecessary. Another advantage is that linkage disequilibrium mapping capitalizes on the cumulative history of recombination events, rather than those occurring within the sampled individuals. A potential limitation of linkage disequilibrium mapping is that linkage equilibrium is likely to prevail in all but the most isolated populations, e.g., those which have recently experienced founder effects or severe population bottlenecks. In order to test the method`s generality, we examined patterns of linkage disequilibrium between pairs of loci within a known genetic map. Two populations were analyzed. The first population, Navajo Indians (N=45), is an isolate that experienced a severe bottleneck in the 1860`s. The second population, Maryland Caucasians (N=45), is cosmopolitan. We expected the Navajo sample to display more linkage disequilibrium than the Caucasian sample, and possibly that the Navajo disequilibrium pattern would reflect the genetic map. Linkage disequilibrium coefficients were estimated between pairs of alleles at different loci using maximum likelihood. The genetic isolate structure of Navajo Indians is confirmed by the DNA typings. Heterozygosity is lower than in the Caucasians, and fewer different alleles are observed. However, a relationship between genetic map distance and linkage disequilibrium could be discerned in neither the Navajo nor the Maryland samples. Slightly more linkage disequilibrium was observed in the Navajos, but both data sets were characterized by very low disequilibrium levels. We tentatively conclude that linkage disequilibrium mapping with dinucleotide repeats will only be useful with close linkage between markers and diseases, even in very isolated populations.

  5. Identification of the novel thio-arsenosugars DMThioAsSugarCarboxyl, DMThioAsSugarCarbamate and DMThioAsSugarAdenine in extracts of giant clam tissues by high-performance liquid chromatography online with electrospray tandem mass spectrometry.

    PubMed

    Nischwitz, Volker; Pergantis, Spiros A

    2006-01-01

    Recently, the identification of four thio-arsenosugars in extracts of marine samples was reported. The biosynthesis of these arsenic species is most likely closely related to the well known oxo-arsenosugars. Due to their symbiotic algae giant clams contain oxo-arsenosugars with Carboxyl-, Carbamate- and Adenine-aglycone in significant amounts. Now we provide evidence for the presence of the thio analogues of these three arsenosugars in tissue extracts of the giant clams Tridacna maxima and Tridacna derasa. Anion-exchange high-performance liquid chromatography (HPLC) online with electrospray tandem mass spectrometry was applied for the identification of the thio-arsenosugars and the indirect quantification via their oxo analogues.

  6. A DNA adenine methylase mutant of Shigella flexneri shows no significant attenuation of virulence.

    PubMed

    Honma, Yasuko; Fernández, Reinaldo E; Maurelli, Anthony T

    2004-04-01

    Mutants of Salmonella defective in DNA adenine methylase (dam) have been reported to be attenuated for virulence and to provide protective immunity when used as vaccine strains. To determine whether these observations could be extended to Shigella, a dam mutant of Shigella flexneri 2a was characterized and examined for the role of dam in pathogenesis. The Shigella dam mutant showed some unique characteristics; however, it retained virulence in vivo as well as in vitro. The mutant invaded cultured L2 monolayer cells as efficiently as the wild-type parent, but its intracellular growth was suppressed up to 7 h post-invasion. Furthermore, the invading dam mutant formed smaller plaques in cell monolayers compared to the parent strain. However, the mutant produced keratoconjunctivitis in the Sereny test in guinea pigs only slightly more slowly than the wild-type. While the effect of the dam mutation on virulence was modest, the rate of spontaneous mutation in the dam mutant was 1000-fold greater compared with the wild-type. The virulence and high mutability displayed by the dam mutant of Sh. flexneri suggest that a general anti-bacterial pathogen vaccine strategy based on mutations in dam needs to be re-evaluated.

  7. Electron impact fragmentation of adenine: partial ionization cross sections for positive fragments

    NASA Astrophysics Data System (ADS)

    van der Burgt, Peter J. M.; Finnegan, Sinead; Eden, Samuel

    2015-07-01

    Using computer-controlled data acquisition we have measured mass spectra of positive ions for electron impact on adenine, with electron energies up to 100 eV. Ion yield curves for 50 ions have been obtained and normalized by comparing their sum to the average of calculated total ionization cross sections. Appearance energies have been determined for 37 ions; for 20 ions for the first time. All appearance energies are consistent with the fragmentation pathways identified in the literature. Second onset energies have been determined for 12 fragment ions (for 11 ions for the first time), indicating the occurrence of more than one fragmentation process e.g. for 39 u (C2HN+) and 70 u (C2H4N3+). Matching ion yield shapes (118-120 u, 107-108 u, 91-92 u, and 54-56 u) provide new evidence supporting closely related fragmentation pathways and are attributed to hydrogen rearrangement immediately preceding the fragmentation. We present the first measurement of the ion yield curve of the doubly charged parent ion (67.5 u), with an appearance energy of 23.5 ± 1.0 eV. Contribution to the Topical Issue "COST Action Nano-IBCT: Nano-scale Processes Behind Ion-Beam Cancer Therapy", edited by Andrey Solov'yov, Nigel Mason, Gustavo García, Eugene Surdutovich.

  8. PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

    PubMed Central

    Tang, Chunlei; Wei, Jinping; Han, Qingmei; Liu, Rui; Duan, Xiaoyuan; Fu, Yanping; Huang, Xueling; Wang, Xiaojie; Kang, Zhensheng

    2015-01-01

    Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies. PMID:26058921

  9. Content of Adenine Nucleotides and Orthophosphate in Exporting and Importing Mature Maize Leaves 1

    PubMed Central

    Eschrich, Walter; Fromm, Joerg

    1985-01-01

    Events of reactivation by re-illumination were studied in predarkened detached mature maize leaves, which were arranged as distal sources and proximal sinks; the latter were kept in CO2-free atmosphere and were either illuminated or darkened. Adenine nucleotide (AdN) content and orthophosphate (Pi) concentrations were measured 10 minutes, 30 minutes, and 2, 7, and 14 hours after the onset of re-illumination. For comparison, mature leaves attached to the plant were analyzed. The sum of AdN increased up to 7 hours of re-illumination, then dark sinks and their sources showed decreasing amounts of AdN, while the increase continued up to 14 hours in sources and illuminated sinks. In leaves attached to the plant, no further increase in AdN level followed the 7-hour mark. The amount of individual AdN (ATP, ADP, AMP) differed considerably in sources and sinks of the detached leaves. Although both the source supplying the illuminated sink and the source supplying the dark sink were treated the same, they showed striking differences in AdN contents. Such relations were also observed, when ATP/ADP ratios and Pi concentrations were compared. The influence a sink can exert on its source suggests a participation of the physiological events in the sink on the regulation of AdN and Pi metabolism in the source. PMID:16664246

  10. Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification

    PubMed Central

    Iyer, Lakshminarayan M.; Zhang, Dapeng

    2015-01-01

    While N6‐methyladenosine (m6A) is a well‐known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well‐studied 5‐methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction‐modification and counter‐restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m6A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m6A‐binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m6A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m6A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract. PMID:26660621

  11. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase*

    PubMed Central

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L.

    2015-01-01

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

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

    PubMed

    Cahn, J K B; Baumschlager, A; Brinkmann-Chen, S; Arnold, F H

    2016-01-01

    NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli. PMID:26512129

  13. DNA Bases Thymine and Adenine in Bio-Organic Light Emitting Diodes

    NASA Astrophysics Data System (ADS)

    Gomez, Eliot F.; Venkatraman, Vishak; Grote, James G.; Steckl, Andrew J.

    2014-11-01

    We report on the use of nucleic acid bases (NBs) in organic light emitting diodes (OLEDs). NBs are small molecules that are the basic building blocks of the larger DNA polymer. NBs readily thermally evaporate and integrate well into the vacuum deposited OLED fabrication. Adenine (A) and thymine (T) were deposited as electron-blocking/hole-transport layers (EBL/HTL) that resulted in increases in performance over the reference OLED containing the standard EBL material NPB. A-based OLEDs reached a peak current efficiency and luminance performance of 48 cd/A and 93,000 cd/m2, respectively, while T-based OLEDs had a maximum of 76 cd/A and 132,000 cd/m2. By comparison, the reference OLED yielded 37 cd/A and 113,000 cd/m2. The enhanced performance of T-based devices is attributed to a combination of energy levels and structured surface morphology that causes more efficient and controlled hole current transport to the emitting layer.

  14. Poly-adenine-based programmable engineering of gold nanoparticles for highly regulated spherical DNAzymes.

    PubMed

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

    2015-11-28

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

  15. Comparison of glycogen and adenine nucleotides as indicators of metabolis stress in mummichogs

    SciTech Connect

    Vetter, R.D.; Hwang, H.M.; Hodson, R.E.

    1986-01-01

    Adenine nucleotide and glycogen concentrations were measured concurrently in white muscle of mummichogs Fundulus heteroclitus after the fish were exposed to stressors that either caused an increase in energy use (metabolic loading) or damaged metabolic function (toxic inhibition). When fish were exposed 4 h to 1% unbleached kraft mill effluent in the presence of 6 mg/L dissolved oxygen, glycogen and AMP concentrations significantly decreased below control values, whereas ATP, ADP, and total adenylate (TA) concentrations as well as the adenylate energy charge (AEC = (ATP + 1/2ADP)/TA) were unchanged. When dissolved oxygen was below 1 mg/L, the effluent caused significant decreases in glycogen, ATP, and TA, but not in ADP, AMP, or the AEC. The combined effect of effluent and hypoxia caused more significant drops in ATP or TA pool. When fish were exposed to 60..mu..g/L DDT for 4 h, none of the measured energy variables changed even though this concentration was lethal after several days. At a concentration of 100 ..mu..g/L DDT, all variables except ADP decreased significantly from control values, which may have reflected energy depletion of the muscle in response to nerve spasms rather than a direct toxic effect on the muscle itself.

  16. Enzyme activities and adenine nucleotide content in aorta, heart muscle and skeletal muscle from uraemic rats.

    PubMed Central

    Krog, M.; Ejerblad, S.; Agren, A.

    1986-01-01

    A prominent feature of arterial and myocardial lesions in uraemia is necrosis of the smooth muscle cells. In this study the possibility of detecting metabolic disturbances before necroses appear was investigated. The investigation was made on rats with moderate uraemia (mean serum creatinine 165 mumol/l) of 12 weeks duration. Enzyme activities and concentrations of adenine nucleotides were measured in aorta, heart and skeletal muscles. Histological examination disclosed no changes in these organs. Hexokinase, an important glycolytic enzyme, showed decreased activity in the skeletal muscle and aorta, whereas the hexosemonophosphate shunt enzyme glucose-6-phosphate dehydrogenase remained unchanged. The aspartate aminotransferase was increased in the skeletal muscle. Fat metabolism was not disturbed as reflected by unchanged activity of hydroxyacyl-CoA-dehydrogenase. Adenylatekinase which is important for the energy supply showed markedly increased activities in all tissues examined from the uraemic rats. Decreased ATP levels were found in the heart muscle and the aorta of the uraemic animals, whereas the total pool of adenosine phosphates remained unchanged in all tissues. The animal model described offers a useful means of detecting early changes in uraemia and should be useful for studying the effects of different treatments of uraemic complications. PMID:3718844

  17. DNA Adenine Methylase Mutants of Salmonella Typhimurium and a Novel Dam-Regulated Locus

    PubMed Central

    Torreblanca, J.; Casadesus, J.

    1996-01-01

    Mutants of Salmonella typhimurium lacking DNA adenine methylase were isolated; they include insertion and deletion alleles. The dam locus maps at 75 min between cysG and aroB, similar to the Escherichia coli dam gene. Dam(-) mutants of S. typhimurium resemble those of E. coli in the following phenotypes: (1) increased spontaneous mutations, (2) moderate SOS induction, (3) enhancement of duplication segregation, (4) inviability of dam recA and dam recB mutants, and (5) suppression of the inviability of the dam recA and dam recB combinations by mutations that eliminate mismatch repair. However, differences between S. typhimurium and E. coli dam mutants are also found: (1) S. typhimurium dam mutants do not show increased UV sensitivity, suggesting that methyl-directed mismatch repair does not participate in the repair of UV-induced DNA damage in Salmonella. (2) S. typhimurium dam recJ mutants are viable, suggesting that the Salmonella RecJ function does not participate in the repair of DNA strand breaks formed in the absence of Dam methylation. We also describe a genetic screen for detecting novel genes regulated by Dam methylation and a locus repressed by Dam methylation in the S. typhimurium virulence (or ``cryptic'') plasmid. PMID:8878670

  18. High-mobility Group Box-1 Protein Promotes Granulomatous Nephritis in Adenine-induced nephropathy

    PubMed Central

    Oyama, Yoko; Hashiguchi, Teruto; Taniguchi, Noboru; Tancharoen, Salunya; Uchimura, Tomonori; Biswas, Kamal K.; Kawahara, Ko-ichi; Nitanda, Takao; Umekita, Yoshihisa; Lotz, Martin; Maruyama, Ikuro

    2011-01-01

    Granulomatous nephritis can be triggered by diverse factors and results in kidney failure. However, despite accumulating data about granulomatous inflammation, pathogenetic mechanisms in nephritis remain unclear. The DNA-binding high-mobility group box-1 protein (HMGB1) initiates and propagates inflammation when released by activated macrophages, functions as an “alarm cytokine” signaling tissue damage. In this study, we demonstrated elevated HMGB1 expression in renal granulomas in rats with crystal-induced granulomatous nephritis caused by feeding an adenine-rich diet. HMGB1 levels were also raised in urine and serum, as well as monocyte chemoattractant protein-1 (MCP-1), a mediator of granulomatous inflammation. Injection of HMGB1 worsened renal function and upregulated MCP-1 in rats with crystal-induced granulomatous nephritis. HMGB1 also induced MCP-1 secretion through mitogen-activated protein kinase (MAPK) and phosphoinositide-3-kinase (PI3K) pathways in rat renal tubular epithelial cells in vitro. Hmgb1+/− mice with crystal-induced nephritis displayed reduced MCP-1 expression in the kidneys and in urine and the number of macrophages in the kidneys was significantly decreased. We conclude that HMGB1 is a new mediator involved in crystal-induced nephritis that amplifies granulomatous inflammation in a cycle where MCP-1 attracts activated macrophages, resulting in excessive and sustained HMGB1 release. HMGB1 could be a novel target for inhibiting chronic granulomatous diseases. PMID:20231821

  19. Partial purification of a 6-methyladenine mRNA methyltransferase which modifies internal adenine residues.

    PubMed Central

    Tuck, M T

    1992-01-01

    Two forms of a 6-methyladenine mRNA methyltransferase have been partially purified using a T7 transcript coding for mouse dihydrofolate reductase as an RNA substrate. Both enzyme forms modify internal adenine residues within the RNA substrate. The enzymes were purified 357- and 37-fold respectively from nuclear salt extracts prepared from HeLa cells using DEAE-cellulose and phosphocellulose chromatography. The activity of the first form of the enzyme eluted from DEAE-cellulose (major form) was at least 3-fold greater than that of the second (minor form). H.p.l.c. analysis of the hydrolysed, methylated mRNA substrates demonstrated that both forms of the enzyme produced only 6-methyladenine. The two forms of the enzyme differed in their RNA substrate specificity as well as in the dependence for a 5' cap structure. The 6-methyladenine mRNA methyltransferase activity was found to be elevated in HeLa nuclei as compared with nuclear extracts from rat kidney and brain. Enzymic activity could not be detected in nuclei from either normal rat liver or regenerating rat liver. In the case of the HeLa cell, activity could only be detected in nuclear extracts, with a small amount in the ribosomal fraction. Other HeLa subcellular fractions were void of activity. PMID:1445268

  20. DNA Bases Thymine and Adenine in Bio-Organic Light Emitting Diodes

    PubMed Central

    Gomez, Eliot F.; Venkatraman, Vishak; Grote, James G.; Steckl, Andrew J.

    2014-01-01

    We report on the use of nucleic acid bases (NBs) in organic light emitting diodes (OLEDs). NBs are small molecules that are the basic building blocks of the larger DNA polymer. NBs readily thermally evaporate and integrate well into the vacuum deposited OLED fabrication. Adenine (A) and thymine (T) were deposited as electron-blocking/hole-transport layers (EBL/HTL) that resulted in increases in performance over the reference OLED containing the standard EBL material NPB. A-based OLEDs reached a peak current efficiency and luminance performance of 48 cd/A and 93,000 cd/m2, respectively, while T-based OLEDs had a maximum of 76 cd/A and 132,000 cd/m2. By comparison, the reference OLED yielded 37 cd/A and 113,000 cd/m2. The enhanced performance of T-based devices is attributed to a combination of energy levels and structured surface morphology that causes more efficient and controlled hole current transport to the emitting layer. PMID:25417819

  1. Structural Basis for Avoidance of Promutagenic DNA Repair by MutY Adenine DNA Glycosylase.

    PubMed

    Wang, Lan; Lee, Seung-Joo; Verdine, Gregory L

    2015-07-10

    The highly mutagenic A:oxoG (8-oxoguanine) base pair in DNA most frequently arises by aberrant replication of the primary oxidative lesion C:oxoG. This lesion is particularly insidious because neither of its constituent nucleobases faithfully transmit genetic information from the original C:G base pair. Repair of A:oxoG is initiated by adenine DNA glycosylase, which catalyzes hydrolytic cleavage of the aberrant A nucleobase from the DNA backbone. These enzymes, MutY in bacteria and MUTYH in humans, scrupulously avoid processing of C:oxoG because cleavage of the C residue in C:oxoG would actually promote mutagenic conversion to A:oxoG. Here we analyze the structural basis for rejection of C:oxoG by MutY, using a synthetic crystallography approach to capture the enzyme in the process of inspecting the C:oxoG anti-substrate, with which it ordinarily binds only fleetingly. We find that MutY uses two distinct strategies to avoid presentation of C to the enzyme active site. Firstly, MutY possesses an exo-site that serves as a decoy for C, and secondly, repulsive forces with a key active site residue prevent stable insertion of C into the nucleobase recognition pocket within the enzyme active site. PMID:25995449

  2. Wolbachia Prophage DNA Adenine Methyltransferase Genes in Different Drosophila-Wolbachia Associations

    PubMed Central

    Saridaki, Aggeliki; Sapountzis, Panagiotis; Harris, Harriet L.; Batista, Philip D.; Biliske, Jennifer A.; Pavlikaki, Harris; Oehler, Stefan; Savakis, Charalambos; Braig, Henk R.; Bourtzis, Kostas

    2011-01-01

    Wolbachia is an obligatory intracellular bacterium which often manipulates the reproduction of its insect and isopod hosts. In contrast, Wolbachia is an essential symbiont in filarial nematodes. Lately, Wolbachia has been implicated in genomic imprinting of host DNA through cytosine methylation. The importance of DNA methylation in cell fate and biology calls for in depth studing of putative methylation-related genes. We present a molecular and phylogenetic analysis of a putative DNA adenine methyltransferase encoded by a prophage in the Wolbachia genome. Two slightly different copies of the gene, met1 and met2, exhibit a different distribution over various Wolbachia strains. The met2 gene is present in the majority of strains, in wAu, however, it contains a frameshift caused by a 2 bp deletion. Phylogenetic analysis of the met2 DNA sequences suggests a long association of the gene with the Wolbachia host strains. In addition, our analysis provides evidence for previously unnoticed multiple infections, the detection of which is critical for the molecular elucidation of modification and/or rescue mechanism of cytoplasmic incompatibility. PMID:21573076

  3. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs.

    PubMed

    Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga; Polonskaia, Anna; Malinina, Lucy; Phan, Anh Tuân; Hobartner, Claudia; Micura, Ronald; Breaker, Ronald R; Patel, Dinshaw J

    2004-12-01

    Metabolite-sensing mRNAs, or "riboswitches," specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing "aptamer" modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytosine, respectively. PMID:15610857

  4. Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine-Sensing mRNAs

    PubMed Central

    Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga; Polonskaia, Anna; Malinina, Lucy; Phan, Anh Tuân; Hobartner, Claudia; Micura, Ronald; Breaker, Ronald R.; Patel, Dinshaw J.

    2015-01-01

    Summary Metabolite-sensing mRNAs, or “riboswitches,” specifically interact with small ligands and direct expression of the genes involved in their metabolism. Ribo-switches contain sensing “aptamer” modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uri-dine and cytosine, respectively. PMID:15610857

  5. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.

    NASA Technical Reports Server (NTRS)

    Yang, C. C.; Oro, J.

    1971-01-01

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

  6. Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli.

    PubMed

    Reschke, Stefan; Sigfridsson, Kajsa G V; Kaufmann, Paul; Leidel, Nils; Horn, Sebastian; Gast, Klaus; Schulzke, Carola; Haumann, Michael; Leimkühler, Silke

    2013-10-11

    The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

  7. Sterols of the fungi - Distribution and biosynthesis

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1973-01-01

    The importance of sterols in the growth and reproduction in fungi is becoming increasingly apparent. This article concerns the composition and biosynthesis of ergosterol in these organisms. Comparison to plant and animal sterol formation are made.

  8. Sterols of the fungi - Distribution and biosynthesis.

    NASA Technical Reports Server (NTRS)

    Weete, J. D.

    1973-01-01

    The importance of sterols in the growth and reproduction in fungi is becoming increasingly apparent. This article concerns the composition and biosynthesis of ergosterol in these organisms. Comparison to plant and animal sterol formation are made.

  9. Identification of the major lesion from the reaction of an acridine-targeted aniline mustard with DNA as an adenine N1 adduct.

    PubMed

    Boritzki, T J; Palmer, B D; Coddington, J M; Denny, W A

    1994-01-01

    DNA adducts of two acridine-linked aniline half-mustards have been isolated and identified. The compound where the half-mustard is attached to the DNA-targeting acridine moiety by a short linker chain alkylates both double- and single-stranded DNA exclusively at guanine N7, as do the majority of known aromatic and aliphatic nitrogen mustards. The longer-chain analogue, also containing a more reactive half-mustard, shows a strikingly different pattern, alkylating double-stranded DNA to yield primarily (> 90%) the adenine N1 adduct, together with < 10% of the adenine N3 adduct and only trace amounts of the guanine N7 adduct. In the presence of MgCl2 (which is known not to inhibit the interaction of drugs at minor groove sites), the adenine N3 adduct is the major product. The latter compound is the first known aniline mustard (and apparently the first known alkylating agent of any type) to preferentially alkylate adenine at the N1 position in duplex DNA. These results are consistent with previous work [Prakash et al. (1990) Biochemistry 29, 9799-9807], which showed that the preferred site of DNA alkylation by the corresponding long-chain acridine-linked aniline bis-mustards in general was at major groove sites of adenines and identifies the major site of alkylation as adenine N1 and not N7. This selectivity for adenine N1 alkylation is suggested to result from a preference for the acridine mustard side chain of these compounds to project into the major groove following intercalation of the acridine, coupled with structural distortion of the DNA helix to make the N1 positions of adenines adjacent to the intercalation sites more accessible.

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

    PubMed

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

    2016-03-15

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

  11. Carotenoid Biosynthesis in Daucus carota.

    PubMed

    Simpson, Kevin; Cerda, Ariel; Stange, Claudia

    2016-01-01

    Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota. PMID:27485223

  12. Carotenoid Biosynthesis in Daucus carota.

    PubMed

    Simpson, Kevin; Cerda, Ariel; Stange, Claudia

    2016-01-01

    Carrot (Daucus carota) is one of the most important vegetable cultivated worldwide and the main source of dietary provitamin A. Contrary to other plants, almost all carrot varieties accumulate massive amounts of carotenoids in the root, resulting in a wide variety of colors, including those with purple, yellow, white, red and orange roots. During the first weeks of development the root, grown in darkness, is thin and pale and devoid of carotenoids. At the second month, the thickening of the root and the accumulation of carotenoids begins, and it reaches its highest level at 3 months of development. This normal root thickening and carotenoid accumulation can be completely altered when roots are grown in light, in which chromoplasts differentiation is redirected to chloroplasts development in accordance with an altered carotenoid profile. Here we discuss the current evidence on the biosynthesis of carotenoid in carrot roots in response to environmental cues that has contributed to our understanding of the mechanism that regulates the accumulation of carotenoids, as well as the carotenogenic gene expression and root development in D. carota.

  13. Biosynthesis of trichothecenes and apotrichothecenes.

    PubMed

    Zamir, L O; Nikolakakis, A; Sauriol, F; Mamer, O

    1999-05-01

    Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described.

  14. Salicylic Acid Biosynthesis and Metabolism

    PubMed Central

    Dempsey, D'Maris Amick; Vlot, A. Corina; Wildermuth, Mary C.; Klessig, Daniel F.

    2011-01-01

    Salicylic acid (SA) has been shown to regulate various aspects of growth and development; it also serves as a critical signal for activating disease resistance in Arabidopsis thaliana and other plant species. This review surveys the mechanisms involved in the biosynthesis and metabolism of this critical plant hormone. While a complete biosynthetic route has yet to be established, stressed Arabidopsis appear to synthesize SA primarily via an isochorismate-utilizing pathway in the chloroplast. A distinct pathway utilizing phenylalanine as the substrate also may contribute to SA accumulation, although to a much lesser extent. Once synthesized, free SA levels can be regulated by a variety of chemical modifications. Many of these modifications inactivate SA; however, some confer novel properties that may aid in long distance SA transport or the activation of stress responses complementary to those induced by free SA. In addition, a number of factors that directly or indirectly regulate the expression of SA biosynthetic genes or that influence the rate of SA catabolism have been identified. An integrated model, encompassing current knowledge of SA metabolism in Arabidopsis, as well as the influence other plant hormones exert on SA metabolism, is presented. PMID:22303280

  15. Biosynthesis of trichothecenes and apotrichothecenes.

    PubMed

    Zamir, L O; Nikolakakis, A; Sauriol, F; Mamer, O

    1999-05-01

    Fusarium culmorum produces two major trichothecenes, 3-acetyldeoxynivalenol and sambucinol, and some minor apotrichothecenes. It was desired to investigate if during their biosynthesis a C-11-keto intermediate was involved. To verify this postulate, trichodiene, a known precursor to trichothecenes, was synthesized with two deuteriums at C-11 and one at C-15. It was then fed to F. culmorum cultures, and the derived metabolites were purified and analyzed. The results ruled out the involvement of an 11-keto intermediate but revealed two novel apotrichothecenes. The characterization of their structures suggested that one of the 2-hydroxy-11alpha-apotrichothecene stereoisomers (2alpha or 2beta) could be converted to sambucinol. These apotrichothecenes were therefore synthesized labeled specifically with two deuteriums at C-4 and C-15 and fed to F. culmorum cultures. Indeed, the result established for the first time that 2alpha-hydroxy-11alpha-apotrichothecene was a precursor to sambucinol. A biosynthetic scheme for the production of trichothecenes and apotrichothecenes is described. PMID:10552458

  16. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue.

    PubMed

    Stanley, Robert J; Hou, Zhanjia; Yang, Aiping; Hawkins, Mary E

    2005-03-01

    6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes.

  17. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue.

    PubMed

    Stanley, Robert J; Hou, Zhanjia; Yang, Aiping; Hawkins, Mary E

    2005-03-01

    6MAP is a fluorescent analogue of adenine that undergoes Watson-Crick base pairing and base stacking in double-stranded DNA. The one-photon absorption spectrum of 6MAP is characterized by a maximum around 330 nm with moderate quantum yield fluorescence centered at about 420 nm. To take advantage of this probe for confocal and single-molecule microscopy, it would be advantageous to be able to excite the analogue via two photons. We report the first determination of the two-photon excitation cross section and spectrum for 6MAP from 614 to 700 nm. The power dependence of the fluorescence indicates that emission results from the absorption of two photons. The one-photon and two-photon emission line shapes are identical within experimental error. A study of the concentration dependence of the fluorescence yield for one-photon excitation shows no measurable quenching up to about 5 microM. The maximum in the two-photon excitation spectrum gives a two-photon cross section, delta(TPE), of 3.4 +/- 0.1 Goeppert-Mayer (G.M.) at 659 nm, which correlates well with the one-photon absorption maximum. This compares quite favorably with cross sections of various naturally fluorescent biological molecules such as flavins and nicotiamide. In addition, we have also obtained the two-photon-induced fluorescence emission spectrum of quinine sulfate. It is approximately the same as that for one-photon excitation, suggesting that two-photon excitation of quinine sulfate may be used for calibration purposes. PMID:16851408

  18. Inhibition of the adenine nucleotide translocator by N-acetyl perfluorooctane sulfonamides in vitro

    SciTech Connect

    O'Brien, Timothy M. Oliveira, Paulo J.; Wallace, Kendall B.

    2008-03-01

    N-alkyl perfluorooctane sulfonamides have been widely used as surfactants on fabrics and papers, fire retardants, and anti-corrosion agents, among many other commercial applications. The global distribution and environmental persistence of these compounds has generated considerable interest regarding potential toxic effects. We have previously reported that perfluorooctanesulfonamidoacetate (FOSAA) and N-ethylperfluorooctanesulfonamidoacetate (N-EtFOSAA) induce the mitochondrial permeability transition (MPT) in vitro. In this study we tested the hypothesis that FOSAA and N-EtFOSAA interact with the adenine nucleotide translocator (ANT) resulting in a functional inhibition of the translocator and induction of the MPT. Respiration and membrane potential of freshly isolated liver mitochondria from Sprague-Dawley rats were measured using an oxygen electrode and a tetraphenylphosphonium-selective (TPP{sup +}) electrode, respectively. Mitochondrial swelling was measured spectrophotometrically. The ANT ligands bongkregkic acid (BKA) and carboxyatractyloside (cATR) inhibited uncoupling of mitochondrial respiration caused by 10 {mu}M N-EtFOSAA, 40 {mu}M FOSAA, and the positive control 8 {mu}M oleic acid. ADP-stimulated respiration and depolarization of mitochondrial membrane potential were inhibited by cATR, FOSAA, N-EtFOSAA, and oleic acid, but not by FCCP. BKA inhibited calcium-dependent mitochondrial swelling induced by FOSAA, N-EtFOSAA, and oleic acid. Seventy-five micromolar ADP also inhibited swelling induced by the test compounds, but cATR induced swelling was not inhibited by ADP. Results of this investigation indicate that N-acetyl perfluorooctane sulfonamides interact directly with the ANT to inhibit ADP translocation and induce the MPT, one or both of which may account for the metabolic dysfunction observed in vivo.

  19. Sodium thiosulfate protects brain in rat model of adenine induced vascular calcification.

    PubMed

    Subhash, N; Sriram, R; Kurian, Gino A

    2015-11-01

    Vascular bed calcification is a common feature of ends stage renal disease that may lead to a complication in cardiovascular and cerebrovascular beds, which is a promoting cause of myocardial infarction, stroke, dementia and aneurysms. Sodium thiosulfate (STS) due to its multiple properties such as antioxidant and calcium chelation has been reported to prevent vascular calcification in uremic rats, without mentioning its impact on cerebral function. Moreover, the previous studies have not explored the effect of STS on the mitochondrial dysfunction, one of the main pathophysiological features associated with the disease and the main site for STS metabolism. The present study addresses this limitation by using a rat model where 0.75% adenine was administered to induce vascular calcification and 400 mg/kg b wt. of STS was given as preventive and curative agent. The blood and urine chemistries along with histopathology of aorta confirms the renal protective effect of STS in two modes of administration. The brain oxidative stress assessment was made through TBARS level, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, found to be in the near normal level. STS administration not only reduced the mitochondrial oxidative stress (measured by TBARS, SOD, GPx and CAT) but also preserved the mitochondrial respiratory enzyme activities (NADH dehydrogenase, Succinate dehydrogenase and Malate dehydrogenase) and its physiology (measured by P/O ratio and RCR). In fact, the protective effect of STS was prominent, when it was administered as a curative agent, where low H2S and high thiosulfate level was observed along with low cystathionine β synthase activity, confirms thiosulfate mediated renal protection. In conclusion, STS when given after induction of calcification is protective to the brain by preserving its mitochondria, compared to the treatment given concomitantly. PMID:26363090

  20. Mitochondrial permeability transition as induced by cross-linking of the adenine nucleotide translocase.

    PubMed

    Zazueta, C; Reyes-Vivas, H; Zafra, G; Sánchez, C A; Vera, G; Chávez, E

    1998-04-01

    Mitochondrial permeability transition is caused by the opening of a transmembrane pore whose chemical nature has not been well established yet. The present work was aimed to further contribute to the knowledge of the membrane entity comprised in the formation of the non-specific channel. The increased permeability was established by analyzing the inability of rat kidney mitochondria to take up and accumulate Ca2+, as well as their failure to build up a transmembrane potential, after the cross-linking of membrane proteins by copper plus ortho-phenanthroline. To identify the cross-linked proteins, polyacrylamide gel electrophoresis was performed. The results are representative of at least three separate experiments. It is indicated that 30 microM Cu2+ induced the release of 4.3 nmol Ca2+ per mg protein. However, in the presence of 100 microM ortho-phenanthroline only 2 microM Cu2+ was required to attain the total release of the accumulated Ca2+; it should be noted that such a reaction is not inhibited by cyclosporin. The increased permeability corresponds to cross-linking of membrane proteins in which approximately 4 nmol thiol groups per mg protein appear to be involved. Such a linking process is inhibited by carboxyatractyloside. By using the fluorescent probe eosin-5-maleimide the label was found in a cross-linking 60 kDa dimer of two 30 kDa monomers. From the data presented it is concluded that copper-o-phenanthroline induces the intermolecular cross-linking of the adenine nucleotide translocase which in turn is converted to non-specific pore. PMID:9675885

  1. Sodium thiosulfate protects brain in rat model of adenine induced vascular calcification.

    PubMed

    Subhash, N; Sriram, R; Kurian, Gino A

    2015-11-01

    Vascular bed calcification is a common feature of ends stage renal disease that may lead to a complication in cardiovascular and cerebrovascular beds, which is a promoting cause of myocardial infarction, stroke, dementia and aneurysms. Sodium thiosulfate (STS) due to its multiple properties such as antioxidant and calcium chelation has been reported to prevent vascular calcification in uremic rats, without mentioning its impact on cerebral function. Moreover, the previous studies have not explored the effect of STS on the mitochondrial dysfunction, one of the main pathophysiological features associated with the disease and the main site for STS metabolism. The present study addresses this limitation by using a rat model where 0.75% adenine was administered to induce vascular calcification and 400 mg/kg b wt. of STS was given as preventive and curative agent. The blood and urine chemistries along with histopathology of aorta confirms the renal protective effect of STS in two modes of administration. The brain oxidative stress assessment was made through TBARS level, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities, found to be in the near normal level. STS administration not only reduced the mitochondrial oxidative stress (measured by TBARS, SOD, GPx and CAT) but also preserved the mitochondrial respiratory enzyme activities (NADH dehydrogenase, Succinate dehydrogenase and Malate dehydrogenase) and its physiology (measured by P/O ratio and RCR). In fact, the protective effect of STS was prominent, when it was administered as a curative agent, where low H2S and high thiosulfate level was observed along with low cystathionine β synthase activity, confirms thiosulfate mediated renal protection. In conclusion, STS when given after induction of calcification is protective to the brain by preserving its mitochondria, compared to the treatment given concomitantly.

  2. Adenine Nucleotide Translocase 4 Is Expressed Within Embryonic Ovaries and Dispensable During Oogenesis

    PubMed Central

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

    2015-01-01

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

  3. Development and Evaluation of Solid Lipid Nanoparticles of N-6-Furfuryl Adenine for Prevention of Photoaging.

    PubMed

    Goindi, Shishu; Guleria, Ankita; Aggarwal, Nidhi

    2015-10-01

    N-6-furfuryl adenine (N6FA) also known as "kinetin" is a biologically active natural phytochemical. It belongs to the category of cytokinins, the natural plant growth hormones that promote cell division and play role in cell differentiation. Overall, N6FA aids in increasing the plant's life span. Human cells also contain.small quantities of N6FA. Scientists are trying to understand its function in humans. N6FA is being investigated for its properties such as antiplatelet, antioxidant, antiproliferative and anti-aging effects on human cells. The aim of the present investigation was to prepare solid lipid nanoparticle (SLN) based topical formulations of N6FA and to evaluate its efficacy against ultraviolet (UV) radiation induced skin photodamage. SLNs were prepared by hot microemulsion technique and optimized for the type and concentration of lipid and surfactant(s). The optimized SLN formulation was characterized in terms of particle size, drug entrapment efficiency, zeta potential and pH; evaluated for stability, spreadability, ex-vivo skin permeation and photoprotective effects against UV induced skin damage. The cumulative amount of drug permeated through mice skin using SLNs was 3 folds higher than from conventional cream base. The results of biochemical and histopathological investigations of skin treated with N6FA loaded SLNs clearly demonstrated the efficacy of optimized formulation in preventing photodamage (lesions, ulcers and changes in skin integrity) due to chronic UV exposure. The effects were comparable with widely used marketed formulation, Garnier wrinkle lift anti-aging cream. Results suggested that N6FA incorporated into SLNs may provide therapeutic as well as cosmeceutical benefits. PMID:26502637

  4. Synthesis and in vivo evaluation of prodrugs of 9-[2-(phosphonomethoxy)ethoxy]adenine.

    PubMed

    Serafinowska, H T; Ashton, R J; Bailey, S; Harnden, M R; Jackson, S M; Sutton, D

    1995-04-14

    A number of esters and amides of the anti-HIV nucleotide analogue 9-[2-(phosphonomethoxy)-ethoxy]adenine (1) have been synthesized as potential prodrugs and evaluated for oral bioavailability in mice. Dialkyl esters 17-20 were prepared via a Mitsunobu coupling of alcohols 8-11 with 9-hydroxypurine 12 whereas (acyloxy)alkyl esters 25-33 and bis-[(alkoxycarbonyl)methyl] and bis(amidomethyl) esters 34-39 were obtained by reaction of 1 with a suitable alkylating agent. Phosphonodichloridate chemistry was employed for the preparation of dialkyl and diaryl esters 42-65, and bis(phosphonoamidates) 66 and 67. Following oral administration to mice, most of the dialkyl esters 17-20 were well-absorbed and then converted to the corresponding monoesters, but minimal further metabolism to 1 occurred. Bis[(pivaloyloxy)methyl] ester 25 displayed an oral bioavailability of 30% that was 15-fold higher than the bioavailability observed after dosing of 1. Methyl substitution at the alpha carbon of the bis[(pivaloyloxy)methyl] ester 25 (33) increased the oral bioavailability of 1 to 74%. Some of the diaryl esters also showed improved absorption properties in comparison with that of 1. In particular, the crystalline hydrochloride salt of diphenyl ester 55 was well-absorbed and efficiently converted to the parent compound with an oral bioavailability of 50%. On the basis of these results as well as the physicochemical properties of the prodrugs and their stability in mouse duodenal contents, the hydrochloride salt of diphenyl ester 55 was identified as the preferred prodrug of 1. PMID:7731022

  5. Modulation by adenine nucleotides of epileptiform activity in the CA3 region of rat hippocampal slices

    PubMed Central

    Ross, F M; Brodie, M J; Stone, T W

    1998-01-01

    Hippocampal slices (450 μm) generate epileptiform bursts of an interictal nature when perfused with a zero magnesium medium containing 4-aminopyridine (50 μM). The effect of adenine nucleotides on this activity was investigated.ATP and adenosine depressed this epileptiform activity in a concentration-dependent manner, with both purines being equipotent at concentrations above 10 μM.Adenosine deaminase 0.2 u ml−1, a concentration that annuls the effect of adenosine (50 μM), did not significantly alter the depression of activity caused by ATP (50 μM).8-Cyclopentyl-1, 3-dimethylxanthine (CPT), an A1 receptor antagonist, enhanced the discharge rate significantly and inhibited the depressant effect of both ATP and adenosine such that the net effect of ATP or adenosine plus CPT was excitatory.Several ATP analogues were also tested: α, β-methyleneATP (α, β-meATP), 2-methylthioATP (2-meSATP) and uridine triphosphate (UTP). Only α, β-meATP (10 μM) produced an increase in the frequency of spontaneous activity which suggests a lack of involvement of P2Y or P2U receptors.Suramin and pyridoxalphosphate-6-azophenyl-2′, 4′-disulphonic acid (PPADS), P2 receptor antagonists, failed to inhibit the depression produced by ATP (50 μM). The excitatory effect of α, β-meATP (10 μM) was inhibited by suramin (50 μM) and PPADS (5 μM).ATP therefore depresses epileptiform activity in this model in a manner which is not consistent with the activation of known P1 or P2 receptors, suggesting the involvement of a xanthine-sensitive nucleotide receptor. The results are also indicative of an excitatory P2X receptor existing in the hippocampal CA3 region. PMID:9484856

  6. The adenine nucleotide translocase type 1 (ANT1): a new factor in mitochondrial disease.

    PubMed

    Sharer, J Daniel

    2005-09-01

    Mitochondrial disorders of oxidative phosphorylation (OXPHOS) comprise a growing list of potentially lethal diseases caused by mutations in either mitochondrial (mtDNA) or nuclear DNA (nDNA). Two such conditions, autosomal dominant progressive external ophthalmoplegia (adPEO) and Senger's Syndrome, are associated with dysfunction of the heart and muscle-specific isoform of the adenine nucleotide translocase (ANT1), a nDNA gene product that facilitates transport of ATP and ADP across the inner mitochondrial membrane. AdPEO is a mtDNA deletion disorder broadly characterized by pathology involving the eyes, skeletal muscle, and central nervous system. In addition to ANT1, mutations in at least two other nuclear genes, twinkle and POLG, have been shown to cause mtDNA destabilization associated with adPEO. Senger's syndrome is an autosomal recessive condition characterized by congenital heart defects, abnormalities of skeletal muscle mitochondria, cataracts, and elevated circulatory levels of lactic acid. This syndrome is associated with severe depletion of ANT1, which may be the result of an as yet unidentified ANT1-specific transcriptional or translational processing error. ANT1 has also been associated with a third condition, autosomal dominant facioscapulohumeral muscular dystrophy (FSHD), an adult onset disorder characterized by variable muscle weakness in the face, feet, shoulders, and hips. FSHD patients possess specific DNA deletions on chromosome 4, which appear to cause derepression of several nearby genes, including ANT1. Early development of FSHD may involve mitochondrial dysfunction and increased oxidative stress, possibly associated with overexpression of ANT1. PMID:16203679

  7. An alternative membrane transport pathway for phosphate and adenine nucleotides in mitochondria and its possible function.

    PubMed

    Reynafarje, B; Lehninger, A L

    1978-10-01

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

  8. Inhibition of RNA synthesis in vitro by 9-aminoacridine carboxamide antitumor agents. Effects on overall RNA synthesis and synthesis of the initiating dinucleotide.

    PubMed

    Piestrzeniewicz, M K; Czyz, M; Denny, W A; Gniazdowski, M

    1990-01-01

    A series of 9-aminoacridine carboxamide derivatives of systematically varied structure was assayed in an RNA synthesis in vitro system. Escherichia coli DNA-dependent RNA polymerase and DNA derived from phage T7 or calf thymus were used to measure the effect of the drugs on overall RNA and the initiating dinucleotide (pppApU) syntheses. By means of multiple linear regression analysis it was shown that the inhibition of these reactions depends both on the drug equilibrium binding constant and kinetic parameters of dissociation of drug-DNA complexes. PMID:1705740

  9. Veratrole biosynthesis in white campion.

    PubMed

    Akhtar, Tariq A; Pichersky, Eran

    2013-05-01

    White campion (Silene latifolia) is a dioecious plant that emits 1,2-dimethoxybenzene (veratrole), a potent pollinator attractant to the nocturnal moth Hadena bicruris. Little is known about veratrole biosynthesis, although methylation of 2-methoxyphenol (guaiacol), another volatile emitted from white campion flowers, has been proposed. Here, we explore the biosynthetic route to veratrole. Feeding white campion flowers with [(13)C9]l-phenylalanine increased guaiacol and veratrole emission, and a significant portion of these volatile molecules contained the stable isotope. When white campion flowers were treated with the phenylalanine ammonia lyase inhibitor 2-aminoindan-2-phosphonic acid, guaiacol and veratrole levels were reduced by 50% and 63%, respectively. Feeding with benzoic acid (BA) or salicylic acid (SA) increased veratrole emission 2-fold, while [(2)H5]BA and [(2)H6]SA feeding indicated that the benzene ring of both guaiacol and veratrole is derived from BA via SA. We further report guaiacol O-methyltransferase (GOMT) activity in the flowers of white campion. The enzyme was purified to apparent homogeneity, and the peptide sequence matched that encoded by a recently identified complementary DNA (SlGOMT1) from a white campion flower expressed sequence tag database. Screening of a small population of North American white campion plants for floral volatile emission revealed that not all plants emitted veratrole or possessed GOMT activity, and SlGOMT1 expression was only observed in veratrole emitters. Collectively these data suggest that veratrole is derived by the methylation of guaiacol, which itself originates from phenylalanine via BA and SA, and therefore implies a novel branch point of the general phenylpropanoid pathway.

  10. Biosynthesis of gold nanoparticles: A green approach.

    PubMed

    Ahmed, Shakeel; Annu; Ikram, Saiqa; Yudha S, Salprima

    2016-08-01

    Nanotechnology is an immensely developing field due to its extensive range of applications in different areas of technology and science. Different types of methods are employed for synthesis of nanoparticles due to their wide applications. The conventional chemical methods have certain limitations with them either in the form of chemical contaminations during their syntheses procedures or in later applications and use of higher energy. During the last decade research have been focussed on developing simple, clean, non-toxic, cost effective and eco-friendly protocols for synthesis of nanoparticles. In order to get this objective, biosynthesis methods have been developed in order to fill this gap. The biosynthesis of nanoparticles is simple, single step, eco-friendly and a green approach. The biochemical processes in biological agents reduce the dissolved metal ions into nano metals. The various biological agents like plant tissues, fungi, bacteria, etc. are used for biosynthesis for metal nanoparticles. In this review article, we summarised recent literature on biosynthesis of gold nanoparticles which have revolutionised technique of synthesis for their applications in different fields. Due to biocompatibility of gold nanoparticles, it has find its applications in biomedical applications. The protocol and mechanism of biosynthesis of gold nanoparticles along with various applications have also been discussed. PMID:27236049

  11. Biosynthesis of gold nanoparticles: A green approach.

    PubMed

    Ahmed, Shakeel; Annu; Ikram, Saiqa; Yudha S, Salprima

    2016-08-01

    Nanotechnology is an immensely developing field due to its extensive range of applications in different areas of technology and science. Different types of methods are employed for synthesis of nanoparticles due to their wide applications. The conventional chemical methods have certain limitations with them either in the form of chemical contaminations during their syntheses procedures or in later applications and use of higher energy. During the last decade research have been focussed on developing simple, clean, non-toxic, cost effective and eco-friendly protocols for synthesis of nanoparticles. In order to get this objective, biosynthesis methods have been developed in order to fill this gap. The biosynthesis of nanoparticles is simple, single step, eco-friendly and a green approach. The biochemical processes in biological agents reduce the dissolved metal ions into nano metals. The various biological agents like plant tissues, fungi, bacteria, etc. are used for biosynthesis for metal nanoparticles. In this review article, we summarised recent literature on biosynthesis of gold nanoparticles which have revolutionised technique of synthesis for their applications in different fields. Due to biocompatibility of gold nanoparticles, it has find its applications in biomedical applications. The protocol and mechanism of biosynthesis of gold nanoparticles along with various applications have also been discussed.

  12. Light-controlled flavonoid biosynthesis in fruits

    PubMed Central

    Zoratti, Laura; Karppinen, Katja; Luengo Escobar, Ana; Häggman, Hely; Jaakola, Laura

    2014-01-01

    Light is one of the most important environmental factors affecting flavonoid biosynthesis in plants. The absolute dependency of light to the plant development has driven evolvement of sophisticated mechanisms to sense and transduce multiple aspects of the light signal. Light effects can be categorized in photoperiod (duration), intensity (quantity), direction and quality (wavelength) including UV-light. Recently, new information has been achieved on the regulation of light-controlled flavonoid biosynthesis in fruits, in which flavonoids have a major contribution on quality. This review focuses on the effects of the different light conditions on the control of flavonoid biosynthesis in fruit producing plants. An overview of the currently known mechanisms of the light-controlled flavonoid accumulation is provided. R2R3 MYB transcription factors are known to regulate by differential expression the biosynthesis of distinct flavonoids in response to specific light wavelengths. Despite recent advances, many gaps remain to be understood in the mechanisms of the transduction pathway of light-controlled flavonoid biosynthesis. A better knowledge on these regulatory mechanisms is likely to be useful for breeding programs aiming to modify fruit flavonoid pattern. PMID:25346743

  13. Apd1(+), a Gene Required for Red Pigment Formation in Ade6 Mutants of Schizosaccharomyces Pombe, Encodes an Enzyme Required for Glutathione Biosynthesis: A Role for Glutathione and a Glutathione-Conjugate Pump

    PubMed Central

    Chaudhuri, B.; Ingavale, S.; Bachhawat, A. K.

    1997-01-01

    Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a genetic investigation into the nature and cause of pigmentation of ade6 mutants of S. pombe and have discovered that one of these pigmentation defective mutants, apd1 (adenine pigmentation defective), is a strict glutathione auxotroph. The gene apd1(+) was found to encode the first enzyme in glutathione biosynthesis, γ-glutamylcysteine synthetase, gcs1(+). This gene when expressed in the mutant could confer both glutathione prototrophy and the characteristic red pigmentation, and disruption of the gene led to a loss in both phenotypes. Supplementation of glutathione in the medium, however, could only restore growth but not the pigmentation because the cells were unable to achieve sufficient intracellular levels of glutathione. Disruption of the second enzyme in glutathione biosynthesis, glutathione synthetase, gsh2(+), also led to glutathione auxotrophy, but only a partial defect in pigment formation. A reevaluation of the major amino acids previously reported to be present in the pigment indicated that the pigment is probably a glutathione conjugate. The ability of vanadate to inhibit pigment formation indicated that the conjugate was transported into the vacuole through a glutathione-conjugate pump. This was further confirmed using strains of S. cerevisiae bearing disruptions in the recently identified glutathione-conjugate pump, YCF1, where a significant reduction in pigment formation was observed. The pump of S. pombe is distinct from the previously identified vacuolar pump, hmt1p, for transporting cadystin peptides into vacuoles of S. pombe. PMID:9017391

  14. Alkaline Phosphatase, Soluble Extracellular Adenine Nucleotides, and Adenosine Production after Infant Cardiopulmonary Bypass

    PubMed Central

    Davidson, Jesse A.; Urban, Tracy; Tong, Suhong; Twite, Mark; Woodruff, Alan

    2016-01-01

    Rationale Decreased alkaline phosphatase activity after infant cardiac surgery is associated with increased post-operative cardiovascular support requirements. In adults undergoing coronary artery bypass grafting, alkaline phosphatase infusion may reduce inflammation. Mechanisms underlying these effects have not been explored but may include decreased conversion of extracellular adenine nucleotides to adenosine. Objectives 1) Evaluate the association between alkaline phosphatase activity and serum conversion of adenosine monophosphate to adenosine after infant cardiac surgery; 2) assess if inhibition/supplementation of serum alkaline phosphatase modulates this conversion. Methods and Research Pre/post-bypass serum samples were obtained from 75 infants <4 months of age. Serum conversion of 13C5-adenosine monophosphate to 13C5-adenosine was assessed with/without selective inhibition of alkaline phosphatase and CD73. Low and high concentration 13C5-adenosine monophosphate (simulating normal/stress concentrations) were used. Effects of alkaline phosphatase supplementation on adenosine monophosphate clearance were also assessed. Changes in serum alkaline phosphatase activity were strongly correlated with changes in 13C5-adenosine production with or without CD73 inhibition (r = 0.83; p<0.0001). Serum with low alkaline phosphatase activity (≤80 U/L) generated significantly less 13C5-adenosine, particularly in the presence of high concentration 13C5-adenosine monophosphate (10.4μmol/L vs 12.9μmol/L; p = 0.0004). Inhibition of alkaline phosphatase led to a marked decrease in 13C5-adenosine production (11.9μmol/L vs 2.7μmol/L; p<0.0001). Supplementation with physiologic dose human tissue non-specific alkaline phosphatase or high dose bovine intestinal alkaline phosphatase doubled 13C5-adenosine monophosphate conversion to 13C5-adenosine (p<0.0001). Conclusions Alkaline phosphatase represents the primary serum ectonucleotidase after infant cardiac surgery and low post

  15. Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate.

    PubMed

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

    2016-05-23

    The decay channels of singlet excited adenine uracil monophosphate (ApU) in water are studied with CASPT2//CASSCF:MM potential energy calculations and simulation of the 2D-UV spectroscopic fingerprints with the aim of elucidating the role of the different electronic states of the stacked conformer in the excited state dynamics. The adenine (1) La state can decay without a barrier to a conical intersection with the ground state. In contrast, the adenine (1) Lb and uracil S(U) states have minima that are separated from the intersections by sizeable barriers. Depending on the backbone conformation, the CT state can undergo inter-base hydrogen transfer and decay to the ground state through a conical intersection, or it can yield a long-lived minimum stabilized by a hydrogen bond between the two ribose rings. This suggests that the (1) Lb , S(U) and CT states of the stacked conformer may all contribute to the experimental lifetimes of 18 and 240 ps. We have also simulated the time evolution of the 2D-UV spectra and provide the specific fingerprint of each species in a recommended probe window between 25 000 and 38 000 cm(-1) in which decongested, clearly distinguishable spectra can be obtained. This is expected to allow the mechanistic scenarios to be discerned in the near future with the help of the corresponding experiments. Our results reveal the complexity of the photophysics of the relatively small ApU system, and the potential of 2D-UV spectroscopy to disentangle the photophysics of multichromophoric systems. PMID:27113273

  16. High-spin ferric ions in Saccharomyces cerevisiae vacuoles are reduced to the ferrous state during adenine-precursor detoxification.

    PubMed

    Park, Jinkyu; McCormick, Sean P; Cockrell, Allison L; Chakrabarti, Mrinmoy; Lindahl, Paul A

    2014-06-24

    The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) Fe(III) complexes with polyphosphate-related ligands. Some Fe(III) oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS Fe(III). Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 μM Fe(III) citrate) accumulated two major NHHS Fe(II) species as the vacuolar NHHS Fe(III) species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS Fe(II) complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS Fe(III) with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS Fe(III) species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated. PMID:24919141

  17. Multiple Decay Mechanisms and 2D-UV Spectroscopic Fingerprints of Singlet Excited Solvated Adenine-Uracil Monophosphate.

    PubMed

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

    2016-05-23

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

  18. Quantitative Subtractively Normalized Interfacial Fourier Transform Infrared Reflection Spectroscopy Study of the Adsorption of Adenine on Au(111) Electrodes.

    PubMed

    Prieto, Francisco; Su, Zhangfei; Leitch, J Jay; Rueda, Manuela; Lipkowski, Jacek

    2016-04-26

    Quantitative subtractively normalized interfacial Fourier transform infrared reflection spectroscopy (SNIFTIRS) was used to determine the molecular orientation and identify the metal-molecular interactions responsible for the adsorption of adenine from the bulk electrolyte solution onto the surface of the Au(111) electrode. The recorded p-polarized IR spectra of the adsorbed species were subtracted from the collected s-polarized IR spectra to remove the IR contributions of the vibrational bands of the desorbed molecules that are located within the thin layer cavity of the spectroelectrochemical cell. The intense IR band around 1640 cm(-1), which is assigned to the pyrimidine ring stretching vibrations of the C5-C6 and C6-N10 bonds, and the IR band at 1380 cm(-1), which results from a combination of the ring stretching vibration of the C5-C7 bond and the in-plane CH bending vibration, were selected for the quantitative analysis measurements. The transition dipoles of these bands were evaluated by DFT calculations. Their orientations differed by 85 ± 5°. The tilt angles of adsorbed adenine molecules were calculated from the intensity of these two vibrations at different potentials. The results indicate that the molecular plane is tilted at an angle of 40° with respect to the surface normal of the electrode and rotates by 16° around its normal axis with increasing electrode potential. This orientation results from the chemical interaction between the N10 and gold atoms coupled with the π-π parallel stacking interactions between the adjacent adsorbed molecules. Furthermore, the changes in the molecular plane rotation with the electric field suggests that the N1 atom of adenine must also participate in the interaction between the molecule and metal.

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

    PubMed

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

    2014-03-15

    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 < 0.05). Similarly, a high-NaCl diet significantly increased SAPV in the low-frequency range only in ACRF animals. To conclude, a 2-wk period of a high-NaCl diet in ACRF rats significantly impaired dynamic RBFA in the frequency range of the myogenic response and increased SAPV in the low-frequency range. These abnormalities may increase the susceptibility to hypertensive end-organ injury and progressive renal failure by facilitating pressure transmission to the microvasculature.

  20. Modelling mutational and selection pressures on dinucleotides in eukaryotic phyla –selection against CpG and UpA in cytoplasmically expressed RNA and in RNA viruses

    PubMed Central

    2013-01-01

    Background Loss of CpG dinucleotides in genomic DNA through methylation-induced mutation is characteristic of vertebrates and plants. However, these and other eukaryotic phyla show a range of other dinucleotide frequency biases with currently uncharacterized underlying mutational or selection mechanisms. We developed a parameterized Markov process to identify what neighbour context-dependent mutations best accounted for patterns of dinucleotide frequency biases in genomic and cytoplasmically expressed mRNA sequences of different vertebrates, other eukaryotic groups and RNA viruses that infect them. Results Consistently, 11- to 14-fold greater frequencies of the methylation-associated mutation of C to T upstream of G (depicted as C→T,G) than other transitions best modelled dinucleotide frequencies in mammalian genomic DNA. However, further mutations such as G→T,T (5-fold greater than the default transversion rate) were required to account for the full spectrum of dinucleotide frequencies in mammalian sequence datasets. Consistent with modeling predictions for these two mutations, instability of both CpG and CpT dinucleotides was identified through SNP frequency analysis of human DNA sequences. Different sets of context-dependent mutations were modelled in other eukaryotes with non-methylated genomic DNA. In contrast to genomic DNA, best-fit models of dinucleotide frequencies in transcribed RNA sequences expressed in the cytoplasm from all organisms were dominated by mutations that eliminated UpA dinucleotides, observations consistent with cytoplasmically driven selection for mRNA stability. Surprisingly, mRNA sequences from organisms with methylated genomes showed evidence for additional selection against CpG through further context-dependent mutations (eg. C→A,G). Similar mutation or selection processes were identified among single-stranded mammalian RNA viruses; these potentially account for their previously described but unexplained under-representations of

  1. A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3',3'-cGAMP.

    PubMed

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

    2014-06-01

    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.

  2. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  3. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  4. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

  5. An ab initio Study of Decay Mechanism of Adenine: the Facile Path of the Amino NH Bond Cleavage

    NASA Astrophysics Data System (ADS)

    Conti, Irene; Garavelli, Marco; Orlandi, Giorgio

    2007-12-01

    A comprehensive study of the radiationless decay processes of the lowest excited singlet states in the isolated 9H-Adenine has been performed at the CASPT2//CASSCF level. The minimum energy paths of the La, Lb and nπ* singlet states along different skeletal distortions have been computed and the Conical Intersections (CIs) involving these states have been determined. The fast deactivation path of La along a skeletal deformation, which leads to a S0/La CI, as previously discussed, is confirmed. Moreover, low-lying CIs between S0 and πσ* singlet states have been characterized, where σ* is the antibonding orbital localized on a N-H bond of the amino (πσNH2*) or of the azine group (πσN9H*). We have found that the repulsive πσNH2* state associated with an amino N-H bond can be populated through a barrierless way. Therefore, the decay path shows a bifurcation leading to two possible ways of radiationless deactivation: on one hand a non-photochemical decay through the S0/La or S0/nπ* CIs and on the other hand a photochemical process via the possible access to the S0/πσNH2* CI that produces N-H cleavage. In this way, we can explain the H atom loss found upon UV excitation. We have considered also the decay of higher energy bright states. We have found that these states can decay also by converting to the repulsive πσN9H* state associated with the azine NH bond. This new channel suggests an increase of H-atom photoproduction yield by excitating Adenine with lower wavelength radiations. The study of the decay processes of an Adenine molecule in the double strand d(A)10ṡd(T)10 in water solvent is currently underway: Adenine is treated by the Quantum Mechanical (QM) approach and the remaining molecules are described at the Molecular Mechanics (MM) level. We use the COBRAMM program that is a tunable QM/MM approach to complex molecular architectures developed by our research group.

  6. Kinetics and Thermodynamics of the Reaction between the (•)OH Radical and Adenine: A Theoretical Investigation.

    PubMed

    Milhøj, Birgitte O; Sauer, Stephan P A

    2015-06-18

    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 ωB97X-D/6-311++G(2df,2pd) level with Eckart tunneling corrections. First the computational method is validated by considering the hydrogen abstraction from the heterocyclic N9 nitrogen in adenine as a test system. Geometries for all molecules in the reaction are optimized with four different DFT exchange-correlation functionals (B3LYP, BHandHLYP, M06-2X, and ωB97X-D), in combination with Pople and Dunning basis sets, all of which have been employed in similar investigations in the literature. Improved energies are obtained through single point calculations with CCSD(T) and the same basis sets, and reaction rate constants are calculated for all methods both without tunneling corrections and with the Wigner, Bell, and Eckart corrections. In comparison to CCSD(T)//BHandHLYP/aug-cc-pVTZ reference results, the ωB97X-D/6-311++G(2df,2pd) method combined with Eckart tunneling corrections provides a sensible compromise between accuracy and time. Using this method, all subreactions of the reaction between adenine and the (•)OH radical are investigated. The total rate constants for hydrogen abstraction and addition for adenine are predicted with this method to be 1.06 × 10(-12) and 1.10 × 10(-12) cm(3) molecules(-1) s(-1), respectively. Abstractions of H61 and H62 contribute the most, while only addition to the C8 carbon is found to be of any significance, in contrast to previous claims that addition is the dominant reaction pathway. The overall rate constant for the complete reaction is found to be 2.17 × 10(-12) cm(3) molecules(-1) s(-1), which agrees exceptionally well with experimental results.

  7. Regioselective Palmitoylation of 9-(2,3-Dihydroxy- propyl)adenine Catalyzed by a Glycopolymer-enzyme Conjugate.

    PubMed

    Brabcová, Jana; Blažek, Jiří; Krečmerová, Marcela; Vondrášek, Jiří; Palomo, Jose M; Zarevúcka, Marie

    2016-05-16

    The enzymatic regioselective monopalmitoylation of racemic 9-(2,3-dihydroxypropyl)- adenine (DHPA), an approved antiviral agent, has been performed by an immobilized form of Candida antarctica B lipase (CAL-B) using a 4:1 DMF/hexane mixture as the reaction medium. To improve the chemical yield of the desired monopalmitoylation reaction, solid-phase chemical modifications of the lipase were evaluated. The reaction yield was successfully increased obtaining 100% product after a second treatment of the product solution with fresh immobilised chemically glycosylated-CAL-B.

  8. Abscisic acid: biosynthesis, inactivation, homoeostasis and signalling.

    PubMed

    Dong, Ting; Park, Youngmin; Hwang, Inhwan

    2015-01-01

    The phytohormone abscisic acid (ABA) plays crucial roles in numerous physiological processes during plant growth and abiotic stress responses. The endogenous ABA level is controlled by complex regulatory mechanisms involving biosynthesis, catabolism, transport and signal transduction pathways. This complex regulatory network may target multiple levels, including transcription, translation and post-translational regulation of genes involved in ABA responses. Most of the genes involved in ABA biosynthesis, catabolism and transport have been characterized. The local ABA concentration is critical for initiating ABA-mediated signalling during plant development and in response to environmental changes. In this chapter we discuss the mechanisms that regulate ABA biosynthesis, catabolism, transport and homoeostasis. We also present the findings of recent research on ABA perception by cellular receptors, and ABA signalling in response to cellular and environmental conditions.

  9. Bacterial exopolysaccharides: biosynthesis pathways and engineering strategies

    PubMed Central

    Schmid, Jochen; Sieber, Volker; Rehm, Bernd

    2015-01-01

    Bacteria produce a wide range of exopolysaccharides which are synthesized via different biosynthesis pathways. The genes responsible for synthesis are often clustered within the genome of the respective production organism. A better understanding of the fundamental processes involved in exopolysaccharide biosynthesis and the regulation of these processes is critical toward genetic, metabolic and protein-engineering approaches to produce tailor-made polymers. These designer polymers will exhibit superior material properties targeting medical and industrial applications. Exploiting the natural design space for production of a variety of biopolymer will open up a range of new applications. Here, we summarize the key aspects of microbial exopolysaccharide biosynthesis and highlight the latest engineering approaches toward the production of tailor-made variants with the potential to be used as valuable renewable and high-performance products for medical and industrial applications. PMID:26074894

  10. Unconventional membrane lipid biosynthesis in Xanthomonas campestris.

    PubMed

    Aktas, Meriyem; Narberhaus, Franz

    2015-09-01

    All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) is well understood in model bacteria such as Escherichia coli. It recently emerged, however, that the diversity of bacterial membrane lipids is huge and that not yet explored biosynthesis pathways exist, even for the common PLs. A good example is the plant pathogen Xanthomonas campestris pv. campestris. It contains PE, PG and CL as major lipids and small amounts of the N-methylated PE derivatives monomethyl PE and phosphatidylcholine (PC = trimethylated PE). Xanthomonas campestris uses a repertoire of canonical and non-canonical enzymes for the synthesis of its membrane lipids. In this minireview, we briefly recapitulate standard pathways and integrate three recently discovered pathways into the overall picture of bacterial membrane biosynthesis.

  11. The Terpenoid Biosynthesis Toolkit of Trichoderma.

    PubMed

    Bansal, Ravindra; Mukherjee, Prasun Kumar

    2016-04-01

    The widely used biotechnologically important fungi belonging to the genus Trichoderma are rich sources of secondary metabolites. Even though the genomes of several Trichoderma spp. have been published, and data are available on the genes involved in biosynthesis of non-ribosomal peptide synthetases and polyketide synthases, no genome-wide data are available for the terpenoid biosynthesis machinery in these organisms. In the present study, we have identified the genes involved in terpene biosynthesis in the genomes of three Trichoderma spp., viz., T. virens, T. atroviride and T. reesei. While the genes involved in the condensation steps are highly conserved across the three species, these fungi differed in the number and organization of terpene cyclases. T. virens genome harbours eleven terpene cyclases, while T. atroviride harbours seven, and T. reeseisix in their genomes; seven, three and two being part of putative secondary metabolism related gene clusters.

  12. Ethylene Biosynthesis-Inducing Xylanase 1

    PubMed Central

    Dean, Jeffrey F. D.; Gross, Kenneth C.; Anderson, James D.

    1991-01-01

    Induction of ethylene biosynthesis in tobacco (Nicotiana tabacum cv Xanthi) leaf discs by the ethylene biosynthesis-inducing xylanase (EIX) isolated from Cellulysin or xylan-grown cultures of Trichoderma viride was dependent upon the concentration of xylanase applied and upon the length of incubation. Arrhenius activation energies of 9,100 and 10,500 calories for the Cellulysin and T. viride EIX xylanase activities, respectively, were derived from the Km and Vmax values determined for each enzyme at several temperatures. The two xylanases digested xylan in a strictly endo fashion, releasing neither xylobiose nor free xylose, and no debranching activity was associated with either enzyme. The xylanases released polysaccharides from ground corn cobs, but little or no carbohydrate was released from tobacco mesophyll cell walls incubated with EIX. No heat-stable products capable of inducing ethylene biosynthesis in tobacco leaf discs were found in EIX digests of purified xylans. PMID:16668223

  13. Triterpenoid Biosynthesis and Engineering in Plants

    PubMed Central

    Sawai, Satoru; Saito, Kazuki

    2011-01-01

    Triterpenoid saponins are a diverse group of natural products in plants and are considered defensive compounds against pathogenic microbes and herbivores. Because of their various beneficial properties for humans, saponins are used in wide-ranging applications in addition to medicinally. Saponin biosynthesis involves three key enzymes: oxidosqualene cyclases, which construct the basic triterpenoid skeletons; cytochrome P450 monooxygenases, which mediate oxidations; and uridine diphosphate-dependent glycosyltransferases, which catalyze glycosylations. The discovery of genes committed to saponin biosynthesis is important for the stable supply and biotechnological application of these compounds. Here, we review the identified genes involved in triterpenoid biosynthesis, summarize the recent advances in the biotechnological production of useful plant terpenoids, and discuss the bioengineering of plant triterpenoids. PMID:22639586

  14. Biosynthesis and metabolism of salicylic acid

    SciTech Connect

    Lee, H.; Leon, J.; Raskin, I.

    1995-05-09

    Pathways of salicylic acid (SA) biosynthesis and metabolism in tobacco have been recently identified. SA, an endogenous regulator of disease resistance, is a product of phenylpropanoid metabolism formed via decarboxylation of trans-cinnamic acid to benzoic acid and its subsequent 2-hydroxylation to SA. In tobacco mosaic virus-inoculated tobacco leaves, newly synthesized SA is rapidly metabolized to SA O-{beta}-D-glucoside and methyl salicylate. Two key enzymes involved in SA biosynthesis and metabolism: benzoic acid 2-hydroxylase, which converts benzoic acid to SA, and UDPglucose:SA glucosyltransferase (EC 2.4.1.35), which catalyzes conversion of SA to SA glucoside have been partially purified and characterized. Progress in enzymology and molecular biology of SA biosynthesis and metabolism will provide a better understanding of signal transduction pathway involved in plant disease resistance. 62 refs., 1 fig.

  15. The Terpenoid Biosynthesis Toolkit of Trichoderma.

    PubMed

    Bansal, Ravindra; Mukherjee, Prasun Kumar

    2016-04-01

    The widely used biotechnologically important fungi belonging to the genus Trichoderma are rich sources of secondary metabolites. Even though the genomes of several Trichoderma spp. have been published, and data are available on the genes involved in biosynthesis of non-ribosomal peptide synthetases and polyketide synthases, no genome-wide data are available for the terpenoid biosynthesis machinery in these organisms. In the present study, we have identified the genes involved in terpene biosynthesis in the genomes of three Trichoderma spp., viz., T. virens, T. atroviride and T. reesei. While the genes involved in the condensation steps are highly conserved across the three species, these fungi differed in the number and organization of terpene cyclases. T. virens genome harbours eleven terpene cyclases, while T. atroviride harbours seven, and T. reeseisix in their genomes; seven, three and two being part of putative secondary metabolism related gene clusters. PMID:27396184

  16. Triterpenoid biosynthesis in Euphorbia lathyris latex

    SciTech Connect

    Hawkins, D.R.

    1987-11-01

    The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed. The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I/sub 50/ concentration of 3.2 ..mu..M. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I/sub 50/ of 4 ..mu..M. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4-/sup 3/H-mevalonic acid and incubating latex with a mixture of this and /sup 14/C-mevalonic acid. From the /sup 3/H//sup 14/C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs.

  17. The expanding universe of alkaloid biosynthesis.

    PubMed

    De Luca, V; Laflamme, P

    2001-06-01

    Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

  18. Combinatorial Biosynthesis of Polyketides – A Perspective

    PubMed Central

    Wong, Fong T.; Khosla, Chaitan

    2012-01-01

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make “unnatural” natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms. PMID:22342766

  19. Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

    PubMed

    Niu, Guoqing; Tan, Huarong

    2015-02-01

    The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics.

  20. The structural biology of phenazine biosynthesis

    PubMed Central

    Blankenfeldt, Wulf; Parsons, James F.

    2014-01-01

    The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis. PMID:25215885

  1. Biosynthesis and biodegradation of wood components

    SciTech Connect

    Higuchi, T.

    1985-01-01

    A textbook containing 22 chapters by various authors covers the structure of wood, the localization of polysaccharides and lignins in wood cell walls, metabolism and synthetic function of cambial tissue, cell organelles and their function in the biosynthesis of cell wall components, biosynthesis of plant cell wall polysaccharides, lignin, cutin, suberin and associated waxes, phenolic acids and monolignols, quinones, flavonoids, tannins, stilbenes and terpenoid wood extractives, the occurrence of extractives, the metabolism of phenolic acids, wood degradation by micro-organisms and fungi, and biodegradation of cellulose, hemicelluloses, lignin, and aromatic extractives of wood. An index is included.

  2. Combinatorial biosynthesis of polyketides--a perspective.

    PubMed

    Wong, Fong T; Khosla, Chaitan

    2012-04-01

    Since their discovery, polyketide synthases have been attractive targets of biosynthetic engineering to make 'unnatural' natural products. Although combinatorial biosynthesis has made encouraging advances over the past two decades, the field remains in its infancy. In this enzyme-centric perspective, we discuss the scientific and technological challenges that could accelerate the adoption of combinatorial biosynthesis as a method of choice for the preparation of encoded libraries of bioactive small molecules. Borrowing a page from the protein structure prediction community, we propose a periodic challenge program to vet the most promising methods in the field, and to foster the collective development of useful tools and algorithms.

  3. Reactivity of nitrogen atoms in adenine and (Ade)2Cu complexes towards ribose and 2-furanmethanol: Formation of adenosine and kinetin.

    PubMed

    Nashalian, Ossanna; Yaylayan, Varoujan A

    2017-01-15

    To explore the interaction of nucleosides and nucleobases in the context of the Maillard reaction and to identify the selectivity of purine nitrogen atoms towards various electrophiles, model systems composed of adenine or adenosine, glycine, ribose and/or 2-furanmethanol (with and without copper) were studied in aqueous solutions heated at 110°C for 2h and subsequently analyzed by ESI/qTOF/MS/MS in addition to isotope labelling techniques. The results indicated that ribose selectively formed mono-ribosylated N(6) adenine, but in the presence of (Ade)2Cu complex the reaction mixture generated mono-, di- and tri-substituted sugar complexes and their hydrolysis products of mono-ribosylated N(6) and N(9) adenine adducts and di-ribosylated N(6,9) adenine. Furthermore, the reaction of 2-furanmethanol with adenine in the presence of ribose generated kinetin and its isomer, while its reaction with adenosine generated kinetin riboside, as confirmed by comparing the MS/MS profiles of these adducts to those of commercial standards. PMID:27542499

  4. Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

    PubMed Central

    Abbas, Charles A.; Sibirny, Andriy A.

    2011-01-01

    Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

  5. Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

    PubMed

    Abbas, Charles A; Sibirny, Andriy A

    2011-06-01

    Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B₂] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

  6. Inhibition of Abscisic Acid Biosynthesis in Cercospora rosicola by Inhibitors of Gibberellin Biosynthesis and Plant Growth Retardants

    PubMed Central

    Norman, Shirley M.; Poling, Stephen M.; Maier, Vincent P.; Orme, Edward D.

    1983-01-01

    The fungus Cercospora rosicola produces abscisic acid (ABA) as a secondary metabolite. We developed a convenient system using this fungus to determine the effects of compounds on the biosynthesis of ABA. Inasmuch as ABA and the gibberellins (GAs) both arise via the isoprenoid pathway, it was of interest to determine if inhibitors of GA biosynthesis affect ABA biosynthesis. All five putative inhibitors of GA biosynthesis tested inhibited ABA biosynthesis. Several plant growth retardants with poorly understood actions in plants were also tested; of these, six inhibited ABA biosynthesis to varying degrees and two had no effect. Effects of plant growth retardants on various branches of the isoprenoid biosynthetic pathway may help to explain some of the diverse and unexpected results reported for these compounds. Knowledge that certain inhibitors of GA biosynthesis also have the ability to inhibit ABA biosynthesis in C. rosicola indicates the need for further studies in plants on the mode of action of these compounds. PMID:16662775

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

    PubMed

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

    2016-01-01

    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence. PMID:27195796

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

    PubMed Central

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

    2016-01-01

    DNA methyltransferases have been implicated in the regulation of virulence genes in a number of pathogens. Relapsing fever Borrelia species harbor a conserved, putative DNA methyltransferase gene on their chromosome, while no such ortholog can be found in the annotated genome of the Lyme disease agent, Borrelia burgdorferi. In the relapsing fever species Borrelia hermsii, the locus bh0463A encodes this putative DNA adenine methyltransferase (dam). To verify the function of the BH0463A protein product as a Dam, the gene was cloned into a Dam-deficient strain of Escherichia coli. Restriction fragment analysis subsequently demonstrated that complementation of this E. coli mutant with bh0463A restored adenine methylation, verifying bh0463A as a Dam. The requirement of bh0463A for B. hermsii viability, infectivity, and persistence was then investigated by genetically disrupting the gene. The dam- mutant was capable of infecting immunocompetent mice, and the mean level of spirochetemia in immunocompetent mice was not significantly different from wild type B. hermsii. Collectively, the data indicate that dam is dispensable for B. hermsii viability, infectivity, and persistence. PMID:27195796

  9. BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism.

    PubMed

    Zhang, Si; Hu, Liang; Du, Hongguang; Guo, Yan; Zhang, Yan; Niu, Haixia; Jin, Jianguo; Zhang, Jian; Liu, Junling; Zhang, Xiaohui; Kunapuli, Satya P; Ding, Zhongren

    2010-10-01

    Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug. PMID:20806121

  10. Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

    PubMed Central

    Quay, S C; Oxender, D L; Tsuyumu, S; Umbarger, H E

    1975-01-01

    Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears

  11. Modified Iterative Extended Hueckel. 2: Application to the interaction of Na(+), Na(+)(aq.), Mg(+)-2(aq.) with adenine and thymine

    NASA Technical Reports Server (NTRS)

    Aronowitz, S.; Macelroy, R.; Chang, S.

    1980-01-01

    Modified Iterative Extended Hueckel, which includes explicit effective internuclear and electronic interactions, is applied to the study of the energetics of Na(+),Mg(+), Na(+) (aqueous), and Mg(+2) (aqueous) ions approaching various possible binding sites on adenine and thymine. Results for the adenine + ion and thymine + ion are in good qualitative agreement with ab initio work on analogous systems. Energy differences between competing sites are in excellent agreement. Hydration appears to be a critical factor in determining favorable binding sites. That the adenine Nl and N3 sites cannot displace a water molecule from the hydrated cation indicates that they are not favorable binding sites in aqueous media. Of those sites investigated, 04 was the most favorable binding site on the thymine for the bare Na(+). However, the 02 site was the most favorable binding site for either hydrated cation.

  12. DNA sequence-specific adenine alkylation by the novel antitumor drug tallimustine (FCE 24517), a benzoyl nitrogen mustard derivative of distamycin.

    PubMed Central

    Broggini, M; Coley, H M; Mongelli, N; Pesenti, E; Wyatt, M D; Hartley, J A; D'Incalci, M

    1995-01-01

    FCE 24517, a novel distamycin derivative possessing potent antitumor activity, is under initial clinical investigation in Europe. In spite of the presence of a benzoyl nitrogen mustard group this compound fails to alkylate the N7 position of guanine, the major site of alkylation by conventional nitrogen mustards. Characterisation of DNA-drug adducts revealed only a very low level of adenine adduct formation. Using a modified Maxam-Gilbert sequencing method the consensus sequence for FCE 24517-adenine adduct formation was found to be 5'-TTTTGA-3'. A single base modification in the hexamer completely abolishes the alkylation of adenine. Using a Taq polymerase stop assay alkylations were confirmed at the A present in the hexamer TTTTGA and, in addition, in one out of three TTTTAA sequences present in the plasmid utilized. The sequence specificity of alkylation by FCE 24517 is therefore the most striking yet observed for an alkylating agent of small molecular weight. Images PMID:7870593

  13. Biosynthesis of coenzyme Q in eukaryotes.

    PubMed

    Kawamukai, Makoto

    2015-01-01

    Coenzyme Q (CoQ) is a component of the electron transport chain that participates in aerobic cellular respiration to produce ATP. In addition, CoQ acts as an electron acceptor in several enzymatic reactions involving oxidation-reduction. Biosynthesis of CoQ has been investigated mainly in Escherichia coli and Saccharomyces cerevisiae, and the findings have been extended to various higher organisms, including plants and humans. Analyses in yeast have contributed greatly to current understanding of human diseases related to CoQ biosynthesis. To date, human genetic disorders related to mutations in eight COQ biosynthetic genes have been reported. In addition, the crystal structures of a number of proteins involved in CoQ synthesis have been solved, including those of IspB, UbiA, UbiD, UbiX, UbiI, Alr8543 (Coq4 homolog), Coq5, ADCK3, and COQ9. Over the last decade, knowledge of CoQ biosynthesis has accumulated, and striking advances in related human genetic disorders and the crystal structure of proteins required for CoQ synthesis have been made. This review focuses on the biosynthesis of CoQ in eukaryotes, with some comparisons to the process in prokaryotes.

  14. Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

    PubMed

    DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

    2015-05-01

    Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases.

  15. The lipid biosynthesis hole in the rickettsiales

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Using a complementation assay in E. coli, we have shown that the propionyl-CoA carboxylase complex (PCC) from Wolbachia pipientis wMel, order Rickettsiales, provides for lipid biosynthesis through malonyl-CoA production. Normally, the prototypical prokaryote fatty acid synthesis (FASII) initiation ...

  16. Biosynthesis of sphinganine-analog mycotoxins.

    PubMed

    Du, L; Zhu, X; Gerber, R; Huffman, J; Lou, L; Jorgenson, J; Yu, F; Zaleta-Rivera, K; Wang, Q

    2008-06-01

    Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5'-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.

  17. Erythro-9-(2-hydroxy-3-nonyl) Adenine alone and in combination with 9-beta-D-arabinofuranosyladenine in treatment of systemic herpesvirus infections in mice.

    PubMed Central

    Shannon, W M; Arnett, G; Schabel, F M; North, T W; Cohen, S S

    1980-01-01

    Although the antiviral activity of erythro-9-(2-hydroxy-3-nonyl)adenine, a potent adenosine deaminase inhibitor, against herpes simplex virus type 1 in cell culture was readily confirmed, the compound was found to be totally ineffective in the treatment of experimentally induced systemic herpes simplex virus type 1 infections in Swiss mice. Data were obtained, however, which clearly indicated that the antiviral potency of 9-beta-D-arabinofuranosyladenine in vivo could be enhanced by the co-administration of low, nontoxic doses of erythro-9-(2-hydroxy-3-nonyl)adenine. PMID:6255863

  18. Data on cell viability of human lung fibroblasts treated with polyphenols-rich extract from Plinia trunciflora (O. Berg) Kausel).

    PubMed

    Calloni, Caroline; Silva Santos, Luciana Fernandes; Martínez, Luana Soares; Salvador, Mirian

    2016-03-01

    Jaboticaba (Plinia trunciflora (O. Berg) Kausel) is a Brazilian native berry, which presents high levels of polyphenols. Here we provide data related to the effects of the polyphenols-rich extract from jaboticaba on the cell viability, mitochondrial complex I (nicotinamide adenine dinucleotide/CoQ oxidoreductase) activity and ATP biosynthesis of human lung fibroblast cells (MRC-5) treated with amiodarone. The data presented in this article demonstrate that the polyphenols-rich extract from jaboticaba was able to reduce cell death as well as the decrease in complex I activity and ATP biosynthesis caused by amiodarone in MRC-5 cells.

  19. Data on cell viability of human lung fibroblasts treated with polyphenols-rich extract from Plinia trunciflora (O. Berg) Kausel)

    PubMed Central

    Calloni, Caroline; Silva Santos, Luciana Fernandes; Martínez, Luana Soares; Salvador, Mirian

    2016-01-01

    Jaboticaba (Plinia trunciflora (O. Berg) Kausel) is a Brazilian native berry, which presents high levels of polyphenols. Here we provide data related to the effects of the polyphenols-rich extract from jaboticaba on the cell viability, mitochondrial complex I (nicotinamide adenine dinucleotide/CoQ oxidoreductase) activity and ATP biosynthesis of human lung fibroblast cells (MRC-5) treated with amiodarone. The data presented in this article demonstrate that the polyphenols-rich extract from jaboticaba was able to reduce cell death as well as the decrease in complex I activity and ATP biosynthesis caused by amiodarone in MRC-5 cells. PMID:26870757

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

    SciTech Connect

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

    1994-08-01

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

  1. The recurrent causal mutation for osteogenesis imperfecta type V occurs at a highly methylated CpG dinucleotide within the IFITM5 gene

    PubMed Central

    Corradi, Massimiliano; Monti, Elena; Venturi, Giacomo; Gandini, Alberto; Mottes, Monica; Antoniazzi, Franco

    2014-01-01

    Recent studies have identified the molecular defect underlying autosomal dominant osteogenesis imperfecta (OI) type V. Unlike all other OI types, which are characterized by high genetic heterogeneity, OI type V appears consistently associated to a unique de novo C>T transition within the 5′ UTR of the IFITM5 gene. Although the precise frequency of OI type V is not known, this recurrent base substitution may well represent a mutational hotspot in the human genome. We show that it occurs at a CpG dinucleotide that is highly methylated in several tissues and particularly in the sperm DNA, suggesting a mutational mechanism common to other de novo recurrent dominant mutations. PMID:27625865

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

    SciTech Connect

    Vanyukov, M.M.; Moss, H.B.; Tarter, R.E.

    1995-04-24

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

  3. iRSpot-DACC: a computational predictor for recombination hot/cold spots identification based on dinucleotide-based auto-cross covariance.

    PubMed

    Liu, Bingquan; Liu, Yumeng; Jin, Xiaopeng; Wang, Xiaolong; Liu, Bin

    2016-09-19

    Meiotic recombination presents an uneven distribution across the genome. Genomic regions that exhibit at relatively high frequencies of recombination are called hotspots, whereas those with relatively low frequencies of recombination are called coldspots. Therefore, hotspots and coldspots would provide useful information for the study of the mechanism of recombination. In this study, we proposed a computational predictor called iRSpot-DACC to predict hot/cold spots across the yeast genome. It combined Support Vector Machines (SVMs) and a feature called dinucleotide-based auto-cross covariance (DACC), which is able to incorporate the global sequence-order information and fifteen local DNA properties into the predictor. Combined with Principal Component Analysis (PCA), its performance was further improved. Experimental results on a benchmark dataset showed that iRSpot-DACC can achieve an accuracy of 82.7%, outperforming some highly related methods.

  4. iRSpot-DACC: a computational predictor for recombination hot/cold spots identification based on dinucleotide-based auto-cross covariance.

    PubMed

    Liu, Bingquan; Liu, Yumeng; Jin, Xiaopeng; Wang, Xiaolong; Liu, Bin

    2016-01-01

    Meiotic recombination presents an uneven distribution across the genome. Genomic regions that exhibit at relatively high frequencies of recombination are called hotspots, whereas those with relatively low frequencies of recombination are called coldspots. Therefore, hotspots and coldspots would provide useful information for the study of the mechanism of recombination. In this study, we proposed a computational predictor called iRSpot-DACC to predict hot/cold spots across the yeast genome. It combined Support Vector Machines (SVMs) and a feature called dinucleotide-based auto-cross covariance (DACC), which is able to incorporate the global sequence-order information and fifteen local DNA properties into the predictor. Combined with Principal Component Analysis (PCA), its performance was further improved. Experimental results on a benchmark dataset showed that iRSpot-DACC can achieve an accuracy of 82.7%, outperforming some highly related methods. PMID:27641752

  5. A new dinucleotide repeat polymorphism at the telomere of chromosome 21q reveals a significant difference between male and female rates of recombination

    SciTech Connect

    Blouin, J.L.; Gos, A.; Morris, M.A.

    1995-08-01

    We have used a half-YAC containing the human chromosome 21 long-arm telomere to clone, map, and characterize a new dinucleotide repeat polymorphism (D21S1575) close to 21qter. The marker is <120 kb from the telomeric (TTAGGG){sub n} sequences and is the most distal highly polymorphic marker on chromosome 21q. This marker has a heterozygosity of 71% because of a variable (TA){sub n} repeat embedded within a long interspersed element (LINE) element. Genotyping of the CEPH families and linkage analysis provided a more accurate determination of the full length of the chromosome 21 genetic map. A highly significant difference was detected between male and female recombination rates in the telomeric region: in the most telomeric 2.3 Mb of chromosome 21q, recombination was only observed in male meioses. 35 refs., 4 figs., 2 tabs.

  6. iRSpot-DACC: a computational predictor for recombination hot/cold spots identification based on dinucleotide-based auto-cross covariance

    PubMed Central

    Liu, Bingquan; Liu, Yumeng; Jin, Xiaopeng; Wang, Xiaolong; Liu, Bin

    2016-01-01

    Meiotic recombination presents an uneven distribution across the genome. Genomic regions that exhibit at relatively high frequencies of recombination are called hotspots, whereas those with relatively low frequencies of recombination are called coldspots. Therefore, hotspots and coldspots would provide useful information for the study of the mechanism of recombination. In this study, we proposed a computational predictor called iRSpot-DACC to predict hot/cold spots across the yeast genome. It combined Support Vector Machines (SVMs) and a feature called dinucleotide-based auto-cross covariance (DACC), which is able to incorporate the global sequence-order information and fifteen local DNA properties into the predictor. Combined with Principal Component Analysis (PCA), its performance was further improved. Experimental results on a benchmark dataset showed that iRSpot-DACC can achieve an accuracy of 82.7%, outperforming some highly related methods. PMID:27641752

  7. The recurrent causal mutation for osteogenesis imperfecta type V occurs at a highly methylated CpG dinucleotide within the IFITM5 gene.

    PubMed

    Corradi, Massimiliano; Monti, Elena; Venturi, Giacomo; Gandini, Alberto; Mottes, Monica; Antoniazzi, Franco

    2014-03-01

    Recent studies have identified the molecular defect underlying autosomal dominant osteogenesis imperfecta (OI) type V. Unlike all other OI types, which are characterized by high genetic heterogeneity, OI type V appears consistently associated to a unique de novo C>T transition within the 5' UTR of the IFITM5 gene. Although the precise frequency of OI type V is not known, this recurrent base substitution may well represent a mutational hotspot in the human genome. We show that it occurs at a CpG dinucleotide that is highly methylated in several tissues and particularly in the sperm DNA, suggesting a mutational mechanism common to other de novo recurrent dominant mutations. PMID:27625865

  8. Tissue-specific methylation of individual CpG dinucleotides in the 5{prime} upstream region of the mouse catalase gene (Cas-1)

    SciTech Connect

    Pillay, I.L.; Singh, S.M.

    1994-09-01

    The intracellular antioxidant enzyme, catalase, is encoded by a gene whose level of expression in different organisms, including humans, varies with tissue-type. The {open_quotes}TATA-less{close_quotes} 5{prime} upstream region of the catalase gene, in mice and humans, contains a CpG island. Such CG-rich regions are target sites for cytosine methylation and have been implicated in tissue-specific gene expression. However, the methylation status of individual CpG dinucleotides and their significance in gene expression has not been established. A 275 bp fragment within the 5{prime} region of Cas-1 was evaluated for CpG methylation. HpaII digestion of genomic DNA, followed by polymerase chain reaction amplification (HpaII-PCR), suggests that at least one of three CCGG is not methylated in nine different somatic tissues that express this enzyme at various levels. In contrast, all three CCGG sites are methylated in DNA from sperm and spleen. Further examination of the methylation specificity of individual CCGG sites was conducted using sodium bisulfite modification of genomic DNA followed by HPaII-PCR. Sodium bisulfite modifies non-methylated cytosines to uracils, changing a CG to a TG dinucleotide. This nucleotide substitution eliminates HpaII sites and allows the methylation status of each of the CCGG sites to be assessed. The ability to discern the number and combination of methylated sites within the 5{prime} region of a gene permits the determination of a possible correlation between differential methylation patterns and temporal/spatial gene regulation. Analysis of differential methylation, using the mouse catalase gene as a model, provides further insight into CpG methylation as one mechanism of mammalian gene regulation.

  9. Acid-base and metal-ion binding properties of the RNA dinucleotide uridylyl-(5'-->3')-[5']uridylate (pUpU3-).

    PubMed

    Knobloch, Bernd; Suliga, Danuta; Okruszek, Andrzej; Sigel, Roland K O

    2005-07-01

    It is well known that Mg2+ and other divalent metal ions bind to the phosphate groups of nucleic acids. Subtle differences in the coordination properties of these metal ions to RNA, especially to ribozymes, determine whether they either promote or inhibit catalytic activity. The ability of metal ions to coordinate simultaneously with two neighboring phosphate groups is important for ribozyme structure and activity. However, such an interaction has not yet been quantified. Here, we have performed potentiometric pH titrations to determine the acidity constants of the protonated dinucleotide H2(pUpU)-, as well as the binding properties of pUpU3- towards Mg2+, Mn2+, Cd2+, Zn2+, and Pb2+. Whereas Mg2+, Mn2+, and Cd2+ only bind to the more basic 5'-terminal phosphate group, Pb2+, and to a certain extent also Zn2+, show a remarkably enhanced stability of the [M(pUpU)]- complex. This can be attributed to the formation of a macrochelate by bridging the two phosphate groups within this dinucleotide by these metal ions. Such a macrochelate is also possible in an oligonucleotide, because the basic structural units are the same, despite the difference in charge. The formation degrees of the macrochelated species of [Zn(pUpU)]- and [Pb(pUpU)]- amount to around 25 and 90 %, respectively. These findings are important in the context of ribozyme and DNAzyme catalysis, and explain, for example, why the leadzyme could be selected in the first place, and why this artificial ribozyme is inhibited by other divalent metal ions, such as Mg2+.

  10. Effects of increased heart work on glycolysis and adenine nucleotides in the perfused heart of normal and diabetic rats

    PubMed Central

    Opie, L. H.; Mansford, K. R. L.; Owen, Patricia

    1971-01-01

    1. In the isolated perfused rat heart, the contractile activity and the oxygen uptake were varied by altering the aortic perfusion pressure, or by the atrial perfusion technique (`working heart'). 2. The maximum increase in the contractile activity brought about an eightfold increase in the oxygen uptake. The rate of glycolytic flux rose, while tissue contents of hexose monophosphates, citrate, ATP and creatine phosphate decreased, and contents of ADP and AMP rose. 3. The changes in tissue contents of adenine nucleotides during increased heart work were time-dependent. The ATP content fell temporarily (30s and 2min) after the start of left-atrial perfusion; at 5 and 10min values were normal; and at 30 and 60min values were decreased. ADP and AMP values were increased in the first 15min, but were at control values 30 or 60min after the onset of increased heart work. 4. During increased heart work changes in the tissue contents of adenine nucleotide and of citrate appeared to play a role in altered regulation of glycolysis at the level of phosphofructokinase activity. 5. In recirculation experiments increased heart work for 30min was associated with increased entry of [14C]glucose (11.1mm) and glycogen into glycolysis and a comparable increase in formation of products of glycolysis (lactate, pyruvate and 14CO2). There was no major accumulation of intermediates. Glycogen was not a major fuel for respiration. 6. Increased glycolytic flux in Langendorff perfused and working hearts was obtained by the addition of insulin to the perfusion medium. The concomitant increases in the tissue values of hexose phosphates and of citrate contrasted with the decreased values of hexose monophosphates and of citrate during increased glycolytic flux obtained by increased heart work. 7. Decreased glycolytic flux in Langendorff perfused hearts was obtained by using acute alloxan-diabetic and chronic streptozotocin-diabetic rats; in the latter condition there were decreased tissue

  11. Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE/MCP-1, and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor-alpha and immunoglobulin G. Evidence for involvement of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidase.

    PubMed

    Satriano, J A; Shuldiner, M; Hora, K; Xing, Y; Shan, Z; Schlondorff, D

    1993-09-01

    The potential involvement of reactive oxygen species in the expression of genes involved in immune response was examined in mesangial cells. Tumor necrosis factor (TNF-alpha) and aggregated (aggr.) IgG increased mRNA levels for the monocyte chemoattractant protein, JE/MCP-1, and the colony-stimulating factor, CSF-1. Scavengers for free radicals such as di- and tetra-methylthiourea (DMTU and TMTU) attenuated the increase in mRNA levels in response to TNF-alpha and aggr. IgG. Generation of superoxide anion by xanthine oxidase and hypoxanthine increased mRNA levels of these genes, but exogenous H2O2 did not. Addition of NADPH to activate a membrane-bound NADPH-oxidase generated superoxide and caused a dose-dependent increase in mRNA levels and further enhanced the stimulation by TNF-alpha or aggr. IgG. An inhibitor of NADPH-dependent oxidase 4'-hydroxy-3'-methoxy-acetophenone attenuated the rise in mRNA levels in response to TNF-alpha and aggr. IgG. By nuclear run-on experiments TNF-alpha, aggr. IgG and NADPH increased the transcription rates for JE/MCP-1 and CSF-1, effects inhibited by TMTU. We conclude that generation of reactive oxygen species, possibly by NADPH-dependent oxidase, are involved in the induction of the JE/MCP-1 and CSF-1 genes by TNF-alpha and IgG complexes. The concerted expression of leukocyte-directed cytokines represents a general response to tissue injury.

  12. Adenine phosphoribosyltransferase (APRT) deficiency: a new genetic mutation with early recurrent renal stone disease in kidney transplantation

    PubMed Central

    Micheli, Vanna; Massarino, Fabio; Jacomelli, Gabriella; Bertelli, Matteo; Corradi, Maria Rita; Guerrini, Andrea; Cucchiara, Antonino; Ravetti, Jean Louis; Negretti, Laura; Cannella, Giuseppe

    2010-01-01

    Adenine phosphoribosyltransferase (APRT) deficiency, a rare inborn error inherited as an autosomic recessive trait, presents with 2,8-dihydroxyadenine (2,8-DHA) crystal nephropathy. We describe clinical, biochemical and molecular findings in a renal transplant recipient with renal failure, 2,8-DHA stones and no measurable erythrocyte APRT activity. Homozygous C > G substitution at −3 in the splicing site of exon 2 (IVS2 −3 c > g) was found in the APRT gene. The patient’s asymptomatic brother was heterozygous for such mutation, and his APRT activity was 23% of controls. A splicing alteration leading to incorrect gene transcription and virtually absent APRT activity is seemingly associated with the newly identified mutation. PMID:25984046

  13. Adenine phosphoribosyltransferase (APRT) deficiency: a new genetic mutation with early recurrent renal stone disease in kidney transplantation.

    PubMed

    Micheli, Vanna; Massarino, Fabio; Jacomelli, Gabriella; Bertelli, Matteo; Corradi, Maria Rita; Guerrini, Andrea; Cucchiara, Antonino; Ravetti, Jean Louis; Negretti, Laura; Cannella, Giuseppe

    2010-10-01

    Adenine phosphoribosyltransferase (APRT) deficiency, a rare inborn error inherited as an autosomic recessive trait, presents with 2,8-dihydroxyadenine (2,8-DHA) crystal nephropathy. We describe clinical, biochemical and molecular findings in a renal transplant recipient with renal failure, 2,8-DHA stones and no measurable erythrocyte APRT activity. Homozygous C > G substitution at -3 in the splicing site of exon 2 (IVS2 -3 c > g) was found in the APRT gene. The patient's asymptomatic brother was heterozygous for such mutation, and his APRT activity was 23% of controls. A splicing alteration leading to incorrect gene transcription and virtually absent APRT activity is seemingly associated with the newly identified mutation.

  14. Determination of Plaque Inhibitory Activity of Adenine Arabinoside (9-β-d-Arabinofuranosyladenine) for Herpesviruses Using an Adenosine Deaminase Inhibitor

    PubMed Central

    Bryson, Yvonne; Connor, James D.; Sweetman, Lawrence; Carey, Sharen; Stuckey, Margaret A.; Buchanan, Robert

    1974-01-01

    The in vitro susceptibility of type 1 and type 2 strains of Herpesvirus hominis to 9-β-d-arabinofuranosyladenine (adenine arabinoside, ara-A) was measured in a system where deamination was inhibited. Under these conditions, it was possible to measure the activity of low concentrations of ara-A. It was determined that plaque inhibitory concentration for type 1 viruses was less than 3 μg/ml for all strains tested. The plaque inhibitory concentration for 7 of 10 type 2 strains was also less than 3 μg/ml. The method used identified and controlled the interaction between antiviral agent (ara-A) and the indicator system, human skin fibroblastic cells. Otherwise, metabolism of ara-A resulted in rapid enzymatic degradation and loss of antiviral activity. PMID:15828177

  15. ISOLATION, SYNTHESIS AND BIOLOGICAL ACTIVITY OF APHROCALLISTIN, AN ADENINE SUBSTITUTED BROMOTYRAMINE METABOLITE FROM THE HEXACTINELLIDA SPONGE APHROCALLISTES BEATRIX

    PubMed Central

    Wright, Amy E.; Roth, Gregory P.; Hoffman, Jennifer K.; Divlianska, Daniela B.; Pechter, Diana; Sennett, Susan H.; Guzmán, Esther A.; Linley, Patricia; McCarthy, Peter J.; Pitts, Tara P.; Pomponi, Shirley A.; Reed, John K.

    2010-01-01

    A new adenine substituted bromotyrosine derived metabolite designated as aphrocallistin (1) has been isolated from the deep-water Hexactinellida sponge Aphrocallistes beatrix beatrix Gray, 1858 (Order Hexactinosida, Family Aphrocallistidae). Its structure was elucidated on the basis of spectral data and confirmed through a convergent, modular total synthetic route that is amenable towards future analog preparation. Aphrocallistin inhibits the growth of a panel of human tumor cell lines with IC50 values ranging from 7.5 to >100 μM and has been shown to induce G1 cell cycle arrest in the PANC-1 pancreatic carcinoma cell line. Aphrocallistin has been fully characterized in the NCI cancer cell line panel and has undergone in vitro ADME pharmacological profiling. PMID:19459694

  16. Hydrogen peroxide formation photoinduced by near-UV radiation in aqueous solutions of adenine derivatives at 77 K

    NASA Astrophysics Data System (ADS)

    Lozinova, T. A.; Lobanov, A. V.; Lander, A. V.

    2015-08-01

    An estimate of the content of free radicals in aqueous solutions of adenosine (Ado), adenosine-5'-diphosphate (ADP) and guanosine-5'-monophosphate (GMP) irradiated with near-UV radiation at 77 K is obtained by interpreting EPR spectra. It is established that in the presence of NaCl (0.1 M), the total number of peroxyl radicals O{2/-·} and HO{2/·} in samples of the studied compounds was 15-45% of the total quantity of produced free radicals and was affected by the conditions of exposure. The estimates are compared with the results from hydrogen peroxide (H2O2) determination in the same samples after thawing. Although the number of peroxyl radicals in the samples of adenine derivatives (A) and GMP are comparable, the formation of H2O2 is observed only in the case of A derivatives, but not in GMP. Possible reasons for these differences are discussed.

  17. Changes in the adenine nucleotide content of beef-heart mitochondrial F1 ATPase during ATP synthesis in dimethyl sulfoxide.

    PubMed

    Beharry, S; Bragg, P D

    1992-01-31

    Beef-heart mitochondrial F1 ATPase can be induced to synthesize ATP from ADP and inorganic phosphate in 30% Me2SO. We have analyzed the adenine nucleotide content of the F1 ATPase during the time-course of ATP synthesis, in the absence of added medium nucleotide, and in the absence and presence of 10 mM inorganic phosphate. The enzyme used in these investigations was either pretreated or not pretreated with ATP to produce F1 with a defined nucleotide content and catalytic or noncatalytic nucleotide-binding site occupancy. We show that the mechanism of ATP synthesis in Me2SO involves (i) an initial rapid loss of bound nucleotide(s), this process being strongly influenced by inorganic phosphate; (ii) a rebinding of lost nucleotide; and (iii) synthesis of ATP from bound ADP and inorganic phosphate.

  18. Structural basis for phosphatidylinositol-phosphate biosynthesis

    NASA Astrophysics Data System (ADS)

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-10-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

  19. [Peculiarities of Proteus mirabilis extracellular metalloproteinase biosynthesis].

    PubMed

    Zamaliutdinova, N M; Sharipova, M R; Bogomol'naia, L M; Bozhokina, E S; Mardanova, A M

    2015-01-01

    Biosynthesis of metalloproteinase by the Proteus mirabilis 5127-1 strain on different media and the influence of glucose and urea on biosynthesis were studied. It was found that the P. mirabilis 5127-1 bacteria secretes metalloproteinase in the medium in two isoforms (52 and 50 kDa). It was established that proteinase synthesis is completely suppressed during the growth of bacteria on synthetic media, as well as in the presence of LB glucose in the medium. It was demonstrated that addition of urea in the medium results in an increase of the culture productivity in the proteinase synthesis. Maximal culture productivity in the proteinase synthesis was found in the medium with natural urine. During the growth of bacteria on artificial urine, proteinase appeared in the medium only after 12 hours of growth as a single isoform. PMID:25872397

  20. Ceramide biosynthesis and metabolism in trophoblast syncytialization.

    PubMed

    Singh, Ambika T; Dharmarajan, Arunasalam; Aye, Irving L M H; Keelan, Jeffrey A

    2012-10-15

    Sphingolipid mediators such as ceramide are pleiotropic regulators of cellular growth, differentiation and apoptosis. We investigated the role of ceramide biosynthesis, metabolism and actions in term human cytotrophoblasts syncytialized over 7 days in culture. Intracellular C16 ceramide levels increased modestly after 3 days in culture, then declined. Ceramidase was present at particularly high levels in syncytialized trophoblasts; inhibition of ceramidase reduced the degree of cell fusion. Exposure to short chain C8 ceramide or aSMase enhanced secretion of the differentiation marker hCG without affecting fusion or cell viability. In contrast, pharmacological inhibition of ceramidase reduced the extent of fusion. Inhibition of the ceramide-responsive JNK and PP2A pathways did not abolish the effects of ceramide, and JNK phosphorylation was unresponsive to ceramide; however, ceramide significantly inhibited phosphorylation of Akt. This study suggests that changes in ceramide biosynthesis and metabolism play a differential role in the biochemical and morphological features of trophoblast differentiation.

  1. Circular Bacteriocins: Biosynthesis and Mode of Action

    PubMed Central

    Brede, Dag A.; Nes, Ingolf F.; Diep, Dzung B.

    2014-01-01

    Circular bacteriocins are a group of N-to-C-terminally linked antimicrobial peptides, produced by Gram-positive bacteria of the phylum Firmicutes. Circular bacteriocins generally exhibit broad-spectrum antimicrobial activity, including against common food-borne pathogens, such as Clostridium and Listeria spp. These peptides are further known for their high pH and thermal stability, as well as for resistance to many proteolytic enzymes, properties which make this group of bacteriocins highly promising for potential industrial applications and their biosynthesis of particular interest as a possible model system for the synthesis of highly stable bioactive peptides. In this review, we summarize the current knowledge on this group of bacteriocins, with emphasis on the recent progress in understanding circular bacteriocin genetics, biosynthesis, and mode of action; in addition, we highlight the current challenges and future perspectives for the application of these peptides. PMID:25172850

  2. Chemical genetics to examine cellulose biosynthesis

    PubMed Central

    Brabham, Chad; DeBolt, Seth

    2013-01-01

    Long-term efforts to decode plant cellulose biosynthesis via molecular genetics and biochemical strategies are being enhanced by the ever-expanding scale of omics technologies. An alternative approach to consider are the prospects for inducing change in plant metabolism using exogenously supplied chemical ligands. Cellulose biosynthesis inhibitors (CBIs) have been identified among known herbicides, during diverse combinatorial chemical libraries screens, and natural chemical screens from microbial agents. In this review, we summarize the current knowledge of the inhibitory effects of CBIs and further group them by how they influence fluorescently tagged cellulose synthase A proteins. Additional attention is paid to the continuing development of the CBI toolbox to explore the cell biology and genetic mechanisms underpinning effector molecule activity. PMID:23372572

  3. Complete biosynthesis of opioids in yeast

    PubMed Central

    Galanie, Stephanie; Thodey, Kate; Trenchard, Isis J.; Interrante, Maria Filsinger; Smolke, Christina D.

    2016-01-01

    Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. Here, we engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof-of-principle, and major hurdles remain before optimization and scale up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds. PMID:26272907

  4. Functional specialization in proline biosynthesis of melanoma.

    PubMed

    De Ingeniis, Jessica; Ratnikov, Boris; Richardson, Adam D; Scott, David A; Aza-Blanc, Pedro; De, Surya K; Kazanov, Marat; Pellecchia, Maurizio; Ronai, Ze'ev; Osterman, Andrei L; Smith, Jeffrey W

    2012-01-01

    Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of (13)C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis.

  5. Biosynthesis and toxicological effects of patulin.

    PubMed

    Puel, Olivier; Galtier, Pierre; Oswald, Isabelle P

    2010-04-01

    Patulin is a toxic chemical contaminant produced by several species of mold, especially within Aspergillus, Penicillium and Byssochlamys. It is the most common mycotoxin found in apples and apple-derived products such as juice, cider, compotes and other food intended for young children. Exposure to this mycotoxin is associated with immunological, neurological and gastrointestinal outcomes. Assessment of the health risks due to patulin consumption by humans has led many countries to regulate the quantity in food. A full understanding of the molecular genetics of patulin biosynthesis is incomplete, unlike other regulated mycotoxins (aflatoxins, trichothecenes and fumonisins), although the chemical structures of patulin precursors are now known. The biosynthetic pathway consists of approximately 10 steps, as suggested by biochemical studies. Recently, a cluster of 15 genes involved in patulin biosynthesis was reported, containing characterized enzymes, a regulation factor and transporter genes. This review includes information on the current understanding of the mechanisms of patulin toxinogenesis and summarizes its toxicological effects.

  6. Combinatorial biosynthesis--potential and problems.

    PubMed

    Floss, Heinz G

    2006-06-25

    Because of their ecological functions, natural products have been optimized in evolution for interaction with biological systems and receptors. However, they have not necessarily been optimized for other desirable drug properties and thus can often be improved by structural modification. Using examples from the literature, this paper reviews the opportunities for increasing structural diversity among natural products by combinatorial biosynthesis, i.e., the genetic manipulation of biosynthetic pathways. It distinguishes between combinatorial biosynthesis in a narrower sense to generate libraries of modified structures, and metabolic engineering for the targeted formation of specific structural analogs. Some of the problems and limitations encountered with these approaches are also discussed. Work from the author's laboratory on ansamycin antibiotics is presented which illustrates some of the opportunities and limitations.

  7. Functional Specialization in Proline Biosynthesis of Melanoma

    PubMed Central

    Richardson, Adam D.; Scott, David A.; Aza-Blanc, Pedro; De, Surya K.; Kazanov, Marat; Pellecchia, Maurizio; Ronai, Ze'ev; Osterman, Andrei L.; Smith, Jeffrey W.

    2012-01-01

    Proline metabolism is linked to hyperprolinemia, schizophrenia, cutis laxa, and cancer. In the latter case, tumor cells tend to rely on proline biosynthesis rather than salvage. Proline is synthesized from either glutamate or ornithine; both are converted to pyrroline-5-carboxylate (P5C), and then to proline via pyrroline-5-carboxylate reductases (PYCRs). Here, the role of three isozymic versions of PYCR was addressed in human melanoma cells by tracking the fate of 13C-labeled precursors. Based on these studies we conclude that PYCR1 and PYCR2, which are localized in the mitochondria, are primarily involved in conversion of glutamate to proline. PYCRL, localized in the cytosol, is exclusively linked to the conversion of ornithine to proline. This analysis provides the first clarification of the role of PYCRs to proline biosynthesis. PMID:23024808

  8. Structural basis for phosphatidylinositol-phosphate biosynthesis

    PubMed Central

    Clarke, Oliver B.; Tomasek, David; Jorge, Carla D.; Dufrisne, Meagan Belcher; Kim, Minah; Banerjee, Surajit; Rajashankar, Kanagalaghatta R.; Shapiro, Lawrence; Hendrickson, Wayne A.; Santos, Helena; Mancia, Filippo

    2015-01-01

    Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis. PMID:26510127

  9. Complete biosynthesis of opioids in yeast.

    PubMed

    Galanie, Stephanie; Thodey, Kate; Trenchard, Isis J; Filsinger Interrante, Maria; Smolke, Christina D

    2015-09-01

    Opioids are the primary drugs used in Western medicine for pain management and palliative care. Farming of opium poppies remains the sole source of these essential medicines, despite diverse market demands and uncertainty in crop yields due to weather, climate change, and pests. We engineered yeast to produce the selected opioid compounds thebaine and hydrocodone starting from sugar. All work was conducted in a laboratory that is permitted and secured for work with controlled substances. We combined enzyme discovery, enzyme engineering, and pathway and strain optimization to realize full opiate biosynthesis in yeast. The resulting opioid biosynthesis strains required the expression of 21 (thebaine) and 23 (hydrocodone) enzyme activities from plants, mammals, bacteria, and yeast itself. This is a proof of principle, and major hurdles remain before optimization and scale-up could be achieved. Open discussions of options for governing this technology are also needed in order to responsibly realize alternative supplies for these medically relevant compounds. PMID:26272907

  10. The effect of dimethylsulfoxide on adenine nucleotide binding and ATP synthesis by beef-heart mitochondrial F1 ATPase.

    PubMed

    Beharry, S; Bragg, P D

    1991-04-01

    Dimethylsulfoxide (Me2SO; 30%, v/v) promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F1 ATPase. The effects of this solvent on the adenine nucleotide binding properties of beef-heart mitochondrial F1 ATPase were examined. The ATP analog adenylyl-5'-imidodiphosphate bound to F1 at 1.9 and 1.0 sites in aqueous and Me2SO systems, respectively, with a KD value of 2.2 microM. Lower affinity sites were present also. Binding of ATP or adenylyl-5'-imidodiphosphate at levels near equimolar with the enzyme occurred to a greater extent in the absence of Me2SO. Addition of ATP to the nucleotide-loaded enzyme resulted in exchange of about one-half of the bound ATP. This occurred only in an entirely aqueous medium. ATP bound in Me2SO medium was not released by exogenous ATP. Comparison of the effect of different concentrations of Me2SO on ADP binding to F1 and ATP synthesis by the enzyme showed that binding of ADP was diminished by concentrations of Me2SO lower than those required to support ATP synthesis. However, one site could still be filled by ADP at concentrations of Me2SO optimal for ATP synthesis. This site is probably a noncatalytic site, since the nucleotide bound there was not converted to ATP in 30% Me2SO. The ATP synthesized by F1 in Me2SO originated from endogenous bound ADP. We conclude that 30% Me2SO affects the adenine nucleotide binding properties of the enzyme. The role of this in the promotion of the formation of ATP from ADP and phosphate is discussed.

  11. Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation.

    PubMed

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

    2015-09-14

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

  12. Endogenously elevated bilirubin modulates kidney function and protects from circulating oxidative stress in a rat model of adenine-induced kidney failure

    PubMed Central

    Boon, Ai-Ching; Lam, Alfred K.; Gopalan, Vinod; Benzie, Iris F.; Briskey, David; Coombes, Jeff S.; Fassett, Robert G.; Bulmer, Andrew C.

    2015-01-01

    Mildly elevated bilirubin is associated with a reduction in the presence and progression of chronic kidney disease and related mortality, which may be attributed to bilirubin’s antioxidant properties. This study investigated whether endogenously elevated bilirubin would protect against adenine-induced kidney damage in male hyperbilirubinaemic Gunn rats and littermate controls. Animals were orally administered adenine or methylcellulose solvent (vehicle) daily for 10 days and were then monitored for 28 days. Serum and urine were assessed throughout the protocol for parameters of kidney function and antioxidant/oxidative stress status and kidneys were harvested for histological examination upon completion of the study. Adenine-treated animals experienced weight-loss, polyuria and polydipsia; however, these effects were significantly attenuated in adenine-treated Gunn rats. No difference in the presence of dihydroadenine crystals, lymphocytic infiltration and fibrosis were noted in Gunn rat kidneys versus controls. However, plasma protein carbonyl and F2-isoprostane concentrations were significantly decreased in Gunn rats versus controls, with no change in urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine or kidney tissue F2-isoprostane concentrations. These data indicated that endogenously elevated bilirubin specifically protects from systemic oxidative stress in the vascular compartment. These data may help to clarify the protective relationship between bilirubin, kidney function and cardiovascular mortality in clinical investigations. PMID:26498893

  13. Influence of the incorporation of (S)-9-(3,4-dihydroxybutyl)adenine on the enzymatic stability and base-pairing properties of oligodeoxynucleotides.

    PubMed Central

    Augustyns, K; Van Aerschot, A; Van Schepdael, A; Urbanke, C; Herdewijn, P

    1991-01-01

    (S)-9-(3,4-dihydroxybutyl)adenine was used at several positions as nucleoside substitute in the synthesis of dimers and 13-mers. Therefore we used the phosporamidite and the H-phosphonate chemistry. The nuclease susceptibilities and the base-pairing properties of these oligomers have been evaluated. PMID:2041735

  14. Dynamics of dipole- and valence bound anions in iodide-adenine binary complexes: A time-resolved photoelectron imaging and quantum mechanical investigation

    SciTech Connect

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

    2015-09-14

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

  15. Effects of low-molecular-weight-chitosan on the adenine-induced chronic renal failure rats in vitro and in vivo

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  16. DNA-directed aniline mustards with high selectivity for adenine or guanine bases: mutagenesis in a variety of Salmonella typhimurium strains differing in DNA-repair capability.

    PubMed

    Ferguson, L R; Denny, W A; Boritzki, T J

    1994-04-01

    Two closely-related aniline monomustards (1 and 2), linked to a DNA-targeting acridine chromophore by a linker chain of different length, show high selectivity for alkylation of polymer DNA. The shorter-chain derivative (2) alkylates mainly at guanine N7 sites, while the longer-chain analogue (1) reacts almost exclusively at adenine N1. The biological effects of these compounds have been studied in standard Ames Salmonella typhimurium strains in order to determine the mutagenic consequences of such well-defined DNA lesions, and the effect of DNA-repair systems on them. Both compounds caused detectable mutations in strains TA1537, TA98 or TA100 and some related strains. Mutation rates were greatly enhanced in strains carrying either a uvrB deletion or the plasmid pKM101. Frameshift mutagenesis by both compounds was completely eliminated by recA deletion, in both the presence or absence of the plasmid. The adenine-selective compound (1) appeared more sensitive to the DNA-repair defects than the guanine-selective derivative (2). Additionally, only the adenine-selective compound (1) caused statistically significant levels of detectable mutation in the repair-proficient strains TA102, TA4001 or TA4006. The bacterial mutagenesis evidence suggests that a bulky, major groove-residing adenine lesion may be more readily recognised by DNA-repair systems, and more likely to lead to a wider range of mutagenic events, than a similar guanine lesion.

  17. β-alanine biosynthesis in Methanocaldococcus jannaschii.

    PubMed

    Wang, Yu; Xu, Huimin; White, Robert H

    2014-08-01

    One efficient approach to assigning function to unannotated genes is to establish the enzymes that are missing in known biosynthetic pathways. One group of such pathways is those involved in coenzyme biosynthesis. In the case of the methanogenic archaeon Methanocaldococcus jannaschii as well as most methanogens, none of the expected enzymes for the biosynthesis of the β-alanine and pantoic acid moieties required for coenzyme A are annotated. To identify the gene(s) for β-alanine biosynthesis, we have established the pathway for the formation of β-alanine in this organism after experimentally eliminating other known and proposed pathways to β-alanine from malonate semialdehyde, l-alanine, spermine, dihydrouracil, and acryloyl-coenzyme A (CoA). Our data showed that the decarboxylation of aspartate was the only source of β-alanine in cell extracts of M. jannaschii. Unlike other prokaryotes where the enzyme producing β-alanine from l-aspartate is a pyruvoyl-containing l-aspartate decarboxylase (PanD), the enzyme in M. jannaschii is a pyridoxal phosphate (PLP)-dependent l-aspartate decarboxylase encoded by MJ0050, the same enzyme that was found to decarboxylate tyrosine for methanofuran biosynthesis. A Km of ∼0.80 mM for l-aspartate with a specific activity of 0.09 μmol min(-1) mg(-1) at 70°C for the decarboxylation of l-aspartate was measured for the recombinant enzyme. The MJ0050 gene was also demonstrated to complement the Escherichia coli panD deletion mutant cells, in which panD encoding aspartate decarboxylase in E. coli had been knocked out, thus confirming the function of this gene in vivo.

  18. Investigating the Elusive Mechanism of Glycosaminoglycan Biosynthesis*

    PubMed Central

    Victor, Xylophone V.; Nguyen, Thao K. N.; Ethirajan, Manivannan; Tran, Vy M.; Nguyen, Khiem V.; Kuberan, Balagurunathan

    2009-01-01

    Glycosaminoglycan (GAG) biosynthesis requires numerous biosynthetic enzymes and activated sulfate and sugar donors. Although the sequence of biosynthetic events is resolved using reconstituted systems, little is known about the emergence of cell-specific GAG chains (heparan sulfate, chondroitin sulfate, and dermatan sulfate) with distinct sulfation patterns. We have utilized a library of click-xylosides that have various aglycones to decipher the mechanism of GAG biosynthesis in a cellular system. Earlier studies have shown that both the concentration of the primers and the structure of the aglycone moieties can affect the composition of the newly synthesized GAG chains. However, it is largely unknown whether structural features of aglycone affect the extent of sulfation, sulfation pattern, disaccharide composition, and chain length of GAG chains. In this study, we show that aglycones can switch not only the type of GAG chains, but also their fine structures. Our findings provide suggestive evidence for the presence of GAGOSOMES that have different combinations of enzymes and their isoforms regulating the synthesis of cell-specific combinatorial structures. We surmise that click-xylosides are differentially recognized by the GAGOSOMES to generate distinct GAG structures as observed in this study. These novel click-xylosides offer new avenues to profile the cell-specific GAG chains, elucidate the mechanism of GAG biosynthesis, and to decipher the biological actions of GAG chains in model organisms. PMID:19628873

  19. Moss cell walls: structure and biosynthesis

    PubMed Central

    Roberts, Alison W.; Roberts, Eric M.; Haigler, Candace H.

    2012-01-01

    The genome sequence of the moss Physcomitrella patens has stimulated new research examining the cell wall polysaccharides of mosses and the glycosyl transferases that synthesize them as a means to understand fundamental processes of cell wall biosynthesis and plant cell wall evolution. The cell walls of mosses and vascular plants are composed of the same classes of polysaccharides, but with differences in side chain composition and structure. Similarly, the genomes of P. patens and angiosperms encode the same families of cell wall glycosyl transferases, yet, in many cases these families have diversified independently in each lineage. Our understanding of land plant evolution could be enhanced by more complete knowledge of the relationships among glycosyl transferase functional diversification, cell wall structural and biochemical specialization, and the roles of cell walls in plant adaptation. As a foundation for these studies, we review the features of P. patens as an experimental system, analyses of cell wall composition in various moss species, recent studies that elucidate the structure and biosynthesis of cell wall polysaccharides in P. patens, and phylogenetic analysis of P. patens genes potentially involved in cell wall biosynthesis. PMID:22833752

  20. Polyketides from dinoflagellates: origins, pharmacology and biosynthesis.

    PubMed

    Rein, K S; Borrone, J

    1999-10-01

    Dinoflagellates, unicellular marine protists, produce some of the largest and most complex polyketides identified to date. The biological activities of these compounds are quite diverse. Compounds having potential therapeutic value as anti-cancer agents as well as deadly neurotoxins, whose production has resulted in severe public health hazards and economic hardships, are represented in this group of secondary metabolites. Stable isotope feeding experiments have firmly established the polyketide origins of representative compounds from each of the three structural classes, the polyether ladders, the macrocycles and the linear polyethers. Yet some unusual labeling patterns have been observed in each class. Pendant methyl groups are most often derived from C-2 of acetate and deletions of C-1 of acetate are common. Studies on the biosynthesis of dinoflagellate derived polyketides at the genomic level have not been reported, in part due to the peculiarities of the dinoflagellate nucleus and the lack of a dinoflagellate transformation system. Nevertheless, a fundamental understanding of the genetics of polyketide biosynthesis by dinoflagellates could be the catalyst for developing several fruitful avenues of research. Dinoflagellate derived polyketides are reviewed with special emphasis on pharmacology and biosynthesis.