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Sample records for adenine nucleotide concentration

  1. Applications of adenine nucleotide measurements in oceanography

    NASA Technical Reports Server (NTRS)

    Holm-Hansen, O.; Hodson, R.; Azam, F.

    1975-01-01

    The methodology involved in nucleotide measurements is outlined, along with data to support the premise that ATP concentrations in microbial cells can be extrapolated to biomass parameters. ATP concentrations in microorganisms and nucleotide analyses are studied.

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

    PubMed

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

    2004-08-01

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

  3. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

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

    1995-03-01

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

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

    SciTech Connect

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

    1990-11-01

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

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

    PubMed

    Sereda, Michal J

    2010-08-01

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

  6. Mathematical Model for Shear Stress Dependent NO and Adenine Nucleotide Production from Endothelial Cells

    PubMed Central

    Kirby, Patrick; Buerk, Donald G.; Parikh, Jaimit; Barbee, Kenneth A.; Jaron, Dov

    2015-01-01

    We developed a mass transport model for a parallel-plate flow chamber apparatus to predict the concentrations of nitric oxide (NO) and adenine nucleotides (ATP, ADP) produced by cultured endothelial cells (ECs) and investigated how the net rates of production, degradation, and mass transport for these three chemical species vary with changes in wall shear stress (τw). These simulations provide an improved understanding of experimental results obtained with parallel-plate flow chambers and allows quantitative analysis of the relationship between τw, adenine nucleotide concentrations, and NO produced by ECs. Experimental data obtained after altering ATP and ADP concentrations with apyrase were analyzed to quantify changes in the rate of NO production (RNO). The effects of different isoforms of apyrase on ATP and ADP concentrations and nucleotide-dependent changes in RNO could be predicted with the model. A decrease in ATP was predicted with apyrase, but an increase in ADP was simulated due to degradation of ATP. We found that a simple proportional relationship relating a component of RNO to the sum of ATP and ADP provided a close match to the fitted curve for experimentally measured changes in RNO with apyrase. Estimates for the proportionality constant ranged from 0.0067 to 0.0321 μM/s increase in RNO per nM nucleotide concentration, depending on which isoform of apyrase was modeled, with the largest effect of nucleotides on RNO at low τw (< 6 dyn/cm2). PMID:26529478

  7. Adenine nucleotides stimulate migration in wounded cultures of kidney epithelial cells.

    PubMed Central

    Kartha, S; Toback, F G

    1992-01-01

    Adenine nucleotides speed structural and functional recovery when administered after experimental renal injury in the rat and stimulate proliferation of kidney epithelial cells. As cell migration is a component of renal regeneration after acute tubular necrosis, we have used an in vitro model of wound healing to study this process. High density, quiescent monkey kidney epithelial cultures were wounded by mechanically scraping away defined regions of the monolayer to simulate the effect of cell loss after tubular necrosis and the number of cells that migrated into the denuded area was counted. Migration was independent of cell proliferation. Provision of adenosine, adenine nucleotides, or cyclic AMP increased the number of migrating cells and accelerated repair of the wound. Other purine and pyrimidine nucleotides were not effective. Arginine-glycine-aspartic acid-serine peptide, which blocks the binding of extracellular fibronectin to its cell surface receptor, completely inhibited migration in the presence or absence of ADP. Very low concentrations of epidermal growth factor (K0.5 approximately 0.3 ng/ml) stimulated migration, whereas transforming growth factor-beta 2 was inhibitory (Ki approximately 0.2 ng/ml). Thus, adenosine and/or adenine nucleotides released from injured or dying renal cells, or administered exogenously, may stimulate surviving cells in the wounded nephron to migrate along the basement membrane, thereby rapidly restoring tubular structure and function. Images PMID:1634617

  8. Brain Injury Alters Ectonucleotidase Activities and Adenine Nucleotide Levels in Rat Serum

    PubMed Central

    Laketa, Danijela; Savić, Jasmina; Bjelobaba, Ivana; Lavrnja, Irena; Vasić, Vesna; Stojiljković, Mirjana; Nedeljković, Nadežda

    2015-01-01

    Summary Background Cortical stab injury (CSI) induces changes in the activity, expression and cellular distribution of specific ectonucleotidases at the injury site. Also, several experimentally induced neuropathologies are associated with changes in soluble ectonucleotidase activities in the plasma and serum, whilst various insults to the brain alter purine compounds levels in cerebrospinal fluid, but also in serum, indicating that insults to the brain may induce alterations in nucleotides release and rate of their hydrolysis in the vascular system. Since adenine nucleotides and adenosine regulate diverse cellular functions in the vascular system, including vascular tone, platelet aggregation and inflammatory responses of lymphocytes and macrophages, alterations of ectonucleotidase activities in the vascular system may be relevant for the clinical outcome of the primary insult. Methods We explored ectonucleotidase activities using specific enzyme assays and determined adenine nucleotides concentrations by the UPLC method in the rat serum after cortical stab injury. Results At 4-h post-injury, ATP and AMP hydrolysis increased by about 60% and 40%, respectively, while phosphodiesterase activity remained unchanged. Also, at 4-h post-injury a marked decrease in ATP concentration and more than 2-fold increase in AMP concentration were recorded. Conclusions CSI induces rapid up-regulation of nucleotide catabolizing soluble ectonucleotidases in rat serum, which leads to the observed shift in serum nucleotide levels. The results obtained imply that ectonucleotidases and adenine nucleotides participate in the communication between the brain and the vascular system in physiological and pathological conditions and thereby may be involved in the development of various human neuropathologies.

  9. 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.

  10. The adsorption and reaction of adenine nucleotides on montmorillonite

    NASA Technical Reports Server (NTRS)

    Ferris, James P.; Hagan, William J., Jr.

    1986-01-01

    The binding of AMP to Zn(2+)-montmorillonite is investigated in the presence of salts and Good's zwitterion buffers, PIPES and MES. The initial concentrations of nucleotide and the percent adsorbtion are used to calculate the adsorption isotherms, and the Langmuir adsorption equation is used for the analysis of data. The adsorption coefficient was found to be three times greater in the presence of 0.2 M PIPES than in its absence. In addition, basal spacings measured by X-ray diffraction were increased by the buffer. These results are interpreted in terms of a model in which the adsorption of AMP is mediated by a Zn(2+) complex of PIPES in different orientations in the interlamellar region of the montmorillonite. Mixed ligand complexes of this type are reminiscent of the complexes observed between metal ions and biological molecules in living systems.

  11. The basal proton conductance of mitochondria depends on adenine nucleotide translocase content

    PubMed Central

    2005-01-01

    The basal proton conductance of mitochondria causes mild uncoupling and may be an important contributor to metabolic rate. The molecular nature of the proton-conductance pathway is unknown. We show that the proton conductance of muscle mitochondria from mice in which isoform 1 of the adenine nucleotide translocase has been ablated is half that of wild-type controls. Overexpression of the adenine nucleotide translocase encoded by the stress-sensitive B gene in Drosophila mitochondria increases proton conductance, and underexpression decreases it, even when the carrier is fully inhibited using carboxyatractylate. We conclude that half to two-thirds of the basal proton conductance of mitochondria is catalysed by the adenine nucleotide carrier, independently of its ATP/ADP exchange or fatty-acid-dependent proton-leak functions. PMID:16076285

  12. High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation.

    PubMed

    Azzu, Vian; Parker, Nadeene; Brand, Martin D

    2008-07-15

    Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.

  13. De novo synthesis of adenine nucleotides in different skeletal muscle fiber types

    SciTech Connect

    Tullson, P.C.; John-Alder, H.B.; Hood, D.A.; Terjung, R.L.

    1988-09-01

    Management of adenine nucleotide catabolism differs among skeletal muscle fiber types. This study evaluated whether there are corresponding differences in the rates of de novo synthesis of adenine nucleotide among fiber type sections of skeletal muscle using an isolated perfused rat hindquarter preparation. Label incorporation into adenine nucleotides from the (1-14C)glycine precursor was determined and used to calculate synthesis rates based on the intracellular glycine specific radioactivity. Results show that intracellular glycine is closely related to the direct precursor pool. Rates of de novo synthesis were highest in fast-twitch red muscle (57.0 +/- 4.0, 58.2 +/- 4.4 nmol.h-1.g-1; deep red gastrocnemius and vastus lateralis), relatively high in slow-twitch red muscle (47.0 +/- 3.1; soleus), and low in fast-twitch white muscle (26.1 +/- 2.0 and 21.6 +/- 2.3; superficial white gastrocnemius and vastus lateralis). Rates for four mixed muscles were intermediate, ranging between 32.3 and 37.3. Specific de novo synthesis rates exhibited a strong correlation (r = 0.986) with muscle section citrate synthase activity. Turnover rates (de novo synthesis rate/adenine nucleotide pool size) were highest in high oxidative muscle (0.82-1.06%/h), lowest in low oxidative muscle (0.30-0.35%/h), and intermediate in mixed muscle (0.44-0.55%/h). Our results demonstrate that differences in adenine nucleotide management among fiber types extends to the process of de novo adenine nucleotide synthesis.

  14. Diminution in adenine nucleotide hydrolysis by platelets and serum from rats submitted to Walker 256 tumour.

    PubMed

    Buffon, Andréia; Ribeiro, Vanessa B; Schanoski, Alessandra S; Sarkis, João J F

    2006-01-01

    Extracellular adenine nucleotide hydrolysis in the circulation is mediated by the action of an NTPDase (CD39, apyrase) and of a 5'-nucleotidase (CD73), presenting as a final product, adenosine. Among other properties described for adenine nucleotides, an anti-cancer activity is suggested, since ATP is considered a cytotoxic molecule in several tumour cell systems. Conversely, some studies demonstrate that adenosine presents a tumour-promoting activity. In this study, we evaluated the pattern of adenine nucleotide hydrolysis by serum and platelets from rats submitted to the Walker 256 tumour model. Extracellular adenine nucleotide hydrolysis by blood serum and platelets obtained from rats at, 6, 10 and 15 days after the subcutaneous Walker 256 tumour inoculation, was evaluated. Our results demonstrate a significant reduction in ATP, ADP and AMP hydrolysis in blood serum at 6, 10 and 15 days after tumour induction. In platelets, a significant reduction in ATP and AMP hydrolysis was observed at 10 and 15 days after tumour induction, while an inhibition of ADP hydrolysis was observed at all times studied. Based on these results, it is possible to suggest a physiologic protection mechanism against the tumoral process in circulation. The inhibition in nucleotide hydrolysis observed probably maintains ATP levels elevated (cytotoxic compound) and, at the same time, reduces the adenosine production (tumour-promoting molecule) in the circulation.

  15. Thiaminylated adenine nucleotides. Chemical synthesis, structural characterization and natural occurrence.

    PubMed

    Frédérich, Michel; Delvaux, David; Gigliobianco, Tiziana; Gangolf, Marjorie; Dive, Georges; Mazzucchelli, Gabriel; Elias, Benjamin; De Pauw, Edwin; Angenot, Luc; Wins, Pierre; Bettendorff, Lucien

    2009-06-01

    Thiamine and its three phosphorylated derivatives (mono-, di- and triphosphate) occur naturally in most cells. Recently, we reported the presence of a fourth thiamine derivative, adenosine thiamine triphosphate, produced in Escherichia coli in response to carbon starvation. Here, we show that the chemical synthesis of adenosine thiamine triphosphate leads to another new compound, adenosine thiamine diphosphate, as a side product. The structure of both compounds was confirmed by MS analysis and 1H-, 13C- and 31P-NMR, and some of their chemical properties were determined. Our results show an upfield shifting of the C-2 proton of the thiazolium ring in adenosine thiamine derivatives compared with conventional thiamine phosphate derivatives. This modification of the electronic environment of the C-2 proton might be explained by a through-space interaction with the adenosine moiety, suggesting U-shaped folding of adenosine thiamine derivatives. Such a structure in which the C-2 proton is embedded in a closed conformation can be located using molecular modeling as an energy minimum. In E. coli, adenosine thiamine triphosphate may account for 15% of the total thiamine under energy stress. It is less abundant in eukaryotic organisms, but is consistently found in mammalian tissues and some cell lines. Using HPLC, we show for the first time that adenosine thiamine diphosphate may also occur in small amounts in E. coli and in vertebrate liver. The discovery of two natural thiamine adenine compounds further highlights the complexity and diversity of thiamine biochemistry, which is not restricted to the cofactor role of thiamine diphosphate.

  16. Adenine Nucleotides Control Proliferation In Vivo of Rat Retinal Progenitors by P2Y1 Receptor.

    PubMed

    de Almeida-Pereira, Luana; Magalhães, Camila Feitosa; Repossi, Marinna Garcia; Thorstenberg, Maria Luiza Prates; Sholl-Franco, Alfred; Coutinho-Silva, Robson; Ventura, Ana Lucia Marques; Fragel-Madeira, Lucianne

    2016-08-24

    Previous studies demonstrated that exogenous ATP is able to regulate proliferation of retinal progenitor cells (RPCs) in vitro possibly via P2Y1 receptor, a G protein-coupled receptor. Here, we evaluated the function of adenine nucleotides in vivo during retinal development of newborn rats. Intravitreal injection of apyrase, an enzyme that hydrolyzes nucleotides, reduced cell proliferation in retinas at postnatal day 2 (P2). This decrease was reversed when retinas were treated together with ATPγ-S or ADPβ-S, two hydrolysis-resistant analogs of ATP and ADP, respectively. During early postnatal days (P0 to P5), an increase in ectonucleotidase (E-NTPDase) activity was observed in the retina, suggesting a decrease in the availability of adenine nucleotides, coinciding with the end of proliferation. Interestingly, intravitreal injection of the E-NTPDase inhibitor ARL67156 increased proliferation by around 60 % at P5 rats. Furthermore, immunolabeling against P2Y1 receptor was observed overall in retina layers from P2 rats, including proliferating Ki-67-positive cells in the neuroblastic layer (NBL), suggesting that this receptor could be responsible for the action of adenine nucleotides upon proliferation of RPCs. Accordingly, intravitreal injection of MRS2179, a selective antagonist of P2Y1 receptors, reduced cell proliferation by approximately 20 % in P2 rats. Moreover, treatment with MRS 2179 caused an increase in p57(KIP2) and cyclin D1 expression, a reduction in cyclin E and Rb phosphorylated expression and in BrdU-positive cell number. These data suggest that the adenine nucleotides modulate the proliferation of rat RPCs via activation of P2Y1 receptors regulating transition from G1 to S phase of the cell cycle.

  17. Neonatal hypothyroidism affects the adenine nucleotides metabolism in astrocyte cultures from rat brain.

    PubMed

    Braganhol, Elizandra; Bruno, Alessandra Nejar; Bavaresco, Luci; Barreto-Chaves, Maria Luiza M; Sarkis, João José Freitas; Battastini, Ana Maria Oliveira

    2006-04-01

    Neonatal hypothyroidism is associated with multiple and severe brain alterations. We recently demonstrated a significant increase in hydrolysis of AMP to adenosine in brain of hypothyroid rats at different ages. However, the origin of this effect was unclear. Considering the effects of adenine nucleotides to brain functions and the harmful effects of neonatal hypothyroidism to normal development of the central nervous system, in this study we investigated the metabolism of adenine nucleotides in hippocampal, cortical and cerebellar astrocyte cultures from rats submitted to neonatal hypothyroidism. ATP and AMP hydrolysis were enhanced by 52 and 210%, respectively, in cerebellar astrocytes from hypothyroid rats. In hippocampus of hypothyroid rats, the 47% increase in AMP hydrolysis was significantly reverted when the astrocytes were treated with T3. Therefore, the imbalance in the ATP and adenosine levels in astrocytes, during brain development, may contribute to some of the effects described in neonatal hypothyroidism.

  18. White spot syndrome virus VP12 interacts with adenine nucleotide translocase of Litopenaeus vannamei.

    PubMed

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

    2014-05-01

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

  19. Interrelationships between hydrogen-supplying reactions, respiration rate and extramitochondrial adenine nucleotide pattern.

    PubMed

    Böhme, G; Schönfeld, P; Bohnensack, R; Küster, U; Kunz, W

    1982-01-01

    1. The influence of a diminished hydrogen supply on the regulation of oxidative phosphorylation of isolated rat liver mitochondria in dependence on the extramitochondrial (ATP)/(ADP) ratio was investigated. 2. The hydrogen supply was diminished by using various (beta-hydroxybutyrate)/(acetoacetate) ratios as a redox buffer and the results were compared with those of experiments using perifusion of immobilized mitochondria with non-saturating substrate concentrations. 3. In both experimental approaches the influence of a diminished hydrogen pressure on the maximum (ATP)/(ADP) ratio at minimum flux was low. An extreme decrease in the (beta-hydroxybutyrate)/(acetoacetate) ratio by more than two orders of magnetitude causes the (APT)/(ADP) ratio to decrease by about 50%. 4. The load capacity of oxidative phosphorylation (maximum flux) is considerably decreased by diminished hydrogen pressure. 5. The borderline cases of purely kinetic and thermodynamic limitations of hydrogen supply were calculated by computer simulation with respect to the regulating behaviour of oxidative phosphorylation and changes in the control strength of adenine nucleotide translocator and hydrogen supply in the overall reaction. 6. A prevalent thermodynamic influence of hydrogen supply on oxidative energy transformation in the cell is discussed in the light of experimental data.

  20. The adsorption and reaction of adenine nucleotides on montmorillonite

    NASA Astrophysics Data System (ADS)

    Ferris, James P.; Hagan, William J.

    1986-03-01

    The binding of AMP to Zn2+-montmorillonite was investigated in the presence of buffers and salts. Good's buffers, piperazine-N,N'-bis(2-ethanesulfonate) [PIPES] and morpholine-N-2-ethanesulfonate [MES], perturbed the exchangeable cations to a lesser extent (only 9% of Zn2+ displaced by 0.2 M buffer) than was observed with imidazole and lutidine buffers or NaCl and KCl salts (up to 80% of Zn2+ displaced). AMP adsorption isotherms measured in the presence of 0.2 M PIPES, MES or Na2SO4 exhibited normal Langmuir-type behavior. The adsorption coefficient, KL, is 3-fold greater in the presence of HEPES or PIPES than it is in the absence of buffers. Basal spacings measured by X-ray diffraction for Zn2+-montmorillonite are 13 and 15 Å in the presence of PIPES, while a value of 12.8 Å was determined in the absence of PIPES. These data are interpreted in a model in which the adsorption of AMP is mediated by a Zn2+ complex of PIPES in different orientations in the interlamellar region of the montmorillonite. The type of exchangeable cation does not affect the ability of the lattice-bound Fe3+ in the montmorillonite to oxidize diaminomaleonitrile (DAMN). Exchangeable Cu2+ oxidizes DAMN, but exchangeable Fe3+ is nearly ineffective as an oxidant. The addition if DISN to 3'-AMP bound to Zn2+-montmorillonite in the presence of 0.2 M PIPES resulted in a higher yield of 2', 3'-cAMP than is observed with a comparable concentration of Zn2+, a result which implicates surface catalystis by the montmorillonite.

  1. Comparison of the effects of adenine-ribose with adenosine for maintenance of ATP concentrations in 5-day hypothermically perfused dog kidneys.

    PubMed

    McAnulty, J F; Southard, J H; Belzer, F O

    1988-10-01

    The quality of preservation of kidneys is dependent upon a number of factors, one of which may be the concentration of adenine nucleotides in the tissue during long-term perfusion preservation. In this study we have investigated how adenine (5 mM) and ribose (5 mM) in combination affect the concentration of adenine nucleotides in dog kidney cortical tissue after 5 days of continuous hypothermic perfusion preservation. These results were compared to kidneys perfused with adenosine and without any added purine precursors of adenine nucleotide synthesis. Additionally, we investigated how these conditions affected renal tissue slice function after 5 days of preservation and how adenine plus ribose affected renal function after autotransplantation in the dog. Adenosine is nearly completely degraded during 5 days of perfusion but there was little loss of adenine (10%). The adenosine triphosphate concentration in kidney cortical tissue was higher in adenine/ribose-perfused kidneys (1.41 +/- 0.19 mumol/g) than in adenosine-perfused kidneys (0.71 +/- 0.1 mumol/g) after 5 days of preservation. Tissue slices prepared from kidneys preserved in the presence of adenine plus ribose were metabolically more functional (slice volume control and electrolyte pump activity) than slices from adenosine-perfused kidneys. Adenine plus ribose had no detrimental effects on kidneys preserved for 3 days as tested in the autotransplant model but did not yield successful 5-day preservation. Because of some potentially detrimental factors in using adenosine as an adenine nucleotide synthesis precursor, we have now switched to the combination of adenine and ribose for perfusion preservation of kidneys both in the laboratory and in the clinic.

  2. Inhibitory and Restorative Effects of Adenine Nucleotides on Rickettsial Adsorption and Hemolysis

    PubMed Central

    Winkler, Herbert H.

    1974-01-01

    The adenine nucleotides, adenosine diphosphate, adenosine triphosphate, (ATP), and the methylene-bridge analogues are inhibitors of rickettsial adsorption to and the hemolysis of sheep erythrocytes. Other nucleotides, adenosine monophosphate, cyclic adenosine monophosphate, cytosine triphosphate, and guanosine triphosphate, are without effect. Adsorption and hemolysis require the generation of energy by the rickettsiae which is usually derived from glutamate. When the generation of energy from the metabolism of glutamate is inhibited by arsenite or cyanide, the addition of ATP can supply the energy to restore hemolysis. However, in the presence of the uncouplers, ATP can not restore hemolysis. Even when functioning in a restorative role, ATP still has its inhibitory properties. These results suggest that a high-energy intermediate (X ∼ I), rather than ATP itself, is the energy source. The interactions of inhibitory nucleotides suggest that these compounds share a common transport system. PMID:4357933

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

    PubMed Central

    Dascombe, M J; Milton, A S

    1975-01-01

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

  4. Adenine Nucleotide Levels in the Cytosol, Chloroplasts, and Mitochondria of Wheat Leaf Protoplasts 1

    PubMed Central

    Stitt, Mark; Lilley, Ross McC.; Heldt, Hans W.

    1982-01-01

    Recently, a new method has been described, in which membrane filtration is used to allow the levels of adenine nucleotides in the chloroplast stroma, the cytosol, and the mitochondrial matrix to be measured. This method is now used to investigate the effect of illumination, of respiratory inhibitors, and of uncouplers on the distribution of ATP, ADP, and AMP in wheat (Triticum aestivum var. `Timmo') leaf protoplasts. (a) The adenine nucleotides are apparently equilibrated by adenylate kinase in the stroma and the cytosol, but not in the mitochondrial matrix. (b) The ATP/ADP quotient in the cytosol is considerably higher than that in the mitochondrial matrix or the chloroplast stroma. (c) A large gradient exists between the ATP/ADP quotients in the cytosol and the mitochondrial matrix in the dark, with a very low ATP/ADP quotient in the mitochondria. This gradient is lowered by uncouplers or respiratory inhibitors showing that, as in animal tissues, it reflects the energization of the mitochondria. (d) In the dark, the stromal ATP/ADP is lower than in the light, and appears to be maintained, at least in part, by import from the cytosol. (e) The cytosolic ATP/ADP, however, actually decreases in the light. This contradicts the widespread assumption, that export of photosynthetically produced ATP from the chloroplast leads to an increase in the cytosolic ATP/ADP, which then inhibits oxidative phosphorylation in the mitochondria. (f) The mitochondrial ATP/ADP increases in the light, and the gradient between the cytosol and mitochondrial matrix falls. This is also difficult to understand in terms of an inhibition of oxidative phosphorylation in the light due to a lack of ADP in the cytosol. (g) The significance of the measured variations in the adenine nucleotide pools are discussed with respect to the diurnal carbohydrate metabolism in a leaf, and to the metabolic function of the chloroplast, the cytosol and the mitochondria. PMID:16662653

  5. Effect of 1-aminoadamantanes on adenine nucleotide and serotonin storage in blood platelets.

    PubMed

    Wesemann, W; Muschalek, G; Stöltzing, H; von Pusch, I; Paul, N

    1981-12-01

    The platelet release reaction, the liberation of adenine nucleotides and serotonin (5-HT) from osmiophilic dense granules, can be induced in human platelets by C-alkyl-derivatives of the antiviral and anti-Parkinson drug 1-aminoadamantane (D 1). The release-inducing activity is enhanced with increasing chain length and with the number of C-alkylsubstituents, respectively. The parent compound, D 1, liberates only 5-HT. Analyses of LDH activity in the incubation medium of the platelets and electron micrographs of platelets treated with 1-aminoadamantanes support the assumption that ATP, ADP, and 5-HT are released from the organelles by an exocytosis-like process rather than by destruction of the plasma and organelle membranes. The number of osmiophilic dense granules in platelets isolated from human and rabbit blood is decreased by the action of the drugs. This is in contrast to other subcellular structures where no morphological changes can be observed. The ADP-stimulated platelet aggregation is inhibited by preincubation with 1-aminoadamantanes. The inhibitory activity of the drugs on platelet aggregation parallels the effects observed after induction of the release reaction: the inhibition of platelet aggregation is enhanced with increasing C-alkylation. Hence, the inhibition of ADP-induced platelet aggregation can be used to screen for the releasing activity of 1-aminoadamantanes which have, as far as tested, similar effects on 5-HT storage in blood platelets and in the CNS. The time-, temperature, and concentration-dependent 5-HT uptake by platelets (K = 0.75 micro M; V max = 0.22 nmoles/min X 10 9 cells) is noncompetitively inhibited by the drugs with K1-values varying from 10 to 100 micro M depending on the degree of C-alkylation.

  6. Thyroid hormone action: identification of the mitochondrial thyroid hormone receptor as adenine nucleotide translocase.

    PubMed

    Sterling, K

    1991-01-01

    A preliminary report from our laboratory suggested that the thyroid hormone triiodothyronine (T3) is bound with an association constant (Ka) approximating 2 x 10(11) M-1 by adenine nucleotide translocase (AdNT) purified from beef heart mitochondria. We now report that [125I]T3 is capable of photoaffinity labeling not only purified AdNT but also the carrier in intact beef heart mitochondria. Photoaffinity labeling in intact mitochondria was appreciably greater than that observed with purified AdNT. The covalently labeled AdNT was identified by 2-dimensional electrophoresis with pI of 10 on electrofocusing and M(r) of 31,000 on SDS gel. Identification of the covalently labeled protein as authentic AdNT was substantiated by its interaction with a specific monoclonal antibody preparation.

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

    PubMed

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

    2011-03-01

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

  8. [Characteristics of adenine nucleotide translocator in mitochondria of rat cerebral cortex during hypobaric hypoxia exposure].

    PubMed

    Chen, Li-Fen; Liu, Jun-Ze; Li, Bing

    2006-02-25

    The purpose of the present study was to explore the effects of hypoxic exposure on mitochondrial adenine nucleotide translocator (ANT) activity and its characteristics. Male Wistar rats were exposed to hypoxia in a hypobaric chamber simulating high altitude at 5 000 m for 1, 5, 15 and 30 d. Control rats were fed outside the hypobaric chamber. Rats were sacrificed by decapitation and mitochondria from the cerebral cortex were isolated by differential centrifugation at each time point. The ANT activity was detected by the atractyloside (ATR)-inhibitor stop technique. Mitochondria was initiated by addition of (3)H-ADP and terminated after 12 s by quick addition of ATR. The radioactivity was measured in a liquid scintillation counter. Nonspecific binding of (3)H-ADP to mitochondria was estimated by incubation of mitochondrial samples with ATR prior to the addition of (3)H-ADP. This blank was substracted from the measured radioactivities. The activity of ANT was expressed as nanomoles (3)H-ADP per minute per milligram protein. The ANT density was determined by titrating the rate of state 3 respiration with increasing concentrations of carboxyatractyloside (CAT). Mitochondria were pre-incubated with CAT in a respiratory medium before ADP addition to initiate state 3 respiration. Plots of O2 consumption versus CAT appeared biphasic with an increasing inhibitory segment followed by a steady respiration, indicating that state 3 respiration was completely inhibited. The density of ANT was determined by the amount of CAT required to completely inhibit state 3 respiration, assuming a 1:1 binding stoichiometry, which was expressed as ANT density per milligram mitochondria protein. (ATP+ADP) in mitochondria was measured by high performance liquid chromatography (HPLC). The results showed that there was an obvious decrease in the ANT activity during hypoxic exposure. The lowest ANT activity was seen in 5 d group. Partial recovery of ANT activity was observed in 15 and 30 d groups

  9. Homo- and heteroexchange of adenine nucleotides and nucleosides in rat hippocampal slices by the nucleoside transport system

    PubMed Central

    Sperlágh, Beáta; Szabó, Gábor; Erdélyi, Ferenc; Baranyi, Mária; Sylvester Vizi, E

    2003-01-01

    Here, we investigated how nucleotides and nucleosides affect the release of tritiated purines and endogenous adenosine 5′-triphosphate (ATP) from superfused rat hippocampal slices. ATP elicited concentration-dependent [3H]purine efflux from slices preloaded with [3H]adenosine. High-performance liquid chromatography analysis of the effluent showed that the tritium label represented the whole set of adenine nucleotides and nucleosides, and ATP significantly increased the outflow of [3H]ATP. Adenosine 5′-diphosphate, adenosine, uridine, uridine 5′-triphosphate, α,β-methylene-ATP and 3′-O-(4-benzoylbenzoyl)-ATP were also active in eliciting [3H]purine release. Adenosine (300 μM) also evoked endogenous ATP efflux from the hippocampal slices. Reverse transcription-coupled-polymerase chain reaction analysis revealed that mRNAs encoding a variety of P2X and P2Y receptor proteins are expressed in the rat hippocampus. Nevertheless, neither P2 receptor (i.e. pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid, 30 μM, suramin, 300 μM and reactive blue 2, 10 μM), nor adenosine receptor (8-cyclopentyl-1,3-dipropylxanthine, 250 nM and dimethyl-1-propargylxanthine, 250 nM) antagonists modified the effect of ATP (300 μM) to evoke [3H]purine release. The nucleoside transport inhibitors, dipyridamole (10 μM), nitrobenzylthioinosine (10 μM) and adenosine deaminase (2–10 U ml−1), but not the ecto-adenylate kinase inhibitor diadenosine pentaphosphate (200 μM) significantly reduced ATP-evoked [3H]purine efflux. In summary, we found that ATP and other nucleotides and nucleosides promote the release of one another and themselves by the nucleoside transport system. This action could have relevance during physiological and pathological elevation of extracellular purine levels high enough to reverse the nucleoside transporter. PMID:12788822

  10. Biapigenin modulates the activity of the adenine nucleotide translocator in isolated rat brain mitochondria.

    PubMed

    Silva, Bruno A; Oliveira, Paulo J; Cristóvão, Armando; Dias, Alberto C P; Malva, João O

    2010-01-01

    In this study, we investigated the effects of biapigenin, a biflavone present in the extracts of Hypericum perforatum, in rat brain mitochondrial bioenergetics and calcium homeostasis. We found that biapigenin significantly decreased adenosine diphosphate (ADP)-induced membrane depolarization and increased repolarization (by 68 and 37%, respectively). These effects were blocked by atractyloside and bongkrekic acid, but not oligomycin. In the presence of biapigenin, an ADP-stimulated state 3 respiration was still noticeable, which did not happen in the presence of adenine nucleotide translocator (ANT) inhibitors. Taking in consideration the relevance of the ANT in the modulation of the mitochondrial permeability transition pore (mPTP), mitochondrial calcium homeostasis was evaluated alone or in the presence of biapigenin. We found that biapigenin reduces mitochondrial calcium retention by increasing calcium efflux, an effect that was blocked by ADP plus oligomycin, an efficient blocker of the mPTP in brain mitochondria. Taken together, the results in this article suggest that biapigenin modulates mPTP opening, possibly by modulating ANT function, contributing for enhanced mitochondrial calcium efflux, thereby reducing calcium burden and contributing for neuroprotection against excitotoxicity.

  11. 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

  12. Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides.

    PubMed Central

    Cunha, R. A.; Correia-de-Sá, P.; Sebastião, A. M.; Ribeiro, J. A.

    1996-01-01

    1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings. PMID:8886406

  13. 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.

  14. 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.

  15. Adenine nucleotide levels in a closed enzymatic digestion system for porcine islet isolation.

    PubMed

    Oshibe, Ikuro; Saito, Takuro; Sato, Yoshihiro; Saito, Takaharu; Tsukada, Manabu; Ise, Kazuya; Kenjo, Akira; Kimura, Takashi; Anazawa, Takayuki; Suzuki, Shigeya; Hashimoto, Yasuhiro; Gotoh, Mitusukazu

    2012-01-01

    Obtaining viable islets is a crucial step for successful islet transplantation. Adenosine triphosphate (ATP) is a marker of cell viability. However, little is known about any changes in the energy status of the tissues that are being digested during the digestion phase. We herein examined whether the ATP content in serially digested pancreatic tissue samples could be specific objective parameters that signal the optimal point to stop the digestion process. We obtained partial pancreata (body to tail) from 4- to 5-year-old pigs from a slaughterhouse. The tissue samples were preserved in M-Kyoto solution for less than 3 h. They were digested using an automated enzymatic and mechanical dissociation system at 37°C for 90 min following intraductal injection of Liberase HI. Samples were collected from the digestive circuit every 5 or 10 min to determine the ATP level, total adenine nucleotide (TAN) level, islet count (count/g), and yield of islet equivalent (IEQ) in the serial digestive fluids. The ATP and TAN levels, IEQ and islet count were increased and then decreased during digestion process. The profile of these parameters differed from case to case. However, when ATP changing ratio (respective value/precedent value) was compared with IEQ changing ratio, a greater than threefold increase in the ATP changing ratio followed by an increase in the islet count changing ratio within 5 min was consistently observed, indicating the optimal time to stop the digestion. The ATP levels of the handpicked islets in the digested samples were lower in the overdigested phase in comparison to those in the earlier digested phase. These results indicate that the ATP level in digested fluid could be an effective indicator to estimate the viability of cells as well as determine the optimal time to terminate the digestion process in order to obtain viable islets.

  16. Deficiency in the mouse mitochondrial adenine nucleotide translocator isoform 2 gene is associated with cardiac noncompaction.

    PubMed

    Kokoszka, Jason E; Waymire, Katrina G; Flierl, Adrian; Sweeney, Katelyn M; Angelin, Alessia; MacGregor, Grant R; Wallace, Douglas C

    2016-08-01

    The mouse fetal and adult hearts express two adenine nucleotide translocator (ANT) isoform genes. The predominant isoform is the heart-muscle-brain ANT-isoform gene 1 (Ant1) while the other is the systemic Ant2 gene. Genetic inactivation of the Ant1 gene does not impair fetal development but results in hypertrophic cardiomyopathy in postnatal mice. Using a knockin X-linked Ant2 allele in which exons 3 and 4 are flanked by loxP sites combined in males with a protamine 1 promoter driven Cre recombinase we created females heterozygous for a null Ant2 allele. Crossing the heterozygous females with the Ant2(fl), PrmCre(+) males resulted in male and female ANT2-null embryos. These fetuses proved to be embryonic lethal by day E14.5 in association with cardiac developmental failure, immature cardiomyocytes having swollen mitochondria, cardiomyocyte hyperproliferation, and cardiac failure due to hypertrabeculation/noncompaction. ANTs have two main functions, mitochondrial-cytosol ATP/ADP exchange and modulation of the mitochondrial permeability transition pore (mtPTP). Previous studies imply that ANT2 biases the mtPTP toward closed while ANT1 biases the mtPTP toward open. It has been reported that immature cardiomyocytes have a constitutively opened mtPTP, the closure of which signals the maturation of cardiomyocytes. Therefore, we hypothesize that the developmental toxicity of the Ant2 null mutation may be the result of biasing the cardiomyocyte mtPTP to remain open thus impairing cardiomyocyte maturation and resulting in cardiomyocyte hyperproliferation and failure of trabecular maturation. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

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

    PubMed

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

    2015-01-01

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

  18. Adenine nucleotide translocase 4 is expressed within embryonic ovaries and dispensable during oogenesis.

    PubMed

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

    2015-02-01

    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.

  19. ADENINE NUCLEOTIDE TRANSLOCASE-1 INDUCES CARDIOMYOCYTE DEATH THROUGH UPREGULATION OF THE PRO-APOPTOTIC PROTEIN BAX

    PubMed Central

    Baines, Christopher P.; Molkentin, Jeffery D.

    2009-01-01

    Overexpression of the adenine nucleotide translocase (ANT) has been shown to be cytotoxic in several cell types. Although ANT was originally proposed to be a critical component of the mitochondrial permeability transition (MPT) pore, recent data have suggested that this may not be the case. We therefore hypothesized that the cytotoxic actions of ANT are through an alternative mechanism, independent of the MPT pore. Infection of cultured neonatal cardiomyocytes with an ANT1-encoding adenovirus induced a gene dosage-dependent increase in cell death. However, ANT1 overexpression failed to induce MPT, and neither pharmacological nor genetic inhibition of the MPT pore was able to prevent ANT1-induced cell death. These data suggested that ANT1-induced death progressed through an MPT pore-independent pathway. Somewhat surprisingly, we observed that protein levels of Bax, a pro-apoptotic Bcl protein, were consistently elevated in ANT1-infected cardiomyocytes. Membranes isolated from ANT1-infected myocytes exhibited significantly increased amounts of membrane-inserted Bax, and immunocytochemistry revealed increased Bax activation in ANT1-infected myocytes. Co-expression with the Bax antagonist Bcl2 was able to greatly reduce the degree of ANT1-induced cell death. Furthermore, Bax/Bak-deficient fibroblasts were resistant to the cytotoxic effects of ANT1 overexpression. Interestingly, ANT1 overexpression was also associated with enhanced production of reactive oxygen species (ROS), and the antioxidant MnTBAP was able to significantly attenuate both the ANT1-induced upregulation of Bax and cell death. Taken together, these data indicate that ANT mediates cell death, not through the MPT pore, but rather via a ROS-dependent upregulation and activation of Bax. PMID:19452617

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

    PubMed Central

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

    2015-01-01

    Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4. PMID:25742135

  1. A new regulatory principle for in vivo biochemistry: pleiotropic low affinity regulation by the adenine nucleotides--illustrated for the glycolytic enzymes of Saccharomyces cerevisiae.

    PubMed

    Mensonides, Femke I C; Bakker, Barbara M; Cremazy, Frederic; Messiha, Hanan L; Mendes, Pedro; Boogerd, Fred C; Westerhoff, Hans V

    2013-09-02

    Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction.

  2. Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells.

    PubMed

    Onetti, C G; Lara, J; García, E

    1996-05-01

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

  3. Evidence for the presence and role of tightly bound adenine nucleotides in phospholipid-free purified Micrococcus lysodeikticus adenosine triphosphatase.

    PubMed

    Muñoz, C; Palacios, P; Muñoz, E

    1977-10-01

    [32P]-labeled ATPase was isolated in a highly purified state from Micrococcus lysodeikticus strain PNB grown in medium supplemented with [32P]orthophosphate. Selective extraction procedures allowed us to determine that at least 25% of the firmly bound label belonged to adenine nucleotides, ATP and ADP being present in equimolar amounts. However, no 32P label was found to be part of phospholipids. This was confirmed by purification of the ATPase from cells fed with [2-3H]glycerol. Using the luciferin-luciferase assay we estimated that ATPase freshly isolated by Sephadex chromatography (specific activity 10-14 micromole substrate transformed x min(-1) x mg protein(-1)) contained 2 moles ATP/mole of enzyme. The ratio fell with the age of enzyme and its purification by gel electrophoresis and this was paralleled by a loss of ATPase activity. The endogenous nucleotides were readily exchanged by added ADP or ATP. This result suggests that the sites for tight binding of adenine nucleotides are equivalent, although ADP seems to have a higher affinity for them. The last properties represent a peculiar characteristic of this bacterial ATPase as compared with other bacterial and organelle energy-transducing proteins.

  4. Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

    PubMed Central

    Chinopoulos, Christos; Konràd, Csaba; Kiss, Gergely; Metelkin, Eugeniy; Töröcsik, Beata; Zhang, Steven F.; Starkov, Anatoly A.

    2011-01-01

    Cyclophilin D was recently shown to bind to and decrease the activity of F0F1-ATP synthase in submitochondrial particles and permeabilized mitochondria (Giorgio et al. 2009, J Biol Chem, 284:33982). Cyclophilin D binding decreased both the ATP synthesis and hydrolysis rates. Here, we reaffirm these findings by demonstrating that in intact mouse liver mitochondria energized by ATP, absence of cyclophilin D or presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria an increase in F0F1-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident as slightly increased respiration rates during arsenolysis. However, the modulation of F0F1-ATP synthase by cyclophilin D did not increase the ANT-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ~2.2 times lower flux control coefficient of the F0F1-ATP synthase than that of ANT, deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that a ~30% change in F0F1-ATP synthase activity in fully energized or fully deenergized mitochondria affects ADP-ATP exchange rate mediated by the ANT in the range of 1.38-1.7%. We conclude that in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or inhibition of its binding to F0F1-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels. PMID:21281446

  5. Hypothesis on Skeletal Muscle Aging: Mitochondrial Adenine Nucleotide Translocator Decreases Reactive Oxygen Species Production While Preserving Coupling Efficiency

    PubMed Central

    Diolez, Philippe; Bourdel-Marchasson, Isabelle; Calmettes, Guillaume; Pasdois, Philippe; Detaille, Dominique; Rouland, Richard; Gouspillou, Gilles

    2015-01-01

    Mitochondrial membrane potential is the major regulator of mitochondrial functions, including coupling efficiency and production of reactive oxygen species (ROS). Both functions are crucial for cell bioenergetics. We previously presented evidences for a specific modulation of adenine nucleotide translocase (ANT) appearing during aging that results in a decrease in membrane potential - and therefore ROS production—but surprisingly increases coupling efficiency under conditions of low ATP turnover. Careful study of the bioenergetic parameters (oxidation and phosphorylation rates, membrane potential) of isolated mitochondria from skeletal muscles (gastrocnemius) of aged and young rats revealed a remodeling at the level of the phosphorylation system, in the absence of alteration of the inner mitochondrial membrane (uncoupling) or respiratory chain complexes regulation. We further observed a decrease in mitochondrial affinity for ADP in aged isolated mitochondria, and higher sensitivity of ANT to its specific inhibitor atractyloside. This age-induced modification of ANT results in an increase in the ADP concentration required to sustain the same ATP turnover as compared to young muscle, and therefore in a lower membrane potential under phosphorylating—in vivo—conditions. Thus, for equivalent ATP turnover (cellular ATP demand), coupling efficiency is even higher in aged muscle mitochondria, due to the down-regulation of inner membrane proton leak caused by the decrease in membrane potential. In the framework of the radical theory of aging, these modifications in ANT function may be the result of oxidative damage caused by intra mitochondrial ROS and may appear like a virtuous circle where ROS induce a mechanism that reduces their production, without causing uncoupling, and even leading in improved efficiency. Because of the importance of ROS as therapeutic targets, this new mechanism deserves further studies. PMID:26733871

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

    PubMed

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

    2009-07-01

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

  7. 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

  8. Modulation of murine dendritic cell function by adenine nucleotides and adenosine: involvement of the A(2B) receptor.

    PubMed

    Ben Addi, Abduelhakem; Lefort, Anne; Hua, Xiaoyang; Libert, Frédérick; Communi, Didier; Ledent, Catherine; Macours, Pascale; Tilley, Stephen L; Boeynaems, Jean-Marie; Robaye, Bernard

    2008-06-01

    Adenosine triphosphate has previously been shown to induce semi-mature human monocyte-derived dendritic cells (DC). These are characterized by the up-regulation of co-stimulatory molecules, the inhibition of IL-12 and the up-regulation of some genes involved in immune tolerance, such as thrombospondin-1 and indoleamine 2,3-dioxygenase. The actions of adenosine triphosphate are mediated by the P2Y(11) receptor; since there is no functional P2Y(11) gene in the murine genome, we investigated the action of adenine nucleotides on murine DC. Adenosine 5'-(3-thiotriphosphate) and adenosine inhibited the production of IL-12p70 by bone marrow-derived DC (BMDC). These inhibitions were relieved by 8-p-sulfophenyltheophylline, an adenosine receptor antagonist. The use of selective ligands and A(2B) (-/-) BMDC indicated the involvement of the A(2B) receptor. A microarray experiment, confirmed by quantitative PCR, showed that, in presence of LPS, 5'-(N-ethylcarboxamido) adenosine (NECA, the most potent A(2B) receptor agonist) regulated the expression of several genes: arginase I and II, thrombospondin-1 and vascular endothelial growth factor were up-regulated whereas CCL2 and CCL12 were down-regulated. We further showed that NECA, in combination with LPS, increased the arginase I enzymatic activity. In conclusion, the described actions of adenine nucleotides on BMDC are mediated by their degradation product, adenosine, acting on the A(2B) receptor, and will possibly lead to an impairment of Th1 response or tolerance.

  9. Biospecific affinity chromatography of an adenosine 3′:5′-cyclic monophosphate-stimulated protein kinase (protamine kinase from trout testis) by using immobilized adenine nucleotides

    PubMed Central

    Jergil, Bengt; Guilford, Hugh; Mosbach, Klaus

    1974-01-01

    1. Two adenine nucleotides, 8-(6-aminohexyl)aminoadenosine 3′:5′-cyclic monophosphate and 8-(6-aminohexyl)amino-AMP, were synthesized. Their structures were established in particular by using mass spectroscopy. 2. Free cyclic AMP and 8-(6-aminohexyl)amino cyclic AMP both stimulate protamine kinase activity at low concentrations, but are inhibitory at concentrations above 0.1mm. AMP is an inhibitor of enzymic activity, whereas neither 8-(6-aminohexyl)amino-AMP nor the earlier synthesized N6-(6-aminohexyl)-AMP is inhibitory. 3. The nucleotides were coupled to Sepharose 4B and used for biospecific chromatography of partially purified protamine kinase. Enzyme applied at high buffer concentrations to the cyclic AMP–Sepharose material was retarded and thereby purified tenfold. At low buffer concentrations the enzyme was adsorbed to the affinity material, and was subsequently released by a pulse of the inhibitor AMP, yielding a 50–100-fold purification. Enzyme applied to immobilized 8-(6-aminohexyl)amino-AMP or N6-(6-aminohexyl)-AMP was eluted together with the main protein peak in the void volume. 4. Protamine kinase eluted from 8-(6-aminohexyl)amino cyclic AMP–Sepharose was no longer activated by cyclic AMP. Results from sucrose gradient centrifugation suggest that a dissociation of the enzyme took place on the immobilized nucleotide. 5. Further information on the mass spectroscopy has been deposited as Supplementary Publication SUP 50026 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5. PMID:4374933

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

    PubMed

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

    2009-12-11

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

  11. Genetic mapping of human heart-skeletal muscle adenine nucleotide translocator and its relationship to the facioscapulohumeral muscular dystrophy locus

    SciTech Connect

    Haraguchi, Y.; Chung, A.B.; Torroni, A.; Stepien, G.; Shoffner, J.M.; Costigan, D.A.; Polak, M.; Wasmuth, J.J.; Altherr, M.R.; Winokur, S.T.

    1993-05-01

    The mitochondrial heart-skeletal muscle adenine nucleotide translocator (ANT1) was regionally mapped to 4q35-qter using somatic cell hybrids containing deleted chromosome 4. The regional location was further refined through family studies using ANT1 intron and promoter nucleotide polymorphisms recognized by the restriction endonucleases MboII, NdeI, and HaeIII. Two alleles were found, each at a frequency of 0.5. The ANT1 locus was found to be closely linked to D4S139, D4S171, and the dominant skeletal muscle disease locus facioscapulohumeral muscular dystrophy (FSHD). A crossover that separated D4S171 and ANT1 from D4S139 was found. Since previous studies have established the chromosome 4 map order as centromere-D4S171-D4S139-FSHD, it was concluded that ANT1 is located on the side of D4S139, that is opposite from FSHD. This conclusion was confirmed by sequencing the exons and analyzing the transcripts of ANT1 from several FSHD patients and finding no evidence of aberration. 35 refs., 5 figs., 1 tab.

  12. Enzymes that hydrolyze adenine nucleotides in platelets and polymorphisms in the alpha2 gene of integrin alpha2beta1 in patients with von Willebrand disease.

    PubMed

    Santos, Karen Freitas; Battisti, Vanessa; Corrêa, Maísa de Carvalho; Mann, Thaís Rapachi; Pereira, Renata da Silva; Araújo, Maria do Carmo; Brülê, Alice Odete; Schetinger, Maria Rosa Chitolina; Morsch, Vera Maria

    2010-07-01

    Von Willebrand disease (VWD) is one of the most common inherited bleeding diseases caused by a qualitative or quantitative deficiency of the von Willebrand factor (FvW). FvW is a multimeric glycoprotein synthesized by megakaryocytes and endothelial cells and it is present in the subendothelial matrix, blood plasma, platelets, and endothelium. This glycoprotein plays an important role in thrombus formation by initiating platelet adhesion to sites of injury as well as platelet aggregation. The aim of this study was to evaluate the activities of enzymes that hydrolyze adenine nucleotides in platelets, ristocetin-induced platelet aggregation (RIPA), and polymorphisms of the alpha2 gene of alpha2beta1 integrin from VWD patients. Platelet nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) activities were verified in 14 VWD patients. For RIPA determination, a final concentration of 1.25 mg/ml of ristocetin was used. Polymorphisms of the alpha2 gene were analyzed through PCR. Platelet NTPDase and E-NPP were decreased in VWD patients. 5'-Nucleotidase activity was not statistically significant between controls and VWD patients. RIPA was significantly reduced, with an allelic frequency of 78.57% for 807C in VWD patients. Our results indicated reduced platelet NTPDase and E-NPP activities which might be related to the low platelet adhesiveness. The prevalence of the 807C allele might account for the variability in bleeding in VWD.

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

    PubMed Central

    Whittam, R.; Wiley, J. S.

    1968-01-01

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

  14. Receptor-promoted exocytosis of airway epithelial mucin granules containing a spectrum of adenine nucleotides.

    PubMed

    Kreda, Silvia M; Seminario-Vidal, Lucia; van Heusden, Catharina A; O'Neal, Wanda; Jones, Lisa; Boucher, Richard C; Lazarowski, Eduardo R

    2010-06-15

    Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. We now demonstrate that protease-activated-receptor (PAR) agonists also stimulate the simultaneous release of mucins and ATP from airway epithelial cells. PAR-mediated mucin and ATP release were dependent on intracellular Ca(2+) and actin cytoskeleton reorganization since BAPTA AM, cytochalasin D, and inhibitors of Rho and myosin light chain kinases blocked both responses. To test the hypothesis that ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of isolated mucin granules purified based on their MUC5AC and VAMP-8 content by density gradients. Mucin granules contained ATP, but the levels of ADP and AMP within granules exceeded by nearly 10-fold that of ATP. Consistent with this finding, apical secretions from PAR-stimulated cells contained relatively high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and also are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules is likely to provide a major source of airway surface adenosine to signal in a paracrine faction ciliated cell A(2b) receptors to activate ion/water secretion and appropriately hydrate goblet cell-released mucins.

  15. Receptor-promoted exocytosis of airway epithelial mucin granules containing a spectrum of adenine nucleotides

    PubMed Central

    Kreda, Silvia M; Seminario-Vidal, Lucia; van Heusden, Catharina A; O’Neal, Wanda; Jones, Lisa; Boucher, Richard C; Lazarowski, Eduardo R

    2010-01-01

    Purinergic regulation of airway innate defence activities is in part achieved by the release of nucleotides from epithelial cells. However, the mechanisms of airway epithelial nucleotide release are poorly understood. We have previously demonstrated that ATP is released from ionomycin-stimulated airway epithelial goblet cells coordinately with mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. We now demonstrate that protease-activated-receptor (PAR) agonists also stimulate the simultaneous release of mucins and ATP from airway epithelial cells. PAR-mediated mucin and ATP release were dependent on intracellular Ca2+ and actin cytoskeleton reorganization since BAPTA AM, cytochalasin D, and inhibitors of Rho and myosin light chain kinases blocked both responses. To test the hypothesis that ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of isolated mucin granules purified based on their MUC5AC and VAMP-8 content by density gradients. Mucin granules contained ATP, but the levels of ADP and AMP within granules exceeded by nearly 10-fold that of ATP. Consistent with this finding, apical secretions from PAR-stimulated cells contained relatively high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and also are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules is likely to provide a major source of airway surface adenosine to signal in a paracrine faction ciliated cell A2b receptors to activate ion/water secretion and appropriately hydrate goblet cell-released mucins. PMID:20421285

  16. [Closure of Ca2+-dependent pores by cyclosporin A: the role of magnesium ions, adenine nucleotides, and conformation status of the ADP/ATP antiporter].

    PubMed

    Andreev, A Iu; Mikhaĭlova, L M; Starkov, A A

    1994-10-01

    Effects of ADP and Mg2+ on the ability of cyclosporin A to "reseal" mitochondria permeabilized by Ca2+ and P(i) have been studied. Cyclosporin A was completely ineffective, when ADP and Mg2+ were not included into the incubation medium. Both ADP and Mg2+ used at high concentrations potentiated the effect of cyclosporin A and prevented it reversal by carboxyatractylate. Data on the influence of different concentrations of ADP and Mg2+ on the resealing efficiency of cyclosporin A suggest that the true effector modulating the state of the Ca(2+)-dependent pore is the ADP-Mg2+ complex, but not ADP or Mg2+ used separately. The ability of non-hydrolyzable analogs of adenine nucleotides, ADP-S and ATP-S, to potentiate the resealing action of cyclosporin on mitochondria permeabilized by loading of different Ca2+ concentrations to that of ADP was compared. ATP-S was ineffective when the pore was induced by high concentrations of Ca2+. The results obtained are discussed in terms of hypothesis on the direct involvement of the ADP/ATP antiporter in regulation of the inner mitochondrial membrane Ca(2+)-dependent pore state.

  17. Alterations of adenine nucleotide metabolism and function of blood platelets in patients with diabetes.

    PubMed

    Michno, Anna; Bielarczyk, Hanna; Pawełczyk, Tadeusz; Jankowska-Kulawy, Agnieszka; Klimaszewska, Joanna; Szutowicz, Andrzej

    2007-02-01

    Increased activity of blood platelets contributes to vascular complications in patients with diabetes. The aim of this work was to investigate whether persisting hyperglycemia in diabetic patients generates excessive accumulation of ATP/ADP, which may underlie platelet hyperactivity. Platelet ATP and ADP levels, thiobarbituric acid-reactive species synthesis, and aggregation of platelets from patients with diabetes were 18-82% higher than in platelets from healthy participants. In patients with diabetes, platelet stimulation with thrombin caused about two times greater release of ATP and ADP than in the healthy group while decreasing intraplatelet nucleotide content to similar levels in both groups. This indicates that the increased content of adenylate nucleotides in the releasable pool in the platelets of diabetic patients does not affect their level in metabolic cytoplasmic/mitochondrial compartments. Significant correlations between platelet ATP levels and plasma fructosamine, as well as between platelet ATP/ADP and platelet activities, have been found in diabetic patients. In conclusion, chronic hyperglycemia-evoked elevations of ATP/ADP levels and release from blood platelets of patients with diabetes may be important factors underlying platelet hyperactivity in the course of the disease.

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

    PubMed

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

    1985-07-15

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

  19. Direct thyroid hormone activation of mitochondria: identification of adenine nucleotide translocase (AdNT) as the hormone receptor.

    PubMed

    Sterling, K

    1987-01-01

    Earlier we presented preliminary data suggesting that the thyroid hormone triiodothyronine (T3) is bound with an association constant (Ka) approximating 2 X 10(11) M-1 by the ADP/ATP carrier, adenine nucleotide translocase (AdNT) purified from beef heart mitochondria (Endocrinology 110: 292, 1986). We now report that [125I] T3 is capable of photoaffinity labeling not only purified AdNT but also the carrier in intact beef heart mitochondria. The identity of the covalently labeled AdNT was corroborated by two dimensional electrophoresis (O'Farrell) with pI approximately 10 on electrofocusing (first dimension) and Mr approximately 31,000 on SDS gel (second dimension). Further identification of the covalently labeled material as authentic AdNT was afforded by recognition by specific monoclonal antibodies. Moreover, we found that addition of excess nonradioactive T3 to intact mitochondria or to mitochondrial protein solution prior to photoaffinity labeling resulted in inhibition of formation of labeled AdNT, compatible with saturation of limited capacity binding sites rather than nonspecific labeling with the ligand [125I] T3. It was considered highly significant that labeling in intact mitochondria was at least an order of magnitude greater than that observed with purified AdNT. This finding is compatible with our concept of an important role of the lipid microenvironment in the intact mitochondrial membrane in T3 binding.

  20. Caffeic acid treatment alters the extracellular adenine nucleotide hydrolysis in platelets and lymphocytes of adult rats.

    PubMed

    Anwar, Javed; Spanevello, Roselia Maria; Pimentel, Victor Camera; Gutierres, Jessié; Thomé, Gustavo; Cardoso, Andreia; Zanini, Daniela; Martins, Caroline; Palma, Heloisa Einloft; Bagatini, Margarete Dulce; Baldissarelli, Jucimara; Schmatz, Roberta; Leal, Cláudio Alberto Martins; da Costa, Pauline; Morsch, Vera Maria; Schetinger, Maria Rosa Chitolina

    2013-06-01

    This study evaluated the effects of caffeic acid on ectonucleotidase activities such as NTPDase (nucleoside triphosphate diphosphohydrolase), Ecto-NPP (nucleotide pyrophosphatase/phosphodiesterase), 5'-nucleotidase and adenosine deaminase (ADA) in platelets and lymphocytes of rats, as well as in the profile of platelet aggregation. Animals were divided into five groups: I (control); II (oil); III (caffeic acid 10 mg/kg); IV (caffeic acid 50 mg/kg); and V (caffeic acid 100 mg/kg). Animals were treated with caffeic acid diluted in oil for 30 days. In platelets, caffeic acid decreased the ATP hydrolysis and increased ADP hydrolysis in groups III, IV and V when compared to control (P<0.05). The 5'-nucleotidase activity was decreased, while E-NPP and ADA activities were increased in platelets of rats of groups III, IV and V (P<0.05). Caffeic acid reduced significantly the platelet aggregation in the animals of groups III, IV and V in relation to group I (P<0.05). In lymphocytes, the NTPDase and ADA activities were increased in all groups treated with caffeic acid when compared to control (P<0.05). These findings demonstrated that the enzymes were altered in tissues by caffeic acid and this compound decreased the platelet aggregation suggesting that caffeic acid should be considered a potentially therapeutic agent in disorders related to the purinergic system.

  1. Changes in the expression of the human adenine nucleotide translocase isoforms condition cellular metabolic/proliferative status

    PubMed Central

    Mampel, Teresa; Viñas, Octavi

    2016-01-01

    Human cells express four mitochondrial adenine nucleotide translocase (hANT) isoforms that are tissue-specific and developmentally regulated. hANT1 is mainly expressed in terminally differentiated muscle cells; hANT2 is growth-regulated and is upregulated in highly glycolytic and proliferative cells; and hANT3 is considered to be ubiquitous and non-specifically regulated. Here, we studied how the expression of hANT isoforms is regulated by proliferation and in response to metabolic stimuli, and examined the metabolic consequences of their silencing and overexpression. In HeLa and HepG2 cells, expression of hANT3 was upregulated by shifting metabolism towards oxidation or by slowed growth associated with contact inhibition or growth-factor deprivation, indicating that hANT3 expression is highly regulated. Under these conditions, changes in hANT2 mRNA expression were not observed in either HeLa or HepG2 cells, whereas in SGBS preadipocytes (which, unlike HeLa and HepG2 cells, are growth-arrest-sensitive cells), hANT2 mRNA levels decreased. Additionally, overexpression of hANT2 promoted cell growth and glycolysis, whereas silencing of hANT3 decreased cellular ATP levels, limited cell growth and induced a stress-like response. Thus, cancer cells require both hANT2 and hANT3, depending on their proliferation status: hANT2 when proliferation rates are high, and hANT3 when proliferation slows. PMID:26842067

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

    PubMed

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

    2014-04-01

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

  3. Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues.

    PubMed Central

    Dümmler, K; Müller, S; Seitz, H J

    1996-01-01

    Thyroid hormone (T3)-dependent gene expression of the adenine nucleotide translocase (ANT) and the FAD-linked glycerol 3-phosphate dehydrogenase (mGPDH) was investigated in several rat tissues. Both proteins provide an important link between cytosolic and mitochondrial metabolic pathways and seem to be involved in the stimulation of mitochondrial oxygen consumption in response to T3. Here we show that two ANT isoforms are expressed in rat, the muscle-specific ANT1 form and the ubiquitous ANT2 form. The expression of ANT1 mRNA is not sensitive to T3 whereas the amount of ANT2 mRNA is increased 7-9-fold in liver and heart within 12-48 h after T3 application. Little or no effect of T3 on ANT2 mRNA was observed in kidney and brain. The mRNA changes are paralleled by an increase in ANT protein, thus explaining the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats. The key role of ANT2 in the control of hyperthyroid metabolism is evident because the expression of the mersalyl-sensitive phosphate carrier and the mitochondrial creatine kinase mRNA, which are functionally linked to ANT, did not respond to T3. Similarly to the ADP/ATP exchange, the transfer of cytosolic NADH to the respiratory chain via the glycerophosphate shuttle is very sensitive to T3. Recently we demonstrated the 10-15-fold induction of mGPDH mRNA in rat liver after administration of T3 [Müller and Seitz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10581-10585]. Here we show that, in contrast with ANT2, the time course of induction is fast (4-6 h). Furthermore, mGPDH mRNA is induced 6-fold by T3 in heart and 4-fold in kidney. From these results we conclude that the T3-mediated transcriptional induction leading to increased activity of ANT2 and mGPDH contributes considerably to the increase in mitochondrial oxygen consumption in rat tissues. PMID:8760382

  4. The mitochondrial BKCa channel cardiac interactome reveals BKCa association with the mitochondrial import receptor subunit Tom22, and the adenine nucleotide translocator.

    PubMed

    Zhang, Jin; Li, Min; Zhang, Zhu; Zhu, Ronghui; Olcese, Riccardo; Stefani, Enrico; Toro, Ligia

    2017-03-01

    Mitochondrial BKCa channel, mitoBKCa, regulates mitochondria function in the heart but information on its protein partnerships in cardiac mitochondria is missing. A directed proteomic approach discovered the novel interaction of BKCa with Tom22, a component of the mitochondrion outer membrane import system, and the adenine nucleotide translocator (ANT). The expressed protein partners co-immunoprecipitated and co-segregated into mitochondrial fractions in HEK293T cells. The BKCa 50 amino acid splice insert, DEC, facilitated BKCa interaction with ANT. Further, BKCa transmembrane domain was required for the association with both Tom22 and ANT. The results serve as a working framework to understand mitoBKCa import and functional relationships.

  5. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    SciTech Connect

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. )

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  6. Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on cape cod

    USGS Publications Warehouse

    Metge, D.W.; Brooks, M.H.; Smith, R.L.; Harvey, R.W.

    1993-01-01

    Changes in adenylate energy charge (EC(A)) and in total adenine nucleotides (A(T)) and DNA content (both normalized to the abundance of free- living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. EC(A)s (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated EC(A)s (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although EC(A) and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture. These data suggest that the DNA content of groundwater bacteria may be more sensitive than their A(T) to the degree of carbon loading, which may have significant ramifications in the use of nucleic acids and adenine nucleotides for estimating the metabolic status of bacterial communities within more highly contaminated aquifers.

  7. Forward operation of adenine nucleotide translocase during F0F1-ATPase reversal: critical role of matrix substrate-level phosphorylation

    PubMed Central

    Chinopoulos, Christos; Gerencser, Akos A.; Mandi, Miklos; Mathe, Katalin; Töröcsik, Beata; Doczi, Judit; Turiak, Lilla; Kiss, Gergely; Konràd, Csaba; Vajda, Szilvia; Vereczki, Viktoria; Oh, Richard J.; Adam-Vizi, Vera

    2010-01-01

    In pathological conditions, F0F1-ATPase hydrolyzes ATP in an attempt to maintain mitochondrial membrane potential. Using thermodynamic assumptions and computer modeling, we established that mitochondrial membrane potential can be more negative than the reversal potential of the adenine nucleotide translocase (ANT) but more positive than that of the F0F1-ATPase. Experiments on isolated mitochondria demonstrated that, when the electron transport chain is compromised, the F0F1-ATPase reverses, and the membrane potential is maintained as long as matrix substrate-level phosphorylation is functional, without a concomitant reversal of the ANT. Consistently, no cytosolic ATP consumption was observed using plasmalemmal KATP channels as cytosolic ATP biosensors in cultured neurons, in which their in situ mitochondria were compromised by respiratory chain inhibitors. This finding was further corroborated by quantitative measurements of mitochondrial membrane potential, oxygen consumption, and extracellular acidification rates, indicating nonreversal of ANT of compromised in situ neuronal and astrocytic mitochondria; and by bioluminescence ATP measurements in COS-7 cells transfected with cytosolic- or nuclear-targeted luciferases and treated with mitochondrial respiratory chain inhibitors in the presence of glycolytic plus mitochondrial vs. only mitochondrial substrates. Our findings imply the possibility of a rescue mechanism that is protecting against cytosolic/nuclear ATP depletion under pathological conditions involving impaired respiration. This mechanism comes into play when mitochondria respire on substrates that support matrix substrate-level phosphorylation.—Chinopoulos, C., Gerencser, A. A., Mandi, M., Mathe, K., Töröcsik, B., Doczi, J., Turiak, L., Kiss, G., Konràd, C., Vajda, S., Vereczki, V., Oh, R. J., Adam-Vizi, V. Forward operation of adenine nucleotide translocase during F0F1-ATPase reversal: critical role of matrix substrate-level phosphorylation. PMID

  8. SERS detection of low-concentration adenine by a patterned silver structure immersion plated on a silicon nanoporous pillar array.

    PubMed

    Feng, Fei; Zhi, Gang; Jia, He Shun; Cheng, Liang; Tian, Yong Tao; Li, Xin Jian

    2009-07-22

    A patterned Ag structure was grown on a Si nanoporous pillar array (Si-NPA) by an immersion plating method, and its surface-enhanced Raman scattering (SERS) activity toward adenine was studied. It was shown that two kinds of Ag structures were grown on Si-NPA, a continuous film covering the Si-NPA substrate and composed of Ag nanocrystallites (nc-Ag), and a quasi-regular, interconnected network composed of loop-chains of sub-micron Ag crystallites surrounding the porous Si pillars. The SERS detection of low-concentration adenine solution was performed by using Ag/Si-NPA as active substrates, in which significantly enhanced Raman signals were observed. The SERS enhancement was attributed to the active spacing sites formed between the Ag particles and the nc-Ag which met the optimal size for causing a SERS effect. Based on the measured SERS spectra, the adsorption mode of adenine molecules on Ag particles was deduced. These results indicated that Ag/Si-NPA might be a promising active substrate for SERS detection of low-concentration bio-molecules.

  9. Identification and Characterization of a Plastidic Adenine Nucleotide Uniporter (OsBT1-3) Required for Chloroplast Development in the Early Leaf Stage of Rice

    PubMed Central

    Hu, Daoheng; Li, Yang; Jin, Wenbin; Gong, Hanyu; He, Qiong; Li, Yangsheng

    2017-01-01

    Chloroplast development is an important subject in botany. In this study, a rice (Oryza sativa) mutant exhibiting impairment in early chloroplast development (seedling leaf albino (sla)) was isolated from a filial generation via hybridization breeding. The sla mutant seedlings have an aberrant form of chloroplasts, which resulted in albinism at the first and second leaves; however, the leaf sheath was green. The mutant gradually turned green after the two-leaf stage, and the third leaf was a normal shade of green. Map-based cloning indicated that the gene OsBT1-3, which belongs to the mitochondrial carrier family (MCF), is responsible for the sla mutant phenotype. OsBT1-3 expression was high in the young leaves, decreased after the two-leaf stage, and was low in the sheath, and these findings are consistent with the recovery of a number of chloroplasts in the third leaf of sla mutant seedlings. The results also showed that OsBT1-3-yellow fluorescent protein (YFP) was targeted to the chloroplast, and a Western blot assay using a peptide-specific antibody indicated that OsBT1-3 localizes to the chloroplast envelope. We also demonstrated that OsBT1-3 functions as a unidirectional transporter of adenine nucleotides. Based on these findings, OsBT1-3 likely acts as a plastid nucleotide uniporter and is essential for chloroplast development in rice leaves at the young seedling stage. PMID:28134341

  10. New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes.

    PubMed

    Hiratsuka, T

    1983-02-15

    The synthesis of fluorescent derivatives of nucleosides and nucleotides, by reaction with isatoic anhydride in aqueous solution at mild pH and temperature, yielding their 3'-O-anthraniloyl derivatives, is here described. The N-methylanthraniloyl derivatives were also synthesized by reaction with N-methylisatoic anhydride. Upon excitation at 330-350 nm these derivatives exhibited maximum fluorescence emission at 430-445 nm in aqueous solution with quantum yields of 0.12-0.24. Their fluorescence was sensitive to the polarity of the solvent; in N,N-dimethylformamide the quantum yields were 0.83-0.93. The major differences between the two fluorophores were the longer wavelength of the emission maximum of the N-methylanthraniloyl group and its greater quantum yield in water. All anthraniloyl derivatives, as well as the N-methylanthraniloyl ones, had virtually identical fluorescent properties, regardless of their base structures. The ATP derivatives showed considerable substrate activity as a replacement of ATP with adenylate kinase, guanylate kinase, glutamine synthetase, myosin ATPase and sodium-potassium transport ATPase. The ADP derivatives were good substrates for creatine kinase and glutamine synthetase (gamma-glutamyl transfer activity). The GMP and adenosine derivatives were substrates for guanylate kinase and adenosine deaminase, respectively. All derivatives had only slightly altered Km values for these enzymes. While more fluorescent in water, the N-methylanthraniloyl derivatives were found to show relatively low substrate activities against some of these enzymes. The results indicate that these ribose-modified nucleosides and nucleotides can be versatile fluorescent substrate analogs for various enzymes.

  11. Dietary fiber suppresses elevation of uric acid and urea nitrogen concentrations in serum of rats with renal dysfunction induced by dietary adenine.

    PubMed

    Koguchi, Takashi; Koguchi, Hiromi; Nakajima, Hisao; Takano, Saburo; Yamamoto, Yuji; Innami, Satoshi; Maekawa, Akio; Tadokoro, Tadahiro

    2004-07-01

    This study was conducted to examine the effects of several kinds of dietary fiber (DF) with different physical properties on the elevation of uric acid and urea nitrogen concentrations in serum of rats induced by dietary adenine. DF decreased an uptake of 14C-labeled adenine in the rat jejunum in vitro, but the reduction varied with the physical property of DF. Male Wistar rats (3 weeks old) were fed a diet with or without a 0.4% adenine and a 5% DF (cellulose, chitin, chitosan, or xanthan gum) for 20 days. Feeding of adenine in the fiber-free group elevated the concentrations of uric acid, creatinine, and urea nitrogen in serum, but decreased the excretions of these compounds into urine and increased the amounts of 2,8-dihydroxyadenine (2,8-DHA) in kidney and urine. The test DF was found to suppress the elevation of uric acid, creatinine, and urea nitrogen concentrations in serum induced by dietary adenine, and to mitigate the decreased excretions of these compounds into urine and the increased retention of 2,8-DHA in kidney and urine. This phenomenon was remarkable in the xanthan gum group. These results suggest that DF suppresses the elevation of uric acid and urea nitrogen concentrations in serum by attenuating the absorption of dietary adenine.

  12. 2-O-β-D-Glucopyranosyl-carboxyatractyligenin from Coffea L. inhibits adenine nucleotide translocase in isolated mitochondria but is quantitatively degraded during coffee roasting.

    PubMed

    Lang, Roman; Fromme, Tobias; Beusch, Anja; Wahl, Anika; Klingenspor, Martin; Hofmann, Thomas

    2013-09-01

    Atractyloside (1) and carboxyatractyloside (2) are well-known inhibitors of the adenine nucleotide translocase (ANT) in mitochondria, thus effectively blocking oxidative phosphorylation. Structurally related derivatives atractyligenin (3), 2-O-β-D-glucopyranosyl-atractyligenin (4), 3'-O-β-D-glucopyranosyl-2'-O-isovaleryl-2β-(2-desoxy-atractyligenin)-β-D-glucopyranoside (5), and 2-O-β-D-glucopyranosyl-carboxyatractyligenin (6) were isolated from raw beans of Coffea L. and the impact of 1-6 on ANT activity was evaluated in isolated mitochondria. Among the coffee components, 6 significantly inhibited ANT activity leading to reduced respiration. Quantitative analysis in commercial coffees, experimental roastings of coffee, and model experiments using purified compound 6 consistently revealed a complete degradation during thermal treatment. In comparison, raw coffee extracts were found to contain high levels of 6, which are therefore expected to be present in food products enriched with raw coffee extracts. This implies the necessity of analytically controlling the levels of 6 in raw coffee extracts when used as additives for food products.

  13. Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes

    PubMed Central

    2016-01-01

    The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein–protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid–protein interactions. PMID:27786441

  14. To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M; Konovalova, Svetlana A; Brailovskaya, Irina V; Saris, Nils-Erik L

    2016-04-01

    The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.

  15. Adenosine produced from adenine nucleotides through an interaction between apoptotic cells and engulfing macrophages contributes to the appearance of transglutaminase 2 in dying thymocytes.

    PubMed

    Sándor, Katalin; Pallai, Anna; Duró, Edina; Legendre, Pascal; Couillin, Isabelle; Sághy, Tibor; Szondy, Zsuzsa

    2017-03-01

    Transglutaminase 2 (TG2) has been known for a long time to be associated with the in vivo apoptosis program of various cell types, including T cells. Though the expression of the enzyme is strongly induced in mouse thymocytes following apoptosis induction in vivo, no significant induction of TG2 can be detected, when thymocytes are induced to die by the same stimuli in vitro indicating that signals arriving from the tissue environment are required for the proper in vivo induction of the enzyme. Previous studies from our laboratory have demonstrated that two of these signals, transforming growth factor-β (TGF-β) and retinoids, are produced by macrophages engulfing apoptotic cells. However, in addition to TGF-β and retinoids, engulfing macrophages produce adenosine as well. Here, we show that in vitro adenosine, adenosine, and retinoic acid or adenosine, TGF-β and retinoic acids together can significantly enhance the TG2 mRNA expression in dying thymocytes. The effect of adenosine is mediated via adenosine A2A receptors (A2ARs) and the A2AR-triggered adenylate cyclase signaling pathway. In accordance, loss of A2ARs in A2AR null mice significantly attenuates the in vivo induction of TG2 following apoptosis induction in the thymus indicating that adenosine indeed contributes in vivo to the apoptosis-related appearance of the enzyme. We also demonstrate that adenosine is produced extracellularly during engulfment of apoptotic thymocytes, partly from adenine nucleotides released via thymocyte pannexin-1 channels. Our data reveal a novel crosstalk between macrophages and apoptotic cells, in which apoptotic cell uptake-related adenosine production contributes to the appearance of TG2 in the dying thymocytes.

  16. Role of Mg(2+) in Ca(2+)-induced Ca(2+) release through ryanodine receptors of frog skeletal muscle: modulations by adenine nucleotides and caffeine.

    PubMed Central

    Murayama, T; Kurebayashi, N; Ogawa, Y

    2000-01-01

    Mg(2+) serves as a competitive antagonist against Ca(2+) in the high-affinity Ca(2+) activation site (A-site) and as an agonist of Ca(2+) in the low-affinity Ca(2+) inactivation site (I-site) of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). This paper presents the quantitative determination of the affinities for Ca(2+) and Mg(2+) of A- and I-sites of RyR in frog skeletal muscles by measuring [(3)H]ryanodine binding to purified alpha- and beta-RyRs and CICR activity in skinned fibers. There was only a minor difference in affinity at most between alpha- and beta-RyRs. The A-site favored Ca(2+) 20- to 30-fold over Mg(2+), whereas the I-site was nonselective between the two cations. The RyR in situ showed fivefold higher affinities for Ca(2+) and Mg(2+) of both sites than the purified alpha- and beta-RyRs with unchanged cation selectivity. Adenine nucleotides, whose stimulating effect was found to be indistinguishable between free and complexed forms, did not alter the affinities for cations in either site, except for the increased maximum activity of RyR. Caffeine increased not only the affinity of the A-site for Ca(2+) alone, but also the maximum activity of RyR with otherwise minor changes. The results presented here suggest that the rate of CICR in frog skeletal muscles appears to be too low to explain the physiological Ca(2+) release, even though Mg(2+) inhibition disappears. PMID:10733962

  17. Specific and nonspecific metal ion-nucleotide interactions at aqueous/solid interfaces functionalized with adenine, thymine, guanine, and cytosine oligomers.

    PubMed

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

    2011-03-02

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

  18. Influence of local sequence context on damaged base conformation in human DNA polymerase iota: molecular dynamics studies of nucleotide incorporation opposite a benzo[a]pyrene-derived adenine lesion.

    PubMed

    Donny-Clark, Kerry; Broyde, Suse

    2009-11-01

    Human DNA polymerase iota is a lesion bypass polymerase of the Y family, capable of incorporating nucleotides opposite a variety of lesions in both near error-free and error-prone bypass. With undamaged templating purines polymerase iota normally favors Hoogsteen base pairing. Polymerase iota can incorporate nucleotides opposite a benzo[a]pyrene-derived adenine lesion (dA*); while mainly error-free, the identity of misincorporated bases is influenced by local sequence context. We performed molecular modeling and molecular dynamics simulations to elucidate the structural basis for lesion bypass. Our results suggest that hydrogen bonds between the benzo[a]pyrenyl moiety and nearby bases limit the movement of the templating base to maintain the anti glycosidic bond conformation in the binary complex in a 5'-CAGA*TT-3' sequence. This facilitates correct incorporation of dT via a Watson-Crick pair. In a 5'-TTTA*GA-3' sequence the lesion does not form these hydrogen bonds, permitting dA* to rotate around the glycosidic bond to syn and incorporate dT via a Hoogsteen pair. With syn dA*, there is also an opportunity for increased misincorporation of dGTP. These results expand our understanding of the versatility and flexibility of polymerase iota and its lesion bypass functions in humans.

  19. Role of base sequence context in conformational equilibria and nucleotide excision repair of benzo[a]pyrene diol epoxide-adenine adducts.

    PubMed

    Yan, Shixiang; Wu, Min; Buterin, Tonko; Naegeli, Hanspeter; Geacintov, Nicholas E; Broyde, Suse

    2003-03-04

    We investigate the influence of base sequence context on the conformations of the 10S (+)- and 10R (-)-trans-anti-[BP]-N(6)-dA adducts through molecular dynamics (MD) simulations with free energy calculations, and relate the structural findings to results of nucleotide excision repair (NER) assays in human cell extracts. In previous studies, these adducts were studied in the CA*A sequence context, and here we report results for the CA*C sequence. Our simulations indicate that the base sequence context affects the syn-anti conformational equilibrium in the 10S (+) adduct by modulating the barrier heights between these states on the energy surface, with a higher barrier in the CA*C case. Our nucleotide excision repair assay finds greater NER susceptibilities in the 10S (+) adduct for the CA*C sequence context. A structural rationale ties together these results. A sequence specific hydrogen bond, accompanied by a significantly increased roll and consequent bending in the 10S (+) adduct, has been found in our simulations for the CA*C sequence, which could account for the enhanced nucleotide excision repair as well as the syn-anti equilibrium difference we observe in this isomer and sequence. Such sequence specific differential repair could contribute to the existence of mutational hotspots and thereby contribute to the complexity of cancer initiation.

  20. Adenine and adenosine salvage in Leishmania donovani.

    PubMed

    Boitz, Jan M; Ullman, Buddy

    2013-08-01

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

  1. 2-Substitution of adenine nucleotide analogues containing a bicyclo[3.1.0]hexane ring system locked in a northern conformation: enhanced potency as P2Y1 receptor antagonists.

    PubMed

    Kim, Hak Sung; Ohno, Michihiro; Xu, Bin; Kim, Hea Ok; Choi, Yongseok; Ji, Xiao D; Maddileti, Savitri; Marquez, Victor E; Harden, T Kendall; Jacobson, Kenneth A

    2003-11-06

    Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.

  2. Photo-excitation of adenine cation radical [A•+] in the near UV-vis region produces sugar radicals in Adenosine and in its nucleotides

    PubMed Central

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

    2011-01-01

    In this study, we report the formation of ribose sugar radicals in high yields (85 – 100%) via photo-excitation of adenine cation radical (A•+) in Ado and its ribonucleotides. Photo-excitation of A•+ at low temperatures in homogenous aqueous glassy samples of Ado, 2′-AMP, 3′-AMP and 5′-AMP forms sugar radicals predominantly at C5′- and also at C3′-sites. The C5′• and C3′• sugar radicals were identified employing Ado deuterated at specific carbon sites: C1′, C2′, and at C5′. Phosphate substitution is found to deactivate sugar radical formation at the site of substitution. Thus, in 5′-AMP, C3′• is observed to be the main radical formed via photo-excitation at ca. 143 K whereas in 3′-AMP, C5′• is the only species found. These results were supported by results obtained employing 5′-AMP with specific deuteration at C5′-site (i.e., 5′,5′-D,D-5′-AMP). Moreover, contrary to the C5′• observed in 3′-dAMP, we find that C5′• in 3′-AMP shows a clear pH dependent conformational change as evidenced by a large increase in the C4′ β–hyperfine coupling on increasing the pH from 6 to 9. Calculations performed employing DFT (B3LYP/6-31G*) for C5′• in 3′-AMP show that the two conformations of C5′• result from strong hydrogen bond formation between the O5′-H and the 3′-phosphate dianion at higher pHs. Employing time-dependent density functional theory [TD-DFT, B3LYP/6-31G(d)] we show that in the excited state, the hole transfers to the sugar moiety and has significant hole localization at the C5′-site in a number of allowed transitions. This hole localization is proposed to lead to the formation of the neutral C5′-radical (C5′•) via deprotonation. PMID:19367991

  3. 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.

  4. Sensory attributes of dishes containing shrimp paste with different concentrations of glutamate and 5'-nucleotides.

    PubMed

    Jinap, S; Ilya-Nur, A R; Tang, S C; Hajeb, P; Shahrim, K; Khairunnisak, M

    2010-10-01

    The shrimp paste called belacan is a traditional umami taste condiment extensively used in Malaysia that is rich in glutamate and 5'-nucleotides. The aim of this study was to determine the concentration of glutamate and 5'-nucleotides of various types of foods prepared with belacan and to measure their sensory attributes. The concentration of free glutamic acid found in different brands of belacan was 180-530mg/100g and in local dishes 601-4207mg/100g. The total amount of 5'-nucleotides in belacan samples ranged from 0.85 to 42.25μg/g. A Quantitative Descriptive Analysis (QDA) using a list of 17 sensory attributes showed a good correlation between belacan concentration in the final food and a range of positive sensory attributes, except for bitter, sweet, sour taste and astringency. Belacan also contains bitter, sweet and sour compounds that change the positive attributes of belacan at higher concentrations. The highest aroma attributes were linked to nasi goreng belacan (belacan fried rice) while the highest flavour attributes were found in sambal belacan. There was a 32 folds significant increase of umami attributes with the addition of belacan to final foods. The optimum amount of belacan was 0.45% for asam pedas (tamarind flavoured dish with belacan), 18% for sambal belacan (chilli belacan), 1.5-2.5% for kangkong goreng belacan (stir fried water convolous with belacan), and 2% for nasi goreng belacan.

  5. Thiolated pyrimidine nucleotides may interfere thiol groups concentrated at lipid rafts of HIV-1 infected cells.

    PubMed

    Kanizsai, Szilvia; Ongrádi, Joseph; Aradi, János; Nagy, Károly

    2014-12-01

    Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29. Selective cytotoxicity of thiolated pyrimidines on HIV-1 infected cells were observed. Results indicate that thiolated pyrimidine derivates may interfere with -SH (thiol) groups concentrated in lipid rafts of cell membrane and interacts HIV-1 infected (activated) cells resulting in a selective cytotoxicity of HIV-1 infected cells, and reducing HIV-1 entry.

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

    PubMed

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

    2007-12-27

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

  7. 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.

  8. 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.

  9. Adenine formation without HCN.

    PubMed

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

    2014-05-22

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

  10. Bimodal oscillations of cyclic nucleotide concentrations in the circadian system of the Madeira cockroach Rhyparobia maderae.

    PubMed

    Schendzielorz, Julia; Schendzielorz, Thomas; Arendt, Andreas; Stengl, Monika

    2014-10-01

    Pigment-dispersing factor (PDF) is the most important coupling factor of the circadian system in insects, comparable to its functional ortholog vasoactive intestinal polypeptide of the mammalian circadian clock. In Drosophila melanogaster, PDF signals via activation of adenylyl cyclases, controlling circadian locomotor activity rhythms at dusk and dawn. In addition, PDF mediates circadian rhythms of the visual system and is involved in entrainment to different photoperiods. We examined whether PDF daytime-dependently elevates cAMP levels in the Madeira cockroach Rhyparobia maderae and whether cAMP mimics PDF effects on locomotor activity rhythms. To determine time windows of PDF release, we searched for circadian rhythms in concentrations of cAMP and its functional opponent cGMP in the accessory medulla (AMe), the insect circadian pacemaker controlling locomotor activity rhythms, and in the optic lobes, as the major input and output area of the circadian clock. Enzyme-linked immunosorbent assays detected PDF-dependent increases of cAMP in optic lobes and daytime-dependent oscillations of cAMP and cGMP baseline levels in the AMe, both with maxima at dusk and dawn. Although these rhythms disappeared at the first day in constant conditions (DD1), cAMP but not cGMP oscillations returned at the second day in constant conditions (DD2). Whereas in light-dark cycles the cAMP baseline level remained constant in other optic lobe neuropils, it oscillated in phase with the AMe at DD2. To determine whether cAMP and cGMP mimic PDF-dependent control of locomotor activity rhythms, both cyclic nucleotides were injected at different times of the circadian day using running-wheel assays. Whereas cAMP injections generated delays at dusk and advances at dawn, cGMP only delayed locomotor activity at dusk. For the first time we found PDF-dependent phase advances at dawn in addition to previously described phase delays at dusk. Thus, we hypothesize that PDF release at dusk and dawn

  11. Adenine suppresses IgE-mediated mast cell activation.

    PubMed

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

    2015-06-01

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

  12. Adenine phosphoribosyltransferase deficiency.

    PubMed

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

    2012-09-01

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

  13. OmpF, a nucleotide-sensing nanoprobe, computational evaluation of single channel activities

    NASA Astrophysics Data System (ADS)

    Abdolvahab, R. H.; Mobasheri, H.; Nikouee, A.; Ejtehadi, M. R.

    2016-09-01

    The results of highthroughput practical single channel experiments should be formulated and validated by signal analysis approaches to increase the recognition precision of translocating molecules. For this purpose, the activities of the single nano-pore forming protein, OmpF, in the presence of nucleotides were recorded in real time by the voltage clamp technique and used as a means for nucleotide recognition. The results were analyzed based on the permutation entropy of current Time Series (TS), fractality, autocorrelation, structure function, spectral density, and peak fraction to recognize each nucleotide, based on its signature effect on the conductance, gating frequency and voltage sensitivity of channel at different concentrations and membrane potentials. The amplitude and frequency of ion current fluctuation increased in the presence of Adenine more than Cytosine and Thymine in milli-molar (0.5 mM) concentrations. The variance of the current TS at various applied voltages showed a non-monotonic trend whose initial increasing slope in the presence of Thymine changed to a decreasing one in the second phase and was different from that of Adenine and Cytosine; e.g., by increasing the voltage from 40 to 140 mV in the 0.5 mM concentration of Adenine or Cytosine, the variance decreased by one third while for the case of Thymine it was doubled. Moreover, according to the structure function of TS, the fractality of current TS differed as a function of varying membrane potentials (pd) and nucleotide concentrations. Accordingly, the calculated permutation entropy of the TS, validated the biophysical approach defined for the recognition of different nucleotides at various concentrations, pd's and polarities. Thus, the promising outcomes of the combined experimental and theoretical methodologies presented here can be implemented as a complementary means in pore-based nucleotide recognition approaches.

  14. Effect of interferon on concentrations of cyclic nucleotides in cultured cells.

    PubMed Central

    Tovey, M G; Rochette-Egly, C; Castagna, M

    1979-01-01

    Constant intracellular concentrations of both adenosine 3',5'-cyclic-monophosphate (cyclic AMP) and guanosine 3',5'-cyclic-monophosphate (cyclic GMP) were obtained when leukemia L1210 cells were cultivated under steady-state conditions in the chemostat. In this sensitive and controlled system addition of mouse interferon resulted in a rapid (5-10 min) increase in the intracellular concentration of cyclic GMP, which preceded by several hours an increase in the intracellular concentration of cyclic AMP. In contrast to the effect of interferon, addition of prostaglandin E1 induced a rapid increase in the intracellular concentration of cyclic AMP without markedly affecting the intracellular concentration of cyclic GMP. It is suggested that the rapid effect of interferon on cyclic GMP plays a role in mediating some of the effects of interferon on cells. PMID:226987

  15. Nickel(II), copper(II) and zinc(II) complexes of 9-[2- (phosphonomethoxy)ethyl]-8-azaadenine (9,8aPMEA), the 8-aza derivative of the antiviral nucleotide analogue 9-[2-(phosphonomethoxy)ethyl] adenine (PMEA). Quantification of four isomeric species in aqueous solution.

    PubMed

    Gómez-Coca, Raquel B; Holý, Antonín; Vilaplana, Rosario A; González-Vílchez, Francisco; Sigel, Helmut

    2004-01-01

    The acidity constants of the twofold protonated acyclic nucleotide analogue 9-[2-(phosphonomethoxy)- ethyl]-8-azaadenine, H(2)(9,8aPMEA)(+/-), as well as the stability constants of the M(H;9,8aPMEA)(+) and M(9,8aPMEA) complexes with the metal ions M(2+) =Ni(2+), Cu(2+) or Zn(2+), have been determined by potentiometric pH titrations in aqueous solution at I=0.1 M (NaNO(3)) and 25. The result for the release of the first proton from H(2)(9,8aPMEA)(+) (pK(a)= 2.73), which originates from the (N1)H(+) site, was confirmed by UV-spectrophotometric measurements. Application of previously determined straight-line plots of log KMM(R-PO(3)) versus PKH(3)(R-HPO(3))' for simple phosph(on)ate ligands, R- PO-, where R represents a residue without an affinity for metal ions, proves that the primary binding site of 9,8aPMEA(2-) is the phosphonate group for all three metal ions studied. By stability constant comparisons with related ligands it is shown, in agreement with conclusions reached earlier for the Cu(PMEA) system [PMEA(2-)=dianion of 9-[2- (phosphonomethoxy)ethyl]adenine], that in total four different isomers are in equilibrium with each other, i.e. (i) an open isomer with a sole phosphonate coordination, M(PA)(op), where PA(2-)=PMEA(2-)or 9,8aPMEA(2-), (ii) an isomer with a 5-membered chelate involving the ether oxygen, M(PA)cl/o, (iii) an isomer which contains 5- and 7-membered chelates formed by coordination of the phosphonate group, the ether oxygen and the N3 site of the adenine residue, M(PA)(cl/O/N3), and finally (iv) a macrochelated isomer involving N7, M(PA)(cl/]N7). The Cu(2+) systems of PMEA(2-) and 9,8aPMEA(2-) behave quite alike; the formation degrees for Cu(PA)(op), CuM(PA)(cl/O), Cu(PA)(cl/O/N3) and Cu(PA)(cl/N3) are approximately 16, 32, 45 and 7%, respectively, which shows that Cu(PA)(cl/N7) is a minority species. In the Ni(2+) and Zn(2+) systems the open isomer is the dominating one followed by M(PA)(cl/O), but there are indications that the other two

  16. Caffeine biosynthesis and adenine metabolism in transgenic Coffea canephora plants with reduced expression of N-methyltransferase genes.

    PubMed

    Ashihara, Hiroshi; Zheng, Xin-Qiang; Katahira, Riko; Morimoto, Masayuki; Ogita, Shinjiro; Sano, Hiroshi

    2006-05-01

    In anti-sense and RNA interference transgenic plants of Coffea canephora in which the expression of CaMXMT1 was suppressed, caffeine biosynthesis from [8-(14)C]adenine was investigated, together with the overall metabolism of [8-(14)C]adenine. Compared with wild type control plants, total purine alkaloid biosynthesis from adenine and conversion of theobromine to caffeine were both reduced in the transgenic plants. As found previously, [8-(14)C]adenine was metabolised to salvage products (nucleotides and RNA), to degradation products (ureides and CO(2)) and to purine alkaloids (theobromine and caffeine). In the transgenic plants, metabolism of [8-(14)C]adenine shifted from purine alkaloid synthesis to purine catabolism or salvage for nucleotides. HPLC analysis revealed a significantly reduced caffeine content in the transgenic plants. A small quantity (less than 20 nmol g(-1) fresh weight) of xanthosine had accumulated in at least one of the transgenic plants.

  17. Protein modification by adenine propenal.

    PubMed

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

    2014-10-20

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

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

    PubMed

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

    2006-08-09

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

  19. Mammalian mismatches in nucleotide metabolism: implications for xenotransplantation.

    PubMed

    Khalpey, Zain; Yuen, Ada H Y; Lavitrano, Marialuisa; McGregor, Christopher G A; Kalsi, Kameljit K; Yacoub, Magdi H; Smolenski, Ryszard T

    2007-10-01

    Acute humoral rejection (AHR) limits the clinical application of animal organs for xenotransplantation. Mammalian disparities in nucleotide metabolism may contribute significantly to the microvascular component in AHR; these, however remain ill-defined. We evaluated the extent of species-specific differences in nucleotide metabolism. HPLC analysis was performed on venous blood samples (nucleotide metabolites) and heart biopsies (purine enzymes) from wild type mice, rats, pigs, baboons, and human donors.Ecto-5'-nucleotidase (E5'N) activities were 4-fold lower in pigs and baboon hearts compared to human and mice hearts while rat activity was highest. Similar differences between pigs and humans were also observed with kidneys and endothelial cells. More than 10-fold differences were observed with other purine enzymes. AMP deaminase (AMPD) activity was exceptionally high in mice but very low in pig and baboon hearts. Adenosine deaminase (ADA) activity was highest in baboons. Adenosine kinase (AK) activity was more consistent across different species. Pig blood had the highest levels of hypoxanthine, inosine and adenine. Human blood uric acid concentration was almost 100 times higher than in other species studied. We conclude that species-specific differences in nucleotide metabolism may affect compatibility of pig organs within a human metabolic environment. Furthermore, nucleotide metabolic mismatches may affect clinical relevance of animal organ transplant models. Supplementation of deficient precursors or application of inhibitors of nucleotide metabolism (e.g., allopurinol) or transgenic upregulation of E5'N may overcome some of these differences.

  20. Was adenine the first purine?

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  1. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    SciTech Connect

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; Melcher, Karsten

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

  2. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    DOE PAGES

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; ...

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

  3. Associations between single nucleotide polymorphisms in folate uptake and metabolizing genes with blood folate, homocysteine and DNA uracil concentrations

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Folate is an essential nutrient which supports nucleotide synthesis and biological methylation reactions. Diminished folate status results in chromosome breakage and is associated with several diseases including colorectal cancer. Folate status is also inversely related to plasma homocys...

  4. Onset of chiral adenine surface growth.

    PubMed

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

    2013-10-07

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

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

    PubMed

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

    2014-12-01

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

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

    PubMed

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

    2017-04-01

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

  7. Relative Reactivity of Ribosyl 2'-OH vs. 3'-OH in Concentrated Aqueous Solutions of Phosphoimidazolide Activated Nucleotides

    NASA Astrophysics Data System (ADS)

    Kanavarioti, Anastassia; Lee, Lynn F.; Gangopadhyay, Sumana

    1999-10-01

    Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN^2'pN and pN^3'pN) and the pyrophosphate-linked (N^5'ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2'-OH vs. 3'-OH was determined from the ratio of the yields of pN^2'pN vs. pN^3'pN. Experiments were performed at 23 °C in the range 7.2 <= pH <= 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg^2+ or Mn^2+) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN^2'pN: pN^3'pN = 2'-5': 3'-5' was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2'-OH is about 2 to 3 times more reactive than the 3'-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN^2'pN: pN^3'pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN^2'pN: pN^3'pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5'= 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3'-OH of one molecule to the reactive P-N bond of

  8. Relative reactivity of ribosyl 2'-OH vs. 3'-OH in concentrated aqueous solutions of phosphoimidazolide activated nucleotides

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Lee, L. F.; Gangopadhyay, S.

    1999-01-01

    Phosphoimidazolide activated ribomononucleotides (*pN, see structure) are useful substrates for the non-enzymatic synthesis of oligonucleotides. In the presence of metal ions, aqueous solutions of *pN yield primarily the two internucleotide-linked (pN2' pN and pN3' pN) and the pyrophosphate-linked (N5' ppN) dimers. Small amounts of cyclic dimers and higher oligomers are also produced. In this study the relative reactivity of 2'-OH vs. 3'-OH was determined from the ratio of the yields of pN2' pN vs. pN3' pN. Experiments were performed at 23 degrees C in the range 7.2 < or = pH < or = 8.4 with substrates that differ in nucleobase (guanosine (G), cytidine (C), uridine (U), and adenosine (A)) and leaving group (imidazole (Im), 2-methylimidazole (2-MeIm) and 2,4-dimethylimidazole (2,4-diMeIm)). Two metal ions (Mg2+ or Mn2+) were employed as catalysts. The conditions used here, i.e. a substrate concentration in the range 0.1 M to 1.0 M and metal ion concentration in the range 0.05 M to 0.2 M, favor base-stacking interactions. The ratio pN2' pN: pN3' pN = 2'-5': 3'-5' was found independent of nucleobase and typically varied between 2 to 3 indicating that the 2'-OH is about 2 to 3 times more reactive than the 3'-OH. *pN with Im, compared to 2-MeIm and 2,4-diMeIm leaving group, produce lower yields of internucleotide linked dimers, and a higher pN2' pN: pN3' pN ratio. Trends in the data, observed with all three leaving groups, suggest an increase in pN2' pN: pN3' pN ratio with decreasing substrate concentration (up to 5.47 with 0.051 M ImpG). The observations are in accord with earlier studies reporting a relative reactivity 2'-5': 3'-5' = 6 to 9 obtained with Im as the leaving group, in dilute nucleotide solutions and under conditions that disfavor stacking. It is speculated that the concentration induced change in the relative reactivity is the result of self-association via base-stacking that enhances selectively the proximity of the 3'-OH of one molecule to the reactive

  9. 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.

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

    PubMed

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

    2013-05-01

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

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

    PubMed

    Rajendiran, N; Thulasidhasan, J

    2015-06-05

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

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

    PubMed

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

    2015-04-01

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

  13. Studies on yeast nucleoside triphosphate-nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). IV. Steady-state kinetic properties with thymidine nucleotides (including 3'-azido-3'-deoxythymidine analogues).

    PubMed

    Kuby, S A; Fleming, G; Alber, T; Richardson, D; Takenaka, H; Hamada, M

    1991-01-01

    A study of the steady-state kinetics of the crystalline brewer's yeast (Saccharomyces carlsbergensis) nucleoside diphosphokinase, with the magnesium complexes of the adenine and thymidine nucleotides as reactants, has led to a postulated kinetic mechanism which proceeds through a substituted enzyme. This agrees with the earlier conclusions of Garces and Cleland [Biochemistry 1969; 8:633-640] who characterized a reaction between the magnesium complexes of the adenine and uridine nucleotides. An advantage of using thymidine nucleotides as reactants is that they permit accurate, rapid and continuous assays of the enzymatic activity in coupled-enzymatic tests. Through measurements of the initial velocities and product inhibition studies, the Michaelis constants, maximum velocities, and inhibition constants could be evaluated for the individual substrates. Competitive substrate inhibition was encountered at relatively high substrate concentrations, which also permitted an evaluation of their ability to act as 'dead-end' inhibitors. The Michaelis constants for the 3'-azido-3'-deoxythymidine (AzT) analogues were also evaluated and, although these values were only somewhat higher than those of their natural substrates, the Km's for the adenine nucleotides as paired substrates were lower and the Vmax's were drastically reduced. The pharmacological implications of these observations are touched upon and extrapolated to the cases where therapeutic doses of AzT may be employed.

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

    PubMed

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

    2012-01-20

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

  15. Dietary nucleotides enhance the liver redox state and protein synthesis in cirrhotic rats.

    PubMed

    Pérez, María José; Sánchez-Medina, Fermín; Torres, Maribel; Gil, Angel; Suárez, Antonio

    2004-10-01

    Cirrhosis is characterized by altered lipid and protein metabolism and an excessive accumulation of extracellular matrix components. The aim of this work was to determine the effect of dietary nucleotide intake on the intracellular pools of nucleic acids and nucleotides, hepatic redox state, and protein synthesis during cirrhosis. Rats were given 300 mg/L thioacetamide (TAA) in drinking water and were fed diets without (TAA-Nt) or with nucleotides (Nt) (TAA+Nt, 3 g each of AMP, inosine 5'-monophosphate, CMP, GMP, and UMP per kg diet) for 4 mo. The degree of liver histological injury was less in group TAA+Nt than in TAA-Nt. The intake of nucleotides significantly increased the hepatic concentration of total nucleotides, adenine nucleotides, and ATP+ADP+AMP. Interestingly, the concentration of CDP-choline, a nucleotide necessary for phospholipid synthesis, was significantly higher in TAA+Nt than in TAA-Nt. The hepatic pyruvate:lactate (P = 0.075) and acetoacetate:beta-hydrodybutyrate (P < 0.05) ratios, indicators of cytosolic and mitochondrial redox states, were lower in TAA-Nt than in TAA+Nt. The total protein concentration was higher in the livers of TAA+Nt than in TAA-Nt. Although there were no differences in the expression of the albumin gene, the hepatic albumin concentration was significantly higher in TAA+Nt than in TAA-Nt. These data indicate that the reduction of liver injury in nucleotide-supplemented rats may be due to the increased intracellular availability of key metabolic nucleotides, the restoration of mitochondrial function, and the augmentation of protein synthesis.

  16. Nucleotide capacitance calculation for DNA sequencing

    SciTech Connect

    Lu, Jun-Qiang; Zhang, Xiaoguang

    2008-01-01

    Using a first-principles linear response theory, the capacitance of the DNA nucleotides, adenine, cytosine, guanine and thymine, are calculated. The difference in the capacitance between the nucleotides is studied with respect to conformational distortion. The result suggests that although an alternate current capacitance measurement of a single-stranded DNA chain threaded through a nano-gap electrodes may not sufficient to be used as a stand alone method for rapid DNA sequencing, the capacitance of the nucleotides should be taken into consideration in any GHz-frequency electric measurements and may also serve as an additional criterion for identifying the DNA sequence.

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

    PubMed

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

    2016-09-01

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

  18. Bound Anionic States of Adenine

    SciTech Connect

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

    2007-03-20

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Anionic states of nucleic acid bases are involved in DNA damage by low-energy electrons and in charge transfer through DNA. Previous gas phase studies of free, unsolvated nucleic acid base parent anions probed only dipole-bound states, which are not present in condensed phase environments, but did not observe valence anionic states, which for purine bases are thought to be adiabatically unbound. Contrary to this expectation, we have demonstrated that some thus far ignored tautomers of adenine, which result from enamine-imine transformations, support valence anionic states with electron vertical detachment energies as large as 2.2 eV, and at least one of these anionic tautomers is adiabatically bound. Moreover, we predict that the new anionic tautomers should also dominate in solutions and should be characterized by larger values of electron vertical detachment energy than the canonical valence anion. All of the newfound anionic tautomers might be formed in the course of dissociative electron attachment followed by a hydrogen atom attachment to a carbon atom, and they might affect the structure and properties of DNA and RNA exposed to low-energy electrons. The new valence states observed here, unlike the dipole-bound state, could exist in condensed phases and might be relevant to radiobiological damage. The discovery of these valence anionic states of adenine was facilitated by the development of (i) an experimental method for preparing parent anions of nucleic acid bases for photoelectron experiments, and (it) a combinatorial/quantum chemical approach for identification of the most stable tautomers of organic molecules.

  19. Adenine Aminohydrolase from Leishmania donovani

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2015-09-01

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

  1. Influence of sprint training on human skeletal muscle purine nucleotide metabolism.

    PubMed

    Stathis, C G; Febbraio, M A; Carey, M F; Snow, R J

    1994-04-01

    To examine the effect of sprint training on human skeletal muscle purine nucleotide metabolism, eight active untrained subjects completed a maximal 30-s sprint bout on a cycle ergometer before and after 7 wk of sprint training. Resting muscle ATP and total adenine nucleotide content were reduced (P < 0.05) by 19 and 18%, respectively, after training. Training resulted in a 52% attenuation (P < 0.05) in the magnitude of ATP depletion after exercise and a similar reduction (P < 0.05) in the accumulation of inosine 5'-monophosphate and ammonia. During recovery, muscle inosine 5'-monophosphate (P < 0.05) and inosine (P < 0.01) content were reduced after training, as was the accumulation of inosine (P < 0.05). Plasma ammonia was higher (P < 0.05) after training early in recovery; in contrast, plasma hypoxanthine concentrations were reduced (P < 0.05) during the latter stages of recovery. The attenuated resting ATP and total adenine nucleotide contents after training probably result from the acute effects of prior training sessions. The reduction in the magnitude of ATP depletion during a 30-s sprint bout after training must reflect an improved balance between ATP hydrolysis and resynthesis. It is unclear which mechanism(s) is responsible for the reduction in the magnitude of ATP degradation after training.

  2. Glibenclamide improves kidney and heart structure and function in the adenine-diet model of chronic kidney disease.

    PubMed

    Diwan, Vishal; Gobe, Glenda; Brown, Lindsay

    2014-01-01

    The development of chronic kidney disease (CKD) and associated cardiovascular disease involves free radical damage and inflammation. Addition of adenine to the diet induces inflammation followed by CKD and cardiovascular disease. NOD-like receptor protein-3 (NLRP-3) is pro-inflammatory in the kidney; glibenclamide inhibits production of NLRP-3. Male Wistar rats were fed either control rat food or adenine (0.25%) in this food for 16 weeks. Glibenclamide (10 mg/kg/day) was administered to two groups with and without adenine for the final 8 weeks. Kidney function (blood urea nitrogen/BUN, plasma creatinine/PCr, plasma uric acid, proteinuria), kidney structure (fibrosis, inflammation), cardiovascular parameters (blood pressure, left ventricular stiffness, vascular responses and echocardiography) and protein expression of markers for oxidative stress (HO-1), and inflammation (TNF-α, NLRP-3) were assessed. In adenine-fed rats, glibenclamide decreased BUN (controls: 6±0.6; adenine: 56.6±5.4; adenine+glibenclamide: 19.4±2.7 mmol/L), PCr (controls: 42±2.8; adenine: 268±23; adenine+glibenclamide: 81±10 μmol/L), proteinuria (controls: 150±7.4; adenine: 303±19; adenine+glibenclamide: 220±13 μmol/L) (all p<0.05). Glibenclamide decreased infiltration of chronic inflammatory cells, fibrosis, tubular damage and expression of HO-1, TNF-α and NLRP-3 in the kidney. Glibenclamide did not alter plasma uric acid concentrations (controls: 38±1; adenine: 63±4; adenine+glibenclamide: 69±14 μmol/L). Cardiovascular changes included decreased systolic blood pressure and improved vascular responses although cardiac fibrosis, left ventricular stiffness and hypertrophy were not reduced. Glibenclamide improved kidney structure and function in CKD and decreased some cardiovascular parameters. Inflammatory markers and cell populations were attenuated by glibenclamide in kidneys.

  3. Chemical probing of adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA

    SciTech Connect

    Rairkar, A.; Rubino, H.M.; Lockard, R.E.

    1988-01-26

    The location of unpaired adenine residues within the secondary structure of rabbit /sup 18/S ribosomal RNA was determined by chemical probing. Naked /sup 18/S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants. Adenines within naked /sup 18/S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides. Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of /sup 32/P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase. The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in /sup 18/S rRNA determined by comparative sequence analysis. The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA. The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit /sup 18/S rRNA than for E. coli /sup 16/S rRNA. Some of these adenines were found clustered in specific helices. Such differences suggest a greater irregularity of many of the helical elements within mammalian /sup 18/S rRNA, as compared with prokaryotic /sup 16/S rRNA. These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

  4. 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

  5. Flavin nucleotides in human lens: regional distribution in brunescent cataracts.

    PubMed

    Bhat, K S; Nayak, S

    1998-12-01

    The biochemical mechanism(s) underlying brunescent cataracts remain unclear. Oxidative stress due to reactive oxygen species may have a role in the pigmentation process in eye lens. We have analysed human cataractous lenses for flavins by high-performance liquid chromatography (HPLC), since flavins are light sensitive and act as endogenous sensitizers generating reactive oxygen species in the eye. The most significant observation in this study is that higher levels of flavin nucleotides occur in brown lens compared to yellow lens. The concentration of flavin nucleotides (flavin monouncleotide, FMN + flavin adenine dinucleotide, FAD) was highest in the nuclear region of the lens followed by the cortical and capsule-epithelial regions. However, the ratio of FAD/FMN was lowest in the nuclear region of the lens followed by other regions. On the other hand, riboflavin was not detected in any of the lens (cataractous) regions. These results suggest that the observed increase in flavin nucleotides in the ocular tissue could contribute towards deepening of lens pigmentation.

  6. Nicotinamide adenine dinucleotide biosynthesis promotes liver regeneration.

    PubMed

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

    2017-02-01

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

  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.

  8. Intramolecular interactions in aminoacyl nucleotides: Implications regarding the origin of genetic coding and protein synthesis

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Watkins, C. L.; Hall, L. M.

    1986-01-01

    Cellular organisms store information as sequences of nucleotides in double stranded DNA. This information is useless unless it can be converted into the active molecular species, protein. This is done in contemporary creatures first by transcription of one strand to give a complementary strand of mRNA. The sequence of nucleotides is then translated into a specific sequence of amino acids in a protein. Translation is made possible by a genetic coding system in which a sequence of three nucleotides codes for a specific amino acid. The origin and evolution of any chemical system can be understood through elucidation of the properties of the chemical entities which make up the system. There is an underlying logic to the coding system revealed by a correlation of the hydrophobicities of amino acids and their anticodonic nucleotides (i.e., the complement of the codon). Its importance lies in the fact that every amino acid going into protein synthesis must first be activated. This is universally accomplished with ATP. Past studies have concentrated on the chemistry of the adenylates, but more recently we have found, through the use of NMR, that we can observe intramolecular interactions even at low concentrations, between amino acid side chains and nucleotide base rings in these adenylates. The use of this type of compound thus affords a novel way of elucidating the manner in which amino acids and nucleotides interact with each other. In aqueous solution, when a hydrophobic amino acid is attached to the most hydrophobic nucleotide, AMP, a hydrophobic interaction takes place between the amino acid side chain and the adenine ring. The studies to be reported concern these hydrophobic interactions.

  9. Single nucleotide polymorphisms in the vitamin D pathway associating with circulating concentrations of vitamin D metabolites and non-skeletal health outcomes: Review of genetic association studies.

    PubMed

    Jolliffe, David A; Walton, Robert T; Griffiths, Christopher J; Martineau, Adrian R

    2016-11-01

    Polymorphisms in genes encoding proteins involved in vitamin D metabolism and transport are recognised to influence vitamin D status. Syntheses of genetic association studies linking these variants to non-skeletal health outcomes are lacking. We therefore conducted a literature review to identify reports of statistically significant associations between single nucleotide polymorphisms (SNP) in 11 vitamin D pathway genes (DHCR7, CYP2R1, CYP3A4, CYP27A1, DBP, LRP2, CUB, CYP27B1, CYP24A1, VDR and RXRA) and non-bone health outcomes and circulating levels of 25-hydroxyvitamin D (25[OH]D and 1,25-dihydroxyvitamin D (1,25[OH]2D). A total of 120 genetic association studies reported positive associations, of which 44 investigated determinants of circulating 25(OH)D and/or 1,25(OH)2D concentrations, and 76 investigated determinants of non-skeletal health outcomes. Statistically significant associations were reported for a total of 55 SNP in the 11 genes investigated. There was limited overlap between genetic determinants of vitamin D status and those associated with non-skeletal health outcomes: polymorphisms in DBP, CYP2R1 and DHCR7 were the most frequent to be reported to associate with circulating concentrations of 25(OH)D, while polymorphisms in VDR were most commonly reported to associate with non-skeletal health outcomes, among which infectious and autoimmune diseases were the most represented.

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

    PubMed

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

    1965-06-25

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

  11. One-pot synthesis of fluorescent polysaccharides: adenine grafted agarose and carrageenan.

    PubMed

    Oza, Mihir D; Prasad, Kamalesh; Siddhanta, A K

    2012-08-01

    New fluorescent polysaccharides were synthesized by grafting the nucleobase adenine on to the backbones of agarose and κ-carrageenan, which were characterized by FT-IR, (13)C NMR, TGA, XRD, UV, and fluorescence properties. The synthesis involved a rapid water based potassium persulfate (KPS) initiated method under microwave irradiation. The emission spectra of adenine grafted agarose and κ-carrageenan were recorded in aqueous (5×10(-5) M) solution, exhibiting λ(em,max) 347 nm by excitation at 261 nm, affording ca. 30% and 40% enhanced emission intensities, respectively compared to that of pure adenine solution in the same concentration. Similar emission intensity was recorded in the pure adenine solution at its molar equivalent concentrations present in the 5×10(-5) M solution of the agarose and carrageenan grafted products, that is, 3.28×10(-5) M and 4.5×10(-5) M respectively. These fluorescent adenine grafted products may have potential utility in various sensor applications.

  12. Electrochemical studies on the oxidation of guanine and adenine at cyclodextrin modified electrodes.

    PubMed

    Abbaspour, Abdolkarim; Noori, Abolhassan

    2008-12-01

    An electrochemical sensor for guanine and adenine using cyclodextrin-modified poly(N-acetylaniline) (PNAANI) on a carbon paste electrode has been developed. The oxidation mechanism of guanine and adenine on the surface of the electrode was investigated by cyclic voltammetry. It was found that the electrode processes are irreversible, pH dependent, and involve several reaction products. The electron transfer process occurs in consecutive steps with the formation of a strongly adsorbed intermediate on the electrode surface. Also, a new method for estimating the apparent formation constants of guanine and adenine with the immobilized cyclodextrins, through the change of surface coverage of studied analytes has been reported. Both guanine and adenine showed linear concentrations in the range of 0.1-10 microM by using differential pulse voltammetry, with an experimental limit of detection down to 0.05 microM. Linear concentration ranges of 2-150 microM for guanine and 6-104 microM for adenine have been found when cyclic voltammetry was used for determination of both analytes.

  13. Nucleotide release provides a mechanism for airway surface liquid homeostasis.

    PubMed

    Lazarowski, Eduardo R; Tarran, Robert; Grubb, Barbara R; van Heusden, Catharina A; Okada, Seiko; Boucher, Richard C

    2004-08-27

    Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca(2+) -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A(2b) adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N(6)-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A(2b) adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A(2b) receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis.

  14. Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis*

    PubMed Central

    Lazarowski, Eduardo R.; Tarran, Robert; Grubb, Barbara R.; van Heusden, Catharina A.; Okada, Seiko; Boucher, Richard C.

    2010-01-01

    Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca2+- and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A2b adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N6-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolayers that endogenously express a luminal A2b adenosine receptor, we found that basal as well as forskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A2b receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis. PMID:15210701

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

    PubMed

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

    2015-05-01

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

  16. Effect of gum arabic on oxidative stress and inflammation in adenine-induced chronic renal failure in rats.

    PubMed

    Ali, Badreldin H; Al-Husseni, Isehaq; Beegam, Sumyia; Al-Shukaili, Ahmed; Nemmar, Abderrahim; Schierling, Simone; Queisser, Nina; Schupp, Nicole

    2013-01-01

    Inflammation and oxidative stress are known to be involved in the pathogenesis of chronic kidney disease in humans, and in chronic renal failure (CRF) in rats. The aim of this work was to study the role of inflammation and oxidative stress in adenine-induced CRF and the effect thereon of the purported nephroprotective agent gum arabic (GA). Rats were divided into four groups and treated for 4 weeks as follows: control, adenine in feed (0.75%, w/w), GA in drinking water (15%, w/v) and adenine+GA, as before. Urine, blood and kidneys were collected from the rats at the end of the treatment for analysis of conventional renal function tests (plasma creatinine and urea concentration). In addition, the concentrations of the pro-inflammatory cytokine TNF-α and the oxidative stress markers glutathione and superoxide dismutase, renal apoptosis, superoxide formation and DNA double strand break frequency, detected by immunohistochemistry for γ-H2AX, were measured. Adenine significantly increased the concentrations of urea and creatinine in plasma, significantly decreased the creatinine clearance and induced significant increases in the concentration of the measured inflammatory mediators. Further, it caused oxidative stress and DNA damage. Treatment with GA significantly ameliorated these actions. The mechanism of the reported salutary effect of GA in adenine-induced CRF is associated with mitigation of the adenine-induced inflammation and generation of free radicals.

  17. 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.

  18. 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.

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

    PubMed

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

    2016-01-05

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

  20. Graphene-Enhanced Raman Scattering from the Adenine Molecules

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  1. Suppression of glycine-15N incorporation into urinary uric acid by adenine-8-13C in normal and gouty subjects

    PubMed Central

    Seegmiller, J. Edwin; Klinenberg, James R.; Miller, John; Watts, R. W. E.

    1968-01-01

    Adenine inhibited the de novo synthesis of purines in both normal and gouty man as shown by inhibition of the incorporation of glycine-15N into urinary uric acid without altering the incorporation of glycine-15N into urinary creatinine. The diminished purine synthesis did not result in a diminution in the 24 hr excretion of uric acid. This observation was explainable in part by the prompt conversion of adenine to uric acid. In addition to this direct conversion, adenine-8-13C provided a slow and prolonged contribution to urinary uric acid. A feedback inhibition of purine synthesis by nucleotides derived from adenine provides the best interpretation of these results. PMID:5645862

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

    SciTech Connect

    Lee, Seongmin; Verdine, Gregory L.

    2010-01-14

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

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

    PubMed Central

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

    1977-01-01

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

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

    SciTech Connect

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

    2007-07-01

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

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

    PubMed

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

    2014-03-01

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

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

    PubMed Central

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

    1973-01-01

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

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

    PubMed

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

    1973-01-01

    A9145 is a basic, water-soluble, antifungal antibiotic which is produced in a complex organic medium by Streptomyces griseolus. The metabolite has a molecular weight of 510, and contains adenine as well as sugar hydroxyl and amino groups. Although glucose, fructose, glucose polymers, and some long-chain fatty acid methyl esters supported biosynthesis, oils were superior, with cottonseed oil being preferred. Several ions and salts, especially Co(2+), PO(4) (3-), and CaCO(3), were stimulatory. Adenine, nucleosides, and some amino acids increased the accumulation of A9145 in shaken-flask fermentors. Enrichment of the culture medium with tyrosine afforded maximal enhancement of antibiotic production in both flask and tank fermentors. Control of the dissolved O(2) level was also critical, the optimal concentration being 3 x 10(-2) to 4.5 x 10(-2) mumole of O(2)/ml. Optimization of various fermentation parameters increased antibiotic titers approximately 135-fold in shaken flask fermentors and 225-fold in stirred vessels.

  8. 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.

  9. 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

  10. Ameliorative effect of chrysin on adenine-induced chronic kidney disease in rats.

    PubMed

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

    2015-01-01

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

  11. Purine metabolite and energy charge analysis of Trypanosoma brucei cells in different growth phases using an optimized ion-pair RP-HPLC/UV for the quantification of adenine and guanine pools.

    PubMed

    Graven, Patricia; Tambalo, Margherita; Scapozza, Leonardo; Perozzo, Remo

    2014-06-01

    Human African Trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. Although trypanosomes are well-studied model organisms, only little is known about their adenine and guanine nucleotide pools. Besides being building blocks of RNA and DNA, these nucleotides are also important modulators of diverse biochemical cellular processes. Adenine nucleotides also play an important role in the regulation of metabolic energy. The energetic state of cells is evaluated by the energy charge which gives information about how much energy is available in form of high energy phosphate bonds of adenine nucleotides. A sensitive and reproducible ion-pair RP-HPLC/UV method was developed and optimized, allowing the quantification of guanine and adenine nucleosides/nucleotides in T. brucei. With this method, the purine levels and their respective ratios were investigated in trypanosomes during logarithmic, stationary and senescent growth phases. Results of this study showed that all adenine and guanine purines under investigation were in the low mM range. The energy charge was found to decrease from logarithmic to static and to senescent phase whereas AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, the AMP/ATP ratio varied as the square of the ADP/ATP ratio, indicating AMP to be the key energy sensor molecule in trypanosomes.

  12. Classification of pseudo pairs between nucleotide bases and amino acids by analysis of nucleotide-protein complexes.

    PubMed

    Kondo, Jiro; Westhof, Eric

    2011-10-01

    Nucleotide bases are recognized by amino acid residues in a variety of DNA/RNA binding and nucleotide binding proteins. In this study, a total of 446 crystal structures of nucleotide-protein complexes are analyzed manually and pseudo pairs together with single and bifurcated hydrogen bonds observed between bases and amino acids are classified and annotated. Only 5 of the 20 usual amino acid residues, Asn, Gln, Asp, Glu and Arg, are able to orient in a coplanar fashion in order to form pseudo pairs with nucleotide bases through two hydrogen bonds. The peptide backbone can also form pseudo pairs with nucleotide bases and presents a strong bias for binding to the adenine base. The Watson-Crick side of the nucleotide bases is the major interaction edge participating in such pseudo pairs. Pseudo pairs between the Watson-Crick edge of guanine and Asp are frequently observed. The Hoogsteen edge of the purine bases is a good discriminatory element in recognition of nucleotide bases by protein side chains through the pseudo pairing: the Hoogsteen edge of adenine is recognized by various amino acids while the Hoogsteen edge of guanine is only recognized by Arg. The sugar edge is rarely recognized by either the side-chain or peptide backbone of amino acid residues.

  13. 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

  14. Concentrations of Pro-Inflammatory Cytokines Are Not Associated with Senescence Marker p16INK4a or Predictive of Intracellular Emtricitabine/Tenofovir Metabolite and Endogenous Nucleotide Exposures in Adults with HIV Infection

    PubMed Central

    Maas, Brian M.; Francis, Owen; Mollan, Katie R.; Lee, Cynthia; Cottrell, Mackenzie L.; Prince, Heather M. A.; Sykes, Craig; Trezza, Christine; Torrice, Chad; White, Nicole; Malone, Stephanie; Hudgens, Michael G.; Sharpless, Norman E.; Dumond, Julie B.

    2016-01-01

    Objectives As the HIV-infected population ages, the role of cellular senescence and inflammation on co-morbid conditions and pharmacotherapy is increasingly of interest. p16INK4a expression, a marker for aging and senescence in T-cells, is associated with lower intracellular concentrations of endogenous nucleotides (EN) and nucleos(t)ide reverse transcriptase inhibitors (NRTIs). This study expands on these findings by determining whether inflammation is contributing to the association of p16INK4a expression with intracellular metabolite (IM) exposure and endogenous nucleotide concentrations. Methods Samples from 73 HIV-infected adults receiving daily tenofovir/emtricitabine (TFV/FTC) with either efavirenz (EFV) or atazanavir/ritonavir (ATV/r) were tested for p16INK4a expression, and plasma cytokine and intracellular drug concentrations. Associations between p16INK4a expression and cytokine concentrations were assessed using maximum likelihood methods, and elastic net regression was applied to assess whether cytokines were predictive of intracellular metabolite/endogenous nucleotide exposures. Results Enrolled participants had a median age of 48 years (range 23–73). There were no significant associations between p16INK4a expression and cytokines. Results of the elastic net regression showed weak relationships between IL-1Ra and FTC-triphosphate and deoxyadenosine triphosphate exposures, and MIP-1β, age and TFV-diphosphate exposures. Conclusions In this clinical evaluation, we found no relationships between p16INK4a expression and cytokines, or cytokines and intracellular nucleotide concentrations. While inflammation is known to play a role in this population, it is not a major contributor to the p16INK4a association with decreased IM/EN exposures in these HIV-infected participants. PMID:28036343

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

    PubMed

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

    2015-10-29

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

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

    PubMed

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

    2017-02-24

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

  17. In vitro selection of adenine-dependent ribozyme against Tpl2/Cot oncogene.

    PubMed

    Li, Yan-Li; Vergne, Jacques; Torchet, Claire; Maurel, Marie-Christine

    2009-01-01

    Hairpin ribozymes possess the properties of RNA sequence-specific recognition and site-specific cleavage. These properties make them a powerful extension of the antisense approach for the inhibition of gene expression. From a randomized RNA pool of hairpin ribozymes, using the systematic evolution of ligands by exponential enrichment, we have obtained an adenine-dependent hairpin ribozyme, Tpl2/Cot (tumour progression locus 2) ribozyme, which cleaves the Tpl2/Cot kinase mRNA sequence at nucleotides A225/G226 relative to the start codon of translation. This serine/threonine kinase activates the mitogen-activated protein kinase pathway implicated in cell proliferation in cancer. The selected 'Tpl2/Cot-YL ribozyme' efficiently cleaves its target sequence in cis and in trans; furthermore, the ribozyme efficiently cleaves a longer target sequence of 54 nucleotides in trans, as well as the full-length mRNA.

  18. Effect of atracylodes rhizome polysaccharide in rats with adenine-induced chronic renal failure.

    PubMed

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

    2015-01-01

    The aim of the study was to elucidate the therapeutic effects of Atracylodes rhizome polysaccharide on adenine-induced chronic renal failure in rats. Fifty male Sprague Dawley rats were selected and randomly divided in to 5 groups (n=10 rats per group): The normal control group, the chronic renal failure pathological control group, the dexamethasone treatment group and two Atracylodes rhizome polysaccharide treatment groups, treated with two different concentrations of the polysaccharide, the Atracylodes rhizome polysaccharide high group and the Atracylodes rhizome polysaccharide low group. All the rats, except those in the normal control group were fed adenine-enriched diets, containing 10 g adenine per kg food for 3 weeks. After being fed with adenine, the dexamethasone treatment group, Atracylodes rhizome polysaccharide high group and Atracylodes rhizome polysaccharide low group rats were administered the drug orally for 2 weeks. On day 35, the kidney coefficient of the rats and the serum levels of creatinine, blood urea nitrogen, total protein and hemalbumin were determined. Subsequent to experimentation on a model of chronic renal failure in rats, the preparation was proven to be able to reduce serum levels of creatinine, blood urea nitrogen and hemalbumin levels (P<0.05) and improve renal function. Atracylodes rhizome polysaccharide had reversed the majority of the indices of chronic renal failure in rats.

  19. Hydrogen exchange in nucleosides and nucleotides. Measurement of hydrogen exchange by stopped-flow and ultraviolet difference spectroscopy.

    PubMed

    Cross, D G

    1975-01-28

    Time-dependent changes in the ultraviolet absorbance of the adenine chromophore are observed in the stopped-flow spectrophotometer when adenosine and its analogs are rapidly transferred from protium oxide to deuterium oxide. These absorbance changes are shown to result from hydrogen exchange in the exocyclic amino groups of the purine ribonucleosides by using derivatives of adenosine in which methyl groups replace exchangeable hydrogens and by showing that the general characteristics of hydrogen exchange in adenosine analogs agree with those found here. A study of the dependence of hydrogen-exchange rate constants on adenosine, AMP, and phosphate concentration showed there is a second-order dependence on AMP concentration which is primarily due to intermolecular catalysis by the phosphate group of the nucleotide. The deuterium oxide perturbation difference spectrum, obtained at equilibrium, was found to contain two components that result from blue shifts of the adenine chromophore absorbance: (1) a shift cause by the substitution of deuterium for protium in the ring (N1) nitrogen and exocyclic nitrogens, and (2) a shift associated with a change in the polarizability of the medium. Since the theory of solvent perturbation, which is used to measure the relative "exposure" of chromophores in macromolecules, assumes that the spectral shifts observed are solely due to (2) above, the use of deuterium oxide as a measure of chromophore exposure to perturbants the size of water must be reexamined.

  20. Adenine oxidation by pyrite-generated hydroxyl radicals.

    PubMed

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

    2010-04-26

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

  1. Adenine and guanine 8CH exchange in nucleic acids: resolution and measurement by Raman optical multichannel analysis.

    PubMed

    Lamba, O P; Becka, R; Thomas, G J

    Deuterium exchange of 8C protons of adenine and guanine in nucleic acids is conveniently monitored by laser Raman spectrophotometry, and the average exchange rate so determined [kA + kG] can be exploited as a dynamic probe of the secondary structure of DNA or RNA [J. M. Benevides and G. J. Thomas, Jr. (1985) Biopolymers 24, 667-682]. The present work describes a rapid Raman procedure, based upon optical multichannel analysis, which permits discrimination of the different 8CH exchange rates, kA of adenine and kG of guanine, in a single experimental protocol. For this procedure, simultaneous measurements are made of the intensity decay or frequency shift in separately resolved Raman bands of adenine and guanine, each of which is sensitive only to 8C deuteration of its respective purine. Resolution of the rates kA and kG is demonstrated for the mononucleotide mixtures, 5'-rAMP + 5'-rGMP and 5'-dAMP + 5'-dGMP, for the polynucleotides poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), for calf thymus DNA, and for the 17 base-pair operator OR3. We show that the different exchange rates of adenine and guanine, in nucleotide mixtures and in DNA, may also be calculated independently from intensity decay of the composite 1481-cm-1 band, comprising overlapped adenine and guanine components, over a time domain that encompasses two distinct regimes: (1) a relatively more rapid exchange of guanine, and (2) a concurrent slower exchange of adenine. Both methods developed here yield consistent results. We find, first, that exchange of guanine is approximately twofold more rapid than that of adenine when both purines are present in the same structure and solvent environment, presumably a consequence of the greater basicity of the 7N site of guanine. Second, we find that adenine suffers greater retardation of exchange than guanine when both purines are incorporated into a "classical" B-DNA secondary structure, such as that of calf thymus DNA. This finding suggests different

  2. Adenine deaminase is encoded by Tad1 and participates in copper accumulation in Trichoderma reesei.

    PubMed

    Fu, Kehe; Fan, Lili; Yu, Chuangjing; Li, Yingying; Gao, Shigang; Li, Yaqian; Chen, Jie

    2014-02-01

    We cloned a novel Tad1 gene and demonstrated that this gene is closely involved in copper bioaccumulation in Trichoderma reesei. Tad1 gene encodes a 510 amino acids protein of the amidohydrolase superfamily which belongs to COG0402. We found that adenine was the most efficient substrate of Tad1 protein among the substrates used in this study. Gene function was also investigated by overexpression and RNA interference. Results showed that copper accumulation increased in mutant cells when Tad1 was overexpressed; by contrast, copper accumulation significantly decreased when Tad1 was inhibited. To investigate the function of Tad1 in copper bioaccumulation, we determined adenine, hypoxanthine, and xanthine concentrations by reversed phase HPLC. Tad1 overexpression induced a substantial production of xanthine, which functions in binding numerous copper ions and reducing copper concentration. We further compared the gene expression profile of AT01 with that of a wild-type T. reesei strain grown in a medium containing 1.0mM Cu(2+) by performing DNA microarray. Several upregulated genes in the mutant were associated with adenine or copper metabolism.

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

    PubMed

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

    2015-11-07

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

  4. 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.

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

    PubMed

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

    2011-12-07

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

  6. Uncovering the polymerase-induced cytotoxicity of an oxidized nucleotide

    NASA Astrophysics Data System (ADS)

    Freudenthal, Bret D.; Beard, William A.; Perera, Lalith; Shock, David D.; Kim, Taejin; Schlick, Tamar; Wilson, Samuel H.

    2015-01-01

    Oxidative stress promotes genomic instability and human diseases. A common oxidized nucleoside is 8-oxo-7,8-dihydro-2'-deoxyguanosine, which is found both in DNA (8-oxo-G) and as a free nucleotide (8-oxo-dGTP). Nucleotide pools are especially vulnerable to oxidative damage. Therefore cells encode an enzyme (MutT/MTH1) that removes free oxidized nucleotides. This cleansing function is required for cancer cell survival and to modulate Escherichia coli antibiotic sensitivity in a DNA polymerase (pol)-dependent manner. How polymerases discriminate between damaged and non-damaged nucleotides is not well understood. This analysis is essential given the role of oxidized nucleotides in mutagenesis, cancer therapeutics, and bacterial antibiotics. Even with cellular sanitizing activities, nucleotide pools contain enough 8-oxo-dGTP to promote mutagenesis. This arises from the dual coding potential where 8-oxo-dGTP(anti) base pairs with cytosine and 8-oxo-dGTP(syn) uses its Hoogsteen edge to base pair with adenine. Here we use time-lapse crystallography to follow 8-oxo-dGTP insertion opposite adenine or cytosine with human pol β, to reveal that insertion is accommodated in either the syn- or anti-conformation, respectively. For 8-oxo-dGTP(anti) insertion, a novel divalent metal relieves repulsive interactions between the adducted guanine base and the triphosphate of the oxidized nucleotide. With either templating base, hydrogen-bonding interactions between the bases are lost as the enzyme reopens after catalysis, leading to a cytotoxic nicked DNA repair intermediate. Combining structural snapshots with kinetic and computational analysis reveals how 8-oxo-dGTP uses charge modulation during insertion that can lead to a blocked DNA repair intermediate.

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

    PubMed

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

    2006-08-25

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

  8. Reverse transcriptase incorporation of 1,5-anhydrohexitol nucleotides

    PubMed Central

    Vastmans, Karen; Froeyen, Matheus; Kerremans, Luc; Pochet, Sylvie; Herdewijn, Piet

    2001-01-01

    Several reverse transcriptases were studied for their ability to accept anhydrohexitol triphosphates, having a conformationally restricted six-membered ring, as substrate for template-directed synthesis of HNA. It was found that AMV, M-MLV, M-MLV (H–), RAV2 and HIV-1 reverse transcriptases were able to recognise the anhydrohexitol triphosphate as substrate and to efficiently catalyse the incorporation of one non-natural anhydrohexitol nucleotide opposite a natural complementary nucleotide. However, only the dimeric enzymes, the RAV2 and HIV-1 reverse transcriptases, seemed to be able to further extend the primer with another anhydrohexitol building block. Subsequently, several HIV-1 mutants (4×AZT, 4×AZT/L100I, L74V, M184V and K65A) were likewise analysed, resulting in selection of K65A and, in particular, M184V as the most succesful mutant HIV-1 reverse transcriptases capable of elongating a DNA primer with several 1,5-anhydrohexitol adenines in an efficient way. Results of kinetic experiments in the presence of this enzyme revealed that incorporation of one anhydrohexitol nucleotide of adenine or thymine gave an increased (for 1,5-anhydrohexitol-ATP) and a slightly decreased (for 1,5-anhydrohexitol-TTP) Km value in comparison to that of their natural counterparts. However, no more than four analogues could be inserted under the experimental conditions required for selective incorporation. Investigation of incorporation of the altritol anhydrohexitol nucleotide of adenine in the presence of M184V and Vent (exo–) DNA polymerase proved that an adjacent hydroxyl group on C3 of 1,5-anhydrohexitol-ATP has a detrimental effect on the substrate activity of the six-ring analogue. These results could be rationalised based on the X-ray structure of HIV-1 reverse transcriptase. PMID:11470872

  9. 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.

  10. Radiolysis of aqueous adenine (vitamin B4) and 8-hydroxyadenine

    NASA Astrophysics Data System (ADS)

    Hartmann, J.; Quint, R. M.; Getoff, N.

    2007-05-01

    The radiolysis of adenine (vitamin B4) was studied in aqueous solution (pH˜7.4) saturated either with argon (operating radicals: 44% e -aq, 46% OH, 10% H) or with air (46% OH, 54% O 2rad - ) and with N 2O (90% OH, 10% H), respectively. The obtained initial Gi-values are: 0.88, 1.16 and 1.45. As main radiolytic product was determined 8-hydroxyadenine (8-HOA), whose yield depends on the OH concentration in the reacting media. Hence, under the same experimental conditions the Gi-values are in media saturated with argon: 0.1, in air: 0.15 and in N 2O: 0.29. In aerated solution also a mixture of aldehydes as well as of carboxylic acids were formed, but they were not identified. 8-HOA is of some biological interest; therefore, its radiolysis was also investigated under the same conditions. The determined Gi(-8HOA)-values were in airfree solution negligible, in aerated solutions: 3.1 and in the presence of N 2O: 4.0. For explanation of the product formation some probable reaction mechanisms were given.

  11. Absence of Ca2+-Induced Mitochondrial Permeability Transition but Presence of Bongkrekate-Sensitive Nucleotide Exchange in C. crangon and P. serratus

    PubMed Central

    Konrad, Csaba; Kiss, Gergely; Torocsik, Beata; Adam-Vizi, Vera; Chinopoulos, Christos

    2012-01-01

    Mitochondria from the embryos of brine shrimp (Artemia franciscana) do not undergo Ca2+-induced permeability transition in the presence of a profound Ca2+ uptake capacity. Furthermore, this crustacean is the only organism known to exhibit bongkrekate-insensitive mitochondrial adenine nucleotide exchange, prompting the conjecture that refractoriness to bongkrekate and absence of Ca2+-induced permeability transition are somehow related phenomena. Here we report that mitochondria isolated from two other crustaceans, brown shrimp (Crangon crangon) and common prawn (Palaemon serratus) exhibited bongkrekate-sensitive mitochondrial adenine nucleotide transport, but lacked a Ca2+-induced permeability transition. Ca2+ uptake capacity was robust in the absence of adenine nucleotides in both crustaceans, unaffected by either bongkrekate or cyclosporin A. Transmission electron microscopy images of Ca2+-loaded mitochondria showed needle-like formations of electron-dense material strikingly similar to those observed in mitochondria from the hepatopancreas of blue crab (Callinectes sapidus) and the embryos of Artemia franciscana. Alignment analysis of the partial coding sequences of the adenine nucleotide translocase (ANT) expressed in Crangon crangon and Palaemon serratus versus the complete sequence expressed in Artemia franciscana reappraised the possibility of the 208-214 amino acid region for conferring sensitivity to bongkrekate. However, our findings suggest that the ability to undergo Ca2+-induced mitochondrial permeability transition and the sensitivity of adenine nucleotide translocase to bongkrekate are not necessarily related phenomena. PMID:22768139

  12. 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.

  13. 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

  14. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  16. 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

  17. 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

  18. Updating Our View of Organelle Genome Nucleotide Landscape

    PubMed Central

    Smith, David Roy

    2012-01-01

    Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits. PMID:22973299

  19. High-mobility group box-1 protein in adenine-induced chronic renal failure and the influence of gum arabic thereon.

    PubMed

    Ali, B H; Al Za'abi, M; Al Shukaili, A; Nemmar, A

    2015-01-01

    Pathogenesis of adenine-induced chronic renal failure may involve inflammatory, immunological and/or oxidant mechanisms. Gum arabic (GA) is a complex polysaccharide that acts as an anti-oxidant which can modulate inflammatory and/or immunological processes. Therefore, we tested here the effect of GA treatment (15 % in the drinking water for 4 weeks) in plasma and urine of rats, on a novel cytokine that has been shown to be pro-inflammatory, viz, DNA-binding high-mobility group box-1 protein (HMGB1). Adenine (0.75 % in the feed, 4 weeks) significantly increased indoxyl sulphate, urea and creatinine concentrations in plasma, and significantly decreased the creatinine clearance. GA significantly abated these effects. The concentrations of HMGB1 in urine before the start of the experiment were similar in all four groups. However, 24 h after the last treatment, adenine treatment increased significantly the concentration of HMGB1 when compared with the control. GA treatment did not affect the HMGB1 concentration in urine. Moreover, the concentration of HMGB1 in plasma obtained 24 h after the last treatment in rats treated with adenine was drastically reduced compared with the control group. This may explain its significant rise in urine. In conclusion, HMGB1 can be considered a potentially useful biomarker in adenine induced CRF and its treatment.

  20. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.

    PubMed

    Wigand, R

    1979-01-01

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

  1. Metabolism of Benzyladenine is Impaired in a Mutant of Arabidopsis thaliana Lacking Adenine Phosphoribosyltransferase Activity 1

    PubMed Central

    Moffatt, Barbara; Pethe, Claude; Laloue, Michel

    1991-01-01

    Formation of the riboside-5′-monophosphate is a general feature of the metabolism of cytokinins in plants. As part of a study of the biological significance of the nucleotide form of cytokinins, we analyzed a mutant of Arabidopsis thaliana deficient in adenine phosphoribosyltransferase (APRT) activity for its ability to metabolize N6-benzyladenine (BA). Formation of N6-benzyladenosine-5′-monophosphate (BAMP) was assayed in vivo, by feeding tritiated BA to wild-type and mutant plantlets, and in crude plantlet extracts. Metabolites were separated by high performance liquid chromatography and quantitated by on-line liquid scintillation spectrometry. BA was rapidly absorbed by A. thaliana plantlets and primarily converted to BAMP and to BA 7- and 9-glucosides. BA was also rapidly absorbed by APRT-deficient plantlets, but its conversion to BAMP was strongly reduced. Formation of BAMP from N6-benzyladenosine was not affected in the mutant plantlets. In vitro conversion of BA to its nucleoside-5′-monophosphate was detected in crude extracts of wild-type plantlets, but not in extracts of APRT-deficient plantlets. Therefore, results of both assays indicate that APRT-deficient tissue does not convert BA to BAMP to a significant extent. Further, nondenaturing isoelectric focusing analysis of APRT activity in leaf extracts indicated that the enzyme activities which metabolize adenine and BA into their corresponding riboside-5′-monophosphate in extracts of wild-type plantlets have the same apparent isoelectric point. These activities were not detected in extracts prepared from APRT-deficient plantlets. Thus, these results demonstrate that APRT is the main enzyme which converts BA to its nucleotide form in young A. thaliana plants and that the ribophosphorylation of BA is not a prerequisite of its absorption by the plantlets. Images Figure 4 PMID:16668070

  2. Kidney Disease in Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2015-08-03

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

  5. 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.

  6. Single nucleotide polymorphisms of APOA1 gene and their relationship with serum apolipoprotein A-I concentrations in the native population of Assam

    PubMed Central

    Bora, Kaustubh; Pathak, Mauchumi Saikia; Borah, Probodh; Hussain, Md. Iftikar; Das, Dulmoni

    2015-01-01

    Background There is a growing interest in the role of allelic variants of the APOA1 gene in relation to a number of disorders. We described two common polymorphisms of the APOA1 gene, G-75A and C+83T and investigated their potential influence on the serum apolipoprotein A-I (apo A-I) levels in the native population of Assam — a region that is ethnically distinct and from where no information is hitherto available. Methods Blood samples were collected from 150 healthy volunteers. Apo A-I levels were estimated by immunoturbidometry. Genotyping was done by a PCR-RFLP method that involved DNA extraction from whole blood, followed by polymerase chain reaction and digestion of the PCR product by MspI restriction enzyme, and analysis of fragment sizes in 12% polyacrylamide gel. Results The GG variant at G-75A locus and CC variant at C+83T locus were the most prevalent. GG/CC was the most common combination. Homozygous TT genotype was not detected in any of the subjects. The rare allele frequencies for the G-75A and C+83T sites were found to be 0.22 and 0.06 respectively, which significantly differed from those reported in some other populations in neighbouring regions. Serum apo A-I concentrations did not vary significantly across the detected genotypes. These findings were consistent in both sexes. Conclusion We described the distribution of the G-75A and C+83T polymorphisms of the APOA1 gene in the population of Assam for the first time. These polymorphisms were not found to directly influence apo A-I concentrations in this population either individually or synergistically. PMID:26702398

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

    PubMed

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

    2006-06-06

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

  8. AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B

    PubMed Central

    Johanns, M.; Lai, Y.-C.; Hsu, M.-F.; Jacobs, R.; Vertommen, D.; Van Sande, J.; Dumont, J. E.; Woods, A.; Carling, D.; Hue, L.; Viollet, B.; Foretz, M; Rider, M H

    2016-01-01

    Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation. PMID:26952277

  9. Negative ion formation in potassium-adenine collisions

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

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

  10. DNA aptamer selection in methanolic media: Adenine-aptamer as proof-of-concept.

    PubMed

    Chaou, Thinhinane; Vialet, Brune; Azéma, Laurent

    2016-03-15

    The major objective of this study is to investigate the usefulness of aptamers as in situ detection tool in organic solvents, which are often used for environmental extraction. But two problems related to the use of methanol-containing buffers have to be addressed. Firstly, the folding of nucleic acids can be impaired, because of weaker hydrogen bonding interactions. Secondly, the affinity of aptamers selected in aqueous buffers can be altered by the presence of methanol. Thus, in order to improve hydrophobicity of the DNA pool, nucleotide with hydrophobic modification 5-(octa1,7-diynyl)-2'-deoxyuridine (ODT) has been chosen instead of thymidine. As a proof of concept, an adenine aptamer operating in presence 25% of methanol has been selected. We have shown that the modified nucleotide is essential for target binding in organic media, in addition to essential structural pattern as proposed through analysing truncated sequences analysis. The strategy described in this paper offers preliminary insight on the adaptability of the implementation of aptamers as key instrument for in situ detection. It could be broaden to identify other aptamers directed against other chemical species after alcoholic extraction or for monitoring by-product traces in drugs production.

  11. 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.

  12. 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

  13. 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

  14. Nucleotide correlations and electronic transport of DNA sequences

    NASA Astrophysics Data System (ADS)

    Albuquerque, E. L.; Vasconcelos, M. S.; Lyra, M. L.; de Moura, F. A. B. F.

    2005-02-01

    We use a tight-binding formulation to investigate the transmissivity and wave-packet dynamics of sequences of single-strand DNA molecules made up from the nucleotides guanine G , adenine A , cytosine C , and thymine T . In order to reveal the relevance of the underlying correlations in the nucleotides distribution, we compare the results for the genomic DNA sequence with those of two artificial sequences: (i) the Rudin-Shapiro one, which has long-range correlations; (ii) a random sequence, which is a kind of prototype of a short-range correlated system, presented here with the same first-neighbor pair correlations of the human DNA sequence. We found that the long-range character of the correlations is important to the persistence of resonances of finite segments. On the other hand, the wave-packet dynamics seems to be mostly influenced by the short-range correlations.

  15. Plant Cyclic Nucleotide Signalling

    PubMed Central

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc

    2007-01-01

    The presence of the cyclic nucleotides 3′,5′-cyclic adenyl monophosphate (cAMP) and 3′,5′-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed. PMID:19704553

  16. The Slc35d3 gene, encoding an orphan nucleotide sugar transporter, regulates platelet-dense granules.

    PubMed

    Chintala, Sreenivasulu; Tan, Jian; Gautam, Rashi; Rusiniak, Michael E; Guo, Xiaoli; Li, Wei; Gahl, William A; Huizing, Marjan; Spritz, Richard A; Hutton, Saunie; Novak, Edward K; Swank, Richard T

    2007-02-15

    Platelet dense granules are lysosome-related organelles which contain high concentrations of several biologically important low-molecular-weight molecules. These include calcium, serotonin, adenine nucleotides, pyrophosphate, and polyphosphate, which are necessary for normal blood hemostasis. The synthesis of dense granules and other lysosome-related organelles is defective in inherited diseases such as Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS). HPS and CHS mutations in 8 human and at least 16 murine genes have been identified. Previous studies produced contradictory findings for the function of the murine ashen (Rab27a) gene in platelet-dense granules. We have used a positional cloning approach with one line of ashen mutants to establish that a new mutation in a second gene, Slc35d3, on mouse chromosome 10 is the basis of this discrepancy. The platelet-dense granule defect is rescued in BAC transgenic mice containing the normal Slc35d3 gene. Thus, Slc35d3, an orphan member of a nucleotide sugar transporter family, specifically regulates the contents of platelet-dense granules. Unlike HPS or CHS genes, it has no apparent effect on other lysosome-related organelles such as melanosomes or lysosomes. The ash-Roswell mouse mutant is an appropriate model for human congenital-isolated delta-storage pool deficiency.

  17. B3LYP, BLYP and PBE DFT band structures of the nucleotide base stacks

    NASA Astrophysics Data System (ADS)

    Szekeres, Zs; Bogár, F.; Ladik, J.

    DFT crystal orbital (band structure) calculations have been performed for the nucleotide base stacks of cytosine, thymine, adenine, and guanine arranged in DNA B geometry. The band structures obtained with PBE, BLYP, and B3LYP functionals are presented and compared to other related experimental and theoretical results. The influence of the quality of the basis set on the fundamental gap values was also investigated using Clementi's double ζ, 6-31G and 6-31G* basis sets.

  18. In vitro and cellular effects of 4-pyridone-3-carboxamide riboside on enzymes of nucleotide metabolism.

    PubMed

    Slominska, Ewa M; Borkowski, Tomasz; Rybakowska, Iwona; Abramowicz-Glinka, Magdalena; Orlewska, Czesława; Smolenski, Ryszard T

    2014-01-01

    4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that has recently been identified as a substrate for intracellular phosphorylation to form nucleotide derivatives. Low level of 4PYR is normally present in human plasma, but 4PYR massively accumulates in patients with renal failure. This study aimed to evaluate effects of 4PYR and its monophosphate derivative (4PYMP) on several enzymes of nucleotide metabolism in homogenates and intact cells. Activities of adenosine monophosphate deaminase (AMPD), adenosine deaminase, ecto-5'-nucleotidase (e5NT), adenine phosphoribosyltransferase (APRT), hypoxanthine/guanine phosphoribosyltransferase, purine nucleoside phosphorylase, and S-adenosylhomocysteine hydrolase (SAHH) were evaluated in erythrocyte lysates, rat heart homogenates, and in the intact rat cardiomyocytes by high performance liquid chromatography-based assays. 4PYMP caused significant inhibition of AMPD in both erythrocyte lysate and heart homogenate with 50% inhibitory concentration (IC50) of 74 and 55 μM, respectively. Inhibition of e5NT in heart homogenates was also noted with IC50 of 63 μM. 4PYMP slightly inhibited APRT and 4PYR caused moderate activation of SAHH. No effects on other enzymes studied were noted. Inhibition of AMPD by 4PYMP in homogenates was confirmed in the intact cell experiments with isolated cardiomyocytes that were allowed to accumulate 4PYMP by incubation with 4PYR. We conclude that among pathways studied, most important is the effect of 4PYMP on AMPD and that such effect could be one of the consequences of elevated plasma 4PYR concentration.

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

    PubMed

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

    2010-07-01

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

  20. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  1. Molecular and Subcellular-Scale Modeling of Nucleotide Diffusion in the Cardiac Myofilament Lattice

    PubMed Central

    Kekenes-Huskey, Peter M.; Liao, Tao; Gillette, Andrew K.; Hake, Johan E.; Zhang, Yongjie; Michailova, Anushka P.; McCulloch, Andrew D.; McCammon, J. Andrew

    2013-01-01

    Contractile function of cardiac cells is driven by the sliding displacement of myofilaments powered by the cycling myosin crossbridges. Critical to this process is the availability of ATP, which myosin hydrolyzes during the cross-bridge cycle. The diffusion of adenine nucleotides through the myofilament lattice has been shown to be anisotropic, with slower radial diffusion perpendicular to the filament axis relative to parallel, and is attributed to the periodic hexagonal arrangement of the thin (actin) and thick (myosin) filaments. We investigated whether atomistic-resolution details of myofilament proteins can refine coarse-grain estimates of diffusional anisotropy for adenine nucleotides in the cardiac myofibril, using homogenization theory and atomistic thin filament models from the Protein Data Bank. Our results demonstrate considerable anisotropy in ATP and ADP diffusion constants that is consistent with experimental measurements and dependent on lattice spacing and myofilament overlap. A reaction-diffusion model of the half-sarcomere further suggests that diffusional anisotropy may lead to modest adenine nucleotide gradients in the myoplasm under physiological conditions. PMID:24209858

  2. Nucleotide-protectable labeling of sulfhydryl groups in subunit I of the ATPase from Halobacterium saccharovorum

    NASA Technical Reports Server (NTRS)

    Sulzner, Michael; Stan-Lotter, Helga; Hochstein, Lawrence I.

    1992-01-01

    The membrane ATPase from Halobacterium saccharovorum was purified as described by Hochstein et al. (1987) and was incubated with C-14 labeled N-ethylmaleimide (NEM), with and without adenine nucleotides, to determine the effect of nucleotides on the enzyme labeling. It was found that NEM incorporates into the 87,000-Da subunit (subunit I) of the enzyme and that the conditions for enzyme modification are similar to those which result in the inhibition of the enzyme activity. The presence of ATP, ADP, and AMP was found to reduce both the inhibitor incorporation and enzyme inhibition. It was shown that the reaction involves a modification of thiol groups.

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

    PubMed

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

    2016-06-02

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

  4. 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.

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

    PubMed

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

    2015-05-07

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

  6. Advances in targeting cyclic nucleotide phosphodiesterases

    PubMed Central

    Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  7. Asymmetric Structure of the Yeast F[subscript 1] ATPase in the Absence of Bound Nucleotides

    SciTech Connect

    Kabaleeswaran, Venkataraman; Shen, Hong; Symersky, Jindrich; Walker, John E.; Leslie, Andrew G.W.; Mueller, David M.

    2009-05-11

    The crystal structure of nucleotide-free yeast F{sub 1} ATPase has been determined at a resolution of 3.6 {angstrom}. The overall structure is very similar to that of the ground state enzyme. In particular, the {beta}{sub DP} and {beta}{sub TP} subunits both adopt the closed conformation found in the ground state structure despite the absence of bound nucleotides. This implies that interactions between the {gamma} and {beta} subunits are as important as nucleotide occupancy in determining the conformational state of the {beta} subunits. Furthermore, this result suggests that for the mitochondrial enzyme, there is no state of nucleotide occupancy that would result in more than one of the {beta} subunits adopting the open conformation. The adenine-binding pocket of the {beta}{sub TP} subunit is disrupted in the apoenzyme, suggesting that the {beta}{sub DP} subunit is responsible for unisite catalytic activity.

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

    PubMed Central

    Roon, Robert J.; Even, Harvey L.

    1973-01-01

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

  9. Probing the ATP site of GRP78 with nucleotide triphosphate analogs

    DOE PAGES

    Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; ...

    2016-05-04

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATPmore » analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other nucleotides tested, even in the presence of Mg++. The 2'-deoxyATP structure showed the conformation of

  10. Characterization of Nucleotide Misincorporation Patterns in the Iceman's Mitochondrial DNA

    PubMed Central

    Olivieri, Cristina; Ermini, Luca; Rizzi, Ermanno; Corti, Giorgio; Bonnal, Raoul; Luciani, Stefania; Marota, Isolina; De Bellis, Gianluca; Rollo, Franco

    2010-01-01

    Background The degradation of DNA represents one of the main issues in the genetic analysis of archeological specimens. In the recent years, a particular kind of post-mortem DNA modification giving rise to nucleotide misincorporation (“miscoding lesions”) has been the object of extensive investigations. Methodology/Principal Findings To improve our knowledge regarding the nature and incidence of ancient DNA nucleotide misincorporations, we have utilized 6,859 (629,975 bp) mitochondrial (mt) DNA sequences obtained from the 5,350–5,100-years-old, freeze-desiccated human mummy popularly known as the Tyrolean Iceman or Ötzi. To generate the sequences, we have applied a mixed PCR/pyrosequencing procedure allowing one to obtain a particularly high sequence coverage. As a control, we have produced further 8,982 (805,155 bp) mtDNA sequences from a contemporary specimen using the same system and starting from the same template copy number of the ancient sample. From the analysis of the nucleotide misincorporation rate in ancient, modern, and putative contaminant sequences, we observed that the rate of misincorporation is significantly lower in modern and putative contaminant sequence datasets than in ancient sequences. In contrast, type 2 transitions represent the vast majority (85%) of the observed nucleotide misincorporations in ancient sequences. Conclusions/Significance This study provides a further contribution to the knowledge of nucleotide misincorporation patterns in DNA sequences obtained from freeze-preserved archeological specimens. In the Iceman system, ancient sequences can be clearly distinguished from contaminants on the basis of nucleotide misincorporation rates. This observation confirms a previous identification of the ancient mummy sequences made on a purely phylogenetical basis. The present investigation provides further indication that the majority of ancient DNA damage is reflected by type 2 (cytosine→thymine/guanine→adenine) transitions and

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

    PubMed

    Matthews, Elizabeth A; Dickenson, Anthony H

    2004-02-19

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

  12. 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.

  13. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model.

    PubMed

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

    2016-07-07

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

  14. Approach to the unfolding and folding dynamics of add A-riboswitch upon adenine dissociation using a coarse-grained elastic network model

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  15. Endogenously elevated bilirubin modulates kidney function and protects from circulating oxidative stress in a rat model of adenine-induced kidney failure.

    PubMed

    Boon, Ai-Ching; Lam, Alfred K; Gopalan, Vinod; Benzie, Iris F; Briskey, David; Coombes, Jeff S; Fassett, Robert G; Bulmer, Andrew C

    2015-10-26

    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.

  16. 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

  17. Fluorometric Determination of Adenosine Nucleotide Derivatives as Measures of the Microfouling, Detrital, and Sedimentary Microbial Biomass and Physiological Status

    PubMed Central

    Davis, William M.; White, David C.

    1980-01-01

    Adenosine, adenine, cyclic adenosine monophosphate (AMP), AMP, nicotinamide adenine dinucleotide, adenosine diphosphate, and adenosine triphosphate (ATP) were recovered quantitatively from aqueous portions of lipid extracts of microfouling, detrital, and sedimentary microbial communities. These could be detected quantitatively in the picomolar range by forming their 1-N6-etheno derivatives and analyzing by high-pressure liquid chromatography with fluorescence detection. Lipid extraction and subsequent analysis allowed the simultaneous measurement of the microbial community structure, total microbial biomass with the quantitative recovery of the adenine-containing cellular components, which were protected from enzymatic destruction. This extraction and fluorescent derivatization method showed equivalency with the luciferin-luciferase method for bacterial ATP measurements. Quick-freezing samples in the field with dry ice-acetone preserved the ATP and energy charge (a ratio of adenosine nucleotides) for analysis at remote laboratories. The metabolic lability of ATP in estuarine detrital and microfouling communities, as well as bacterial monocultures of constant biomass, showed ATP to be a precarious measure of biomass under some conditions. Combinations of adenosine and adenine nucleotides gave better correlations with microbial biomass measured as extractable lipid phosphate in the detrital and microfouling microbial communities than did ATP alone. Stresses such as anoxia or filtration are reflected in the rapid accumulation of intracellular adenosine and the excretion of adenosine and AMP into the surrounding milieu. Increases in AMP and adenosine may prove to be more sensitive indicators of metabolic status than the energy charge. PMID:16345633

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

    PubMed

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

    2013-08-01

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

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

    PubMed Central

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

    1990-01-01

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

  20. Simultaneous determination of adenine and guanine in ruminant bacterial pellets by ion-pair HPLC.

    PubMed

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

    2005-11-05

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

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

    PubMed

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

    2016-05-15

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

  2. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-11-01

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

  3. Nucleotide signalling during inflammation

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Eltzschig, Holger K.

    2014-01-01

    Inflammatory conditions are associated with the extracellular release of nucleotides, particularly ATP. In the extracellular compartment, ATP predominantly functions as a signalling molecule through the activation of purinergic P2 receptors. Metabotropic P2Y receptors are G-protein-coupled, whereas ionotropic P2X receptors are ATP-gated ion channels. Here we discuss how signalling events through P2 receptors alter the outcomes of inflammatory or infectious diseases. Recent studies implicate a role for P2X/P2Ysignalling in mounting appropriate inflammatory responses critical for host defence against invading pathogens or tumours. Conversely, P2X/P2Y signalling can promote chronic inflammation during ischaemia and reperfusion injury, inflammatory bowel disease or acute and chronic diseases of the lungs. Although nucleotide signalling has been used clinically in patients before, research indicates an expanding field of opportunities for specifically targeting individual P2 receptors for the treatment of inflammatory or infectious diseases. PMID:24828189

  4. PA0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine

    SciTech Connect

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

    2011-08-02

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

  5. Pa0148 from Pseudomonas aeruginosa Catalyzes the Deamination of Adenine

    SciTech Connect

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

    2011-12-31

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

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

    PubMed

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

    2012-04-05

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

  7. 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.

  8. Nucleotide cleaving agents and method

    DOEpatents

    Que, Jr., Lawrence; Hanson, Richard S.; Schnaith, Leah M. T.

    2000-01-01

    The present invention provides a unique series of nucleotide cleaving agents and a method for cleaving a nucleotide sequence, whether single-stranded or double-stranded DNA or RNA, using and a cationic metal complex having at least one polydentate ligand to cleave the nucleotide sequence phosphate backbone to yield a hydroxyl end and a phosphate end.

  9. KATP channels process nucleotide signals in muscle thermogenic response

    PubMed Central

    Reyes, Santiago; Park, Sungjo; Terzic, Andre; Alekseev, Alexey E.

    2014-01-01

    Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K+ (KATP) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, KATP channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how KATP channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the KATP channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with KATP channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal KATP channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of KATP channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of KATP channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders. PMID:20925594

  10. Different mechanisms of extracellular adenosine accumulation by reduction of the external Ca(2+) concentration and inhibition of adenosine metabolism in spinal astrocytes.

    PubMed

    Eguchi, Ryota; Akao, Sanae; Otsuguro, Ken-ichi; Yamaguchi, Soichiro; Ito, Shigeo

    2015-05-01

    Extracellular adenosine is a neuromodulator in the central nervous system. Astrocytes mainly participate in adenosine production, and extracellular adenosine accumulates under physiological and pathophysiological conditions. Inhibition of intracellular adenosine metabolism and reduction of the external Ca(2+) concentration ([Ca(2+)]e) participate in adenosine accumulation, but the precise mechanisms remain unclear. This study investigated the mechanisms underlying extracellular adenosine accumulation in cultured rat spinal astrocytes. The combination of adenosine kinase and deaminase (ADK/ADA) inhibition and a reduced [Ca(2+)]e increased the extracellular adenosine level. ADK/ADA inhibitors increased the level of extracellular adenosine but not of adenine nucleotides, which was suppressed by inhibition of equilibrative nucleoside transporter (ENT) 2. Unlike ADK/ADA inhibition, a reduced [Ca(2+)]e increased the extracellular level not only of adenosine but also of ATP. This adenosine increase was enhanced by ENT2 inhibition, and suppressed by sodium polyoxotungstate (ecto-nucleoside triphosphate diphosphohydrolase inhibitor). Gap junction inhibitors suppressed the increases in adenosine and adenine nucleotide levels by reduction of [Ca(2+)]e. These results indicate that extracellular adenosine accumulation by ADK/ADA inhibition is due to the adenosine release via ENT2, while that by reduction of [Ca(2+)]e is due to breakdown of ATP released via gap junction hemichannels, after which ENT2 incorporates adenosine into the cells.

  11. Regulation of innate immunity by extracellular nucleotides

    PubMed Central

    Gorini, Stefania; Gatta, Lucia; Pontecorvo, Laura; Vitiello, Laura; la Sala, Andrea

    2013-01-01

    Extracellular ATP (eATP) is the most abundant among extracellular nucleotides and is commonly considered as a classical danger signal, which stimulates immune responses in the presence of tissue injury. In fact, increased nucleotide concentration in the extracellular space is generally closely associated with tissue stress or damage. However non-lytic nucleotide release may also occur in many cell types under a variety of conditions. Extracellular nucleotides are sensed by a class of plasma membrane receptors called P2 purinergic receptors (P2Rs). P2 receptors are expressed by all immunological cells and their activation elicits different responses. Extracellular ATP can act as an initiator or terminator of immune responses being able to induce different effects on immune cells depending on the pattern of P2 receptors engaged, the duration of the stimulus and its concentration in the extracellular milieu. Millimolar (high) concentrations of extracellular ATP, induce predominantly proinflammatory effects, while micromolar (low) doses exert mainly tolerogenic/immunosuppressive action. Moreover small, but significant differences in the pattern of P2 receptor expression in mice and humans confer diverse capacities of ATP in regulating the immune response. PMID:23358447

  12. De novo synthesis of purine nucleotides in different fiber types of rat skeletal muscle

    SciTech Connect

    Tullson, P.C.; John-Alder, H.; Hood, D.A.; Terjung, R.L.

    1986-03-01

    The contribution of de novo purine nucleotide synthesis to nucleotide metabolism in skeletal muscles is not known. The authors have determined rates of de novo synthesis in soleus (slow-twitch red), red gastrocnemius (fast-twitch red), and white gastrocnemius (fast-twitch white) using the perfused rat hindquarter. /sup 14/C glycine incorporation into ATP was linear after 1 and 2 hours of perfusion with 0.2 mM added glycine. The intracellular (I) and extracellular (E) specific activity of /sup 14/C glycine was determined by HPLC of phenylisothiocyanate derivatives of neutralized PCA extracts. The rates of de novo synthesis when expressed relative to muscle ATP content show slow and fast-twitch red muscles to be similar and about twice as great as fast-twitch white muscles. This could represent a greater turnover of the adenine nucleotide pool in more oxidative red muscle types.

  13. The Nucleotide-Binding Sites of SUR1: A Mechanistic Model

    PubMed Central

    Vedovato, Natascia; Ashcroft, Frances M.; Puljung, Michael C.

    2015-01-01

    ATP-sensitive potassium (KATP) channels comprise four pore-forming Kir6.2 subunits and four modulatory sulfonylurea receptor (SUR) subunits. The latter belong to the ATP-binding cassette family of transporters. KATP channels are inhibited by ATP (or ADP) binding to Kir6.2 and activated by Mg-nucleotide interactions with SUR. This dual regulation enables the KATP channel to couple the metabolic state of a cell to its electrical excitability and is crucial for the KATP channel’s role in regulating insulin secretion, cardiac and neuronal excitability, and vascular tone. Here, we review the regulation of the KATP channel by adenine nucleotides and present an equilibrium allosteric model for nucleotide activation and inhibition. The model can account for many experimental observations in the literature and provides testable predictions for future experiments. PMID:26682803

  14. IRE1α nucleotide sequence cleavage specificity in the unfolded protein response.

    PubMed

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J; Tirasophon, Witoon

    2017-01-01

    Inositol-requiring enzyme 1 (IRE1) is a conserved sensor of the unfolded protein response that has protein kinase and endoribonuclease (RNase) enzymatic activities and thereby initiates HAC1/XBP1 splicing. Previous studies demonstrated that human IRE1α (hIRE1α) does not cleave Saccharomyces cerevisiae HAC1 mRNA. Using an in vitro cleavage assay, we show that adenine to cytosine nucleotide substitution at the +1 position in the 3' splice site of HAC1 RNA is required for specific cleavage by hIRE1α. A similar restricted nucleotide specificity in the RNA substrate was observed for XBP1 splicing in vivo. Together these findings underscore the essential role of cytosine nucleotide at +1 in the 3' splice site for determining cleavage specificity of hIRE1α.

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    1977-01-01

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

  17. 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.

  18. Ultraviolet absorption and luminescence of matrix-isolated adenine

    SciTech Connect

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

    2011-10-01

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

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

    PubMed Central

    2015-01-01

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

  20. Vibrational signatures of Watson-Crick base pairing in adenine-thymine mimics.

    PubMed

    Nosenko, Yevgeniy; Kunitski, Maksim; Stark, Tina; Göbel, Michael; Tarakeshwar, Pilarisetty; Brutschy, Bernhard

    2013-07-21

    The vibrational fingerprints of hydrogen-bonding associated with the adenine-thymine (A-T) Watson-Crick (WC) base pair have been identified in an infrared study of the A-T mimics 4-aminopyrimidine-1-methylthymine (4APM-1MT) and 4-aminopyrimidine-6-methyl-4-pyrimidinone (4APM-M4PMN) in the gas-phase. The IR vibrational spectra were measured via a double resonance scheme utilizing femtosecond multiphoton ionization. The changes in the molecular structure, anharmonic vibrational parameters, and the assignment of the observed vibrational spectra in the NH/CH stretch region were investigated by carrying out high-level theoretical calculations of the anharmonic spectra. The experimental observations and theoretical calculations indicate that the hydrogen bonds associated with WC base-pairing are relatively stronger than those associated with reverse WC (rWC) base pairing. This is manifested in a more pronounced red-shift of the H-bonded vibrational modes associated with the WC as compared with the rWC base-pairing. An analysis of the factors contributing to the anharmonicity of the vibrational modes associated with H-bonding reveals that the magnitude of the off-diagonal anharmonic coupling of the H-bonded -NH2 stretch and the -NH2 bend is much smaller in WC base-pairing than in the corresponding rWC base-pairing. The chemical and biological implications of these results, especially in the context of using vibrational spectroscopy as a tool for identifying the signatures of nucleotide base vibrations is addressed.

  1. Nucleotide Selectivity of Antibiotic Kinases▿

    PubMed Central

    Shakya, Tushar; Wright, Gerard D.

    2010-01-01

    Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3′)-IIIa, APH(2″)-Ib, and MPH(2′)-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3′)-IIIa exclusively uses ATP, MPH(2′)-I exclusively uses GTP, and APH(2″)-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance. PMID:20231391

  2. Nucleotide selectivity of antibiotic kinases.

    PubMed

    Shakya, Tushar; Wright, Gerard D

    2010-05-01

    Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3')-IIIa, APH(2'')-Ib, and MPH(2')-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3')-IIIa exclusively uses ATP, MPH(2')-I exclusively uses GTP, and APH(2'')-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.

  3. Template polymerization of nucleotide analogues

    NASA Technical Reports Server (NTRS)

    Orgel, L. E.

    1991-01-01

    Recent work on the template-directed reactions of the natural D-nucleotides has made it clear that l-nucleotides and nucleotide-like derivatives of other sugars would strongly inhibit the formation of long oligonucleotides. Consequently, attention is focusing on molecules simpler than nucleotides that might have acted as monomers of an information transfer system. We have begun a general exploration of the template directed reactions of diverse peptide analogues. I will present work by Dr. Taifeng Wu on oxidative oligomerization of phosphorothioates and of Dr. Mary Tohidi on the cyclic polymerization of nucleoside and related cyclic pyrophosphates.

  4. High-spin ferric ions in Saccharomyces cerevisiae vacuoles are reduced to the ferrous state during adenine-precursor detoxification.

    PubMed

    Park, Jinkyu; McCormick, Sean P; Cockrell, Allison L; Chakrabarti, Mrinmoy; Lindahl, Paul A

    2014-06-24

    The majority of Fe in Fe-replete yeast cells is located in vacuoles. These acidic organelles store Fe for use under Fe-deficient conditions and they sequester it from other parts of the cell to avoid Fe-associated toxicity. Vacuolar Fe is predominantly in the form of one or more magnetically isolated nonheme high-spin (NHHS) Fe(III) complexes with polyphosphate-related ligands. Some Fe(III) oxyhydroxide nanoparticles may also be present in these organelles, perhaps in equilibrium with the NHHS Fe(III). Little is known regarding the chemical properties of vacuolar Fe. When grown on adenine-deficient medium (A↓), ADE2Δ strains of yeast such as W303 produce a toxic intermediate in the adenine biosynthetic pathway. This intermediate is conjugated with glutathione and shuttled into the vacuole for detoxification. The iron content of A↓ W303 cells was determined by Mössbauer and EPR spectroscopies. As they transitioned from exponential growth to stationary state, A↓ cells (supplemented with 40 μM Fe(III) citrate) accumulated two major NHHS Fe(II) species as the vacuolar NHHS Fe(III) species declined. This is evidence that vacuoles in A↓ cells are more reducing than those in adenine-sufficient cells. A↓ cells suffered less oxidative stress despite the abundance of NHHS Fe(II) complexes; such species typically promote Fenton chemistry. Most Fe in cells grown for 5 days with extra yeast-nitrogen-base, amino acids and bases in minimal medium was HS Fe(III) with insignificant amounts of nanoparticles. The vacuoles of these cells might be more acidic than normal and can accommodate high concentrations of HS Fe(III) species. Glucose levels and rapamycin (affecting the TOR system) affected cellular Fe content. This study illustrates the sensitivity of cellular Fe to changes in metabolism, redox state and pH. Such effects broaden our understanding of how Fe and overall cellular metabolism are integrated.

  5. Zwitterionic character of nucleotides: possible significance in the evolution of nucleic acids.

    PubMed Central

    Sundaralingam, M; Prusiner, P

    1978-01-01

    X-ray crystallography has shown that the free acids of adenosine 5'- and 3'-monophosphates and of cytidine 5'- and 3'-monophosphates exist as zwiterions in the solid state with protonation of the adenine base at the N(1) site and of the cytosine base at the corresponding site N(3) and the phosphate group negatively charged. In this paper, evidence is presented for the zwitterionic character of the free acids of the monomeric nucleotides guanosine 5'-monophosphate and inosine 5'-monophosphate with protonation of the base at the N(7) site of the imidazole moiety. PMID:724518

  6. Slow deactivation channels in UV-photoexcited adenine DNA.

    PubMed

    Chen, Xuebo; Fang, Weihai; Wang, Haobin

    2014-03-07

    The molecular mechanism for removing the excess energy in DNA bases is responsible for the high photostability of DNA and is thus the subject of intense theoretical/computational investigation. To understand why the excited state decay of the stacked bases is significantly longer than that of the monomers, we carried out electronic structure calculations on an adenine monomer and an aqueous (dA)5 oligonucleotide employing the CASPT2//CASSCF and CASPT2//CASSCF/AMBER levels of theory. The newly-found bright excited state pair Sstack1((1)ππ*) and Sstack2((1)ππ*) of d(A)5, originated from base stacking, is of intra-base charge transfer nature and occurs in different stacked bases with charge transfer along opposite directions. Two slow deactivation channels of d(A)5 were proposed as a result of the sizable barriers along the relaxation paths starting from the FC point of the Sstack1((1)ππ*) state. The SN1P((1)nπ*) state of d(A)5 serves as an intermediate state in one relaxation channel, to which a nonadiabatic decay from the Sstack1((1)ππ*) state occurs in an energy degeneracy region. A relatively high barrier in this state is found and attributed to the steric hindrance of the DNA environment due to the large NH2 group twisting, which gives a weak and red-shifted fluorescence. Another direct relaxation channel, induced by the C2-H2 bond twisting motion, is found to go through a conical intersection between the Sstack1((1)ππ*) and the ground state. The barrier found here enables fluorescence from the Sstack1((1)ππ*) state and may explain the bright state emission observed in the fluorescence upconversion measurements. The inter-molecular SCT((1)ππ*) state may be involved in the slow relaxation process of the photoexcited adenine oligomers through efficient internal conversion to the intra-base Sstack1((1)ππ*) state.

  7. 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.

  8. Design of laser pulses for selective vibrational excitation of the N6-H bond of adenine and adenine-thymine base pair using optimal control theory.

    PubMed

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

    2009-06-01

    Time dependent quantum dynamics and optimal control theory are used for selective vibrational excitation of the N6-H (amino N-H) bond in free adenine and in the adenine-thymine (A-T) base pair. For the N6-H bond in free adenine we have used a one dimensional model while for the hydrogen bond, N6-H(A)...O4(T), present in the A-T base pair, a two mathematical dimensional model is employed. The conjugate gradient method is used for the optimization of the field dependent cost functional. Optimal laser fields are obtained for selective population transfer in both the model systems, which give virtually 100% excitation probability to preselected vibrational levels. The effect of the optimized laser field on the other hydrogen bond, N1(A)...H-N3(T), present in A-T base pair is also investigated.

  9. 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

  10. 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.

  11. Phosphorylation and nucleotide-dependent dephosphorylation of hepatic polypeptides related to the plasma cell differentiation antigen PC-1.

    PubMed Central

    Uriarte, M; Stalmans, W; Hickman, S; Bollen, M

    1993-01-01

    A glycoprotein fraction was isolated from rat liver membranes by affinity chromatography on immobilized wheat-germ lectin. Incubation of this fraction with MgATP or MgGTP resulted in a sequential phosphorylation and dephosphorylation of a complex of three polypeptides (118, 128 and 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each polypeptide was recognized by polyclonal antibodies against recombinant plasma cell differentiation antigen PC-1. The relationship of the 118 kDa and 128 kDa polypeptides with PC-1 was confirmed by observations that they are linked by disulphide bonds into a larger protein, and that they are exclusively phosphorylated on Thr residues. Phosphorylation of p118, p128 and p197 only occurred after a lag period (up to 90 min at 30 degrees C), which lasted until most of the ATP had been converted to adenosine and Pi, with ADP and AMP as intermediate products. The length of the latency period increased with the concentration of initially added ATP (5-1000 microM) and could be prolonged by a second addition of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alpha beta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, emerge as the direct determinants of the latency. After further purification of the lectin-purified membrane fraction on anion-exchange and molecular-sieve columns, the complex of p118, p128 and p197 was still capable of autophosphorylation and dephosphorylation. The dephosphorylation was not affected by classical inhibitors (NaF, okadaic acid, EDTA, EGTA, phenylalanine). It was stimulated about 20-fold by various adenine nucleotides and analogues, with the same order of efficiency as noted for the induction of the latency. A similar stimulation of dephosphorylation was caused by 0.5 mM Na3VO4, which also prevented the phosphorylation of the three polypeptides. The likely explanation for the latency that precedes the

  12. Synthesis and Characterization of Oligodeoxyribonucleotides Modified with 2′-Amino-α-L-LNA Adenine Monomers: High-affinity Targeting of Single-Stranded DNA

    PubMed Central

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

    2014-01-01

    Development of conformationally restricted nucleotide building blocks continues to attract considerable interest due to their successful use within antisense, antigene and other gene-targeting strategies. Locked nucleic acid (LNA) and its diastereomer α-L-LNA are two interesting examples hereof. Oligonucleotides modified with these units display greatly increased affinity toward nucleic acid targets, improved binding specificity and enhanced enzymatic stability relative to unmodified strands. Here, we present the synthesis and biophysical characterization of oligodeoxyribonucleotides (ONs) modified with 2′-amino-α-L-LNA adenine monomers W–Z. The synthesis of target phosphoramidites 1–4 initiates from pentafuranose 5, which upon Vorbrüggen glycosylation, O2′-deacylation, O2′-activation and C2′-azide introduction yields nucleoside 8. A one-pot tandem Staudinger/intramolecular nucleophilic substitution converts 8 into 2′-amino-α-L-LNA adenine intermediate 9, which after a series of non-trivial protecting group manipulations affords key intermediate 15. Subsequent chemoselective N2′-functionalization and O3′-phosphitylation gives targets 1–4 in ~1–3% overall yield over eleven steps from 5. ONs modified with pyrene-functionalized 2′-amino-α-L-LNA adenine monomers X-Z display greatly increased affinity toward DNA targets (ΔTm/modification up to +14 °C). Results from absorption and fluorescence spectroscopy suggest that the duplex stabilization is a result of pyrene intercalation. These characteristics render N2′-pyrene-functionalized 2′-amino-α-L-LNA of considerable interest for DNA-targeting applications. PMID:24304240

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

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

    1986-01-14

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

  15. Neutrophil gelatinase-associated lipocalin in a triphasic rat model of adenine-induced kidney injury.

    PubMed

    Gil, Amnon; Brod, Vera; Awad, Hoda; Heyman, Samuel N; Abassi, Zaid; Frajewicki, Victor

    2016-10-01

    The aim of this study is to investigate whether NGAL, given its advantages over traditional biomarkers, can be used to describe the dynamic characteristics of the renal tubulointerstitial insult caused by adenine. Subsequently, it will be possible to assess NGAL as a biomarker of any acute kidney injury, on top of chronic interstitial disease, if NGAL levels are stable through the chronic phase of our adenine model. Study group rats were fed an adenine diet, and control group rats were fed a regular diet only. Blood and urine samples for urea, creatinine and NGAL were drawn from each rat at the beginning of the study and after 1, 3, 4, 5, 6, 7 and 8 weeks. Kidney slices from these rats were stained with Hematoxylin-eosin (HE) and β-actin stainings. Serum urea, creatinine and NGAL levels and urinary NGAL/creatinine ratio in the study group were higher than baseline and than in the control group; these differences were statistically significant in some of the intervals. Tubulointerstitial changes and adenine crystals were evident in the study group rats. In the rats fed adenine, serum urea, creatinine and NGAL levels and urinary NGAL/creatinine ratio followed a triphasic pattern of kidney injury: an acute phase while on the adenine diet, a partial recovery phase after switching to the regular diet and a chronic kidney disease phase after stabilization of renal function. NGAL can serve a biomarker for acute kidney injury and possibly for chronic kidney disease in the tubulointerstitial rat model.

  16. Improved growth and stress tolerance in the Arabidopsis oxt1 mutant triggered by altered adenine metabolism.

    PubMed

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

    2012-11-01

    Plants perceive and respond to environmental stresses with complex mechanisms that are often associated with the activation of antioxidant defenses. A genetic screen aimed at isolating oxidative stress-tolerant lines of Arabidopsis thaliana has identified oxt1, a line that exhibits improved tolerance to oxidative stress and elevated temperature but displays no apparent deleterious growth effects under non-stress conditions. Oxt1 harbors a mutation that arises from the altered expression of a gene encoding adenine phosphoribosyltransferase (APT1), an enzyme that converts adenine to adenosine monophosphate (AMP), indicating a link between purine metabolism, whole-plant growth responses, and stress acclimation. The oxt1 mutation results in decreased APT1 expression that leads to reduced enzymatic activity. Correspondingly, oxt1 plants possess elevated levels of adenine. Decreased APT enzyme activity directly correlates with stress resistance in transgenic lines that ectopically express APT1. The metabolic alteration in oxt1 plants also alters the expression of several antioxidant defense genes and the response of these genes to oxidative challenge. Finally, it is shown that manipulation of adenine levels can induce stress tolerance to wild-type plants. Collectively, these results show that alterations in cellular adenine levels can trigger stress tolerance and improve growth, leading to increases in plant biomass. The results also suggest that adenine might play a part in the signals that modulate responses to abiotic stress and plant growth.

  17. 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.

  18. Labeled nucleotide phosphate (NP) probes

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2009-02-03

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  19. Nucleotide Accumulation Induced in Staphylococcus aureus by Glycine

    PubMed Central

    Strominger, Jack L.; Birge, Claire H.

    1965-01-01

    Strominger, Jack L. (Washington University School of Medicine, St. Louis, Mo.), and Claire H. Birge. Nucleotide accumulation induced in Staphylococcus aureus by glycine. J. Bacteriol. 89:1124–1127. 1965.—High concentrations of glycine induce accumulation of four uridine nucleotides in Staphylococcus aureus. Investigations of their structure suggest that these compounds are uridine diphosphate (UDP)-acetylmuramic acid, UDP-acetylmuramyl-gly-d-glu-l-lys, UDP-acetylmuramyl-l-ala-d-glu-l-lys and UDP-acetylmuramyl-gly-d-glu-l-lys-d-ala-d-ala. The mechanism by which glycine may induce uridine nucleotide accumulation and protoplast formation is discussed. Images PMID:14276106

  20. Dependence of the E. coli promoter strength and physical parameters upon the nucleotide sequence

    PubMed Central

    Berezhnoy, Andrey Y.; Shckorbatov, Yuriy G.

    2005-01-01

    The energy of interaction between complementary nucleotides in promoter sequences of E. coli was calculated and visualized. The graphic method for presentation of energy properties of promoter sequences was elaborated on. Data obtained indicated that energy distribution through the length of promoter sequence results in picture with minima at −35, −8 and +7 regions corresponding to areas with elevated AT (adenine-thymine) content. The most important difference from the random sequences area is related to −8. Four promoter groups and their energy properties were revealed. The promoters with minimal and maximal energy of interaction between complementary nucleotides have low strengths, the strongest promoters correspond to promoter clusters characterized by intermediate energy values. PMID:16252339

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

    PubMed

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

    2004-11-19

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

  2. 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

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

    PubMed Central

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

    2002-01-01

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

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

    PubMed

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

    2015-03-01

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

  5. Probing the ATP site of GRP78 with nucleotide triphosphate analogs

    SciTech Connect

    Hughes, Scott J.; Antoshchenko, Tetyana; Chen, Yun; Lu, Hua; Pizarro, Juan C.; Park, Hee -Won

    2016-05-04

    GRP78, a member of the ER stress protein family, can relocate to the surface of cancer cells, playing key roles in promoting cell proliferation and metastasis. GRP78 consists of two major functional domains: the ATPase and protein/peptide-binding domains. The protein/peptide-binding domain of cell-surface GRP78 has served as a novel functional receptor for delivering cytotoxic agents (e.g., a apoptosis-inducing peptide or taxol) across the cell membrane. Here, we report our study on the ATPase domain of GRP78 (GRP78ATPase), whose potential as a transmembrane delivery system of cytotoxic agents (e.g., ATP-based nucleotide triphosphate analogs) remains unexploited. As the binding of ligands (ATP analogs) to a receptor (GRP78ATPase) is a pre-requisite for internalization, we determined the binding affinities and modes of GRP78ATPase for ADP, ATP and several ATP analogs using surface plasmon resonance and x-ray crystallography. The tested ATP analogs contain one of the following modifications: the nitrogen at the adenine ring 7-position to a carbon atom (7-deazaATP), the oxygen at the beta-gamma bridge position to a carbon atom (AMPPCP), or the removal of the 2'-OH group (2'-deoxyATP). We found that 7-deazaATP displays an affinity and a binding mode that resemble those of ATP regardless of magnesium ion (Mg++) concentration, suggesting that GRP78 is tolerant to modifications at the 7-position. By comparison, AMPPCP's binding affinity was lower than ATP and Mg++-dependent, as the removal of Mg++ nearly abolished binding to GRP78ATPase. The AMPPCP-Mg++ structure showed evidence for the critical role of Mg++ in AMPPCP binding affinity, suggesting that while GRP78 is sensitive to modifications at the β-γ bridge position, these can be tolerated in the presence of Mg++. Furthermore, 2'-deoxyATP's binding affinity was significantly lower than those for all other

  6. [Nucleotide receptors in learning and neuronal plasticity].

    PubMed

    Czajkowski, Rafał

    2014-01-01

    Nucleotide signalling plays an important role in neuronal plasticity and learning. Nucleotides are released at the synaptic terminals and may act pre- and postsynaptically by activating Pland P2 receptors. The A1 receptor, activated tonically by resting concentration of adenosine regulates basal neurotransmission. The A2A receptor is activated by increased adenosine levels and participates in plastic changes. ATP may act as an independent neurotransmitter on the P2X1 receptor, or via P2X3 subtype as a neuromodulator that affects NMDA receptor signalling. The G protein coupled P2Y receptors also evoke neuromodulatory effect on the neuronal plasticity, inhibiting LTD in prefrontal cortex. P2X7 receptor is responsible for communication between astrocytes and for synchronizing their activity. ATP and adenosine released by astrocytes act as neuromodulators both at the release site and heterosynaptically. Taken together, these multiple actions of nucleotides constitute a mechanism regulating homeostatic processes that are necessary for proper brain functioning: synaptic scaling and metaplasticity.

  7. The biased nucleotide composition of the HIV genome: a constant factor in a highly variable virus.

    PubMed

    van der Kuyl, Antoinette C; Berkhout, Ben

    2012-11-06

    Viruses often deviate from their hosts in the nucleotide composition of their genomes. The RNA genome of the lentivirus family of retroviruses, including human immunodeficiency virus (HIV), contains e.g. an above average percentage of adenine (A) nucleotides, while being extremely poor in cytosine (C). Such a deviant base composition has implications for the amino acids that are encoded by the open reading frames (ORFs), both in the requirement of specific tRNA species and in the preference for amino acids encoded by e.g. A-rich codons. Nucleotide composition does obviously affect the secondary and tertiary structure of the RNA genome and its biological functions, but it does also influence phylogenetic analysis of viral genome sequences, and possibly the activity of the integrated DNA provirus. Over time, the nucleotide composition of the HIV-1 genome is exceptionally conserved, varying by less than 1% per base position per isolate within either group M, N, or O during 1983-2009. This extreme stability of the nucleotide composition may possibly be achieved by negative selection, perhaps conserving semi-stable RNA secondary structure as reverse transcription would be significantly affected for a less A-rich genome where secondary structures are expected to be more stable and thus more difficult to unfold.This review will discuss all aspects of the lentiviral genome composition, both of the RNA and of its derived double-stranded DNA genome, with a focus on HIV-1, the nucleotide composition over time, the effects of artificially humanized codons as well as contributions of immune system pressure on HIV nucleotide bias.

  8. DNA Adenine Methyltransferase Influences the Virulence of Aeromonas hydrophila

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2015-06-18

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

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

    PubMed

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

    2016-01-15

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

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

    SciTech Connect

    Fraser, Marie E.; Cherney, Maia M.; Marcato, Paola; Mulvey, George L.; Armstrong, Glen D.; James, Michael N. G.

    2006-07-01

    Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active as an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

  13. Alkylation by propylene oxide of deoxyribonucleic acid, adenine, guanosine and deoxyguanylic acid

    PubMed Central

    Lawley, P. D.; Jarman, M.

    1972-01-01

    1. Propylene oxide reacts with DNA in aqueous buffer solution at about neutral pH to yield two principal products, identified as 7-(2-hydroxypropyl)guanine and 3-(2-hydroxypropyl)adenine, which hydrolyse out of the alkylated DNA at neutral pH values at 37°C. 2. These products were obtained in quantity by reactions between propylene oxide and guanosine or adenine respectively. 3. The reactions between propylene oxide and adenine in acetic acid were parallel to those between dimethyl sulphate and adenine in neutral aqueous solution; the alkylated positions in adenine in order of decreasing reactivity were N-3, N-1 and N-9. A method for separating these alkyladenines is described. 4. Deoxyguanylic acid sodium salt was alkylated at N-7 by propylene oxide in neutral aqueous solution. 5. The nature of the side chain in the principal alkylation products was established by mass spectrometry, and the nature of the products is consistent with their formation by the bimolecular reaction mechanism. PMID:5073240

  14. Metalated nucleotide chemisorption on hydroxyapatite.

    PubMed

    Benedetti, Michele; Antonucci, Daniela; De Castro, Federica; Girelli, Chiara R; Lelli, Marco; Roveri, Norberto; Fanizzi, Francesco P

    2015-12-01

    The experiments here reported evidence on the importance of the residual charge of a nucleotide derivative, for the adsorption on nHAP (hydroxyapatite nanocrystals), in water solution. We found that the simple presence of phosphates on the nucleotide derivative does not guarantee adsorption on nHAP. On the other hand, we demonstrated that a cationic or neutral charge on a nucleotide derivative produces a strongly reduced chemical adsorption (chemisorption) whereas, in the presence of a net negative charge, relevant adsorption on nHAP is observed. The number of phosphates can only modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. Previous considerations are important to model the binding ability of phosphate bearing nucleotide derivatives or molecules on hydroxyapatite. The findings reported in the present paper could be relevant in bone tissue targeting or nHAP mediated drug delivery.

  15. Bioenergetics and gene silencing approaches for unraveling nucleotide recognition by the human EIF2C2/Ago2 PAZ domain.

    PubMed

    Kandeel, Mahmoud; Al-Taher, Abdullah; Nakashima, Remi; Sakaguchi, Tomoya; Kandeel, Ali; Nagaya, Yuki; Kitamura, Yoshiaki; Kitade, Yukio

    2014-01-01

    Gene silencing and RNA interference are major cellular processes that control gene expression via the cleavage of target mRNA. Eukaryotic translation initiation factor 2C2 (EIF2C2, Argonaute protein 2, Ago2) is considered to be the major player of RNAi as it is the core component of RISC complexes. While a considerable amount of research has focused on RNA interference and its associated mechanisms, the nature and mechanisms of nucleotide recognition by the PAZ domain of EIF2C2/Ago2 have not yet been characterized. Here, we demonstrate that the EIF2C2/Ago2 PAZ domain has an inherent lack of binding to adenine nucleotides, a feature that highlights the poor binding of 3'-adenylated RNAs with the PAZ domain as well as the selective high trimming of the 3'-ends of miRNA containing adenine nucleotides. We further show that the PAZ domain selectively binds all ribonucleotides (except adenosine), whereas it poorly recognizes deoxyribonucleotides. In this context, the modification of dTMP to its ribonucleotide analogue gave a drastic improvement of binding enthalpy and, hence, binding affinity. Additionally, higher in vivo gene silencing efficacy was correlated with the stronger PAZ domain binders. These findings provide new insights into the nature of the interactions of the EIF2C2/Ago2 PAZ domain.

  16. Dietary nucleotides prevent decrease in cellular immunity in ground-based microgravity analog

    NASA Technical Reports Server (NTRS)

    Yamauchi, Keiko; Hales, Nathan W.; Robinson, Sandra M.; Niehoff, Michael L.; Ramesh, Vani; Pellis, Neal R.; Kulkarni, Anil D.

    2002-01-01

    Microgravity and stress of spaceflights result in immune dysfunction. The role of nutrition, especially nucleotide supplementation, has become an area of intensive research and significant interest in immunomodulation for maintenance of cellular immune responses. The studies presented here evaluate the plausibility of administering nucleotides to obviate immune dysfunction in an Earth-based in vivo analog of microgravity as studied in anti-orthostatic tail suspension (AOS) of mice. Mice were divided into three housing groups: group, isolation, and AOS. Mice were fed either control chow diet (CD), or RNA-, adenine-, or uracil-supplemented CD for the 1-wk duration of the experiments. In AOS mice, supplemental nucleotides significantly increased in vivo lymph node proliferation and ex vivo lymphoproliferation response to alloantigen and mitogens, respectively, and interleukin-2 and interferon-gamma production. A lower corticosterone level was observed in uracil-supplemented CD compared with CD. These results suggest that exogenous nucleotide supplementation, especially uracil, of normal diet is beneficial in the maintenance and restoration of the immune response during the microgravity analog conditions.

  17. Excited-state lifetime of adenine near the first electronic band origin

    NASA Astrophysics Data System (ADS)

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

    2010-10-01

    The excited-state lifetime of supersonically cooled adenine was measured in the gas phase by femtosecond pump-probe transient ionization as a function of excitation energy between 36 100 and 37 500 cm-1. The excited-state lifetime of adenine is ˜2 ps around the 0-0 band of the L1b ππ ∗ state (36 105 cm-1). The lifetime drops to ˜1 ps when adenine is excited to the L1a ππ ∗ state with the pump energy at 36 800 cm-1 and above. The excited-state lifetimes of L1a and L1b ππ∗ states are differentiated in accordance with previous frequency-resolved and computational studies.

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

    PubMed

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

    2014-04-01

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

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

    PubMed Central

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

    1991-01-01

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

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

    PubMed

    Hartmann, K; Kuffer, M; Balzarini, J; Naesens, L; Goldberg, M; Erfle, V; Goebel, F D; De Clercq, E; Jindrich, J; Holy, A; Bischofberger, N; Kraft, W

    1998-02-01

    The acyclic nucleoside phosphonates (S)-9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA) were evaluated for their efficacy and side effects in a double-blind placebo-controlled trial using naturally occurring feline immunodeficiency virus (FIV)-infected cats. This natural retrovirus animal model is considered highly relevant for the pathogenesis and chemotherapy of HIV in humans. Both PMEA and FPMPA proved effective in ameliorating the clinical symptoms of FIV-infected cats, as measured by several clinical parameters including the incidence and severity of stomatitis, Karnofsky's score, immunologic parameters such as relative and absolute CD4+ lymphocyte counts, and virologic parameters including proviral DNA levels in peripheral blood mononuclear cells (PBMC) of drug-treated animals. In contrast with PMEA, FPMPA showed no hematologic side effects at a dose that was 2.5-fold higher than PMEA.

  1. Ricin Activity Assay by Direct Analysis in Real Time Mass Spectrometry Detection of Adenine Release

    DTIC Science & Technology

    2010-02-01

    direct analysis in real time mass spectrometry. The release of adenine from the inhomo- geneous substrate herring sperm DNA by ricin was determined to...chain catalyzes cleavage at adenosine 4324 (in rat RNA) of 28S rRNA to release adenine.10 This action inhibits protein synthesis, leading to cell...death. In addition to RNA, herring sperm DNA (hsDNA) is a substrate for ricin.11 We chose to employ hsDNA for this assay because it is relatively stable

  2. The two-photon excitation cross section of 6MAP, a fluorescent adenine analogue.

    PubMed

    Stanley, Robert J; Hou, Zhanjia; Yang, Aiping; Hawkins, Mary E

    2005-03-03

    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.

  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.

  4. Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

    PubMed

    Shetty, Kartika N; Latha, Vakada Lavanya; Rao, Rameshwaram Nagender; Nadimpalli, Siva Kumar; Suguna, Kaza

    2013-07-01

    Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

  5. Synthesis, cyclopolymerization and cyclo-copolymerization of 9-(2-diallylaminoethyl)adenine and its hydrochloride salt.

    PubMed

    Bouhadir, Kamal H; Abramian, Lara; Ezzeddine, Alaa; Usher, Karyn; Vladimirov, Nikolay

    2012-11-08

    We report herein the synthesis and characterization of 9-(2-diallylaminoethyl) adenine. We evaluated two different synthetic routes starting with adenine where the optimal route was achieved through coupling of 9-(2-chloroethyl)adenine with diallylamine. The cyclopolymerization and cyclo-copolymerization of 9-(2-diallylaminoethyl)adenine hydrochloride salt resulted in low molecular weight oligomers in low yields. In contrast, 9-(2-diallylaminoethyl)adenine failed to cyclopolymerize, however, it formed a copolymer with SO₂ in relatively good yields. The molecular weights of the cyclopolymers were around 1,700-6,000 g/mol, as estimated by SEC. The cyclo-copolymer was stable up to 226 °C. To the best of our knowledge, this is the first example of a free-radical cyclo-copolymerization of a neutral alkyldiallylamine derivative with SO₂. These polymers represent a novel class of carbocyclic polynucleotides.

  6. The Effect of Adenine Repeats on G-quadruplex/hemin Peroxidase Mimicking DNAzyme Activity.

    PubMed

    Chen, Jielin; Guo, Yuehua; Zhou, Jun; Ju, Huangxian

    2017-03-23

    The catalytic activity of G-quadruplex/hemin is much lower than that of proteinous enzymes, so it is very important to increase its activity. Very recently, flanking sequences, which can be regarded as an external part of G-quadruplexes, were found to enhance the activity of G-quadruplex/hemin DNAzyme. However, little is known about the effect of internal parts, such as loop sequences and linkers, on the activity. In the present study, adenine repeats were incorporated into several designed G-quadruplex structures either in the loops, bulges, or linkers, and the constructed G-quadruplex/hemin DNAzyme exhibit about fivefold improvement in peroxidase-mimicking activity in some cases. The enhancement effect may result from the formation of compound I, protoporphyrin⋅Fe(IV) =O(.+) , accelerated by dA repeats, which was demonstrated by H2 O2 decay kinetics and pH dependency analysis. The novel enhancement methods described here may help in the development of high-activity DNAzymes, illustrated by a dimer G-quadruplex with flanking adenine at one end, a relatively long adenine run in one loop, and another adenine run in the linker.

  7. 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

  8. Effects of genetic deletion of soluble 5'-nucleotidases NT5C1A and NT5C2 on AMPK activation and nucleotide levels in contracting mouse skeletal muscles.

    PubMed

    Kviklyte, Samanta; Vertommen, Didier; Yerna, Xavier; Andersén, Harriet; Xu, Xiufeng; Gailly, Philippe; Bohlooly-Y, Mohammad; Oscarsson, Jan; Rider, Mark H

    2017-03-21

    AMP-activated protein kinase (AMPK) plays a key role in energy homeostasis and is activated in response to contraction-induced ATP depletion in skeletal muscle via a rise in intracellular AMP/ADP concentrations. AMP can be deaminated by AMP-deaminase to IMP, which is hydrolysed to inosine by cytosolic 5'-nucleotidase-II (NT5C2). AMP can also be hydrolysed to adenosine by cytosolic 5'-nucleotidase-IA (NT5C1A). Previous gene silencing and overexpression studies indicated control of AMPK activation by NT5C enzymes. In the present study using gene knockout mouse models, we investigated effects of NT5C1A and NT5C2 deletion on intracellular adenine nucleotide levels and AMPK activation in electrically stimulated skeletal muscles. Surprisingly, NT5C enzyme knockout did not lead to enhanced AMP or ADP concentrations in response to contraction, with no potentiation of increases in AMPK activity in extensor digitorum longus (EDL) and soleus mouse muscles. Moreover, dual blockade of AMP metabolism in EDL using an AMPD inhibitor combined with NT5C1A deletion did not enhance rises in AMP and ADP or increased AMPK activation by electrical stimulation. The results on muscles from the NT5C knockout mice contradict previous findings where AMP levels and AMPK activity were shown to be modulated by NT5C enzymes.

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

    PubMed

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

    2015-06-18

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

  10. 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).

  11. 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

  12. The Single Nucleotide Polymorphism Consortium

    NASA Technical Reports Server (NTRS)

    Morgan, Michael

    2003-01-01

    I want to discuss both the Single Nucleotide Polymorphism (SNP) Consortium and the Human Genome Project. I am afraid most of my presentation will be thin on law and possibly too high on rhetoric. Having been engaged in a personal and direct way with these issues as a trained scientist, I find it quite difficult to be always as objective as I ought to be.

  13. Guanine nucleotide regulation of receptor binding of thyrotropin-releasing hormone (TRH) in rat brain regions, retina and pituitary.

    PubMed

    Sharif, N A; Burt, D R

    1987-10-29

    Guanine nucleotides inhibited the specific binding of [3H](3-Me-His2)thyrotropin-releasing hormone ([3H]MeTRH) to receptors for TRH in washed homogenates of rat anterior pituitary gland in a dose-related manner. The order of potency (at 100 and 500 microM final) was Gpp(NH)p (a stable analog of GTP) greater than GTP much greater than GDP much greater than cGMP (with the adenine nucleotides being inactive) in the pituitary and various brain regions. Gpp(NH)p at 1 mM caused 17-35% inhibition of [3H]MeTRH binding to different tissues including the pituitary, hypothalamus, retina and nucleus accumbens. A statistically significant nucleotide effect was not observed, however, in the olfactory bulb and medulla/pons membranes. Gpp(NH)p (1 mM) increased the dissociation constants for [3H]MeTRH binding by 1.9- to 2.4-fold in the pituitary, n. accumbens and retinal preparations without altering the apparent binding capacity. These data suggest that TRH receptor binding can be allosterically regulated by guanine nucleotides and provide further evidence for the existence of guanine nucleotide binding protein(s) coupled to the TRH receptor.

  14. Cloning and characterization of two tandemly arranged DNA methyltransferase genes of Neisseria lactamica: an adenine-specific M.NlaIII and a cytosine-type methylase.

    PubMed

    Labbé, D; Höltke, H J; Lau, P C

    1990-10-01

    The gene encoding the Neisseria lactamica III DNA methyltransferase (M.NlaIII) which recognizes the sequence CATG has been cloned and expressed in Escherichia coli. DNA sequencing of a 3.125 kb EcoRI-PstI fragment localizes the M. NlaIII gene to a 334 codon open reading frame (ORF) and identifies, 468 bp downstream, a second ORF of 313 amino acids, which is referred to as M.NlaX. Both proteins are detectable in the E. coli coupled in vitro transcription-translation system; they are apparently expressed from separate N. lactamica promoters. The N-terminal half of the previously characterized M.FokI, which methylates adenine in one of the DNA strands with its asymmetric recognition sequence (GGATG), is found to have 41% sequence identity and a further 11.7% sequence similarity with M.NlaIII. Among the conserved amino acids is the wellknown DPPY sequence motif. With one exception, analysis of the nucleotides coding for the DP dipeptide in all known DPPY sequences shows the presence of an inherent DNA adenine methylation (dam) recognition site of GATC. A low level of expression of M.NlaX in E. coli prevents the elucidation of its sequence recognition specificity. Sequence analysis of M.NlaX shows that it is closely related to the group of monospecific 5-methylcytosine DNA methyltransferases (M.EcoRII, Dcm, M.HpaII and M.HhaI) which all have a modified cytosine at the second position of the recognition sequences. Both M.EcoRII and Dcm amino acid sequences are about 50% identical with M.NlaX; a considerable degree of sequence identity is found in the so-called variable region which is believed to be responsible for sequence recognition specificity. M.NlaX is probably the counterpart to the E. coli Dcm in N. lactamica.

  15. Supramolecular polymeric chemosensor for biomedical applications: design and synthesis of a luminescent zinc metallopolymer as a chemosensor for adenine detection.

    PubMed

    Chow, Cheuk-Fai

    2012-11-01

    Adenine is an important bio-molecule that plays many crucial roles in food safety and biomedical diagnostics. Differentiating adenine from a mixture of adenosine and other nucleic bases (guanine, thymine, cytosine, and uracil) is particularly important for both biological and clinical applications. A neutral Zn(II) metallosupramolecular polymer based on acyl hydrazone derived coordination centres (P1) were generated through self-assembly polymerization. It is a linear coordination polymer that behaves like self-standing film. The synthesis, (1)H-NMR characterization, and spectroscopic properties of this supramolecular material are reported. P1 was found to be a chemosensor specific to adenine, with a luminescent enhancement. The binding properties of P1 with common nucleic bases and nucleosides reveal that this supramolecular polymer is very selective to adenine molecules (~20 to 420 times more selectivity than other nucleic bases). The formation constant (K) of P1 to adenine was found to be log K = 4.10 ± 0.02. This polymeric chemosensor produces a specific response to adenine down to 90 ppb. Spectrofluorimetric and (1)H-NMR titration studies showed that the P1 polymer allows each Zn(II) coordination centre to bind to two adenine molecules through hydrogen bonding with their imine and hydrazone protons.

  16. Expression of microRNAs in Horse Plasma and Their Characteristic Nucleotide Composition

    PubMed Central

    Lee, Seungwoo; Hwang, Seungwoo; Yu, Hee Jeong; Oh, Dayoung; Choi, Yu Jung; Kim, Myung-Chul; Kim, Yongbaek; Ryu, Doug-Young

    2016-01-01

    MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition. PMID:26731407

  17. 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.

  18. Electrical detection and quantification of single and mixed DNA nucleotides in suspension

    NASA Astrophysics Data System (ADS)

    Ahmad, Mahmoud Al; Panicker, Neena G.; Rizvi, Tahir A.; Mustafa, Farah

    2016-09-01

    High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution.

  19. Development of a nucleotide sugar purification method using a mixed mode column & mass spectrometry detection.

    PubMed

    Eastwood, Heather; Xia, Fang; Lo, Mei-Chu; Zhou, Jing; Jordan, John B; McCarter, John; Barnhart, Wesley W; Gahm, Kyung-Hyun

    2015-11-10

    Analysis of nucleotide sugars, nucleoside di- and triphosphates and sugar-phosphates is an essential step in the process of understanding enzymatic pathways. A facile and rapid separation method was developed to analyze these compounds present in an enzymatic reaction mixture utilized to produce nucleotide sugars. The Primesep SB column explored in this study utilizes hydrophobic interactions as well as electrostatic interactions with the phosphoric portion of the nucleotide sugars. Ammonium formate buffer was selected due to its compatibility with mass spectrometry. Negative ion mode mass spectrometry was adopted for detection of the sugar phosphate (fucose-1-phophate), as the compound is not amenable to UV detection. Various mobile phase conditions such as pH, buffer concentration and organic modifier were explored. The semi-preparative separation method was developed to prepare 30mg of the nucleotide sugar. (19)F NMR was utilized to determine purity of the purified fluorinated nucleotide sugar. The collected nucleotide sugar was found to be 99% pure.

  20. Electrical detection and quantification of single and mixed DNA nucleotides in suspension

    PubMed Central

    Ahmad, Mahmoud Al; Panicker, Neena G.; Rizvi, Tahir A.; Mustafa, Farah

    2016-01-01

    High speed sequential identification of the building blocks of DNA, (deoxyribonucleotides or nucleotides for short) without labeling or processing in long reads of DNA is the need of the hour. This can be accomplished through exploiting their unique electrical properties. In this study, the four different types of nucleotides that constitute a DNA molecule were suspended in a buffer followed by performing several types of electrical measurements. These electrical parameters were then used to quantify the suspended DNA nucleotides. Thus, we present a purely electrical counting scheme based on the semiconductor theory that allows one to determine the number of nucleotides in a solution by measuring their capacitance-voltage dependency. The nucleotide count was observed to be similar to the multiplication of the corresponding dopant concentration and debye volume after de-embedding the buffer contribution. The presented approach allows for a fast and label-free quantification of single and mixed nucleotides in a solution. PMID:27677329

  1. European Nucleotide Archive in 2016

    PubMed Central

    Toribio, Ana Luisa; Alako, Blaise; Amid, Clara; Cerdeño-Tarrága, Ana; Clarke, Laura; Cleland, Iain; Fairley, Susan; Gibson, Richard; Goodgame, Neil; ten Hoopen, Petra; Jayathilaka, Suran; Kay, Simon; Leinonen, Rasko; Liu, Xin; Martínez-Villacorta, Josué; Pakseresht, Nima; Rajan, Jeena; Reddy, Kethi; Rosello, Marc; Silvester, Nicole; Smirnov, Dmitriy; Vaughan, Daniel; Zalunin, Vadim; Cochrane, Guy

    2017-01-01

    The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena) offers a rich platform for data sharing, publishing and archiving and a globally comprehensive data set for onward use by the scientific community. With a broad scope spanning raw sequencing reads, genome assemblies and functional annotation, the resource provides extensive data submission, search and download facilities across web and programmatic interfaces. Here, we outline ENA content and major access modalities, highlight major developments in 2016 and outline a number of examples of data reuse from ENA. PMID:27899630

  2. Antiherpesvirus Activity in Human Sera and Urines after Administration of Adenine Arabinoside

    PubMed Central

    Champney, Keith J.; Lauter, Carl B.; Bailey, Elizabeth J.; Lerner, A. Martin

    1978-01-01

    The minimum inhibitory concentration (MIC) of adenine arabinoside (ara-A) in rabbit kidney microtiter tissue cultures (RK-13) to a prototype strain of herpes simplex virus, type 1 (E115) based upon inhibition of cytopathic effects is 1.5 μg/ml. In this system, the MIC of arabinosylhypoxanthine (ara-Hx), the major in vivo metabolic derivative of ara-A, is 75 μg/ml. Inhibition of cytopathic effects of herpes simplex virus, type 1 (HSV-1) in microtiter wells of RK-13 cells varies directly with the concentrations of ara-A or ara-Hx, and inversely with residual HSV-1. The MIC of ara-A for HSV-1 in RK-13 cells is 5-20 times lower than similar measures with vero renal, mouse embryo, or human foreskin cultures. With RK-13 tissue cultures in microtiter plates, an assay for “ara-A equivalents” in human body fluids was developed which compares in sensitivity with high pressure liquid chromatography and has the advantage of simultaneously measuring combined antiherpesvirus effects of ara-A and its major metabolic derivative, ara-Hx. In vitro checkerboard studies in RK-13 cells confirmed that ara-A and ara-Hx in combination had antiviral effects which are synergistic. The total of the fractional MIC of ara-A plus ara-Hx in combination also varies inversely with residual HSV-1 in microtiter wells. Because virus adsorption is complete at 2 h before specimens to be tested are added in this assay, and because human interferon is not measured in rabbit cells, the antiviral assay is not affected by the presence of type-specific antiherpesvirus antibody or human interferon. Antiviral activity (AVA) was assayed as ara-A equivalents in sera and urines from 10 patients with serious herpesvirus infections who received 2.5-20 mg/kg daily of ara-A by intramuscular or intravenous routes. When a dosage schedule of 10 mg/kg per day or more was used, sustained concentrations of AVA that ranged from 0.8 to 14.4 μg/ml were found. When an inhibitor of adenosine deaminase (covidarabine) was

  3. New nucleotide analogues with enhanced signal properties.

    PubMed

    Cherkasov, Dmitry; Biet, Thorsten; Bäuml, Englbert; Traut, Walther; Lohoff, Michael

    2010-01-01

    We describe synthesis and testing of a novel type of dye-modified nucleotides which we call macromolecular nucleotides (m-Nucs). Macromolecular nucleotides comprise a nucleotide moiety, a macromolecular linear linker, and a large macromolecular ligand carrying multiple fluorescent dyes. With incorporation of the nucleotide moiety into the growing nucleic acid strand during enzymatic synthesis, the macromolecular ligand together with the coupled dyes is bound to the nucleic acid. By the use of this new class of modified nucleotides, signals from multiple dye molecules can be obtained after a single enzymatic incorporation event. The modified nucleotides are considered especially useful in the fields of nanobiotechnology, where signal stability and intensity is a limiting factor.

  4. Particular behavior of the adenine and guanine ring-breathing modes upon the DNA conformational transitions.

    PubMed

    Ghomi, M; Letellier, R; Taillandier, E

    1988-06-01

    Harmonic dynamics calculations performed on the deoxyguanosine (dG) and deoxyadenosine (dA) residues, based on a reliable force field, show that the breathing motions of both guanine and adenine residues are involved in two different vibration modes (750-500 cm-1 spectral region). The calculated results reveal a strong coupling of these modes with the sugar pucker motions. This effect has been verified for the dG residue by the Raman spectra of polyd(G-C). As far as the dA residue is concerned, the particular behavior of the adenine residue breathing mode predicted by these calculations, has been confirmed by Raman spectra of polyd(A-T) undergoing a B----Z conformational transition.

  5. 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.

  6. From formamide to adenine: a self-catalytic mechanism for an abiotic approach.

    PubMed

    Wang, Jing; Gu, Jiande; Nguyen, Minh Tho; Springsteen, Greg; Leszczynski, Jerzy

    2013-11-14

    Mechanisms for abiotic reaction pathways from formamide (H2NCHO) to adenine are presented herein. Formamide is a simple C1 building block hypothesized to be a precursor to many protometabolic compounds. On the basis of a step-by-step mechanism of the reaction pathways, formamide is suggested to be more reactive in addition reactions than HCN. In addition to its simplicity, the formamide self-catalyzed mechanism is energetically (kinetically) more viable than either a water-catalyzed mechanism or noncatalyzed processes. Moreover, this self-catalyzed mechanism accounts for the yields of purine and adenine previously observed in experiments. This mechanism may elucidate processes that were vital for the emergence of life on the early earth.

  7. Critical appraisal of excited state nonadiabatic dynamics simulations of 9H-adenine.

    PubMed

    Barbatti, Mario; Lan, Zhenggang; Crespo-Otero, Rachel; Szymczak, Jaroslaw J; Lischka, Hans; Thiel, Walter

    2012-12-14

    In spite of the importance of nonadiabatic dynamics simulations for the understanding of ultrafast photo-induced phenomena, simulations based on different methodologies have often led to contradictory results. In this work, we proceed through a comprehensive investigation of on-the-fly surface-hopping simulations of 9H-adenine in the gas phase using different electronic structure theories (ab initio, semi-empirical, and density functional methods). Simulations that employ ab initio and semi-empirical multireference configuration interaction methods predict the experimentally observed ultrafast deactivation of 9H-adenine with similar time scales, however, through different internal conversion channels. Simulations based on time-dependent density functional theory with six different hybrid and range-corrected functionals fail to predict the ultrafast deactivation. The origin of these differences is analyzed by systematic calculations of the relevant reaction pathways, which show that these discrepancies can always be traced back to topographical features of the underlying potential energy surfaces.

  8. 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.

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

    PubMed

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

    2008-07-01

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

  10. Nucleotide Metabolism and DNA Replication.

    PubMed

    Warner, Digby F; Evans, Joanna C; Mizrahi, Valerie

    2014-10-01

    The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.

  11. Geometric consequences of electron delocalization for adenine tautomers in aqueous solution.

    PubMed

    Raczyńska, Ewa D; Makowski, Mariusz

    2014-06-01

    Geometric consequences of electron delocalization were studied for all possible adenine tautomers in aqueous solution by means of ab initio methods {PCM(water)//DFT(B3LYP)/6-311+G(d,p)} and compared to those in the gas phase {DFT(B3LYP)/6-311+G(d,p)}. To measure the consequences of any type of resonance conjugation (π-π, n-π, and σ-π), the geometry-based harmonic oscillator model of electron delocalization (HOMED) index, recently extended to the isolated (DFT) and hydrated (PCM//DFT) molecules, was applied to the molecular fragments (imidazole, pyrimidine, 4-aminopyrimidine, and purine) and also to the whole tautomeric system. For individual tautomers, the resonance conjugations and consequently the bond lengths strongly depend on the position of the labile protons. The HOMED indices are larger for tautomers (or their fragments) possessing the labile proton(s) at the N rather than C atom. Solvent interactions with adenine tautomers slightly increase the resonance conjugations. Consequently, they slightly shorten the single bonds and lengthen the double bonds. When going from the gas phase to water solution, the HOMED indices increase (by less than 0.15 units). There is a good relation between the HOMED indices estimated in water solution and those in the gas phase for the neutral and ionized forms of adenine. Subtle effects, being a consequence of intramolecular interactions between the neighboring groups, are so strongly reduced by solvent that the relation between the HOMED indices and the relative energies for the neutral adenine tautomers seems to be better in water solution than in the gas phase.

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

    NASA Astrophysics Data System (ADS)

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

    2016-06-01

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

  13. DNA Bases Thymine and Adenine in Bio-Organic Light Emitting Diodes

    DTIC Science & Technology

    2014-11-24

    DNA Bases Thymine and Adenine in Bio-Organic Light Emitting Diodes Eliot F. Gomez1, Vishak Venkatraman1, James G. Grote2 & Andrew J. Steckl1...45433-7707 USA. We report on the use of nucleic acid bases (NBs) in organic light emitting diodes (OLEDs). NBs are small molecules that are the basic...polymer has been a frequent natural material integrated in electronic devices. DNA has been used in organic light - emitting diodes (OLEDs)4,5,7–14

  14. Dietary L-lysine prevents arterial calcification in adenine-induced uremic rats.

    PubMed

    Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Isaka, Yoshitaka; Rakugi, Hiromi

    2014-09-01

    Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC.

  15. Synthesis of rigid homo- and heteroditopic nucleobase-terminated molecules incorporating adenine and/or thymine.

    PubMed

    Jacobsen, Mikkel F; Andersen, Casper S; Knudsen, Martin M; Gothelf, Kurt V

    2007-07-19

    A series of homo- and heteroditopic thymine- and/or adenine-terminated molecules incorporating rigid aryl or oligo(phenylene ethynylene) linkers has been efficiently synthesized. The key steps involved in the synthesis are the construction of the N-arylated nucleobases using the Chan-Lam-Evans-modified Ullman coupling and their further elaboration using the Sonogashira coupling. Furthermore, the synthesis of a rigid tripodal thymine derivative is reported.

  16. Dietary l-Lysine Prevents Arterial Calcification in Adenine-Induced Uremic Rats

    PubMed Central

    Shimomura, Akihiro; Matsui, Isao; Hamano, Takayuki; Ishimoto, Takuya; Katou, Yumiko; Takehana, Kenji; Inoue, Kazunori; Kusunoki, Yasuo; Mori, Daisuke; Nakano, Chikako; Obi, Yoshitsugu; Fujii, Naohiko; Takabatake, Yoshitsugu; Nakano, Takayoshi; Tsubakihara, Yoshiharu; Rakugi, Hiromi

    2014-01-01

    Vascular calcification (VC) is a life-threatening complication of CKD. Severe protein restriction causes a shortage of essential amino acids, and exacerbates VC in rats. Therefore, we investigated the effects of dietary l-lysine, the first-limiting amino acid of cereal grains, on VC. Male Sprague-Dawley rats at age 13 weeks were divided randomly into four groups: low-protein (LP) diet (group LP), LP diet+adenine (group Ade), LP diet+adenine+glycine (group Gly) as a control amino acid group, and LP diet+adenine+l-lysine·HCl (group Lys). At age 18 weeks, group LP had no VC, whereas groups Ade and Gly had comparable levels of severe VC. l-Lysine supplementation almost completely ameliorated VC. Physical parameters and serum creatinine, urea nitrogen, and phosphate did not differ among groups Ade, Gly, and Lys. Notably, serum calcium in group Lys was slightly but significantly higher than in groups Ade and Gly. Dietary l-lysine strongly suppressed plasma intact parathyroid hormone in adenine rats and supported a proper bone-vascular axis. The conserved orientation of the femoral apatite in group Lys also evidenced the bone-protective effects of l-lysine. Dietary l-lysine elevated plasma alanine, proline, arginine, and homoarginine but not lysine. Analyses in vitro demonstrated that alanine and proline inhibit apoptosis of cultured vascular smooth muscle cells, and that arginine and homoarginine attenuate mineral precipitations in a supersaturated calcium/phosphate solution. In conclusion, dietary supplementation of l-lysine ameliorated VC by modifying key pathways that exacerbate VC. PMID:24652795

  17. 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.

  18. Mechanism of charge separation in DNA by hole transfer through consecutive adenines.

    PubMed

    Kawai, Kiyohiko; Osakada, Yasuko; Fujitsuka, Mamoru; Majima, Tetsuro

    2008-01-01

    To investigate the mechanism of charge separation in DNA with consecutive adenines adjacent to a photosensitizer (Sens), a series of naphthalimide (NI) and 5-bromouracil ((br)U)-modified DNAs were prepared, and the quantum yields of formation of the charge-separated states (Phi) upon photo-excitation of the Sens NI in DNA were measured. The Phi was modulated by the incorporation site of (br)U, which changes the oxidation potential of its complementary A through hydrogen bonding and the hole-transfer rates between adenines. The results were interpreted as charge separation by means of the initial charge transfer between NI in the singlet excited state and the second- and third-nearest adenine to the NI. In addition, the oxidation of the A nearest to NI leads to the rapid charge recombination within a contact ion pair. This suggests that the charge-separation process can be refined to maximize the Phi by putting a redox-inactive spacer base pair between a photosensitizer and an A-T stretch.

  19. Identification of a Campylobacter coli methyltransferase targeting adenines at GATC sites.

    PubMed

    Dutta, Vikrant; Altermann, Eric; Crespo, Maria D; Olson, Jonathan W; Siletzky, Robin M; Kathariou, Sophia

    2016-12-02

    Campylobacter coli can infect humans and colonize multiple other animals but its host-associated genes or adaptations are poorly understood. Adenine methylation at GATC sites, resulting in MboI resistance of genomic DNA, was earlier frequently detected among C. coli from swine but not among turkey-derived isolates. The underlying genetic basis has remained unknown. Comparative genome sequence analyses of C. coli 6461, a swine-derived strain with MboI-resistant DNA, revealed two chromosomal ORFs, 0059 and 0060, encoding a putative DNA methyltransferase and a conserved hypothetical protein, respectively, which were lacking from the genome of the turkey-derived C. coli strain 11601, which had MboI-susceptible DNA. To determine whether the ORF0059 mediated MboI resistance and hence encoded a putative N6-adenine DNA methyltransferase, the gene was cloned immediately upstream of a chloramphenicol resistance cassette (cat) and a PCR fragment harboring ORF0059-cat was transformed into C. coli 11601. The transformants had MboI-resistant DNA, suggesting a direct role of this gene in methylation of adenines at GATC sites. In-silico analyses suggested that the ORF0059-ORF0060 cassette was more frequent among C. coli from swine than certain other sources (e.g. cattle, humans). Potential impacts of ORF0059-mediated methylation on C. coli host preference and other adaptations remain to be elucidated.

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

    NASA Astrophysics Data System (ADS)

    Nielsen, Lisbeth Munksgaard; Pedersen, Sara Øvad; Kirketerp, Maj-Britt Suhr; Nielsen, Steen Brøndsted

    2012-02-01

    The degree of electronic coupling between DNA bases is a topic being up for much debate. Here we report on the intrinsic electronic properties of isolated DNA strands in vacuo free of solvent, which is a good starting point for high-level excited states calculations. Action spectra of DNA single strands of adenine reveal sign of exciton coupling between stacked bases from blueshifted absorption bands (˜3 nm) relative to that of the dAMP mononucleotide (one adenine base). The bands are blueshifted by about 10 nm compared to those of solvated strands, which is a shift similar to that for the adenine molecule and the dAMP mononucleotide. Desolvation has little effect on the bandwidth, which implies that inhomogenous broadening of the absorption bands in aqueous solution is of minor importance compared to, e.g., conformational disorder. Finally, at high photon energies, internal conversion competes with electron detachment since dissociation of the bare photoexcited ions on the microsecond time scale is measured.

  1. 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.

  2. Geometrical Characterization of Adenine And Guanine on Cu(110) By NEXAFS, XPS, And DFT Calculation

    SciTech Connect

    Furukawa, M.; Yamada, T.; Katano, S.; Kawai, M.; Ogasawara, H.; Nilsson, A.; /SLAC, SSRL /Stockholm U.

    2009-04-30

    Adsorption of purine DNA bases (guanine and adenine) on Cu(1 1 0) was studied by X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine-structure spectroscopy (NEXAFS), and density-functional theory (DFT) calculation. At coverages near 0.2 monolayers, Angular-resolved NEXAFS analysis revealed that adenine adsorbates lie almost flat and that guanine adsorbates are tilted up on the surface with the purine ring parallel to the atom rows of Cu(1 1 0). Referring to the previous studies on pyrimidine DNA bases [M. Furukawa, H. Fujisawa, S. Katano, H. Ogasawara, Y. Kim, T. Komeda, A. Nilsson, M. Kawai, Surf. Sci. 532-535 (2003) 261], the isomerization of DNA bases on Cu(1 1 0) was found to play an important role in the adsorption geometry. Guanine, thymine and cytosine adsorption have an amine-type nitrogen next to a carbonyl group, which is dehydrogenated into imine nitrogen on Cu(1 1 0). These bases are bonded by the inherent portion of - NH-CO - altered by conversion into enolic form and dehydrogenation. Adenine contains no CO group and is bonded to Cu(1 1 0) by participation of the inherent amine parts, resulting in nearly flatly-lying position.

  3. Heptacopper(II) and dicopper(II)-adenine complexes: synthesis, structural characterization, and magnetic properties

    DOE PAGES

    Leite Ferreira, B. J. M.; Brandão, Paula; Dos Santos, A. M.; ...

    2015-07-13

    The syntheses, crystal structures, and magnetic properties of two new copper(II) complexes with molecular formulas [Cu7(μ2-OH2)6(μ3-O)6(adenine)6(NO3)26H2O (1) and [Cu2(μ2-H2O)2(adenine)2(H2O)4](NO3)42H2O (2) are reported. We composed the heptanuclear compound of a central octahedral CuO6 core sharing edges with six adjacent copper octahedra. In 2, the copper octahedra shares one equatorial edge. In both compounds, these basic copper cluster units are further linked by water bridges and bridging adenine ligands through N3 and N9 donors. All copper(II) centers exhibit Jahn-Teller distorted octahedral coordination characteristic of a d9 center. Our study of the magnetic properties of the heptacopper complex revealed a dominant ferromagnetic intra-clustermore » interaction, while the dicopper complex exhibits antiferromagnetic intra-dimer interactions with weakly ferromagnetic inter-dimer interaction.« less

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

    PubMed

    Anumukonda, Lakshmi N; Young, Avery; Lynn, David G; Buckley, Ragan; Warrayat, Amena; Graves, Christina L; Bean, Heather D; Hud, Nicholas V

    2011-12-01

    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.

  5. 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

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

    PubMed Central

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

    1999-01-01

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

  7. Heptacopper(II) and dicopper(II)-adenine complexes: synthesis, structural characterization, and magnetic properties

    SciTech Connect

    Leite Ferreira, B. J. M.; Brandão, Paula; Dos Santos, A. M.; Gai, Z.; Cruz, C.; Reis, M. S.; Santos, T. M.; Félix, V.

    2015-07-13

    The syntheses, crystal structures, and magnetic properties of two new copper(II) complexes with molecular formulas [Cu72-OH2)63-O)6(adenine)6(NO3)26H2O (1) and [Cu22-H2O)2(adenine)2(H2O)4](NO3)42H2O (2) are reported. We composed the heptanuclear compound of a central octahedral CuO6 core sharing edges with six adjacent copper octahedra. In 2, the copper octahedra shares one equatorial edge. In both compounds, these basic copper cluster units are further linked by water bridges and bridging adenine ligands through N3 and N9 donors. All copper(II) centers exhibit Jahn-Teller distorted octahedral coordination characteristic of a d9 center. Our study of the magnetic properties of the heptacopper complex revealed a dominant ferromagnetic intra-cluster interaction, while the dicopper complex exhibits antiferromagnetic intra-dimer interactions with weakly ferromagnetic inter-dimer interaction.

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

    PubMed Central

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

    1966-01-01

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

  9. Photoinduced formation of hydrogen peroxide in aqueous solutions of adenine derivatives at 77 K

    NASA Astrophysics Data System (ADS)

    Lozinova, T. A.; Lobanov, A. V.; Lander, A. V.

    2016-11-01

    The amount of hydrogen peroxide in aqueous solutions of adenine (A), adenosine (Ado), cytidine (Cyt), and thymine (T) containing 0.1 M NaCl and irradiated with near-UV light at 77 K is determined. It is established by comparing the results to data obtained earlier that the amount of H2O2 detected in the defrosted samples following identical irradiation falls in the order Ado > adenosine-5'-diphosphate (ADP) > A >> Cyt. The formation of H2O2 was not detected for T. The formation of H2O2 in solutions of adenine derivatives was observed when the samples were irradiated with light having wavelengths in the ranges λ = 240-400 nm and 290-450 nm. The latter covers only the long wave absorption range of these compounds. It is shown that the change in the intensity of irradiation that strongly affected the intensity of EPR signals of irradiated samples prior to defrosting affected the amount of detected H2O2 only slightly, and the effect was not unidirectional. The results from determining H2O2 in the samples of adenine derivatives are compared to estimates of the content of free peroxyl radicals, obtained by analyzing EPR spectra. Plausible mechanisms of the processes are discussed.

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

    PubMed

    Rose, Nicholas D; Regan, John M

    2015-12-01

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

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

    PubMed

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

    2014-05-21

    Nucleic acid bases play important roles in living beings. Thus, their interaction with salts the prebiotic Earth could be an important issue for the understanding of origin of life. In this study, the effect of pH and artificial seawaters on the structure of adenine and thymine was studied via parallel determinations using FT-IR, Raman spectroscopy and theoretical calculations. Thymine and adenine lyophilized in solutions at basic and acidic conditions showed characteristic bands of the enol-imino tautomer due to the deprotonation and the hydrochloride form due to protonation, respectively. The interaction of thymine and adenine with different seawaters representative of different geological periods on Earth was also studied. In the case of thymine a strong interaction with Sr(2+) promoted changes in the Raman and infrared spectra. For adenine changes in infrared and Raman spectra were observed in the presence of salts from all seawaters tested. The experimental results were compared to theoretical calculations, which showed structural changes due to the presence of ions Na(+), Mg(2+), Ca(2+) and Sr(2+) of artificial seawaters. For thymine the bands arising from C4=C5 and C6=O stretching were shifted to lower values, and for adenine, a new band at 1310cm(-1) was observed. The reactivity of adenine and thymine was studied by comparing changes in nucleophilicity and energy of the HOMO orbital.

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

    NASA Astrophysics Data System (ADS)

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

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

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

    PubMed

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

    2016-06-01

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

  14. Mosaic organization of DNA nucleotides

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Havlin, S.; Simons, M.; Stanley, H. E.; Goldberger, A. L.

    1994-01-01

    Long-range power-law correlations have been reported recently for DNA sequences containing noncoding regions. We address the question of whether such correlations may be a trivial consequence of the known mosaic structure ("patchiness") of DNA. We analyze two classes of controls consisting of patchy nucleotide sequences generated by different algorithms--one without and one with long-range power-law correlations. Although both types of sequences are highly heterogenous, they are quantitatively distinguishable by an alternative fluctuation analysis method that differentiates local patchiness from long-range correlations. Application of this analysis to selected DNA sequences demonstrates that patchiness is not sufficient to account for long-range correlation properties.

  15. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process.

  16. Nucleotide excision repair in humans

    PubMed Central

    Spivak, Graciela

    2015-01-01

    The demonstration of DNA damage excision and repair replication by Setlow, Howard-Flanders, Hanawalt and their colleagues in the early 1960s, constituted the discovery of the ubiquitous pathway of nucleotide excision repair (NER). The serial steps in NER are similar in organisms from unicellular bacteria to complex mammals and plants, and involve recognition of lesions, adducts or structures that disrupt the DNA double helix, removal of a short oligonucleotide containing the offending lesion, synthesis of a repair patch copying the opposite undamaged strand, and ligation, to restore the DNA to its original form. The transcription-coupled repair (TCR) subpathway of NER, discovered nearly two decades later, is dedicated to the removal of lesions from the template DNA strands of actively transcribed genes. In this review I will outline the essential factors and complexes involved in NER in humans, and will comment on additional factors and metabolic processes that affect the efficiency of this important process. PMID:26388429

  17. Running out of time: the decline of channel activity and nucleotide activation in adenosine triphosphate-sensitive K-channels

    PubMed Central

    Proks, Peter; Puljung, Michael C.; Vedovato, Natascia; Sachse, Gregor; Mulvaney, Rachel; Ashcroft, Frances M.

    2016-01-01

    KATP channels act as key regulators of electrical excitability by coupling metabolic cues—mainly intracellular adenine nucleotide concentrations—to cellular potassium ion efflux. However, their study has been hindered by their rapid loss of activity in excised membrane patches (rundown), and by a second phenomenon, the decline of activation by Mg-nucleotides (DAMN). Degradation of PI(4,5)P2 and other phosphoinositides is the strongest candidate for the molecular cause of rundown. Broad evidence indicates that most other determinants of rundown (e.g. phosphorylation, intracellular calcium, channel mutations that affect rundown) also act by influencing KATP channel regulation by phosphoinositides. Unfortunately, experimental conditions that reproducibly prevent rundown have remained elusive, necessitating post hoc data compensation. Rundown is clearly distinct from DAMN. While the former is associated with pore-forming Kir6.2 subunits, DAMN is generally a slower process involving the regulatory sulfonylurea receptor (SUR) subunits. We speculate that it arises when SUR subunits enter non-physiological conformational states associated with the loss of SUR nucleotide-binding domain dimerization following prolonged exposure to nucleotide-free conditions. This review presents new information on both rundown and DAMN, summarizes our current understanding of these processes and considers their physiological roles. This article is part of the themed issue ‘Evolution brings Ca2+ and ATP together to control life and death’. PMID:27377720

  18. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  19. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

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

    PubMed

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

    2012-01-03

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

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

    PubMed

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

    2011-03-01

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

  2. 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.

  3. Quantitative UPLC-MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency.

    PubMed

    Thorsteinsdottir, Margret; Thorsteinsdottir, Unnur A; Eiriksson, Finnur F; Runolfsdottir, Hrafnhildur L; Agustsdottir, Inger M Sch; Oddsdottir, Steinunn; Sigurdsson, Baldur B; Hardarson, Hordur K; Kamble, Nilesh R; Sigurdsson, Snorri Th; Edvardsson, Vidar O; Palsson, Runolfur

    2016-11-15

    Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for quantification of urinary DHA. The UPLC-MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NH4OH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, respectively. A rapid and robust UPLC-MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.

  4. Toward electrochemical resolution of two genes on one electrode: using 7-deaza analogues of guanine and adenine to prepare PCR products with differential redox activity.

    PubMed

    Yang, Ivana V; Ropp, Patricia A; Thorp, H Holden

    2002-01-15

    The 7-deaza analogues of guanine and adenine were incorporated into polymerase chain reaction (PCR) products by substitution of the appropriate nucleotide triphosphates into the reaction. These PCR products can be immobilized on ITO electrodes and detected by catalytic cyclic voltammetry with ruthenium polypyridyl complexes. Immobilization on indium tin oxide (ITO) electrodes of 330- and 1200-base pair (bp) PCR amplicons from the E. coli dacA gene containing one or both of the 7-deazapurines was effected by precipitation from a 9:1 DMF/acetate solution. Amplicons containing the 7-deazaguanine base were detected by observing current enhancement in the cyclic voltammogram of Ru(dmb)3(3)+/2+ (dmb = 4,4'-dimethyl-2,2'-bipyridine) due to the selective oxidation of the modified base by this mediator. Oxidation of incorporated 7-deazaadenine bases in addition to native guanines gives rise to a higher current enhancement in the cyclic voltammogram of Ru(bpy)3(3)+/2+ (bpy = 2,2'-bipyridine) compared to the enhancement observed in the presence of guanine only. This strategy was employed to simultaneously detect the 330-bp sequence containing 7-deazaadenine and the 1200-bp sequence containing 7-deazaguanine on the same ITO electrode. Such a strategy may provide a means for detecting multiple genes on a single microlocation and may thereby lead to more highly multiplexed gene assays.

  5. Essential role of vesicular nucleotide transporter in vesicular storage and release of nucleotides in platelets

    PubMed Central

    Hiasa, Miki; Togawa, Natsuko; Miyaji, Takaaki; Omote, Hiroshi; Yamamoto, Akitsugu; Moriyama, Yoshinori

    2014-01-01

    Abstract Nucleotides are stored in the dense granules of platelets. The release of nucleotides triggers one of the first steps in a series of cascades responsible for blood coagulation. However, the mechanism of how the nucleotides are accumulated in the granules is still far less understood. The transporter protein responsible for storage of nucleotides in the neuroendocrine cells has been identified and characterized. We hypothesized that the vesicular nucleotide transporter (VNUT) is also involved in the vesicular storage of nucleotides in platelets. In this article, we present three lines of evidence that VNUT is responsible for the vesicular storage of nucleotides in platelets and that vesicular ATP transport is crucial for platelet function, detection and characterization of VNUT activity in platelets isolated from healthy humans and MEG‐01 cells, RNA interference experiments on MEG‐01 cells, and studies on nucleotide transport and release with a selective inhibitor. PMID:24907298

  6. Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.

    PubMed

    Menezes, Camila Braz; Durgante, Juliano; de Oliveira, Rafael Rodrigues; Dos Santos, Victor Hugo Jacks Mendes; Rodrigues, Luiz Frederico; Garcia, Solange Cristina; Dos Santos, Odelta; Tasca, Tiana

    2016-05-01

    Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final

  7. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions.

    PubMed

    Coari, Kristin M; Martin, Rebecca C; Jain, Kopal; McGown, Linda B

    2017-02-03

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  8. Nucleotide Selectivity in Abiotic RNA Polymerization Reactions

    NASA Astrophysics Data System (ADS)

    Coari, Kristin M.; Martin, Rebecca C.; Jain, Kopal; McGown, Linda B.

    2017-02-01

    In order to establish an RNA world on early Earth, the nucleotides must form polymers through chemical rather than biochemical reactions. The polymerization products must be long enough to perform catalytic functions, including self-replication, and to preserve genetic information. These functions depend not only on the length of the polymers, but also on their sequences. To date, studies of abiotic RNA polymerization generally have focused on routes to polymerization of a single nucleotide and lengths of the homopolymer products. Less work has been done the selectivity of the reaction toward incorporation of some nucleotides over others in nucleotide mixtures. Such information is an essential step toward understanding the chemical evolution of RNA. To address this question, in the present work RNA polymerization reactions were performed in the presence of montmorillonite clay catalyst. The nucleotides included the monophosphates of adenosine, cytosine, guanosine, uridine and inosine. Experiments included reactions of mixtures of an imidazole-activated nucleotide (ImpX) with one or more unactivated nucleotides (XMP), of two or more ImpX, and of XMP that were activated in situ in the polymerization reaction itself. The reaction products were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify the lengths and nucleotide compositions of the polymerization products. The results show that the extent of polymerization, the degree of heteropolymerization vs. homopolymerization, and the composition of the polymeric products all vary among the different nucleotides and depend upon which nucleotides and how many different nucleotides are present in the mixture.

  9. DNA sequence selective adenine alkylation, mechanism of adduct repair, and in vivo antitumor activity of the novel achiral seco-amino-cyclopropylbenz[e]indolone analogue of duocarmycin AS-I-145.

    PubMed

    Kiakos, Konstantinos; Sato, Atsushi; Asao, Tetsuji; McHugh, Peter J; Lee, Moses; Hartley, John A

    2007-10-01

    AS-I-145 is a novel achiral seco-amino-cyclopropylbenz[e]indolone (seco-amino-CBI) analogue of duocarmycin that has evolved from an alternative strategy of designing CC-1065/duocarmycin agents lacking the characteristic chiral center of the natural agents. The sequence specificity of this compound was assessed by a Taq polymerase stop assay, identifying the sites of covalent modification on plasmid DNA. The adenine-N3 adducts were confirmed at AT-rich sequences using a thermally induced strand cleavage assay. These studies reveal that this compound retains the inherent sequence selectivity of the related natural compounds. The AS-I-145 sensitivity of yeast mutants deficient in excision and post-replication repair (PRR) pathways was assessed. The sensitivity profile suggests that the sequence-specific adenine-N3 adducts are substrates for nucleotide excision repair (NER) but not base excision repair (BER). Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution in yeast cells. Sequence specificity was preserved in intact cells, and adduct elimination occurred in a transcription-coupled manner and was dependent on a functional NER pathway and Rad18. The involvement of NER as the predominant excision pathway was confirmed in mammalian DNA repair mutant cells. AS-I-145 showed good in vivo antitumor activity in the National Cancer Institute standard hollow fiber assay and was active against the human breast MDA-MD-435 xenograft when administered i.v. or p.o. Its novel structure and in vivo activity renders AS-I-145 a new paradigm in the design of novel achiral analogues of CC-1065 and the duocarmycins.

  10. Automated Identification of Nucleotide Sequences

    NASA Technical Reports Server (NTRS)

    Osman, Shariff; Venkateswaran, Kasthuri; Fox, George; Zhu, Dian-Hui

    2007-01-01

    STITCH is a computer program that processes raw nucleotide-sequence data to automatically remove unwanted vector information, perform reverse-complement comparison, stitch shorter sequences together to make longer ones to which the shorter ones presumably belong, and search against the user s choice of private and Internet-accessible public 16S rRNA databases. ["16S rRNA" denotes a ribosomal ribonucleic acid (rRNA) sequence that is common to all organisms.] In STITCH, a template 16S rRNA sequence is used to position forward and reverse reads. STITCH then automatically searches known 16S rRNA sequences in the user s chosen database(s) to find the sequence most similar to (the sequence that lies at the smallest edit distance from) each spliced sequence. The result of processing by STITCH is the identification of the most similar well-described bacterium. Whereas previously commercially available software for analyzing genetic sequences operates on one sequence at a time, STITCH can manipulate multiple sequences simultaneously to perform the aforementioned operations. A typical analysis of several dozen sequences (length of the order of 103 base pairs) by use of STITCH is completed in a few minutes, whereas such an analysis performed by use of prior software takes hours or days.

  11. Plastid sequence evolution: a new pattern of nucleotide substitutions in the Cucurbitaceae.

    PubMed

    Decker-Walters, Deena S; Chung, Sang-Min; Staub, Jack E

    2004-05-01

    Nucleotide substitutions (i.e., point mutations) are the primary driving force in generating DNA variation upon which selection can act. Substitutions called transitions, which entail exchanges between purines (A = adenine, G = guanine) or pyrimidines (C = cytosine, T = thymine), typically outnumber transversions (e.g., exchanges between a purine and a pyrimidine) in a DNA strand. With an increasing number of plant studies revealing a transversion rather than transition bias, we chose to perform a detailed substitution analysis for the plant family Cucurbitaceae using data from several short plastid DNA sequences. We generated a phylogenetic tree for 19 taxa of the tribe Benincaseae and related genera and then scored conservative substitution changes (e.g., those not exhibiting homoplasy or reversals) from the unambiguous branches of the tree. Neither the transition nor (A+T)/(G+C) biases found in previous studies were supported by our overall data. More importantly, we found a novel and symmetrical substitution bias in which Gs had been preferentially replaced by A, As by C, Cs by T, and Ts by G, resulting in the G-->A-->C-->T-->G substitution series. Understanding this pattern will lead to new hypotheses concerning plastid evolution, which in turn will affect the choices of substitution models and other tree-building algorithms for phylogenetic analyses based on nucleotide data.

  12. Nucleotide variability in the 5-enolpyruvylshikimate-3-phosphate synthase gene from Eleusine indica (L.) Gaertn.

    PubMed

    Chong, J L; Wickneswari, R; Ismail, B S; Salmijah, S

    2008-02-01

    This study reports the results of the partial DNA sequence analysis of the 5-enolpyruvyl-shikimate-3-phosphate synthase (EPSPS) gene in glyphosate-resistant (R) and glyphosate-susceptible (S) biotypes of Eleusine indica (L.) Gaertn from Peninsular Malaysia. Sequencing results revealed point mutation at nucleotide position 875 in the R biotypes of Bidor, Chaah and Temerloh. In the Chaah R population, substitution of cytosine (C) to adenine (A) resulted in the change of threonine (Thr106) to proline (Pro106) and from C to thymidine (T) in the Bidor R population, leading to serine (Ser106) from Pro106. As for the Temerloh R, C was substituted by T resulting in the change of Pro106 to Ser106. A new mutation previously undetected in the Temerloh R was revealed with C being substituted with A, resulting in the change of Pro106 to Thr106 indicating multiple founding events rather than to the spread of a single resistant allele. There was no point mutation recorded at nucleotide position 875 previously demonstrated to play a pivotal role in conferring glyphosate resistance to E. indica for the Lenggeng, Kuala Selangor, Melaka R populations. Thus, there may be another resistance mechanism yet undiscovered in the resistant Lenggeng, Kuala Selangor and Melaka populations.

  13. Long-range correlations in nucleotide sequences

    NASA Technical Reports Server (NTRS)

    Peng, C. K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-01-01

    DNA sequences have been analysed using models, such as an n-step Markov chain, that incorporate the possibility of short-range nucleotide correlations. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  14. Long-range correlations in nucleotide sequences

    NASA Astrophysics Data System (ADS)

    Peng, C.-K.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Sciortino, F.; Simons, M.; Stanley, H. E.

    1992-03-01

    DNA SEQUENCES have been analysed using models, such as an it-step Markov chain, that incorporate the possibility of short-range nucleotide correlations1. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  15. 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.

  16. 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.

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

    PubMed

    Kuppuraj, Gopi; Kruise, Dennis; Yura, Kei

    2014-11-26

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

  18. Simultaneous determination of adenine nucleotides, creatine phosphate and creatine in rat liver by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

    PubMed

    Jiang, Yang; Sun, Chengjun; Ding, Xueqin; Yuan, Ding; Chen, Kefei; Gao, Bo; Chen, Yi; Sun, Aimin

    2012-07-01

    A high performance liquid chromatography-electrospray ionization-tandem mass spectrometric method (HPLC-ESI-MS/MS) was developed for simultaneous determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), creatine phosphate (CP), and creatine in rat liver. After extraction with pre-cooled (4 °C) methanol/water (1:1, v/v), the analytes were separated on a porous graphitic carbon (Hypercarb) column (2.1 mm × 150 mm, 5 μm) using a programmed gradient elution with a mobile phase consisting of 2 mmol/L ammonium acetate in water and 2 mmol/L ammonium acetate in acetonitrile (pH=10.0). The analytes were detected in a way of multiple reaction monitoring (MRM) under negative scan mode by a triple quadrupole mass spectrometer with electrospray ionization (ESI). An external calibration method with linear ranges from 10 to 5000 ng/mL for the five target compounds was used for quantification with a correlation coefficients≥0.9973. The limits of detection and limits of quantification for all analytes were in ranges from 0.50 to 1.5 ng/mL and 1.6 to 0.5 ng/mL, respectively. The average recoveries spiked in three levels were from 77.2% to 102% and precisions expressed in RSDs were from 0.2% to 4.8%. The established method was successfully applied to determination of ATP, ADP, AMP, CP and creatine in liver tissue.

  19. In vitro studies of immunoglobulin heavy-chain binding protein (BiP, GRP78). Interactions of BiP with newly synthesized proteins and adenine nucleotides

    SciTech Connect

    Kassenbrock, C.K.

    1988-01-01

    Here we examine the interaction of BiP with newly synthesized polypeptides in an in vitro protein translations-translocation system. We find that BiP forms tight complexes with nonglycosylated yeast invertase and incorrectly disulfide-bonded prolactin but not with glycosylated invertase or correctly disulfide-bonded prolactin. Moreover, BiP associates detectably only with completed chains of prolactin, not with chains undergoing synthesis. We conclude that BiP recognizes and binds with high affinity to aberrantly folded or aberrantly glycosylated polypeptides in vitro, but not to all nascent chains as they are folding. BiP also binds APT and can be purified by APT affinity chromatography. We show that submicromolar levels of ATP or ADP decrease the rate of absorption of {sup 125}I-BiP to nitrocellulose filters coated with protein or nonionic detergents. ATP and ADP also protect portions of BiP from proteolytic degradation. In contrast, micromolar levels of AMP increase the rate of adsorption and the rate of proteolytic degradation of BiP. We also show that an ATPase activity co-purifies with BiP, but its slow turnover number suggests a regulatory, rather than a functional role. The BiP-associated ATPase shares several properties with the related cytoplasmic protein, HSC70/clathrin uncoating ATPase.

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

    PubMed Central

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

    1973-01-01

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

  1. 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.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-25

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

  5. 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.

  6. 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.

  7. Single nucleotide polymorphism analysis using different colored dye dimer probes

    NASA Astrophysics Data System (ADS)

    Marmé, Nicole; Friedrich, Achim; Denapaite, Dalia; Hakenbeck, Regine; Knemeyer, Jens-Peter

    2006-09-01

    Fluorescence quenching by dye dimer formation has been utilized to develop hairpin-structured DNA probes for the detection of a single nucleotide polymorphism (SNP) in the penicillin target gene pbp2x, which is implicated in the penicillin resistance of Streptococcus pneumoniae. We designed two specific DNA probes for the identification of the pbp2x genes from a penicillin susceptible strain R6 and a resistant strain Streptococcus mitis 661 using green-fluorescent tetramethylrhodamine (TMR) and red-fluorescent DY-636, respectively. Hybridization of each of the probes to its respective target DNA sequence opened the DNA hairpin probes, consequently breaking the nonfluorescent dye dimers into fluorescent species. This hybridization of the target with the hairpin probe achieved single nucleotide specific detection at nanomolar concentrations via increased fluorescence.

  8. 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

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

    PubMed Central

    Egberink, H; Borst, M; Niphuis, H; Balzarini, J; Neu, H; Schellekens, H; De Clercq, E; Horzinek, M; Koolen, M

    1990-01-01

    The acyclic purine nucleoside analogue 9-(2-phosphonomethoxyethyl)adenine [PMEA; formerly referred to as 9-(2-phosphonylmethoxyethyl)adenine] is a potent and selective inhibitor of human immunodeficiency virus replication in vitro and of Moloney murine sarcoma virus-induced tumor formation in mice. In the latter system PMEA has stronger antiretroviral potency and selectivity than 3'-azido-3'-thymidine (AZT). We have now investigated the effect of the drug in cats infected with the feline immunodeficiency virus (FIV). In vitro, PMEA was found to efficiently block FIV replication in feline thymocytes (50% effective dose, 0.6 microM). When administered to cats at doses of 20, 5, or 2 mg/kg per day, PMEA caused a dose-dependent suppression of FIV replication and virus-specific antibody production. Seropositive field cats with signs of opportunistic infection (gingivitis, stomatitis, and diarrhea) showed clinical improvement during PMEA therapy (5 mg/kg per day) and recurrence of the disease after treatment was discontinued. Thus, FIV infection in cats is an excellent model to test the efficacy of selective anti-human immunodeficiency virus agents in vivo. Images PMID:2158102

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

    PubMed

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

    2002-04-01

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

  11. 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.

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

    PubMed

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

    2015-07-31

    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.

  13. 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.

  14. Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors.

    PubMed

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

    2017-02-20

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

  15. Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors

    NASA Astrophysics Data System (ADS)

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

    2017-02-01

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

  16. The impact of adenine and inventory utilization decisions on blood inventory management.

    PubMed

    Cohen, M A; Pierskalla, W P; Sassetti, R J

    1983-01-01

    The use of citrate-phosphate-dextrose-adenine as an anticoagulant for whole blood increases the storage period permitted for whole blood and red cells from 21 to 35 days. A simulation model was used to analyze the possible consequences for outdates and shortages of the addition of adenine. The model accepts as input (1) the maximum age (21 or 35 days), (2) parameters describing the demand and supply distributions, and (3) parameters describing inventory control (crossmatch recycle period, transfusion fraction, deviation from optimal target inventory levels). These parameters were varied over wide ranges, and a full factorial design was carried out. The observed shortage and outdate rates were then related (via multiple regression) to the parameter values. The resulting shortage and outdate functions indicated the effect of parameter changes, including extending the lifetime from 21 to 35 days, and the joint effect of changing more than one parameter. Conclusions indicate that, while the contribution of an increased lifetime to reducing shortages and outdates can be substantial, this contribution can be easily dissipated by relaxing the tightness of other inventory management controls.

  17. Adenine-functionalized Spongy Graphene for Green and High-Performance Supercapacitors

    PubMed Central

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

    2017-01-01

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

  18. DNA adenine methylation of sams1 gene in symbiont-bearing Amoeba proteus.

    PubMed

    Jeon, Taeck J

    2008-10-01

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

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

    NASA Astrophysics Data System (ADS)

    Alparone, Andrea

    2014-07-01

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

  20. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2013-11-01

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

  2. 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.

  3. Absolute doubly differential cross sections for ionization of adenine by 1.0-MeV protons

    SciTech Connect

    Iriki, Y.; Kikuchi, Y.; Imai, M.; Itoh, A.

    2011-09-15

    Double-differential ionization cross sections of adenine (C{sub 5}H{sub 5}N{sub 5}) by 1.0-MeV protons have been measured using a vapor-phase adenine target. Ejected electrons were analyzed by a 45 deg. parallel-plate electrostatic spectrometer in an electron energy range from 1 to 1000 eV at electron emission angles from 15 deg. to 165 deg. The effective target thickness of adenine was determined by a Rutherford forward scattering method and a vapor deposition method. Present data are in good agreement with recent calculations. Comparisons were made with other data on various hydrocarbon molecules. It was found that the ionization cross sections of these molecules can be scaled fairly well in terms of the total number of valence electrons.

  4. N(7)-protonation-induced conformational flipping in hypermodified nucleic acid base N 6-(N-glycylcarbonyl) adenine

    NASA Astrophysics Data System (ADS)

    Tewari, Ravindra

    1995-06-01

    Protonation-induced conformational changes are studied in the hypermodified nucleic acid base N 6-(N-glycylcarbonyl) adenine, gc 6Ade, using the quantum chemical perturbative configuration interaction using localised orbitals method. Protonation at the N(7) position of adenine in gc 6Ade induces reorientation of the N 6 substituent, so as to allow stabilisation through an intramolecular hydrogen bond involving N(7)H and the carbonyl oxygen in the glycylcarbonyl substituent. The relative orientation of the carboxyl group with respect to the carbonyl group in the ureido HNCONH linkage is predicted to be similar to that in unprotonated gc 6Ade. The theoretically preferred proximal conformation of N(7)-protonated gc 6Ade restores the participation of N(6)H and N(1) in the Watson-Crick base pairing similar to that in unmodified adenine. This unique orientation can be significant for altering the reading frame in codon-anticodon interactions.

  5. Plant cyclic nucleotide signalling: facts and fiction.

    PubMed

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc; Maathuis, Frans Jm

    2007-11-01

    The presence of the cyclic nucleotides 3',5'-cyclic adenyl monophosphate (cAMP) and 3',5'-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed.

  6. Single Nucleotide Polymorphisms and Osteoarthritis

    PubMed Central

    Wang, Ting; Liang, Yuting; Li, Hong; Li, Haibo; He, Quanze; Xue, Ying; Shen, Cong; Zhang, Chunhua; Xiang, Jingjing; Ding, Jie; Qiao, Longwei; Zheng, Qiping

    2016-01-01

    Abstract Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage and is largely attributed to genetic risk factors. Single nucleotide polymorphisms (SNPs) are common DNA variants that have shown promising and efficiency, compared with positional cloning, to map candidate genes of complex diseases, including OA. In this study, we aim to provide an overview of multiple SNPs from a number of genes that have recently been linked to OA susceptibility. We also performed a comprehensive meta-analysis to evaluate the association of SNP rs7639618 of double von Willebrand factor A domains (DVWA) gene with OA susceptibility. A systematic search of studies on the association of SNPs with susceptibility to OA was conducted in PubMed and Google scholar. Studies subjected to meta-analysis include human and case-control studies that met the Hardy–Weinberg equilibrium model and provide sufficient data to calculate an odds ratio (OR). A total of 9500 OA cases and 9365 controls in 7 case-control studies relating to SNP rs7639618 were included in this study and the ORs with 95% confidence intervals (CIs) were calculated. Over 50 SNPs from different genes have been shown to be associated with either hip (23), or knee (20), or both (13) OA. The ORs of these SNPs for OA and the subtypes are not consistent. As to SNP rs7639618 of DVWA, increased knee OA risk was observed in all genetic models analyzed. Specifically, people from Asian with G-allele showed significantly increased risk of knee OA (A versus G: OR = 1.28, 95% CI 1.13–1.46; AA versus GG: OR = 1.60, 95% CI 1.25–2.05; GA versus GG: OR = 1.31, 95% CI 1.18–1.44; AA versus GA+GG: OR = 1.34, 95% CI 1.12–1.61; AA+GA versus GG: OR = 1.40, 95% CI 1.19–1.64), but not in Caucasians or with hip OA. Our results suggest that multiple SNPs play different roles in the pathogenesis of OA and its subtypes; SNP rs7639618 of DVWA gene is associated with a significantly increased

  7. Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5‧ monophosphate (AMP)

    NASA Astrophysics Data System (ADS)

    Hammami, K.; Feki, H. El; Marsan, O.; Drouet, C.

    2015-10-01

    This work investigates the interaction between the nucleotide adenosine 5‧ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads ≅ -22 kJ/mol) intermediate between "physisorption" and "chemisorption" was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

  8. 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

  9. Studies on the preferred uracil-adenine base pair at the cleavage site of 10-23 DNAzyme by functional group modifications on adenine.

    PubMed

    Zhu, Junfei; Li, Zhiwen; Yang, Zhenjun; He, Junlin

    2015-08-01

    10-23 DNAzyme is capable of catalytically cleaving RNA substrates with the preferred cleavage sites rAU and rGU, in which the common base pair U-dA0 forms between the substrate and the DNAzyme in the cleavage reaction. Here its conservation was studied with base modifications on dA and extra functional groups introduced. The nitrogen atom at 7- or 8-position of adenine was demonstrated to be equally important for the cleavage reaction, although it is not related to the thermal stability of the base pair. Deletion of 6-amino group led to decreased stability of the base pair and a slight slower reaction rate. Extra functional groups through 6-amino group were not favorably accommodated in the cleavage site. From these modifications at the level of functional groups, it demonstrated that the base pair U-dA0 not only contributes to the recognition and binding stability, but also it is involved in the active catalytic center by its functional groups and base stacking. This kind of chemical modifications with 7-substituted 8-aza-7-deaza-2'-deoxyadenosine at dA0 is favorable for the introduction of signal molecules for mechanistic studies and biological applications, without significant loss of the catalytic function and structural destruction.

  10. Hydrolytic and alcoholytic dephosphorylation of nucleotides by acid phosphatase in the presence of ethanol.

    PubMed

    Tomaszewski, M; Buchowicz, J

    1971-08-01

    The effect of ethanol on the activity of acid phosphatase from wheat germ was studied, by using ribonucleoside monophosphates as the enzyme substrates. The nucleotides were effectively degraded to the corresponding nucleosides in the presence of ethanol at all concentrations tested, including a 96% (v/v) solution. However, the nucleotide dephosphorylation was accompanied by the liberation of orthophosphate only when the concentration of ethanol in the assay mixture did not exceed 15%. No inorganic phosphate was liberated when ethanol was present at higher concentrations. Instead, monoethyl phosphate was formed in quantities expected for orthophosphate. The results are explained in terms of phosphatase-catalysed alcoholysis.

  11. Extreme accumulation of nucleotides in simulated hydrothermal pore systems

    PubMed Central

    Baaske, Philipp; Weinert, Franz M.; Duhr, Stefan; Lemke, Kono H.; Russell, Michael J.; Braun, Dieter

    2007-01-01

    We simulate molecular transport in elongated hydrothermal pore systems influenced by a thermal gradient. We find extreme accumulation of molecules in a wide variety of plugged pores. The mechanism is able to provide highly concentrated single nucleotides, suitable for operations of an RNA world at the origin of life. It is driven solely by the thermal gradient across a pore. On the one hand, the fluid is shuttled by thermal convection along the pore, whereas on the other hand, the molecules drift across the pore, driven by thermodiffusion. As a result, millimeter-sized pores accumulate even single nucleotides more than 108-fold into micrometer-sized regions. The enhanced concentration of molecules is found in the bulk water near the closed bottom end of the pore. Because the accumulation depends exponentially on the pore length and temperature difference, it is considerably robust with respect to changes in the cleft geometry and the molecular dimensions. Whereas thin pores can concentrate only long polynucleotides, thicker pores accumulate short and long polynucleotides equally well and allow various molecular compositions. This setting also provides a temperature oscillation, shown previously to exponentially replicate DNA in the protein-assisted PCR. Our results indicate that, for life to evolve, complicated active membrane transport is not required for the initial steps. We find that interlinked mineral pores in a thermal gradient provide a compelling high-concentration starting point for the molecular evolution of life. PMID:17494767

  12. A type of nucleotide motif that distinguishes tobamovirus species more efficiently than nucleotide signatures.

    PubMed

    Gibbs, A J; Armstrong, J S; Gibbs, M J

    2004-10-01

    The complete genomic sequences of forty-eight tobamoviruses were classified and found to form at least twelve species clusters. Individual species were not conveniently defined by 'nucleotide signatures' (i.e. strings of one or more nucleotides unique to a taxon) as these were scattered sparsely throughout the genomes and were mostly single nucleotides. By contrast all the species were concisely and uniquely distinguished by short nucleotide motifs consisting of conserved genus-specific sites intercalated with variable sites that provided species-specific combinations of nucleotides (nucleotide combination motifs; NC-motifs). We describe the procedure for finding NC-motifs in a convenient and phylogenetically conserved region of the tobamovirus RNA polymerase gene, the '4404-50 motif'. NC-motifs have been found in other sets of homologous sequences, and are convenient for use in published taxonomic descriptions.

  13. Pyridine nucleotide cycling and control of intracellular redox state in relation to poly (ADP-ribose) polymerase activity and nuclear localization of glutathione during exponential growth of Arabidopsis cells in culture.

    PubMed

    Pellny, Till K; Locato, Vittoria; Vivancos, Pedro Diaz; Markovic, Jelena; De Gara, Laura; Pallardó, Federico V; Foyer, Christine H

    2009-05-01

    Pyridine nucleotides, ascorbate and glutathione are major redox metabolites in plant cells, with specific roles in cellular redox homeostasis and the regulation of the cell cycle. However, the regulation of these metabolite pools during exponential growth and their precise functions in the cell cycle remain to be characterized. The present analysis of the abundance of ascorbate, glutathione, and pyridine nucleotides during exponential growth of Arabidopsis cells in culture provides evidence for the differential regulation of each of these redox pools. Ascorbate was most abundant early in the growth cycle, but glutathione was low at this point. The cellular ascorbate to dehydroascorbate and reduced glutathione (GSH) to glutathione disulphide ratios were high and constant but the pyridine nucleotide pools were largely oxidized over the period of exponential growth and only became more reduced once growth had ceased. The glutathione pool increased in parallel with poly (ADP-ribose) polymerase (PARP) activities and with increases in the abundance of PARP1 and PARP2 mRNAs at a time of high cell cycle activity as indicated by transcriptome information. Marked changes in the intracellular partitioning of GSH between the cytoplasm and nucleus were observed. Extension of the exponential growth phase by dilution or changing the media led to increases in the glutathione and nicotinamide adenine dinucleotide, oxidized form (NAD)-plus-nicotinamide adenine dinucleotide, reduced form (NADH) pools and to higher NAD/NADH ratios but the nicotinamide adenine dinucleotide phosphate, oxidized form (NADP)-plus-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) pool sizes, and NAPD/NADPH ratios were much less affected. The ascorbate, glutathione, and pyridine nucleotide pools and PARP activity decreased before the exponential growth phase ended. We conclude that there are marked changes in intracellular redox state during the growth cycle but that redox homeostasis is

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

    SciTech Connect

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

    1987-05-01

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

  15. 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.

  16. 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

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

    PubMed

    Park, Ki Soo; Park, Hyun Gyu

    2015-02-15

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

  18. Cyclic Nucleotide Phosphodiesterases: important signaling modulators and therapeutic targets

    PubMed Central

    Ahmad, Faiyaz; Murata, Taku; Simizu, Kasumi; Degerman, Eva; Maurice, Donald; Manganiello, Vincent

    2014-01-01

    By catalyzing hydrolysis of cAMP and cGMP, cyclic nucleotide phosphodiesterases are critical regulators of their intracellular concentrations and their biological effects. Since these intracellular second messengers control many cellular homeostatic processes, dysregulation of their signals and signaling pathways initiate or modulate pathophysiological pathways related to various disease states, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication, chronic obstructive pulmonary disease, and psoriasis. Alterations in expression of PDEs and PDE-gene mutations (especially mutations in PDE6, PDE8B, PDE11A and PDE4) have been implicated in various diseases and cancer pathologies. PDEs also play important role in formation and function of multi-molecular signaling/regulatory complexes called signalosomes. At specific intracellular locations, individual PDEs, together with pathway-specific signaling molecules, regulators, and effectors, are incorporated into specific signalosomes, where they facilitate and regulate compartmentalization of cyclic nucleotide signaling pathways and specific cellular functions. Currently, only a limited number of PDE inhibitors (PDE3, PDE4, PDE5 inhibitors) are used in clinical practice. Future paths to novel drug discovery include the crystal structure-based design approach, which has resulted in generation of more effective family-selective inhibitors, as well as burgeoning development of strategies to alter compartmentalized cyclic nucleotide signaling pathways by selectively targeting individual PDEs and their signalosome partners. PMID:25056711

  19. SVOP Is a Nucleotide Binding Protein

    PubMed Central

    Yao, Jia; Bajjalieh, Sandra M.

    2009-01-01

    Background Synaptic Vesicle Protein 2 (SV2) and SV2-related protein (SVOP) are transporter-like proteins that localize to neurotransmitter-containing vesicles. Both proteins share structural similarity with the major facilitator (MF) family of small molecule transporters. We recently reported that SV2 binds nucleotides, a feature that has also been reported for another MF family member, the human glucose transporter 1 (Glut1). In the case of Glut1, nucleotide binding affects transport activity. In this study, we determined if SVOP also binds nucleotides and assessed its nucleotide binding properties. Methodology/Principal Findings We performed in vitro photoaffinity labeling experiments with the photoreactive ATP analogue, 8-azido-ATP[γ] biotin and purified recombinant SVOP-FLAG fusion protein. We found that SVOP is a nucleotide-binding protein, although both its substrate specificity and binding site differ from that of SV2. Within the nucleotides tested, ATP, GTP and NAD show same level of inhibition on SVOP-FLAG labeling. Dose dependent studies indicated that SVOP demonstrates the highest affinity for NAD, in contrast to SV2, which binds both NAD and ATP with equal affinity. Mapping of the binding site revealed a single region spanning transmembrane domains 9–12, which contrasts to the two binding sites in the large cytoplasmic domains in SV2A. Conclusions/Significance SVOP is the third MF family member to be found to bind nucleotides. Given that the binding sites are unique in SVOP, SV2 and Glut1, this feature appears to have arisen separately. PMID:19390693

  20. Structural and thermodynamic studies on the adenine.guanine mismatch in B-DNA.

    PubMed Central

    Leonard, G A; Booth, E D; Brown, T

    1990-01-01

    The structure of the synthetic dodecamer d(CGCAAATTGGCG) has been shown by single crystal X-ray diffraction methods to be that of a B-DNA helix containing two A(anti).G(syn) base pairs. The refinement, based on data to a resolution of 2.25 A shows that the mismatch base pairs are held together by two hydrogen bonds. The syn-conformation of the guanine base of the mismatch is stabilised by hydrogen bonding to a network of solvent molecules in both the major and minor grooves. A pH-dependent ultraviolet melting study indicates that the duplex is stabilised by protonation, suggesting that the bases of the A.G mispair are present in their most common tautomeric forms and that the N(1)-atom of adenine is protonated. The structure refinement shows that there is some disorder in the sugar-phosphate backbone. PMID:2216754

  1. 8-(2-Furyl)adenine derivatives as A₂A adenosine receptor ligands.

    PubMed

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Thomas, Ajiroghene; Klotz, Karl-Norbert; Federico, Stephanie; Cacciari, Barbara; Spalluto, Giampiero; Volpini, Rosaria

    2013-01-01

    Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2-furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A₂A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N(6)-position takes to different effects on the A₂A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A₂A receptor crystal structures provides an interpretation of the obtained biological results.

  2. 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.

  3. 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.

  4. Surface-Enhanced Hyper-Raman Spectra of Adenine, Guanine, Cytosine, Thymine, and Uracil

    PubMed Central

    2016-01-01

    Using picosecond excitation at 1064 nm, surface-enhanced hyper-Raman scattering (SEHRS) spectra of the nucleobases adenine, guanine, cytosine, thymine, and uracil with two different types of silver nanoparticles were obtained. Comparing the SEHRS spectra with SERS data from the identical samples excited at 532 nm and with known infrared spectra, the major bands in the spectra are assigned. Due to the different selection rules for the one- and two-photon excited Raman scattering, we observe strong variation in relative signal strengths of many molecular vibrations obtained in SEHRS and SERS spectra. The two-photon excited spectra of the nucleobases are found to be very sensitive with respect to molecule–nanoparticle interactions. Using both the SEHRS and SERS data, a comprehensive vibrational characterization of the interaction of nucleobases with silver nanostructures can be achieved. PMID:28077982

  5. 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.

  6. 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.

  7. 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.

  8. Theoretical investigation of hydrogen transfer mechanism in the adenine thymine base pair

    NASA Astrophysics Data System (ADS)

    Villani, Giovanni

    2005-09-01

    We have studied the quantum dynamics of the hydrogen bonds in the adenine-thymine base pair. Due to the position of hydrogen atoms, different tautomers are possible: the stable Watson-Crick A-T, the imino-enol A*-T* and the zwitterionic (the form with charge separation) A +-T - and A --T + structures. The common idea in the literature is that only A-T exists either because the difference of energy among this tautomer and the others is large or because the other structures are transformed quickly in A-T. Here, we show a detailed theoretical study that suggests the following conclusion: A-T is the stablest tautomer, a partially charged system is important and a small amount of the imino-enol A*-T* tautomer is present at any time. The mechanism of passage from A-T tautomer to the others has also been investigated.

  9. 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)).

  10. 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.

  11. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  13. 3-Methyl-2-butenal: an enzymatic degradation product of the cytokinin, N-6-(delta-2 isopentenyl)adenine.

    PubMed

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

    1975-01-01

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

  14. Highly sensitive and synergistic detection of guanine and adenine based on poly(xanthurenic acid)-reduced graphene oxide interface.

    PubMed

    Yang, Tao; Kong, Qianqian; Li, Qianhe; Wang, Xinxing; Chen, Lihua; Jiao, Kui

    2014-07-23

    In order to achieve the large direct electrochemical signals of guanine and adenine, an urgent request to explore novel electrode materials and interfaces has been put forward. In this paper, a poly(xanthurenic acid, Xa)-reduced graphene oxide (PXa-ERGNO) interface, which has rich negatively charged active sites and accelerated electron transfer ability, was fabricated for monitoring the positively charged guanine and adenine. Scanning electron microscopy, Fourier transform infrared spectroscopy, Raman spectra, X-ray photoelectron spectroscopy, cyclic voltammetry, electrochemical impedance spectroscopy, and differential pulse voltammetry were adopted to characterize the morphology and prove the electrochemical properties of the prepared interface. The PXa-ERGNO interface with rich negative charge and large electrode surface area was an excellent sensing platform to prompt the adsorption of the positively charged guanine and adenine via strong π-π* interaction or electrostatic adsorption. The PXa-ERGNO interface exhibited prominent synergistic effect and good electrocatalytic activity for sensitive determination of guanine and adenine compared with sole PXa or ERGNO modified electrode. The sensing platform we built could be further applied in the adsorption and detection of other positively charged biomolecules or aromatic molecules.

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

    PubMed

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

    2016-06-01

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

  16. 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.

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

    PubMed Central

    Biris, Alexandru R; Pruneanu, Stela; Pogacean, Florina; Lazar, Mihaela D; Borodi, Gheorghe; Ardelean, Stefania; Dervishi, Enkeleda; Watanabe, Fumiya; Biris, Alexandru S

    2013-01-01

    This work describes the synthesis of few-layer graphene sheets embedded with various amounts of gold nanoparticles (Gr-Au-x) over an Aux/MgO catalytic system (where × = 1, 2, or 3 wt%). The sheet-like morphology of the Gr-Au-x nanostructures was confirmed by transmission electron microscopy and high resolution transmission electron microscopy, which also demonstrated that the number of layers within the sheets varied from two to seven. The sample with the highest percentage of gold nanoparticles embedded within the graphitic layers (Gr-Au-3) showed the highest degree of crystallinity. This distinct feature, along with the large number of edge-planes seen in high resolution transmission electron microscopic images, has a crucial effect on the electrocatalytic properties of this material. The reaction yields (40%–50%) and the final purity (96%–98%) of the Gr-Au-x composites were obtained by thermogravimetric analysis. The Gr-Au-x composites were used to modify platinum substrates and subsequently to detect adenine, one of the DNA bases. For the bare electrode, no oxidation signal was recorded. In contrast, all of the modified electrodes showed a strong electrocatalytic effect, and a clear peak for adenine oxidation was recorded at approximately +1.05 V. The highest increase in the electrochemical signal was obtained using a platinum/Gr-Au-3-modified electrode. In addition, this modified electrode had an exchange current density (I0, obtained from the Tafel plot) one order of magnitude higher than that of the bare platinum electrode, which also confirmed that the transfer of electrons took place more readily at the Gr-Au-3-modified electrode. PMID:23610521

  18. Excretory Function of Intestinal Tract Enhanced in Kidney Impaired Rats Caused by Adenine

    PubMed Central

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

    2016-01-01

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

  19. Adenine phosphoribosyltransferase deficiency in the United Kingdom: two novel mutations and a cross-sectional survey

    PubMed Central

    Arenas-Hernandez, Monica; Escuredo, Emilia; Fairbanks, Lynette; Marinaki, Tony; Mapplebeck, Sarah; Sheaff, Michael; Almond, Michael K.

    2016-01-01

    Background Adenine phosphoribosyltransferase deficiency is an inborn error of metabolism that can cause kidney disease from crystalline nephropathy or kidney stones. Methods We present three cases from a single centre with varied presentations to illustrate how increasing awareness led to better patient identification. We then undertook a cross-sectional survey of all the patients identified from the Purine Research Laboratory in the UK since 1974. Results Our index case presented with recurrent nephrolithiasis and was diagnosed on stone analysis, the second case presented with acute kidney injury and the third case was identified from a biopsy undertaken for acute on chronic kidney injury. Genetic studies identified two novel mutations. Twenty patients were retrospectively identified. The mean age at diagnosis was 25 years (range 2–70); eight were <20 years, seven were 20–40 years and five were >40 years. Five of the 20 patients were deceased, 3 after end-stage renal disease (ESRD). Twelve have normal renal function, one had CKD stage 3, one had severe kidney disease and one was on dialysis. Conclusions Adenine phosphoribosyltransferase deficiency presents in a wide spectrum in all age groups. Patients can be completely asymptomatic and kidney disease may be incorrectly attributed to other conditions. Outcome is poor in late diagnosis and there is a high prevalence of ESRD. Patients with unexplained renal stone disease or deterioration in kidney function should be considered for screening. Identification and surveillance of patients in the UK can improve. There is now a rare disease registry with meetings organized that include patients, families and health care providers to improve awareness. PMID:27994857

  20. Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

    PubMed

    Maurice, Donald H; Wilson, Lindsay S; Rampersad, Sarah N; Hubert, Fabien; Truong, Tammy; Kaczmarek, Milosz; Brzezinska, Paulina; Freitag, Silja I; Umana, M Bibiana; Wudwud, Alie

    2014-04-01

    The cyclic nucleotide second messengers cAMP and cGMP each affect virtually all cellular processes. Although these hydrophilic small molecules readily diffuse throughout cells, it is remarkable that their ability to activate their multiple intracellular effectors is spatially and temporally selective. Studies have identified a critical role for compartmentation of the enzymes which hydrolyse and metabolically inactivate these second messengers, the PDEs (cyclic nucleotide phosphodiesterases), in this specificity. In the present article, we describe several examples from our work in which compartmentation of selected cAMP- or cGMP-hydrolysing PDEs co-ordinate selective activation of cyclic nucleotide effectors, and, as a result, selectively affect cellular functions. It is our belief that therapeutic strategies aimed at targeting PDEs within these compartments will allow greater selectivity than those directed at inhibiting these enzymes throughout the cells.

  1. Polymerization of amino acids containing nucleotide bases

    NASA Technical Reports Server (NTRS)

    Ben Cheikh, Azzouz; Orgel, Leslie E.

    1990-01-01

    The nucleoamino acids 1-(3'-amino,3'-carboxypropyl)uracil (3) and 9-(3'-amino,3'-carboxypropyl)adenine (4) have been prepared as (L)-en-antiomers and as racemic mixtures. When 3 or 4 is suspended in water and treated with N,N'-carbon-yldiimidazole, peptides are formed in good yield. The products formed from the (L)-enantiomers are hydrolyzed to the monomeric amino acids by pronase. Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (L)-enantiomer of 3 did not act as templates to facilitate the oligomerization of 4.

  2. The Immunosuppressant Mycophenolic Acid Alters Nucleotide and Lipid Metabolism in an Intestinal Cell Model

    PubMed Central

    Heischmann, Svenja; Dzieciatkowska, Monika; Hansen, Kirk; Leibfritz, Dieter; Christians, Uwe

    2017-01-01

    The study objective was to elucidate the molecular mechanisms underlying the negative effects of mycophenolic acid (MPA) on human intestinal cells. Effects of MPA exposure and guanosine supplementation on nucleotide concentrations in LS180 cells were assessed using liquid chromatography-mass spectrometry. Proteomics analysis was carried out using stable isotope labeling by amino acids in cell culture combined with gel-based liquid chromatography-mass spectrometry and lipidome analysis using 1H nuclear magnetic resonance spectroscopy. Despite supplementation, depletion of guanosine nucleotides (p < 0.001 at 24 and 72 h; 5, 100, and 250 μM MPA) and upregulation of uridine and cytidine nucleotides (p < 0.001 at 24 h; 5 μM MPA) occurred after exposure to MPA. MPA significantly altered 35 proteins mainly related to nucleotide-dependent processes and lipid metabolism. Cross-reference with previous studies of MPA-associated protein changes widely corroborated these results, but showed differences that may be model- and/or method-dependent. MPA exposure increased intracellular concentrations of fatty acids, cholesterol, and phosphatidylcholine (p < 0.01 at 72 h; 100 μM MPA) which corresponded to the changes in lipid-metabolizing proteins. MPA affected intracellular nucleotide levels, nucleotide-dependent processes, expression of structural proteins, fatty acid and lipid metabolism in LS180 cells. These changes may compromise intestinal membrane integrity and contribute to gastrointestinal toxicity. PMID:28327659

  3. Extracellular modulation of the silkmoth sex pheromone receptor activity by cyclic nucleotides.

    PubMed

    Nakagawa, Tatsuro; Touhara, Kazushige

    2014-01-01

    Odorants and pheromones are essential to insects as chemical cues for finding food or an appropriate mating partner. These volatile compounds bind to olfactory receptors (Ors) expressed by olfactory sensory neurons. Each insect Or functions as a ligand-gated ion channel and is a heteromeric complex that comprises one type of canonical Or and a highly conserved Orco subunit. Because there are many Or types, insect Ors can recognize with high specificity a myriad of chemical cues. Cyclic nucleotides can modulate the activity of insect Or-Orco complexes; however, the mechanism of action of these nucleotides is under debate. Here, we show that cyclic nucleotides, including cAMP and cGMP, interact with the silkmoth sex pheromone receptor complex, BmOr-1-BmOrco, from the outside of the cell and that these nucleotides act as antagonists at low concentrations and weak agonists at high concentrations. These cyclic nucleotides do not compete with the sex pheromone, bombykol, for binding to the BmOr-1 subunit. ATP and GTP also weakly inhibited BmOr-1-BmOrco activity, but D-ribose had no effect; these findings indicated that the purine moiety was crucial for the inhibition. Only the bombykol receptors have been so far shown to be subject to modulation by nucleotide-related compounds, indicating that this responsiveness to these compounds is not common for all insect Or-Orco complexes.

  4. Kinetic approach for the purification of nucleotides with magnetic separation.

    PubMed

    Tural, Servet; Tural, Bilsen; Ece, Mehmet Şakir; Yetkin, Evren; Özkan, Necati

    2014-11-01

    The isolation of β-nicotinamide adenine dinucleotide is of great importance since it is widely used in different scientific and technologic fields such as biofuel cells, sensor technology, and hydrogen production. In order to isolate β-nicotinamide adenine dinucleotide, first 3-aminophenyboronic acid functionalized magnetic nanoparticles were prepared to serve as a magnetic solid support and subsequently they were used for reversible adsorption/desorption of β-nicotinamide adenine dinucleotide in a batch fashion. The loading capacity of the 3-aminophenyboronic acid functionalized nanoparticles for β-nicotinamide adenine dinucleotide adsorption was 13.0 μmol/g. Adsorption kinetic and isotherm studies showed that the adsorption process followed a pseudo-second-order kinetic model and the experimental data can be represented using Langmuir isotherm model. The 3-aminophenyboronic acid functionalized magnetic nanoparticles were proposed as an alternative support for the β-nicotinamide adenine dinucleotide purification. The results elucidated the significance of magnetic separation as a fast, relatively simple, and low-cost technique. Furthermore, the magnetic supports can be reused at least five times for purification processes.

  5. Proofreading of misincorporated nucleotides in DNA transcription

    NASA Astrophysics Data System (ADS)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B.

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighboring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction.

  6. Proofreading of misincorporated nucleotides in DNA transcription

    NASA Astrophysics Data System (ADS)

    Voliotis, Margaritis; Cohen, Netta; Molina-París, Carmen; Liverpool, Tanniemola B.

    2012-06-01

    The accuracy of DNA transcription is crucial for the proper functioning of the cell. Although RNA polymerases demonstrate selectivity for correct nucleotides, additional active mechanisms of transcriptional error correction are required to achieve observed levels of fidelity. Recent experimental findings have shed light on a particular mechanism of transcriptional error correction involving: (i) diffusive translocation of the RNA polymerase along the DNA (backtracking) and (ii) irreversible RNA cleavage. This mechanism achieves preferential cleavage of misincorporated nucleotides by biasing the local rates of translocation. Here, we study how misincorporated nucleotides affect backtracking dynamics and how this effect determines the level of transcriptional fidelity. We consider backtracking as a diffusive process in a periodic, one-dimensional energy landscape, which at a coarse-grained level gives rise to a hopping process between neighbouring local minima. We propose a model for how misincorporated nucleotides deform this energy landscape and hence affect the hopping rates. In particular, we show that this model can be used to derive both the theoretical limit on the fidelity (i.e. the minimum fraction of misincorporated nucleotides) and the actual fidelity relative to this optimum, achieved for specific combinations of the cleavage and polymerization rates. Finally, we study how external factors influencing backtracking dynamics affect transcriptional fidelity. We show that biologically relevant loads, similar to those exerted by nucleosomes or other transcriptional barriers, increase error correction.

  7. Theoretical study of the pre- and post-translational effects of adenine and thymine tautomers and methyl derivatives.

    PubMed

    Gardner, Noel; Magers, David; Hill, Glake

    2013-09-01

    The study of pre-translational effects (ionization, tautomerization) and post-translational effects (methylation) of adenine and thymine has only recently been the focus of some studies. These effects can potentially help regulate gene expression as well as potentially disrupt normal gene function. Because of this wide array of roles, greater insight into these effects in deoxyribonucleic acids (DNA) are paramount. There has been considerable research of each phenomenon (tautomerization, methylation and ionization) individually. In this work, we attempt to shed light upon the pre-translational effects and post translational effects of adenine and thymine by investigating the electron affinities (EAs) and ionization potentials (IPs) of the major and minor tautomers and their methyl derivatives. We performed all calculations using the density functional theory (DFT) B3LYP functional accompanied with 6-311G(d,p), 6-311+G(d,p) and 6-311++G(df,pd) basis sets. Our results reveal that the thymine tautomer has a higher EA and IP than the adenine tautomers. The higher EA suggests that an electron that attaches to the AT base pair would predominately attach to the thymine instead of adenine. The higher IP would suggest that an electron that is removed from the AT base pair would be predominately removed from the adenine within the base pair. Understanding how tautomerization, ionization and methylation differences change effects, discourages, or promotes one another is lacking. In this work, we begin the steps of integrating these effects with one another, to gain a greater understanding of molecular changes in DNA bases.

  8. Electron ionization of the nucleobases adenine and hypoxanthine near the threshold: a combined experimental and theoretical study.

    PubMed

    Dawley, M Michele; Tanzer, Katrin; Cantrell, William A; Plattner, Peter; Brinkmann, Nicole R; Scheier, Paul; Denifl, Stephan; Ptasińska, Sylwia

    2014-12-07

    Electron ionization of the DNA nucleobase, adenine, and the tRNA nucleobase, hypoxanthine, was investigated near the threshold region (∼5-20 eV) using a high-resolution hemispherical electron monochromator and a quadrupole mass spectrometer. Ion efficiency curves of the threshold regions and the corresponding appearance energies (AEs) are presented for the parent cations and the five most abundant fragment cations of each molecule. The experimental ionization energies (IEs) of adenine and hypoxanthine were determined to be 8.70 ± 0.3 eV and 8.88 ± 0.5 eV, respectively. Quantum chemical calculations (B3LYP/6-311+G(2d,p)) yielded a vertical IE of 8.08 eV and an adiabatic IE of 8.07 eV for adenine and a vertical IE of 8.51 eV and an adiabatic IE of 8.36 eV for hypoxanthine, and the lowest energy optimized structures of the fragment cations and their respective neutral species were calculated. The enthalpies of the possible reactions from the adenine and hypoxanthine cations were also obtained computationally, which assisted in determining the most likely electron ionization pathways leading to the major fragment cations. Our results suggest that the imidazole ring is more stable than the pyrimidine ring in several of the fragmentation reactions from both adenine and hypoxanthine. This electron ionization study contributes to the understanding of the biological effects of electrons on nucleobases and to the database of the electronic properties of biomolecules, which is necessary for modeling the damage of DNA in living cells that is induced by ionizing radiation.

  9. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

    PubMed Central

    Sesma, Juliana I.; Kreda, Silvia M.; Okada, Seiko F.; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C.; O'Neal, Wanda K.; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori

    2013-01-01

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins. PMID:23467297

  10. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

    PubMed

    Sesma, Juliana I; Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C; O'Neal, Wanda K; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori; Lazarowski, Eduardo R

    2013-05-15

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

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

    PubMed

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

    2017-02-01

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

  12. The International Nucleotide Sequence Database Collaboration

    PubMed Central

    Cochrane, Guy; Karsch-Mizrachi, Ilene; Takagi, Toshihisa; Sequence Database Collaboration, International Nucleotide

    2016-01-01

    The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org) comprises three global partners committed to capturing, preserving and providing comprehensive public-domain nucleotide sequence information. The INSDC establishes standards, formats and protocols for data and metadata to make it easier for individuals and organisations to submit their nucleotide data reliably to public archives. This work enables the continuous, global exchange of information about living things. Here we present an update of the INSDC in 2015, including data growth and diversification, new standards and requirements by publishers for authors to submit their data to the public archives. The INSDC serves as a model for data sharing in the life sciences. PMID:26657633

  13. The International Nucleotide Sequence Database Collaboration.

    PubMed

    Nakamura, Yasukazu; Cochrane, Guy; Karsch-Mizrachi, Ilene

    2013-01-01

    The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org), one of the longest-standing global alliances of biological data archives, captures, preserves and provides comprehensive public domain nucleotide sequence information. Three partners of the INSDC work in cooperation to establish formats for data and metadata and protocols that facilitate reliable data submission to their databases and support continual data exchange around the world. In this article, the INSDC current status and update for the year of 2012 are presented. Among discussed items of international collaboration meeting in 2012, BioSample database and changes in submission are described as topics.

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

    PubMed

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

    2006-02-01

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

  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. Energy level alignment at the interfaces between typical electrodes and nucleobases: Al/adenine/indium-tin-oxide and Al/thymine/indium-tin-oxide

    SciTech Connect

    Lee, Younjoo; Lee, Hyunbok; Park, Soohyung; Yi, Yeonjin

    2012-12-03

    We investigated the interfacial electronic structures of Al/adenine/indium-tin-oxide (ITO) and Al/thymine/ITO using in situ ultraviolet and x-ray photoemission spectroscopy and density functional theory calculations. Adenine shows both an interface dipole and level bending, whereas thymine shows only an interface dipole in contact with ITO. In addition, thymine possesses a larger ionization energy than adenine. These are understood with delocalized {pi} states confirmed with theoretical calculations. For the interface between nucleobases and Al, both nucleobases show a prominent reduction of the electron injection barrier from Al to each base in accordance with a downward level shift.

  17. Elimination and utilization of oxidized guanine nucleotides in the synthesis of RNA and its precursors.

    PubMed

    Sekiguchi, Takeshi; Ito, Riyoko; Hayakawa, Hiroshi; Sekiguchi, Mutsuo

    2013-03-22

    Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When (14)C-labeled 8-oxo-GTP was applied to CaCl2-permeabilized cells of a mutT(-) mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.

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

    SciTech Connect

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

    2007-02-07

    The photoelectron spectra of the adenine-formic acid (AFA)- and 9-methyladenine-formic acid (MAFA)- anionic complexes have been recorded with 2.540 eV photons. These spectra reveal broad features with maxima at 1.5-1.4 eV that indicate formation of stable valence anions in the gas phase. The neutral and anionic complexes of adenine/9- methyladenine and formic acid were also studied computationally at the B3LYP, second order Møller-Plesset and coupled clusters levels of theory, with the 6-31++G** and aug-cc-pVDZ basis sets. The neutral complexes form cyclic hydrogen bonds and the most stable dimers are bound by 17.7 and 16.0 kcal/mol for AFA and MAFA, respectively. The theoretical results indicate that the excess electron in both (AFA)- and (MAFA)- occupies a p* orbital localized on adenine/9-methyladenine and the adiabatic stability of the most stable anions amounts to 0.67 and 0.54 eV for AFA- and MAFA-, respectively. The excess electron attachment to the complexes induces a barrierfree proton transfer (BFPT) from the carboxylic group of formic acid to a N atom of adenine or 9-mathyladenine. As a result, the most stable structures of the anionic complexes can be characterized as neutral radicals of hydrogenated adenine(9-methyladenine) solvated by a deprotonated formic acid. The BFPT to the N atoms of adenine may be biologically relevant because some of these sites are not involved in the Watson-Crick pairing scheme and are easily accessible in the cellular environment. We suggest that valence anions of purines might be as important as those of pyrimidines in the process of DNA damage by low energy electrons. The calculations were performed at the Academic Computer Center in Gdansk (TASK) and at the Molecular Science Computing Facility (MSCF) in the William R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research and located at the Pacific

  19. Heterogeneous nucleotide occupancy stimulates functionality of phage shock protein F, an AAA+ transcriptional activator.

    PubMed

    Joly, Nicolas; Schumacher, Jörg; Buck, Martin

    2006-11-17

    The catalytic AAA+ domain (PspF1-275) of an enhancer-binding protein is necessary and sufficient to contact sigma54-RNA polymerase holoenzyme (Esigma54), remodel it, and in so doing catalyze open promoter complex formation. Whether ATP binding and hydrolysis is coordinated between subunits of PspF and the precise nature of the nucleotide(s) bound to the oligomeric forms responsible for substrate remodeling are unknown. We demonstrate that ADP stimulates the intrinsic ATPase activity of PspF1-275 and propose that this heterogeneous nucleotide occupancy in a PspF1-275 hexamer is functionally important for specific activity. Binding of ADP and ATP triggers the formation of functional PspF1-275 hexamers as shown by a gain of specific activity. Furthermore, ATP concentrations congruent with stoichiometric ATP binding to PspF1-275 inhibit ATP hydrolysis and Esigma54-promoter open complex formation. Demonstration of a heterogeneous nucleotide-bound state of a functional PspF1-275.Esigma54 complex provides clear biochemical evidence for heterogeneous nucleotide occupancy in this AAA+ protein. Based on our data, we propose a stochastic nucleotide binding and a coordinated hydrolysis mechanism in PspF1-275 hexamers.

  20. 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

  1. 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

  2. Selective Nucleoside Triphosphate Diphosphohydrolase-2 (NTPDase2) Inhibitors: Nucleotide Mimetics Derived from Uridine-5′-carboxamide†

    PubMed Central

    Brunschweiger, Andreas; Iqbal, Jamshed; Umbach, Frank; Scheiff, Anja B.; Munkonda, Mercedes N.; Sévigny, Jean; Knowles, Aileen F.; Müller, Christa E

    2016-01-01

    Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases) dephosphorylate nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present study we synthesized adenine and uracil nucleotide mimetics, in which the phosphate residues were replaced by phosphonic acid esters attached to the nucleoside at the 5′-position by amide linkers. Among the synthesized uridine derivatives, we identified the first potent and selective inhibitors of human NTPDase2. The most potent compound was 19a (PSB-6426), which was a competitive inhibitor of NTPDase2 exhibiting a Ki value of 8.2 μM and selectivity versus other NTPDases. It was inactive toward uracil nucleotide-activated P2Y2, P2Y4, and P2Y6 receptor subtypes. Compound 19a was chemically and metabolically highly stable. In contrast to the few known (unselective) NTPDase inhibitors, 19a is an uncharged molecule and may be perorally bioavailable. NTPDase2 inhibitors have potential as novel cardioprotective drugs for the treatment of stroke and for cancer therapy. PMID:18630897

  3. Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection

    PubMed Central

    Morrow, K. A.; Seifert, R.; Kaever, V.; Britain, A. L.; Sayner, S. L.; Ochoa, C. D.; Cioffi, E. A.; Frank, D. W.; Rich, T. C.

    2015-01-01

    Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY+) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular (PMVECs) endothelial cells both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP ≈ cGMP ≈ cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY+ intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h, when the experiment was terminated. cGMP concentrations increased within 1 h postinfection, whereas cAMP and cUMP concentrations increased within 3 h, and cCMP concentrations increased within 4 h postinfection. In PMVECs, intercellular gaps did not form until 4 h postinfection. Only cGMP and cUMP concentrations increased at 3 and 6 h postinfection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY+ intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation. PMID:26386118

  4. The contribution of adenines in the catalytic core of 10-23 DNAzyme improved by the 6-amino group modifications.

    PubMed

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

    2016-09-15

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

  5. Synthesis of FinP RNA by plasmids F and pSLT is regulated by DNA adenine methylation.

    PubMed Central

    Torreblanca, J; Marqués, S; Casadesús, J

    1999-01-01

    DNA adenine methylase mutants of Salmonella typhimurium contain reduced amounts of FinP, an antisense RNA encoded by the virulence plasmid pSLT. Lowered FinP levels are detected in both Dam- FinO+ and Dam- FinO- backgrounds, suggesting that Dam methylation regulates FinP production rather than FinP half-life. Reduced amounts of F-encoded FinP RNA are likewise found in Dam- mutants of Escherichia coli. A consequence of FinP RNA scarcity in the absence of DNA adenine methylation is that Dam- mutants of both S. typhimurium and E. coli show elevated levels of F plasmid transfer. Inhibition of F fertility by the S. typhimurium virulence plasmid is also impaired in a Dam- background. PMID:10408954

  6. Ultrafast deactivation processes in the 2-aminopyridine dimer and the adenine-thymine base pair: Similarities and differences

    SciTech Connect

    Ai Yuejie; Zhang Feng; Cui Ganglong; Fang Weihai; Luo Yi

    2010-08-14

    2-aminopyridine dimer has frequently been used as a model system for studying photochemistry of DNA base pairs. We examine here the relevance of 2-aminopyridine dimer for a Watson-Crick adenine-thymine base pair by studying UV-light induced photodynamics along two main hydrogen bridges after the excitation to the localized {sup 1}{pi}{pi}* excited-state. The respective two-dimensional potential-energy surfaces have been determined by time-dependent density functional theory with Coulomb-attenuated hybrid exchange-correlation functional (CAM-B3LYP). Different mechanistic aspects of the deactivation pathway have been analyzed and compared in detail for both systems, while the related reaction rates have also be obtained from Monte Carlo kinetic simulations. The limitations of the 2-aminopyridine dimer as a model system for the adenine-thymine base pair are discussed.

  7. Synthesis of metal-adeninate frameworks with high separation capacity on C{sub 2}/C{sub 1} hydrocarbons

    SciTech Connect

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

    2016-06-15

    By introducing isophthalic acid or 2,5-thiophenedicarboxylic acid to assemble with adenine and cadmium salt, two isostructural and anionic porous metal-organic frameworks (1 and 2) possessing the novel (4,8)-connected sqc topology are presented here. 1 shows permanent porosity with Langmuir surface area of 770.1 m{sup 2}/g and exhibits high separation capacity on C{sub 2}/C{sub 1} hydrocarbons. - Graphical abstract: The assembly between isophthalic acid, adenine ligands and Cd{sup 2+} ions leads to an anionic porous metal-organic frameworks, which shows permanent porosity and exhibits high C{sub 2}/C{sub 1} hydrocarbons separation capacity. Display Omitted.

  8. Ultrafast deactivation processes in the 2-aminopyridine dimer and the adenine-thymine base pair: Similarities and differences

    NASA Astrophysics Data System (ADS)

    Ai, Yue-Jie; Zhang, Feng; Cui, Gang-Long; Luo, Yi; Fang, Wei-Hai

    2010-08-01

    2-aminopyridine dimer has frequently been used as a model system for studying photochemistry of DNA base pairs. We examine here the relevance of 2-aminopyridine dimer for a Watson-Crick adenine-thymine base pair by studying UV-light induced photodynamics along two main hydrogen bridges after the excitation to the localized π1π∗ excited-state. The respective two-dimensional potential-energy surfaces have been determined by time-dependent density functional theory with Coulomb-attenuated hybrid exchange-correlation functional (CAM-B3LYP). Different mechanistic aspects of the deactivation pathway have been analyzed and compared in detail for both systems, while the related reaction rates have also be obtained from Monte Carlo kinetic simulations. The limitations of the 2-aminopyridine dimer as a model system for the adenine-thymine base pair are discussed.

  9. 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).

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

    PubMed

    Sasaki, Yo; Araki, Toshiyuki; Milbrandt, Jeffrey

    2006-08-16

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

  11. 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.

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

    PubMed Central

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

    1997-01-01

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

  13. Evaluation of adenine as scaffold for the development of novel P2X3 receptor antagonists.

    PubMed

    Lambertucci, Catia; Sundukova, Mayya; Kachare, Dhuldeo D; Panmand, Deepak S; Dal Ben, Diego; Buccioni, Michela; Marucci, Gabriella; Marchenkova, Anna; Thomas, Ajiroghene; Nistri, Andrea; Cristalli, Gloria; Volpini, Rosaria

    2013-07-01

    Ligands that selectively block P2X3 receptors localized on nociceptive sensory fibres may be useful for the treatment of chronic pain conditions including neuropathic pain, migraine, and inflammatory pain. With the aim at exploring the suitability of adenine moiety as a scaffold for the development of antagonists of this receptor, a series of 9-benzyl-2-aminoadenine derivatives were designed and synthesized. These new compounds were functionally evaluated at rat or human P2X3 receptors expressed in human embryonic kidney (HEK) cells and on native P2X3 receptors from mouse trigeminal ganglion sensory neurons using patch clamp recording under voltage clamp configuration. The new molecules behaved as P2X3 antagonists, as they rapidly and reversibly inhibited (IC50 in the low micromolar range) the membrane currents induced via P2X3 receptor activation by the full agonist α,β-methyleneATP. Introduction of a small lipophilic methyl substituent at the 6-amino group enhanced the activity, in comparison to the corresponding unsubstituted derivative, resulting in the 9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N(6)-methyl-9H-purine-2,6-diamine (24), which appears to be a good antagonist on recombinant and native P2X3 receptors with IC50 = 1.74 ± 0.21 μM.

  14. DNA methyltransferase detection based on digestion triggering the combination of poly adenine DNA with gold nanoparticles.

    PubMed

    Liu, Pei; Wang, Dandan; Zhou, Yunlei; Wang, Haiyan; Yin, Huanshun; Ai, Shiyun

    2016-06-15

    DNA methyltransferase (MTase) has received a large amount of attention due to its catalyzation of DNA methylation in both eukaryotes and prokaryotes, which has a close relationship to cancer and bacterial diseases. Herein, a novel electrochemical strategy based on Dpn I digestion triggering the combination of poly adenine (polyA) DNA with a gold nanoparticles functioned glassy carbon electrode (AuNPs/GCE), is developed for the simple and efficient detection of DNA MTase and inhibitor screening. Only one methylene blue (MB)-labeled DNA hairpin probe and two enzymes are involved in this designed method. In the presence of Dam MTase, the hairpin probe can be methylated and then cleaved by the restriction endonuclease. Thus, a MB-labeled polyA signal-stranded DNA product is introduced to the surface of AuNPs/GCE through the effect between polyA and AuNPs, resulting in an obvious electrochemical signal. On the contrary, in the absence of Dam MTase, the DNA probe cannot be cleaved and a relatively small electrochemical response can be observed. As a result, the as-proposed biosensor offered an efficient way for Dam MTase activity monitoring with a low detection of 0.27 U/mL, a wide linear range and good stability. Additionally, this assay holds great potential for further application in real biological matrices and inhibitors screening, which is expected to be useful in disease diagnosis and drug discovery.

  15. 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.

  16. Excess electron trapping in duplex DNA: long range transfer via stacked adenines.

    PubMed

    Black, Paul J; Bernhard, William A

    2012-11-08

    An understanding of charge transfer (CT) in DNA lies at the root of assessing the risks and benefits of exposure to ionizing radiation. Energy deposition by high-energy photons and fast-charged particles creates holes and excess electrons (EEs) in DNA, and the subsequent reactions determine the complexity of DNA damage and ultimately the risk of disease. Further interest in CT comes from the possibility that hole transfer, excess electron transfer (EET), or both in DNA might be used to develop nanoscale circuits. To study EET in DNA, EPR spectroscopy was used to determine the distribution of EE trapping by oligodeoxynucleotides irradiated and observed at 4 K. Our results indicate that stretches of consecutive adenine bases on the same strand serve as an ideal conduit for intrastrand EET in duplex DNA at 4 K. Specifically, we show that A is an efficient trap for EE at 4 K if, and only if, the A strand of the duplex does not contain one of the other three bases. If there is a T, C, or G on the A strand, then trapping occurs at T or C instead of A. This holds true for stretches up to 32 A's. Whereas T competes effectively against A for the EE, it does not compete effectively against C. Long stretches of T pass the majority of EE to C. Our results show that AT stretches channel EE to cytosine, an end point with significance to both radiation damage and the photochemical repair of pyrimidine dimers.

  17. Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD

    PubMed Central

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

    2015-01-01

    The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis–mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment. PMID:25525179

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

    PubMed Central

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

    2016-01-01

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

  19. DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium▿

    PubMed Central

    Balbontín, Roberto; Rowley, Gary; Pucciarelli, M. Graciela; López-Garrido, Javier; Wormstone, Yvette; Lucchini, Sacha; García-del Portillo, Francisco; Hinton, Jay C. D.; Casadesús, Josep

    2006-01-01

    Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression. PMID:16997949

  20. Salmonella DNA Adenine Methylase Mutants Elicit Protective Immune Responses to Homologous and Heterologous Serovars in Chickens

    PubMed Central

    Dueger, E. L.; House, J. K.; Heithoff, D. M.; Mahan, M. J.

    2001-01-01

    Salmonella DNA adenine methylase (Dam) mutants that lack or overproduce Dam are highly attenuated for virulence in mice and confer protection against murine typhoid fever. To determine whether vaccines based on Dam are efficacious in poultry, a Salmonella Dam− vaccine was evaluated in the protection of chicken broilers against oral challenge with homologous and heterologous Salmonella serovars. A Salmonella enterica serovar Typhimurium Dam− vaccine strain was attenuated for virulence in day-of-hatch chicks more than 100,000-fold. Vaccination of chicks elicited cross-protective immune responses, as evidenced by reduced colonization (10- to 10,000-fold) of the gastrointestinal tract (ileum, cecum, and feces) and visceral organs (bursa and spleen) after challenge with homologous (Typhimurium F98) and heterologous (Enteritidis 4973 and S. enterica O6,14,24: e,h-monophasic) Salmonella serovars that are implicated in Salmonella infection of poultry. The protection conferred was observed for the organ or the maximum CFU/tissue/bird as a unit of analysis, suggesting that Dam mutant strains may serve as the basis for the development of efficacious poultry vaccines for the containment of Salmonella. PMID:11705984

  1. 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.

  2. 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

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

    PubMed

    Mericskay, Mathias

    2016-03-01

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

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

    PubMed Central

    Cahn, J.K.B.; Baumschlager, A.; Brinkmann-Chen, S.; Arnold, F.H.

    2016-01-01

    NAD(P)H-dependent enzymes are ubiquitous in metabolism and cellular processes and are also of great interest for pharmaceutical and industrial applications. Here, we present a structure-guided enzyme engineering strategy for improving catalytic properties of NAD(P)H-dependent enzymes toward native or native-like reactions using mutations to the enzyme's adenine-binding pocket, distal to the site of catalysis. Screening single-site saturation mutagenesis libraries identified mutations that increased catalytic efficiency up to 10-fold in 7 out of 10 enzymes. The enzymes improved in this study represent three different cofactor-binding folds (Rossmann, DHQS-like, and FAD/NAD binding) and utilize both NADH and NADPH. Structural and biochemical analyses show that the improved activities are accompanied by minimal changes in other properties (cooperativity, thermostability, pH optimum, uncoupling), and initial tests on two enzymes (ScADH6 and EcFucO) show improved functionality in Escherichia coli. PMID:26512129

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

    PubMed

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

    2015-06-01

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

  6. 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.

  7. Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

    PubMed

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

    1997-07-01

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

  8. Adenine methylation in eukaryotes: Apprehending the complex evolutionary history and functional potential of an epigenetic modification.

    PubMed

    Iyer, Lakshminarayan M; Zhang, Dapeng; Aravind, L

    2016-01-01

    While N(6) -methyladenosine (m(6) A) is a well-known epigenetic modification in bacterial DNA, it remained largely unstudied in eukaryotes. Recent studies have brought to fore its potential epigenetic role across diverse eukaryotes with biological consequences, which are distinct and possibly even opposite to the well-studied 5-methylcytosine mark. Adenine methyltransferases appear to have been independently acquired by eukaryotes on at least 13 occasions from prokaryotic restriction-modification and counter-restriction systems. On at least four to five instances, these methyltransferases were recruited as RNA methylases. Thus, m(6) A marks in eukaryotic DNA and RNA might be more widespread and diversified than previously believed. Several m(6) A-binding protein domains from prokaryotes were also acquired by eukaryotes, facilitating prediction of potential readers for these marks. Further, multiple lineages of the AlkB family of dioxygenases have been recruited as m(6) A demethylases. Although members of the TET/JBP family of dioxygenases have also been suggested to be m(6) A demethylases, this proposal needs more careful evaluation. Also watch the Video Abstract.

  9. Laser pulse trains for controlling excited state dynamics of adenine in water.

    PubMed

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

    2012-04-14

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

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

    PubMed

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

    2014-09-01

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

  11. Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD.

    PubMed

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

    2015-08-01

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

  12. Cellular localization of adenine receptors in the rat kidney and their functional significance in the inner medullary collecting duct

    PubMed Central

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

    2013-01-01

    The Gi-coupled adenine receptor (AdeR) binds adenine with high affinity and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in the renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex and outer medulla compared with the inner medulla. Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid COOH-terminal sequence of rat AdeR, which we generated, detected two bands between ∼30 and 40 kDa (molecular mass of native protein: 37 kDa) in the cortex, outer medulla, and inner medulla. These bands were ablated by preadsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature, including the glomerular arterioles, and less intense labeling in the cells of the collecting duct system. Confocal immunofluorescence imaging colocalized AdeR with aquaporin-2 protein to the apical plasma membrane in the collecting duct. Functionally, adenine (10 μM) significantly decreased (P < 0.01) 1-deamino-8-d-arginine vasopressin (10 nM)-induced cAMP production in ex vivo preparations of inner medullary collecting ducts, which was reversed by PSB-08162 (20 μM, P < 0.01), a selective antagonist of AdeR. Thus, we demonstrated the expression of AdeR in the renal vasculature and collecting ducts and its functional relevance. This study may open a new avenue for the exploration of autocrine/paracrine regulation of renal vascular and tubular functions by the nucleobase adenine in health and disease. PMID:23986514

  13. Cellular localization of adenine receptors in the rat kidney and their functional significance in the inner medullary collecting duct.

    PubMed

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

    2013-11-01

    The Gi-coupled adenine receptor (AdeR) binds adenine with high affinity and potentially reduces cellular cAMP levels. Since cAMP is an important second messenger in the renal transport of water and solutes, we localized AdeR in the rat kidney. Real-time RT-PCR showed higher relative expression of AdeR mRNA in the cortex and outer medulla compared with the inner medulla. Immunoblots using a peptide-derived and affinity-purified rabbit polyclonal antibody specific for an 18-amino acid COOH-terminal sequence of rat AdeR, which we generated, detected two bands between ∼30 and 40 kDa (molecular mass of native protein: 37 kDa) in the cortex, outer medulla, and inner medulla. These bands were ablated by preadsorption of the antibody with the immunizing peptide. Immunofluorescence labeling showed expression of AdeR protein in all regions of the kidney. Immunoperoxidase revealed strong labeling of AdeR protein in the cortical vasculature, including the glomerular arterioles, and less intense labeling in the cells of the collecting duct system. Confocal immunofluorescence imaging colocalized AdeR with aquaporin-2 protein to the apica