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Sample records for dihydroxypropoxymethyl guanine dhpg

  1. Plasma dihydroxyphenylglycol (DHPG) as an index of diabetic autonomic neuropathy

    DEFF Research Database (Denmark)

    Christensen, N J; Dejgaard, Anders; Hilsted, J

    1988-01-01

    Forearm venous plasma noradrenalin and dihydroxyphenylglycol (DHPG) concentrations were measured in eight diabetic patients with and eight diabetic patients without neuropathy. Plasma noradrenalin was on average the same in patients with and without neuropathy and correlated to serum creatinine...

  2. DHPG Activation of Group 1 mGluRs in BLA Enhances Fear Conditioning

    Science.gov (United States)

    Rudy, Jerry W.; Matus-Amat, Patricia

    2009-01-01

    Group 1 metabotropic glutamate receptors are known to play an important role in both synaptic plasticity and memory. We show that activating these receptors prior to fear conditioning by infusing the group 1 mGluR agonist, (R.S.)-3,5-dihydroxyphenylglycine (DHPG), into the basolateral region of the amygdala (BLA) of adult Sprague-Dawley rats…

  3. Unique pentafluorobenzylation and collision-induced dissociation for specific and accurate GC-MS/MS quantification of the catecholamine metabolite 3,4-dihydroxyphenylglycol (DHPG) in human urine.

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    Zoerner, Alexander A; Heusser, Karsten; Gutzki, Frank M; Mitschke, Anja; Tank, Jens; Stichtenoth, Dirk O; Jordan, Jens; Tsikas, Dimitrios

    2011-05-15

    In the human body, the catecholamine norepinephrine is mainly metabolized to 3,4-dihydroxyphenylglycol (DHPG) which therefore serves as an important biomarker for norepinephrine's metabolism. Most data on DHPG concentrations in human plasma and urine has been generated by using HPLC-ECD or GC-MS technologies. Here, we describe a stable-isotope dilution GC-MS/MS method for the quantitative determination of DHPG in human urine using trideutero-DHPG (d(3)-DHPG) as internal standard and a two-step derivatization process with pentafluorobenzyl bromide (PFB-Br) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). Two pentafluorobenzyl (PFB) trimethylsilyl (TMS) derivatives were obtained and identified, i.e., two isomeric DHPG-PFB-(TMS)(3) derivatives and the later eluting DHPG-tetrafluorobenzyl-(TMS)(2) derivative, i.e., DHPG-TFB-(TMS)(2). To our knowledge the DHPG-TFB-(TMS)(2) derivative and the underlying reaction have not been reported previously. In this reaction both vicinal aromatic hydroxyl groups of DHPG react with PFB-Br to form a heterocyclic seven-membered [1,4]dioxepin compound. The DHPG-TFB-(TMS)(2) derivative was used for quantitative GC-MS/MS analysis in the electron-capturing negative-ion chemical ionization mode by selected-reaction monitoring of m/z 351 from m/z 401 for DHPG and of m/z 352 from m/z 404 for d(3)-DHPG. Validation experiments on human urine samples spiked with DHPG in a narrow (0-33 nM) and a wide range (0-901 nM) revealed high recovery (86-104%) and low imprecision (RSD; 0.01-2.8%). LOD and relative LLOQ (rLLOQ) values of the method for DHPG were determined to be 76 amol and 9.4%, respectively. In urine of 28 patients suffering from chronic inflammatory rheumatic diseases, DHPG was measured at a mean concentration of 238 nM (38.3 μg/g creatinine). The DHPG concentration in the respective control group of 40 healthy subjects was measured to be 328 nM (39.2 μg/g creatinine). Given the unique derivatization reaction and collision

  4. Synthesis of Lipophilic Guanine N-9 Derivatives

    DEFF Research Database (Denmark)

    Wamberg, Michael C; Pedersen, Pernille L; Löffler, Philipp M G

    2017-01-01

    the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid...

  5. 21 CFR 73.1329 - Guanine.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 1 2010-04-01 2010-04-01 false Guanine. 73.1329 Section 73.1329 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR... vary from 3 to 25 percent, depending on the particular fish and tissue from which the crystals are...

  6. Topoisomerase IB of Deinococcus radiodurans resolves guanine ...

    Indian Academy of Sciences (India)

    2015-11-28

    Nov 28, 2015 ... [Kota S and Misra HS 2015 Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro. J. Biosci. 40 833–843] ... known for its efficient DNA double strand break repair. (Zahradka et al. ..... These samples were analysed on 12% native PAGE in KCl buffer (a). For CD ...

  7. Neurodevelopmental disabilities in children with intermediate and premutation range fragile X cytosine-guanine-guanine expansions.

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    Renda, Meredith M; Voigt, Robert G; Babovic-Vuksanovic, Dusica; Highsmith, W Edward; Vinson, Sherry S; Sadowski, Christine M; Hagerman, Randi J

    2014-03-01

    To determine the range of neurodevelopmental diagnoses associated with intermediate (45-54 repeats) and premutation (55-200 repeats) range cytosine-guanine-guanine fragile X expansions, the medical records of children with intermediate or premutation range expansions were retrospectively reviewed, and all neurodevelopmental diagnoses were abstracted. Twenty-nine children (9 female, 20 male; age, 13 months to 17 years) with intermediate (n = 25) or premutation (n = 4) range expansions were identified with neurodevelopmental diagnoses, including global developmental delay/intellectual disability (n = 15), language and learning disorders (n = 9), attention-deficit hyperactivity disorder (n = 5), epilepsy (n = 5), and motor disorders (n = 12), including 2 boys younger than 4 years of age with tremor and ataxia. Thus, children with intermediate or premutation range fragile X cytosine-guanine-guanine expansions may be more susceptible than children without such expansions to other processes, both genetic and environmental, that contribute to neurodevelopmental disability.

  8. Calculation of the Stabilization Energies of Oxidatively Damaged Guanine Base Pairs with Guanine

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    Hiroshi Miyazawa

    2012-06-01

    Full Text Available DNA is constantly exposed to endogenous and exogenous oxidative stresses. Damaged DNA can cause mutations, which may increase the risk of developing cancer and other diseases. G:C-C:G transversions are caused by various oxidative stresses. 2,2,4-Triamino-5(2H-oxazolone (Oz, guanidinohydantoin (Gh/iminoallantoin (Ia and spiro-imino-dihydantoin (Sp are known products of oxidative guanine damage. These damaged bases can base pair with guanine and cause G:C-C:G transversions. In this study, the stabilization energies of these bases paired with guanine were calculated in vacuo and in water. The calculated stabilization energies of the Ia:G base pairs were similar to that of the native C:G base pair, and both bases pairs have three hydrogen bonds. By contrast, the calculated stabilization energies of Gh:G, which form two hydrogen bonds, were lower than the Ia:G base pairs, suggesting that the stabilization energy depends on the number of hydrogen bonds. In addition, the Sp:G base pairs were less stable than the Ia:G base pairs. Furthermore, calculations showed that the Oz:G base pairs were less stable than the Ia:G, Gh:G and Sp:G base pairs, even though experimental results showed that incorporation of guanine opposite Oz is more efficient than that opposite Gh/Ia and Sp.

  9. Synthesis and antiviral activity of some C2-, C4-, and C6-substituted pyrazolo[3,4-D]pyrimidine acyclonucleosides with the alkylating chains of ACV, HBG, and ISO-DHPG.

    Science.gov (United States)

    Moukha-Chafiq, Omar; Taha, Mohamed Labd; Lazrek, Hassan Bihi; Vasseur, Jean-Jacques; Clercq, Erik De

    2006-01-01

    A useful route to obtain trisubstituted pyrazolo[3,4-d]pyrimidines 14-17 is described. Those later were coupled with the alkylating agents 18-20 as in ACV, HBG, and iso-DHPG to give, after deprotection, the desired acylonucleosides 33-44. Almost all of the new compounds were evaluated for their inhibitory effects against the replication of various DNA viruses in culture.

  10. Identification, expression, and characterization of Escherichia coli guanine deaminase.

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    Maynes, J T; Yuan, R G; Snyder, F F

    2000-08-01

    Using the human cDNA sequence corresponding to guanine deaminase, the Escherichia coli genome was scanned using the Basic Local Alignment Search Tool (BLAST), and a corresponding 439-residue open reading frame of unknown function was identified as having 36% identity to the human protein. The putative gene was amplified, subcloned into the pMAL-c2 vector, expressed, purified, and characterized enzymatically. The 50.2-kDa protein catalyzed the conversion of guanine to xanthine, having a K(m) of 15 microM with guanine and a k(cat) of 3.2 s(-1). The bacterial enzyme shares a nine-residue heavy metal binding site with human guanine deaminase, PG[FL]VDTHIH, and was found to contain approximately 1 mol of zinc per mol of subunit of protein. The E. coli guanine deaminase locus is 3' from an open reading frame which shows homology to a bacterial purine base permease.

  11. Guanine quadruplex structures localize to heterochromatin.

    Science.gov (United States)

    Hoffmann, Roland F; Moshkin, Yuri M; Mouton, Stijn; Grzeschik, Nicola A; Kalicharan, Ruby D; Kuipers, Jeroen; Wolters, Anouk H G; Nishida, Kazuki; Romashchenko, Aleksander V; Postberg, Jan; Lipps, Hans; Berezikov, Eugene; Sibon, Ody C M; Giepmans, Ben N G; Lansdorp, Peter M

    2016-01-08

    Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. [Triplet expansion cytosine-guanine-guanine: Three cases of OMIM syndrome in the same family].

    Science.gov (United States)

    González-Pérez, Jesús; Izquierdo-Álvarez, Silvia; Fuertes-Rodrigo, Cristina; Monge-Galindo, Lorena; Peña-Segura, José Luis; López-Pisón, Francisco Javier

    2016-04-01

    The dynamic increase in the number of triplet repeats of cytosine-guanine-guanine (CGG) in the FMR1 gene mutation is responsible for three OMIM syndromes with a distinct clinical phenotype: Fragile X syndrome (FXS) and two pathologies in adult carriers of the premutation (55-200 CGG repeats): Primary ovarian insufficiency (FXPOI) and tremor-ataxia syndrome (FXTAS) associated with FXS. CGG mutation dynamics of the FMR1 gene were studied in DNA samples from peripheral blood from the index case and other relatives of first, second and third degree by TP-PCR, and the percentage methylation. Diagnosis of FXS was confirmed in three patients (21.4%), eight patients (57.1%) were confirmed in the premutation range transmitters, one male patient with full mutation/permutation mosaicism (7.1%) and two patients (14.3%) with normal study. Of the eight permutated patients, three had FXPOI and one male patient had FXTAS. Our study suggests the importance of making an early diagnosis of SXF in order to carry out a family study and genetic counselling, which allow the identification of new cases or premutated patients with FMR1 gene- associated syndromes (FXTAS, FXPOI). Copyright © 2015 Elsevier España, S.L.U. All rights reserved.

  13. Characterization of oxidative guanine damage and repair in mammalian telomeres.

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    Zhilong Wang

    2010-05-01

    Full Text Available 8-oxo-7,8-dihydroguanine (8-oxoG and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG are among the most common oxidative DNA lesions and are substrates for 8-oxoguanine DNA glycosylase (OGG1-initiated DNA base excision repair (BER. Mammalian telomeres consist of triple guanine repeats and are subject to oxidative guanine damage. Here, we investigated the impact of oxidative guanine damage and its repair by OGG1 on telomere integrity in mice. The mouse cells were analyzed for telomere integrity by telomere quantitative fluorescence in situ hybridization (telomere-FISH, by chromosome orientation-FISH (CO-FISH, and by indirect immunofluorescence in combination with telomere-FISH and for oxidative base lesions by Fpg-incision/Southern blot assay. In comparison to the wild type, telomere lengthening was observed in Ogg1 null (Ogg1(-/- mouse tissues and primary embryonic fibroblasts (MEFs cultivated in hypoxia condition (3% oxygen, whereas telomere shortening was detected in Ogg1(-/- mouse hematopoietic cells and primary MEFs cultivated in normoxia condition (20% oxygen or in the presence of an oxidant. In addition, telomere length abnormalities were accompanied by altered telomere sister chromatid exchanges, increased telomere single- and double-strand breaks, and preferential telomere lagging- or G-strand losses in Ogg1(-/- mouse cells. Oxidative guanine lesions were increased in telomeres in Ogg1(-/- mice with aging and primary MEFs cultivated in 20% oxygen. Furthermore, oxidative guanine lesions persisted at high level in Ogg1(-/- MEFs after acute exposure to hydrogen peroxide, while they rapidly returned to basal level in wild-type MEFs. These findings indicate that oxidative guanine damage can arise in telomeres where it affects length homeostasis, recombination, DNA replication, and DNA breakage repair. Our studies demonstrate that BER pathway is required in repairing oxidative guanine damage in telomeres and maintaining telomere integrity

  14. Spatiotemporal regulation of Rap guanine nucleotide exchange factors

    NARCIS (Netherlands)

    Consonni, S.V.

    2014-01-01

    Guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) orchestrate the activity of small G-proteins. In response to extracellular stimuli, GEFs and GAPs activate signaling cascades regulated by G-proteins by controlling their regulation in time and in space. Generally, GEFs

  15. Endogenous melatonin and oxidatively damaged guanine in DNA

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    Poulsen Henrik E

    2009-10-01

    Full Text Available Abstract Background A significant body of literature indicates that melatonin, a hormone primarily produced nocturnally by the pineal gland, is an important scavenger of hydroxyl radicals and other reactive oxygen species. Melatonin may also lower the rate of DNA base damage resulting from hydroxyl radical attack and increase the rate of repair of that damage. This paper reports the results of a study relating the level of overnight melatonin production to the overnight excretion of the two primary urinary metabolites of the repair of oxidatively damaged guanine in DNA. Methods Mother-father-daughter(s families (n = 55 were recruited and provided complete overnight urine samples. Total overnight creatinine-adjusted 6-sulphatoxymelatonin (aMT6s/Cr has been shown to be highly correlated with total overnight melatonin production. Urinary 8-oxo-7,8-dihydro-guanine (8-oxoGua results from the repair of DNA or RNA guanine via the nucleobase excision repair pathway, while urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG may possibly result from the repair of DNA guanine via the nucleotide excision repair pathway. Total overnight urinary levels of 8-oxodG and 8-oxoGua are therefore a measure of total overnight guanine DNA damage. 8-oxodG and 8-oxoGua were measured using a high-performance liquid chromatography-electrospray ionization tandem mass spectrometry assay. The mother, father, and oldest sampled daughter were used for these analyses. Comparisons between the mothers, fathers, and daughters were calculated for aMT6s/Cr, 8-oxodG, and 8-oxoGua. Regression analyses of 8-oxodG and 8-oxoGua on aMT6s/Cr were conducted for mothers, fathers, and daughters separately, adjusting for age and BMI (or weight. Results Among the mothers, age range 42-80, lower melatonin production (as measured by aMT6s/CR was associated with significantly higher levels of 8-oxodG (p Conclusion Low levels of endogenous melatonin production among older individuals may lead to

  16. Guanine base stacking in G-quadruplex nucleic acids

    Science.gov (United States)

    Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2013-01-01

    G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5′–5′ manner based on different accessible tetrad stacking modes at the stacking interfaces of 5′–5′ and 3′–3′ stacked G-quadruplexes. PMID:23268444

  17. Quantum molecular modeling of the interaction between guanine and alkylating agents--2--nitrogen mustard.

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    Hamza, A; Broch, H; Vasilescu, D

    1996-06-01

    The alkylation mechanism of guanine by nitrogen mustard (HN2) was studied by using a supermolecular modeling at the ab initio 6-31G level. Our computations show that interaction of guanine with the aziridinium form of HN2 necessitates a transition state for the N7 alkylation route. The pathway of N7-guanine alkylation by nitrogen and sulfur mustards is discussed on the basis of the Molecular Electrostatic Potential and HOMO-LUMO properties of these molecules.

  18. Interactions of fluoride and guanine nucleotides with thyroid adenylate cyclase.

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    Goldhammer, A; Wolff, J

    1982-02-18

    The activation of bovine thyroid adenylate cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) by Gpp(NH)p has been studied using steady-state kinetic methods. This activation is complex and may be characterized by two Gpp(NH)p binding sites of different affinities with measured constants: Ka1 = 0.1 micro M and Ka2 = 2.9 micro M. GDP beta S does not completely inhibit the Gpp(NH)p activation: analysis of the data is consistent with a single GDP beta S inhibitory site which is competitive with the weaker Gpp(NH)p site. Guanine nucleotide effects upon F- activation of adenylate cyclase have been studied. When App(NH)p is the substrate, 10 micro M GTP along with 10 mM NaF gives higher activity than NaF alone, while GDP together with NaF inhibits the activity by 50% relative to NaF. These features are not observed when the complex is assayed with ATP in the presence of a nucleotide regenerating system or when analogs Gpp)NH)p or GDP beta S are used along with NaF. These effects were studied in three other membrane systems using App(NH)p as substrate: rat liver, rat ovary and turkey erythrocyte. No consistent pattern of guanine nucleotide effects upon fluoride activation could be observed in the different membrane preparations. Previous experiments showed that the size of soluble thyroid adenylate cyclase changed whether membranes were preincubated with Gpp(NH)p or NaF. This size change roughly corresponded to the molecular weight of the nucleotide regulatory protein. This finding, coupled with the present data, suggests that two guanine nucleotide binding sites may be involved in regulating thyroid cyclase and that these sites may be on different protein chains.

  19. Phosphodiester-mediated reaction of cisplatin with guanine in oligodeoxyribonucleotides.

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    Campbell, Meghan A; Miller, Paul S

    2008-12-02

    The cancer chemotherapeutic agent cis-diamminedichloroplatinum(II) or cisplatin reacts primarily with guanines in DNA to form 1,2-Pt-GG and 1,3-Pt-GNG intrastrand cross-links and, to a lesser extent, G-G interstrand cross-links. Recent NMR evidence has suggested that cisplatin can also form a coordination complex with the phosphodiester internucleotide linkage of DNA. We have examined the effects of the phosphodiester backbone on the reactions of cisplatin with oligodeoxyribonucleotides that lack or contain a GTG sequence. Cisplatin forms a stable adduct with TpT that can be isolated by reversed phase HPLC. The cis-Pt-TpT adduct contains a single Pt, as determined by atomic absorption spectroscopy (AAS) and by electrospray ionization mass spectrometry (ESI-MS), and is resistant to digestion by snake venom phosphodiesterase. Treatment of the adduct with sodium cyanide regenerates TpT. Similar adduct formation was observed when T(pT)(8) was treated with cisplatin, but not when the phosphodiester linkages of T(pT)(8) were replaced with methylphosphonate groups. These results suggest that the platinum may be coordinated with the oxygens of the thymine and possibly with those of the phosphodiester group. As expected, reaction of a 9-mer containing a GTG sequence with cisplatin yielded an adduct that contained a 1,3-Pt-GTG intrastrand cross-link. However, we found that the number and placement of phosphodiesters surrounding a GTG sequence significantly affected intrastrand cross-link formation. Increasing the number of negatively charged phosphodiesters in the oligonucleotide increased the amount of GTG platination. Surrounding the GTG sequence with nonionic methylphosphonate linkages inhibited or eliminated cross-link formation. These observations suggest that interactions between cisplatin and the negatively charged phosphodiester backbone may play an important role in facilitating platination of guanine nucleotides in DNA.

  20. Lifetimes and reaction pathways of guanine radical cations and neutral guanine radicals in an oligonucleotide in aqueous solutions.

    Science.gov (United States)

    Rokhlenko, Yekaterina; Geacintov, Nicholas E; Shafirovich, Vladimir

    2012-03-14

    The exposure of guanine in the oligonucleotide 5'-d(TCGCT) to one-electron oxidants leads initially to the formation of the guanine radical cation G(•+), its deptotonation product G(-H)(•), and, ultimately, various two- and four-electron oxidation products via pathways that depend on the oxidants and reaction conditions. We utilized single or successive multiple laser pulses (308 nm, 1 Hz rate) to generate the oxidants CO(3)(•-) and SO(4)(•-) (via the photolysis of S(2)O(8)(2-) in aqueous solutions in the presence and absence of bicarbonate, respectively) at concentrations/pulse that were ∼20-fold lower than the concentration of 5'-d(TCGCT). Time-resolved absorption spectroscopy measurements following single-pulse excitation show that the G(•+) radical (pK(a) = 3.9) can be observed only at low pH and is hydrated within 3 ms at pH 2.5, thus forming the two-electron oxidation product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single pulse excitation, the principal reactive intermediate is G(-H)(•), which, at best, reacts only slowly with H(2)O and lives for ∼70 ms in the absence of oxidants/other radicals to form base sequence-dependent intrastrand cross-links via the nucleophilic addition of N3-thymidine to C8-guanine (5'-G*CT* and 5'-T*CG*). Alternatively, G(-H)(•) can be oxidized further by reaction with CO(3)(•-), generating the two-electron oxidation products 8-oxoG (C8 addition) and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), appear only after a second (or more) laser pulse. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses.

  1. Application of Ammonium Persulfate for Selective Oxidation of Guanines for Nucleic Acid Sequencing

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    Yafen Wang

    2017-07-01

    Full Text Available Nucleic acids can be sequenced by a chemical procedure that partially damages the nucleotide positions at their base repetition. Many methods have been reported for the selective recognition of guanine. The accurate identification of guanine in both single and double regions of DNA and RNA remains a challenging task. Herein, we present a new, non-toxic and simple method for the selective recognition of guanine in both DNA and RNA sequences via ammonium persulfate modification. This strategy can be further successfully applied to the detection of 5-methylcytosine by using PCR.

  2. Quantum molecular modeling of the interaction between guanine and alkylating agents--1--sulfur mustard.

    Science.gov (United States)

    Broch, H; Hamza, A; Vasilescu, D

    1996-06-01

    Interaction between Guanine and the episulfonium form of Sulfur mustard (HD) was studied using the ab initio LCAO-MO method at the HF/6-31G level. The alkylation mechanism on guanine-N7 was analyzed by using a supermolecular modeling. Our stereostructural results associated with the molecular electrostatic potentials and HOMO-LUMO properties, show that in vacuum the alkylation of the N7 of guanine by HD in the aggressive episulfonium form is a direct process without transition state and of which the pathway is determined.

  3. Oligomeric state of hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Eng, Wai Soon; Keough, Dianne T; Hockova, Dana; Winzor, Donald J; Guddat, Luke W

    2017-04-01

    Sedimentation equilibrium and size-exclusion chromatography experiments on Mycobacterium tuberculosis hypoxanthine-guanine phosphoribosyltransferase (MtHGPRT) have established the existence of this enzyme as a reversibly associating mixture of dimeric and tetrameric species in 0.1 M Tris-HCl-0.012 M MgCl 2 , pH 7.4. Displacement of the equilibrium position towards the larger oligomer by phosphate signifies the probable existence of MtHGPRT as a tetramer in the biological environment. These data thus add credibility to the relevance of considering enzyme function in the light of a published tetrameric structure deduced from X-ray crystallography. Failure of 5-phospho-α-d-ribosyl-1-pyrophosphate (PRib-PP) to perturb the dimer-tetramer equilibrium position indicates the equivalence and independence of binding for this substrate (the first to bind in an ordered sequential mechanism) to the two oligomers. By virtue of the displacement of the equilibrium position towards dimer that is affected by removing MgCl 2 from the Tris-HCl buffer, it can be concluded that divalent metal ions, as well as phosphate, can affect the oligomerization. These characteristics of MtHGPRT in solution are correlated with published crystal structures of four enzyme-ligand complexes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  4. Chlamydial entry involves TARP binding of guanine nucleotide exchange factors.

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    B Josh Lane

    2008-03-01

    Full Text Available Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein. TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs, Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K, appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.

  5. Fluorescence enhancement of DNA-silver nanoclusters from guanine proximity

    Energy Technology Data Exchange (ETDEWEB)

    Yeh, Hsin-chih [Los Alamos National Laboratory; Sharma, Jaswinder [Los Alamos National Laboratory; Yoo, Hyojong [Los Alamos National Laboratory; Martinez, Jennifer S [Los Alamos National Laboratory

    2010-01-01

    Oligonucleotide-templated, silver nanoclusters (DNA/Ag NCs) are a versatile set of fluorophores and have already been used for live cell imaging, detection of specific metal ions, and single-nucleotide variation identification. Compared to commonly used organic dyes, these fluorescent nanoclusters have much better photostability and are often a few times brighter. Owing to their small size, simple preparation, and biocompatibility (i.e. made of nontoxic metals), DNA/Ag NCs should find more applications in biological imaging and chemical detection in the years to come. While clearly promising as new fluorophores, DNA/Ag NCs possess a unique and poorly understood dynamic process not shared by organic dyes or photoluminescent nanocrystals - the conversion among different NC species due to silver oxidation/reduction or NC regrouping. While this environmental sensitivity can be viewed as a drawback, in the appropriate context, it can be used as a sensor or reporter. Often reversible, conversions among different NC species have been found to depend upon a number of factors, including time, temperature, oxygen and salt content. In this communication, we report significant fluorescence enhancement of DNA/Ag NCs via interactions with guanine-rich DNA sequences. Moreover, we demonstrated this property can be used for sensitive detection of specific target DNA from a human oncogene (i.e. Braf gene).

  6. Purine nucleotide synthesis from exogenous adenine and guanine in rodent small intestine

    International Nuclear Information System (INIS)

    Gross, C.J.; Karlberg, P.K.; Savaiano, D.A.

    1986-01-01

    14 C-Adenine and 14 C-guanine uptake was studied in isolated guinea pig enterocytes. Cells were incubated in Hank's buffer and separated from the medium by centrifugation through silicone oil into 1M PCA. Uptake was temperature and concentration dependent. Both compounds were incorporated into nucleotides as measured by HPLC and HVE. Adenine was more extensively incorporated into nucleotides than was guanine. Adenine nucleotides accounted for about 70% of the intracellular label after 30 min with a majority being ADP and ATP (medium concentration = 10 μM). Guanine nucleotides accounted for only 30% of the intracellular label after 30 min. Labeled intracellular free adenine or guanine were not detected. Significantly more guanine vs. adenine was converted to uric acid. After 30 min, 11.5 +/- 3.9% (n=3) and 83.0 +/- 8.4% (n=4) of the label was present as uric acid in the medium when adenine and guanine, respectively, were the substrate. After 1 min, 34.8 +/- 3.4% (n=4) of the label in the medium was present as uric acid when guanine was the substrate. Decreasing the concentration of adenine resulted in an increase in the percent of uric acid in the medium. 14 C-adenine (75 nmol) was injected into 1 gm segments of rat jejunum. After 5 min., segments were quickly flushed and the tissue homogenized in 1M PCA. Only uric acid was present after 5 min (n=6). In contrast, in animals fasted 3 to 5 days, less conversion to uric acid was observed in the intestinal content (50-80% of the same dose was still present as adenine after 5 min) and nucleotide formation was observed in the tissue. The results indicate that uric acid and nucleotide synthesis from exogenous adenine and guanine are concentration dependent and affected by nutritional state

  7. Behavior of the guanine base in G-quadruplexes probed by the fluorescent guanine analog, 6-methyl isozanthopterin

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ji Hoon; Chitrapriya, Nataraj; Lee, Hyun Suk; Lee, Young Ae; Kim, Seog K. [Dept. of Chemistry, Yeungnam University, Gyeongsan (Korea, Republic of); Jung, Maeng Joon [Dept. of Chemistry, Kyungpook National University, Daegu (Korea, Republic of)

    2017-02-15

    In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G{sub 3}T{sub 2}G{sub 3}TGTG{sub 3}T{sub 2}G{sub 3} sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

  8. Hypoxanthine-guanine phosophoribosyltransferase (HPRT deficiency: Lesch-Nyhan syndrome

    Directory of Open Access Journals (Sweden)

    Puig Juan G

    2007-12-01

    Full Text Available Abstract Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1/380,000 live births in Canada, and 1/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated with lithiasis and gout. Neurological manifestations include severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit, and self-injurious behaviour. The most severe forms are known as Lesch-Nyhan syndrome (patients are normal at birth and diagnosis can be accomplished when psychomotor delay becomes apparent. Partial HPRT-deficient patients present these symptoms with a different intensity, and in the least severe forms symptoms may be unapparent. Megaloblastic anaemia is also associated with the disease. Inheritance of HPRT deficiency is X-linked recessive, thus males are generally affected and heterozygous female are carriers (usually asymptomatic. Human HPRT is encoded by a single structural gene on the long arm of the X chromosome at Xq26. To date, more than 300 disease-associated mutations in the HPRT1 gene have been identified. The diagnosis is based on clinical and biochemical findings (hyperuricemia and hyperuricosuria associated with psychomotor delay, and enzymatic (HPRT activity determination in haemolysate, intact erythrocytes or fibroblasts and molecular tests. Molecular diagnosis allows faster and more accurate carrier and prenatal diagnosis. Prenatal diagnosis can be performed with amniotic cells obtained by amniocentesis at about 15–18 weeks' gestation, or chorionic villus cells obtained at about 10–12 weeks' gestation. Uric acid overproduction can be managed by allopurinol treatment. Doses must be carefully adjusted to avoid xanthine lithiasis. The

  9. A DFT investigation on interactions between asymmetric derivatives of cisplatin and nucleobase guanine

    Science.gov (United States)

    Tai, Truong Ba; Nhat, Pham Vu

    2017-07-01

    The interactions of hydrolysis products of cisplatin and its asymmetric derivatives cis- and trans-[PtCl2(iPram)(Mepz)] with guanine were studied using DFT methods. These interactions are dominated by electrostatic effects, namely hydrogen bond contributions and there exists a charge flow from H-atoms of ligands to the O-atoms of guanine. The replacement of NH3 moieties by larger functional groups accompanies with a moderate reaction between PtII and guanine molecule, diminishing the cytotoxicity of the drug. The asymmetric and symmetric NH2 stretching modes of complexes having strong hydrogen bond interactions are red shifted importantly as compared to complexes without presence of hydrogen bond interactions.

  10. Synthesis of a Pseudodisaccharide α-C-Glycosidically Linked to an 8-Alkylated Guanine

    Directory of Open Access Journals (Sweden)

    Jan Duchek

    2013-04-01

    Full Text Available The synthesis of stable guanofosfocin analogues has attracted considerable attention in the past 15 years. Several guanofosfocin analogues mimicking the three constitutional elements of mannose, ribose, and guanine were designed and synthesized. Interest in ether-linked pseudodisaccharides and 8-alkylated guanines is increasing, due to their potential applications in life science. In this article, a novel guanofosfocin analogue 6, an ether-linked pseudodisaccharide connected α-C-glycosidically to an 8-alkylated guanine, was synthesized in a 10-longest linear step sequence from known diol 13, resulting in an overall yield of 26%. The key steps involve the ring-opening of cyclic sulfate 8 by alkoxide generated from 7 and a reductive cyclization of 4-N-acyl-2,4-diamino-5-nitrosopyrimidine 19 to form compound 6.

  11. Intramolecular and intermolecular guanine quadruplexes of DNA in aqueous salt and ethanol solutions

    Czech Academy of Sciences Publication Activity Database

    Vorlíčková, Michaela; Bednářová, Klára; Kejnovská, Iva; Kypr, Jaroslav

    2007-01-01

    Roč. 86, č. 1 (2007), s. 1-10 ISSN 0006-3525 Institutional research plan: CEZ:AV0Z50040702 Keywords : guanine quadruplex * G-tetrads * CD spectroscopy Subject RIV: BO - Biophysics Impact factor: 2.389, year: 2007

  12. Circular dichroism spectroscopy of conformers of (guanine + adenine) repeat strands of DNA

    Czech Academy of Sciences Publication Activity Database

    Kejnovská, Iva; Kypr, Jaroslav; Vorlíčková, Michaela

    2003-01-01

    Roč. 15, č. 7 (2003), s. 584-592 ISSN 0899-0042 R&D Projects: GA AV ČR IAA4004201; GA ČR GA204/01/0561 Institutional research plan: CEZ:AV0Z5004920 Keywords : DNA conformation * (guanine + adenine) repeats * homoduplexes Subject RIV: BO - Biophysics Impact factor: 1.793, year: 2003

  13. Ethanol is a better inducer of DNA guanine tetraplexes than potassium cations

    Czech Academy of Sciences Publication Activity Database

    Vorlíčková, Michaela; Bednářová, Klára; Kypr, Jaroslav

    2006-01-01

    Roč. 82, č. 3 (2006), s. 253-260 ISSN 0006-3525 R&D Projects: GA AV ČR(CZ) IAA4004201 Institutional research plan: CEZ:AV0Z50040507 Keywords : DNA * guanine tetraplexes * ethanol Subject RIV: BO - Biophysics Impact factor: 2.480, year: 2006

  14. Partial hypoxanthine-guanine phosphoribosyl transferase deficiency without elevated urinary hypoxanthine excretion

    NARCIS (Netherlands)

    van Dael, C. M. L.; Pierik, L. J. W. M.; Reijngoud, D. J.; Niezen-Koning, K. E.; van Diggelen, O. P.; van Spronsen, F. J.

    Partial hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency, also known as the Kelley-Seegmiller syndrome, can give rise to a wide range of neurological symptoms, and renal insufficiency. Biochemically, it is characterized by high uric acid concentrations in blood, high uric acid and

  15. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    International Nuclear Information System (INIS)

    Sakamoto, C.; Matozaki, T.; Nagao, M.; Baba, S.

    1987-01-01

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125 I-[Tyr 1 ]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg 2+ . When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125 I-[Tyr 1 ]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

  16. The Study of Adsorption of Patulin by Nanocellulose Conjugated with Poly Guanine in Contaminated Apple juice

    Directory of Open Access Journals (Sweden)

    M Ghafori Bidakhavidi

    2016-07-01

    Full Text Available Abstract Introdction: Patulin is a dangerous toxin produced by various fungi. Hence, the current study aimed to evaluate adsorption of Patulin by nanocellulose conjugated with Poly-guanine in contaminated apple juice. Methods: Firstly, nanocellulose was synthesized, and then it was bonded to poly-guanine by a cross-linker. Then, concentration serial of Patulin was prepared in the apple juice, conjugated nanoparticles were added to them, and all were incubated at 37 ºC. After incubation, the Patulin concentration was measured by HPLC, and finally the adsorption percentage was calculated for each tube. Regarding molecular simulation, the initial structures of Patulin and nanocellulose conjugated with Poly-guanine were inserted into Hyperchem software, and their intermolecular energy was calculated during 50 picoseconds. Results: The results of the present study demonstrated that there was a significant direct correlation between the initial concentration of Patulin and the adsorption percentage of toxin. In addition, the adsorption maximum was reported 70±5 %, and the intermolecular energy between two structures was -20.3 Kcal/mol based on the computational simulation. Conclusions: It can be concluded that nanocellulose conjugated with Poly-guanine seems to be a good adsorbent for Patulin, which is demanded to be used in the future studies in regard with its application.

  17. Scaffold-hopping from xanthines to tricyclic guanines: A case study of dipeptidyl peptidase 4 (DPP4) inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Pissarnitski, Dmitri A.; Zhao, Zhiqiang; Cole, David; Wu, Wen-Lian; Domalski, Martin; Clader, John W.; Scapin, Giovanna; Voigt, Johannes; Soriano, Aileen; Kelly, Theresa; Powles, Mary Ann; Yao, Zuliang; Burnett, Duane A. (Merck)

    2016-11-01

    Molecular modeling of unbound tricyclic guanine scaffolds indicated that they can serve as effective bioisosteric replacements of xanthines. This notion was further confirmed by a combination of X-ray crystallography and SAR studies, indicating that tricyclic guanine DPP4 inhibitors mimic the binding mode of xanthine inhibitors, exemplified by linagliptin. Realization of the bioisosteric relationship between these scaffolds potentially will lead to a wider application of cyclic guanines as xanthine replacements in drug discovery programs for a variety of biological targets. Newly designed DPP4 inhibitors achieved sub-nanomolar potency range and demonstrated oral activity in vivo in mouse glucose tolerance test.

  18. Guanine nucleotide binding to the Bateman domain mediates the allosteric inhibition of eukaryotic IMP dehydrogenases

    Science.gov (United States)

    Buey, Rubén M.; Ledesma-Amaro, Rodrigo; Velázquez-Campoy, Adrián; Balsera, Mónica; Chagoyen, Mónica; de Pereda, José M.; Revuelta, José L.

    2015-11-01

    Inosine-5'-monophosphate dehydrogenase (IMPDH) plays key roles in purine nucleotide metabolism and cell proliferation. Although IMPDH is a widely studied therapeutic target, there is limited information about its physiological regulation. Using Ashbya gossypii as a model, we describe the molecular mechanism and the structural basis for the allosteric regulation of IMPDH by guanine nucleotides. We report that GTP and GDP bind to the regulatory Bateman domain, inducing octamers with compromised catalytic activity. Our data suggest that eukaryotic and prokaryotic IMPDHs might have developed different regulatory mechanisms, with GTP/GDP inhibiting only eukaryotic IMPDHs. Interestingly, mutations associated with human retinopathies map into the guanine nucleotide-binding sites including a previously undescribed non-canonical site and disrupt allosteric inhibition. Together, our results shed light on the mechanisms of the allosteric regulation of enzymes mediated by Bateman domains and provide a molecular basis for certain retinopathies, opening the door to new therapeutic approaches.

  19. Benchmark Theoretical and Experimental Study on N-15 NMR Shifts of Oxidatively Damaged Guanine

    Czech Academy of Sciences Publication Activity Database

    Dračínský, Martin; Šála, Michal; Klepetářová, Blanka; Šebera, Jakub; Fukal, Jiří; Holečková, Veronika; Tanaka, Y.; Nencka, Radim; Sychrovský, Vladimír

    2016-01-01

    Roč. 120, č. 5 (2016), s. 915-925 ISSN 1520-6106 R&D Projects: GA ČR GA13-27676S; GA ČR GA15-11223S Institutional support: RVO:61388963 Keywords : NMR spectroscopy * DFT calculations * oxidatively damaged guanine * hOGG1 Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.177, year: 2016

  20. The Mid-IR spectra of 9-ethyl guanine, guanosine, and 2-deoxyguanosine

    Czech Academy of Sciences Publication Activity Database

    Abo-Riziq, A.; Crews, B. O.; Compagnon, I.; Oomens, J.; Meijer, G.; Von Helden, G.; Kabeláč, Martin; Hobza, Pavel; de Vries, M. S.

    2007-01-01

    Roč. 111, č. 31 (2007), s. 7529-7536 ISSN 1089-5639 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0009 Grant - others:NSF(US) CHE-0615401 Institutional research plan: CEZ:AV0Z40550506 Keywords : IR spectrum * guanine * deoxyguanosine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.918, year: 2007

  1. Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

    OpenAIRE

    Hall, James P.; Poynton, Fergus E.; Keane, Paraic M.; Gurung, Sarah P.; Brazier, John A.; Cardin, David J.; Winter, Graeme; Gunnlaugsson, Thorfinnur; Sazanovich, Igor V.; Towrie, Michael; Cardin, Christine J.; Kelly, John M.; Quinn, Susan J.

    2015-01-01

    To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ul...

  2. IR-UV double resonance spectroscopy of guanine-H2O clusters

    Czech Academy of Sciences Publication Activity Database

    Crews, B.; Abo-Riziq, A.; Grace, L.; Callahan, M.; Kabeláč, Martin; Hobza, Pavel; Vries de, M. S.

    2005-01-01

    Roč. 7, - (2005), s. 3015-3020 ISSN 1463-9076 R&D Projects: GA MŠk(CZ) LC512; GA ČR(CZ) GA203/05/0009; GA AV ČR(CZ) KJB400550518 Grant - others:NSF(US) CHE-0244341 Institutional research plan: CEZ:AV0Z40550506 Keywords : guanine * hydration * IR-UV spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.519, year: 2005

  3. Towards a better understanding of the unusual conformations of the alternating guanine-adenine repeat strands of DNA

    Czech Academy of Sciences Publication Activity Database

    Kejnovská, Iva; Kypr, Jaroslav; Vondrušková, Jitka; Vorlíčková, Michaela

    2007-01-01

    Roč. 85, č. 1 (2007), s. 19-27 ISSN 0006-3525 R&D Projects: GA AV ČR(CZ) IAA4004201; GA AV ČR(CZ) IAA5004802 Institutional research plan: CEZ:AV0Z50040702 Keywords : CD spectroscopy * alternating guanine-adenine microsatellite * guanine quadruplex Subject RIV: BO - Biophysics Impact factor: 2.389, year: 2007

  4. Activation of G Proteins by Guanine Nucleotide Exchange Factors Relies on GTPase Activity.

    Science.gov (United States)

    Stanley, Rob J; Thomas, Geraint M H

    2016-01-01

    G proteins are an important family of signalling molecules controlled by guanine nucleotide exchange and GTPase activity in what is commonly called an 'activation/inactivation cycle'. The molecular mechanism by which guanine nucleotide exchange factors (GEFs) catalyse the activation of monomeric G proteins is well-established, however the complete reversibility of this mechanism is often overlooked. Here, we use a theoretical approach to prove that GEFs are unable to positively control G protein systems at steady-state in the absence of GTPase activity. Instead, positive regulation of G proteins must be seen as a product of the competition between guanine nucleotide exchange and GTPase activity--emphasising a central role for GTPase activity beyond merely signal termination. We conclude that a more accurate description of the regulation of G proteins via these processes is as a 'balance/imbalance' mechanism. This result has implications for the understanding of intracellular signalling processes, and for experimental strategies that rely on modulating G protein systems.

  5. Synthesis and antiviral activity of 8-aza analogs of chiral [2-(phosphonomethoxy) propyl]guanines.

    Science.gov (United States)

    Franchetti, P; Sheikha, G A; Cappellacci, L; Grifantini, M; De Montis, A; Piras, G; Loi, A G; La Colla, P

    1995-09-29

    (R)- And (S)-8-aza-9(-)[2-(phosphonomethoxy)propyl]guanine [(R)-and (S)-8-aza-PMPG] were synthesized and tested in vitro for anti-human immunodeficiency virus (HIV) activity. The synthesis of the above compounds and of (R)-9(-)[2-(phosphonomethoxy)propyl]guanine [(R)-PMPG] was carried out through the alkylation of 8-azaguanine or guanine with (R)- and (S)-2-O(-)[(diisopropylphosphono)methyl]-1-O-(tolylsulfonyl) -1,2-propanediol followed by deprotection of the phosphonic moiety. A different, even more convenient synthesis of (R)-8-aza-PMPG starting from 2-amino-6-chloro-5-nitro-4(3H)-pyrimidinone and (R)(-)[2(-)[(diisopropylphosphono)-methoxy]propyl]amine is also reported. Both (R)-8-aza-PMPG and (R)-PMPG demonstrated anti-HIV activity in the MTT assay with EC50 values of 12 and 4.5 microM, respectively. The corresponding S enantiomers were found to be less potent. When evaluated in combination with AZT, ddI, or DABO 603, (R)-8-aza-PMPG gave additive, additive, and synergistic anti-HIV-1 effects, respectively.

  6. Residue propensities, discrimination and binding site prediction of adenine and guanine phosphates

    Directory of Open Access Journals (Sweden)

    Ahmad Zulfiqar

    2011-05-01

    Full Text Available Abstract Background Adenine and guanine phosphates are involved in a number of biological processes such as cell signaling, metabolism and enzymatic cofactor functions. Binding sites in proteins for these ligands are often detected by looking for a previously known motif by alignment based search. This is likely to miss those where a similar binding site has not been previously characterized and when the binding sites do not follow the rule described by predefined motif. Also, it is intriguing how proteins select between adenine and guanine derivative with high specificity. Results Residue preferences for AMP, GMP, ADP, GDP, ATP and GTP have been investigated in details with additional comparison with cyclic variants cAMP and cGMP. We also attempt to predict residues interacting with these nucleotides using information derived from local sequence and evolutionary profiles. Results indicate that subtle differences exist between single residue preferences for specific nucleotides and taking neighbor environment and evolutionary context into account, successful models of their binding site prediction can be developed. Conclusion In this work, we explore how single amino acid propensities for these nucleotides play a role in the affinity and specificity of this set of nucleotides. This is expected to be helpful in identifying novel binding sites for adenine and guanine phosphates, especially when a known binding motif is not detectable.

  7. Glyoxals as in vivo RNA structural probes of guanine base-pairing.

    Science.gov (United States)

    Mitchell, David; Ritchey, Laura E; Park, Hongmarn; Babitzke, Paul; Assmann, Sarah M; Bevilacqua, Philip C

    2018-01-01

    Elucidation of the folded structures that RNA forms in vivo is vital to understanding its functions. Chemical reagents that modify the Watson-Crick (WC) face of unprotected nucleobases are particularly useful in structure elucidation. Dimethyl sulfate penetrates cell membranes and informs on RNA base-pairing and secondary structure but only modifies the WC face of adenines and cytosines. We present glyoxal, methylglyoxal, and phenylglyoxal as potent in vivo reagents that target the WC face of guanines as well as cytosines and adenines. Tests on rice ( Oryza sativa) 5.8S rRNA in vitro read out by reverse transcription and gel electrophoresis demonstrate specific modification of almost all guanines in a time- and pH-dependent manner. Subsequent in vivo tests on rice, a eukaryote, and Bacillus subtilis and Escherichia coli , Gram-positive and Gram-negative bacteria, respectively, showed that all three reagents enter living cells without prior membrane permeabilization or pH adjustment of the surrounding media and specifically modify solvent-exposed guanine, cytosine, and adenine residues. © 2018 Mitchell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  8. Rates of chemical cleavage of DNA and RNA oligomers containing guanine oxidation products.

    Science.gov (United States)

    Fleming, Aaron M; Alshykhly, Omar; Zhu, Judy; Muller, James G; Burrows, Cynthia J

    2015-06-15

    The nucleobase guanine in DNA (dG) and RNA (rG) has the lowest standard reduction potential of the bases, rendering it a major site of oxidative damage in these polymers. Mapping the sites at which oxidation occurs in an oligomer via chemical reagents utilizes hot piperidine for cleaving oxidized DNA and aniline (pH 4.5) for cleaving oxidized RNA. In the present studies, a series of time-dependent cleavages of DNA and RNA strands containing various guanine lesions were examined to determine the strand scission rate constants. The guanine base lesions 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), 5-guanidinohydantoin (Gh), 2,2,4-triamino-2H-oxazol-5-one (Z), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) were evaluated in piperidine-treated DNA and aniline-treated RNA. These data identified wide variability in the chemical lability of the lesions studied in both DNA and RNA. Further, the rate constants for cleaving lesions in RNA were generally found to be significantly smaller than for lesions in DNA. The OG nucleotides were poorly cleaved in DNA and RNA; Sp nucleotides were slowly cleaved in DNA and did not cleave significantly in RNA; Gh and Z nucleotides cleaved in both DNA and RNA at intermediate rates; and 2Ih oligonucleotides cleaved relatively quickly in both DNA and RNA. The data are compared and contrasted with respect to future experimental design.

  9. Voltammetric Determination of Guanine on the Electrode Modified by Gold Deposit and Nafion Film

    Directory of Open Access Journals (Sweden)

    L.G. Shaidarova

    2016-09-01

    Full Text Available Electrodeposited gold and Nafion-gold composite on the surface of glassy carbon electrodes (GCE have shown electrocatalytic activity during guanine oxidation. In comparison with the unmodified electrode, decreasing of the oxidation potential by 100 mV and increasing of the current of organic compound oxidation have been observed. When the Nafion (NF film is applied to the surface of the glassy carbon electrode with electrodeposited gold, a five-fold increase of guanine oxidation current has been achieved compared to its oxidation on the modified electrode without the NF film. Conditions have been found for electrodeposition of gold on the surface of the glassy carbon electrode, including that one covered with the NF film, as well as for registration of the maximum catalytic current on these electrodes. Linear dependence of the electrocatalytic response of the modified electrode from the guanine concentration has been observed in the range from 5·10–6 to 5·10–3 mol·L–1 (for Au GCE and from 5·10–7 to 5·10–3 mol·L–1 (for NF-Au GCE.

  10. Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides.

    Science.gov (United States)

    Boyer, J L; Downes, C P; Harden, T K

    1989-01-15

    Membranes prepared from [3H]inositol-labeled turkey erythrocytes express a phospholipase C that is markedly stimulated by stable analogs of GTP (Harden, T. K., Stephens, L., Hawkins, P. T., and Downes, C. P. (1987) J. Biol. Chem. 262, 9057-9061). We now report that P2-purinergic receptor-mediated regulation of the enzyme occurs in the membrane preparation. The order of potency of a series of ATP and ADP analogs for stimulation of inositol phosphate formation, i.e. 2-methylthioadenosine 5'-triphosphate (2MeSATP) greater than adenosine 5'-O-(2-thiodiphosphate) greater than adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than 5'-adenylyl imidodiphosphate approximately ADP greater than alpha, beta-methyleneadenosine 5'-triphosphate greater than beta, gamma-methyleneadenosine 5'-triphosphate, was consistent with that for the P2Y-purinergic receptor subtype. Agonist-stimulated effects were completely dependent on the presence of guanine nucleotide. Activation of phospholipase C by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) occurred with a considerable time lag. The rate of activation followed first order kinetics and was markedly increased by increasing concentrations of a P2Y receptor agonist; in contrast, the rate of activation at a fixed agonist concentration was independent of guanine nucleotide concentration. Addition of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prior to addition of agonist and GTP, 5'-guanylyl imidodiphosphate (Gpp(NH)p), or GTP gamma S blocked in a concentration-dependent manner the stimulatory effect of guanine nucleotide. GDP beta S, added subsequent to preactivation of membranes with 2MeSATP and GTP gamma S or Gpp(NH)p had only small inhibitory effects on the rate of inositol phosphate production observed over the subsequent 10 min. In contrast, addition of GDP beta S to GTP-preactivated membranes resulted in a rapid return of enzyme activity to the basal state within 60 s. Taken together, the data are consistent with

  11. Increased mobility and on/off ratio in organic field-effect transistors using low-cost guanine-pentacene multilayers

    Science.gov (United States)

    Shi, Wei; Zheng, Yifan; Taylor, André D.; Yu, Junsheng; Katz, Howard E.

    2017-07-01

    Layer-by-layer deposited guanine and pentacene in organic field-effect transistors (OFETs) is introduced. Through adjusting the layer thickness ratio of guanine and pentacene, the tradeoff of two electronic parameters in OFETs, charge carrier mobility and current on/off ratio, was controlled. The charge mobility was enhanced by depositing pentacene over and between guanine layers and by increasing the proportion of pentacene in the layer-by-layer system, while the current on/off ratio was increased via the decreased off current induced by the guanine layers. The tunable device performance was mainly ascribed to the trap and dopant neutralizing properties of the guanine layers, which would decrease the density of free hydroxyl groups in the OFETs. Furthermore, the cost of the devices could be reduced remarkably via the adoption of low-cost guanine.

  12. Effect O6-guanine alkylation on DNA flexibility studied by comparative molecular dynamics simulations.

    Science.gov (United States)

    Kara, Mahmut; Drsata, Tomas; Lankas, Filip; Zacharias, Martin

    2015-01-01

    Alkylation of guanine at the O6 atom is a highly mutagenic DNA lesion because it alters the coding specificity of the base causing G:C to A:T transversion mutations. Specific DNA repair enzymes, e.g. O(6)-alkylguanin-DNA-Transferases (AGT), recognize and repair such damage after looping out the damaged base to transfer it into the enzyme active site. The exact mechanism how the repair enzyme identifies a damaged site within a large surplus of undamaged DNA is not fully understood. The O(6)-alkylation of guanine may change the deformability of DNA which may facilitate the initial binding of a repair enzyme at the damaged site. In order to characterize the effect of O(6)-methyl-guanine (O(6)-MeG) containing base pairs on the DNA deformability extensive comparative molecular dynamics (MD) simulations on duplex DNA with central G:C, O(6)-MeG:C or O(6)-MeG:T base pairs were performed. The simulations indicate significant differences in the helical deformability due to the presence of O(6)-MeG compared to regular undamaged DNA. This includes enhanced base pair opening, shear and stagger motions and alterations in the backbone fine structure caused in part by transient rupture of the base pairing at the damaged site and transient insertion of water molecules. It is likely that the increased opening motions of O(6)-MeG:C or O(6)-MeG:T base pairs play a decisive role for the induced fit recognition or for the looping out of the damaged base by repair enzymes. © 2014 Wiley Periodicals, Inc.

  13. Kinetics and structural aspects of the cisplatin interactions with guanine: A quantum mechanical description

    Science.gov (United States)

    Costa, Luiz Antônio S.; Hambley, Trevor W.; Rocha, Willian R.; Almeida, Wagner B. De; Dos Santos, Hélio F.

    The interaction of cisplatin with guanine DNA bases has been investigated using ab initio Hartree-Fock (HF) and density functional levels of theory in gas phase and aqueous solution. The overall process was divided into three steps: the reaction of the monoaqua [Pt(NH3)2Cl(H2O)]+ species with guanine (G) (reaction 1), the hydrolysis process yielding the adduct [Pt(NH3)2(G) (H2O)]2+ (reaction 2) and the reaction with a second guanine leading to the product [Pt(NH3)2(G)2]2+ (reaction 3). The functionals B3LYP, BHandH, and mPW1PW91 were used in the present study, to develop an understanding of the role of the distinct models. The geometries presented for the intermediate structures were obtained by IRC calculations from the transition state structure for each reaction. The structural analysis for the intermediates and transition states showed that hydrogen bonds with the guanine O6 atom play an important role in stabilizing these species. The geometries were not very sensitive to the level of theory applied with the HF level, giving a satisfactory overall performance. However, the energy barriers and the rate constants were found to be strongly dependent on the level of calculation and basis set, with the DFT calculations giving more accurate results. For reaction 1 the rate constant calculated in aqueous solution at PCM-BHandH/6-311G* (k1 = 7.55 × 10-1 M-1 s-1) was in good agreement with the experiment (5.4 × 10-1 M-1 s-1). The BHandH/6-31G* calculated gas phase rate constants for reactions 2 and 3 were: k2 = 0.9 × 10-6 M-1 s-1 and k3 = 2.99 × 10-4 M-1 s-1 in fairly good accordance with the experimental findings for reaction 2 (1.0 × 10-6 M-1 s-1) and reaction 3 (3.0 × 10-4 M-1 s-1).

  14. Generation of Guanine – Thymidine Cross-links in DNA by Peroxynitrite/Carbon Dioxide

    Science.gov (United States)

    Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir

    2011-01-01

    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and thymine N3 (T*) atoms (Crean et al., Nucleic Acids Res., 2008, 36, 742–755). In this work we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5–7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitroG), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxoG) and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level, and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled 15N,13C-labeled 2′-deoxy oligoribonucleotides 5′-dGpT and 5′-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and tri-oligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction-monitoring mode. The Nim and 8nitroG are the major products formed (~ 0.05% each), and lesser amounts of 8-oxoG (~ 0.02%), and d(G*pT*) and d(G*-T*) enzymatic digestion products (~ 0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both

  15. Silver (I) as DNA glue: Ag+-mediated guanine pairing revealed by removing Watson-Crick constraints

    Science.gov (United States)

    Swasey, Steven M.; Leal, Leonardo Espinosa; Lopez-Acevedo, Olga; Pavlovich, James; Gwinn, Elisabeth G.

    2015-05-01

    Metal ion interactions with DNA have far-reaching implications in biochemistry and DNA nanotechnology. Ag+ is uniquely interesting because it binds exclusively to the bases rather than the backbone of DNA, without the toxicity of Hg2+. In contrast to prior studies of Ag+ incorporation into double-stranded DNA, we remove the constraints of Watson-Crick pairing by focusing on homo-base DNA oligomers of the canonical bases. High resolution electro-spray ionization mass spectrometry reveals an unanticipated Ag+-mediated pairing of guanine homo-base strands, with higher stability than canonical guanine-cytosine pairing. By exploring unrestricted binding geometries, quantum chemical calculations find that Ag+ bridges between non-canonical sites on guanine bases. Circular dichroism spectroscopy shows that the Ag+-mediated structuring of guanine homobase strands persists to at least 90 °C under conditions for which canonical guanine-cytosine duplexes melt below 20 °C. These findings are promising for DNA nanotechnology and metal-ion based biomedical science.

  16. Electrocatalytic activity of oxidation products of guanine and 5'-GMP towards the oxidation of NADH

    Energy Technology Data Exchange (ETDEWEB)

    Santos-Alvarez, Noemi de los; Lobo-Castanon, Maria Jesus; Miranda-Ordieres, Arturo J. [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, Julian Claveria 8, 33006 Oviedo (Spain); Tunon-Blanco, Paulino [Departamento de Quimica Fisica y Analitica, Universidad de Oviedo, Julian Claveria 8, 33006 Oviedo (Spain)], E-mail: ptb@uniovi.es

    2007-12-01

    We have studied the potential electrocatalytic activity towards the oxidation of NADH of several oxidation products of guanine and its derivative guanosine-5'-monophosphate (5'-GMP) on pyrolytic graphite electrodes (PGE). The distribution of products generated strongly depends on the experimental conditions. Our investigations focused on the oxidation products that are adsorbed on the electrode surface, are redox active and, exhibited electrocatalytic activity toward the oxidation of NADH. These compounds were electrochemically and kinetically characterized in terms of dependence of the formal potential on pH and electron transfer rate constant (k{sub s}). The voltammetric and catalytic behavior of both guanine and 5'-GMP oxidation products was compared with that of other guanine derivatives we have previously studied. Some mechanistic aspects concerning the generation of the catalysts are also discussed.

  17. The Emerging Role of Guanine Exchange Factors in ALS and other neurodegenerative diseases

    Directory of Open Access Journals (Sweden)

    Cristian eDroppelmann

    2014-09-01

    Full Text Available Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs, of which two classes: Dbl-related exchange factors and the more recently described Dock family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF in the pathogenesis of amyotrophic lateral sclerosis (ALS. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament (NEFL mRNA 3’UTR to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.

  18. Signalling to actin: role of C3G, a multitasking guanine-nucleotide-exchange factor.

    Science.gov (United States)

    Radha, Vegesna; Mitra, Aninda; Dayma, Kunal; Sasikumar, Kotagiri

    2011-08-01

    C3G (Crk SH3-domain-binding guanine-nucleotide-releasing factor) is a ubiquitously expressed member of a class of molecules called GEFs (guanine-nucleotide-exchange factor) that activate small GTPases and is involved in pathways triggered by a variety of signals. It is essential for mammalian embryonic development and many cellular functions in adult tissues. C3G participates in regulating functions that require cytoskeletal remodelling such as adhesion, migration, maintenance of cell junctions, neurite growth and vesicle traffic. C3G is spatially and temporally regulated to act on Ras family GTPases Rap1, Rap2, R-Ras, TC21 and Rho family member TC10. Increased C3G protein levels are associated with differentiation of various cell types, indicating an important role for C3G in cellular differentiation. In signalling pathways, C3G serves functions dependent on catalytic activity as well as protein interaction and can therefore integrate signals necessary for the execution of more than one cellular function. This review summarizes our current knowledge of the biology of C3G with emphasis on its role as a transducer of signals to the actin cytoskeleton. Deregulated C3G may also contribute to pathogenesis of human disorders and therefore could be a potential therapeutic target.

  19. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  20. Polymerase recognition of 2-thio-iso-guanine·5-methyl-4-pyrimidinone (iGs·P)--A new DD/AA base pair.

    Science.gov (United States)

    Lee, Dong-Kye; Switzer, Christopher

    2016-02-15

    Polymerase specificity is reported for a previously unknown base pair with a non-standard DD/AA hydrogen bonding pattern: 2-thio-iso-guanine·5-methyl-4-pyrimidinone. Our findings suggest that atomic substitution may provide a solution for low fidelity previously associated with enzymatic copying of iso-guanine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Electrochemical behaviors and simultaneous determination of guanine and adenine based on graphene–ionic liquid–chitosan composite film modified glassy carbon electrode

    International Nuclear Information System (INIS)

    Niu Xiuli; Yang Wu; Ren Jie; Guo Hao; Long Shijia; Chen Jiaojiao; Gao Jinzhang

    2012-01-01

    Highlights: ► This work developed a novel electrochemical biosensors for guanine and adenine detection simultaneously. ► A disposable electrode based on graphene sheets, ionic liquid and chitosan was proposed. ► The presented method was also applied to simultaneous determination of guanine and adenine in denatured DNA samples with satisfying results. ► Easy fabrication, high sensitivity, excellent reproducibility and long-term stability. - Abstract: A graphene sheets (GS), ionic liquid (IL) and chitosan (CS) modified electrode was fabricated and the modified electrode displayed excellent electrochemical catalytic activities toward guanine and adenine. The transfer electron number (n) and the charge transfer coefficient (α) were calculated with the result as n = 2, α = 0.58 for guanine, and n = 2, α = 0.51 for adenine, which indicated the electrochemical oxidation of guanine and adenine on GS/IL/CS modified electrode was a two-electron and two-proton process. The oxidation overpotentials of guanine and adenine were decreased significantly compared with those obtained at the bare glassy carbon electrode and multi-walled carbon nanotubes modified electrode. The modified electrode exhibited good analytical performance and was successfully applied for individual and simultaneous determination of guanine and adenine. Low detection limits of 0.75 μM for guanine and 0.45 μM for adenine were obtained, with the linear calibration curves over the concentration range 2.5–150 μM and 1.5–350 μM, respectively. At the same time, the proposed method was successfully applied for the determination of guanine and adenine in denatured DNA samples with satisfying results. Moreover, the GS/IL/CS modified electrode exhibited good sensitivity, long-term stability and reproducibility for the determination of guanine and adenine.

  2. Guanine quadruplex monoclonal antibody 1H6 cross-reacts with restrained thymidine-rich single stranded DNA

    NARCIS (Netherlands)

    Kazemier, Hinke G.; Paeschke, Katrin; Lansdorp, Peter M.

    2017-01-01

    Previously we reported the production and characterization of monoclonal antibody 1H6 raised against (T(4)G(4))(2) intermolecular guanine quadruplex (G4) DNA structures (Henderson A. et al. (2014) Nucleic Acids Res., 42, 860-869; Hoffmann R. F. et al. (2016) Nucleic Acids Res., 44, 152-163). It was

  3. Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras

    NARCIS (Netherlands)

    Medema, R.H.; Vries-Smits, A.M. de; Zon, G.C.M. van der; Maassen, J.A.; Bos, J.L.

    1993-01-01

    A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate

  4. Synthesis and Evaluation of Asymmetric Acyclic Nucleoside Bisphosphonates as Inhibitors of Plasmodium falciparum and Human Hypoxanthine-Guanine-(Xanthine) Phosphoribosyltransferase

    Czech Academy of Sciences Publication Activity Database

    Špaček, Petr; Keough, D. T.; Chavchich, M.; Dračínský, Martin; Janeba, Zlatko; Naesens, L.; Edstein, M. D.; Guddat, L. W.; Hocková, Dana

    2017-01-01

    Roč. 60, č. 17 (2017), s. 7539-7554 ISSN 0022-2623 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : hypoxanthine-guanine phosphoribosyltransferase * 2nd phosphonate group * 6-oxopurine phosphoribosyltransferases Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 6.259, year: 2016

  5. Origin of Difference between One-Electron Redox Potentials of Guanosine and Guanine: Electrochemical and Quantum Chemical Study

    Czech Academy of Sciences Publication Activity Database

    Langmaier, Jan; Samec, Zdeněk; Samcová, E.; Hobza, Pavel; Řeha, David

    2004-01-01

    Roč. 108, č. 40 (2004), s. 15896-15899 ISSN 1520-6106 R&D Projects: GA ČR GA203/01/0653 Institutional research plan: CEZ:AV0Z4040901 Keywords : cyclic voltammetry * guanosine * guanine Subject RIV: CG - Electrochemistry Impact factor: 3.834, year: 2004

  6. Dictyostelium Ric8 is a nonreceptor guanine exchange factor for heterotrimeric G proteins and is important for development and chemotaxis

    NARCIS (Netherlands)

    Kataria, Rama; Xu, Xuehua; Fusetti, Fabrizia; Keizer-Gunnink, Ineke; Jin, Tian; van Haastert, Peter J M; Kortholt, Arjan

    2013-01-01

    Heterotrimeric G proteins couple external signals to the activation of intracellular signal transduction pathways. Agonist-stimulated guanine nucleotide exchange activity of G-protein-coupled receptors results in the exchange of G-protein-bound GDP to GTP and the dissociation and activation of the

  7. The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best.

    Science.gov (United States)

    Zaccaria, F; Paragi, G; Fonseca Guerra, C

    2016-08-21

    The alkali metal ion affinity of guanine quadruplexes has been studied using dispersion-corrected density functional theory (DFT-D). We have done computational investigations in aqueous solution that mimics artificial supramolecular conditions where guanine bases assemble into stacked quartets as well as biological environments in which telomeric quadruplexes are formed. In both cases, an alkali metal cation is needed to assist self-assembly. Our quantum chemical computations on these supramolecular systems are able to reproduce the experimental order of affinity of the guanine quadruplexes for the cations Li(+), Na(+), K(+), Rb(+), and Cs(+). The strongest binding is computed between the potassium cation and the quadruplex as it occurs in nature. The desolvation and the size of alkali metal cations are thought to be responsible for the order of affinity. Until now, the relative importance of these two factors has remained unclear and debated. By assessing the quantum chemical 'size' of the cation, determining the amount of deformation of the quadruplex needed to accommodate the cation and through the energy decomposition analysis (EDA) of the interaction energy between the cation and the guanines, we reveal that the desolvation and size of the alkali metal cation are both almost equally responsible for the order of affinity.

  8. Modified gold electrodes based on thiocytosine/guanine-gold nanoparticles for uric and ascorbic acid determination

    International Nuclear Information System (INIS)

    Vulcu, Adriana; Grosan, Camelia; Muresan, Liana Maria; Pruneanu, Stela; Olenic, Liliana

    2013-01-01

    The present paper describes the preparation of new modified surfaces for electrodes based on guanine/thiocytosine and gold nanoparticles. The gold nanoparticles were analyzed by UV–vis spectroscopy and transmission electron microscopy (TEM) and it was found that they have diameters between 30 and 40 nm. The layers were characterized by specular reflectance infrared spectroscopy (FTIR-RAS) and by atomic force microscopy (AFM). The thickness of layers was found to be approximately 30 nm for TC layers and 300 nm for GU layers. Every layer was characterized as electrochemical sensor (by cyclic voltammetry) both for uric acid and ascorbic acid determinations, separately and in their mixture. The modified sensors have good calibration functions with good sensitivity (between 1.145 and 1.406 mA cm −2 /decade), reproducibility ( t hiocytosine (Au T C) and gold g uanine (Au G U) layers

  9. Structural study and investigation of NMR tensors in interaction of dopamine with Adenine and guanine

    Directory of Open Access Journals (Sweden)

    Lingjia Xu

    2007-04-01

    Full Text Available The interaction of dopamine with adenine and guanine were studied at the Hartree-Fock level theory. The structural and vibrational properties of dopamine-4-N7GUA and dopamine-4-N3ADE were studied at level of HF/6-31G*. Interaction energies (ΔE were calculated to be -11.49 and -11.92 kcal/mol, respectively. Some of bond lengths, angels and tortions are compared. NBO studies were performed to the second-order and perturbative estimates of donor-acceptor interaction have been done. The procedures of gauge-invariant atomic orbital (GIAO and continuous-set-of-gauge-transformation (CSGT were employed to calculate isotropic shielding, chemical shifts anisotropy and chemical shifts anisotropy asymmetry and effective anisotropy using 6-31G* basis set. These calculations yielded molecular geometries in good agreement with available experimental data.

  10. INTRACELLULAR Leishmania amazonensis KILLING INDUCED BY THE GUANINE NUCLEOSIDE 8-BROMOGUANOSINE

    Directory of Open Access Journals (Sweden)

    GIORGIO Selma

    1998-01-01

    Full Text Available In this study we investigated the effect of 8-Bromoguanosine, an immunostimulatory compound, on the cytotoxicity of macrophages against Leishmania amazonensis in an in vitro system. The results showed that macrophages treated with 8-Bromoguanosine before or after infection are capable to reduce parasite load, as monitored by the number of amastigotes per macrophage and the percentage of infected cells (i.e. phagocytic index. Since 8-Bromoguanosine was not directly toxic to the promastigotes, it was concluded that the ribonucleoside induced macrophage activation. Presumably, 8-Bromoguanosine primed macrophages by inducing interferon alpha and beta which ultimately led to L. amazonensis amastigote killing. The results suggest that guanine ribonucleosides may be useful to treat infections with intracellular pathogens.

  11. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Hazelton, Keith Z. [Yeshiva Univ., New York, NY (United States); Ho, Meng-Chaio [Yeshiva Univ., New York, NY (United States); Cassera, Maria B. [Yeshiva Univ., New York, NY (United States); Clinch, Keith [Industrial Research Ltd., Lower Hutt (New Zealand); Crump, Douglas R. [Industrial Research Ltd., Lower Hutt (New Zealand); Rosario Jr., Irving [Yeshiva Univ., New York, NY (United States); Merino, Emilio F. [Yeshiva Univ., New York, NY (United States); Almo, Steve C. [Yeshiva Univ., New York, NY (United States); Tyler, Peter C. [Industrial Research Ltd., Lower Hutt (New Zealand); Schramm, Vern L. [Yeshiva Univ., New York, NY (United States)

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  12. Theoretical study of valence orbital response to guanine tautomerization in coordinate and momentum spaces

    International Nuclear Information System (INIS)

    Yang, Zejin; Duffy, Patrick; Zhu, Quan; Wang, Feng; Takahashi, Masahiko

    2015-01-01

    The binding energy spectra and electron momentum spectra of eight stable guanine tautomers are calculated in the complete valence space. The present results show that the canonical keto (C=O) guanine N(9)H tautomer (GU1) possesses the largest dipole moment, molecular electronic spatial extent, molecular hardness value, and the minimum first vertical ionization potential (VIP). Valence orbital profile investigations find that several orbitals remain almost unchanged during tautomerization, such as frontier highest occupied molecular orbital 39a and 18a. Several orbitals with interchanged order and inverse direction in charge spatial orientations are also detected. Outer valence orbitals (with smaller VIPs) show more complex orbital shapes in the momentum space than those of inner ones (larger VIPs) due mainly to the relatively strong inter-orbital interaction and delocalized electronic distributions. Proton rotation along C–O(H) and C–N(H) axes within hexagonal ring causes smaller influence to orbital profiles than those of proton migration within pentagonal and/or hexagonal rings. Orbital variation trends between enol (GU3–GU5) and keto (GU1, GU2, GU6–GU8) tautomers are observed, including the signature orbitals of enol form, the variation tendency of total orbital intensity, and the variation order of the maximum orbital intensity. In the outer valence momentum space (outside 26a), orbital composed by p z electrons show single peak with a gradual increasing peak site from 0.5 a.u. of inner valence orbital to 1.0 a.u. of outer valence orbital, whereas orbitals composed by p x,y electrons form double peaks with respective sites at about 0.5 and 1.5 a.u., only three p x,y -orbitals present single peaks (33a,34a,36a). The general variation trends in the complete valence space for all the valence orbitals on their intensities, peak sites, and orbital components are concluded. (paper)

  13. Guanine holes are prominent targets for mutation in cancer and inherited disease.

    Science.gov (United States)

    Bacolla, Albino; Temiz, Nuri A; Yi, Ming; Ivanic, Joseph; Cer, Regina Z; Donohue, Duncan E; Ball, Edward V; Mudunuri, Uma S; Wang, Guliang; Jain, Aklank; Volfovsky, Natalia; Luke, Brian T; Stephens, Robert M; Cooper, David N; Collins, Jack R; Vasquez, Karen M

    2013-01-01

    Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

  14. Guanine holes are prominent targets for mutation in cancer and inherited disease.

    Directory of Open Access Journals (Sweden)

    Albino Bacolla

    Full Text Available Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials. Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

  15. Functional modification of the guanine nucleotide regulatory protein after desensitization of turkey erythrocytes by catecholamines.

    Science.gov (United States)

    Briggs, M M; Stadel, J M; Iyengar, R; Lefkowitz, R J

    1983-07-01

    Densensitization of turkey erythrocytes by exposure to the beta-adrenergic agonist (-)isoproterenol leads to decreased activation of adenylate cyclase by agonist, NaF, and guanyl-5'-yl imido diphosphate, with no reduction in the number of beta-adrenergic receptors. Interactions between the receptor and the guanine nucleotide regulatory protein (N protein) also seem to be impaired. These observations suggest that a component distal to the beta-adrenergic receptor may be a locus of modification. Accordingly we examined the N protein to determine whether it was altered by desensitization. The rate at which (-)isoproterenol stimulated the release of [3H]GDP from the N protein was substantially lower in membranes prepared from desensitized cells, providing further evidence for uncoupling of the receptor and the N protein. The amount of N protein in membranes from control and desensitized cells was compared by labeling the 42,000 Mr component of the N protein with [32P]NAD+ and cholera toxin; no significant difference was found. However, significantly more N protein (p less than .001) was solubilized by cholate extraction of desensitized membranes, suggesting an altered association of the N protein with the membrane after desensitization. The functional activity of the N protein was measured by reconstitution of cholate extracts of turkey erythrocyte membranes into S49 lymphoma cyc- membranes. Reconstitution of (-)isoproterenol stimulation of adenylate cyclase activity was reduced significantly (p less than .05) after desensitization. These observations suggest that desensitization of the turkey erythrocyte by (-)isoproterenol results in functional modifications of the guanine nucleotide regulatory protein, leading to impaired interactions with the beta-adrenergic receptor and reduced activation of adenylate cyclase.

  16. The Lack of Mutagenic Potential of a Guanine-Rich Triplex Forming Oligonucleotide in Physiological Conditions.

    Science.gov (United States)

    Saleh, Amer F; Fellows, Mick D; Ying, Liming; Gooderham, Nigel J; Priestley, Catherine C

    2017-01-01

    Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analyzed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.

  17. Highly Sensitive Bacteria Quantification Using Immunomagnetic Separation and Electrochemical Detection of Guanine-Labeled Secondary Beads

    Directory of Open Access Journals (Sweden)

    Harikrishnan Jayamohan

    2015-05-01

    Full Text Available In this paper, we report the ultra-sensitive indirect electrochemical detection of E. coli O157:H7 using antibody functionalized primary (magnetic beads for capture and polyguanine (polyG oligonucleotide functionalized secondary (polystyrene beads as an electrochemical tag. Vacuum filtration in combination with E. coli O157:H7 specific antibody modified magnetic beads were used for extraction of E. coli O157:H7 from 100 mL samples. The magnetic bead conjugated E. coli O157:H7 cells were then attached to polyG functionalized secondary beads to form a sandwich complex (magnetic bead/E. coli secondary bead. While the use of magnetic beads for immuno-based capture is well characterized, the use of oligonucleotide functionalized secondary beads helps combine amplification and potential multiplexing into the system. The antibody functionalized secondary beads can be easily modified with a different antibody to detect other pathogens from the same sample and enable potential multiplexing. The polyGs on the secondary beads enable signal amplification up to 10\\(^{8}\\ guanine tags per secondary bead (\\(7.5\\times10^{6}\\ biotin-FITC per secondary bead, 20 guanines per oligonucleotide bound to the target (E. coli. A single-stranded DNA probe functionalized reduced graphene oxide modified glassy carbon electrode was used to bind the polyGs on the secondary beads. Fluorescent imaging was performed to confirm the hybridization of the complex to the electrode surface. Differential pulse voltammetry (DPV was used to quantify the amount of polyG involved in the hybridization event with tris(2,2'-bipyridineruthenium(II (Ru(bpy\\(_{3}^{2+}\\ as the mediator. The amount of polyG signal can be correlated to the amount of E. coli O157:H7 in the sample. The method was able to detect concentrations of E. coli O157:H7 down to 3 CFU/100 mL, which is 67 times lower than the most sensitive technique reported in literature. The signal to noise ratio for this work was 3

  18. Dictyostelium Ric8 is a nonreceptor guanine exchange factor for heterotrimeric G proteins and is important for development and chemotaxis.

    Science.gov (United States)

    Kataria, Rama; Xu, Xuehua; Fusetti, Fabrizia; Keizer-Gunnink, Ineke; Jin, Tian; van Haastert, Peter J M; Kortholt, Arjan

    2013-04-16

    Heterotrimeric G proteins couple external signals to the activation of intracellular signal transduction pathways. Agonist-stimulated guanine nucleotide exchange activity of G-protein-coupled receptors results in the exchange of G-protein-bound GDP to GTP and the dissociation and activation of the complex into Gα-GTP and a Gβγ dimer. In Dictyostelium, a basal chemotaxis pathway consisting of heterotrimeric and monomeric G proteins is sufficient for chemotaxis. Symmetry breaking and amplification of chemoattractant sensing occurs between heterotrimeric G protein signaling and Ras activation. In a pull-down screen coupled to mass spectrometry, with Gα proteins as bait, we have identified resistant to inhibitors of cholinesterase 8 (Ric8) as a nonreceptor guanine nucleotide exchange factor for Gα-protein. Ric8 is not essential for the initial activation of heterotrimeric G proteins or Ras by uniform chemoattractant; however, it amplifies Gα signaling, which is essential for Ras-mediated symmetry breaking during chemotaxis and development.

  19. Automated quantum chemistry based molecular dynamics simulations of electron ionization induced fragmentations of the nucleobases Uracil, Thymine, Cytosine, and Guanine.

    Science.gov (United States)

    Grimme, Stefan; Bauer, Christopher Alexander

    2015-01-01

    The gas-phase decomposition pathways of electron ionization (EI)-induced radical cations of the nucleobases uracil, thymine, cytosine, and guanine are investigated by means of mixed quantum-classical molecular dynamics. No preconceived fragmentation channels are used in the calculations. The results compare well to a plethora of experimental and theoretical data for these important biomolecules. With our combined stochastic and dynamic approach, one can access in an unbiased way the energetically available decomposition mechanisms. Additionally, we are able to separate the EI mass spectra of different tautomers of cytosine and guanine. Our method (previously termed quantum chemistry electron ionization mass spectra) reproduces free nucleobase experimental mass spectra well and provides detailed mechanistic in-sight into high-energy unimolecular decomposition processes.

  20. Synthesis of adenine, guanine, cytosine, and other nitrogen organic compounds by a Fischer-Tropsch-like process.

    Science.gov (United States)

    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).

  1. Detection of Guanine and Adenine Using an Aminated Reduced Graphene Oxide Functional Membrane-Modified Glassy Carbon Electrode

    Directory of Open Access Journals (Sweden)

    Di Li

    2017-07-01

    Full Text Available A new electrochemical sensor based on a Nafion, aminated reduced graphene oxide and chitosan functional membrane-modified glassy carbon electrode was proposed for the simultaneous detection of adenine and guanine. Fourier transform-infrared spectrometry (FTIR, transmission electron microscopy (TEM, and electrochemical methods were utilized for the additional characterization of the membrane materials. The prepared electrode was utilized for the detection of guanine (G and adenine (A. The anodic peak currents to G and A were linear in the concentrations ranging from 0.1 to 120 μM and 0.2 to 110 μM, respectively. The detection limits were found to be 0.1 μM and 0.2 μM, respectively. Moreover, the modified electrode could also be used to determine G and A in calf thymus DNA.

  2. Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders.

    Science.gov (United States)

    Zuccarini, Mariachiara; Giuliani, Patricia; Frinchi, Monica; Mudò, Giuseppa; Serio, Rosa Maria; Belluardo, Natale; Buccella, Silvana; Carluccio, Marzia; Condorelli, Daniele F; Caciagli, Francesco; Ciccarelli, Renata; Di Iorio, Patrizia

    2018-01-01

    Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment). This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i) in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii) the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii) the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory-related disorders.

  3. Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders

    Directory of Open Access Journals (Sweden)

    Mariachiara Zuccarini

    2018-02-01

    Full Text Available Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson’s and Alzheimer’s diseases. Guanosine (GUO and guanine (GUA are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP and guanine deaminase (GDA. Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptotic and neurotrophic effects, causes a decay of cognitive activities, whereas GUA administration in rats results in working memory improvement (prevented by L-NAME pre-treatment. This study was designed to investigate, in a model of SH-SY5Y neuroblastoma cell line, the signal transduction pathway activated by extracellular GUA. Altogether, our results showed that: (i in addition to an enhanced phosphorylation of ASK1, p38 and JNK, likely linked to a non-massive and transient ROS production, the PKB/NO/sGC/cGMP/PKG/ERK cascade seems to be the main signaling pathway elicited by extracellular GUA; (ii the activation of this pathway occurs in a pertussis-toxin sensitive manner, thus suggesting the involvement of a putative G protein coupled receptor; (iii the GUA-induced NO production, strongly reduced by cell pre-treatment with L-NAME, is negatively modulated by the EPAC-cAMP-CaMKII pathway, which causes the over-expression of GDA that, in turn, reduces the levels of GUA. These molecular mechanisms activated by GUA may be useful to support our previous observation showing that GUA improves learning and memory functions through the stimulation of NO signaling pathway, and underscore the therapeutic potential of oral administration of guanine for treating memory

  4. Formation pathways of a guanine-quadruplex DNA revealed by molecular dynamics and thermodynamic analysis of the substates

    Czech Academy of Sciences Publication Activity Database

    Štefl, R.; Cheatham III, T. E.; Špačková, Naďa; Fadrná, E.; Berger, I.; Koča, J.; Šponer, Jiří

    2003-01-01

    Roč. 85, č. 3 (2003), s. 1787-1804 ISSN 0006-3495 R&D Projects: GA MŠk LN00A016 Grant - others:National Science Foundation(US) CDA9601580; GA FRVŠ(CZ) G604; Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : guanine quadruplex * quadruplex formation * molecular dynamics simulations Subject RIV: BO - Biophysics Impact factor: 4.463, year: 2003

  5. Guanine quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G4T4G4 fragment

    Czech Academy of Sciences Publication Activity Database

    Vondrušková, Jitka; Kypr, Jaroslav; Kejnovská, Iva; Fialová, Markéta; Vorlíčková, Michaela

    2008-01-01

    Roč. 89, č. 10 (2008), s. 797-806 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA204/07/0057; GA AV ČR(CZ) IAA100040701 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA/DNA hybrids * guanine quadruplex * circular dichroism spectroscopy Subject RIV: BO - Biophysics Impact factor: 2.823, year: 2008

  6. Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification.

    Science.gov (United States)

    Lech, Christopher Jacques; Phan, Anh Tuân

    2017-06-20

    Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Arf6 guanine-nucleotide exchange factor cytohesin-2 regulates myelination in nerves.

    Science.gov (United States)

    Torii, Tomohiro; Ohno, Nobuhiko; Miyamoto, Yuki; Kawahara, Kazuko; Saitoh, Yurika; Nakamura, Kazuaki; Takashima, Shou; Sakagami, Hiroyuki; Tanoue, Akito; Yamauchi, Junji

    2015-05-08

    In postnatal development of the peripheral nervous system (PNS), Schwann cells differentiate to insulate neuronal axons with myelin sheaths, increasing the nerve conduction velocity. To produce the mature myelin sheath with its multiple layers, Schwann cells undergo dynamic morphological changes. While extracellular molecules such as growth factors and cell adhesion ligands are known to regulate the myelination process, the intracellular molecular mechanism underlying myelination remains unclear. In this study, we have produced Schwann cell-specific conditional knockout mice for cytohesin-2, a guanine-nucleotide exchange factor (GEF) specifically activating Arf6. Arf6, a member of the Ras-like protein family, participates in various cellular functions including cell morphological changes. Cytohesin-2 knockout mice exhibit decreased Arf6 activity and reduced myelin thickness in the sciatic nerves, with decreased expression levels of myelin protein zero (MPZ), the major myelin marker protein. These results are consistent with those of experiments in which Schwann cell-neuronal cultures were treated with pan-cytohesin inhibitor SecinH3. On the other hand, the numbers of Ki67-positive cells in knockout mice and controls are comparable, indicating that cytohesin-2 does not have a positive effect on cell numbers. Thus, signaling through cytohesin-2 is required for myelination by Schwann cells, and cytohesin-2 is added to the list of molecules known to underlie PNS myelination. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Proteomic analysis of Rac1 signaling regulation by guanine nucleotide exchange factors.

    Science.gov (United States)

    Marei, Hadir; Carpy, Alejandro; Macek, Boris; Malliri, Angeliki

    2016-08-02

    The small GTPase Rac1 is implicated in various cellular processes that are essential for normal cell function. Deregulation of Rac1 signaling has also been linked to a number of diseases, including cancer. The diversity of Rac1 functioning in cells is mainly attributed to its ability to bind to a multitude of downstream effectors following activation by Guanine nucleotide Exchange Factors (GEFs). Despite the identification of a large number of Rac1 binding partners, factors influencing downstream specificity are poorly defined, thus hindering the detailed understanding of both Rac1's normal and pathological functions. In a recent study, we demonstrated a role for 2 Rac-specific GEFs, Tiam1 and P-Rex1, in mediating Rac1 anti- versus pro-migratory effects, respectively. Importantly, via conducting a quantitative proteomic screen, we identified distinct changes in the Rac1 interactome following activation by either GEF, indicating that these opposing effects are mediated through GEF modulation of the Rac1 interactome. Here, we present the full list of identified Rac1 interactors together with functional annotation of the differentially regulated Rac1 binding partners. In light of this data, we also provide additional insights into known and novel signaling cascades that might account for the GEF-mediated Rac1-driven cellular effects.

  9. Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors.

    Science.gov (United States)

    Kulasekaran, Gopinath; Nossova, Nadya; Marat, Andrea L; Lund, Ingrid; Cremer, Christopher; Ioannou, Maria S; McPherson, Peter S

    2015-07-17

    Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.

    Science.gov (United States)

    Marat, Andrea L; McPherson, Peter S

    2010-04-02

    Rabs constitute the largest family of monomeric GTPases, yet for the majority of Rabs relatively little is known about their activation and recruitment to vesicle-trafficking pathways. We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as a guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35, whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2. Characterization of connecdenn 2 reveals binding to the beta2-ear of AP-2 on a site that overlaps with that used by the autosomal recessive hypercholesterolemia protein and betaarrestin, although the sequence used by connecdenn 2 is unique. Loss of connecdenn 2 function through small interference RNA knockdown results in an enlargement of early endosomes, similar to what is observed upon loss of Rab35 activity. Our studies reveal connecdenn DENN domains as generalized GEFs for Rab35 and identify a new AP-2-binding motif, demonstrating a complex link between the clathrin machinery and Rab35 activation.

  11. Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors*

    Science.gov (United States)

    Kulasekaran, Gopinath; Nossova, Nadya; Marat, Andrea L.; Lund, Ingrid; Cremer, Christopher; Ioannou, Maria S.; McPherson, Peter S.

    2015-01-01

    Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction. PMID:26055712

  12. WBSCR16 Is a Guanine Nucleotide Exchange Factor Important for Mitochondrial Fusion

    Directory of Open Access Journals (Sweden)

    Guorui Huang

    2017-07-01

    Full Text Available Regulated inter-mitochondrial fusion/fission is essential for maintaining optimal mitochondrial respiration and control of apoptosis and autophagy. In mammals, mitochondrial fusion is controlled by outer membrane GTPases MFN1 and MFN2 and by inner membrane (IM GTPase OPA1. Disordered mitochondrial fusion/fission contributes to various pathologies, and MFN2 or OPA1 mutations underlie neurodegenerative diseases. Here, we show that the WBSCR16 protein is primarily associated with the outer face of the inner mitochondrial membrane and is important for mitochondrial fusion. We provide evidence of a WBSCR16/OPA1 physical interaction in the intact cell and of a WBSCR16 function as an OPA1-specific guanine nucleotide exchange factor (GEF. Homozygosity for a Wbscr16 mutation causes early embryonic lethality, whereas neurons of mice heterozygous for the mutation have mitochondria with reduced membrane potential and increased susceptibility to fragmentation upon exposure to stress, suggesting roles for WBSCR16 deficits in neuronal pathologies.

  13. The minimal autoinhibited unit of the guanine nucleotide exchange factor intersectin.

    Directory of Open Access Journals (Sweden)

    K Farid Ahmad

    2010-06-01

    Full Text Available Intersectin-1L is a member of the Dbl homology (DH domain guanine nucleotide exchange factors (GEF which control Rho-family GTPase signaling. Intersectin-1L is a GEF that is specific for Cdc42. It plays an important role in endocytosis, and is regulated by several partners including the actin regulator N-WASP. Intact intersectin-1L shows low Cdc42 exchange activity, although the isolated catalytic DH domain shows high activity. This finding suggests that the molecule is autoinhibited. To investigate the mechanism of autoinhibition we have constructed a series of domain deletions. We find that the five SH3 domains of intersectin are important for autoinhibition, with the fifth domain (SH3(E being sufficient for the bulk of the autoinhibitory effect. This SH3 domain appears to primarily interact with the DH domain. We have determined the crystal structure of the SH3(E-DH domain construct, which shows a domain swapped arrangement in which the SH3 from one monomer interacts with the DH domain of the other monomer. Analytical ultracentrifugation and gel filtration, however, show that under biochemical concentrations, the construct is fully monomeric. Thus we propose that the actual autoinhibited structure contains the related intramolecular SH3(E-DH interaction. We propose a model in which this intramolecular interaction may block or distort the GTPase binding region of the DH domain.

  14. EspM2 is a RhoA guanine nucleotide exchange factor.

    Science.gov (United States)

    Arbeloa, Ana; Garnett, James; Lillington, James; Bulgin, Richard R; Berger, Cedric N; Lea, Susan M; Matthews, Steve; Frankel, Gad

    2010-05-01

    We investigated how the type III secretion system WxxxE effectors EspM2 of enterohaemorrhagic Escherichia coli, which triggers stress fibre formation, and SifA of Salmonella enterica serovar Typhimurium, which is involved in intracellular survival, modulate Rho GTPases. We identified a direct interaction between EspM2 or SifA and nucleotide-free RhoA. Nuclear Magnetic Resonance Spectroscopy revealed that EspM2 has a similar fold to SifA and the guanine nucleotide exchange factor (GEF) effector SopE. EspM2 induced nucleotide exchange in RhoA but not in Rac1 or H-Ras, while SifA induced nucleotide exchange in none of them. Mutating W70 of the WxxxE motif or L118 and I127 residues, which surround the catalytic loop, affected the stability of EspM2. Substitution of Q124, located within the catalytic loop of EspM2, with alanine, greatly attenuated the RhoA GEF activity in vitro and the ability of EspM2 to induce stress fibres upon ectopic expression. These results suggest that binding of SifA to RhoA does not trigger nucleotide exchange while EspM2 is a unique Rho GTPase GEF.

  15. Bacterial Guanine Nucleotide Exchange Factors SopE-Like and WxxxE Effectors▿

    Science.gov (United States)

    Bulgin, Richard; Raymond, Benoit; Garnett, James A.; Frankel, Gad; Crepin, Valerie F.; Berger, Cedric N.; Arbeloa, Ana

    2010-01-01

    Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce “the trigger mechanism of cell entry.” Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection. PMID:20123714

  16. Bacterial guanine nucleotide exchange factors SopE-like and WxxxE effectors.

    Science.gov (United States)

    Bulgin, Richard; Raymond, Benoit; Garnett, James A; Frankel, Gad; Crepin, Valerie F; Berger, Cedric N; Arbeloa, Ana

    2010-04-01

    Subversion of Rho family small GTPases, which control actin dynamics, is a common infection strategy used by bacterial pathogens. In particular, Salmonella enterica serovar Typhimurium, Shigella flexneri, enteropathogenic Escherichia coli (EPEC), and enterohemorrhagic Escherichia coli (EHEC) translocate type III secretion system (T3SS) effector proteins to modulate the Rho GTPases RhoA, Cdc42, and Rac1, which trigger formation of stress fibers, filopodia, and lamellipodia/ruffles, respectively. The Salmonella effector SopE is a guanine nucleotide exchange factor (GEF) that activates Rac1 and Cdc42, which induce "the trigger mechanism of cell entry." Based on a conserved Trp-xxx-Glu motif, the T3SS effector proteins IpgB1 and IpgB2 of Shigella, SifA and SifB of Salmonella, and Map of EPEC and EHEC were grouped together into a WxxxE family; recent studies identified the T3SS EPEC and EHEC effectors EspM and EspT as new family members. Recent structural and functional studies have shown that representatives of the WxxxE effectors share with SopE a 3-D fold and GEF activity. In this minireview, we summarize contemporary findings related to the SopE and WxxxE GEFs in the context of their role in subverting general host cell signaling pathways and infection.

  17. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  18. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Carla J Eaton

    Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

  19. The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery*

    Science.gov (United States)

    Marat, Andrea L.; McPherson, Peter S.

    2010-01-01

    Rabs constitute the largest family of monomeric GTPases, yet for the majority of Rabs relatively little is known about their activation and recruitment to vesicle-trafficking pathways. We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as a guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35, whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2. Characterization of connecdenn 2 reveals binding to the β2-ear of AP-2 on a site that overlaps with that used by the autosomal recessive hypercholesterolemia protein and βarrestin, although the sequence used by connecdenn 2 is unique. Loss of connecdenn 2 function through small interference RNA knockdown results in an enlargement of early endosomes, similar to what is observed upon loss of Rab35 activity. Our studies reveal connecdenn DENN domains as generalized GEFs for Rab35 and identify a new AP-2-binding motif, demonstrating a complex link between the clathrin machinery and Rab35 activation. PMID:20154091

  20. Guanine-centric self-assembly of nucleotides in water: an important consideration in prebiotic chemistry.

    Science.gov (United States)

    Cassidy, Lauren M; Burcar, Bradley T; Stevens, Wyatt; Moriarty, Elizabeth M; McGown, Linda B

    2014-10-01

    Investigations of plausible prebiotic chemistry on early Earth must consider not only chemical reactions to form more complex products such as proto-biopolymers but also reversible, molecular self-assembly that would influence the availability, organization, and sequestration of reactant molecules. The self-assembly of guanosine compounds into higher-order structures and lyotropic liquid crystalline "gel" phases through formation of hydrogen-bonded guanine tetrads (G-tetrads) is one such consideration that is particularly relevant to an RNA-world scenario. G-tetrad-based gelation has been well studied for individual guanosine compounds and was recently observed in mixtures of guanosine with 5'-guanosine monophosphate (GMP) as well. The present work investigates the self-assembly of GMP in the presence of the other RNA nucleotides. Effects of the total concentration and relative proportion of the nucleotides in the mixtures, the form (disodium salt vs. free acid) of the nucleotides, temperature, pH, and salt concentration were determined by visual observations and circular dichroism (CD) spectroscopy. The results show that formation of cholesteric G-tetrad phases is influenced by interactions with other nucleotides, likely through association (e.g., intercalation) of the nucleotides with the G-tetrad structures. These interactions affect the structure and stability of the G-tetrad gel phase, as well as the formation of alternate self-assembled GMP structures such as a continuous, hydrogen-bonded GMP helix or dimers and aggregates of GMP. These interactions and multiple equilibria are influenced by the presence of cations, especially in the presence of K(+). This work could have important implications for the emergence of an RNA or proto-RNA world, which would require mixtures of nucleotides at sufficiently high, local concentrations for abiotic polymerization to occur.

  1. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    International Nuclear Information System (INIS)

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-01-01

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the α subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single β subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the α subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub sα/ relative to G/sub ichemical bond/ and G/sub ochemical bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with [ 125 I]protein. Immunohistochemical studies using an antiserum against the β subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the α subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium

  2. QuadBase2: web server for multiplexed guanine quadruplex mining and visualization.

    Science.gov (United States)

    Dhapola, Parashar; Chowdhury, Shantanu

    2016-07-08

    DNA guanine quadruplexes or G4s are non-canonical DNA secondary structures which affect genomic processes like replication, transcription and recombination. G4s are computationally identified by specific nucleotide motifs which are also called putative G4 (PG4) motifs. Despite the general relevance of these structures, there is currently no tool available that can allow batch queries and genome-wide analysis of these motifs in a user-friendly interface. QuadBase2 (quadbase.igib.res.in) presents a completely reinvented web server version of previously published QuadBase database. QuadBase2 enables users to mine PG4 motifs in up to 178 eukaryotes through the EuQuad module. This module interfaces with Ensembl Compara database, to allow users mine PG4 motifs in the orthologues of genes of interest across eukaryotes. PG4 motifs can be mined across genes and their promoter sequences in 1719 prokaryotes through ProQuad module. This module includes a feature that allows genome-wide mining of PG4 motifs and their visualization as circular histograms. TetraplexFinder, the module for mining PG4 motifs in user-provided sequences is now capable of handling up to 20 MB of data. QuadBase2 is a comprehensive PG4 motif mining tool that further expands the configurations and algorithms for mining PG4 motifs in a user-friendly way. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Guanine nucleotide induced conformational change of Cdc42 revealed by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Yang, Sheng-Wei; Ting, Hsiu-Chi; Lo, Yi-Ting; Wu, Ting-Yuan; Huang, Hung-Wei; Yang, Chia-Jung; Chan, Jui-Fen Riva; Chuang, Min-Chieh; Hsu, Yuan-Hao Howard

    2016-01-01

    Cdc42 regulates pathways related to cell division. Dysregulation of Cdc42 can lead to cancer, cardiovascular diseases and neurodegenerative diseases. GTP induced activation mechanism plays an important role in the activity and biological functions of Cdc42. P-loop, Switch I and Switch II are critical regions modulating the enzymatic activity of Cdc42. We applied amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (HDXMS) to investigate the dynamic changes of apo-Cdc42 after GDP, GTP and GMP-PCP binding. The natural substrate GTP induced significant decreases of deuteration in P-loop and Switch II, moderate changes of deuteration in Switch I and significant changes of deuteration in the α7 helix, a region far away from the active site. GTP binding induced similar effects on H/D exchange to its non-hydrolysable analog, GMP-PCP. HDXMS results indicate that GTP binding blocked the solvent accessibility in the active site leading to the decrease of H/D exchange rate surrounding the active site, and further triggered a conformational change resulting in the drastic decrease of H/D exchange rate at the remote α7 helix. Comparing the deuteration levels in three activation states of apo-Cdc42, Cdc42-GDP and Cdc42-GMP-PCP, the apo-Cdc42 has the most flexible structure, which can be stabilized by guanine nucleotide binding. The rates of H/D exchange of Cdc42-GDP are between the GMP-PCP-bound and the apo form, but more closely to the GMP-PCP-bound form. Our results show that the activation of Cdc42 is a process of conformational changes involved with P-loop, Switch II and α7 helix for structural stabilization. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Elimination and Utilization of Oxidized Guanine Nucleotides in the Synthesis of RNA and Its Precursors*

    Science.gov (United States)

    Sekiguchi, Takeshi; Ito, Riyoko; Hayakawa, Hiroshi; Sekiguchi, Mutsuo

    2013-01-01

    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 14C-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. PMID:23376345

  5. Demonstration of binding components specific for 7,8-disubstituted guanine ribonucleosides in murine B lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Goodman, M.G. (Research Institute of Scripps Clinic, La Jolla, CA (USA))

    1990-12-25

    7,8-Disubstituted guanine ribonucleosides are known to be potent intracellular modulators of immune responses. These compounds trigger and modulate a wide variety of lymphocyte responses including effects exerted directly on B cells. However, little is known about their mechanism of action. The current paper describes studies undertaken to evaluate whether binding components specific for these bioactive molecules exist in splenic B lymphocytes. After exposure of cells to labeled nucleoside, two different pools of nucleoside can be distinguished: a rapidly exchangeable nucleoside pool and a slowly exchangeable pool. The material in the latter pool consists of authentic unaltered nucleoside that is complexed to a relatively hydrophobic cellular component with an apparent Mr of 30,000-40,000; binding appears to interfere with free interaction of the nucleoside's cis hydroxyls with a boronate affinity resin. The slowly exchangeable nucleoside pool is seen to localize predominantly to the nucleus in electron microscopic autoradiographs. This pool is maximally bound by 30 min of incubation. Specific, saturable binding is demonstrable, with an apparent Kd of approximately 7 microM. This value correlates well with concentrations at which half-maximal biological activity occurs and suggests that the binding component likely mediates antigen-dependent immunomodulatory activity. Splenic B cells express approximately 2 x 10(4) binding sites/cell, whereas thymic lymphocytes, which do not respond functionally to nucleosides, do not display a measurable number of nucleoside binding sites. Ligand specificity of the binding interaction is confirmed by binding inhibition studies, in which binding inhibitory activity of unlabeled agonistic structural analogs recapitulate their degree of immunobiological activity.

  6. The influence of N-7 guanine modifications on the strength of Watson-Crick base pairing and guanine N-1 acidity: Comparison of gas-phase and condensed-phase trends

    Czech Academy of Sciences Publication Activity Database

    Burda, J. V.; Šponer, Jiří; Hrabáková, J.; Zeizinger, M.; Leszczynski, J.

    2003-01-01

    Roč. 107, č. 22 (2003), s. 5349-5356 ISSN 1520-6106 R&D Projects: GA MŠk ME 517; GA MŠk LN00A016 Grant - others:Wellcome Trust(GB) GR067507MF; ONR(US) N00034-03-1-0116; National Science Foundation(US) CREST 9805465 Institutional research plan: CEZ:AV0Z5004920 Keywords : Watson -Crick base pairing * guanines * gas-phase and condensed-phase trends Subject RIV: BO - Biophysics Impact factor: 3.679, year: 2003

  7. A comparative density functional theory study of guanine chemisorption on Al12N12, Al12P12, B12N12, and B12P12 nano-cages

    International Nuclear Information System (INIS)

    Shokuhi Rad, Ali; Ayub, Khurshid

    2016-01-01

    Density functional theory (DFT) calculations have been performed for adsorption of guanine (a nucleobase) on the surface of Al 12 N 12 (AlN), Al 12 P 12 (AlP), B 12 N 12 (BN), and B 12 P 12 (BP) nano-cages. The interaction of guanine with nano-cages is highly exothermic (chemisorption), and the adsorption energies are in order of AlN > AlP > BN > BP. Although AlN has the highest adsorption energy; however, BN and BP show more changes in electronic property upon adsorption of guanine. The results of charge analysis, density of states (DOSs), and frontier molecular orbital, confirm a distinguished orbital hybridization upon adsorption of guanine, which suggest potential application of BN as biochemical adsorbent for guanine. - Highlights: • The adsorption energies are in order of AlN > AlP > BN > BP upon adsorption of guanine. • BN and BP show more changes in electronic property upon adsorption of guanine. • Distinguished orbital hybridization upon adsorption of guanine. • The strongest interaction energy of guanine was obtained for AlN.

  8. Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

    Science.gov (United States)

    Shukla, P. K.; Mishra, P. C.; Suhai, S.

    Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the

  9. Kinetics of the interactions between yeast elongation factors 1A and 1Balpha, guanine nucleotides, and aminoacyl-tRNA

    DEFF Research Database (Denmark)

    Gromadski, Kirill B; Schümmer, Tobias; Strømgaard, Anne

    2007-01-01

    of guanine nucleotides. At the concentrations of nucleotides and factors prevailing in the cell, the overall exchange rate is expected to be in the range of 6 s(-1), which is compatible with the rate of protein synthesis in the cell. eEF1A.GTP binds Phe-tRNA(Phe) with a K(d) of 3 nm, whereas eEF1A.GDP shows...... no significant binding, indicating that eEF1A has similar tRNA binding properties as its prokaryotic homolog, EF-Tu. Udgivelsesdato: 2007-Dec-7...

  10. Regulation of mitotic spindle formation by the RhoA guanine nucleotide exchange factor ARHGEF10

    Directory of Open Access Journals (Sweden)

    Satoh Takaya

    2009-07-01

    Full Text Available Abstract Background The Dbl family guanine nucleotide exchange factor ARHGEF10 was originally identified as the product of the gene associated with slowed nerve-conduction velocities of peripheral nerves. However, the function of ARHGEF10 in mammalian cells is totally unknown at a molecular level. ARHGEF10 contains no distinctive functional domains except for tandem Dbl homology-pleckstrin homology and putative transmembrane domains. Results Here we show that RhoA is a substrate for ARHGEF10. In both G1/S and M phases, ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa cells. Furthermore, RNA interference-based knockdown of ARHGEF10 resulted in multipolar spindle formation in M phase. Each spindle pole seems to contain a centrosome consisting of two centrioles and the pericentriolar material. Downregulation of RhoA elicited similar phenotypes, and aberrant mitotic spindle formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively activated RhoA. Multinucleated cells were not increased upon ARHGEF10 knockdown in contrast to treatment with Y-27632, a specific pharmacological inhibitor for the RhoA effector kinase ROCK, which induced not only multipolar spindle formation, but also multinucleation. Therefore, unregulated centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10 knockdown-dependent multipolar spindle formation. We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10. Knockdown of KIF3B again caused multipolar spindle phenotypes. The supernumerary centrosome phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of ARHGEF10, RhoA or KIF3B was abrogated by RNA interference. Conclusion Collectively, our results suggest that a novel RhoA-dependent signaling pathway under the control of ARHGEF10 has a pivotal role in the regulation of the cell division cycle. This pathway is not involved in

  11. Simultaneous protection of organic p- and n-channels in complementary inverter from aging and bias-stress by DNA-base guanine/Al2O3 double layer.

    Science.gov (United States)

    Lee, Junyeong; Hwang, Hyuncheol; Min, Sung-Wook; Shin, Jae Min; Kim, Jin Sung; Jeon, Pyo Jin; Lee, Hee Sung; Im, Seongil

    2015-01-28

    Although organic field-effect transistors (OFETs) have various advantages of lightweight, low-cost, mechanical flexibility, and nowadays even higher mobility than amorphous Si-based FET, stability issue under bias and ambient condition critically hinder its practical application. One of the most detrimental effects on organic layer comes from penetrated atmospheric species such as oxygen and water. To solve such degradation problems, several molecular engineering tactics are introduced: forming a kinetic barrier, lowering the level of molecule orbitals, and increasing the band gap. However, direct passivation of organic channels, the most promising strategy, has not been reported as often as other methods. Here, we resolved the ambient stability issues of p-type (heptazole)/or n-type (PTCDI-C13) OFETs and their bias-stability issues at once, using DNA-base small molecule guanine (C5H5N5O)/Al2O3 bilayer. The guanine protects the organic channels as buffer/and H getter layer between the channels and capping Al2O3, whereas the oxide capping resists ambient molecules. As a result, both p-type and n-type OFETs are simultaneously protected from gate-bias stress and 30 days-long ambient aging, finally demonstrating a highly stable, high-gain complementary-type logic inverter.

  12. Higher order structural effects stabilizing the reverse watson-crick guanine-cytosine base pair in functional RNAs

    KAUST Repository

    Chawla, Mohit

    2013-10-10

    The G:C reverse Watson-Crick (W:W trans) base pair, also known as Levitt base pair in the context of tRNAs, is a structurally and functionally important base pair that contributes to tertiary interactions joining distant domains in functional RNA molecules and also participates in metabolite binding in riboswitches. We previously indicated that the isolated G:C W:W trans base pair is a rather unstable geometry, and that dicationic metal binding to the Guanine base or posttranscriptional modification of the Guanine can increase its stability. Herein, we extend our survey and report on other H-bonding interactions that can increase the stability of this base pair. To this aim, we performed a bioinformatics search of the PDB to locate all the occurencies of G:C trans base pairs. Interestingly, 66% of the G:C trans base pairs in the PDB are engaged in additional H-bonding interactions with other bases, the RNA backbone or structured water molecules. High level quantum mechanical calculations on a data set of representative crystal structures were performed to shed light on the structural stability and energetics of the various crystallographic motifs. This analysis was extended to the binding of the preQ1 metabolite to a preQ1-II riboswitch. 2013 The Author(s).

  13. The guanine nucleotide exchange factor Vav3 regulates differentiation of progenitor cells in the developing mouse retina.

    Science.gov (United States)

    Luft, Veronika; Reinhard, Jacqueline; Shibuya, Masabumi; Fischer, Klaus D; Faissner, Andreas

    2015-02-01

    The seven main cell types in the mammalian retina arise from multipotent retinal progenitor cells, a process that is tightly regulated by intrinsic and extrinsic signals. However, the molecular mechanisms that control proliferation, differentiation and cell-fate decisions of retinal progenitor cells are not fully understood yet. Here, we report that the guanine nucleotide exchange factor Vav3, a regulator of Rho-GTPases, is involved in retinal development. We demonstrate that Vav3 is expressed in the mouse retina during the embryonic period. In order to study the role of Vav3 in the developing retina, we generate Vav3-deficient mice. The loss of Vav3 results in an accelerated differentiation of retinal ganglion cells and cone photoreceptors during early and late embryonic development. We provide evidence that more retinal progenitor cells express the late progenitor marker Sox9 in Vav3-deficient mice than in wild-types. This premature differentiation is compensated during the postnatal period and late-born cell types such as bipolar cells and Müller glia display normal numbers. Taken together, our data imply that Vav3 is a regulator of retinal progenitor cell differentiation, thus highlighting a novel role for guanine nucleotide exchange factors in retinogenesis.

  14. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M. (National Institutes of Health, Bethesda, MD (USA))

    1989-08-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A){sup +} RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A){sup +} RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs.

  15. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: Two guanine nucleotide-dependent activators of cholera toxin

    International Nuclear Information System (INIS)

    Bobak, D.A.; Nightingale, M.S.; Murtagh, J.J.; Price, S.R.; Moss, J.; Vaughan, M.

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A) + RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A) + RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFS also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs

  16. Constructing a novel 8-hydroxy-2'-deoxyguanosine electrochemical sensor and application in evaluating the oxidative damages of DNA and guanine.

    Science.gov (United States)

    Guo, Zhipan; Liu, Xiuhui; Liu, Yuelin; Wu, Guofan; Lu, Xiaoquan

    2016-12-15

    8-Hydroxy-2'-deoxyguanosine (8-OHdG) is commonly identified as a biomarker of oxidative DNA damage. In this work, a novel and facile 8-OHdG sensor was developed based on the multi-walled carbon nanotubes (MWCNTs) modified glassy carbon electrode (GCE). It exhibited good electrochemical responses toward the oxidation of 8-OHdG, and the linear ranges were 5.63×10(-8)-6.08×10(-6)M and 6.08×10(-6)-1.64×10(-5)M, with the detection limit of 1.88×10(-8)M (S/N=3). Moreover, the fabricated sensor was applied for the determination of 8-OHdG generated from damaged DNA and guanine, respectively, and the oxidation currents of 8-OHdG increased along with the damaged DNA and guanine within certain concentrations. These results could be used to evaluate the DNA damage, and provide useful information on diagnosing diseases caused by mutation and deficiency of the immunity system. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. A compositional segmentation of the human mitochondrial genome is related to heterogeneities in the guanine mutation rate

    Science.gov (United States)

    Samuels, David C.; Boys, Richard J.; Henderson, Daniel A.; Chinnery, Patrick F.

    2003-01-01

    We applied a hidden Markov model segmentation method to the human mitochondrial genome to identify patterns in the sequence, to compare these patterns to the gene structure of mtDNA and to see whether these patterns reveal additional characteristics important for our understanding of genome evolution, structure and function. Our analysis identified three segmentation categories based upon the sequence transition probabilities. Category 2 segments corresponded to the tRNA and rRNA genes, with a greater strand-symmetry in these segments. Category 1 and 3 segments covered the protein- coding genes and almost all of the non-coding D-loop. Compared to category 1, the mtDNA segments assigned to category 3 had much lower guanine abundance. A comparison to two independent databases of mitochondrial mutations and polymorphisms showed that the high substitution rate of guanine in human mtDNA is largest in the category 3 segments. Analysis of synonymous mutations showed the same pattern. This suggests that this heterogeneity in the mutation rate is partly independent of respiratory chain function and is a direct property of the genome sequence itself. This has important implications for our understanding of mtDNA evolution and its use as a ‘molecular clock’ to determine the rate of population and species divergence. PMID:14530452

  18. Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization.

    Science.gov (United States)

    Peng, Jun; Ling, Jian; Zhang, Xiu-Qing; Bai, Hui-Ping; Zheng, Liyan; Cao, Qiu-E; Ding, Zhong-Tao

    2015-02-25

    In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0-160.0 and 6-240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Dependence of DNA-protein cross-linking via guanine oxidation upon local DNA sequence as studied by restriction endonuclease inhibition.

    Science.gov (United States)

    Madison, Amanda L; Perez, Zitadel A; To, Phuong; Maisonet, Tiffany; Rios, Eunice V; Trejo, Yuri; Ochoa-Paniagua, Carmen; Reno, Anita; Stemp, Eric D A

    2012-01-10

    Oxidative damage plays a causative role in many diseases, and DNA-protein cross-linking is one important consequence of such damage. It is known that GG and GGG sites are particularly prone to one-electron oxidation, and here we examined how the local DNA sequence influences the formation of DNA-protein cross-links induced by guanine oxidation. Oxidative DNA-protein cross-linking was induced between DNA and histone protein via the flash quench technique, a photochemical method that selectively oxidizes the guanine base in double-stranded DNA. An assay based on restriction enzyme cleavage was developed to detect the cross-linking in plasmid DNA. Following oxidation of pBR322 DNA by flash quench, several restriction enzymes (PpuMI, BamHI, EcoRI) were then used to probe the plasmid surface for the expected damage at guanine sites. These three endonucleases were strongly inhibited by DNA-protein cross-linking, whereas the AT-recognizing enzyme AseI was unaffected in its cleavage. These experiments also reveal the susceptibility of different guanine sites toward oxidative cross-linking. The percent inhibition observed for the endonucleases, and their pBR322 cleavage sites, decreased in the order: PpuMI (5'-GGGTCCT-3' and 5'-AGGACCC-3') > BamHI (5'-GGATCC-3') > EcoRI (5'-GAATTC-3'), a trend consistent with the observed and predicted tendencies for guanine to undergo one-electron oxidation: 5'-GGG-3' > 5'-GG-3' > 5'-GA-3'. Thus, it appears that in mixed DNA sequences the guanine sites most vulnerable to oxidative cross-linking are those that are easiest to oxidize. These results further indicate that equilibration of the electron hole in the plasmid DNA occurs on a time scale faster than that of cross-linking.

  20. Translocation of microfilament-associated inhibitory guanine-nucleotide-binding proteins to the plasma membrane in myeloid differentiated human leukemia (HL-60) cells

    NARCIS (Netherlands)

    Meyer zu Heringdorf, D.; Liedel, K.; Kaldenberg-Stasch, S.; Michel, M. C.; Jakobs, K. H.; Wieland, T.

    1996-01-01

    The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to

  1. Estrogen Repression of MicroRNAs Is Associated with High Guanine Content in the Terminal Loop Sequences of Their Precursors

    Directory of Open Access Journals (Sweden)

    Amit Cohen

    2017-08-01

    Full Text Available Widespread microRNA (miRNA repression is a phenomenon observed in mammals after exposure to cigarette smoke and in many types of cancer. A comprehensive reduction in miRNA expression after treatment with the hormone estrogen has also previously been described. Here, we reveal a conserved association of miRNA downregulation after estrogen exposure in zebrafish, mouse, and human breast cancer cell line, with a high guanine content in the terminal loop sequences of their precursors, and offer a possible link between estrogen-related miRNA-adducts formation and carcinogenesis. We also show common gene expression patterns shared by breast cancer tumors and estrogen-treated zebrafish, suggesting that this organism can be used as a powerful model system for the study of human breast cancer.

  2. Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

    Science.gov (United States)

    Marei, Hadir; Carpy, Alejandro; Woroniuk, Anna; Vennin, Claire; White, Gavin; Timpson, Paul; Macek, Boris; Malliri, Angeliki

    2016-01-01

    The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner. PMID:26887924

  3. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Science.gov (United States)

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  4. Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  5. Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

    DEFF Research Database (Denmark)

    Jacobsen, Jack Hummeland; Schmiegelow, Kjeld; Nersting, Jakob

    2012-01-01

    by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10 pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1 fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery...... was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra...... for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified...

  6. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor

    Science.gov (United States)

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G. H.; Fukuhara, Shigetomo; Taylor, Susan S.; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-01-01

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. PMID:26797121

  7. Specific binding of (/sup 3/H)substance P to the rat submaxillary gland. The effects of ions and guanine nucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Bahouth, S.W.; Musacchio, J.M.

    1985-08-01

    (/sup 3/H)Substance P ((/sup 3/H)SP), in a high ionic strength incubation medium, binds to a single class of saturable, noninteracting binding sites on rat submaxillary gland membranes with a KD = 2.8 +/- 0.34 nM and maximum binding (Bmax) = 220 +/- 31 fmol/mg of protein. The rank order of potency of various tachykinins, SP fragments and analogs to compete against (/sup 3/H)SP is correlated with their potency to induce salivation. These findings indicate that, under the conditions described, (/sup 3/H)SP binds to a physiologically relevant tachykinin receptor of the SP-P subtype. (/sup 3/H)SP binding increases by 35% in the presence of optimal concentrations of Mn++ and Mg++ whereas guanine nucleotides reduce (/sup 3/H)SP binding. The effect produced by either divalent cations or guanine nucleotides is due to increasing or decreasing the Bmax, respectively, without changing the affinity of (/sup 3/H)SP. Guanine nucleotides reduce the Bmax of (/sup 3/H)SP to the same level in the presence or absence of divalent cations, indicating that divalent cations increase the population of SP receptors that are sensitive to guanine nucleotides. In low ionic strength media, and when the nonspecific binding is defined by 1 microM SP, (/sup 3/H)SP binds to two sites: a high affinity site with a KD of 0.14 nM and a Bmax of 370 fmol/mg of protein and a low affinity high capacity site. When the nonspecific binding is defined by 1 microM physalaemin, the high affinity is the only detectable site. However, in low ionic strength media, physalaemin has about one-fiftieth the potency of SP in competing with (/sup 3/H)SP.

  8. Role of loops in the guanine quadruplex formation by DNA/RNA hybrid analogs of G4T4G4

    Czech Academy of Sciences Publication Activity Database

    Vondrušková, Jitka; Kypr, Jaroslav; Kejnovská, Iva; Fialová, Markéta; Vorlíčková, Michaela

    2008-01-01

    Roč. 43, č. 5 (2008), s. 463-467 ISSN 0141-8130 R&D Projects: GA ČR(CZ) GA204/07/0057; GA AV ČR(CZ) IAA100040701 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : nucleic acids * RNA /DNA hybrids * guanine quadruplexes Subject RIV: BO - Biophysics Impact factor: 1.867, year: 2008

  9. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

    Science.gov (United States)

    Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

    2015-02-01

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  10. Comparative analysis of urinary N7-(2-hydroxyethyl)guanine for ethylene oxide- and non-exposed workers.

    Science.gov (United States)

    Huang, Chih-Chun Jean; Wu, Chia-Fang; Shih, Wei-Chung; Chen, Ming-Feng; Chen, Chang-Yuh; Chien, Yeh-Chung; Liou, Saou-Hsing; Chiang, Su-Yin; Wu, Kuen-Yuh

    2011-05-10

    Ethylene oxide (EO), a direct alkylating agent and a carcinogen, can attack the nucleophilic sites of DNA bases to form a variety of DNA adducts. The most abundant adduct, N7-(2-hydroxyethyl)guanine (N7-HEG), can be depurinated spontaneously or enzymatically from DNA backbone to form abasic sites. Molecular dosimetry of the excised N7-HEG in urine can serve as an EO exposure and potential risk-associated biomarker. This study was to analyze N7-HEG in urine collected from 89 EO-exposed and 48 nonexposed hospital workers and 20 exposed and 10 nonexposed factory workers by using our newly developed on-line solid-phase extraction isotope-dilution LC-MS/MS method. Statistical analysis of data shows that the exposed factory workers excreted significantly greater concentrations of N7-HEG than both the nonexposed factory workers and hospital workers. Multiple linear regression analysis reveals that the EO-exposed factory workers had a significantly greater post-shift urinary N7-HEG than their nonexposed coworkers and hospital workers. These results demonstrate that analysis of urinary N7-HEG can serve as a biomarker of EO exposure for future molecular epidemiology studies to better understand the role of the EO-induced DNA adduct formation in EO carcinogenicity and certainly for routine surveillance of occupational EO exposure for the study of potential health impacts on workers. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  11. Total inhibition of (1)O2-induced oxidative damage to guanine bases of DNA/RNA by turmeric extracts.

    Science.gov (United States)

    Joshi, Prakash C; Li, Hsin H; Merchant, Monique; Keane, Thomas C

    2014-09-26

    The guanine base of nucleic acids is known to be very reactive towards degradation by (1)O2-induced oxidative stress. Oxidative reactions of DNA are linked to many human diseases including cancer. Among the various forms of reactive O2 species (OH, (1)O2 or O2(-)), the oxidative stress caused by (1)O2 is of particular physiologic importance because of its selectively long life in aqueous medium and its ability to diffuse through a cell membrane. In this study we investigated the degradation of a model compound guanosine (Guo) by (1)O2, which was generated by riboflavin-induced photosensitization and by molybdate ion catalyzed disproportionation of H2O2. We observed the remarkable ability of an aqueous and alcoholic extracts of Turmeric (Curcuma longa) as an extraordinary scavenger of (1)O2 to completely inhibit the degradation of Guo. The alcoholic extracts were more effective in their antioxidant activity than the corresponding water extract. This naturally occurring antioxidant offers a most economical supplement to protect biologically significant molecules from the oxidative stress induced by (1)O2. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Directory of Open Access Journals (Sweden)

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  13. Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

    International Nuclear Information System (INIS)

    Hartley, J.A.; Forrow, S.M.; Souhami, R.L.

    1990-01-01

    Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increase ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specific chemical cleavage technique for DNA sequencing. The result differed with both the nitrogen mustard and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove

  14. Role of a guanine nucleotide-binding protein in α1-adrenergic receptor-mediated Ca2+ mobilization in DDT1 MF-2 cells

    International Nuclear Information System (INIS)

    Cornett, L.E.; Norris, J.S.

    1987-01-01

    In this study the mechanisms involved in α 1 -adrenergic receptor-mediated Ca 2+ mobilization at the level of the plasma membrane were investigated. Stimulation of 45 Ca 2+ efflux from saponin-permeabilized DDT 1 MF-2 cells was observed with the addition of either the α 1 -adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of [ 32 P] NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT 1 , MF-2 cells, possibly the α-subunit of N/sub i/. However, stimulation of unidirectional 45 Ca 2+ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the α 1 -adrenergic receptor to Ca 2+ mobilization in DDT 1 MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family

  15. The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain.

    Science.gov (United States)

    Xu, Zhen; Gakhar, Lokesh; Bain, Fletcher E; Spies, Maria; Fuentes, Ernesto J

    2017-10-27

    T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PH n -CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PH c ) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PH n -PH c ), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PH n -CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PH c domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PH n -PH c fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PH n -CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Guanine nucleotide-binding protein subunit beta-2-like 1, a new Annexin A7 interacting protein

    Energy Technology Data Exchange (ETDEWEB)

    Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu, E-mail: jianwutdlmedu@163.com

    2014-02-28

    Highlights: • RACK1 formed a complex with Annexin A7. • Depletion of RACK1 inhibited the proliferation, migration and invasion. • RACK1 RNAi abolished RACK1-Annexin A7 interaction. • RACK1-Annexin A7 may play a role in regulating the metastatic potentials. - Abstract: We report for the first time that Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) formed a complex with Annexin A7. Hca-F and Hca-P are a pair of syngeneic mouse hepatocarcinoma cell lines established and maintained in our laboratory. Our previous study showed that both Annexin A7 and RACK1 were expressed higher in Hca-F (lymph node metastasis >70%) than Hca-P (lymph node metastasis <30%). Suppression of Annexin A7 expression in Hca-F cells induced decreased migration and invasion ability. In this study, knockdown of RACK1 by RNA interference (RNAi) had the same impact on metastasis potential of Hca-F cells as Annexin A7 down-regulation. Furthermore, by co-immunoprecipitation and double immunofluorescence confocal imaging, we found that RACK1 was in complex with Annexin A7 in control cells, but not in the RACK1-down-regulated cells, indicating the abolishment of RACK1-Annexin A7 interaction in Hca-F cells by RACK1 RNAi. Taken together, these results suggest that RACK1-Annexin A7 interaction may be one of the means by which RACK1 and Annexin A7 influence the metastasis potential of mouse hepatocarcinoma cells in vitro.

  17. Modulation of B-cell receptor and microenvironment signaling by a guanine exchange factor in B-cell malignancies

    International Nuclear Information System (INIS)

    Liao, Wei; Sharma, Sanjai

    2016-01-01

    Objective: Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) cells over-express a guanine exchange factor (GEF), Rasgrf-1. This GEF increases active Ras as it catalyzes the removal of GDP from Ras so that GTP can bind and activate Ras. This study aims to study the mechanism of action of Rasgrf-1 in B-cell malignancies. Methods: N-terminus truncated Rasgrf-1 variants have a higher GEF activity as compared to the full-length transcript therefore a MCL cell line with stable over-expression of truncated Rasgrf-1 was established. The B-cell receptor (BCR) and chemokine signaling pathways were compared in the Rasgrf-1 over-expressing and a control transfected cell line. Results: Cells over-expressing truncated form of Rasgrf-1 have a higher proliferative rate as compared to control transfected cells. BCR was activated by lower concentrations of anti-IgM antibody in Rasgrf-1 over-expressing cells as compared to control cells indicating that these cells are more sensitive to BCR signaling. BCR signaling also phosphorylates Rasgrf-1 that further increases its GEF function and amplifies BCR signaling. This activation of Rasgrf-1 in over-expressing cells resulted in a higher expression of phospho-ERK, AKT, BTK and PKC-alpha as compared to control cells. Besides BCR, Rasgrf-1 over-expressing cells were also more sensitive to microenvironment stimuli as determined by resistance to apoptosis, chemotaxis and ERK pathway activation. Conclusions: This GEF protein sensitizes B-cells to BCR and chemokine mediated signaling and also upregulates a number of other signaling pathways which promotes growth and survival of these cells

  18. Localization of the 5-phospho-alpha-D-ribosyl-1-pyrophosphate binding site of human hypoxanthine-guanine phosphoribosyltransferase.

    Science.gov (United States)

    Keough, D T; Emmerson, B T; de Jersey, J

    1991-02-22

    Human erythrocyte hypoxanthine-guanine phosphoribosyltransferase (HPRT) is inactivated by iodoacetate in the absence, but not in the presence, of the substrate, 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRib-PP). Treatment of HPRT with [14C]iodoacetate followed by tryptic digestion, peptide separation and sequencing has shown that Cys-22 reacts with iodoacetate only in the absence of PRib-PP; this strongly suggests that Cys-22 is in or near the PRib-PP binding site. In contrast, Cys-105 reacts with [14C]iodoacetate both in the presence and absence of PRib-PP. Carboxymethylation of Cys-22 resulted in an increase in the Km for PRib-PP, but no change in Vmax. Storage of HPRT also resulted in an increase in the Km for PRib-PP and a decrease in its susceptibility to inactivation by iodoacetate. Dialysis of stored enzyme against 1 mM dithiothreitol resulted in a marked decrease in Km for PRib-PP. The stoichiometry of the reaction of [14C]iodoacetate with Cys-22 in HPRT leading to inactivation (approx. 1 residue modified per tetramer) showed that, in this preparation of HPRT purified from erythrocytes, only about 25% of the Cys-22 side chains were present as free and accessible thiols. Titration of thiol groups [with 5,5'-dithiobis(2-nitrobenzoic acid)] and the effect of dithiothreitol on Km for PRib-PP indicate that oxidation of thiol groups occurs on storage of HPRT, even in the presence of 1 mM beta-mercaptoethanol.

  19. The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

    Directory of Open Access Journals (Sweden)

    Geldner Niko

    2005-02-01

    Full Text Available Abstract Background Small G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown. Results Based on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7. Conclusion Our analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

  20. Relative abundances of primary ion radicals in γ-irradiated DNA: Cytosine vs thymine anions and guanine vs adenine cations

    International Nuclear Information System (INIS)

    Sevilla, M.D.; Becker, D.; Yan, Mengyao; Summerfield, S.R.

    1991-01-01

    An ESR study of the relative distribution of ion radicals formed in DNA equilibrated with D 2 O and γ-irradiated at 77 K is presented. The ESR spectra of irradiated DNA and polynucleotides (poly[dG]·poly[dC] and poly[dAdT]·poly[dAdT]) were obtained and employed in a computer-assisted analysis for the individual ion-radical distribution. Analysis of spectra as a function of power allowed the separation of the spectra of the pyrimidine anions (T sm-bullet- , C sm-bullet- ) from the spectra of the purine cations (G sm-bullet+ , A sm-bullet+ ). The spectra of the mononucleotide ion radicals, dCMP sm-bullet- , dTMP sm-bullet- , dGMP sm-bullet+ , and dAMP sm-bullet+ , were produced in 8 M LiCl glasses. In addition, the spectra of the ion radicals of all of the mononucleotide ion radicals except dAMP + were simulated by using hyperfine and g tensors from the literature. Basis spectra derived from (1) power saturation experiments, (2) polynucleotide and mononucleotide spectra, (3) spectra of mononucleotides alone, and (4) anisotropic simulations were used to fit the spectra of DNA by use of a linear least-squares analysis. Each of the four separate analyses confirms that the cytosine anion dominates the spectra of DNA at 100 K. Three analyses included the cationic composition, and they strongly favor the guanine cation over the adenine cation. An average of the authors results gives the DNA ion radicals' relative to abundances as ca. 77% C sm-bullet- , 23% T sm-bullet- for the anions and >90% G sm-bullet+ for the cations about equal amounts of anions and cations are present. No difference in results is found for DNA irradiated in frozen D 2 O solutions or simply exchanged at 100% D 2 O humidity

  1. Overexpression of GEFT, a Rho family guanine nucleotide exchange factor, predicts poor prognosis in patients with rhabdomyosarcoma.

    Science.gov (United States)

    Sun, Chao; Liu, Chunxia; Li, Shugang; Li, Hongan; Wang, Yuanyuan; Xie, Yuwen; Li, Bingcheng; Cui, Xiaobin; Chen, Yunzhao; Zhang, Wenjie; Li, Feng

    2014-01-01

    Rhabdomyosarcoma (RMS) is one of the most common soft-tissue sarcomas in children and adolescents with poor prognosis. Yet, there is lack of effective prognostic biomarkers for RMS. The present study, therefore, aimed to explore potential biomarkers for RMS based on our previous findings using array comparative genomic hybridization. We investigated guanine nucleotide exchange factor, GEFT, at expression level in 45 RMS patients and 36 normal striated muscle controls using immunohistochemistry using tissue microarrays. The expression rate of GEFT in RMS samples (42/45, 93.33%) was significantly higher (Prate of GEFT in RMS (31/45, 68.89%) was also significantly higher (P<0.05) than that in normal controls (0/36, 0.00%). Increased expression of GEFT correlated significantly with advanced disease stages (stages III/IV) (P=0.001), lymph node metastasis (P=0.019), and distant metastasis (P=0.004), respectively, in RMS patients. In addition, RMS patients having overexpressed GEFT experienced worse overall survival (OS) than those having low levels of GEFT (P=0.001). GEFT overexpression was determined to be an independent prognostic factor for poor OS in RMS patients (hazard ratio: 3.491, 95% confidence interval: 1.121-10.871, P=0.004). In conclusion, these observations provide the first evidence of GEFT overexpression in RMS and its correlations with disease aggressiveness and metastasis. These findings suggest that GEFT may serve as a promising biomarker predicting poor prognosis in RMS patients, thus implying its potential as a therapeutic target.

  2. Investigation of specificity determinants in bacterial tRNA-guanine transglycosylase reveals queuine, the substrate of its eucaryotic counterpart, as inhibitor.

    Directory of Open Access Journals (Sweden)

    Inna Biela

    Full Text Available Bacterial tRNA-guanine transglycosylase (Tgt catalyses the exchange of the genetically encoded guanine at the wobble position of tRNAs(His,Tyr,Asp,Asn by the premodified base preQ1, which is further converted to queuine at the tRNA level. As eucaryotes are not able to synthesise queuine de novo but acquire it through their diet, eucaryotic Tgt directly inserts the hypermodified base into the wobble position of the tRNAs mentioned above. Bacterial Tgt is required for the efficient pathogenicity of Shigella sp, the causative agent of bacillary dysentery and, hence, it constitutes a putative target for the rational design of anti-Shigellosis compounds. Since mammalian Tgt is known to be indirectly essential to the conversion of phenylalanine to tyrosine, it is necessary to create substances which only inhibit bacterial but not eucaryotic Tgt. Therefore, it seems of utmost importance to study selectivity-determining features within both types of proteins. Homology models of Caenorhabditis elegans Tgt and human Tgt suggest that the replacement of Cys158 and Val233 in bacterial Tgt (Zymomonas mobilis Tgt numbering by valine and accordingly glycine in eucaryotic Tgt largely accounts for the different substrate specificities. In the present study we have created mutated variants of Z. mobilis Tgt in order to investigate the impact of a Cys158Val and a Val233Gly exchange on catalytic activity and substrate specificity. Using enzyme kinetics and X-ray crystallography, we gained evidence that the Cys158Val mutation reduces the affinity to preQ1 while leaving the affinity to guanine unaffected. The Val233Gly exchange leads to an enlarged substrate binding pocket, that is necessary to accommodate queuine in a conformation compatible with the intermediately covalently bound tRNA molecule. Contrary to our expectations, we found that a priori queuine is recognised by the binding pocket of bacterial Tgt without, however, being used as a substrate.

  3. Studies by Near Edge X-ray Absorption Spectroscopies of Bonding Dynamics at the Graphene/Guanine Interface - A Proposal for High Mobility, Organic Graphene Field Effect Transistors

    Science.gov (United States)

    2015-07-01

    C=O   impurities .  In  applications  where  4  ML  of   graphene  is  needed,  NEXAFS  spectra  yields  little...AFRL-AFOSR-UK-TR-2015-0034 Studies by Near Edge X-ray Absorption Spectroscopies of Bonding Dynamics at the Graphene /Guanine...Interface – A Proposal for High Mobility, Organic Graphene Field Effect Transistors Eva Campo BANGOR UNIVERSITY COLLEGE ROAD BANGOR

  4. Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor.

    Science.gov (United States)

    Cooper, C L; Morris, A J; Harden, T K

    1989-04-15

    Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be

  5. The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

    Science.gov (United States)

    Moore, Elaine Ann; Xu, Yao-Zhong

    2017-01-01

    The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T) as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

  6. Voltammetric and electrochemical gravimetric selective detection of interactions between Tl(I) and guanine and the influence on activity of DNA drug-intercalators.

    Science.gov (United States)

    Nowicka, Anna M; Mackiewicz, Marcin; Matysiak, Edyta; Krasnodebska-Ostrega, Beata; Stojek, Zbigniew

    2013-03-15

    The interactions of Tl(I), a well known toxic species, with selected oligonucleotides were examined. The oligonucleotide sequences selected for the investigation were taken from gene hOGG1 responsible for repairing of DNA damage. Cyclic voltammetry was particularly useful, since nitrogen N-7 in guanine can be electrooxidized while its binding with Tl(I) leads to the loss of electroactivity. So, this selected interaction could be quantitatively used in drawing Scatchard's plot and calculating the binding constants and the number of active sites in guanine molecules occupied by one metal ion. Further, we have shown that the presence of Tl(I) leads to a decrease in activity of doxorubicin (DOX), a popular anticancer drug, vs. DNA. The obtained circular dichroism (CD) spectra and the measurements with an electrochemical quartz crystal microbalance (EQCM) led to a conclusion that in the presence of monovalent thallium cations the DNA double helix was neither damaged/oxidized nor its conformation changed substantially. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. The effect of S-substitution at the O6-guanine site on the structure and dynamics of a DNA oligomer containing a G:T mismatch.

    Directory of Open Access Journals (Sweden)

    Elaine Ann Moore

    Full Text Available The effect of S-substitution on the O6 guanine site of a 13-mer DNA duplex containing a G:T mismatch is studied using molecular dynamics. The structure, dynamic evolution and hydration of the S-substituted duplex are compared with those of a normal duplex, a duplex with S-substitution on guanine, but no mismatch and a duplex with just a G:T mismatch. The S-substituted mismatch leads to cell death rather than repair. One suggestion is that the G:T mismatch recognition protein recognises the S-substituted mismatch (GS:T as G:T. This leads to a cycle of futile repair ending in DNA breakage and cell death. We find that some structural features of the helix are similar for the duplex with the G:T mismatch and that with the S-substituted mismatch, but differ from the normal duplex, notably the helical twist. These differences arise from the change in the hydrogen-bonding pattern of the base pair. However a marked feature of the S-substituted G:T mismatch duplex is a very large opening. This showed considerable variability. It is suggested that this enlarged opening would lend support to an alternative model of cell death in which the mismatch protein attaches to thioguanine and activates downstream damage-response pathways. Attack on the sulphur by reactive oxygen species, also leading to cell death, would also be aided by the large, variable opening.

  8. Effective Strategy for Conformer-Selective Detection of Short-Lived Excited State Species: Application to the IR Spectroscopy of the N1H Keto Tautomer of Guanine.

    Science.gov (United States)

    Asami, Hiroya; Tokugawa, Munefumi; Masaki, Yoshiaki; Ishiuchi, Shun-Ichi; Gloaguen, Eric; Seio, Kohji; Saigusa, Hiroyuki; Fujii, Masaaki; Sekine, Mitsuo; Mons, Michel

    2016-04-14

    The ultrafast deactivation processes in the excited state of biomolecules, such as the most stable tautomers of guanine, forbid any state-of-the-art gas phase spectroscopic studies on these species with nanosecond lasers. This drawback can be overcome by grafting a chromophore having a long-lived excited state to the molecule of interest, allowing thus a mass-selective detection by nanosecond R2PI and therefore double resonance IR/UV conformer-selective spectroscopic studies. The principle is presently demonstrated on the keto form of a modified 9-methylguanine, for which the IR/UV double resonance spectrum in the C═O stretch region, reported for the first time, provides evidence for extensive vibrational couplings within the guanine moiety. Such a successful strategy opens up a route to mass-selective IR/UV spectroscopic investigations on molecules exhibiting natural chromophores having ultrashort-lived excited states, such as DNA bases, their complexes as well as peptides containing short-lived aromatic residues.

  9. Oxidation of guanine in G, GG, and GGG sequence contexts by aromatic pyrenyl radical cations and carbonate radical anions: relationship between kinetics and distribution of alkali-labile lesions.

    Science.gov (United States)

    Lee, Young Ae; Durandin, Alexander; Dedon, Peter C; Geacintov, Nicholas E; Shafirovich, Vladimir

    2008-02-14

    Oxidatively generated DNA damage induced by the aromatic radical cation of the pyrene derivative 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT), and by carbonate radicals anions, was monitored from the initial one-electron transfer, or hole injection step, to the formation of hot alkali-labile chemical end-products monitored by gel electrophoresis. The fractions of BPT molecules bound to double-stranded 20-35-mer oligonucleotides with noncontiguous guanines G and grouped as contiguous GG and GGG sequences were determined by a fluorescence quenching method. Utilizing intense nanosecond 355 nm Nd:YAG laser pulses, the DNA-bound BPT molecules were photoionized to BPT*+ radicals by a consecutive two-photon ionization mechanism. The BPT*+ radicals thus generated within the duplexes selectively oxidize guanine by intraduplex electron-transfer reactions, and the rate constants of these reactions follow the trend 5'-..GGG.. > 5'-..GG.. > 5'-..G... In the case of CO3*- radicals, the oxidation of guanine occurs by intermolecular collision pathways, and the bimolecular rate constants are independent of base sequence context. However, the distributions of the end-products generated by CO3*- radicals, as well as by BPT*+, are base sequence context-dependent and are greater than those in isolated guanines at the 5'-G in 5'-...GG... sequences, and the first two 5'- guanines in the 5'-..GGG sequences. These results help to clarify the conditions that lead to a similar or different base sequence dependence of the initial hole injection step and the final distribution of oxidized, alkali-labile guanine products. In the case of the intermolecular one-electron oxidant CO3*-, the rate constant of hole injection is similar for contiguous and isolated guanines, but the subsequent equilibration of holes by hopping favors trapping and product formation at contiguous guanines, and the sequence dependence of these two phenomena are not correlated. In contrast, in the case of the DNA

  10. Causes and consequences of plant radio-resistance. Formation of DNA basis lesions and self-repairing activity of one of them, the 8-oxo-7,8-dihydro-guanine in Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Dany, A.L.

    2001-01-01

    In this research thesis, the author first explains how and why DNA is injured when it is submitted to an oxidizing stress, and describes precisely the formation and the biological consequences of lesions of DNA bases, the 8-oxo-7,8-dihydro-guanine (8-oxoGua). She describes the repairing activities of the oxidized DNA, and more particularly the repairing of 8-oxoGua, in prokaryotes as well as in yeast, mammals and plants. Methodologies used are described, together with the repair activities of the 8-oxo-7,8-dihydro-guanine following a biochemical type approach and a molecular biology approach

  11. Identification of the Structural Features of Guanine Derivatives as MGMT Inhibitors Using 3D-QSAR Modeling Combined with Molecular Docking.

    Science.gov (United States)

    Sun, Guohui; Fan, Tengjiao; Zhang, Na; Ren, Ting; Zhao, Lijiao; Zhong, Rugang

    2016-06-23

    DNA repair enzyme O⁶-methylguanine-DNA methyltransferase (MGMT), which plays an important role in inducing drug resistance against alkylating agents that modify the O⁶ position of guanine in DNA, is an attractive target for anti-tumor chemotherapy. A series of MGMT inhibitors have been synthesized over the past decades to improve the chemotherapeutic effects of O⁶-alkylating agents. In the present study, we performed a three-dimensional quantitative structure activity relationship (3D-QSAR) study on 97 guanine derivatives as MGMT inhibitors using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. Three different alignment methods (ligand-based, DFT optimization-based and docking-based alignment) were employed to develop reliable 3D-QSAR models. Statistical parameters derived from the models using the above three alignment methods showed that the ligand-based CoMFA (Qcv² = 0.672 and Rncv² = 0.997) and CoMSIA (Qcv² = 0.703 and Rncv² = 0.946) models were better than the other two alignment methods-based CoMFA and CoMSIA models. The two ligand-based models were further confirmed by an external test-set validation and a Y-randomization examination. The ligand-based CoMFA model (Qext² = 0.691, Rpred² = 0.738 and slope k = 0.91) was observed with acceptable external test-set validation values rather than the CoMSIA model (Qext² = 0.307, Rpred² = 0.4 and slope k = 0.719). Docking studies were carried out to predict the binding modes of the inhibitors with MGMT. The results indicated that the obtained binding interactions were consistent with the 3D contour maps. Overall, the combined results of the 3D-QSAR and the docking obtained in this study provide an insight into the understanding of the interactions between guanine derivatives and MGMT protein, which will assist in designing novel MGMT inhibitors with desired activity.

  12. From lin-Benzoguanines to lin-Benzohypoxanthines as Ligands for Zymomonas mobilis tRNA-Guanine Transglycosylase: Replacement of Protein-Ligand Hydrogen Bonding by Importing Water Clusters.

    NARCIS (Netherlands)

    Barandun, L.J.; Immekus, F.; Kohler, P.C.; Tonazzi, S.; Wagner, B.; Wendelspiess, S.; Ritschel, T.; Heine, A.; Kansy, M.; Klebe, G.; Diederich, F.

    2012-01-01

    The foodborne illness shigellosis is caused by Shigella bacteria that secrete the highly cytotoxic Shiga toxin, which is also formed by the closely related enterohemorrhagic Escherichia coli (EHEC). It has been shown that tRNA-guanine transglycosylase (TGT) is essential for the pathogenicity of

  13. Guanine nucleotide exchange factor αPIX leads to activation of the Rac 1 GTPase/glycogen phosphorylase pathway in interleukin (IL)-2-stimulated T cells

    DEFF Research Database (Denmark)

    Llavero, Francisco; Urzelai, Bakarne; Osinalde, Nerea

    2015-01-01

    Recently, we have reported that the active form of Rac 1 GTPase binds to the glycogen phosphorylase muscle isoform (PYGM) and modulates its enzymatic activity leading to T cell proliferation. In the lymphoid system, Rac 1 and in general other small GTPases of the Rho family participate...... in the signaling cascades that are activated after engagement of the T cell antigen receptor. However, little is known about the IL-2-dependent Rac 1 activator molecules. For the first time, a signaling pathway leading to the activation of Rac 1/PYGM in response to IL-2-stimulated T cell proliferation is described....... More specifically, αPIX, a known guanine nucleotide exchange factor for the small GTPases of the Rho family, preferentially Rac 1, mediates PYGM activation in Kit 225 T cells stimulated with IL-2. Using directed mutagenesis, phosphorylation of αPIX Rho-GEF serines 225 and 488 is required for activation...

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

    International Nuclear Information System (INIS)

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

    1990-01-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G- 3 H] hypoxanthine (Hy) into [ 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 [ 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

  15. Comparison of two full automatic synthesis methods of 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl)guanine using different chemistry modules

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Se Hun [Molecular Imaging and Therapy Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Department of Chemistry, Sogang University, Seoul 121-742 (Korea, Republic of); Oh, Seung Jun [Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736 (Korea, Republic of)], E-mail: sjoh@amc.seoul.kr; Lee, Sang Ju [Department of Nuclear Medicine, Asan Medical Center, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-gu, Seoul 138-736 (Korea, Republic of); Keon, Wook Kang; Kim, Seok-ki [Molecular Imaging and Therapy Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Department of Nuclear Medicine, National Cancer Center, Goyang 410-769 (Korea, Republic of); Lee, Ho-young [Department of Nuclear Medicine, National Cancer Center, Goyang 410-769 (Korea, Republic of); Lee, Sang Jin [Genitourinary Cancer Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Kim, In Hoo [Molecular Imaging and Therapy Branch, National Cancer Center, Goyang 410-769 (Korea, Republic of); Lee, Won Koo [Department of Chemistry, Sogang University, Seoul 121-742 (Korea, Republic of)

    2009-10-15

    We have developed synthesis methods for 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl)guanine ([{sup 18}F]FHBG) using two commercial automatic chemistry modules, Tracerlab MX and Explora RN, and compared radiochemical yields. Synthesis conditions and sequence programs were modified for two modules because both these modules have different mechanical structures, including heater type, vacuum system, reactor, and tubing size. Synthesis using the Tracerlab MX module showed a 21.0{+-}3.8% yield of radiochemical, which was 98{+-}0.9% pure; the total preparation time was 63.0{+-}5.0 min including an HPLC purification step. In contrast, synthesis using the Explora RN module showed a 32.0{+-}1.2% yield of radiochemical, which was 99.0{+-}0.6% pure; the total preparation time was 38{+-}2 min, using different HPLC purification conditions and without the HPLC solvent evaporation step.

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

    Energy Technology Data Exchange (ETDEWEB)

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. (Fitzsimons Army Medical Center, Aurora, CO (USA))

    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.

  17. Relative Stability of the La and Lb Excited States in Adenine and Guanine: Direct Evidence from TD-DFT Calculations of MCD Spectra.

    Science.gov (United States)

    Santoro, Fabrizio; Improta, Roberto; Fahleson, Tobias; Kauczor, Joanna; Norman, Patrick; Coriani, Sonia

    2014-06-05

    The relative position of La and Lb ππ* electronic states in purine nucleobases is a much debated topic, since it can strongly affect our understanding of their photoexcited dynamics. To assess this point, we calculated the absorption and magnetic circular dichroism (MCD) spectra of adenine, guanine, and their nucleosides in gas-phase and aqueous solution, exploiting recent developments in MCD computational technology within time-dependent density functional theory. MCD spectroscopy allows us to resolve the intense S0→ La transition from the weak S0→ Lb transition. The spectra obtained in water solution, by using B3LYP and CAM-B3LYP functionals and describing solvent effect by cluster models and by the polarizable continuum model (PCM), are in very good agreement with the experimental counterparts, thus providing direct and unambiguous evidence that the energy ordering predicted by TD-DFT, La Lb, is the correct one.

  18. Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3.

    Science.gov (United States)

    Thomas, Celestine J; Tall, Gregory G; Adhikari, Anirban; Sprang, Stephen R

    2008-08-22

    Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3.

  19. Protein Kinase A (PKA) Type I Interacts with P-Rex1, a Rac Guanine Nucleotide Exchange Factor: EFFECT ON PKA LOCALIZATION AND P-Rex1 SIGNALING.

    Science.gov (United States)

    Chávez-Vargas, Lydia; Adame-García, Sendi Rafael; Cervantes-Villagrana, Rodolfo Daniel; Castillo-Kauil, Alejandro; Bruystens, Jessica G H; Fukuhara, Shigetomo; Taylor, Susan S; Mochizuki, Naoki; Reyes-Cruz, Guadalupe; Vázquez-Prado, José

    2016-03-18

    Morphology of migrating cells is regulated by Rho GTPases and fine-tuned by protein interactions and phosphorylation. PKA affects cell migration potentially through spatiotemporal interactions with regulators of Rho GTPases. Here we show that the endogenous regulatory (R) subunit of type I PKA interacts with P-Rex1, a Rac guanine nucleotide exchange factor that integrates chemotactic signals. Type I PKA holoenzyme interacts with P-Rex1 PDZ domains via the CNB B domain of RIα, which when expressed by itself facilitates endothelial cell migration. P-Rex1 activation localizes PKA to the cell periphery, whereas stimulation of PKA phosphorylates P-Rex1 and prevents its activation in cells responding to SDF-1 (stromal cell-derived factor 1). The P-Rex1 DEP1 domain is phosphorylated at Ser-436, which inhibits the DH-PH catalytic cassette by direct interaction. In addition, the P-Rex1 C terminus is indirectly targeted by PKA, promoting inhibitory interactions independently of the DEP1-PDZ2 region. A P-Rex1 S436A mutant construct shows increased RacGEF activity and prevents the inhibitory effect of forskolin on sphingosine 1-phosphate-dependent endothelial cell migration. Altogether, these results support the idea that P-Rex1 contributes to the spatiotemporal localization of type I PKA, which tightly regulates this guanine exchange factor by a multistep mechanism, initiated by interaction with the PDZ domains of P-Rex1 followed by direct phosphorylation at the first DEP domain and putatively indirect regulation of the C terminus, thus promoting inhibitory intramolecular interactions. This reciprocal regulation between PKA and P-Rex1 might represent a key node of integration by which chemotactic signaling is fine-tuned by PKA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Guanine nucleotide-dependent, pertussis toxin-insensitive, stimulation of inositol phosphate formation by carbachol in a membrane preparation from astrocytoma cells

    International Nuclear Information System (INIS)

    Hepler, J.R.; Harden, T.K.

    1986-01-01

    Formation of the inositol phosphates (InsP), InsP 3 , InsP 2 , and InsP 1 was increased in a concentration dependent manner (K/sub 0.5/ ∼ 5 μM) by GTPΣS in washed membranes prepared from 3 H-inositol-prelabelled 1321N1 human astrocytoma cells. Both GTPγS and GppNHp stimulated InsP formation by 2-3 fold over control; GTP and GDP were much less efficacious and GMP had no effect. Although the muscarinic cholinergic receptor agonist carbachol had no effect in the absence of guanine nucleotide, in the presence of 10 μM GTPγS, carbachol stimulated (K/sub 0.5/ ∼ 10 μ M) the formation of InsP above the level achieved with GTPγS alone. The effect of carbachol was completely blocked by atropine. The order of potency for a series of nucleotides for stimulation of InsP formation in the presence of 500 μM carbachol was GTPγS > GppNHp > GTP = GDP. Pertussis toxin, at concentrations that fully ADP-ribosylate and functionally inactivate G/sub i/, had no effect on InsP formation in the presence of GTPγS or GTPγS plus carbachol. Histamine and bradykinin also stimulated InsP formation in the presence of GTPγS in washed membranes from 1321N1 cells. These data are consistent with the idea that a guanine nucleotide regulatory protein that is not G/sub i/ is involved in receptor-mediated stimulation of InsP formation in 1321N1 human astrocytoma cells

  1. Electrochemically-tuned luminescence of a [Ru(bpy) 2(tatp)] 2+-sensitized TiO 2 anode and its applications to photo-stimulated guanine/H 2O 2 fuel cells

    Science.gov (United States)

    Shao, Jiangyang; Sun, Ting; Ji, Shibo; Li, Hong; Lan, Sheng; Li, Weishan

    A phenazine-containing Ru(II) complex [Ru(bpy) 2(tatp)] 2+ (bpy = 2,2‧-bipyridine and tatp = 1,4,8,9-tetra-aza-triphenylene) is first applied to a modification of the nano-TiO 2/indium-tin oxide (ITO) electrode by the method of repetitive voltammetric sweeping. The resulting [Ru(bpy) 2(tatp)] 2+-modified TiO 2 electrode shows two pairs of well-defined redox waves and excellent electrocatalytic activity for the oxidation of guanine. [Ru(bpy) 2(tatp)] 2+ on TiO 2 surfaces exhibits intense absorbance and photoluminescence in visible region, revealed by absorption spectra, emission spectra and fluorescence microscope. While [Ru(bpy) 2(tatp)] 2+-sensitized TiO 2 is functionalized as an anode to combine with a continuous wave green laser via an optical microscope, the luminescence of Ru(II)-based excited states can be enhanced by the oxidation of guanine. Furthermore, the [Ru(bpy) 2(tatp)] 2+-sensitized TiO 2 electrode is used as photoanode and hemoglobin-modified single-walled carbon nanotubes (SWCNTs) as cathode for the elaboration of a photo-stimulated guanine/H 2O 2 fuel cell with a saturated KCl salt-bridge. It becomes evident that the photo-stimulated fuel cell performance depends strongly on the excited states of Ru(II) complex-sensitized anodes as well as the electrocatalytic oxidation of guanine. This study provides an electrochemically-tuned luminescence method for better evaluating contributions of the sensitizer excited states to photo-stimulated fuel cells.

  2. The G-BHQ synergistic effect: Improved double quenching molecular beacons based on guanine and Black Hole Quencher for sensitive simultaneous detection of two DNAs.

    Science.gov (United States)

    Xiang, Dongshan; Li, Fengquan; Wu, Chenyi; Shi, Boan; Zhai, Kun

    2017-11-01

    We designed two double quenching molecular beacons (MBs) with simple structure based on guanine (G base) and Black Hole Quencher (BHQ), and developed a new analytical method for sensitive simultaneous detection of two DNAs by synchronous fluorescence analysis. In this analytical method, carboxyl fluorescein (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) were respectively selected as fluorophore of two MBs, Black Hole Quencher 1 (BHQ-1) and Black Hole Quencher 2 (BHQ-2) were respectively selected as organic quencher, and three continuous nucleotides with G base were connected to organic quencher (BHQ-1 and BHQ-2). In the presence of target DNAs, the two MBs hybridize with the corresponding target DNAs, the fluorophores are separated from organic quenchers and G bases, leading to recovery of fluorescence of FAM and TAMRA. Under a certain conditions, the fluorescence intensities of FAM and TAMRA all exhibited good linear dependence on their concentration of target DNAs (T1 and T2) in the range from 4 × 10 -10 to 4 × 10 -8 molL -1 (M). The detection limit (3σ, n = 13) of T1 was 3 × 10 -10 M and that of T2 was 2×10 -10 M, respectively. Compared with the existing analysis methods for multiplex DNA with MBs, this proposed method based on double quenching MBs is not only low fluorescence background, short analytical time and low detection cost, but also easy synthesis and good stability of MB probes. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Hypoxanthine guanine phosphoribosyltransferase activity is related to 6-thioguanine nucleotide concentrations and thiopurine-induced leukopenia in the treatment of inflammatory bowel disease.

    Science.gov (United States)

    Ding, Liang; Zhang, Fang-bin; Liu, Hui; Gao, Xiang; Bi, Hui-chang; Wang, Xue-ding; Chen, Bai-li; Zhang, Yu; Zhao, Li-zi; Zhong, Guo-ping; Hu, Pin-jin; Chen, Min-hu; Huang, Ming

    2012-01-01

    Thiopurine drugs are widely used in the treatment of inflammatory bowel disease (IBD). The polymorphic enzyme thiopurine S-methyltransferase (TPMT) is of importance for thiopurine metabolism and adverse events occurrence. The role of other thiopurine-metabolizing enzymes is less well known. This study investigated the effects of TPMT and hypoxanthine guanine phosphoribosyltransferase (HPRT) activities on 6-thioguanine nucleotides (6-TGNs) concentrations and thiopurine-induced leukopenia in patients with IBD. Clinical data and blood samples were collected from 120 IBD patients who were receiving azathioprine (AZA)/6-mercaptopurine (6-MP) therapy. Erythrocyte TPMT, HPRT activities and 6-TGNs concentrations were determined. HPRT activity and its correlation with TPMT activity, 6-TGNs level, and leukopenia were evaluated. The HPRT activity of all patients ranged from 1.63-3.33 (2.31 ± 0.36) μmol/min per g Hb. HPRT activity was significantly higher in patients with leukopenia (27, 22.5%) than without (P leukopenia (r = 0.526, P = 0.005). Patients with HPRT activity > 2.70 μmol/min per g Hb could have an increased risk of developing leukopenia (odds ratio = 7.47, P leukopenia. High levels of HPRT activity could be a predictor of leukopenia and unsafe 6-TGN concentrations in patients undergoing AZA/6-MP therapy. This could partly explain the therapeutic response or toxicity that could not be adequately explained by the polymorphisms of TPMT. Copyright © 2011 Crohn's & Crohn's & Colitis Foundation of America, Inc.

  4. Effect of Six Sessions of High Intensity Interval Training on Levels of Hypoxanthine, Xanthine, Hypoxanthine-Guanine phosphoribosyltransferase (HGPRT and Serum Uric Acid in active young men

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    ROHOULLAH HAGHSHENAS GATABI

    2017-01-01

    Full Text Available Abstract Introduction and objectives: long-term sport and physical activity results in compatibility in maintaining purine derivatives but the compatibility achieved within a few sessions is not well investigated. This study aimed to investigate the effect of a 30-seconds high intensity interval training on Hypoxanthine, xanthine, hypoxanthine-guanine phosphoribosyltransferase (HGPRT and serum uric acid in young college men. Methods: In this study, 18 untrained healthy men were divided into two control and training groups after homogenization based on their personal characteristics. Training included six sessions (every other day for two weeks with different intervals (4, 7, 6, 6, 5 & 4, respectively with a fixed four-minute rest between each interval, and with a constant load of .6 on the cycle-ergometer. Blood samples were taken before and 48 hours after the last training session, and were used to analyze hypoxanthine, xanthine, uric acid, and serum HGPRT. Statistical analysis was performed using analysis of covariance (ANCOVA. Results: The results showed that high-intensity interval training for two weeks did not cause significant changes in serum HGPRT (P = .73; likewise, the increase in serum hypoxanthine (P = .170 and serum xanthine (P = .170 was not statistically significant but significant reduction was observed in serum uric acid (P = .025. Discussion and conclusion: The results of this study indicated that two-week HIIT training is likely to enhance athletic performance and recovery of purine nucleotide cycle.

  5. Activation of guanine-{beta}-D-arabinofuranoside and deoxyguanosine to triphosphates by a common pathway blocks T lymphoblasts at different checkpoints

    Energy Technology Data Exchange (ETDEWEB)

    Leanza, Luigi; Miazzi, Cristina; Ferraro, Paola; Reichard, Peter; Bianchi, Vera, E-mail: vbianchi@bio.unipd.it

    2010-12-10

    The deoxyguanosine (GdR) analog guanine-ss-D-arabinofuranoside (araG) has a specific toxicity for T lymphocytes. Also GdR is toxic for T lymphocytes, provided its degradation by purine nucleoside phosphorylase (PNP) is prevented, by genetic loss of PNP or by enzyme inhibitors. The toxicity of both nucleosides requires their phosphorylation to triphosphates, indicating involvement of DNA replication. In cultured cells we found by isotope-flow experiments with labeled araG a rapid accumulation and turnover of araG phosphates regulated by cytosolic and mitochondrial kinases and deoxynucleotidases. At equilibrium their partition between cytosol and mitochondria depended on the substrate saturation kinetics and cellular abundance of the kinases leading to higher araGTP concentrations in mitochondria. dGTP interfered with the allosteric regulation of ribonucleotide reduction, led to highly imbalanced dNTP pools with gradual inhibition of DNA synthesis and cell-cycle arrest at the G1-S boundary. AraGTP had no effect on ribonucleotide reduction. AraG was in minute amounts incorporated into nuclear DNA and stopped DNA synthesis arresting cells in S-phase. Both nucleosides eventually induced caspases and led to apoptosis. We used high, clinically relevant concentrations of araG, toxic for nuclear DNA synthesis. Our experiments do not exclude an effect on mitochondrial DNA at low araG concentrations when phosphorylation occurs mainly in mitochondria.

  6. mRNA:guanine-N7 cap methyltransferases: identification of novel members of the family, evolutionary analysis, homology modeling, and analysis of sequence-structure-function relationships

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    Radlinska Monika

    2001-06-01

    Full Text Available Abstract Background The 5'-terminal cap structure plays an important role in many aspects of mRNA metabolism. Capping enzymes encoded by viruses and pathogenic fungi are attractive targets for specific inhibitors. There is a large body of experimental data on viral and cellular methyltransferases (MTases that carry out guanine-N7 (cap 0 methylation, including results of extensive mutagenesis. However, a crystal structure is not available and cap 0 MTases are too diverged from other MTases of known structure to allow straightforward homology-based interpretation of these data. Results We report a 3D model of cap 0 MTase, developed using sequence-to-structure threading and comparative modeling based on coordinates of the glycine N-methyltransferase. Analysis of the predicted structural features in the phylogenetic context of the cap 0 MTase family allows us to rationalize most of the experimental data available and to propose potential binding sites. We identified a case of correlated mutations in the cofactor-binding site of viral MTases that may be important for the rational drug design. Furthermore, database searches and phylogenetic analysis revealed a novel subfamily of hypothetical MTases from plants, distinct from "orthodox" cap 0 MTases. Conclusions Computational methods were used to infer the evolutionary relationships and predict the structure of Eukaryotic cap MTase. Identification of novel cap MTase homologs suggests candidates for cloning and biochemical characterization, while the structural model will be useful in designing new experiments to better understand the molecular function of cap MTases.

  7. Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1.

    Science.gov (United States)

    Fleming, Ian N; Batty, Ian H; Prescott, Alan R; Gray, Alex; Kular, Gursant S; Stewart, Hazel; Downes, C Peter

    2004-09-15

    Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P(2) to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P(2) breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P(3) concentrations, rather than the closely related lipid, PtdIns(3,4)P(2). Finally, the data demonstrate that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.

  8. Multi-level Quantum Mechanics and Molecular Mechanics Study of Ring Opening Process of Guanine Damage by Hydroxyl Radical in Aqueous Solution.

    Science.gov (United States)

    Liu, Peng; Wang, Qiong; Niu, Meixing; Wang, Dunyou

    2017-08-10

    Combining multi-level quantum mechanics theories and molecular mechanics with an explicit water model, we investigated the ring opening process of guanine damage by hydroxyl radical in aqueous solution. The detailed, atomic-level ring-opening mechanism along the reaction pathway was revealed in aqueous solution at the CCSD(T)/MM levels of theory. The potentials of mean force in aqueous solution were calculated at both the DFT/MM and CCSD(T)/MM levels of the theory. Our study found that the aqueous solution has a significant effect on this reaction in solution. In particular, by comparing the geometries of the stationary points between in gas phase and in aqueous solution, we found that the aqueous solution has a tremendous impact on the torsion angles much more than on the bond lengths and bending angles. Our calculated free-energy barrier height 31.6 kcal/mol at the CCSD(T)/MM level of theory agrees well with the one obtained based on gas-phase reaction profile and free energies of solvation. In addition, the reaction path in gas phase was also mapped using multi-level quantum mechanics theories, which shows a reaction barrier at 19.2 kcal/mol at the CCSD(T) level of theory, agreeing very well with a recent ab initio calculation result at 20.8 kcal/mol.

  9. The radioprotector WR-2721 reduces neutron-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in mouse splenocytes when administered prior to or following irradiation

    International Nuclear Information System (INIS)

    Grdina, D.J.; Basic, I.

    1992-01-01

    An in vitro T-lymphocyte cloning technique has been applied to study the effects of JANUS fission-spectrum neutron irradiation and the radioprotector S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721) on the subsequent development of somatic mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in hybrid B6CF1 male mice. In control studies performed to establish an in vitro cloning technique, the mutant frequencies of splenic T-lymphocytes, as a result of exposure to a 100 cGy dose of neutrons, increased with time from a control level of 9 x 10 -7 to a maximum value of 1.7 x 10 -5 at 56 days following irradiation. Between 56 and 150 days after irradiation, mutant frequencies were observed to plateau and remain stable. All subsequent determinations were performed at 56 days following the experimental treatment of animals. WR-2721 at a dose of 400 mg/kg was effective in protecting against the induction of hprt mutants (i.e. a mutant frequency reduction factor, MFRF) following the largest dose of neutrons used (i.e. 150 cGy). The antimutagenic effectiveness of WR-2721 administered 30 min prior to irradiation was unaffected, even when the dose was reduced to 200 mg/kg. These findings confirm our earlier report using the radioprotector N-(2-mercaptoethyl)-1,2-diaminopropane (WR-1065) under in vitro conditions, and demonstrate that these agents can be used as effective antimutagens even when they are administered up to 3 h following radiation exposure. (Author)

  10. Ric-8a, a guanine nucleotide exchange factor for heterotrimeric G proteins, regulates bergmann glia-basement membrane adhesion during cerebellar foliation.

    Science.gov (United States)

    Ma, Shang; Kwon, Hyo Jun; Huang, Zhen

    2012-10-24

    The cerebellum consists of an intricate array of lobules that arises during the process of foliation. Foliation not only increases surface area, but may also facilitate organization of cerebellar neural circuitry. Defects in cerebellar foliation are associated with a number of diseases. Yet, little is known about how foliation, a process involving large-scale and simultaneous movement of several different cell types, is coordinated by cell-cell signaling at the molecular level. Here we show that Ric-8a, a guanine nucleotide exchange factor in the G-protein-coupled receptor pathway, is specifically required in Bergmann glia during cerebellar foliation. We find that ric-8a mutation in mice results in disorganized Bergmann glial scaffolding, defective granule cell migration, and disrupted Purkinje cell positioning. These abnormalities result from primary defects in Bergmann glia since mutations in granule cells do not show similar effects. They first arise during late embryogenesis, at the onset of foliation, when ric-8a mutant Bergmann glia fail to maintain adhesion to the basement membrane specifically at emerging fissures. This suggests that Ric-8a is essential for the enhanced Bergmann glia-basement membrane adhesion required for fissure formation. Indeed, we find that ric-8a-deficient cerebellar glia show decreased affinity for basement membrane components. We also find that weakening Bergmann glia-basement membrane interaction by β1 integrin deletion results in a similar phenotype. These results thus reveal a novel role of Ric-8a in modulating Bergmann glia-basement membrane adhesion during foliation, and provide new insights into the signaling pathways that coordinate cellular movement during cerebellar morphogenesis.

  11. Rac1 Activation Caused by Membrane Translocation of a Guanine Nucleotide Exchange Factor in Akt2-Mediated Insulin Signaling in Mouse Skeletal Muscle.

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    Nobuyuki Takenaka

    Full Text Available Insulin-stimulated glucose uptake in skeletal muscle is mediated by the glucose transporter GLUT4, which is translocated to the plasma membrane following insulin stimulation. Several lines of evidence suggested that the protein kinase Akt2 plays a key role in this insulin action. The small GTPase Rac1 has also been implicated as a regulator of insulin-stimulated GLUT4 translocation, acting downstream of Akt2. However, the mechanisms whereby Akt2 regulates Rac1 activity remain obscure. The guanine nucleotide exchange factor FLJ00068 has been identified as a direct regulator of Rac1 in Akt2-mediated signaling, but its characterization was performed mostly in cultured myoblasts. Here, we provide in vivo evidence that FLJ00068 indeed acts downstream of Akt2 as a Rac1 regulator by using mouse skeletal muscle. Small interfering RNA knockdown of FLJ00068 markedly diminished GLUT4 translocation to the sarcolemma following insulin administration or ectopic expression of a constitutively activated mutant of either phosphoinositide 3-kinase or Akt2. Additionally, insulin and these constitutively activated mutants caused the activation of Rac1 as shown by immunofluorescent microscopy using a polypeptide probe specific to activated Rac1 in isolated gastrocnemius muscle fibers and frozen sections of gastrocnemius muscle. This Rac1 activation was also abrogated by FLJ00068 knockdown. Furthermore, we observed translocation of FLJ00068 to the cell periphery following insulin stimulation in cultured myoblasts. Localization of FLJ00068 in the plasma membrane in insulin-stimulated, but not unstimulated, myoblasts and mouse gastrocnemius muscle was further affirmed by subcellular fractionation and subsequent immunoblotting. Collectively, these results strongly support a critical role of FLJ00068 in Akt2-mediated Rac1 activation in mouse skeletal muscle insulin signaling.

  12. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5’-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris, semiaquatic (Lontra longicaudis annectens and terrestrial (Sus scrofa

    Directory of Open Access Journals (Sweden)

    Myrna eBarjau Perez-Milicua

    2015-07-01

    Full Text Available Aquatic and semiaquatic mammals have the capacity of breath hold (apnea diving. Northern elephant seals (Mirounga angustirostris have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens can hold their breath for about 30 sec. Such periods of apnea may result in reduced oxygen concentration (hypoxia and reduced blood supply (ischemia to tissues. Production of adenosine 5’-triphosphate (ATP requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa, are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal (n=11, semiaquatic (neotropical river otter (n=4 and terrestrial (domestic pig (n=11. Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT was determined by spectrophotometry, and activity of inosine 5’-monophosphate dehydrogenase (IMPDH and the concentration of hypoxanthine (HX, inosine 5’-monophosphate (IMP, adenosine 5’-monophosphate (AMP, adenosine 5’-diphosphate (ADP, ATP, guanosine 5’-diphosphate (GDP, guanosine 5’-triphosphate (GTP, and xanthosine 5’-monophosphate (XMP were determined by high-performance liquid chromatography (HPLC. The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise, aquatic and semiaquatic mammals have less purine mobilization than their terrestrial counterparts.

  13. Guanine α-carboxy nucleoside phosphonate (G-α-CNP) shows a different inhibitory kinetic profile against the DNA polymerases of human immunodeficiency virus (HIV) and herpes viruses.

    Science.gov (United States)

    Balzarini, Jan; Menni, Michael; Das, Kalyan; van Berckelaer, Lizette; Ford, Alan; Maguire, Nuala M; Liekens, Sandra; Boehmer, Paul E; Arnold, Eddy; Götte, Matthias; Maguire, Anita R

    2017-07-15

    α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class of nucleotide-competing reverse transcriptase (RT) inhibitors (NcRTIs). They were designed to act directly against HIV-1 RT without the need for prior activation (phosphorylation). In this respect, they differ from the nucleoside or nucleotide RTIs [N(t)RTIs] that require conversion to their triphosphate forms before being inhibitory to HIV-1 RT. The guanine derivative (G-α-CNP) has now been synthesized and investigated for the first time. The (L)-(+)-enantiomer of G-α-CNP directly and competitively inhibits HIV-1 RT by interacting with the substrate active site of the enzyme. The (D)-(-)-enantiomer proved inactive against HIV-1 RT. In contrast, the (+)- and (-)-enantiomers of G-α-CNP inhibited herpes (i.e. HSV-1, HCMV) DNA polymerases in a non- or uncompetitive manner, strongly indicating interaction of the (L)-(+)- and the (D)-(-)-G-α-CNPs at a location different from the polymerase substrate active site of the herpes enzymes. Such entirely different inhibition profile of viral polymerases is unprecedented for a single antiviral drug molecule. Moreover, within the class of α-CNPs, subtle differences in their sensitivity to mutant HIV-1 RT enzymes were observed depending on the nature of the nucleobase in the α-CNP molecules. The unique properties of the α-CNPs make this class of compounds, including G-α-CNP, direct acting inhibitors of multiple viral DNA polymerases. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  15. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-01-01

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

  16. The nuclear guanine nucleotide exchange factors Ect2 and Net1 regulate RhoB-mediated cell death after DNA damage.

    Directory of Open Access Journals (Sweden)

    Melissa C Srougi

    2011-02-01

    Full Text Available Commonly used antitumor treatments, including radiation and chemotherapy, function by damaging the DNA of rapidly proliferating cells. However, resistance to these agents is a predominant clinical problem. A member of the Rho family of small GTPases, RhoB has been shown to be integral in mediating cell death after ionizing radiation (IR or other DNA damaging agents in Ras-transformed cell lines. In addition, RhoB protein expression increases after genotoxic stress, and loss of RhoB expression causes radio- and chemotherapeutic resistance. However, the signaling pathways that govern RhoB-induced cell death after DNA damage remain enigmatic. Here, we show that RhoB activity increases in human breast and cervical cancer cell lines after treatment with DNA damaging agents. Furthermore, RhoB activity is necessary for DNA damage-induced cell death, as the stable loss of RhoB protein expression using shRNA partially protects cells and prevents the phosphorylation of c-Jun N-terminal kinases (JNKs and the induction of the pro-apoptotic protein Bim after IR. The increase in RhoB activity after genotoxic stress is associated with increased activity of the nuclear guanine nucleotide exchange factors (GEFs, Ect2 and Net1, but not the cytoplasmic GEFs p115 RhoGEF or Vav2. Importantly, loss of Ect2 and Net1 via siRNA-mediated protein knock-down inhibited IR-induced increases in RhoB activity, reduced apoptotic signaling events, and protected cells from IR-induced cell death. Collectively, these data suggest a mechanism involving the nuclear GEFs Ect2 and Net1 for activating RhoB after genotoxic stress, thereby facilitating cell death after treatment with DNA damaging agents.

  17. Distinctions in beta-adrenergic receptor interactions with the magnesium-guanine nucleotide coupling proteins in turkey erythrocyte and S49 lymphoma membranes.

    Science.gov (United States)

    Vauquelin, G; Cech, S Y; André, C; Strosberg, A D; Maguire, M E

    1982-01-01

    Several homogeneous cell systems contain distinct subpopulations of beta-adrenergic receptors, distinguished by their relative sensitivity to N-ethylmaleimide (NEM) in the presence of agonist but not antagonist (G. Vauquelin and M.E. Maguire (1980) Mol. Pharmacol. 18, 363-369). The sensitivity to agonist/NEM inactivation requires receptor interaction with the magnesium-guanine nucleotide coupling proteins (G/F). We have investigated the effects of agonist/NEM treatment on Mg2+ and GTP modulation of receptor affinity in two such systems, turkey erythrocytes and murine S49 lymphoma cells. In each systems, the agonist/NEM-sensitive beta-receptor subpopulation exhibits both Mg2+ and GTP modulation of beta-receptor affinity for agonist. Further, Mg2+ and GTP are not competitive with regard to alteration of receptor affinity; that is, GTP can block the effect of Mg2+, but not vice versa. In contrast, the agonist/NEM-resistant beta-receptor subpopulation shows distinct differences in Mg2+ and GTP effects when the turkey and S49 systems are compared. The agonist/NEM-resistant population in S49 shows no effect of Mg2+ or GTP on beta-receptor affinity for agonist whereas the resistant beta-receptors of turkey erythrocytes still exhibit modulation by both GTP and Mg2+. Moreover, in this receptor population the actions of GTP and Mg2+ are apparently competitive, with increasing Mg2+ concentrations able to overcome the decrease in affinity induced by GTP. Thus, beta-receptor interaction with the metal/nucleotide coupling proteins may differ significantly in the two systems examined. An additional result of these experiments is the demonstration for S49 beta-receptors that free, unchelated GTP or GDP rather than MgGTP or MgGDP modulates receptor affinity for agonist.

  18. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    International Nuclear Information System (INIS)

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of [ 3 H]GDP binding to plasma membranes suggested a single high affinity site with a K d = 0.24 uM. Competition studies indicated that GTP γ S was 7-fold more potent than GDP β S. Bound GDP could be released by FSH in the presence of GTP γ S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP β S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP β S competitively inhibited GTP γ S-stimulated adenylate cyclase activity with a K i = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP γ S-bound form persisted even if GDP β S previously occupied all available binding sites. Two membrane proteins, M r = 43,000 and 48,000, were ADP·ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP γ S but not by GDP β S. The M r = 43,000 and 48,000 proteins represented variant forms of G S . A single protein of M r = 40,000 (G i ) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC 50 = 0.1 uM. The adenosine analog, N 6 ·phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin

  19. Modeling of Toxicity-Relevant Electrophilic Reactivity for Guanine with Epoxides: Estimating the Hard and Soft Acids and Bases (HSAB) Parameter as a Predictor.

    Science.gov (United States)

    Zhang, Jing; Wang, Chenchen; Ji, Li; Liu, Weiping

    2016-05-16

    According to the electrophilic theory in toxicology, many chemical carcinogens in the environment and/or their active metabolites are electrophiles that exert their effects by forming covalent bonds with nucleophilic DNA centers. The theory of hard and soft acids and bases (HSAB), which states that a toxic electrophile reacts preferentially with a biological macromolecule that has a similar hardness or softness, clarifies the underlying chemistry involved in this critical event. Epoxides are hard electrophiles that are produced endogenously by the enzymatic oxidation of parent chemicals (e.g., alkenes and PAHs). Epoxide ring opening proceeds through a SN2-type mechanism with hard nucleophile DNA sites as the major facilitators of toxic effects. Thus, the quantitative prediction of chemical reactivity would enable a predictive assessment of the molecular potential to exert electrophile-mediated toxicity. In this study, we calculated the activation energies for reactions between epoxides and the guanine N7 site for a diverse set of epoxides, including aliphatic epoxides, substituted styrene oxides, and PAH epoxides, using a state-of-the-art density functional theory (DFT) method. It is worth noting that these activation energies for diverse epoxides can be further predicted by quantum chemically calculated nucleophilic indices from HSAB theory, which is a less computationally demanding method than the exacting procedure for locating the transition state. More importantly, the good qualitative/quantitative correlations between the chemical reactivity of epoxides and their bioactivity suggest that the developed model based on HSAB theory may aid in the predictive hazard evaluation of epoxides, enabling the early identification of mutagenicity/carcinogenicity-relevant SN2 reactivity.

  20. Conserved guanine-guanine stacking in tetraplex and duplex DNA

    Czech Academy of Sciences Publication Activity Database

    Kypr, Jaroslav; Fialová, Markéta; Chládková, Jana; Tůmová, Marcela; Vorlíčková, Michaela

    2001-01-01

    Roč. 30, č. 7 (2001), s. 555-558 ISSN 0175-7571 R&D Projects: GA AV ČR IAA5004802 Institutional research plan: CEZ:AV0Z5004920 Keywords : tetraplex DNA * duplex DNA * circular dichroism spectra Subject RIV: BO - Biophysics Impact factor: 1.917, year: 2001

  1. Oxidation of 5'-site guanine at GG and GGG sequences induced by a metabolite of carcinogenic heterocyclic amine PhIP in the presence of Cu(II) and NADH.

    Science.gov (United States)

    Murata, Mariko; Kawanishi, Shosuke

    2002-05-01

    Adduct formation has been considered to be a major causal factor of DNA damage by carcinogenic heterocyclic amines. By means of experiments with an electrochemical detector coupled to a high-performance liquid chromatograph, we revealed that N-hydroxy metabolite of 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) induced the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in the presence of Cu(II). Addition of an endogenous reductant NADH enhanced the 8-OH-dG formation. Experiments with (32)P-labeled DNA fragments showed that this metabolite [PhIP(NHOH)] caused 8-hydroxylation of guanines in the presence of Cu(II) and NADH, and subsequent treatment with formamidopyrimidine-DNA glycosylase led to chain cleavages at the 5'-site guanine of GG and GGG sequences. Interestingly, antioxidant enzyme SOD enhanced the intensity of DNA damage, and thymine residues were appended to its guanine-predominant cleavage sites. Catalase and bathocuproine, a Cu(I)-specific chelator, inhibited the DNA damage, suggesting the involvement of H(2)O(2) and Cu(I). A UV-visible spectroscopic study indicated that Cu(II) and SOD catalyze the autoxidation of PhIP(NHOH). These results suggest that Cu(II)-dependent autooxidation of PhIP(NHOH) coupled with NADH-mediated reduction of its oxidized product form redox cycle, resulting in oxidative DNA damage by low concentrations of PhIP(NHOH). We conclude that in addition to DNA adduct formation, oxidative DNA damage may be involved in the carcinogenic process of PhIP.

  2. Clinical significance of the methylated cytosine-phosphate-guanine sites of protocadherin-10 promoter for evaluating the prognosis of gastric cancer.

    Science.gov (United States)

    Deng, Jingyu; Liang, Han; Ying, Guoguang; Dong, Qiuping; Zhang, Li; Yu, Jun; Fan, Daiming; Hao, Xishan

    2014-11-01

    Protocadherin-10 (PCDH10) has been identified as a tumor suppressor gene in multiple carcinomas. In this study, we intended to elucidate the clinical applicability of the methylation of CpG sites of PCDH10 promoter for prognostic prediction in gastric cancer (GC). Qualitative and quantitative detections of PCDH10 promoter methylation were performed with methylation-specific polymerase chain reaction (MSP) and bisulphite genomic sequencing, respectively. The methylated statuses of 27 cytosine-phosphate-guanine (CpG) sites in PCDH10 promoter were detected in a series of 458 GC tissues to supply precise information of prognostic prediction. Associations between molecular, clinicopathologic, and survival data were analyzed. Protocadherin-10 promoter methylation was found in 91.92% in all patients. Gastric cancer patients with 5 or more methylated CpG sites of PCDH10 promoter was significantly associated with poorer survival (p = 0.038). Meanwhile, methylation of combined CpG (-115, -108, -13, and +3) sites was also identified to provide elaborate survival discrimination for GC patients (p = 0.044). On multivariate survival analysis, methylation of combined CpG (-115, -108, -13, and +3) sites (hazard ratio [HR] = 1.255; p = 0.049) was identified to be an independent prognostic indicator of GC, as were N stage and T stage. Additionally, the methylation of combined CpG (-115, -108, -13, and +3) sites had smaller Akaike information criterion (AIC) and Bayesian information criterion (BIC) values than the other 2 independent predictors of the survival. Ultimately, we demonstrated that the methylation of combined CpG (-115, -108, -13, and +3) sites was negatively associated with PCDH10 expression in GC tissues. The methylated CpG sites of PCDH10 promoter had significant applicability for clinical evaluation of the prognosis of GC. Copyright © 2014 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

  3. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    LENUS (Irish Health Repository)

    Krause-Gruszczynska, Malgorzata

    2011-12-28

    Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-\\/-, integrin-beta1-\\/-, focal adhesion kinase (FAK)-\\/- and Src\\/Yes\\/Fyn-\\/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1\\/2-\\/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  4. [3H]WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

    International Nuclear Information System (INIS)

    Norman, A.B.; Battaglia, G.; Creese, I.

    1985-01-01

    In the presence of a 30 nM prazosin mask, [ 3 H]-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ([ 3 H]WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for [ 3 H] WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at [ 3 H]WB4101-binding sites in the presence of 30 nM prazosin and [ 3 H] lysergic acid diethylamide ([ 3 H]LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of [ 3 H]WB4101 is significantly lower than the Bmax of [ 3 H]LSD in various brain regions. WB4101 competition for [ 3 H] LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of [ 3 H]WB4101 binding derived from saturation experiments. This suggests that [ 3 H]WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by [ 3 H]LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for [ 3 H]WB4101 but compete for multiple [ 3 H]LSD 5-HT1 binding sites. These data indicate that [ 3 H]WB4101 selectively labels the 5-HT1A serotonin receptor, whereas [ 3 H] LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of [ 3 H]WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of [ 3 H]WB4101 binding

  5. The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

    Directory of Open Access Journals (Sweden)

    Krause-Gruszczynska Malgorzata

    2011-12-01

    Full Text Available Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker

  6. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    Energy Technology Data Exchange (ETDEWEB)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. (Burroughs Wellcome Co., Research Triangle Park, NC (USA))

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  7. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein

    International Nuclear Information System (INIS)

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G.

    1988-01-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the α subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5'-[α- 32 P]triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an α subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera

  8. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα-encoding (GNAS genomic imprinting domain are associated with performance traits

    Directory of Open Access Journals (Sweden)

    Mullen Michael P

    2011-01-01

    Full Text Available Abstract Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486 were located upstream of the GNAS gene, while one SNP (rs41694646 was located in the second intron of the GNAS gene. The final SNP (rs41694656 was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646 is associated (P ≤ 0.05 with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf and gestation length. Association (P ≤ 0.01 with direct calving difficulty (i.e. due to calf size and maternal calving difficulty (i.e. due to the maternal pelvic width size was also observed at the rs

  9. DNA sequence polymorphisms within the bovine guanine nucleotide-binding protein Gs subunit alpha (Gsα)-encoding (GNAS) genomic imprinting domain are associated with performance traits

    Science.gov (United States)

    2011-01-01

    Background Genes which are epigenetically regulated via genomic imprinting can be potential targets for artificial selection during animal breeding. Indeed, imprinted loci have been shown to underlie some important quantitative traits in domestic mammals, most notably muscle mass and fat deposition. In this candidate gene study, we have identified novel associations between six validated single nucleotide polymorphisms (SNPs) spanning a 97.6 kb region within the bovine guanine nucleotide-binding protein Gs subunit alpha gene (GNAS) domain on bovine chromosome 13 and genetic merit for a range of performance traits in 848 progeny-tested Holstein-Friesian sires. The mammalian GNAS domain consists of a number of reciprocally-imprinted, alternatively-spliced genes which can play a major role in growth, development and disease in mice and humans. Based on the current annotation of the bovine GNAS domain, four of the SNPs analysed (rs43101491, rs43101493, rs43101485 and rs43101486) were located upstream of the GNAS gene, while one SNP (rs41694646) was located in the second intron of the GNAS gene. The final SNP (rs41694656) was located in the first exon of transcripts encoding the putative bovine neuroendocrine-specific protein NESP55, resulting in an aspartic acid-to-asparagine amino acid substitution at amino acid position 192. Results SNP genotype-phenotype association analyses indicate that the single intronic GNAS SNP (rs41694646) is associated (P ≤ 0.05) with a range of performance traits including milk yield, milk protein yield, the content of fat and protein in milk, culled cow carcass weight and progeny carcass conformation, measures of animal body size, direct calving difficulty (i.e. difficulty in calving due to the size of the calf) and gestation length. Association (P ≤ 0.01) with direct calving difficulty (i.e. due to calf size) and maternal calving difficulty (i.e. due to the maternal pelvic width size) was also observed at the rs43101491 SNP. Following

  10. Capillary HPLC-accurate mass MS/MS quantitation of N7-(2,3,4-trihydroxybut-1-yl)-guanine adducts of 1,3-butadiene in human leukocyte DNA.

    Science.gov (United States)

    Sangaraju, Dewakar; Villalta, Peter; Goggin, Melissa; Agunsoye, Maria O; Campbell, Colin; Tretyakova, Natalia

    2013-10-21

    1,3-Butadiene (BD) is a high volume industrial chemical commonly used in polymer and rubber production. It is also present in cigarette smoke, automobile exhaust, and urban air, leading to widespread exposure of human populations. Upon entering the body, BD is metabolized to electrophilic epoxides, 3,4-epoxy-1-butene (EB), diepoxybutane (DEB), and 3,4-epoxy-1,2-diol (EBD), which can alkylate DNA nucleobases. The most abundant BD epoxide, EBD, modifies the N7-guanine positions in DNA to form N7-(2, 3, 4-trihydroxybut-1-yl) guanine (N7-THBG) adducts, which can be useful as biomarkers of BD exposure and metabolic activation to DNA-reactive epoxides. In the present work, a capillary HPLC-high resolution ESI⁺-MS/MS (HPLC-ESI⁺-HRMS/MS) methodology was developed for accurate, sensitive, and reproducible quantification of N7-THBG in cell culture and in human white blood cells. In our approach, DNA is subjected to neutral thermal hydrolysis to release N7-guanine adducts from the DNA backbone, followed by ultrafiltration, solid-phase extraction, and isotope dilution HPLC-ESI⁺-HRMS/MS analysis on an Orbitrap Velos mass spectrometer. Following method validation, N7-THBG was quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of DEB and in DNA isolated from blood of smokers, nonsmokers, individuals participating in a smoking cessation program, and occupationally exposed workers. N7-THBG concentrations increased linearly from 31.4 ± 4.84 to 966.55 ± 128.05 adducts per 10⁹ nucleotides in HT1080 cells treated with 1-100 μM DEB. N7-THBG amounts in leukocyte DNA of nonsmokers, smokers, and occupationally exposed workers were 7.08 ± 5.29, 8.20 ± 5.12, and 9.72 ± 3.80 adducts per 10⁹ nucleotides, respectively, suggesting the presence of an endogenous or environmental source for this adduct. The availability of sensitive HPLC-ESI⁺-HRMS/MS methodology for BD-induced DNA adducts in humans will enable future population studies of

  11. True Stabilization Energies for the Optimal Planar Hydrogen-Bonded and Stacked Structures of GuanineĎCytosine, AdenineĎThymine and Their 9- and 1-Methyl Derivatives: Complete Basis Set Calculations at the MP2 and CCSD(T) Levels and Comparison with Experiment

    Czech Academy of Sciences Publication Activity Database

    Jurečka, Petr; Hobza, Pavel

    2003-01-01

    Roč. 125, č. 50 (2003), s. 15608-15613 ISSN 0002-7863 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : H-bonded * guanine * cytosine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 6.516, year: 2003

  12. Mass spectrometric (HPLC/ESI--MS/MS) quantification of pyrimido[1,3-a]purin-10(3H)-one, a guanine adduct formed by reaction of malondialdehyde with DNA.

    Science.gov (United States)

    Hakala, K; Auriola, S; Koivisto, A; Lönnberg, H

    1999-12-01

    A high performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI MS/MS) method has been developed for quantification of pyrimido[1,2-a]purin-10(3H)-one adducts from DNA. The method is based on acid-catalyzed cleavage of the adducts from DNA and the use of [2,3a,10-13C3]pyrimido[1,2-a]purin-10(3H)-one as an internal standard in the analysis. For this purpose the latter compound was prepared. Rate constants for the acid-catalyzed cleavage of pyrimido[1,2-a]purin-10(3H)-one from the corresponding 2'-deoxyribonucleoside were determined, and its hydrolytic stability and possible formation by a cross reaction between guanine and [2,3a,10]pyrimido[1,2-a]purin-10(3H)-one were studied.

  13. Surface amplification of pencil graphite electrode with polypyrrole and reduced graphene oxide for fabrication of a guanine/adenine DNA based electrochemical biosensors for determination of didanosine anticancer drug

    Science.gov (United States)

    Karimi-Maleh, Hassan; Bananezhad, Asma; Ganjali, Mohammad R.; Norouzi, Parviz; Sadrnia, Abdolhossein

    2018-05-01

    Didanosine is nucleoside analog reverse transcriptase inhibitors with many side effects such as nausea and vomiting, stomach pain, tingling, burning and numbness and determination of this drug is very important in biological samples. This paper presents a DNA biosensor for determination of didanosine (DDI) in pharmaceutical samples. A pencil graphite electrode modified with conductive materials such as polypyrrole (PPy) and reduced graphene oxide (rGO) (PGE/PPy/rGO) was used for this goal. The double-stranded DNA was successfully immobilized on PGE/PPy/rGO. The PGE/PPy/rGO was characterized by microscopic and electrochemical methods. Then, the interaction of DDI with DNA was identified by decreases in the oxidation currents of guanine and adenine by differential pulse voltammetric (DPV) method. The dynamic range of DDI identified in the range of 0.02-50.0 μM and this electrode provided a low limit of detection (LOD = 8.0 nM) for DDI. The PGE/PPy/rGO loaded with ds-DNA was utilized for the measurement of DDI in real samples and obtained data were compared with HPLC method. The statistical tests such as F-test and t-test were used for confirming ability of PGE/PPy/rGO loaded with ds-DNA for analysis of DDI in real samples.

  14. Brefeldin A-Inhibited Guanine Nucleotide-Exchange Factor 1 (BIG1 Governs the Recruitment of Tumor Necrosis Factor Receptor-Associated Factor 2 (TRAF2 to Tumor Necrosis Factor Receptor 1 (TNFR1 Signaling Complexes

    Directory of Open Access Journals (Sweden)

    Takuya Noguchi

    2016-11-01

    Full Text Available Tumor necrosis factor receptor-associated factor 2 (TRAF2 is a critical mediator of tumor necrosis factor-α (TNF-α signaling. However, the regulatory mechanisms of TRAF2 are not fully understood. Here we show evidence that TRAF2 requires brefeldin A-inhibited guanine nucleotide-exchange factor 1 (BIG1 to be recruited into TNF receptor 1 (TNFR1 signaling complexes. In BIG1 knockdown cells, TNF-α-induced c-Jun N-terminal kinase (JNK activation was attenuated and the sensitivity to TNF-α-induced apoptosis was increased. Since these trends correlated well with those of TRAF2 deficient cells as previously demonstrated, we tested whether BIG1 functions as an upstream regulator of TRAF2 in TNFR1 signaling. As expected, we found that knockdown of BIG1 suppressed TNF-α-dependent ubiquitination of TRAF2 that is required for JNK activation, and impaired the recruitment of TRAF2 to the TNFR1 signaling complex (complex I. Moreover, we found that the recruitment of TRAF2 to the death-inducing signaling complex termed complex II was also impaired in BIG1 knockdown cells. These results suggest that BIG1 is a key component of the machinery that drives TRAF2 to the signaling complexes formed after TNFR1 activation. Thus, our data demonstrate a novel and unexpected function of BIG1 that regulates TNFR1 signaling by targeting TRAF2.

  15. A guanine nucleotide exchange factor for Rab5 proteins is essential for intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

    Science.gov (United States)

    Fukuda, Masako; Wen, Liuying; Satoh-Cruz, Mio; Kawagoe, Yasushi; Nagamura, Yoshiaki; Okita, Thomas W; Washida, Haruhiko; Sugino, Aya; Ishino, Sonoko; Ishino, Yoshizumi; Ogawa, Masahiro; Sunada, Mariko; Ueda, Takashi; Kumamaru, Toshihiro

    2013-06-01

    Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as a precursor, which are then transported via the Golgi to protein storage vacuoles (PSVs), where they are proteolytically processed into acidic and basic subunits. The glutelin precursor mutant6 (glup6) accumulates abnormally large amounts of proglutelin. Map-base cloning studies showed that glup6 was a loss-of-function mutant of guanine nucleotide exchange factor (GEF), which activates Rab GTPase, a key regulator of membrane trafficking. Immunofluorescence studies showed that the transport of proglutelins and α-globulins to PSV was disrupted in glup6 endosperm. Secreted granules of glutelin and α-globulin were readily observed in young glup6 endosperm, followed by the formation of large dilated paramural bodies (PMBs) containing both proteins as the endosperm matures. The PMBs also contained membrane biomarkers for the Golgi and prevacuolar compartment as well as the cell wall component, β-glucan. Direct evidence was gathered showing that GLUP6/GEF activated in vitro GLUP4/Rab5 as well as several Arabidopsis (Arabidopsis thaliana) Rab5 isoforms to the GTP-bound form. Therefore, loss-of-function mutations in GEF or Rab5 disrupt the normal transport of proglutelin from the Golgi to PSVs, resulting in the initial extracellular secretion of these proteins followed, in turn, by the formation of PMBs. Overall, our results indicate that GLUP6/GEF is the activator of Rab5 GTPase and that the cycling of GTP- and GDP-bound forms of this regulatory protein is essential for the intracellular transport of proglutelin and α-globulin from the Golgi to PSVs and in the maintenance of the general structural organization of the endomembrane system in rice seeds.

  16. Multisite contacts involved in coupling of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein. Structural and functional studies by beta-receptor-site-specific synthetic peptides.

    Science.gov (United States)

    Münch, G; Dees, C; Hekman, M; Palm, D

    1991-06-01

    Synthetic peptides, 12-22 amino acid residues long, comprising the presumed coupling sites of the beta-adrenergic receptor with the stimulatory guanine-nucleotide-binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N-terminal region of the third cytoplasmic loop, and the N-terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone-stimulated receptor for Gs activation with median effector concentrations of 15-35 microM, or 3-4 microM for combinations of two peptides. One peptide, corresponding to the C-terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs-adenylate-cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor-mediated G-protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G-protein heterotrimer, resulting in ligand-induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the alpha subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin-transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878-6882].

  17. CLL2-1, a chemical derivative of orchid 1,4-phenanthrenequinones, inhibits human platelet aggregation through thiol modification of calcium-diacylglycerol guanine nucleotide exchange factor-I (CalDAG-GEFI).

    Science.gov (United States)

    Liao, Chieh-Yu; Lee, Chia-Lin; Wang, Hui-Chun; Liang, Shih-Shin; Kung, Po-Hsiung; Wu, Yang-Chang; Chang, Fang-Rong; Wu, Chin-Chung

    2015-01-01

    CalDAG-GEFI is a guanine nucleotide exchange factor, which actives small GTPase Rap1 and plays an important role in platelet aggregation. Our previous study has shown that CalDAG-GEFI contains redox-sensitive thiols, and its function can be inhibited by thiol modification. In the present study, the effect of CLL2-1, a 1,4-phenanthrenequinone, on CalDAG-GEFI and platelet functions was investigated. In human platelets, CLL2-1 prevented platelet aggregation caused by various stimulators. Flow cytometric analysis revealed that CLL2-1 inhibited GPIIb/IIIa activation and P-selectin secretion. Moreover, CLL2-1 prevented Rap1 activation caused by thrombin, the Ca(2+) ionophore A23187, and the diacylglycerol mimetic phorbol 12-myristate 13-acetate, while only slightly inhibited thrombin-induced increases in [Ca(2+)]i and did not inhibit protein kinase C activation. Western blots after reducing SDS-PAGE showed that treatment of either platelets or platelet lysates with CLL2-1 led to a decrease of monomeric CalDAG-GEFI and appearance of cross-linked oligomers of CalDAG-GEFI, and these effects were inhibited by pretreatment of platelets or lysates with thiol reducing agents prior to the addition of CLL2-1, indicating thiol modification of CalDAG-GEFI by CLL2-1. Furthermore, the thiol reducing agents also prevented the inhibitory effect of CLL2-1 on Rap1 activation, GPIIb/IIIa activation, and platelet aggregation. In CalDAG-GEFI-overexpressing human embryonic kidney 293T cells, CLL2-1 also inhibited CalDAG-GEFI-mediated Rap1 activation. Taken together, our results suggest that the antiplatelet effect of CLL2-1 is due to, at least in part, inhibition of CalDAG-GEFI-mediated Rap1 activation, and provide the basis for development of novel antiplatelet drugs. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation.

    Science.gov (United States)

    Chang, Donald D; Colecraft, Henry M

    2015-12-01

    Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (Ca(V)1.2 currents, Ca(2+) transients, Ca(V)β binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized Rad(S105N) and Rem(T94N), which are the cognate mutants to Ras(S17N), a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either Rad(S105N) or Rem(T94N) strongly inhibited reconstituted Ca(V)1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that Rad(S105N) is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or Rad(S105N) in cardiomyocytes dramatically blocked L-type calcium current (I(Ca,L)) and inhibited Ca(2+)-induced Ca(2+) release, contradicting reports that Rad(S105N) acts as a dominant negative in heart. By contrast, Rem(T94N) was significantly less effective than wt Rem at inhibiting I(Ca,L) and Ca(2+) transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad(S105N) and Rem(T94N) had moderately reduced binding affinity for Ca(V)βs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in Ca(V)1.2 regulation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  19. Nuclear magnetic resonance studies of an N2-guanine adduct derived from the tumorigen dibenzo[a,l]pyrene in DNA: impact of adduct stereochemistry, size, and local DNA sequence on solution conformations.

    Science.gov (United States)

    Rodríguez, Fabián A; Liu, Zhi; Lin, Chin H; Ding, Shuang; Cai, Yuqin; Kolbanovskiy, Alexander; Kolbanovskiy, Marina; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

    2014-03-25

    The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide-DNA adducts formed include the stereoisomeric 14S and 14R trans-anti-DB[a,l]P-N(2)-dG and the stereochemically analogous 10S- and 10R-B[a]P-N(2)-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N(2)-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N(2)-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5'-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure-function relationship in NER.

  20. Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    International Nuclear Information System (INIS)

    Grasso, P.; Reichert, L.E. Jr.

    1990-01-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel

  1. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    Energy Technology Data Exchange (ETDEWEB)

    Grasso, P.; Reichert, L.E. Jr. (Albany Medical College, NY (USA))

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  2. Topoisomerase IB of Deinococcus radiodurans resolves guanine ...

    Indian Academy of Sciences (India)

    DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopolB. This implies that DraTopolB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited ...

  3. Topoisomerase IB of Deinococcus radiodurans resolves guanine ...

    Indian Academy of Sciences (India)

    2015-11-28

    Nov 28, 2015 ... structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in. D. radiodurans. [Kota S and Misra HS 2015 Topoisomerase IB of ..... 2004 Intracellular transcription of G-rich DNAs induces forma- tion of G-loops, novel structures containing G4 DNA. Genes. Dev.

  4. Synthesis of 2-substituted 9-oxa-guanines {5-aminooxazolo[5,4-d]pyrimidin-7(6H-ones} and 9-oxa-2-thio-xanthines {5-mercaptooxazolo[5,4-d]pyrimidin-7(6H-ones}

    Directory of Open Access Journals (Sweden)

    2008-07-01

    Full Text Available Oxazolo[5,4-d]pyrimidines can be considered as 9-oxa-purine analogs of naturally occurring nucleic acid bases. Interest in this ring system has increased due to recent reports of biologically active derivatives. In particular, 5-aminooxazolo[5,4-d]pyrimidine-7(6H-ones (9-oxa-guanines have been shown to inhibit ricin. The preparation of a series of 2-substituted 5-aminooxazolo[5,4-d]pyrimidin-7(6H-ones and related 5-thio-oxazolo[5,4-d]pyrimidines is described, including analogs suitable for further elaboration employing “click” chemistry utilizing copper-catalyzed Huisgen 1,3-dipolar cycloadditions. Two of the compounds prepared were found to inhibit ricin with IC50 ca. 1–3 mM.

  5. Reactivity of chitosan derivatives and their interaction with guanine ...

    Indian Academy of Sciences (India)

    Out of different models for accounting the solva- tion energies, dielectric continuum solvation models have been widely and successfully applied. In the self- ..... 40. Parr R G and Yang W 1989 In Density Functional The- ory of Atoms and Molecules (Oxford University Press: New York). 41. Parthasarathi R, Padmanabhan J, ...

  6. Reactivity of chitosan derivatives and their interaction with guanine ...

    Indian Academy of Sciences (India)

    by red region) spreading only over the N7 and O6 centers which points at a greater inclination of these ... co-workers have studied the behavior of these descrip- tors in presence of solvent media.48. DFRT defines ...... J, Dapprich S, Daniels A D, Farkas Ö., Foresman J B,. Ortiz J V, Cioslowski J and Fox R A D J. 2009 Gaus-.

  7. Oligomeric state of hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis

    Czech Academy of Sciences Publication Activity Database

    Eng, W. S.; Keough, D. T.; Hocková, Dana; Winzor, D. J.; Guddat, L. W.

    2017-01-01

    Roč. 135, Apr (2017), s. 6-14 ISSN 0300-9084 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : enzyme inhibitors * acyclic nucleoside phosphonates * 6-oxopurine phosphoribosyltransferase Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 3.112, year: 2016

  8. Preparation of guanine and diaminopurine from biuret. Part III.

    Science.gov (United States)

    Lagoja, Irene M; Herdewijn, Piet

    2007-04-01

    Because of their potential prebiotic origin and relative chemical stability, urea, biuret, formic acid, and glycine amide might have played a role in the assembly process of purine bases. In this paper, we describe a short reaction path to purine nucleobases from these acyclic precursors. The formation of different purines was verified by UV and NMR spectroscopy, as well as by mass spectrometry.

  9. Crystal structures of Trypanosoma brucei hypoxanthine-guanine-xanthine phosphoribosyltransferase

    Czech Academy of Sciences Publication Activity Database

    Terán, D.; Hocková, Dana; Česnek, Michal; Keough, D. T.; Guddat, L. W.

    2016-01-01

    Roč. 72, Suppl. (2016), S207 ISSN 0108-7673. [European Crystallographic Meeting /30./. 28.08.2016-01.09.2016, Basel] Institutional support: RVO:61388963 Keywords : 6-oxopurine phosphoribosyltransferase * enzyme inhibitors * acyclic nucleoside phosphonates Subject RIV: CC - Organic Chemistry

  10. Endogenous melatonin and oxidatively damaged guanine in DNA

    DEFF Research Database (Denmark)

    Davanipour, Zoreh; Poulsen, Henrik E; Weimann, Allan

    2009-01-01

    levels and either 8-oxodG or 8-oxoGua levels. When the mother and father data were further analyzed using only subjects older than the oldest daughter, the associations became somewhat stronger. CONCLUSION: Low levels of endogenous melatonin production among older individuals may lead to higher levels...

  11. Photochemical selectivity in guanine-cytosine base-pair structures

    Czech Academy of Sciences Publication Activity Database

    Abo-Riziq, A.; Grace, L.; Nir, E.; Kabeláč, Martin; Hobza, Pavel; Vries de, M. S.

    2005-01-01

    Roč. 102, č. 1 (2005), s. 20-23 ISSN 0027-8424 R&D Projects: GA ČR(CZ) GA203/05/0009 Grant - others:NSF(US) CHE-0244341 Institutional research plan: CEZ:AV0Z40550506 Keywords : DNA base pairs * IR-UV spectroscopy * phytochemistry Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 10.231, year: 2005

  12. Circular dichroism spectroscopy reveals invariant conformation of guanine runs in DNA

    Czech Academy of Sciences Publication Activity Database

    Kypr, Jaroslav; Vorlíčková, Michaela

    2002-01-01

    Roč. 67, 4/5 (2002), s. 275-277 ISSN 0006-3525 R&D Projects: GA AV ČR IAA5004802; GA ČR GA301/01/0590; GA ČR GA204/01/0561 Institutional research plan: CEZ:AV0Z5004920 Keywords : circular dichroism * spectroscopy * DNA Subject RIV: BO - Biophysics Impact factor: 2.372, year: 2002

  13. Microinjection of Guanine Nucleotide Analogues into Lily Pollen Tubes Results in Isodiametric Tip Expansion

    Czech Academy of Sciences Publication Activity Database

    Eliáš, M.; Cvrčková, F.; Obermeyer, G.; Žárský, Viktor

    2001-01-01

    Roč. 3, - (2001), s. 489-492 ISSN 1435-8603 R&D Projects: GA AV ČR IAA5038907; GA ČR GA206/99/1138 Institutional research plan: CEZ:AV0Z5038910 Keywords : pollen tube * G-proteins * cell polarity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 1.828, year: 2001

  14. Oxidative damage to guanine nucleosides following combination chemotherapy with 5-fluorouracil and oxaliplatin

    DEFF Research Database (Denmark)

    Afzal, Shoaib; Jensen, Søren Astrup; Sørensen, Jens Benn

    2011-01-01

    PURPOSE: Recent in vitro and animal studies have suggested that the cytotoxicity of 5-fluorouracil and oxaliplatin is linked to increased formation of reactive oxygen species (ROS). This prospective study was undertaken to examine the generation of oxidative stress, in 106 colorectal cancer...... patients, by 5-fluorouracil and oxaliplatin combination (FOLFOX) therapy as measured by urinary excretion of 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydro-guanosine (8-oxoGuo). METHODS: The amounts of 8-oxoGuo and 8-oxodG were measured in 3 spot urine samples from 106 patients by using...... ultra performance liquid chromatography and tandem mass spectrometry. Furthermore, we collected information on other clinical and demographic variables hypothesized to be associated with oxidative stress. Repeated measures linear mixed models were used to model the relationship between urinary...

  15. Theoretical study on the detailed repair of O6-methyl guanine to ...

    Indian Academy of Sciences (India)

    and Parkinson's diseases7–10 can be located on the endo- cylic nitrogen and exocyclic oxygen atoms of DNA bases (N7, N3 and O6 in the purines and O2, O4 and. N3 in the pyrimidines).11,12. Though N7-methylguanine (N7mG) is the most vulnerable site, yet O6-methylguanine (O6mG) has been found to be very toxic, ...

  16. Theoretical study of the guanine → 6-thioguanine substitution in duplexes, triplexes, and tetraplexes

    Czech Academy of Sciences Publication Activity Database

    Špačková, Naďa; Cubero, E.; Šponer, Jiří; Orozco, M.

    2004-01-01

    Roč. 126, č. 44 (2004), s. 14642-14650 ISSN 0002-7863 R&D Projects: GA MŠk LN00A016 Grant - others:European Training Mobility Program(XE) HPRI-CT-1999-00071; Ministry of Science and Technology (ES) BIO2003-06848; Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : 6-thioguanine * molecular dynamics simulations * thermodynamic integration Subject RIV: BO - Biophysics Impact factor: 6.903, year: 2004

  17. Crystal structures and inhibition of Trypanosoma brucei hypoxanthine-guanine phosphoribosyltransferase

    Czech Academy of Sciences Publication Activity Database

    Terán, D.; Hocková, Dana; Česnek, Michal; Zíková, Alena; Naesens, L.; Keough, D. T.; Guddat, L. W.

    2016-01-01

    Roč. 6, Oct 27 (2016), č. článku 35894. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA16-06049S; GA MŠk LL1205 Institutional support: RVO:61388963 ; RVO:60077344 Keywords : enzyme inhibitors * acyclic nucleoside phosphonates * HGPRT Subject RIV: CC - Organic Chemistry; EE - Microbiology, Virology (BC-A) Impact factor: 4.259, year: 2016 http://www.nature.com/articles/srep35894

  18. The guanine-rich fragile X chromosome repeats are reluctant to form tetraplexes

    Czech Academy of Sciences Publication Activity Database

    Fojtík, Petr; Kejnovská, Iva; Vorlíčková, Michaela

    2004-01-01

    Roč. 32, č. 1 (2004), s. 298-306 ISSN 0305-1048 R&D Projects: GA ČR GA204/01/0561; GA AV ČR IAA4004201 Institutional research plan: CEZ:AV0Z5004920 Keywords : fragile X chromosome syndrom * trinucleotide repeats * DNA polymorphism Subject RIV: BO - Biophysics Impact factor: 7.260, year: 2004

  19. Guanine nucleotide binding proteins in zucchini seedlings: Characterization and interactions with the NPA receptor

    International Nuclear Information System (INIS)

    Lindeberg, M.; Jacobs, M.

    1989-01-01

    A microsomal membrane preparation from hypocotyls of dark-grown Cucurbita pepo L. seedlings contains specific high-affinity binding sites for the non-hydrolyzable GTP analog guanosine 5'-[γ-thio] triphosphate (GTP-γ-S). Both the binding affinity and the pattern of binding specificity for GTP and GTP analogs are similar to animal G-proteins, and two zucchini membrane proteins are recognized in western blots by antiserum specific for the σ subunit of platelet G s protein. GTP-γ-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al +3 and F - agents known to activate G-proteins - decreased NPA specific binding by ca. 15%. In tests of in vitro auxin transport employing zucchini plasma membrane vesicles, AlF - 4 strongly inhibited 3 H-indoleacetic acid nor accumulation; GTP-γ-S effects on this system will be discussed

  20. Effect O6-Guanine Alkylation on DNA Flexibility Studied by Comparative Molecular Dynamics Simulations

    Czech Academy of Sciences Publication Activity Database

    Kara, M.; Dršata, Tomáš; Lankaš, Filip; Zacharias, M.

    2015-01-01

    Roč. 103, č. 1 (2015), s. 23-32 ISSN 0006-3525 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA damage * DNA alkylation * DNA repair * molecular simulation * molecular dynamics simulation Subject RIV: BO - Biophysics Impact factor: 2.248, year: 2015

  1. Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

    Science.gov (United States)

    Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

    2018-03-05

    Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

  2. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    Energy Technology Data Exchange (ETDEWEB)

    Di Paolo, T.; Falardeau, P.

    1987-08-31

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p < 0.001). Competition for (/sup 3/H)-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-(..beta..-..gamma..-imino)triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables.

  3. Guanine nucleotide regulation of dopamine receptor agonist affinity states in rat estradiol-induced pituitary tumors

    International Nuclear Information System (INIS)

    Di Paolo, T.; Falardeau, P.

    1987-01-01

    The authors have investigated dopamine (DA) receptor agonist high- and low-affinity states in female rate estradiol-induced prolactin (PRL)-secreting pituitary tumors and intact pituitary tissue. Estradiol treatment increased the anterior pituitary weight 9-fold and plasma prolactin levels 74-fold and these measures are correlated (R = 0.745, n = 73, p 3 H]-spiperone binding to the DA receptor by apomorphine was compared in normal and adenomatous pituitary tissue. The inhibition constants (Ki) and the proportions of the two apomorphine sites are unchanged in tumors compared to intact pituitary tissue. Guanosine 5'-[β-γ-imino]triphosphate (Gpp(NH)p) causes complete conversion of the high into low affinity dopaminergic agonist site in normal pituitary and in tumors. These results suggest that rats with primary estradiol-induced pituitary tumors have normal and functional DA receptors. 9 references, 2 tables

  4. Vibrational circular dichroism study of isotropic and liquid crystalline phases formed by guanine quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Andrushchenko, Valery; Tsankov, D.; Krasteva, M.; Wieser, H.; Bouř, Petr

    2012-01-01

    Roč. 19, č. 1 (2012), s. 3-3 ISSN 1210-8529. [10th Discussions in Structural Molecular Biology. 22.03.2012-24.03.2012, Nové Hrady] R&D Projects: GA ČR GAP208/10/0559; GA ČR GAP208/11/0105 Grant - others:AV ČR M200550902 Institutional research plan: CEZ:AV0Z40550506 Keywords : G-quadruplexes * DNA * liquid crystals * VCD spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry

  5. Selected health and lifestyle factors, cytosine-adenine-guanine status, and phenoconversion in Huntington's disease.

    Science.gov (United States)

    Tanner, Caroline; Marder, Karen; Eberly, Shirley; Biglan, Kevin; Oakes, David; Shoulson, Ira

    2018-03-01

    In Huntington's disease, 60% of the variance in onset age is not explained by the huntingtin gene mutation. Huntington's disease onset was earlier in caffeine users. The objective of this study was to assess the relationship of lifestyle factors with motor phenoconversion among persons at risk for Huntington's disease. The associations of motor phenoconversion and exposure to selected lifestyle and health factors were examined using Cox proportional hazards analyses adjusted for age, gender, and repeat length. Of 247 participants, 36 (14.6%) phenoconverted. Mean follow-up was 4.2 years. Greater caffeinated soda use was associated with an increased hazard of phenoconversion: moderate use hazard ratio 2.26 (95% confidence interval 0.59-8.71), high use hazard ratio 4.05 (95% confidence interval 1.18-13.96). Huntington's disease onset was earlier among consumers of caffeinated soda, but not other caffeinated beverages. This finding may be spurious or not related to caffeine. © 2018 International Parkinson and Movement Disorder Society. © 2018 International Parkinson and Movement Disorder Society.

  6. Guanine nucleotide-binding regulatory proteins in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Jiang, Meisheng; Tran, V.T.; Fong, H.K.W. (Univ. of Southern California, Los Angeles (United States)); Pandey, S. (Doheny Eye Inst., Los Angeles, CA (United States))

    1991-05-01

    The expression of GTP-binding regulatory proteins (G proteins) in retinal pigment epithelial (RPE) cells was analyzed by RNA blot hybridization and cDNA amplification. Both adult and fetal human RPE cells contain mRNA for multiple G protein {alpha} subunits (G{alpha}) including G{sub s}{alpha}, G{sub i-1}{alpha}, G{sub i-2}{alpha}, G{sub i-3}{alpha}, and G{sub z}{alpha} (or G{sub x}{alpha}), where G{sub s} and G{sub i} are proteins that stimulate or inhibit adenylyl cyclase, respectively, and G{sub z} is a protein that may mediate pertussis toxin-insensitive events. Other G{alpha}-related mRNA transcripts were detected in fetal RPE cells by low-stringency hybridization to G{sub i-2}{alpha} and G{sub s}{alpha} protein-coding cDNA probes. The diversity of G proteins in RPE cells was further studied by cDNA amplification with reverse transcriptase and the polymerase chain reaction. This approach revealed that, besides the above mentioned members of the G{alpha} gene family, at least two other G{alpha} subunits are expressed in RPE cells. Human retinal cDNA clones that encode one of the additional G{alpha} subunits were isolated and characterized. The results indicate that this G{alpha} subunit belongs to a separate subfamily of G proteins that may be insensitive to inhibition by pertussis toxin.

  7. Functional reconstitution of prostaglandin E receptor from bovine adrenal medulla with guanine nucleotide binding proteins

    International Nuclear Information System (INIS)

    Negishi, M.; Ito, S.; Yokohama, H.; Hayashi, H.; Katada, T.; Ui, M.; Hayaishi, O.

    1988-01-01

    Prostaglandin E 2 (PEG 2 ) was found to bind specifically to a 100,000 x g pellet prepared from bovine adrenal medulla. The PGE receptor was associated with a GTP-binding protein (G-protein) and could be covalently cross-linked with this G-protein by dithiobis(succinimidyl propionate) in the 100,000 x g pellet. In order to characterize the G-protein associated with the PGE receptor and reconstitute these proteins in phospholipid vesicles, the authors purified the G-protein to apparent homogeneity from the 100,000 x g pellet. The G-protein served as a substrate of pertussis toxin but differed in its α subunit from two known pertussis toxin substrate G-proteins (G/sub i/ and G 0 ) purified from bovine brain. The molecular weight of the α subunit was 40,000, which is between those of G/sub i/ and G 0 . The purified protein was also distinguished immunologically from G/sub i/ and G 0 and was referred to as G/sub am/. Reconstitution of the PGE receptor with pure C/sub am/, G/sub i/, or G 0 in phospholipid vesicles resulted in a remarkable restoration of [ 3 H]PGE 2 binding activity in a GTP-dependent manner. The efficiency of these three G-proteins in this capacity was roughly equal. When pertussis toxin- or N-ethylmaleimide-treated G-proteins, instead of the native ones, were reconstituted into vesicles, the restoration of binding activity was no longer observed. These results indicate that the PGE receptor can couple functionally with G/sub am/, G/sub i/, or G 0 in phospholipid vesicles and suggest that G/sub am/ may be involved in signal transduction of the PGE receptor in bovine adrenal medulla

  8. Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: A new class of antimalarial therapeutics

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Hocková, Dana; Holý, Antonín; Naesens, L.; Skinner-Adams, T. S.; de Jersey, J.; Guddat, L. W.

    2009-01-01

    Roč. 52, č. 14 (2009), s. 4391-4399 ISSN 0022-2623 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * phosphoribosyltransferase * enzyme inhibitors * Plasmodium falciparum Subject RIV: CC - Organic Chemistry Impact factor: 4.802, year: 2009

  9. Theoretical study on the detailed repair of O6-methyl guanine to ...

    Indian Academy of Sciences (India)

    (chemical pollutants and UV radiation) and endoge- nous (the normal metabolic byproducts, especially in the oxidative deamination process) sources of DNA damaging agents.1–6 Methylating agents which lead to a wide range of DNA damage such as aging, chronic inflammatory diseases, mutation, cancer, Alzheimer's.

  10. Voltametric Determination of Adenine, Guanine and DNA Using Liquid Mercury Free Polished Silver Solid Amalgam Electrode

    Czech Academy of Sciences Publication Activity Database

    Fadrná, Renata; Josypčuk, Bohdan; Fojta, Miroslav; Navrátil, Tomáš; Novotný, Ladislav

    2004-01-01

    Roč. 37, č. 3 (2004), s. 399-413 ISSN 0003-2719 R&D Projects: GA AV ČR KSK4040110 Grant - others:GIT(AR) 101/02/U111/CZ Keywords : voltammetry * DNA * polished silver solid amalgam electrode Subject RIV: CG - Electrochemistry Impact factor: 1.165, year: 2004

  11. Study of Adenine and Guanine Oxidation Mechanism by Surface-Enhanced Raman Spectroelectrochemistry

    Czech Academy of Sciences Publication Activity Database

    Ibanez, D.; Santidrian, Ana; Heras, A.; Kalbáč, Martin; Colina, A.

    2015-01-01

    Roč. 119, č. 15 (2015), s. 8191-8198 ISSN 1932-7447 R&D Projects: GA MŠk LL1301 Institutional support: RVO:61388955 Keywords : nucleic-acid bases * electrochemical oxidation * silver electrode Subject RIV: CG - Electrochemistry Impact factor: 4.509, year: 2015

  12. Guanine Bases in DNA G-Quadruplex Adopt Nonplanar Geometries Owing to Solvation and Base Pairing

    Czech Academy of Sciences Publication Activity Database

    Sychrovský, Vladimír; Sochorová Vokáčová, Zuzana; Trantírek, L.

    2012-01-01

    Roč. 116, č. 16 (2012), s. 4144-4151 ISSN 1089-5639 R&D Projects: GA ČR GAP205/10/0228 Institutional research plan: CEZ:AV0Z40550506 Keywords : pyramidalization * nonplanarity * G-quadruplex * DNA * solvent effect Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.771, year: 2012

  13. Combined theoretical and computational study of interstrand DNA guanine-guanine cross-linking by trans-[Pt(pyridine)2] derived from the photoactivated prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2

    Czech Academy of Sciences Publication Activity Database

    Tai, H.-Ch.; Brodbeck, R.; Kašpárková, Jana; Farrer, N.J.; Brabec, Viktor; Sadler, P.J.; Deeth, R.J.

    2012-01-01

    Roč. 51, č. 12 (2012), s. 6830-6841 ISSN 0020-1669 R&D Projects: GA ČR(CZ) GAP301/10/0598 Institutional research plan: CEZ:AV0Z50040702 Keywords : platinum * photoactivation * DNA Subject RIV: BO - Biophysics Impact factor: 4.593, year: 2012

  14. Theoretical study of gas and solvent phase stability and molecular adsorption of noncanonical guanine bases on graphene.

    Science.gov (United States)

    Saikia, Nabanita; Karna, Shashi P; Pandey, Ravindra

    2017-06-28

    The gas and solvent phase stability of noncanonical (Gua) n nucleobases is investigated in the framework of dispersion-corrected density functional theory (DFT). The calculated results strongly support the high tendency for the dimerization of (Gua) n bases in both gas and solvent phases. An interplay between intermolecular and bifurcated H-bonds is suggested to govern the stability of (Gua) n bases which bears a correlation with the description of dispersion correction terms employed in the DFT calculations. For example, a higher polarity is predicted for (Gua) n bases by the dispersion-corrected DFT in contrast to the non-polar nature of (Gua) 3 and (Gua) 4 predicted by the hybrid meta-GGA calculations. This distinct variation becomes significant under physiological conditions as polar (Gua) n is likely to exhibit greater stabilization in the gas phase compared to solvated (Gua) n . Graphene acting as a substrate induces modification in base configurations via maximization of π-orbital overlap between the base and substrate. In solvent, the substrate-induced effects are further heightened with lowering of the dipole moments of (Gua) n as also displayed by the corresponding isosurface of the electrostatic potential. The graphene-induced stability in both gas and solvent phases appears to fulfill one of the prerequisite criteria for molecular self-assembly. The DFT results therefore provide atomistic insights into the stability and molecular assembly of free-standing noncanonical (Gua) n nucleobases which can be extended to understanding the self-assembly process of functional biomolecules on 2D materials for potential biosensing applications.

  15. VEGF-induced Rac1 activation in endothelial cells is regulated by the guanine nucleotide exchange factor Vav2

    NARCIS (Netherlands)

    Garrett, Tiana A.; van Buul, Jaap D.; Burridge, Keith

    2007-01-01

    Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the

  16. Guanine nucleotides in the meiotic maturation of starfish oocytes: regulation of the actin cytoskeleton and of Ca(2+ signaling.

    Directory of Open Access Journals (Sweden)

    Keiichiro Kyozuka

    Full Text Available BACKGROUND: Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA. The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca(2+ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca(2+ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. METHODOLOGY/PRINCIPAL FINDINGS: By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPgammaS or inactive (GDPbetaS forms of G-proteins, we have demonstrated that: i GTPgammaS induces Ca(2+ release that mimics the effect of 1-MA; ii GDPbetaS completely blocks 1-MA-induced Ca(2+; iii GDPbetaS has little effect on the amplitude of the Ca(2+ peak, but significantly expedites the initial Ca(2+ waves induced by InsP(3 photoactivation, iv GDPbetaS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca(2+ release kinetics; v alteration of cortical actin networks with jasplakinolide, GDPbetaS, or actinase E, all led to significant changes of 1-MA-induced Ca(2+ signaling. CONCLUSIONS/SIGNIFICANCE: Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca(2+ release.

  17. Relative Stability of Different DNA Guanine Quadruplex Stem Topologies Derived Using Large-Scale Quantum-Chemical Computations

    Czech Academy of Sciences Publication Activity Database

    Šponer, Jiří (ed.); Mládek, Arnošt; Špačková, Naďa; Cang, X.; Cheatham III, Thomas E.; Grimme, S.

    2013-01-01

    Roč. 135, č. 26 (2013), s. 9785-9796 ISSN 0002-7863 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068; GA ČR(CZ) GAP208/11/1822 Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : DENSITY -FUNCTIONAL THEORY * MOLECULAR-DYNAMICS SIMULATIONS * SUGAR-PHOSPHATE BACKBONE Subject RIV: BO - Biophysics Impact factor: 11.444, year: 2013

  18. Guanine radical reaction processes: a computational description of proton transfer in X-irradiated 9-ethylguanine single crystals.

    Science.gov (United States)

    Jayatilaka, Nayana; Nelson, William H

    2008-12-25

    Computational methods based on DFT procedures have been used to investigate proton-transfer processes in irradiated 9-ethylguanine crystals. Previous experimental results from X-irradiation and study of this system at 10 K found significant concentrations of two main products, R1, formed by N7-hydrogenation of the purine ring, and R2, the primary one-electron oxidation product (Jayatilaka, N.; Nelson, W. H. J. Phys. Chem. B 2007, 111, 7887). The objective of this work is to describe the processes leading to these products using computational methods that take into account molecular packing and bulk dielectric properties. The basic concept is that a proton will transfer following ionization if the net electronic energy of the system, consisting of the donor plus the acceptor plus any intervening molecules, becomes lower. Three approaches were used to investigate this concept, two based on energies computed for single molecules and one based on energies computed for two-molecule clusters arranged as in the crystals. The results are that the methods successfully predict the observed behavior, that it is energetically favorable on one-electron reduction for proton H1 to transfer from a neutral molecule to N7 of the neighbor, forming the N7-hydrogenated product, and that there is virtually no energy advantage for a proton to transfer upon one-electron oxidation. The results also support the proposal that the C8 H-addition radical, found only upon irradiation at 300 K, was the product of intramolecular transfer of the H7 proton to C8 in a process apparently requiring sufficient thermal energy for activation. Finally, the computations predict hyperfine couplings and tensors in very good agreement with those from experiment, thereby providing additional evidence for the success of the computations in describing the experimental observations.

  19. Cytosolic guanine nucledotide binding deficient form of transglutaminase 2 (R580a potentiates cell death in oxygen glucose deprivation.

    Directory of Open Access Journals (Sweden)

    Gozde Colak

    Full Text Available Transglutaminase 2 (TG2 is a hypoxia-responsive protein that is a calcium-activated transamidating enzyme, a GTPase and a scaffolding/linker protein. Upon activation TG2 undergoes a large conformational change, which likely affects not only its enzymatic activities but its non-catalytic functions as well. The focus of this study was on the role of transamidating activity, conformation and localization of TG2 in ischemic cell death. Cells expressing a GTP binding deficient form of TG2 (TG2-R580A with high basal transamidation activity and a more extended conformation showed significantly increased cell death in response to oxygen-glucose deprivation; however, targeting TG2-R580A to the nucleus abrogated its detrimental role in oxygen-glucose deprivation. Treatment of cells expressing wild type TG2, TG2-C277S (a transamidating inactive mutant and TG2-R580A with Cp4d, a reversible TG2 inhibitor, did not affect cell death in response to oxygen-glucose deprivation. These findings indicate that the pro-cell death effects of TG2 are dependent on its localization to the cytosol and independent of its transamidation activity. Further, the conformational state of TG2 is likely an important determinant in cell survival and the prominent function of TG2 in ischemic cell death is as a scaffold to modulate cellular processes.

  20. ITAM signaling by Vav family Rho guanine nucleotide exchange factors regulates interstitial transit rates of neutrophils in vivo.

    Directory of Open Access Journals (Sweden)

    Daniel B Graham

    Full Text Available In response to infection, neutrophils are quickly recruited from the blood into inflamed tissues. The interstitial migration of neutrophils is crucial for the efficient capture and control of rapidly proliferating microbes before microbial growth can overwhelm the host's defenses. However, the molecular mechanisms that regulate interstitial migration are incompletely understood.Here, we use two-photon microscopy (2PM to study discrete steps of neutrophil responses during subcutaneous infection with bacteria. Our study demonstrates that signals emanating from ITAM-containing receptors mediated by Vav family Rho GEFs control the velocity, but not the directionality, of neutrophil migration towards sites of bacterial infection.Here we show that during neutrophil migration towards sites of bacterial infection, signals emanating from ITAM-containing receptors specifically control interstitial neutrophil velocity.

  1. Can We Execute Reliable MM-PBSA Free Energy Computations of Relative Stabilities of Different Guanine Quadruplex Folds?

    Czech Academy of Sciences Publication Activity Database

    Islam, B.; Stadlbauer, Petr; Neidle, S.; Haider, S.; Šponer, Jiří

    2016-01-01

    Roč. 120, č. 11 (2016), s. 2899-2912 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : MOLECULAR- DYNAMICS SIMULATIONS * TELOMERIC G-QUADRUPLEX * AMBER FORCE-FIELD Subject RIV: BO - Biophysics Impact factor: 3.177, year: 2016

  2. Folding of guanine quadruplex molecules-funnel-like mechanism or kinetic partitioning? An overview from MD simulation studies

    Czech Academy of Sciences Publication Activity Database

    Šponer, Jiří; Bussi, G.; Stadlbauer, Petr; Kührová, P.; Banáš, P.; Islam, Barira; Haider, S.; Neidle, S.; Otyepka, M.

    2017-01-01

    Roč. 1861, č. 5 (2017), s. 1246-1263 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : telomeric g-quadruplex * parallel g-quadruplex Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  3. Free-radical intermediates produced from the one-electron reduction of purine, adenine, and guanine derivatives in water

    International Nuclear Information System (INIS)

    Moorthy, P.N.; Hayon, E.

    1975-01-01

    The one-electron reduction of purine (PH), 9-methylpurine (MP), adenosine (A) and l-methylguanosine (MG) in water was studied using the fast-reaction technique of pulse radiolysis and kinetic absorption spectrophotometry. The hydrated electron, e - /sub aq/, and the acetone ketyl radical, (CH 3 ) 2 COH, were used as the reducing agents. The reaction rate constants of the purine derivatives with e - /sub aq/ and the (CH 3 ) 2 COH radical were determined at different pH values, consistent with the pK/sub a/ values of these compounds. The electron transfer reaction from (CH 3 ) 2 COH was found to be strongly dependent on the acid-base properties of the purines and on the nature of the substituents. The transient optical absorption spectra of the free-radical intermediates produced from the reduction of the purine derivatives were determined over the pH range 0 to 14. The extinction coefficients and decay kinetics are also presented. For 9-methylpurine, the MPH 3 . 2+ , MPH 2 . + , and MPH. radicals have pK/sb a/ values of 2.9 plus or minus 0.2, 6.3 plus or minus 0.1, and 13.1 plus or minus 0.2, respectively. For adenosine, the AH 3 . 2+ and AH 2 + radicals have pK/sub a/ values of 4.6 plus or minus 0.1 and 10.5 plus or minus 0.1, respectively, while the ionization of AH. is not observed up to pH 13.6. For l-methylguanosine, two pK/sub a/ (radical) values approximately 7.0 and greater than or equal to 13.0 are observed. These and other results are discussed, and tentative assignments are suggested for the various radical intermediates. (U.S.)

  4. Guanine polynucleotides are self-antigens for human natural autoantibodies and are significantly reduced in the human genome

    Science.gov (United States)

    Fattal, Ittai; Shental, Noam; Ben-Dor, Shifra; Molad, Yair; Gabrielli, Armando; Pokroy-Shapira, Elisheva; Oren, Shirly; Livneh, Avi; Langevitz, Pnina; Zandman-Goddard, Gisele; Sarig, Ofer; Margalit, Raanan; Gafter, Uzi; Domany, Eytan; Cohen, Irun R

    2015-01-01

    In the course of investigating anti-DNA autoantibodies, we examined IgM and IgG antibodies to poly-G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20-mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo-nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti-G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170 000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain-associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti-G20 antibodies; so natural anti-G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti-G20 antibodies in human health and disease and of runs of G20 in the human genome. PMID:26227667

  5. Hydrogen Evolution Facilitates Reduction of DNA Guanine Residues at the Hanging Mercury Drop Electrode: Evidence for a Chemical Mechanism

    Czech Academy of Sciences Publication Activity Database

    Daňhel, Aleš; Havran, Luděk; Trnková, L.; Fojta, Miroslav

    2016-01-01

    Roč. 28, č. 11 (2016), s. 2785-2790 ISSN 1040-0397 R&D Projects: GA ČR GAP206/11/1638; GA ČR(CZ) GA16-01625S Institutional support: RVO:68081707 Keywords : central trinucleotide sequences * cyclic voltammetry * cisplatin Subject RIV: BO - Biophysics Impact factor: 2.851, year: 2016

  6. N-Phosphonocarbonylpyrrolidine Derivatives of Guanine: A New Class of Bi-Substrate Inhibitors of Human Purine Nucleoside Phosphorylase

    Czech Academy of Sciences Publication Activity Database

    Rejman, Dominik; Panova, Natalya; Klener, P.; Maswabi, B.; Pohl, Radek; Rosenberg, Ivan

    2012-01-01

    Roč. 55, č. 4 (2012), s. 1612-1621 ISSN 0022-2623 R&D Projects: GA MŠk 2B06065; GA MŠk(CZ) LC06077; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z40550506 Keywords : nucleoside * phosphonic acid * pyrrolidine * purine nucleoside Subject RIV: CC - Organic Chemistry Impact factor: 5.614, year: 2012

  7. Vanadate stimulates adenylate cyclase via the guanine nucleotide regulatory protein by a mechanism differing from that of fluoride.

    Science.gov (United States)

    Krawietz, W; Downs, R W; Spiegel, A M; Aurbach, G D

    1982-03-01

    Vanadate stimulates adenylate cyclase activity in turkey erythrocyte membranes. The maximal stimulation is 7-fold over basal at 3 mM vanadate; higher concentrations are inhibitory. A suboptimal concentration of fluoride (1 mM) together with vanadate (3 mM) activates adenylate cyclase in a non-additive manner; cyclase activation by optimal fluoride (10 mM) is inhibited by vanadate (3 mM). There is no stimulation by vanadate of adenylate cyclase activity (measured either with Mg2+ or Mn2+) in CYC- S49 lymphoma cell membranes. Vanadate (3 mM) shows no effect on binding of Beta-adrenergic agonists or antagonists to the [3H] (-)-dihydroalprenolol binding site in turkey erythrocyte membranes. These results suggest that the effect of vanadate on Adenylate cyclase is mediated through the nucleotide regulatory protein and may act by a mechanism similar to fluoride. However, in cholera toxic-treated membranes as well as in GDP-beta-S plus isoproterenol-treated membranes, fluoride-stimulated adenylate cyclase activity is significantly reduced, but vanadate stimulation is not. Our results suggest that although the actions of vanadate and fluoride in adenylate cyclase may each involve the nucleotide regulatory unit, the exact mechanisms of activation by the two anions differ.

  8. Strikingly Different Effects of Hydrogen Bonding on the Photodynamics of Individual Nucleobases in DNA: Comparison of Guanine and Cytosine

    Czech Academy of Sciences Publication Activity Database

    Zelený, T.; Ruckenbauer, M.; Aquino, A. J. A.; Müller, T.; Lankaš, Filip; Dršata, Tomáš; Hase, W. L.; Nachtigallová, Dana; Lischka, H.

    2012-01-01

    Roč. 134, č. 33 (2012), s. 13662-13669 ISSN 0002-7863 R&D Projects: GA ČR GAP208/12/1318; GA MŠk(CZ) LH11021 Grant - others:GA ČR(CZ) GD203/09/H046 Program:GD Institutional support: RVO:61388963 Keywords : excited-state dynamics * ultrafast internal-conversion * photoinduced nonadiabatic dynamics * nonradiative decay mechanisms * MR-CI level * ab initio Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 10.677, year: 2012

  9. Effect of Guanine to Inosine Substitution on Stability of Canonical DNA and RNA Duplexes: Molecular Dynamics Thermodynamics Integration Study

    Czech Academy of Sciences Publication Activity Database

    Krepl, Miroslav; Otyepka, M.; Banáš, Pavel; Šponer, Jiří

    2013-01-01

    Roč. 117, č. 6 (2013), s. 1872-1879 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GBP305/12/G034 Grant - others:GA ČR(CZ) GPPP301/11/P558; GA MŠk(CZ) ED1.1.00/02.0068 Program:ED Institutional research plan: CEZ:AV0Z50040702 Institutional support: RVO:68081707 Keywords : FREE-ENERGY CALCULATIONS * PARTICLE MESH EWALD * ACID BASE-PAIRS Subject RIV: BO - Biophysics Impact factor: 3.377, year: 2013

  10. Group I metabotropic glutamate receptors reduce excitotoxic injury and may facilitate neurogenesis

    DEFF Research Database (Denmark)

    Baskys, Andrius; Bayazitov, Ildar; Fang, Liwei

    2005-01-01

    neuroprotective activation of group I metabotropic glutamate receptors. Brain Research, Molecular Brain Research 117, 196-205.]. In the present study, we used organotypic hippocampal culture preparation to examine specific phospholipase C (PLC) inhibitor U73122 effects on DHPG-induced neuroprotection, changes......-CA1 pathway. The fEPSP depression was not affected by the PLC inhibitor U73122. In contrast, prolonged (2-h) treatment of cultures with DHPG induced a significant protective effect that was blocked by a PLC inhibitor U73122 but not by its inactive analog U73343. Voltage-clamp measurements...... a PLC involvement. Since activation of PLC is thought to be associated with cell proliferation, we investigated whether group I mGluR agonist DHPG or subtype antagonists LY367385 and MPEP have an effect on dentate granule cells expressing immature neuronal marker TOAD-64. DHPG (100 microM, 72 h...

  11. Plasma Catechols in Familial Dysautonomia: A Long-term Follow-up Study

    Science.gov (United States)

    Holmes, Courtney; Axelrod, Felicia B.

    2017-01-01

    This study tested whether familial dysautonomia (FD) involves progressive loss of noradrenergic nerves. Plasma levels of catechols, including dihydroxyphenylglycol (DHPG), norepinephrine (NE), dopamine (DA), and DOPA, were measured in 7 adult patients with FD and 50 healthy control subjects. FD patients were re-tested after a mean follow-up period of 13 years. Compared to controls, FD patients had low plasma levels of DHPG (P < 0.001), high DOPA and DA levels (P = 0.01, P = 0.0002), and high NE:DHPG (P < 0.0001), DA:NE (P = 0.0003), and DOPA:DHPG (P < 0.0001) ratios. At follow-up there were no changes in plasma levels of individual catechols; however, there were further increases in DOPA:DHPG ratios (mean 24 ± 7%, P = 0.01). In FD, plasma catechol profiles are sufficiently stable, at least over a decade, to be used as a biomarker of disease involvement. An increasing DOPA:DHPG ratio suggests slight but consistent, progressive loss of noradrenergic neurons. PMID:18357519

  12. Tyrosine dephosphorylation regulates AMPAR internalisation in mGluR-LTD.

    Science.gov (United States)

    Gladding, Clare M; Collett, Valerie J; Jia, Zhengping; Bashir, Zafar I; Collingridge, Graham L; Molnár, Elek

    2009-02-01

    Long-term depression (LTD) can be induced at hippocampal CA1 synapses by activation of either NMDA receptors (NMDARs) or group I metabotropic glutamate receptors (mGluRs), using their selective agonists NMDA and (RS)-3,5-dihydroxyphenylglycine (DHPG), respectively. Recent studies revealed that DHPG-LTD is dependent on activation of postsynaptic protein tyrosine phosphatases (PTPs), which transiently dephosphorylate tyrosine residues in AMPA receptors (AMPARs). Here we show that while both endogenous GluR2 and GluR3 AMPAR subunits are tyrosine phosphorylated at basal activity, only GluR2 is dephosphorylated in DHPG-LTD. The tyrosine dephosphorylation of GluR2 does not occur in NMDA-LTD. Conversely, while NMDA-LTD is associated with the dephosphorylation of GluR1-serine-845, DHPG-LTD does not alter the phosphorylation of this site. The increased AMPAR endocytosis in DHPG-LTD is PTP-dependent and involves tyrosine dephosphorylation of cell surface AMPARs. Together, these results indicate that the subunit selective tyrosine dephosphorylation of surface GluR2 regulates AMPAR internalisation in DHPG-LTD but not in NMDA-LTD in the hippocampus.

  13. ARF6 Activated by the LHCG Receptor through the Cytohesin Family of Guanine Nucleotide Exchange Factors Mediates the Receptor Internalization and Signaling*

    Science.gov (United States)

    Kanamarlapudi, Venkateswarlu; Thompson, Aiysha; Kelly, Eamonn; López Bernal, Andrés

    2012-01-01

    The luteinizing hormone chorionic gonadotropin receptor (LHCGR) is a Gs-coupled GPCR that is essential for the maturation and function of the ovary and testis. LHCGR is internalized following its activation, which regulates the biological responsiveness of the receptor. Previous studies indicated that ADP-ribosylation factor (ARF)6 and its GTP-exchange factor (GEF) cytohesin 2 regulate LHCGR internalization in follicular membranes. However, the mechanisms by which ARF6 and cytohesin 2 regulate LHCGR internalization remain incompletely understood. Here we investigated the role of the ARF6 signaling pathway in the internalization of heterologously expressed human LHCGR (HLHCGR) in intact cells using a combination of pharmacological inhibitors, siRNA and the expression of mutant proteins. We found that human CG (HCG)-induced HLHCGR internalization, cAMP accumulation and ARF6 activation were inhibited by Gallein (βγ inhibitor), Wortmannin (PI 3-kinase inhibitor), SecinH3 (cytohesin ARF GEF inhibitor), QS11 (an ARF GAP inhibitor), an ARF6 inhibitory peptide and ARF6 siRNA. However, Dynasore (dynamin inhibitor), the dominant negative mutants of NM23-H1 (dynamin activator) and clathrin, and PBP10 (PtdIns 4,5-P2-binding peptide) inhibited agonist-induced HLHCGR and cAMP accumulation but not ARF6 activation. These results indicate that heterotrimeric G-protein, phosphatidylinositol (PI) 3-kinase (PI3K), cytohesin ARF GEF and ARF GAP function upstream of ARF6 whereas dynamin and clathrin act downstream of ARF6 in the regulation of HCG-induced HLHCGR internalization and signaling. In conclusion, we have identified the components and molecular details of the ARF6 signaling pathway required for agonist-induced HLHCGR internalization. PMID:22523074

  14. Theoretical study on the structure, stability, and electronic properties of the guanine-Zn-cytosine base pair in M-DNA

    Czech Academy of Sciences Publication Activity Database

    Fuentes-Cabrera, M.; Sumpter, B.G.; Šponer, Judit E.; Šponer, Jiří; Petit, L.; Wells, J.C.

    2007-01-01

    Roč. 111, č. 4 (2007), s. 870-879 ISSN 1520-6106 R&D Projects: GA AV ČR(CZ) 1QS500040581; GA ČR(CZ) GA203/05/0388; GA ČR(CZ) GA203/05/0009; GA MŠk(CZ) LC512 Institutional research plan: CEZ:AV0Z50040702; CEZ:AV0Z40550506 Keywords : metalated DNA * ab initio * electronic properties of DNA Subject RIV: BO - Biophysics Impact factor: 4.086, year: 2007

  15. Serine34 phosphorylation of RHO guanine dissociation inhibitor (RHOGDI{alpha}) links signaling from conventional protein kinase C to RHO GTPase in cell adhesion

    DEFF Research Database (Denmark)

    Dovas, Athanassios; Choi, Youngsil; Yoneda, Atsuko

    2010-01-01

    , resulting in a specific decrease in affinity for RhoA, but not Rac1 or cdc42. The mechanism of RhoGDIalpha phosphorylation is distinct, requiring PKCalpha and phosphatidylinositol 4,5 bisphosphate, consistent with recent evidence that the inositide can activate, localize and orient PKCalpha in membranes...

  16. Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72.

    Science.gov (United States)

    Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B

    2018-01-22

    An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. 5-METHYLCYTOSINE IN CPG SITES AND THE REACTIVITY OF NEAREST NEIGHBORING GUANINES TOWARD THE CARCINOGEN AFLATOXIN B1-8,9-EPOXIDE. (R825809)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  18. Synthesis and antiviral activities of hexadecyloxypropyl prodrugs of acyclic nucleoside phosphonates containing guanine or hypoxanthine and a (S)-HPMP or PEE acyclic moiety

    Czech Academy of Sciences Publication Activity Database

    Tichý, Tomáš; Andrei, G.; Snoeck, R.; Balzarini, J.; Dračínský, Martin; Krečmerová, Marcela

    2012-01-01

    Roč. 55, Sep (2012), s. 307-314 ISSN 0223-5234 R&D Projects: GA ČR GAP207/11/0108; GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : antivirals * prodrugs * acyclic nucleoside phosphonate * phosphonate ester Subject RIV: CC - Organic Chemistry Impact factor: 3.499, year: 2012

  19. Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

    Energy Technology Data Exchange (ETDEWEB)

    Monaco, L.; Murtagh, J.J.; Newman, K.B.; Tsai, Su-Chen; Moss, J.; Vaughan, M. (National Institutes of Health, Bethesda, MD (USA))

    1990-03-01

    ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and used to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    Two open reading frames in the genome of Sulfolobus solfataricus (SSO2341 and SSO2424) were cloned and expressed in E. coli. The protein products were purified and their enzymatic activity characterized. Although SSO2341 was annotated as a gene (gpT-1) encoding a 6-oxopurine phosphoribosyltransfe...

  1. Bacillus subtilis guanine deaminase is encoded by the yknA gene and is induced during growth with purines as the nitrogen source

    DEFF Research Database (Denmark)

    Nygaard, P.; Bested, S. M.; Andersen, K. A. K.

    2000-01-01

    amino acid polypeptide and was preceded by a promoter sequence that is recognized by the A form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular...

  2. Theoretical study on the structure, stability, and electronic properties of the guanine-Zn-cytosine base pair in M-DNA

    International Nuclear Information System (INIS)

    Fuentes-Cabrera, Miguel A.; Sumpter, Bobby G.; Sponer, Judit; Sponer, Jiri; Petit, Leon; Wells, Jack C.

    2007-01-01

    M-DNA is a type of metalated DNA that forms at high pH and in the presence of Zn, Ni, and Co, with the metals placed in between each base pair, as in G-Zn-C. Experiments have found that M-DNA could be a promising candidate for a variety of nanotechnological applications, as it is speculated that the metal d-states enhance the conductivity, but controversy still clouds these findings. In this paper, we carry out a comprehensive ab initio study of eight G-Zn-C models in the gas phase to help discern the structure and electronic properties of Zn-DNA. Specifically, we study whether a model prefers to be planar and has electronic properties that correlate with Zn-DNA having a metallic-like conductivity. Out of all the studied models, there is only one which preserves its planarity upon full geometry optimization. Nevertheless, starting from this model, one can deduce a parallel Zn-DNA architecture only. This duplex would contain the imino proton, in contrast to what has been proposed experimentally. Among the nonplanar models, there is one that requires less than 8 kcal/mol to flatten (both in gas and solvent conditions), and we propose that it is a plausible model for building an antiparallel duplex. In this duplex, the imino proton would be replaced by Zn, in accordance with experimental models. Neither planar nor nonplanar models have electronic properties that correlate with Zn-DNA having a metallic-like conductivity due to Zn d-states. To understand whether density functional theory (DFT) can describe appropriately the electronic properties of M-DNAs, we have investigated the electronic properties of G-Co-C base pairs. We have found that when self-interaction corrections (SIC) are not included the HOMO state contains Co d-levels, whereas these levels are moved below the HOMO state when SIC are considered. This result indicates that caution should be exercised when studying the electronic properties of M-DNAs with functionals that do not account for strong electronic correlations

  3. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha1-adrenergic system

    International Nuclear Information System (INIS)

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha 1 -adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha 1 -adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha 1 -selective radioligand [ 125 I]-I-2-[β-(4-hydroxphenyl)ethylaminomethyl]tetralone ([ 125 I]-IBE 2254) demonstrated that the alpha 1 -adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha 1 -adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha 1 -adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha 1 -mediated positive chronotropic response in the neonate, whereas in the adult the alpha 1 -mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha 1 -adrenergic receptor to its effector under certain circumstances such as high potassium (K + ) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha 1 -response

  4. Guanine nucleotide-binding protein (Gα) endocytosis by a cascade of ubiquitin binding domain proteins is required for sustained morphogenesis and proper mating in yeast.

    Science.gov (United States)

    Dixit, Gauri; Baker, Rachael; Sacks, Carly M; Torres, Matthew P; Dohlman, Henrik G

    2014-05-23

    Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109-amino acid insert within the α-helical domain that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1, and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: Evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Česnek, Michal; Hocková, Dana; Holý, Antonín; Dračínský, Martin; Baszczyňski, Ondřej; de Jersey, J.; Keough, D. T.; Guddat, L. W.

    2012-01-01

    Roč. 20, č. 2 (2012), s. 1076-1089 ISSN 0968-0896 R&D Projects: GA MŠk 1M0508; GA ČR GAP207/11/0108 Institutional research plan: CEZ:AV0Z40550506 Keywords : Plasmodium * malaria * acyclic nucleoside phosphonates Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

  6. The effect of novel [3-fluoro-(2-phosphonoethoxy)propyl]purines on the inhibition of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Baszczyňski, Ondřej; Hocková, Dana; Janeba, Zlatko; Holý, Antonín; Jansa, Petr; Dračínský, Martin; Keough, D. T.; Guddat, L. W.

    2013-01-01

    Roč. 67, Sep (2013), s. 81-89 ISSN 0223-5234 R&D Projects: GA MV VG20102015046; GA ČR GAP207/11/0108 Grant - others:National Health and Madical Research Council(AU) 1030353; National Health and Medical Research Council(AU) 569703 Institutional support: RVO:61388963 Keywords : Malaria * nucleoside phosphonates * fluorine * purines * FPMP Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.432, year: 2013

  7. Alperujo extract, hydroxytyrosol, and 3,4-dihydroxyphenylglycol are bioavailable and have antioxidant properties in vitamin E-deficient rats-a proteomics and network analysis approach

    NARCIS (Netherlands)

    Rodríguez-Gutiérrez, G.; Duthie, G.G.; Wood, S.; Morrice, P.; Nicol, F.; Reid, M.; Cantlay, L.L.; Kelder, T.; Horgan, G.W.; Fernández-Bolaños Guzmán, J.; Roos, B. de

    2012-01-01

    Scope: Olive products are rich in phenolic compounds, which are natural antioxidants in vitro. We tested the in vivo effects of alperujo, an olive production by-product, as well as hydroxytyrosol and 3,4-dihydroxyphenylglycol (DHPG) isolated from alperujo, on indices and pathways of oxidative and

  8. Hypoxanthine Guanine Phosphoribosyl Transferase Is the Most Stable Reference Gene for Gene Expression Analysis by Quantitative PCR in Peripheral Blood Mononuclear Cells from Women with the Polycystic Ovary Syndrome

    Directory of Open Access Journals (Sweden)

    Milutinović Danijela Vojnović

    2014-09-01

    Full Text Available Background: The polycystic ovary syndrome (PCOS is a frequent endocrine disorder that affects women of reproductive age. As the syndrome is strongly associated with obesity, it is of interest to examine the gene expression diffe rences that accompany its development and the associ a ted metabolic disturbances. Real-time RT PCR is a standard method for studying changes in gene expression. However, to obtain accurate and reliable results, validation of reference genes is obligatory. The aim of this study was to identify a suitable reference for the normalization of gene expression in peripheral blood mononuclear cells (PBMCs from obese and normal-weight women with PCOS.

  9. NanoHPLC-nanoESI(+)-MS/MS quantitation of bis-N7-guanine DNA-DNA cross-links in tissues of B6C3F1 mice exposed to subppm levels of 1,3-butadiene.

    Science.gov (United States)

    Sangaraju, Dewakar; Goggin, Melissa; Walker, Vernon; Swenberg, James; Tretyakova, Natalia

    2012-02-07

    1,3-Butadiene (BD) is an important industrial chemical and a common environmental pollutant present in urban air. BD is classified as a human carcinogen based on epidemiological evidence for an increased incidence of leukemia in workers occupationally exposed to BD and its potent carcinogenicity in laboratory mice. A diepoxide metabolite of BD, 1,2,3,4-diepoxybutane (DEB), is considered the ultimate carcinogenic species of BD due to its ability to form genotoxic DNA-DNA cross-links. We have previously employed capillary HPLC-ESI(+)-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) methods to quantify DEB-induced DNA-DNA conjugates, e.g. 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD), 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and 1,N(6)-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N(6)-HMHP-dA), in tissues of laboratory mice exposed to 6.25-625 ppm BD (Goggin et al. Cancer Res. 2009, 69(6), 2479-2486). However, typical BD human exposure levels are 0.01 to 3.2 ppb in urban air and 1-2.0 ppm in an occupational setting, requiring greater detection sensitivity for these critical lesions. In the present study, a nanoHPLC-nanoESI(+)-MS/MS method was developed for ultrasensitive, accurate, and precise quantitation of bis-N7G-BD in tissues of laboratory mice treated with low ppm and subppm concentrations of BD. The LOD value of the new method is 0.5 fmol/100 μg DNA, and the LOQ is 1.0 fmol/100 μg DNA, making it possible to quantify bis-N7G-BD adducts present at concentrations of 3 per 10(9) nucleotides. Bis-N7G-BD adduct amounts in liver tissues of mice exposed to 0.5, 1.0, and 1.5 ppm BD for 2 weeks were 5.7 ± 3.3, 9.2 ± 1.5, and 18.6 ± 6.9 adducts per 10(9) nucleotides, respectively, suggesting that bis-N7G-BD adduct formation is more efficient under low exposure conditions. To our knowledge, this is the first quantitative analysis of DEB specific DNA adducts following low ppm and subppm exposure to BD. © 2012 American Chemical Society

  10. 4-Nitrobenzyloxycarbonyl Derivatives of O6-Benzylguanine as Hypoxia-Activated Prodrug Inhibitors of O6-Alkylguanine-DNA Alkyltransferase (AGT) which Produces Resistance to Agents Targeting the O-6 Position of DNA Guanine

    Science.gov (United States)

    Zhu, Rui; Liu, Mao-Chin; Luo, Mei-Zhen; Penketh, Philip G.; Baumann, Raymond P.; Shyam, Krishnamurthy; Sartorelli, Alan C.

    2011-01-01

    A series of 4-nitrobenzyloxycarbonyl prodrug derivatives of O6-benzylguanine (O6-BG), conceived as prodrugs of O6-BG, an inhibitor of the resistance protein O6-alkylguanine-DNA alkyltransferase (AGT), were synthesized and evaluated for their ability to undergo bioreductive activation by reductase enzymes under oxygen deficiency. Three agents of this class, 4-nitrobenzyl (6-(benzyloxy)-9H-purin-2-yl)carbamate (1), and its monomethyl (2) and gem-dimethyl analogues (3) were tested for activation by reductase enzyme systems under oxygen deficient conditions. Compound 3, the most water-soluble of these agents, gave the highest yield of O6-BG following reduction of the nitro group trigger. Compound 3 was also evaluated for its ability to sensitize 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(methylamino)carbonyl]hydrazine (laromustine)-resistant DU145 human prostate carcinoma cells, which express high levels of AGT, to the cytotoxic effects of this agent under normoxic and oxygen deficient conditions. While 3 had little or no effect on laromustine cytotoxicity under aerobic conditions, significant enhancement occurred under oxygen deficiency, providing evidence for the preferential release of the AGT inhibitor O6-BG under hypoxia. PMID:21955333

  11. DNA adducts of acrolein: site-specific synthesis of an oligodeoxynucleotide containing 6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one, an acrolein adduct of guanine.

    Science.gov (United States)

    Nechev, Lubomir V; Kozekov, Ivan D; Brock, Angela K; Rizzo, Carmelo J; Harris, Thomas M

    2002-05-01

    3-(2-Deoxy-beta-D-erythro-pentofuranosyl)-6-hydroxy-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one is formed in low yield by the reaction of acrolein with 2'-deoxyguanosine. The nucleoside and an oligodeoxynucleotide containing it have been synthesized. For preparation of the nucleoside 2'-deoxyguanosine was alkylated at the N1 position using 1-bromo-3-butene to give 1-(3-butenyl)-2'-deoxyguanosine. Oxidation with OsO(4) and N-methylmorpholine-N-oxide to give the 3,4-dihydroxybutyl adduct followed by oxidation with NaIO(4) gave the 1-(3-oxopropyl) adduct which cyclized spontaneously to yield the title compound as a rapidly epimerizing mixture of two diastereomers. Reduction of the nucleoside with NaBH(4) gave the unfunctionalized compound plus 1-(3-hydroxypropyl)-2'-deoxyguanosine showing that epimerization was occurring via both the imine and the 1-(3-oxopropyl) adduct. Reduction with NaCNBH(3) gave exclusively unfunctionalized 3-(2-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one. The phosphoramidite reagent needed for preparation of oligonucleotides was prepared from 1-(3-butenyl)-2'-deoxyguanosine by glycolation after protection of the 3' and 5' hydroxyl groups as silyl derivatives. Acetylation of the vicinal hydroxyl groups and the exocyclic amino group followed by removal of silyl protection gave the protected nucleoside. Protection of the 5' hydroxyl group as the 4,4'-dimethoxytrityl ether followed by phosphitylation with 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite gave the prosphoramidite reagent which was used to prepare a 12-mer oligodeoxynucleotide.

  12. Theoretical Calculation of the NMR Spin-Spin Coupling Constants and the NMR Shifts Allow Distinguishability between the Specific Direct and the Water-Mediated Binding of a Divalent Metal Cation to Guanine

    Czech Academy of Sciences Publication Activity Database

    Sychrovský, Vladimír; Šponer, Jiří; Hobza, Pavel

    2004-01-01

    Roč. 126, č. 2 (2004), s. 663-672 ISSN 0002-7863 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : nuclear-magnetic-resonance * ion binding * base-pairs Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 6.903, year: 2004

  13. Channeling through Two Stacked Guanine Quartets of One and Two Alkali Cations in the Li+, Na+, K+, and Rb+ Series. Assessment of the Accuracy of the SIBFA Anisotropic Polarizable Molecular Mechanics Potential

    Czech Academy of Sciences Publication Activity Database

    Gresh, N.; Naseem-Khan, S.; Lagardere, L.; Piquemal, J.P.; Šponer, Judit E.; Šponer, Jiří

    2017-01-01

    Roč. 121, č. 16 (2017), s. 3997-4014 ISSN 1520-6106 R&D Projects: GA ČR(CZ) GA16-13721S; GA MŠk(CZ) LQ1601 Institutional support: RVO:68081707 Keywords : intermolecular interaction energies * quantum -chemical computations * classical drude oscillator * kit promoter quadruplex Subject RIV: BO - Biophysics OBOR OECD: Physical chemistry Impact factor: 3.177, year: 2016

  14. Conversion of 3, 4-dihydroxyphenylalanine and deuterated 3, 4-dihydroxyphenylalanine to alcoholic metabolites of catecholamines in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, D.J.; Rizk, M.

    1981-05-01

    We have investigated the effects of 3, 4-dihydroxyphenylalanine (L-DOPA) and its deuterated analogue on the concentrations of alcoholic metabolites of catecholamines in rat brain by means of gas chromatography/mass spectrometry with selected-ion monitoring. Whole brain concentrations of the two neutral norepinephrine metabolites, 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG) and 3, 4-dihydroxyphenylethyleneglycol (DHPG), were significantly increased in a dose-dependent manner by a single intraperitoneal injection of L-DOPA. Both MHPG and DHPG, as well as the corresponding dopamine metabolites, reached a maximum 1 h after injection. Brain MHPG and DHPG concentrations were elevated by 78 and 134% respectively, 1 h after injection of 150 mg/kg L-DOPA. Analyses of discrete brain regions revealed that concentrations of the norepinephrine metabolites were elevated uniformly in all regions, except that MHPG showed a greater increase in the cerebellum than in other regions. The latter result appeared to be explained by the finding that 52% of the total MHPG in the cerebellum was unconjugated (compared to 15% in the whole brain). L-DOPA caused a proportionately greater increase in free MHPG than in total MHPG in the cerebellum and brain stem. By using deuterated L-DOPA in place of L-DOPA and measuring both the deuterated and nondeuterated norepinephrine metabolites, reseachers demonstrated that virtually all of the increases in MHPG and DHPG were due to the conversion of the exogenous L-DOPA to norepinephrine. Thus, the effects of norepinephrine metabolism need to be considered in attempts to understand clinical and behavioral effects of L-DOPA.

  15. New approaches to evaluate sympathoadrenal system activity in experiments on Earth and in space

    Science.gov (United States)

    Kvetnansky, R.; Noskov, V. B.; Blazicek, P.; Macho, L.; Grigoriev, A. I.; Goldstein, D. S.; Kopin, I. J.

    In previous studies the activity of the sympathoadrenal system (SAS) in cosmonauts during space flights was evaluated by measuring plasma catecholamines (CA) levels and urinary CA and their metabolites concentrations. Plasma CA levels are accepted indicators of SAS activity, however, they are determined by the plasma clearances as well as the rates of CA release (spillover-SO) into the bloodstream. Nowadays methods are available which evaluate not only plasma levels of CA but also their release, spillover, uptake, reuptake, degradation and also CA synthesis in vivo measured by plasma levels of dihydroxyphenylalanine (DOPA). Plasma concentrations of DOPA, the CA noradrenaline (NE), adrenaline (ADR), and dopamine (DA), the deaminated catechol metabolites dihydroxyphenylglycol (DHPG) and dihydroxyphenylacetic acid (DOPAC), and the O-methylated metabolites methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured during immobilization stress (IMO) in conscious rats. Radiotracer methods were used to measure NE SO. IMO markedly increased arterial NE levels but NE SO was less elevated bacause the NE clearance was slightly reduced in IMO rats. Simultaneous measurements of plasma CA and their metabolites provide another means to obtain information about SAS function. For instance, dissociation between changes of plasma DHPG and NE levels can indicate changes in neuronal reuptake of NE. We found marked parallel increases in plasma NE and DHPG levels during acute IMO; however after repeated IMO, plasma NE levels were increased but DHPG responses were less pronounced suggesting a reduced NE reuptake. DOPA, the CA precursor, circulates in plasma at a concentration higher than NE. During stress, increased sympathoneural outflow stimulates DOPA synthesis and release into the circulation supporting the view that changes in plasma DOPA levels during stress reflect in vivo changes in the rate of CA synthesis. We propose to measure the new plasma indicators of SAS

  16. Type-1, but Not Type-5, Metabotropic Glutamate Receptors are Coupled to Polyphosphoinositide Hydrolysis in the Retina.

    Science.gov (United States)

    Romano, Maria Rosaria; Di Menna, Luisa; Scarselli, Pamela; Mascio, Giada; Madonna, Michele; Notartomaso, Serena; Puliti, Aldamaria; Bruno, Valeria; Battaglia, Giuseppe; Nicoletti, Ferdinando

    2016-04-01

    mGlu1 and mGlu5 metabotropic glutamate receptors are expressed in the vertebrate retina, and are co-localized in some retinal neurons. It is believed that both receptors are coupled to polyphosphoinositide (PI) hydrolysis in the retina and their function may diverge in some cells because of a differential engagement of downstream signaling molecules. Here, we show that it is only the mGlu1 receptor that is coupled to PI hydrolysis in the retina. We used either bovine retinal slices or intact mouse retinas challenged with the mixed mGlu1/5 receptor agonist, DHPG. In both models, DHPG-stimulated PI hydrolysis was abrogated by the selective mGlu1 receptor antagonist, JNJ16259685, but was insensitive to the mGlu5 receptor antagonist, MPEP. In addition, the PI response to DHPG was unchanged in the retina of mGlu5(-/-) mice but was abolished in the retina of crv4 mice lacking mGlu1 receptors. Stimulation of the mitogen-activated protein kinase pathway by DHPG in intact mouse retinas were also entirely mediated by mGlu1 receptors. Our data provide the first example of a tissue in which a biochemically detectable PI response is mediated by mGlu1, but not mGlu5, receptors. Hence, bovine retinal slices might be used as a model for the functional screening of mGlu1 receptor ligands. In addition, the mGlu1 receptor caters the potential as a drug target in the experimental treatment of degenerative disorders of the retina.

  17. Effects of Regulatory BC1 RNA Deletion on Synaptic Plasticity, Learning, and Memory

    Science.gov (United States)

    Chung, Ain; Dahan, Nessy; Alarcon, Juan Marcos; Fenton, André A.

    2017-01-01

    Nonprotein coding dendritic BC1 RNA regulates translation of mRNAs in neurons. We examined two lines of BC1 knockout mice and report that loss of BC1 RNA exaggerates group I mGluR-stimulated LTD of the Schaffer collateral synapse, with one of the lines showing a much more enhanced DHPG-induced LTD than the other. When the animals were given the…

  18. Dual effect of 17β-estradiol on NMDA-induced neuronal death: involvement of metabotropic glutamate receptor 1.

    Science.gov (United States)

    Spampinato, Simona Federica; Merlo, Sara; Molinaro, Gemma; Battaglia, Giuseppe; Bruno, Valeria; Nicoletti, Ferdinando; Sortino, Maria Angela

    2012-12-01

    Pretreatment with 10 nm 17β-estradiol (17βE2) or 100 μm of the metabotropic glutamate 1 receptor (mGlu1R) agonist, dihydroxyphenylglycine (DHPG), protected neurons against N-methyl-d-aspartate (NMDA) toxicity. This effect was sensitive to blockade of both estrogen receptors and mGlu1R by their respective antagonists. In contrast, 17βE2 and/or DHPG, added after a low-concentration NMDA pulse (45 μm), produced an opposite effect, i.e. an exacerbation of NMDA toxicity. Again this effect was prevented by both receptor antagonists. In support of an interaction of estrogen receptors and mGlu1R in mediating a neurotoxic response, exacerbation of NMDA toxicity by 17βE2 disappeared when cultures were treated with DHPG prior to NMDA challenge, and conversely, potentiation of NMDA-induced cell death by DHPG was prevented by pretreatment with 17βE2. Addition of calpain III inhibitor (10 μm), 2 h before NMDA, prevented the increased damage induced by the two agonists, an affect that can be secondary to cleavage of mGlu1R by calpain. Accordingly, NMDA stimulation reduced expression of the full-length (140 kDa) mGluR1, an effect partially reversed by calpain inhibitor. Finally, in the presence of NMDA, the ability of 17βE2 to stimulate phosphorylation of AKT and ERK was impaired. Pretreatment with calpain inhibitor prevented the reduction of phosphorylated ERK but had no significant effect on phosphorylated AKT. Accordingly, the inhibition of ERK signaling by U0126 (1 μm) counteracted the effect of calpain inhibition on 17βE2-induced exacerbation of NMDA toxicity. The present data confirm the dual role of estrogens in neurotoxicity/neuroprotection and highlight the role of the timing of exposure to estrogens.

  19. Activated Müller Cells Involved in ATP-Induced Upregulation of P2X7 Receptor Expression and Retinal Ganglion Cell Death

    Directory of Open Access Journals (Sweden)

    Ying Xue

    2016-01-01

    Full Text Available P2X7 receptor (P2X7R, an ATP-gated ion channel, plays an important role in glaucomatous retinal ganglion cell (RGC apoptotic death, in which activated retinal Müller glial cells may be involved by releasing ATP. In the present study, we investigated whether and how activated Müller cells may induce changes in P2X7R expression in RGCs by using immunohistochemistry and Western blot techniques. Intravitreal injection of DHPG, a group I metabotropic glutamate receptor (mGluR I agonist, induced upregulation of GFAP expression, suggestive of Müller cell activation (gliosis, as we previously reported. Accompanying Müller cell activation, P2X7R protein expression was upregulated, especially in the cells of ganglion cell layer (GCL, which was reversed by coinjection of brilliant blue G (BBG, a P2X7R blocker. In addition, intravitreal injection of ATP also induced upregulation of P2X7R protein expression. Similar results were observed in cultured retinal neurons by ATP treatment. Moreover, both DHPG and ATP intravitreal injection induced a reduction in the number of fluorogold retrogradely labeled RGCs, and the DHPG effect was partially rescued by coinjection of BBG. All these results suggest that activated Müller cells may release ATP and, in turn, induce upregulation of P2X7R expression in the cells of GCL, thus contributing to RGC death.

  20. Endocannabinoids control vesicle release mode at midbrain periaqueductal grey inhibitory synapses.

    Science.gov (United States)

    Aubrey, Karin R; Drew, Geoffrey M; Jeong, Hyo-Jin; Lau, Benjamin K; Vaughan, Christopher W

    2017-01-01

    The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition. The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress-induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)-3,5-dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca 2+ , and when release probability was reduced by lowering Ca 2+ . However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca 2+ . Lowering external Ca 2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca 2+ . The low affinity GABA A receptor antagonist TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca 2+ . Together these findings indicate that the normal mode of GABA release is

  1. Optical properties of nucleobase thin films as studied by attenuated total reflection and surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Kim, MinSuk; Ham, Won Kyu; Kim, Wonyoung; Hwangbo, Chang Kwon; Choi, Eun Ha; Lee, Geon Joon

    2018-04-01

    Optical properties of nucleobase thin films were studied by attenuated total reflection (ATR) and surface-enhanced Raman spectroscopy (SERS). Adenine and guanine films were deposited on fused silica and silver at room temperature by thermal evaporation, and the normal dispersion of refractive indices of transparent adenine and guanine films in the visible and near-infrared regions were analyzed. The measured ATR spectra of adenine (guanine) films and numerical simulations by optical transfer matrix formalism demonstrate that the shift of surface plasmon resonance (SPR) wavelength is approximately linearly proportional to the adenine (guanine) film thickness, indicating that SPR can be used for quantitative measurements of biomaterials. The Raman spectra indicated that the adenine (guanine) films can be deposited by thermal evaporation. The adenine (guanine) films on silver exhibited Raman intensity enhancement as compared to those on glass, which was attributed to the SPR effect of silver platform and might play a role as a hot plate for SERS detection of biomaterials.

  2. Dicty_cDB: Contig-U16413-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 2( AL355142 |pid:none) Human DNA sequence from clone RP11... 57 5e-06 (Q9QX73) RecName: Full=Rho guanine nucleotide exchange...e exchange factor for Rac 30; 117 4e-24 (Q55E26) RecName: Full=Rac guanine nucleotide exchange...112926 |pid:none) Homo sapiens Rho guanine nucleotid... 77 5e-12 ( O15013 ) RecName: Full=Rho guanine nucleotide exchange...e nucleotid... 75 2e-11 (Q8C033) RecName: Full=Rho guanine nucleotide exchange factor 10; 75 2e-11...-1; AltName: Full=Alpha-acti... 72 1e-10 (Q29RM4) RecName: Full=Rho guanine nucleotide exchange

  3. Cocaine Withdrawal Impairs mGluR5-Dependent Long-Term Depression in Nucleus Accumbens Shell Neurons of Both Direct and Indirect Pathways.

    Science.gov (United States)

    Huang, Chiung-Chun; Liang, Ying-Ching; Lee, Cheng-Che; Hsu, Kuei-Sen

    2015-12-01

    We previously reported that animals withdrawn from repeated cocaine exposure exhibited a selective deficit in the ability to elicit metabotropic glutamate receptor 5 (mGluR5)-dependent long-term depression (LTD) in the nucleus accumbens (NAc) shell. To determine whether such impairment occurs in the NAc in a cell-type-specific manner, we used bacterial artificial chromosome (BAC) transgenic mice expressing enhanced green fluorescent protein (eGFP) under the control of gene regulatory elements for the dopamine D1 receptor (Drd1) or dopamine D2 receptor (Drd2) to identify distinct subpopulations of medium spiny neurons (MSNs). We found that bath application of group I mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) reliably induced LTD in both NAc shell and core MSNs of wild-type, hemizygous Drd1-eGFP, and Drd2-eGFP mice. Confirming our previous results, cocaine withdrawal selectively impaired DHPG-LTD in NAc shell Drd1-expressing direct and Drd2-expressing indirect pathway MSNs. We also found that the expression of DHPG-LTD in NAc MSNs was not affected by the Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 1-naphthyl acetyl spermine. Furthermore, systemic administration of mGluR5-negative allosteric modulator fenobam before the daily injection of cocaine preserved mGluR5 function and significantly reduced the expression of cocaine-induced behavioral sensitization. These results reveal that withdrawal from repeated cocaine exposure may result in the impairment of NAc mGluR5-LTD in a subregion- but not cell-type-specific manner and suggests that pharmacological antagonism of mGluR5 may represent a potential strategy for reducing cocaine-induced addictive behaviors.

  4. NMDAR NR2A and NR2B specific PKC-dependent regulation of mGluR is defective in the Fragile X Syndrome mouse model

    DEFF Research Database (Denmark)

    Banke, Tue G.; Toft, Anna Karina; Lundbye, Camilla Johanne

    ). Similar results were observed when mature KO slices were preincubated with the protein kinase C activator (PMA), or inactivator (chelerythrine), suggesting that NMDAR activation leads to PKC regulation of mGluR-LTD. Surprisingly, preincubation WT with the NR2B specific NMDAR inhibitor CP-101.......606 completely blocked DHPG-induced LTD. This blockage was reversed by either preincubation with PMA or by co-application of the NR2A antagonist TCN-201. In contrast, application of TCN-201 alone had no apparent effects. Our data suggest a model where NMDARs regulate mGluR-LTD through regulation of PKC...

  5. Reality and non-reality of the spectrum of PT -symmetric operators ...

    Indian Academy of Sciences (India)

    (8) if and only if φp solves Hp(g)φp = λφp where Hp(g) is the operator in L2(0, 2π) formally given by. Hp(g)u = (. −i d dx. + p. )2 u + qu + gW u, u ∈ D(Hp(g)),. (9) with periodic boundary conditions. Then σ(H(g)) = ⋃ p∈]−1/2,1/2] σ(Hp(g)). Since the spectrum of Hp(g) is discrete ∀p, σ(Hp(g)): = {λn(g; p): n = 0, 1,...}, it suffices to ...

  6. Radiolabeled guanine derivatives for the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase: 6-(4-[(18)F]Fluoro-benzyloxy)-9H-purin-2-ylamine and 6-(3-[(131)I]Iodo-benzyloxy)-9H-purin-2-ylamine.

    Science.gov (United States)

    Vaidyanathan, G; Affleck, D J; Cavazos, C M; Johnson, S P; Shankar, S; Friedman, H S; Colvin, M O; Zalutsky, M R

    2000-01-01

    Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O(6)-benzylguanine; BG) potentially useful in the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[(18)F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [(131)I]7 in about 70% overall radiochemical yield. The IC(50) values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [(18)F]6 and [(131)I]7 to purified AGT was specific and saturable with both exhibiting similar IC(50) values (5-6 microM).

  7. Extent of intramolecular pi stacks in aqueous solution in mixed-ligand copper(II) complexes formed by heteroaromatic amines and the anticancer and antivirally active 9[2-(phosphonomethoxy)ethyl]guanine (PMEG). A comparison with related acyclic nucleotide analogues

    Czech Academy of Sciences Publication Activity Database

    Blindauer, C. A.; Sigel, A.; Operschall, B. P.; Griesser, R.; Holý, Antonín; Sigel, H.

    2016-01-01

    Roč. 103, Jan 8 (2016), s. 248-260 ISSN 0277-5387 Institutional support: RVO:61388963 Keywords : anticancer activity * antivirals * aromatic-ring stacking * isomeric equilibria * nucleotide analogues Subject RIV: CC - Organic Chemistry Impact factor: 1.926, year: 2016

  8. Yeast Interacting Proteins Database: YOL126C, YDL100C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available t as prey (0) YDL100C GET3 Guanine nucleotide exchange factor for Gpa1p; amplifies...ey ORF YDL100C Prey gene name GET3 Prey description Guanine nucleotide exchange factor for Gpa1p; amplifies

  9. Yeast Interacting Proteins Database: YDL121C, YDL100C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available as bait (1) Rows with this bait as prey (0) YDL100C GET3 Guanine nucleotide exchange factor for Gpa1p; amplifies...ion Guanine nucleotide exchange factor for Gpa1p; amplifies G protein signaling; subunit of the GET complex,

  10. Amyloid Beta Peptides Differentially Affect Hippocampal Theta Rhythms In Vitro

    Directory of Open Access Journals (Sweden)

    Armando I. Gutiérrez-Lerma

    2013-01-01

    Full Text Available Soluble amyloid beta peptide (Aβ is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble Aβ alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different Aβ peptides, we also compared Aβ25−35 and Aβ1−42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μM. We found that Aβ25−35 reduces, with less potency than Aβ1−42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ25−35 but was reduced by Aβ1−42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.

  11. Alperujo extract, hydroxytyrosol, and 3,4-dihydroxyphenylglycol are bioavailable and have antioxidant properties in vitamin E-deficient rats--a proteomics and network analysis approach.

    Science.gov (United States)

    Rodríguez-Gutiérrez, Guillermo; Duthie, Garry G; Wood, Sharon; Morrice, Phil; Nicol, Fergus; Reid, Martin; Cantlay, Louise L; Kelder, Thomas; Horgan, Graham W; Fernández-Bolaños Guzmán, Juan; de Roos, Baukje

    2012-07-01

    Olive products are rich in phenolic compounds, which are natural antioxidants in vitro. We tested the in vivo effects of alperujo, an olive production by-product, as well as hydroxytyrosol and 3,4-dihydroxyphenylglycol (DHPG) isolated from alperujo, on indices and pathways of oxidative and metabolic stress in a vitamin E-deficient rat model. Rats were fed a vitamin E-deficient diet for 10 weeks, followed by this diet supplemented with either 100 mg/kg diet dα-tocopherol, alperujo extract, hydroxytyrosol, or 10 mg/kg diet DHPG, for a further 2 weeks. We detected alperujo phenolics in tissues and blood, indicating they are bioavailable. Alperujo extract partially ameliorated elevated plasma levels of thiobarbituric acid reactive substances and also lowered plasma cholesterol levels, whereas hydroxytyrosol increased plasma triglyceride levels. Proteomics and subsequent network analysis revealed that hepatic mitochondrial aldehyde dehydrogenase (ALDH2), of which protein and activity levels were regulated by dα-tocopherol and olive phenolics, represents a novel central regulatory protein hub affected by the dietary interventions. The in vivo free radical scavenging properties of olive phenolics appear relatively modest in our model. But alternative mechanisms, including regulation of ALDH2, may represent relevant antioxidant mechanisms by which dietary olive phenolics could have beneficial impact on cardiovascular health. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Familial orthostatic tachycardia due to norepinephrine transporter deficiency

    Science.gov (United States)

    Robertson, D.; Flattem, N.; Tellioglu, T.; Carson, R.; Garland, E.; Shannon, J. R.; Jordan, J.; Jacob, G.; Blakely, R. D.; Biaggioni, I.

    2001-01-01

    Orthostatic intolerance (OI) or postural tachycardia syndrome (POTS) is a syndrome primarily affecting young females, and is characterized by lightheadedness, palpitations, fatigue, altered mentation, and syncope primarily occurring with upright posture and being relieved by lying down. There is typically tachycardia and raised plasma norepinephrine levels on upright posture, but little or no orthostatic hypotension. The pathophysiology of OI is believed to be very heterogeneous. Most studies of the syndrome have focused on abnormalities in norepinephrine release. Here the hypothesis that abnormal norepinephrine transporter (NET) function might contribute to the pathophysiology in some patients with OI was tested. In a proband with significant orthostatic symptoms and tachycardia, disproportionately elevated plasma norepinephrine with standing, impaired systemic, and local clearance of infused tritiated norepinephrine, impaired tyramine responsiveness, and a dissociation between stimulated plasma norepinephrine and DHPG elevation were found. Studies of NET gene structure in the proband revealed a coding mutation that converts a highly conserved transmembrane domain Ala residue to Pro. Analysis of the protein produced by the mutant cDNA in transfected cells demonstrated greater than 98% reduction in activity relative to normal. NE, DHPG/NE, and heart rate correlated with the mutant allele in this family. CONCLUSION: These results represent the first identification of a specific genetic defect in OI and the first disease linked to a coding alteration in a Na+/Cl(-)-dependent neurotransmitter transporter. Identification of this mechanism may facilitate our understanding of genetic causes of OI and lead to the development of more effective therapeutic modalities.

  13. Group I mGlu receptors potentiate synaptosomal [3H]glutamate release independently of exogenously applied arachidonic acid

    International Nuclear Information System (INIS)

    Reid, M.E.; Toms, N.J.; Bedingfield, J.S.; Roberts, P.J.

    1999-01-01

    In the current study, we have characterized group I metabotropic glutamate (mGlu) receptor enhancement of 4-aminopyridine (4AP)-evoked [ 3 H]glutamate release from rat cerebrocortical synaptosomes. The broad spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD, 10 μM) increased 4AP-evoked [ 3 H]glutamate release (143.32±2.73% control) only in the presence of exogenously applied arachidonic acid; an effect reversed by the inclusion of bovine serum albumin (BSA, fatty acid free). In contrast, the selective group I mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) potentiated (EC 50 =1.60±0.25 μM; E max =147.61±10.96% control) 4AP-evoked [ 3 H]glutamate release, in the absence of arachidonic acid. This potentiation could be abolished by either the selective mGlu 1 receptor antagonist (R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) or the selective PKC inhibitor (Ro 31-8220, 10 μM) and was BSA-insensitive. The selective mGlu 5 receptor agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG, 300μM) was without effect. DHPG (100 μM) also potentiated both 30 mM and 50 mM K + -evoked [ 3 H]glutamate release (121.60±12.77% and 121.50±4.45% control, respectively). DHPG (100 μM) failed to influence both 4AP-stimulated 45 Ca 2+ influx and 50 mM K + -induced changes in synaptosomal membrane potential. Possible group I mGlu receptor suppression of tonic adenosine A 1 receptor, group II/III mGlu receptors or GABA B receptor activity is unlikely since 4AP-evoked [ 3 H]glutamate release was insensitive to the selective inhibitory receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine, (R,S)-α-cyclopropyl-4-phosphonophenylglycine or CGP55845A, respectively. These data suggest an 'mGlu 1 receptor-like' receptor potentiates [ 3 H]glutamate release from cerebrocortical synaptosomes in the absence of exogenously applied arachidonic acid. This PKC dependent effect is unlikely to be via modulation of synaptosomal membrane

  14. Presidential Green Chemistry Challenge: 2000 Greener Synthetic Pathways Award

    Science.gov (United States)

    Presidential Green Chemistry Challenge 2000 award winner, Roche Colorado, developed a greener synthesis for gancyclovir (Cytovene, a potent antiviral drug) that uses a second-generation Guanine Triester (GTE) process.

  15. Mild Lesch-Nyhan Disease in a Boy with a Null Mutation inHPRT1

    DEFF Research Database (Denmark)

    Bayat, Allan; Christensen, Mette; Wibrand, Flemming

    2015-01-01

    Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency results in a continuous spectrum of clinical phenotypes though all include overproduction of uric acid with hyperuricaemia, urate nephrolithiasis and gout. HPRT1 mutations that result in very low or no HPRT enzyme activities...

  16. Low Melatonin Production During Adulthood - Phase 2: Association with Levels of Hydroxyl Radical Scavenging and DNA Damage

    National Research Council Canada - National Science Library

    Sobel, Eugene L; Davanipour, Zoreh; Poulsen, Henrik

    2004-01-01

    The primary purpose of the proposed study is to develop cross-sectional evidence concerning whether or not lower melatonin production levels are associated with increased oxidative DNA guanine damage...

  17. ORF Alignment: NT_033779 [GENIUS II[Archive

    Lifescience Database Archive (English)

    Full Text Available rEMBL::g2674107:GUANINE NUCLEOTIDE-EXCHANGE ... PROTEIN. organism:BOS TAURUS (BOVINE). dbxref:GenBank...; ... AF023451; g2674107; -.'', species:''BOS TAURUS ... Length = 185 ... Query: 586 METGIELFNRKP

  18. Genetics Home Reference: Hermansky-Pudlak syndrome

    Science.gov (United States)

    ... mediate protein trafficking to lysosome-related organelles. J Biol Chem. 2012 Jun 1;287(23):19550-63. ... a Rab32/38 guanine nucleotide exchange factor. Curr Biol. 2012 Nov 20;22(22):2135-9. doi: ...

  19. Dicty_cDB: Contig-U14335-1 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available 149644 |pid:none) Mus musculus bone marrow macrophag... 41 0.083 (Q9UHV5) RecName: Full=Rap guanine nucleotide exchange...e nucleotide exchange factor 4;... 46 0.003 AF465264_1( AF465264 |pid:none) Rattus norvegicus ras guanin...( AY160107 ) Dictyostelium discoideum nucleotide exchange fact... 1427 0.0 2 ( AC115682 ) Dictyostelium discoideum chromosome 2 ma...2e-24 A37488( A37488 )Ras guanine nucleotide exchange factor son-of-seve... 69 4e-24 AM920436_178( AM920436 ...one) Kluyveromyces lactis strain NRRL... 70 2e-16 (Q9USU1) RecName: Full=Ras guanine nucleotide exchange

  20. Gene Variants Reduce Opioid Risks

    Science.gov (United States)

    ... morphine or methadone treatment to wean them off opioid dependence, and 21 (24 percent) were treated with additional medications to alleviate withdrawal symptoms. However, those infants with a guanine rs1799971 ...

  1. The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca2+]i

    Directory of Open Access Journals (Sweden)

    Stephanie N. Blandford

    2016-01-01

    Full Text Available Calcium-imaging techniques were used to determine if mouse retinal astrocytes in situ respond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG; trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration (Ca2+i increased in response to these glutamate agonists. The one exception was AMPA that increased Ca2+i in some, but not all, mouse retinal astrocytes in situ. However, AMPA did not increase Ca2+i in mouse retinal astrocytes in vitro, suggesting that the effect of AMPA in situ may be indirect.

  2. The Effect of Glutamate Receptor Agonists on Mouse Retinal Astrocyte [Ca(2+)]i.

    Science.gov (United States)

    Blandford, Stephanie N; Baldridge, William H

    2016-01-01

    Calcium-imaging techniques were used to determine if mouse retinal astrocytes in situ respond to agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA; N-methyl-D-aspartate, NMDA) and metabotropic (S-3,5-dihydroxyphenylglycine, DHPG; trans-1-amino-1,3-cyclopentanedicarboxylic acid, ACPD) glutamate receptors. In most cases we found no evidence that retinal astrocyte intracellular calcium ion concentration ([Ca(2+)]i) increased in response to these glutamate agonists. The one exception was AMPA that increased [Ca(2+)]i in some, but not all, mouse retinal astrocytes in situ. However, AMPA did not increase [Ca(2+)]i in mouse retinal astrocytes in vitro, suggesting that the effect of AMPA in situ may be indirect.

  3. Metabolic Diversity for Degradation, Detection, and Synthesis of Nitro Compounds and Toxins

    Science.gov (United States)

    2012-07-08

    bacteria but xanthine oxidase , which catalyzes the first step in the guanine degradation pathway, did not attack 8NG. We are currently determing the...the 8NG concentration but did not relieve the inhibition. Guanine supports growth of the bacteria but xanthine oxidase , which catalyzes the first...nitropropane dioxygenase from P. aeruginosa PAO1 (in red) and for nitroalkane oxidase from Streptomyces ansochromogenes (red). No sequences are available for

  4. Nucleobases functionalized quantum dots and gold nanoparticles bioconjugates as a fluorescence resonance energy transfer (FRET) system - Synthesis, characterization and potential applications.

    Science.gov (United States)

    Rodzik-Czałka, Łucja; Lewandowska-Łańcucka, Joanna; Gatta, Viviana; Venditti, Iole; Fratoddi, Ilaria; Szuwarzyński, Michał; Romek, Marek; Nowakowska, Maria

    2018-03-15

    Fluorescence resonance energy transfer (FRET) system based on functionalized CdTe-guanine and AuNPs-cytosine bioconjugates for the model nucleobase - guanine detection was developed. Thioglycolic acid coated cadmium telluride quantum dots (QDs) conjugated with guanine and sodium 3-mercapto-1-propanesulfonate stabilized gold nanoparticles (AuNPs) capped by cytosine were obtained and fully characterized. Successful formation of the materials was confirmed by UV-Vis, fluorescence and FTIR spectroscopies. Composition of the conjugates was also characterized with elemental analysis and XPS. By employing a guanine-cytosine interaction the bonding between these complementary nucleobases attached to the nanoparticles leads to the formation of QDs-guanine-AuNPs-cytosine assembly, with the size about 7 nm as demonstrated using atomic force microscopy. That enables an effective FRET from functionalized QDs to AuNPs since both, the required distance and the spectral characteristics of donor-acceptor pair were secured. However, it was shown that in the presence of guanine-model molecule which inhibits the interaction between conjugated QDs and AuNPs the FRET is efficiently hampered. Thus monitoring the changes in the restoring fluorescence signal allows to assay the free guanine concentration. Importantly, we have demonstrated the sensitivity and selectivity of the obtained FRET-based system towards guanine. Moreover, in order to confirm the feasibility of the proposed material for nucleobase detection in the real biological samples the developed nanoparticles were also evaluated under simulated urine conditions. The presented strategy of FRET-based conjugated system preparation might be easily used for the development of another nucleobases selective detection and thus opens many possibilities for the determination of biomolecules in the real samples. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. [Mass spectrometry of triterpene glycosides molecular complexation with purine bases of nucleic acids].

    Science.gov (United States)

    Lekar', A V; Vetrova, E V; Borisenko, N I; Iakovishin, L A; Grishkovets, V I

    2011-01-01

    The molecular complexation of adenine and guanine with hederagenin 3-O-alpha-L-rhamnopyranosyl-(1-->2)-O-alpha-L-arabinopyranoside (alpha-hederin) and its 28-O-alpha-L-rhamnopyranosyl-(1-->4)-O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl ester (hederasaponin C) was investigated for the first time using the method of electrospray ionization mass spectrometry. Guanine forms complexes more diverse in composition than adenine.

  6. Acute food deprivation enhances fear extinction but inhibits long-term depression in the lateral amygdala via ghrelin signaling.

    Science.gov (United States)

    Huang, Chiung-Chun; Chou, Dylan; Yeh, Che-Ming; Hsu, Kuei-Sen

    2016-02-01

    Fear memory-encoding thalamic input synapses to the lateral amygdala (T-LA) exhibit dynamic efficacy changes that are tightly correlated with fear memory strength. Previous studies have shown that auditory fear conditioning involves strengthening of synaptic strength, and conversely, fear extinction training leads to T-LA synaptic weakening and occlusion of long-term depression (LTD) induction. These findings suggest that the mechanisms governing LTD at T-LA synapses may determine the behavioral outcomes of extinction training. Here, we explored this hypothesis by implementing food deprivation (FD) stress in mice to determine its effects on fear extinction and LTD induction at T-LA synapses. We found that FD increased plasma acylated ghrelin levels and enhanced fear extinction and its retention. Augmentation of fear extinction by FD was blocked by pretreatment with growth hormone secretagogue receptor type-1a antagonist D-Lys(3)-GHRP-6, suggesting an involvement of ghrelin signaling. Confirming previous findings, two distinct forms of LTD coexist at thalamic inputs to LA pyramidal neurons that can be induced by low-frequency stimulation (LFS) or paired-pulse LFS (PP-LFS) paired with postsynaptic depolarization, respectively. Unexpectedly, we found that FD impaired the induction of PP-LFS- and group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD, but not LFS-induced LTD. Ghrelin mimicked the effects of FD to impair the induction of PP-LFS- and DHPG-induced LTD at T-LA synapses, which were blocked by co-application of D-Lys(3)-GHRP-6. The sensitivity of synaptic transmission to 1-naphthyl acetyl spermine was not altered by either FD or ghrelin treatment. These results highlight distinct features of fear extinction and LTD at T-LA synapses. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Long-term activation of group I metabotropic glutamate receptors increases functional TRPV1-expressing neurons in mouse dorsal root ganglia

    Directory of Open Access Journals (Sweden)

    Takayoshi eMasuoka

    2016-03-01

    Full Text Available Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1 in dorsal root ganglion (DRG neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC, a transient receptor potential ankyrin type 1 (TRPA1 agonist. Increase in the proportion was suppressed by phospholipase C, protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.

  8. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing.

    Science.gov (United States)

    Memon, Farhat N; Owen, Anne M; Sanchez-Graillet, Olivia; Upton, Graham J G; Harrison, Andrew P

    2010-01-15

    A tetramer quadruplex structure is formed by four parallel strands of DNA/ RNA containing runs of guanine. These quadruplexes are able to form because guanine can Hoogsteen hydrogen bond to other guanines, and a tetrad of guanines can form a stable arrangement. Recently we have discovered that probes on Affymetrix GeneChips that contain runs of guanine do not measure gene expression reliably. We associate this finding with the likelihood that quadruplexes are forming on the surface of GeneChips. In order to cope with the rapidly expanding size of GeneChip array datasets in the public domain, we are exploring the use of cloud computing to replicate our experiments on 3' arrays to look at the effect of the location of G-spots (runs of guanines). Cloud computing is a recently introduced high-performance solution that takes advantage of the computational infrastructure of large organisations such as Amazon and Google. We expect that cloud computing will become widely adopted because it enables bioinformaticians to avoid capital expenditure on expensive computing resources and to only pay a cloud computing provider for what is used. Moreover, as well as financial efficiency, cloud computing is an ecologically-friendly technology, it enables efficient data-sharing and we expect it to be faster for development purposes. Here we propose the advantageous use of cloud computing to perform a large data-mining analysis of public domain 3' arrays.

  9. Purine metabolism in Toxoplasma gondii

    International Nuclear Information System (INIS)

    Krug, E.C.; Marr, J.J.; Berens, R.L.

    1989-01-01

    We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides

  10. Sensitive and specific detection of ligands using engineered riboswitches.

    Science.gov (United States)

    Morse, Daniel P; Nevins, Colin E; Aggrey-Fynn, Joana; Bravo, Rick J; Pfaeffle, Herman O I; Laney, Jess E

    2018-03-05

    Riboswitches are RNA elements found in non-coding regions of messenger RNAs that regulate gene expression through a ligand-triggered conformational change. Riboswitches typically bind tightly and specifically to their ligands, so they have the potential to serve as highly effective sensors in vitro. In B. subtilis and other gram-positive bacteria, purine nucleotide synthesis is regulated by riboswitches that bind to guanine. We modified the xpt-pbuX guanine riboswitch for use in a fluorescence quenching assay that allowed us to specifically detect and quantify guanine in vitro. Using this assay, we reproducibly detected as little as 5 nM guanine. We then produced sensors for 2'-deoxyguanosine and cyclic diguanylate (c-diGMP) by appending the P1 stem of the guanine riboswitch to the ligand-binding domains of a 2'-deoxyguanosine riboswitch and a c-diGMP riboswitch. These hybrid sensors could detect 15 nM 2'-deoxyguanosine and 3 nM c-diGMP, respectively. Each sensor retained the ligand specificity of its corresponding natural riboswitch. In order to extend the utility of our approach, we developed a strategy for the in vitro selection of sensors with novel ligand specificity. Here we report a proof-of-principle experiment that demonstrated the feasibility of our selection strategy. Published by Elsevier B.V.

  11. Designing a New Class of Bases for Nucleic Acid Quadruplexes and Quadruplex-Active Ligands.

    Science.gov (United States)

    Bazzi, Sophia; Novotný, Jan; Yurenko, Yevgen P; Marek, Radek

    2015-06-22

    A new class of quadruplex nucleobases, derived from 3-deazaguanine, has been designed for various applications as smart quadruplex ligands as well as quadruplex-based aptamers, receptors, and sensors. An efficient strategy for modifying the guanine quadruplex core has been developed and tested by using quantum chemistry methods. Several potential guanine derivatives modified at the 3- or 8-position or both are analyzed, and the results compared to reference systems containing natural guanine. Analysis of the formation energies (BLYP-D3(BJ)/def2-TZVPP level of theory, in combination with the COSMO model for water) in model systems consisting of two and three stacked tetrads with Na(+) /K(+) ion(s) inside the internal channel indicates that the formation of structures with 3-halo-3-deazaguanine bases leads to a substantial gain in energy, as compared to the corresponding reference guanine complexes. The results cast light on changes in the noncovalent interactions (hydrogen bonding, stacking, and ion coordination) in a quadruplex stem upon modification of the guanine core. In particular, the enhanced stability of the modified quadruplexes was shown to originate mainly from increased π-π stacking. Our study suggests the 3-halo-3-deazaguanine skeleton as a potential building unit for quadruplex systems and smart G-quadruplex ligands. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Direct measurement of interaction forces between a platinum dichloride complex and DNA molecules.

    Science.gov (United States)

    Muramatsu, Hiroshi; Shimada, Shogo; Okada, Tomoko

    2017-09-01

    The interaction forces between a platinum dichloride complex and DNA molecules have been studied using atomic force microscopy (AFM). The platinum dichloride complex, di-dimethylsulfoxide-dichloroplatinum (II) (Pt(DMSO) 2 Cl 2 ), was immobilized on an AFM probe by coordinating the platinum to two amino groups to form a complex similar to Pt(en)Cl 2 , which is structurally similar to cisplatin. The retraction forces were measured between the platinum complex and DNA molecules immobilized on mica plates using force curve measurements. The histogram of the retraction force for λ-DNA showed several peaks; the unit retraction force was estimated to be 130 pN for a pulling rate of 60 nm/s. The retraction forces were also measured separately for four single-base DNA oligomers (adenine, guanine, thymine, and cytosine). Retraction forces were frequently observed in the force curves for the DNA oligomers of guanine and adenine. For the guanine DNA oligomer, the most frequent retraction force was slightly lower than but very similar to the retraction force for λ-DNA. A higher retraction force was obtained for the adenine DNA oligomer than for the guanine oligomer. This result is consistent with a higher retraction activation energy of adenine with the Pt complex being than that of guanine because the kinetic rate constant for retraction correlates to exp(FΔx - ΔE) where ΔE is an activation energy, F is an applied force, and Δx is a displacement of distance.

  13. Intrinsic nucleic acid dynamics modulates HIV-1 nucleocapsid protein binding to its targets.

    Directory of Open Access Journals (Sweden)

    Ali Bazzi

    Full Text Available HIV-1 nucleocapsid protein (NC is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using (13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.

  14. Intrinsic Nucleic Acid Dynamics Modulates HIV-1 Nucleocapsid Protein Binding to Its Targets

    Science.gov (United States)

    Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; De Rocquigny, Hugues; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2012-01-01

    HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using 13C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome. PMID:22745685

  15. Binding of organometallic ruthenium(II) anticancer compounds to nucleobases: a computational study.

    Science.gov (United States)

    Gossens, Christian; Tavernelli, Ivano; Rothlisberger, Ursula

    2009-10-29

    The reaction of the anticancer compound [(eta(6)-benzene)Ru(en)(OH(2))](2+) (1) toward the nucleobases guanine, adenine, and cytosine is studied computationally using DFT/BP86 calculations. The aqua leaving group of such compounds is known to undergo ligand exchange reactions with nucleophilic centers in DNA and preferentially with the N7 atom of guanine, N7(G). Our results show that an H-bonded reactant adduct with nucleobases is formed via either the aqua ligand (cis adduct) or the en (ethylenediamine) ligand (trans adduct) of 1. All studied nucleobases favor an H-bonded cis adduct. Only guanine forms also a trans reactant adduct in the gas phase. The guanine N7 and O6 atoms in this trans adduct are situated in an ideal position to form each a strong H-bond to both amino groups of the en ligand of 1. A docking study shows that this unique recognition pattern is also plausible for the interaction with double stranded DNA. For the reaction of 1 with guanine, we identified three different reaction pathways: (i) A cis (G)N7-Ru-OH(2) transition state (TS). (ii) A direct trans reaction pathway. (iii) A 2-step trans mechanism. The activation energies for the cis pathway are smaller than for the trans pathways. The ultimately formed Ru-N7(G) product is characterized by a thermally stable H-bond between the O6(G) and a diamine-NH(2) hydrogen.

  16. Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

    Science.gov (United States)

    Barankiewicz, J; Paszkowski, J

    1980-01-15

    The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.

  17. High-throughput screening assay of hepatitis C virus helicase inhibitors using fluorescence-quenching phenomenon

    International Nuclear Information System (INIS)

    Tani, Hidenori; Akimitsu, Nobuyoshi; Fujita, Osamu; Matsuda, Yasuyoshi; Miyata, Ryo; Tsuneda, Satoshi; Igarashi, Masayuki; Sekiguchi, Yuji; Noda, Naohiro

    2009-01-01

    We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential inhibitors.

  18. Cloning of monomeric human papillomavirus type 16 DNA integrated within cell DNA from a cervical carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Matsukura, T.; Kanda, T.; Furuno, A.; Yoshikawa, H.; Kawana, T.; Yoshiike, K.

    1986-06-01

    The authors have molecularly cloned and characterized monomeric human papillomavirus type 16 DNA with flanking cell DNA sequences from a cervical carcinoma. Determination of nucleotide sequence around the junctions of human papillomavirus and cell DNAs revealed that at the site of integration within cell DNA the cloned viral DNA had a deletion between nucleotides 1284 and 4471 (numbering system from K. Seedorf, G. Kraemmer, M. Duerst, S. Suhai, and W.G. Roewkamp), which includes the greater part of E1 gene and the entire E2 gene. In the remaining part of the E1 gene, three guanines were found at the location where two guanines at nucleotides 1137 and 1138 have been recorded. This additional guanine shifted the reading frame and erased an interruption in the E1 gene. The data strongly suggest that, like other papillomaviruses, human papillomavirus type 16 has an uninterrupted E1 gene.

  19. The damage and repair of DNA in teleosts after administration of N-methyl-N-nitrosourea

    International Nuclear Information System (INIS)

    Krupitza, G.H.G.

    1986-01-01

    14 C-MNU, dissolved in a DMSO-citratebuffer solution, was given intraperitoneally to Black Mollies (B.M.) and Poecilia formosa (P.f.). Gills, liver, tailfinmuscles, intestine, gonads and brain were removed from each fish and DNA was isolated by phenol extraction. The DNA was hydrolysed and then the purines were separated using HPLC. Methylation of purines was determined by a liquid scintillation counter. Maximum methylation was formed in the N-7 position of guanine in the DNA from intestine of B.M. The highest content of O 6 -methyl guanine was found in the DNA of tailfinmuscles of B.M. whereas DNA from brain of B.M. showed the maximum methylation in N-3 position of adenine. The methylation of the purines from B.M. showed the similar pattern as in P.f. but was quantitatively double the amount as that found in P.f. The methylation of O 6 -position of guanine and N-3 position of adenine occured earlier in P.f. than in B.M. Maximum methylation of purines from each of the organs investigated was found to occur after 1/2 to 8 hours. The amount of methylation as low as 10% of the maximum was observed in a period from 1/2 to 16 hours after the application of 14 C-MNU. Excision repair seems to be responsible for removal of N-3 methyl adenine and N-7 methyl guanine whereas O 6 -methyl guanine seems to be repaired by methyltransferases. Removal of methylgroups from O 6 -position of guanine and the excision repair known to exist in mammals and bacteria probably play a role in these two species of teleosts as well. (Author)

  20. Crystal structure of four-stranded Oxytricha telomeric DNA

    Science.gov (United States)

    Kang, C.; Zhang, X.; Ratliff, R.; Moyzis, R.; Rich, A.

    1992-01-01

    The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

  1. A theoretical investigation on bio-transformation of third generation anti-cancer drug Heptaplatin and its interaction with DNA purine bases

    Science.gov (United States)

    Reddy B., Venkata P.; Mukherjee, Subhajit; Mitra, Ishani; Moi, Sankar Ch.

    2017-12-01

    Heptaplatin is an approved platinum based cytostatic drug for the treatment of gastric cancers. The hydrolytic bio-transformation of Heptaplatin and the platination processes of guanine (G) and adenine (A) with resulting mono and di-aquated species of Heptaplatin have been investigated using density functional theory (DFT) combined with the conductor like dielectric continuum model (CPCM) approach, to spotlight the drug activation energy profiles and their binding mechanisms. The stationary points on the potential energy surfaces were fully optimized and characterized. The mono-functional binding of Heptaplatin, guanine as target over adenine due to electronic factors and more favorable hydrogen-bonds pattern.

  2. Conformational change and biocatalysis-triggered spectral shift of single Au nanoparticles.

    Science.gov (United States)

    Zhao, Yun; He, Ya-Kai; Zhang, Jing; Wang, Feng-Bin; Wang, Kang; Xia, Xing-Hua

    2014-05-28

    Spectral shift of localized plasmon resonance scattering of guanine-rich DNA modified single Au nanoparticles is observed under a dark field microscope equipped with a spectrometer. The spectra continuously red-shift with the conformational change of the guanine-rich DNA upon associating with K(+), hemin and the biocatalytic growth of the polymer. The scattering spectrum of single nanoparticles is proved to be sensitive both to a subtle conformational change and the biocatalysis process. 20 mM K(+) or 100 μM H2O2 can trigger a detectable peak shift. The present study paves a new and efficient way to extract chemical information from micro/nanospace.

  3. Metabolism and distribution of guanosine given intraperitoneally: implications for spinal cord injury.

    Science.gov (United States)

    Jiang, Shucui; Fischione, Gemma; Giuliani, Patricia; Guiliani, Patricia; Romano, Silvia; Caciagli, Francesco; Di Iorio, Patrizia; Diiorio, Patrizia

    2008-06-01

    Intraperitoneal administration of guanosine to rats with chronic spinal cord injury stimulates remyelination and functional recovery. If guanosine produced its effects in the nervous system, it should enter it and elevate endogenous concentrations. [(3)H]-guanosine (8 mg/kg) was administered intraperitoneally to rats and its distribution and concentration in different sites determined. Guanosine rapidly entered all tissues; its concentration peaked at about 15 minutes except in adipose tissue and CNS where it continued to rise for 30 minutes. Its chief metabolic product in all sites was guanine with over twice as much guanine as guanosine present in CNS after 30 minutes.

  4. Multi-stress resistance in Lactococcus lactis is actually escape from purine-induced stress sensitivity

    DEFF Research Database (Denmark)

    Ryssel, Mia; Hviid, Anne-Mette Meisner; Dawish, Mohamed S.

    2014-01-01

    in the rich and complex M17 medium. When salvage of purines and subsequent conversion to GTP was permitted in various genetic backgrounds of L. lactis MG1363, the cells became sensitive to acid stress, indicating that an excess of guanine nucleotides induces stress sensitivity. The addition of phosphate...... nucleotides is formed as a result of an improved conversion of guanosine in the salvage pathway. Based upon our findings, we suggest that L. lactis MG1363 is naturally multi-stress resistant in habitats devoid of any purine source. However, any exogenous purine that results in increased guanine nucleotide...

  5. Apparent involvement of purines in the control of expression of Salmonella typhimurium pyr genes: analysis of a leaky guaB mutant resistant to pyrimidine analogs

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank

    1979-01-01

    A leaky guaB mutant of Salmonella typhimurium LT-2 was obtained during a selection for mutants resistant to a combination of the two pyrimidine analogs, 5-fluorouracil and 5-fluorouridine. In the absence of exogenous guanine compounds, the growth rate of this mutant is limited by the endogenous s......%, respectively. The pyrimidine metabolism of the guaB mutant is restored completely by addition of guanine (or xanthine) to the growth medium. The data indicate purine nucleotide involvement in the regulation of expression of the pyr genes of S. typhimurium....

  6. Huntingtin gene repeat size variations affect risk of lifetime depression

    NARCIS (Netherlands)

    Gardiner, Sarah L.; van Belzen, Martine J.; Boogaard, Merel W.; van Roon-Mom, Willeke M. C.; Rozing, Maarten P.; van Hemert, Albert M.; Smit, Johannes H.; Beekman, Aartjan T. F.; van Grootheest, Gerard; Schoevers, Robert A.; Voshaar, Richard C. Oude; Roos, Raymund A. C.; Comijs, Hannie C.; Penninx, Brenda W. J. H.; van der Mast, Roos C.; Aziz, N. Ahmad

    2017-01-01

    Huntington disease (HD) is a severe neuropsychiatric disorder caused by a cytosine-adenine-guanine (CAG) repeat expansion in the HTT gene. Although HD is frequently complicated by depression, it is still unknown to what extent common HTT CAG repeat size variations in the normal range could affect

  7. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Sandra Witz

    Full Text Available Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  8. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    Science.gov (United States)

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  9. Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

    Science.gov (United States)

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  10. The Rac1 hypervariable region in targeting and signaling: a tail of many stories

    NARCIS (Netherlands)

    Lam, B. Daniel; Hordijk, Peter L.

    2013-01-01

    Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control

  11. Progressive osseous heteroplasia (POH): an Egyptian patient | El ...

    African Journals Online (AJOL)

    Progressive osseous heteroplasia is a rare genetic disorder characterized by cutaneous ossification during infancy and progressive ossification of subcutaneous and deep connective tissue including muscle and fascia during childhood. It is at the severe end of a spectrum of Guanine Nucleotide-binding protein, ...

  12. Radioresistance of GGG sequences to prompt strand break formation from direct-type radiation damage.

    Science.gov (United States)

    Black, Paul J; Miller, Adam S; Hayes, Jeffrey J

    2016-11-01

    As humans, we are constantly exposed to ionizing radiation from natural, man-made and cosmic sources which can damage DNA, leading to deleterious effects including cancer incidence. In this work, we introduce a method to monitor strand breaks resulting from damage due to the direct effect of ionizing radiation and provide evidence for sequence-dependent effects leading to strand breaks. To analyze only DNA strand breaks caused by radiation damage due to the direct effect of ionizing radiation, we combined an established technique to generate dehydrated DNA samples with a technique to analyze single-strand breaks on short oligonucleotide sequences via denaturing gel electrophoresis. We find that direct damage primarily results in a reduced number of strand breaks in guanine triplet regions (GGG) when compared to isolated guanine (G) bases with identical flanking base context. In addition, we observe strand break behavior possibly indicative of protection of guanine bases when flanked by pyrimidines and sensitization of guanine to strand break when flanked by adenine (A) bases in both isolated G and GGG cases. These observations provide insight into the strand break behavior in GGG regions damaged via the direct effect of ionizing radiation. In addition, this could be indicative of DNA sequences that are naturally more susceptible to strand break due to the direct effect of ionizing radiation.

  13. Deciphering the four-letter code : The genetic basis of complex traits and common disease

    NARCIS (Netherlands)

    Pulit, S.L.

    2016-01-01

    Deoxyribonucleic acid (DNA) is made up of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T). Assembled in a strategic fashion, these bases code for the unique genomes of all walks of life, from viruses, to rodents, to primates. The human genome, mapped completely for the first time

  14. High-Resolution Scanning Tunneling Microscopy Studies of Molecular Nanostructures on Surfaces

    DEFF Research Database (Denmark)

    Song, Xin

    . First, to study the role of hydrogen bonding in self-assembly, we investigate the monomolecular self-assembled system of pyrene-4,5,9,10-tetrone and phenanthrene- 9,10-dione molecules on Au(111) and HOPG surface respectively and the binary molecular self-assembled system of stearic acid and guanine...

  15. Inhibition of the Escherichia coli 6-oxopurine phosphoribosyltransferases by nucleoside phosphonates: potential for new antibacterial agents

    Czech Academy of Sciences Publication Activity Database

    Keough, D. T.; Hocková, Dana; Rejman, Dominik; Špaček, Petr; Vrbková, Silvie; Krečmerová, Marcela; Eng, W. S.; Jans, H.; West, N. P.; Naesens, L. M. J.; de Jersey, J.; Guddat, L. W.

    2013-01-01

    Roč. 56, č. 17 (2013), s. 6967-6984 ISSN 0022-2623 R&D Projects: GA ČR GAP207/11/0108 Institutional support: RVO:61388963 Keywords : nucleoside phosphonates * antibacterial agents * hypoxanthine-guanine phosphoribosyltransferase * state analog inhibitor * antimalarial chemotherapy Subject RIV: CC - Organic Chemistry Impact factor: 5.480, year: 2013

  16. The construction of cosmid libraries which can be used to transform eukaryotic cells.

    NARCIS (Netherlands)

    F.G. Grosveld (Frank); T. Lund; E.J. Murray; A.L. Mellor; H.H.M. Dahl; R.A. Flavell (Richard)

    1982-01-01

    textabstractCosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase

  17. AcEST: DK949606 [AcEST

    Lifescience Database Archive (English)

    Full Text Available |GNL3_CAEEL Guanine nucleotide-binding protein-like 3 h... 37 0.099 sp|P17453|BPI_BOVIN Bactericidal permeab...RRKACNVGNLPGITKEIQ--EVELDKNIRLIDSPGVILVSQKDLD-PIEVALKNAIR 334 >sp|P17453|BPI_BOVIN Bactericidal permeability

  18. Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2

    NARCIS (Netherlands)

    Vila-Bedmar, Rocio; Cruces-Sande, Marta; Lucas, Elisa; Willemen, Hanneke L D M; Heijnen, Cobi J; Kavelaars, Annemieke; Mayor, Federico; Murga, Cristina

    2015-01-01

    Insulin resistance is a common feature of obesity and predisposes individuals to various prevalent pathological conditions. G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor kinase 2 (GRK2) integrates several signal transduction pathways and is emerging as a

  19. Yeast Interacting Proteins Database: YHL004W, YDL100C [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available it as bait (3) Rows with this bait as prey (0) YDL100C GET3 Guanine nucleotide exchange factor for Gpa1p; amplifies...exchange factor for Gpa1p; amplifies G protein signaling; subunit of the GET complex, which is involved in G

  20. Graphite Composite Electrode in Voltammetry

    Czech Academy of Sciences Publication Activity Database

    Šebková, Světlana; Navrátil, Tomáš; Kopanica, M.

    2005-01-01

    Roč. 38, - (2005), s. 1747-1758 ISSN 0003-2719 Institutional research plan: CEZ:AV0Z40400503 Keywords : 2-aminonaphtalene * adenine * guanine * manganese * EVLS Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.036, year: 2005

  1. Enhanced H-bonding and pi-stacking in DNA: a potent duplex-stabilizing and mismatch sensing nucleobase analogue

    DEFF Research Database (Denmark)

    Lou, Chenguang; Dallmann, Andre; Marafini, Pietro

    2014-01-01

    X-pyrene is a new nucleic acid duplex stabilizing cytosine analogue that combines enhanced pi-stacking, hydrogen bonding and electrostatic interactions to greatly increase the stability of bulged DNA duplexes and DNA/RNA hybrids. X-pyrene is highly selective for guanine as a partner and duplex...

  2. Progressive osseous heteroplasia

    African Journals Online (AJOL)

    Progressive osseous heteroplasia is a rare genetic disorder characterized by cu- taneous ossification during infancy and progressive ossification of subcutane- ous and deep connective tissue including muscle and fascia during childhood. It is at the severe end of a spectrum of Guanine Nucleotide-binding protein,.

  3. Discriminating DNA mismatches by electrochemical and gravimetric techniques.

    Science.gov (United States)

    Mazouz, Zouhour; Fourati, Najla; Zerrouki, Chouki; Ommezine, Asma; Rebhi, Lamia; Yaakoubi, Nourdin; Kalfat, Rafik; Othmane, Ali

    2013-10-15

    A silicon nitride functionalized electrode and a 104 MHz lithium tantalate (LiTaO₃) surface acoustic wave (SAW) sensor have been used to investigate target-probe recognition processes. Electrochemical and gravimetric measurements have been considered to monitor hybridization of single base mismatch (SBM) in synthetic oligonucleotides and single-nucleotide polymorphisms ApoE in real clinical genotypes. Obvious discrimination of SBM in nucleotides has been shown by both gravimetric and electrochemical techniques, without labeling nor amplification. Investigations on mismatches nature and position have also been considered. For guanine-adenine (GA), guanine-thymine (GT) and guanine-guanine (GG) mismatches, the sensors responses present a dependence upon positions. Considering the capacitance variations and hybridization rates, results showed that gravimetric transduction is more sensitive than electrochemical one. Moreover, the highest value of GT hybridization rate (in the middle position) was found in accordance with the nearest-neighbor model, where the considered configuration appears as the most thermodynamically stable. For the real samples, where the electrochemical transduction, by combining capacitance and flat-band potential measurements, were found more sensitive, the results show that the realized sensor permits an unambiguous discrimination of recognition between fully complementary, non-complementary and single base mismatched targets, and even between the combination of differently matched strands. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Structural Nonrigitidy of Nucleid Acid Bases. Post-Hartree-Fock ab initio Study

    Czech Academy of Sciences Publication Activity Database

    Shishkin, O. V.; Gorb, L.; Hobza, Pavel; Leszczynski, J.

    2000-01-01

    Roč. 80, 4/5 (2000), s. 1116-1124 ISSN 0020-7608 Grant - others:NSF(US) 94-4-756-01; NIH(US) G12RR13459 Institutional research plan: CEZ:AV0Z4040901 Keywords : uracyl * thymine * isocytosine guanine and adenine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 1.317, year: 2000

  5. Spectroscopic insights into quadruplexes of five-repeat telomere DNA sequences upon G-block damage

    Czech Academy of Sciences Publication Activity Database

    Dvořáková, Zuzana; Vorlíčková, Michaela; Renčiuk, Daniel

    2017-01-01

    Roč. 1861, č. 11 (2017), s. 2750-2757 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GJ17-19170Y Institutional support: RVO:68081707 Keywords : k+ solution * guanine quadruplexes Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  6. Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers

    DEFF Research Database (Denmark)

    Loft, S; Poulsen, H E; Vistisen, K

    1999-01-01

    '-deoxyguanosine (8-oxodG), a repair product of the highly mutagenic oxidation of guanine in DNA or the cellular pool of GTP. CYP1A2 activity was estimated from the urinary excretion of metabolites of dietary caffeine. The DNA repair was estimated by unscheduled DNA synthesis (UDS) in mononuclear cells isolated...

  7. CHAPTER 1

    African Journals Online (AJOL)

    Dr Olaleye

    form of natural DNA. .... either covalent or non-covalent bonding. In a covalent binding the reactive part of the compounds interacts with a nitrogen base of DNA such as guanine N7 e.g., ... electrostatic and groove binding of cationic metal.

  8. Positive Relationship between Abdominal Coloration and Dermal Melanin Density in Phrynosomatid Lizards

    Science.gov (United States)

    Vanessa S. Quinn; Diana K. Hews

    2003-01-01

    Phrynosomatid lizards show considerable variation among species in the occurrence of a secondary sexual trait, blue abdominal coloration. The production of blue skin may be controlled by at least two cellular components, melanin in melanophores, and guanine in iridophores. To examine the hypothesis that a mechanism producing variation in abdominal coloration is...

  9. Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding

    NARCIS (Netherlands)

    Canault, Matthias; Ghalloussi, Dorsaf; Grosdidier, Charlotte; Guinier, Marie; Perret, Claire; Chelghoum, Nadjim; Germain, Marine; Raslova, Hana; Peiretti, Franck; Morange, Pierre E.; Saut, Noemie; Pillois, Xavier; Nurden, Alan T.; Cambien, François; Pierres, Anne; van den Berg, Timo K.; Kuijpers, Taco W.; Alessi, Marie-Christine; Tregouet, David-Alexandre

    2014-01-01

    The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1

  10. Attenuated Variants of Lesch-Nyhan Disease

    Science.gov (United States)

    Jinnah, H. A.; Ceballos-Picot, Irene; Torres, Rosa J.; Visser, Jasper E.; Schretlen, David J.; Verdu, Alfonso; Larovere, Laura E.; Chen, Chung-Jen; Cossu, Antonello; Wu, Chien-Hui; Sampat, Radhika; Chang, Shun-Jen; de Kremer, Raquel Dodelson; Nyhan, William; Harris, James C.; Reich, Stephen G.; Puig, Juan G.

    2010-01-01

    Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The classic form of the disease is described by a characteristic syndrome that includes overproduction of uric acid, severe generalized dystonia, cognitive disability and self-injurious behaviour. In addition to the…

  11. Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Kjaersgård, I V; Wiborg, O

    1995-01-01

    All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches. Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism. Gly94 and Gly126...

  12. Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc

    NARCIS (Netherlands)

    Kranenburg, O.; Verlaan, I.; Hordijk, P. L.; Moolenaar, W. H.

    1997-01-01

    Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known

  13. Progressive osseous heteroplasia

    African Journals Online (AJOL)

    ous and deep connective tissue including muscle and fascia during childhood. It is at the severe end of a spectrum of Guanine ... and extensive bone formation in deep muscle and fascia. Dermal lesions co- alesce rapidly to form ... web-like calcification surrounding all the upper and lower limbs and sparing only the soft ...

  14. Sequence Classification: 894359 [

    Lifescience Database Archive (English)

    Full Text Available ondrial translation elongation factor Tu; comprises both GTPase and guanine nucleotide exchange factor activities, while these activi...ties are found in separate proteins in S. pombe and humans; Tuf1p || http://www.ncbi.nlm.nih.gov/protein/6324761 ...

  15. Nye molekylaere markører ved de kroniske myeloproliferative sygdomme

    DEFF Research Database (Denmark)

    Larsen, Thomas Stauffer; Pallisgaard, Niels; Christensen, Jacob Haaber

    2006-01-01

    The Philadelphia-negative chronic myeloproliferative disorders feature autonomous myeloid hyperproliferation and hypersensitivity to a number of growth factors, which most recently have been shown to be explained by a guanine-to-thymidine mutation in the Janus tyrosine kinase (JAK2) gene, implica...

  16. Urinary markers of nucleic acid oxidation and cancer in type 2 diabetes

    DEFF Research Database (Denmark)

    Broedbaek, Kasper; Siersma, Volkert; Henriksen, Trine Foged

    2015-01-01

    AIMS/HYPOTHESIS: We investigated whether urinary markers of nucleic acid oxidation are associated with an increased risk of cancer in type 2 diabetes patients. METHODS: Urine samples from 1381 newly diagnosed diabetes patients were assayed for the oxidatively modified guanine nucleosides 8-oxo-7,...

  17. Membrane association of the Arabidopsis ARF exchange factor GNOM involves interaction of conserved domains

    DEFF Research Database (Denmark)

    Anders, Nadine; Nielsen, Michael M.; Keicher, Jutta

    2008-01-01

    The GNOM protein plays a fundamental role in Arabidopsis thaliana development by regulating endosome-to-plasma membrane trafficking required for polar localization of the auxin efflux carrier PIN1. GNOM is a family member of large ARF guanine nucleotide exchange factors (ARF-GEFs), which regulate...

  18. The role of Rap1 in cell adhesion and migration

    NARCIS (Netherlands)

    Raaijmakers, J.H.

    2009-01-01

    cAMP is a pivotal second messenger that regulates a wide range of cellular processes. Besides protein kinase A, cAMP also activates the guanine nucleotide exchange factor (GEF) Epac that can subsequently activate the small GTPase Rap1. Rap1 is an important regulator of E-cadherin mediated cell-cell

  19. Measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-guanosine in cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry

    DEFF Research Database (Denmark)

    Weimann, Allan; Simonsen, Anja Hviid; Poulsen, Henrik E

    2018-01-01

    Increased levels of nucleosides modified by oxidation in human cerebrospinal fluid (CSF) have several times been reported in Alzheimer patients and patients suffering from Parkinson's disease. The focus has especially been on nucleosides containing the 8-hydroxylation of guanine. Only few reports...

  20. Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

    OpenAIRE

    Barankiewicz, J; Paszkowski, J

    1980-01-01

    The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed fro...

  1. Degradation of nucleic acids with ozone. II. Degradation of yeast RNA, yeast phenylalanine tRNA and tobacco mosaic virus RNA.

    Science.gov (United States)

    Shinriki, N; Ishizaki, K; Ikehata, A; Yoshizaki, T; Nomura, A; Miura, K; Mizuno, Y

    1981-10-27

    The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.

  2. Clenbuterol-Stimulated Glucose Uptake Activates both GS and GI ...

    African Journals Online (AJOL)

    In the later, β2-AR induces PKA-catalysed phosphorylation of the receptor, which intends couples to Gi, at high concentration. We proposed that, clenbuterol ... relieved the inhibitory effect. Keywords: PTX, Pertusis toxin, G-proteins, Guanine nucleotide binding proteins, β-AR, beta adrenoceptor, M2, muscarinic receptors ...

  3. O-6-benzylguanine potentiates BCNU but not busulfan toxicity in hematopoietic stem cells

    NARCIS (Netherlands)

    Westerhof, GR; Down, JD; Blokland, [No Value; Wood, M; Boudewijn, A; Watson, AJ; McGown, AT; Ploemacher, RE; Margison, GP

    Objective. Busulfan (BU) is often used in conditioning regimens prior to bone marrow transplantation, but its mechanism of action remains to be resolved. We have examined the possibility that BU may exert part of its toxic effects via DNA alkylation at the Oh position of guanine as this might

  4. Influence of solitons on the conductance properties of double ...

    Indian Academy of Sciences (India)

    bonds between guanine (G) and cytosine (C) bases, on the electronic conduction properties of poly(dG)–poly(dC) DNA in metal/DNA/metal system. According to our model, in the presence of a sublattice of solitons, the band gap of the semicon- ductor structure of DNA molecule may be suppressed and at room temperature,.

  5. One-pot build-up procedure for the synthesis of variously substituted purine derivatives

    Czech Academy of Sciences Publication Activity Database

    Dejmek, Milan; Kovačková, Soňa; Zborníková, Eva; Hřebabecký, Hubert; Šála, Michal; Dračínský, Martin; Nencka, Radim

    2012-01-01

    Roč. 2, č. 17 (2012), s. 6970-6980 ISSN 2046-2069 R&D Projects: GA MV VG20102015046 Institutional support: RVO:61388963 Keywords : purines * heterocycles * adenine * guanine Subject RIV: CC - Organic Chemistry Impact factor: 2.562, year: 2012

  6. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F. E.; Moutinho, C.; Falcomatà, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; de Braud, F.; Rudà, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; de Witt Hamer, P.; Pietrantonio, F.; Siena, S.; Esteller, M.; Sartore-Bianchi, A.; di Nicolantonio, Federica

    2015-01-01

    Background: O6-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. Patients and methods: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and

  7. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F. E.; Moutinho, C.; Falcomatà, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; de Braud, F.; Rudà, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; de Witt Hamer, P.; Pietrantonio, F.; Siena, S.; Esteller, M.; Sartore-Bianchi, A.; Di Nicolantonio, F.

    2015-01-01

    O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA

  8. Developmental stage-specific contribution of LGR5(+) cells to basal and luminal epithelial lineages in the postnatal mammary gland

    NARCIS (Netherlands)

    de Visser, K.E.; Ciampricotti, M.; Michalak, E.M.; Tan, D.W.; Speksnijder, E.N.; Hau, C.S.; Clevers, H.; Barker, N.; Jonkers, J.

    2012-01-01

    The leucine-rich repeat-containing heterotrimeric guanine nucleotide-binding protein-coupled receptor 5 (LGR5) has been identified as a marker of cycling stem cells in several epithelial tissues, including small intestine, colon, stomach and hair follicle. To investigate whether LGR5 also marks

  9. Download this PDF file

    African Journals Online (AJOL)

    trimethylxanthinc) Was first isolated from coffee in. 1820(Arnand, 1984). It was subsequently identified in cocoa ... compounds ~ adenine and guanine, a property that is relevant to their biosynthesis and physiological ... properties and the slight degree of bitterness that it imparts (Harold, 1992). The purpose of this work is.

  10. Watson-Crick hydrogen bonds. Nature and role in DNA replication

    NARCIS (Netherlands)

    Guerra, Célia Fonseca; Bickelhaupt, F. Matthias

    2006-01-01

    Genetic information is found in the DNA. This information is found to be on adenine (A), thymine (T), guanine (G), and cytosine (C) occurs in a DNA strand. The two strands of the DNA double helix are held together by the hydrogen bonds of the Watson-Crick base pairs that form selectively between A

  11. Isolation of deletion alleles by G4 DNA-induced mutagenesis

    NARCIS (Netherlands)

    Pontier, Daphne B; Kruisselbrink, Evelien; Guryev, Victor; Tijsterman, Marcel

    Metazoan genomes contain thousands of sequence tracts that match the guanine-quadruplex (G4) DNA signature G(3)N(x)G(3)N(x)G(3)N(x)G(3), a motif that is intrinsically mutagenic, probably because it can form secondary structures during DNA replication. Here we show how and to what extent this feature

  12. The extraordinary specificity of xanthine phosphoribosyltransferase from Bacillus subtilis elucidated by reaction kinetics, ligand binding, and crystallography

    DEFF Research Database (Denmark)

    Arent, Susan; Kadziola, Anders; Larsen, Sine

    2006-01-01

    xanthine. The XPRTase is able also to react with guanine and hypoxanthine albeit at much lower (10-4-fold) catalytic efficiency. Different pKa values for the bases and variations in their electrostatic potential can account for these catalytic differences. The unique base specificity of XPRTase has been...

  13. Unigene BLAST: CBRC-HSAP-20-0016 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-HSAP-20-0016 gnl|UG|Hs#S29965912 Homo sapiens Rho guanine nucleotide exchange ...factor (GEF) 10-like, mRNA (cDNA clone IMAGE:5745823), with apparent retained intron /gb=BC111058 /gi=84040272 /ug=Hs.443460 /len=4973 0.074 28% ...

  14. Large normal-range TBP and ATXN7 CAG repeat lengths are associated with increased lifetime risk of depression

    DEFF Research Database (Denmark)

    Gardiner, S. L.; van Belzen, M. J.; Boogaard, M. W.

    2017-01-01

    Depression is one of the most prevalent and debilitating psychiatric disorders worldwide. Recently, we showed that both relatively short and relatively long cytosine–adenine–guanine (CAG) repeats in the huntingtin gene (HTT) are associated with an increased risk of lifetime depression. However, t...

  15. Huntingtin gene repeat size variations affect risk of lifetime depression

    DEFF Research Database (Denmark)

    Gardiner, Sarah L.; van Belzen, Martine J.; Boogaard, Merel W.

    2017-01-01

    Huntington disease (HD) is a severe neuropsychiatric disorder caused by a cytosine-adenine-guanine (CAG) repeat expansion in the HTT gene. Although HD is frequently complicated by depression, it is still unknown to what extent common HTT CAG repeat size variations in the normal range could affect...

  16. Sequence Classification: 893846 [

    Lifescience Database Archive (English)

    Full Text Available ds zinc, found both on membranes and in the cytosol; guanine nucleotide dissociation stimulator; Dss4p || http://www.ncbi.nlm.nih.gov/protein/6325274 ... ...tide release factor functioning in the post-Golgi secretory pathway, required for ER-to-Golgi transport, bin

  17. Sequence Classification: 785961 [

    Lifescience Database Archive (English)

    Full Text Available hways (157.5 kD) (aex-3) || http://www.ncbi.nlm.nih.gov/protein/25147747 ... ...ulsion defective AEX-3, Rab3 Guanine nucleotide Exchange Factor, regulator of presynaptic activity, regulates the rab-3 and cab-1 pat

  18. Characterization of genetic miscoding lesions caused by postmortem damage

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Hansen, Anders J; Willerslev, Eske

    2002-01-01

    The spectrum of postmortem damage in mitochondrial DNA was analyzed in a large data set of cloned sequences from ancient human specimens. The most common forms of damage observed are two complementary groups of transitions, termed "type 1" (adenine-->guanine/thymine-->cytosine) and "type 2" (cyto...

  19. HMOX1 promoter polymorphism modulates the relationship between disease activity and joint damage in rheumatoid arthritis.

    NARCIS (Netherlands)

    Wagener, F.A.D.T.G.; Toonen, E.J.M.; Wigman, L.; Fransen, J.; Creemers, M.C.W.; Radstake, T.R.D.J.; Coenen, M.J.H.; Barrera Rico, P.; Riel, P.L.C.M. van; Russel, F.G.M.

    2008-01-01

    OBJECTIVE: The guanine-thymidine (GT)n repeat in the HMOX1 promoter determines the level of induction of the heme-degrading enzyme heme oxygenase 1 (HO-1), which protects against inflammatory and oxidative stress. In individuals with short (GT)n repeats (where n < 25; SS genotype), higher levels of

  20. Structure-function relationship of a plant NCS1 member - Homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from arabidopsis

    KAUST Repository

    Witz, Sandra

    2014-03-12

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. 2014 Witz et al.

  1. Purine synthetic capacities of de novo and salvage pathways in rat hepatoma 3924A cells

    International Nuclear Information System (INIS)

    Natsumeda, Y.; Ikegami, T.; Weber, G.

    1986-01-01

    This study compares the potential synthetic capacities of purine de vovo and salvage pathways in neoplastic cells as reflected in enzyme specific activities and incorporation rates of C 14-formate, C 14-adenine, C 14-hypoxanthine and C 14-guanine into purine nucleotides and nucleic acids

  2. A New HPLC Purine Assay for Quantifying Microbial Flow

    Science.gov (United States)

    A HPLC method was developed to quantify the purines adenine and guanine and their metabolites xanthine and hypoxanthine in hydrolysates of isolated bacteria and omasal digesta and to assess the effect of using either purines only, or purines plus metabolites, as microbial markers for estimating micr...

  3. Cloning, expression and antigenicity of the L. donovani reductase

    DEFF Research Database (Denmark)

    Jensen, A T; Kemp, K; Theander, T G

    2001-01-01

    . We have cloned the reductase gene from L donovani and have shown that it differs in only one nucleotide from the L. major homologue, resulting in one amino acid change. A cytosine (C) to guanine (G) transposition in the coding sequence leads to a nonconserved substitution of asparagine (N) for lysine...

  4. Purine Bases in Blood Plasma of Patients with Chronic Pulmonary Diseases

    Directory of Open Access Journals (Sweden)

    Larissa E. Muravluyova

    2012-09-01

    Full Text Available The article is focused on the study of purine bases and intermediates of purine catabolism in plasma of patients with chronic obstructive bronchitis and idiopathic interstitial pneumonia. Decrease of adenine and hypoxantine in plasma of patients with idiopathic interstitial pneumonia was registered. Increase of guanine in plasma of patients with chronic obstructive pulmonary disease was established.

  5. Kinetics of hydrogen-deuterium exchange in guanosine 5'-monophosphate and guanosine 3':5'-monophosphate determined by laser-Raman spectroscopy.

    Science.gov (United States)

    Lane, M J; Thomas, G J

    1979-09-04

    Pseudo-first-order rate constants governing the deuterium exchange of 8-CH groups in guanosine 5'-monophosphate (5'-rGMP) and guanosine 3':5'-monophosphate (cGMP) were determined as a function of temperature in the range 30-80 degrees C by means of laser-Raman spectroscopy. For each guanine nucleotide the logarithm of the rate constant exhibits a strictly linear dependence on reciprocal temperature: i.e., k psi = Ae-Ea/RT with A = 8.84 X 10(14) h-1 and Ea = 24.6 kcal/mol for 5'-rGMP and A = 3.33 X 10(13) h-1 and Ea = 22.2 kcal/mol for cGMP. Exchange of the 8-CH groups in guanine nucleotides is generally 2-3 times more rapid than in adenine nucleotides [cf. g. j. thomas, Jr., & J. Livramento (1975) Biochemistry 14, 5210-5218]. As in the case of adenine nucleotides, cyclic and 5' nucleotides of guanine exchange at markedly different rates at lower temperatures, with exchange in the cyclic nucleotide being the more facile. Each of the guanine nucleotides was prepared in four different isotopic modifications for Raman spectral analysis. The Raman frequency shifts resulting from the various isotopic substitutions have been tabulated, and assignments have been given for most of the observed vibrational frequencies.

  6. Comparison on the interaction of Al3+/nano-Al13 with calf thymus DNA /salmon sperm DNA

    Science.gov (United States)

    Ma, Fei; Ma, Yue; Du, Changwen; Yang, Xiaodi; Shen, Renfang

    2015-11-01

    The conformation change, binding mode and binding site between Al3+/nano-Al13 and calf thymus DNA/salmon sperm DNA were investigated by UV-vis absorption, FTIR spectra, Raman spectroscopy and CD spectra, as well as melting curves measurement. The UV-vis spectra and circular dichroism spectra results suggested that the phosphate group structure was changed when Al3+ interacted with DNA, while the double-helix was distorted when nano-Al13 interacted with DNA. The FTIR and Raman spectroscopy revealed that the binding sites were Al3+ … PO2, Al3+ … N7/guanine PO2 … Al13 … N7-C8/guanine with calf thymus DNA, and Al3+ … N3-O2/cytosine, Al3+ … N7-C8/guanine, PO2 … Al13 … N7-C8/guanine, PO2 … Al13 … N1/adenine with salmon sperm DNA, respectively. The electrostatic binding was existed between Al3+ and DNA, and the electrostatic binding and complexing were found between nano-Al13 and DNA.

  7. Formation of platinated GG cross-links on DNA by photoactivation of a Pt(IV) azide complex

    Czech Academy of Sciences Publication Activity Database

    Kašpárková, Jana; Mackay, F. S.; Brabec, Viktor; Sadler, P. J.

    2003-01-01

    Roč. 8, č. 7 (2003), s. 741-745 ISSN 0949-8257 R&D Projects: GA AV ČR IAA5004101; GA AV ČR KJB5004301 Institutional research plan: CEZ:AV0Z5004920 Keywords : cross-links * DNA * guanine Subject RIV: BO - Biophysics Impact factor: 3.905, year: 2003

  8. Site-directed mutagenesis of Arg58 and Asp86 of elongation factor Tu from Escherichia coli: effects on the GTPase reaction and aminoacyl-tRNA binding

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.

    1996-01-01

    Elongation factor Tu from Escherichia coli was mutated separately at positions Asp86 and Arg58, in order to shed light both on the GTPase mechanism of elongation factor Tu and on the binding of aminoacyl-tRNA. In addition, the binding of guanine nucleotides was investigated by determination...

  9. 6-Oxopurine Phosphoribosyltransferase: A Target for the Development of Antimalarial Drugs

    Czech Academy of Sciences Publication Activity Database

    de Jersey, J.; Holý, Antonín; Hocková, Dana; Naesens, L.; Keough, D. T.; Guddat, L. W.

    2011-01-01

    Roč. 11, č. 16 (2011), s. 2085-2102 ISSN 1568-0266 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : Malaria * acyclic nucleoside phosphonates * hypoxanthine * guanine phosphoribosyl transferase Subject RIV: CC - Organic Chemistry Impact factor: 4.174, year: 2011

  10. Synthesis and spectroscopy of clay intercalated Cu(II) bio-monomer complexes: coordination of Cu(II) with purines and nucleotides

    NARCIS (Netherlands)

    Weckhuysen, B.M.; Leeman, H.; Schoonheydt, R.A.

    1999-01-01

    The spectroscopic properties of Cu(bio-monomer)nm+ complexes [BM=bio-monomer (purine, adenine, guanine, hypoxanthine, 5-ADP and 5-GMP)] in saponite clays have been investigated by diffuse reflectance spectroscopy (DRS) in the UV-Vis-NIR region and electron paramagnetic resonance (EPR) at X-band.

  11. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F.E.; Moutinho, C.; Falcomata, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; Braud, F. De; Ruda, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; Hamer, P. de Witt; Pietrantonio, F.; Siena, S. Di; Esteller, M.; Sartore-Bianchi, A.; Nicolantonio, F. Di

    2015-01-01

    BACKGROUND: O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. PATIENTS AND METHODS: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor

  12. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models

    NARCIS (Netherlands)

    Huang, Yunhong; Skwarek-Maruszewska, Aneta; Horré, Katrien; Vandewyer, Elke; Wolfs, Leen; Snellinx, An; Saito, Takashi; Radaelli, Enrico; Corthout, Nikky; Colombelli, Julien; Lo, Adrian C; Van Aerschot, Leen; Callaerts-Vegh, Zsuzsanna; Trabzuni, Daniah; Bossers, Koen; Verhaagen, Joost; Ryten, Mina; Munck, Sebastian; D'Hooge, Rudi; Swaab, Dick F; Hardy, John; Saido, Takaomi C; De Strooper, Bart; Thathiah, Amantha

    2015-01-01

    The orphan G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer's disease (AD). However, given the vast

  13. Response to Comment on "Positive Selection of Tyrosine Loss in Metazoan Evolution"

    DEFF Research Database (Denmark)

    Tan, Chris Soon Heng; Schoof, Erwin; Creixell, Pau

    2011-01-01

    Su et al. claim guanine-cytosine (GC) content variation can largely explain the observed tyrosine frequency variation, independent of adaptive evolution of cell-signaling complexity. We found that GC content variation, in the absence of selection for amino acid changes, can only maximally account...

  14. Epigenetic Vestiges of Early Developmental Adversity: Childhood Stress Exposure and DNA Methylation in Adolescence

    Science.gov (United States)

    Essex, Marilyn J.; Boyce, W. Thomas; Hertzman, Clyde; Lam, Lucia L.; Armstrong, Jeffrey M.; Neumann, Sarah M. A.; Kobor, Michael S.

    2013-01-01

    Fifteen-year-old adolescents (N = 109) in a longitudinal study of child development were recruited to examine differences in DNA methylation in relation to parent reports of adversity during the adolescents' infancy and preschool periods. Microarray technology applied to 28,000 cytosine-guanine dinucleotide sites within DNA derived from buccal…

  15. Development of a Novel Fluorescence Assay Based on the Use of the Thrombin-Binding Aptamer for the Detection of O6-Alkylguanine-DNA Alkyltransferase Activity

    Directory of Open Access Journals (Sweden)

    Maria Tintoré

    2010-01-01

    Full Text Available Human O6-alkylguanine-DNA alkyltransferase (hAGT is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O6 position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA. The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O6-methyl-guanine. The sequence also contains a fluorophore (fluorescein and a quencher (dabsyl attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O6-methyl group.

  16. RESEARCH ARTICLE Genetic Variations in Influx Transporter Gene ...

    Indian Academy of Sciences (India)

    Admin

    C480G (rs683369) of SLC22A1 in mediating resistance and/or good response to IM among. 278 Malaysian CML ... involves nucleotide change from cytosine (C) to guanine (G) which leads to an amino acid change from phenyl ... By referring to European Leukemia Net: Guideline for managing CML patients [9] hematologic ...

  17. Surface-enhanced Raman spectroscopy of DNA bases

    NARCIS (Netherlands)

    Otto, Cornelis; van den Tweel, T.J.J.; de Mul, F.F.M.; Greve, Jan

    1986-01-01

    A Raman microprobe has been used to measure the surface-enhanced Raman spectra of adenine, guanine, cytosine and thymine. Comparison of the SERS spectrum with solution spectra shows that some line positions are not influenced by the adsorption process while others show large shifts. In the SERS

  18. Group I Metabotropic Glutamate Receptors

    DEFF Research Database (Denmark)

    Erichsen, Julie Ladeby; Blaabjerg, Morten; Bogetofte Thomasen, Helle

    2015-01-01

    is, however, needed to realise their therapeutic potential. Glutamate and group I metabotropic glutamate receptors (mGluRs) affect proliferation and survival of rodent NSCs both during embryonic and postnatal development. To investigate the role of group I mGluRs (mGluR1 and mGluR5) on human NSCs, we...... differentiated an immortalized, forebrain-derived stem cell line in the presence or absence of glutamate and with addition of either the group I mGluR agonist DHPG or the selective antagonists; MPEP (mGluR5) and LY367385 (mGluR1). Characterization of differentiated cells revealed that both mGluR1 and mGluR5 were...... present on the cells. Addition of glutamate to the growth medium significantly increased cell proliferation and reduced cell death, resulting in increased cell numbers. In the presence of glutamate, selective activation of group I mGluRs reduced gliogenesis, whereas selective inhibition of group I m...

  19. mGluR-mediated calcium signalling in the thalamic reticular nucleus.

    Science.gov (United States)

    Neyer, Christina; Herr, David; Kohmann, Denise; Budde, Thomas; Pape, Hans-Christian; Coulon, Philippe

    2016-06-01

    The thalamic reticular nucleus (TRN) plays a major role in modulating the transfer of information from the thalamus to the cortex. GABAergic inhibition via the TRN is differentially regulated by metabotropic glutamate receptors (mGluRs) and the effect of mGluRs on the membrane potential, on ion channels, and on the plasticity of electrical coupling of TRN neurons has been studied previously. Although mGluRs are generally known to trigger Ca(2+) transients, mGluR-mediated Ca(2+)-transients in TRN neurons have not yet been investigated. In this study, we show that mGluRs can trigger Ca(2+)-transients in TRN neurons, that these transients depend on intracellular Ca(2+)-stores, and are mediated by IP3 receptors. Ca(2+) transients caused by the group I mGluR agonist DHPG elicit a current that is sensitive to flufenamic acid and has a reversal potential around -40mV. Our results add mGluR-mediated Ca(2+)-signalling in the TRN to the state-dependent modulators of the thalamocortical system. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Developing chiral-technologies. Chiral technology that Alice in the mirror land gave a birth to; Hattensuru kiraru technology. Kagami no kuni no Alice ga unda kiraru technology

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, T. [Kanegafuchi Chemical Industry Co. Ltd., Osaka (Japan)

    1998-05-01

    Since the theory for optical isomers has been advocated by Van`t Hoff et al, studies were developed on correlation between structures and functions of optically active bodies through separation of 1-tartaric acid by Pasteur. Technologies to create freely the optically active bodies has contributed to advancement of chemistry. This paper describes improvement in the method of manufacturing D-amino acid by using biomass. Kaneka Co., Ltd. uses an enzyme method to produce D-parahydroxyphenylglycin (D-HPG) which is an important chiral block in manufacturing amoxicillin, a constituent substance. Its prescription is represented by a method to covert hydantoin compound of racemic modification into D-amino acid through hydrolysis by utilizing two kinds of enzymes. Heat resistance was imparted to elevate stability of the parent decarbamylase. The decarbamylase genes originated from the 712 bacteria were introduced into a phage, which were randomly mutated by using a mutant. These mutated genes were introduced into colibacilli to select bacteria having decarbamylase whose heat resistance was increased by using recombined colibacilli. 2 refs., 2 figs., 3 tabs.

  1. Multiple Drug Treatments That Increase cAMP Signaling Restore Long-Term Memory and Aberrant Signaling in Fragile X Syndrome Models

    Science.gov (United States)

    Choi, Catherine H.; Schoenfeld, Brian P.; Bell, Aaron J.; Hinchey, Joseph; Rosenfelt, Cory; Gertner, Michael J.; Campbell, Sean R.; Emerson, Danielle; Hinchey, Paul; Kollaros, Maria; Ferrick, Neal J.; Chambers, Daniel B.; Langer, Steven; Sust, Steven; Malik, Aatika; Terlizzi, Allison M.; Liebelt, David A.; Ferreiro, David; Sharma, Ali; Koenigsberg, Eric; Choi, Richard J.; Louneva, Natalia; Arnold, Steven E.; Featherstone, Robert E.; Siegel, Steven J.; Zukin, R. Suzanne; McDonald, Thomas V.; Bolduc, Francois V.; Jongens, Thomas A.; McBride, Sean M. J.

    2016-01-01

    Fragile X is the most common monogenic disorder associated with intellectual disability (ID) and autism spectrum disorders (ASD). Additionally, many patients are afflicted with executive dysfunction, ADHD, seizure disorder and sleep disturbances. Fragile X is caused by loss of FMRP expression, which is encoded by the FMR1 gene. Both the fly and mouse models of fragile X are also based on having no functional protein expression of their respective FMR1 homologs. The fly model displays well defined cognitive impairments and structural brain defects and the mouse model, although having subtle behavioral defects, has robust electrophysiological phenotypes and provides a tool to do extensive biochemical analysis of select brain regions. Decreased cAMP signaling has been observed in samples from the fly and mouse models of fragile X as well as in samples derived from human patients. Indeed, we have previously demonstrated that strategies that increase cAMP signaling can rescue short term memory in the fly model and restore DHPG induced mGluR mediated long term depression (LTD) in the hippocampus to proper levels in the mouse model (McBride et al., 2005; Choi et al., 2011, 2015). Here, we demonstrate that the same three strategies used previously with the potential to be used clinically, lithium treatment, PDE-4 inhibitor treatment or mGluR antagonist treatment can rescue long term memory in the fly model and alter the cAMP signaling pathway in the hippocampus of the mouse model. PMID:27445731

  2. Site-specific oxidation at GG and GGG sequences in double-stranded DNA by benzoyl peroxide as a tumor promoter.

    Science.gov (United States)

    Kawanishi, S; Oikawa, S; Murata, M; Tsukitome, H; Saito, I

    1999-12-21

    Benzoyl peroxide (BzPO), a free-radical generator, has tumor-promoting activity. As a method for approaching the mechanism of tumor promoter function, the ability of oxidative DNA damage by BzPO was investigated by using (32)P-labeled DNA fragments obtained from the human p53 tumor suppressor gene and c-Ha-ras-1 protooncogene. BzPO induced piperidine-labile sites at the 5'-site guanine of GG and GGG sequences of double-stranded DNA in the presence of Cu(I), whereas the damage occurred at single guanine residues of single-stranded DNA. Both methional and dimethyl sulfoxide (DMSO) inhibited DNA damage induced by BzPO and Cu(I), but typical hydroxyl radical ((*)OH) scavengers, superoxide dismutase (SOD) and catalase, did not inhibit it. On the other hand, H(2)O(2) induced piperidine-labile sites at cytosine and thymine residues of double-stranded DNA in the presence of Cu(I). Phenylhydrazine, which is known to produce phenyl radicals, induced Cu(I)-dependent damage at thymine residues but not at guanine residues. These results suggest that the BzPO-derived reactive species causing DNA damage is different from (*)OH and phenyl radicals generated from benzoyloxyl radicals. BzPO/Cu(I) induced 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in double-stranded DNA more effectively than that in single-stranded DNA. Furthermore, we observed that BzPO increased the amount of 8-oxodG in human cultured cells. Consequently, it is concluded that benzoyloxyl radicals generated by the reaction of BzPO with Cu(I) may oxidize the 5'-guanine of GG and GGG sequences in double-stranded DNA to lead to 8-oxodG formation and piperidine-labile guanine lesions, and the damage seems to be relevant to the tumor-promoting activity of BzPO.

  3. Reaction mechanism of Ru(II) piano-stool complexes: umbrella sampling QM/MM MD study.

    Science.gov (United States)

    Futera, Zdeněk; Burda, Jaroslav V

    2014-07-15

    Biologically relevant interactions of piano-stool ruthenium(II) complexes with ds-DNA are studied in this article by hybrid quantum mechanics-molecular mechanics (QM/MM) computational technique. The whole reaction mechanism is divided into three phases: (i) hydration of the [Ru(II) (η(6) -benzene)(en)Cl](+) complex, (ii) monoadduct formation between the resulting aqua-Ru(II) complex and N7 position of one of the guanines in the ds-DNA oligomer, and (iii) formation of the intrastrand Ru(II) bridge (cross-link) between two adjacent guanines. Free energy profiles of all the reactions are explored by QM/MM MD umbrella sampling approach where the Ru(II) complex and two guanines represent a quantum core, which is described by density functional theory methods. The combined QM/MM scheme is realized by our own software, which was developed to couple several quantum chemical programs (in this study Gaussian 09) and Amber 11 package. Calculated free energy barriers of the both ruthenium hydration and Ru(II)-N7(G) DNA binding process are in good agreement with experimentally measured rate constants. Then, this method was used to study the possibility of cross-link formation. One feasible pathway leading to Ru(II) guanine-guanine cross-link with synchronous releasing of the benzene ligand is predicted. The cross-linking is an exergonic process with the energy barrier lower than for the monoadduct reaction of Ru(II) complex with ds-DNA. Copyright © 2014 Wiley Periodicals, Inc.

  4. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

    Directory of Open Access Journals (Sweden)

    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  5. Acidity and proton affinity of hypoxanthine in the gas phase versus in solution: intrinsic reactivity and biological implications.

    Science.gov (United States)

    Sun, Xuejun; Lee, Jeehiun K

    2007-08-17

    Hypoxanthine is a mutagenic purine base that most commonly arises from the oxidative deamination of adenine. Damaged bases such as hypoxanthine are associated with carcinogenesis and cell death. This inevitable damage is counteracted by glycosylase enzymes, which cleave damaged bases from DNA. Alkyladenine DNA glycosylase (AAG) is the enzyme responsible for excising hypoxanthine from DNA in humans. In an effort to understand the intrinsic properties of hypoxanthine, we examined the gas-phase acidity and proton affinity using quantum mechanical calculations and gas-phase mass spectrometric experimental methods. In this work, we establish that the most acidic site of hypoxanthine has a gas-phase acidity of 332 +/- 2 kcal mol-1, which is more acidic than hydrochloric acid. We also bracket a less acidic site of hypoxanthine at 368 +/- 3 kcal mol-1. We measure the proton affinity of the most basic site of hypoxanthine to be 222 +/- 3 kcal mol-1. DFT calculations of these values are consistent with the experimental data. We also use calculations to compare the acidic and basic properties of hypoxanthine with those of the normal bases adenine and guanine. We find that the N9-H of hypoxanthine is more acidic than that of adenine and guanine, pointing to a way that AAG could discriminate damaged bases from normal bases. We hypothesize that AAG may cleave certain damaged nucleobases as anions and that the active site may take advantage of a nonpolar environment to favor deprotonated hypoxanthine as a leaving group versus deprotonated adenine or guanine. We also show that an alternate mechanism involving preprotonation of hypoxanthine is energetically less attractive, because the proton affinity of hypoxanthine is less than that of adenine and guanine. Last, we compare the acidity in the gas phase versus that in solution and find that a nonpolar environment enhances the differences in acidity among hypoxanthine, adenine, and guanine.

  6. Protection of riboflavin and UVB sensitized degradation of DNA and RNA bases by natural antioxidants.

    Science.gov (United States)

    Joshi, Prakash C; Gray, Thomas A; Keane, Thomas C

    2012-04-01

    Riboflavin (RF) is a potent photosensitizer producing extensive degradation of purine and pyrimidine derivatives of nucleic acids under UVA, UVB and sunlight. In this study we have demonstrated that reactive O(2) species generated by photosensitized RF under UVB were responsible for the degradation of DNA and RNA bases. While (1)O(2) accounted for the degradation of adenine, guanine, thymine and uracil, O(2)(-·)also contributed to partial degradation of adenine. Cytosine remained unaffected by the synergistic action of RF and UVB. Ascorbic acid, glutathione, glycolic acid and quercetin showed remarkable protection (88-100%) against photodegradation of bases. Sorbitol was effective in preventing photodegradation of guanine. These naturally occurring antioxidants are potential candidates for prevention against oxidative stress caused by photosensitization. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. Predicting the effects of basepair mutations in DNA-protein complexes by thermodynamic integration.

    Science.gov (United States)

    Beierlein, Frank R; Kneale, G Geoff; Clark, Timothy

    2011-09-07

    Thermodynamically rigorous free energy methods in principle allow the exact computation of binding free energies in biological systems. Here, we use thermodynamic integration together with molecular dynamics simulations of a DNA-protein complex to compute relative binding free energies of a series of mutants of a protein-binding DNA operator sequence. A guanine-cytosine basepair that interacts strongly with the DNA-binding protein is mutated into adenine-thymine, cytosine-guanine, and thymine-adenine. It is shown that basepair mutations can be performed using a conservative protocol that gives error estimates of ∼10% of the change in free energy of binding. Despite the high CPU-time requirements, this work opens the exciting opportunity of being able to perform basepair scans to investigate protein-DNA binding specificity in great detail computationally. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Investigating the Fundamentals of Molecular Depth Profiling Using Strong-field Photoionization of Sputtered Neutrals.

    Science.gov (United States)

    Willingham, D; Brenes, D A; Winograd, N; Wucher, A

    2011-01-01

    Molecular depth profiles of model organic thin films were performed using a 40 keV C 60 + cluster ion source in concert with TOF-SIMS. Strong-field photoionization of intact neutral molecules sputtered by 40 keV C 60 + primary ions was used to analyze changes in the chemical environment of the guanine thin films as a function of ion fluence. Direct comparison of the secondary ion and neutral components of the molecular depth profiles yields valuable information about chemical damage accumulation as well as changes in the molecular ionization probability. An analytical protocol based on the erosion dynamics model is developed and evaluated using guanine and trehalose molecular secondary ion signals with and without comparable laser photoionization data.

  9. Light reflection control in biogenic micro-mirror by diamagnetic orientation.

    Science.gov (United States)

    Iwasaka, Masakazu; Mizukawa, Yuri

    2013-04-02

    As has become known, most materials, such as proteins and DNA, show orientation under strong magnetic fields. However, the critical threshold for the magnetic field of the magnetomechanical phenomena is still unknown. We demonstrate that a thin micro-mirror from a fish scale with high reflectivity exhibits a distinct magnetic response at 100 mT. A dramatic event under a magnetic field is the decrease of light scattering from guanine crystals as well as rapid rotation against the applied magnetic field. Enhancement of light scattering intensity is also observed when the three vectors of light incidence, magnetic field, and observation are orthogonally directed. The results indicate that biogenic guanine crystals have a large diamagnetic anisotropy along the surface parallel and normal directions. The micrometer to submicrometer scale of thin biogenic plates can act as a noninvasively, magnetically controlled micro-mirror for light irradiation control in the micrometer-scale region.

  10. Mutation spectrum resulting in M13mp2 phage DNA exposed to N-nitrosoproline with UVA irradiation.

    Science.gov (United States)

    Horai, Yumi; Ando, Yoshiko; Kimura, Sachiko; Arimoto-Kobayashi, Sakae

    2017-09-01

    N-nitrosoproline (NPRO) is endogenously formed from proline and nitrite. In an effort to delineate the mechanism of NPRO-induced photomutagenicity, we investigated the mutagenic spectrum of NPRO on M13mp2 DNA with UVA irradiation. Following exposure to NPRO and UVA, the mutation frequency increased significantly in an NPRO and UVA dose-dependent manner. The sequence data derived from seventy of the mutants indicated that mutagenesis resulted mainly from an increase in single-base substitutions, the most frequent being GC to CG transversions. Non-clustering of the GC to CG mutations suggests that NPRO+UVA damage to DNA is random. These transversions may be caused by guanine adducts in DNA or in part by oxidatively modified guanine in DNA exposed to NPRO and UVA. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Biological (molecular and cellular) markers of toxicity

    International Nuclear Information System (INIS)

    Shugart, L.R.; D'Surney, S.J.; Gettys-Hull, C.; Greeley, M.S. Jr.

    1991-01-01

    Several molecular and cellular markers of genotoxicity were adapted for measurement in the Medaka (Oryzias latipes), and were used to describe the effects of treatment of the organism with diethylnitrosamine (DEN). NO 6 -ethyl guanine adducts were detected, and a slight statistically significant, increase in DNA strand breaks was observed. These results are consistent with the hypothesis that prolonged exposure to high levels of DEN induced alkyltransferase activity which enzymatically removes any O 6 -ethyl guanine adducts but does not result in strand breaks or hypomethylation of the DNA such as might be expected from excision repair of chemically modified DNA. Following a five week continuous DEN exposure with 100 percent renewal of DEN-water every third day, the F values (DNA double strandedness) increased considerably and to similar extent in fish exposed to 25, 50, and 100 ppM DEN. This has been observed also in medaka exposed to BaP

  12. Microfabricated electrochemical sensor for the detection of radiation-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, J.; Rivas, G.; Ozsoz, M.; Grant, D.H.; Cai, X.; Parrado, C. [New Mexico State Univ., Las Cruces, NM (United States)

    1997-04-01

    An electrochemical biosensor protocol for the detection of radiation-induced DNA damage is described. The procedure employs a dsDNA-coated screen-printed electrode and relies on changes in the guanine-DNA oxidation signal upon exposure to ultraviolet radiation. The decreased signal is ascribed primarily to conformational changes in the DNA and to the photoconversion of the guanine-DNA moiety to a nonelectroactive monomeric base product. Factors influencing the response of these microfabricated DNA sensors, such as irradiation time, wavelength, and distance, are explored, and future prospects are discussed. Similar results are given for the use of bare strip electrodes in connection with irradiated DNA solutions. 8 refs., 4 figs.

  13. Ionic liquid-functionalized graphene as modifier for electrochemical and electrocatalytic improvement: comparison of different carbon electrodes.

    Science.gov (United States)

    Du, Meng; Yang, Tao; Ma, Suyan; Zhao, Changzhi; Jiao, Kui

    2011-04-01

    Electrochemical activities of typically electrochemical targets at three kinds of modified carbon electrodes, i.e. carbon ionic liquid electrode (CILE), graphene/carbon paste electrode (CPE), and ionic liquid-functionalized graphene (IL-graphene)/CPE, were compared in detail. The redox processes of the probes at IL-graphene/CPE were faster than those at CILE and graphene/CPE from cyclic voltammetry. An electrochemical method for the simultaneous determination of guanine and adenine was described with detection limits of 6.5×10(-8) mol L(-1) (guanine) and 3.2×10(-8) mol L(-1) (adenine). Single A→G mutation of sequence-specific DNA could be discriminated by the IL-graphene/CPE. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. The image-forming mirror in the eye of the scallop

    Science.gov (United States)

    Palmer, Benjamin A.; Taylor, Gavin J.; Brumfeld, Vlad; Gur, Dvir; Shemesh, Michal; Elad, Nadav; Osherov, Aya; Oron, Dan; Weiner, Steve; Addadi, Lia

    2017-12-01

    Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop’s habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.

  15. Current approaches to improve the anticancer chemotherapy with alkylating agents: state of the problem in world and Ukraine.

    Directory of Open Access Journals (Sweden)

    Iatsyshyna A. P.

    2012-01-01

    Full Text Available Alkylating agents are frequently used in many established anticancer chemotherapies. They alkylate the genomic DNA at various sites. Alkylation of the guanine at the O6-position is cytotoxic, it has the strongest mutagenic potential, as well as can cause the tumor development. Alkyl groups at the O6-position of guanine are removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT. The effectiveness of alkylating chemotherapy is limited by MGMT in cancer cells and adverse toxic side effects in normal cells. Different approaches consisting in the modulation of the MGMT expression and activity are under development now to improve the cancer chemotherapy. They include two main directions, in particular, the increase in chemosensitivity of cancer cells to alkylating drugs and the protection of normal cells from the toxic side effects of chemotherapy. This review is focused on current attempts to improve the alkylating chemotherapy of malignant tumours worldwide and state of the issue in Ukraine

  16. DNA minor groove alkylating agents.

    Science.gov (United States)

    Denny, W A

    2001-04-01

    Recent work on a number of different classes of anticancer agents that alkylate DNA in the minor groove is reviewed. There has been much work with nitrogen mustards, where attachment of the mustard unit to carrier molecules can change the normal patterns of both regio- and sequence-selectivity, from reaction primarily at most guanine N7 sites in the major groove to a few adenine N3 sites at the 3'-end of poly(A/T) sequences in the minor groove. Carrier molecules discussed for mustards are intercalators, polypyrroles, polyimidazoles, bis(benzimidazoles), polybenzamides and anilinoquinolinium salts. In contrast, similar targeting of pyrrolizidine alkylators by a variety of carriers has little effect of their patterns of alkylation (at the 2-amino group of guanine). Recent work on the pyrrolobenzodiazepine and cyclopropaindolone classes of natural product minor groove binders is also reviewed.

  17. Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay.

    Science.gov (United States)

    Sun, Daekyu; Hurley, Laurence H

    2010-01-01

    The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.

  18. Sequences with high propensity to form G-quartet structures in kinetoplast DNA from Phytomonas serpens.

    Science.gov (United States)

    Sá-Carvalho, D; Traub-Cseko, Y M

    1995-06-01

    Naturally occurring sequences containing repetitive guanine motifs have the potential to form tetraplex DNA. Phytomonas serpens minicircle DNA shows some regions where one strand is composed mainly of G and T (GT regions). These regions contain several stretches of contiguous guanines. An oligonucleotide was constructed with the sequence corresponding to one of these regions (Phyto-GT). It was demonstrated by native gel electrophoresis and methylation protection that Phyto-GT forms tetramolecular (G4), bimolecular (G'2) and unimolecular (G4') structures stabilized through G-quartets. Tetraplex DNA formation by this sequence could have biological relevance as it can be formed in physiological conditions and GT regions comprise approximately one-third of P. serpens and Crithidia oncopelti minicircles.

  19. Inverting the G-Tetrad Polarity of a G-Quadruplex by Using Xanthine and 8-Oxoguanine.

    Science.gov (United States)

    Cheong, Vee Vee; Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân

    2016-01-04

    G-quadruplexes are four-stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G-tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8-oxoguanine (O), within a single G-tetrad of a G-quadruplex was recently shown to lead to the formation of a stable G⋅G⋅X⋅O tetrad. Herein, a judicious introduction of X and O into a human telomeric G-quadruplex-forming sequence is shown to reverse the hydrogen-bond polarity of the modified G-tetrad while preserving the original folding topology. The control exerted over G-tetrad polarity by joint X⋅O modification will be valuable for the design and programming of G-quadruplex structures and their properties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Biological (molecular and cellular) markers of toxicity. Final report, September 15, 1988 - September 14, 1991

    Energy Technology Data Exchange (ETDEWEB)

    Shugart, L. R.; D' Surney, S. J.; Gettys-Hull, C.; Greeley, Jr, M. S.

    1991-12-15

    Several molecular and cellular markers of genotoxicity were adapted for measurement in the Medaka (Oryzias latipes), and were used to describe the effects of treatment of the organism with diethylnitrosamine (DEN). NO{sup 6}-ethyl guanine adducts were detected, and a slight statistically significant, increase in DNA strand breaks was observed. These results are consistent with the hypothesis that prolonged exposure to high levels of DEN induced alkyltransferase activity which enzymatically removes any O{sup 6}-ethyl guanine adducts but does not result in strand breaks or hypomethylation of the DNA such as might be expected from excision repair of chemically modified DNA. Following a five week continuous DEN exposure with 100 percent renewal of DEN-water every third day, the F values (DNA double strandedness) increased considerably and to similar extent in fish exposed to 25, 50, and 100 ppM DEN. This has been observed also in medaka exposed to BaP.

  1. Kennedy's disease and partial androgen insensitivity syndrome. Report of 4 cases and literature review.

    Science.gov (United States)

    Valera Yepes, Rocío; Virgili Casas, Maria; Povedano Panades, Monica; Guerrero Gual, Mireia; Villabona Artero, Carles

    2015-05-01

    Kennedy's disease, also known as bulbospinal muscular atrophy, is a rare, X-linked recessive neurodegenerative disorder affecting adult males. It is caused by expansion of an unstable cytosine-adenine-guanine tandem-repeat in exon 1 of the androgen-receptor gene on chromosome Xq11-12, and is characterized by spinal motor neuron progressive degeneration. Endocrinologically, these patients often have the features of hypogonadism associated to the androgen insensitivity syndrome, particularly its partial forms. We report 4 cases with the typical neurological presentation, consisting of slowly progressing generalized muscle weakness with atrophy and bulbar muscle involvement; these patients also had several endocrine manifestations; the most common non-neurological manifestation was gynecomastia. In all cases reported, molecular analysis showed an abnormal cytosine-adenine-guanine triplet repeat expansion in the androgen receptor gene. Copyright © 2014 SEEN. Published by Elsevier España, S.L.U. All rights reserved.

  2. Repair of DNA treated with γ-irradiation and chemical carcinogens. Comprehensive report of entire period of ERDA support from June 1, 1975--January 15, 1978

    International Nuclear Information System (INIS)

    Goldthwait, D.A.

    1978-01-01

    A partially purified enzyme fraction isolated from E. coli showed an N-glycosidase activity as well as a phosphodiesterase activity on DNA treated with methylnitrosourea, and with 7-bromomethylbenz(a)anthracene and a phosphodiesterase activity against γ-irradiated DNA. Both 0-6 methyl guanine and 3-methyladenine were released from DNA treated with MNU; the adenine and guanine derivatives from the DNA treated with 7-bromomethyl-12-methylbenz(a)anthracene were also liberated. Progress is also reported on studies on Endonucleases II and VI and Exonuclease III of E. coli; methods for assay and for synthesis of substrates; attempts at purification of repair enzymes from mammalian tissues; and β-propiolactone reactions with deoxynucleosides and with DNA

  3. Factors that influence telomeric oxidative base damage and repair by DNA glycosylase OGG1

    DEFF Research Database (Denmark)

    Rhee, David B; Ghosh, Avik; Lu, Jian

    2011-01-01

    Telomeres are nucleoprotein complexes at the ends of linear chromosomes in eukaryotes, and are essential in preventing chromosome termini from being recognized as broken DNA ends. Telomere shortening has been linked to cellular senescence and human aging, with oxidative stress as a major...... contributing factor. 7,8-Dihydro-8-oxogaunine (8-oxodG) is one of the most abundant oxidative guanine lesions, and 8-oxoguanine DNA glycosylase (OGG1) is involved in its removal. In this study, we examined if telomeric DNA is particularly susceptible to oxidative base damage and if telomere-specific factors...... affect the incision of oxidized guanines by OGG1. We demonstrated that telomeric TTAGGG repeats were more prone to oxidative base damage and repaired less efficiently than non-telomeric TG repeats in vivo. We also showed that the 8-oxodG-incision activity of OGG1 is similar in telomeric and non...

  4. Thesaurus of DDC Descriptors

    Science.gov (United States)

    1966-06-01

    NT DISSOCIATION ELECTROLYSIS HYDROLYSIS PHOTOLYSIS PYROLYSIS DECAY SCHEMES 18 08 (TRANSFORMATION SCHEMES BY WHICH RADIO- ACTIVE DECAY OF A...06 01 UF FRUCTOSE FRUCTOSE DERIVATIVES BT CARdOHYDRATES >HEXüSES FRUITS U6 06 UF APRICOTS BAMAWAS CITRUS FRUITS TOMATOES bT FOOD FUEL...PYROGENS 06 IS PUklNES 06 01 UF AMINOPURINES NT «OEHINt GUANINE PYROLYSIS 07 OH bT CHEMICAL REACTIONS DECOMPOSITION PURSUIT COURSEj 01

  5. Are radical cation states delocalized over GG and GGG hole traps in DNA?

    Science.gov (United States)

    Voityuk, Alexander A

    2005-06-02

    Recently, Kurnikov et al. (J. Phys. Chem. B 2002, 106, 7) have shown that solvation of DNA duplexes destabilizes holes of sizes larger than three base pairs. In this paper, we consider the effects of solvation and internal reorganization on the hole charge distribution in sequences 5'-X-GG-Y-3'. Radical cation states in DNA are found to be localized to a single guanine site independent of the nature of adjacent base pairs.

  6. GNG Motifs Can Replace a GGG Stretch during G-Quadruplex Formation in a Context Dependent Manner.

    Science.gov (United States)

    Das, Kohal; Srivastava, Mrinal; Raghavan, Sathees C

    2016-01-01

    G-quadruplexes are one of the most commonly studied non-B DNA structures. Generally, these structures are formed using a minimum of 4, three guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In this study, guanines along with bulges, also referred to as GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the contribution from continuous G-tracts may be dispensible during G-quadruplex formation when such motifs are flanked by GNGs. Importantly, the positioning and number of GNG/GNGNG can also influence the formation of G-quadruplexes. Further, we assessed three genomic regions from HIF1 alpha, VEGF and SHOX gene for G-quadruplex formation using GNG motifs. We show that HIF1 alpha sequence harbouring GNG motifs can fold into intramolecular G-quadruplex. In contrast, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two continuous stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX), it can fold into an intramolecular G-quadruplex. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody, BG4 to SHOX G-quadruplex. Overall, our study provides novel insights into the role of GNG motifs in G-quadruplex structure formation which may have both physiological and pathological implications.

  7. GNG Motifs Can Replace a GGG Stretch during G-Quadruplex Formation in a Context Dependent Manner.

    Directory of Open Access Journals (Sweden)

    Kohal Das

    Full Text Available G-quadruplexes are one of the most commonly studied non-B DNA structures. Generally, these structures are formed using a minimum of 4, three guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In this study, guanines along with bulges, also referred to as GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the contribution from continuous G-tracts may be dispensible during G-quadruplex formation when such motifs are flanked by GNGs. Importantly, the positioning and number of GNG/GNGNG can also influence the formation of G-quadruplexes. Further, we assessed three genomic regions from HIF1 alpha, VEGF and SHOX gene for G-quadruplex formation using GNG motifs. We show that HIF1 alpha sequence harbouring GNG motifs can fold into intramolecular G-quadruplex. In contrast, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two continuous stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX, it can fold into an intramolecular G-quadruplex. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody, BG4 to SHOX G-quadruplex. Overall, our study provides novel insights into the role of GNG motifs in G-quadruplex structure formation which may have both physiological and pathological implications.

  8. Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release

    DEFF Research Database (Denmark)

    Leonardsen, L; DeClue, J E; Lybaek, H

    1996-01-01

    The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino ac...... and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive....

  9. Human N6-Methyl-AMP/dAMP aminohydrolase (abacavir 5’-monophosphate deaminase) is capable of metabolizing N6-substituted purine acyclic nucleoside phosphonates

    Czech Academy of Sciences Publication Activity Database

    Schinkmanová, Markéta; Votruba, Ivan; Shibata, R.; Han, B.; Liu, X.; Cihlař, T.; Holý, Antonín

    2008-01-01

    Roč. 73, č. 2 (2008), s. 275-291 ISSN 0010-0765 R&D Projects: GA MŠk 1M0508; GA AV ČR 1QS400550501 Institutional research plan: CEZ:AV0Z40550506 Keywords : guanine * acyclic nucleoside phosphonates * cPrPMEDAP * abacavir 5'-phosphate Subject RIV: CC - Organic Chemistry Impact factor: 0.784, year: 2008

  10. 1,1’,1’’,1’’’-[Porphyrin-5,10,15,20-tetrayltetrakis(3,1-phenylenemethylene]tetraquinolinium Tetrabromide

    Directory of Open Access Journals (Sweden)

    Satoshi Fujii

    2010-11-01

    Full Text Available Cationic porphyrins interact strongly with guanine quadruplex DNA (G-quadruplex. We herein report the preparation of a cationic porphyrin bearing quinolinium side arms, 1,1’,1’’,1’’’-[porphyrin-5,10,15,20-tetrayltetrakis(3,1-phenylenemethylene]­tetra­quinolinium tetrabromide (mQu, as a potential G-quadruplex ligand.

  11. Guanine–aspartic acid interactions probed with IR–UV resonance Spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Crews, B. O.; Abo-Riziq, A.; Pluháčková, Kristýna; Thompson, P.; Hill, G.; Hobza, Pavel; de Vries, M. S.

    2010-01-01

    Roč. 12, č. 14 (2010), s. 3597-3605 ISSN 1463-9076 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550510 Grant - others:NSF(US) CHE-0911564 Institutional research plan: CEZ:AV0Z40550506 Keywords : double resonance spectroscopy * correlated ab initio calculations * guanine-aspartic acid complexes Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.454, year: 2010

  12. Das DNA-Puzzle

    Science.gov (United States)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  13. Ontogenetic changes in contribution of calcium sensitization and calcium entry to blood pressure maintenance of Wistar–Kyoto and spontaneously hypertensive rats

    Czech Academy of Sciences Publication Activity Database

    Behuliak, Michal; Vavřínová, Anna; Bencze, Michal; Polgárová, Kamila; Ergang, Peter; Kuneš, Jaroslav; Vaněčková, Ivana; Zicha, Josef

    2015-01-01

    Roč. 33, č. 12 (2015), s. 2443-2454 ISSN 0263-6352 R&D Projects: GA ČR(CZ) GAP304/12/0259; GA ČR(CZ) GP14-16225P Institutional support: RVO:67985823 Keywords : Ca2+ influx * Ca2+ sensitization * CPI-17 * fasudil * isolated arteries * nifedipine * Rho guanine nucleotide exchange factors * RhoA/Rho kinase Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 5.062, year: 2015

  14. Structure of 2,4-Diaminopyrimidine-Theobromine Alternate Base Pairs

    Czech Academy of Sciences Publication Activity Database

    Gengeliczki, Z.; Callahan, M. P.; Kabeláč, Martin; Rijs, A. M.; de Vries, M. S.

    2011-01-01

    Roč. 115, č. 41 (2011), s. 11423-11427 ISSN 1089-5639 R&D Projects: GA MŠk LC512; GA AV ČR IAA400550808 Institutional research plan: CEZ:AV0Z40550506 Keywords : ab-initio calculations * double resonance spectroscopy * basis-set * guanine * cytosine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.946, year: 2011

  15. Electrochemical monitoring of the interaction between mitomycin C and DNA at chitosan--carbon nanotube composite modified electrodes

    OpenAIRE

    CANAVAR, Pembe Ece; EKŞİN, Ece; ERDEM, Arzum

    2015-01-01

    Single-walled carbon nanotube (CNT) and chitosan composite (chitosan*CNT) based sensors were developed as DNA biosensors, and then they were applied for electrochemical investigation of the interaction between the anticancer drug mitomycin C (MC) and DNA. The oxidation signals of MC and guanine were monitored before and after the interaction process by differential pulse voltammetry (DPV). The DPV results were in good agreement with those of electrochemical impedance spectroscopy (EIS)....

  16. Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis

    OpenAIRE

    Sameni, Sara; Malacrida, Leonel; Tan, Zhiqun; Digman, Michelle A.

    2018-01-01

    Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here...

  17. Synthesis of 9,9′-[1,2-Ethanediylbis(oxymethylene]bis-2-amino-1,9-dihydro-6H-purin-6-one, an Impurity of Acyclovir

    Directory of Open Access Journals (Sweden)

    Juan José Vaquero

    2012-07-01

    Full Text Available The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene]bis-2-amino-1,9-dihydro-6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-ylacetamide, the process uses an acid catalysed phase transfer catalysis (PTC process to produce the selective alkylation at the 9 position of the guanine ring.

  18. Mammalian phospholipase D: activation by ammonium sulfate and nucleotides.

    OpenAIRE

    Nakamura, S; Shimooku, K; Akisue, T; Jinnai, H; Hitomi, T; Kiyohara, Y; Ogino, C; Yoshida, K; Nishizuka, Y

    1995-01-01

    Phospholipase D (PLD) associated with the rat kidney membrane was activated by guanine 5'-[gamma-thio]triphosphate and a cytosol fraction that contained ADP-ribosylation factor. When assayed by measuring the phosphatidyl transfer reaction to ethanol with exogenously added radioactive phosphatidylcholine as substrate, the PLD required a high concentration (1.6 M) of ammonium sulfate to exhibit high enzymatic activity. Other salts examined were far less effective or practically inactive, and th...

  19. Alternate Splicing of CD44 Messenger RNA in Prostate Cancer Growth

    Science.gov (United States)

    2009-10-01

    University of Colorado Health Science Center); to the physical necessities of the move; and to the need to recruit by advertising ; and to administrative...Chien J, Wong E, Nikes E, Noble MJ, Pantazis CG, Shah GV. Constitutive activation of stimulatory guanine nucleotide binding protein (Gsα-QL)-medicated...for cyclic AMP- dependent protein kinase A in calcitonin action. Int J Cancer 2005, 117:551-560. 17. Chien J, Wong E, Nikes E, Noble MJ, Pantazis CG

  20. Adenosine deaminase organic effect in normal and abnormal cerebrospinal fluid

    International Nuclear Information System (INIS)

    Hamad, A.M.; Samarai, M.A.

    2007-01-01

    To study the effect of the organic substances on adenosine deaminase (ADA) activity in normal and abnormal cerebrospinal fluid (CSF). Various concentrations of 2-mercaptopurine, Ame-tycine, Adenosine analogues (Guanine, Thymine) and ATP were tested to see their effect on ADA activity in normal and abnormal CSF. ADA activity in normal and abnormal CSF was remarkably decreased with the increasing of concentrations of substances tested. These effects may have important therapeutic implications. (author)

  1. Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

    OpenAIRE

    Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A.; Nakamura, Jun

    2016-01-01

    Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that ...

  2. The development of in vitro mutagenicity testing systems using T-lymphocytes. Research progress report, November 1, 1989--April 30, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Albertini, R.J.

    1992-05-01

    This work has focused on the development of in vitro T-cell mutation assays. Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes. This assay is a parallel to our in vivo hprt assay, in that the same cells are utilized. However, the in vitro assay allows for carefully controlled dose response studies. 21 refs., 16 figs., 13 tabs.

  3. The development of in vitro mutagenicity testing systems using T-lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Albertini, R.J.

    1992-05-01

    This work has focused on the development of in vitro T-cell mutation assays. Conditions have been defined to measure the in vitro induction of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in human T-lymphocytes. This assay is a parallel to our in vivo hprt assay, in that the same cells are utilized. However, the in vitro assay allows for carefully controlled dose response studies. 21 refs., 16 figs., 13 tabs.

  4. Production of Hemolysin and Bacteriolysin in a Synthetic Medium by Streptococcus faecalis var. zymogenes.

    Science.gov (United States)

    Appelbaum, B; Zimmerman, L N

    1974-11-01

    A synthetic medium has been developed in which Streptococcus faecalis var. zymogenes X-14 elaborates a lysin. The medium consists of 18 amino acids, lactose, six vitamins, adenine, guanine, uracil, and six salts. Glucose, and K(2)HPO(4) in excess of 0.5% (wt/vol), were inhibitory to lysin production; increasing concentrations of l-arginine-hydrochloride gave increasing yields of lytic activity.

  5. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    2012-06-25

    Jun 25, 2012 ... 2008). In the LC-MS/MS study performed in both aerobic and anaerobic conditions, it has been further found that due to radical formation at the C5 site of cytosine, it can be covalently bonded to the C8 position of guanine of same strand to form G[C8-C5]C intra-strand crosslink product. (scheme 6d) (Box et ...

  6. Revaluation of biomass-derived furfuryl alcohol derivatives for the synthesis of carbocyclic nucleoside phosphonate analogues

    OpenAIRE

    Sidi Mohamed, Bemba; P?rigaud, Christian; Math?, Christophe

    2017-01-01

    The racemic synthesis of new carbocyclic nucleoside methylphosphonate analogues bearing purine bases (adenine and guanine) was accomplished using bio-sourced furfuryl alcohol derivatives. All compounds were prepared using a Mitsunobu coupling between the heterocyclic base and an appropriate carbocyclic precursor. After deprotection, the compounds were evaluated for their activity against a large number of viruses. However, none of them showed significant antiviral activity or cytotoxicity.

  7. Chemical Carcinogen-Induced Changes in tRNA Metabolism in Human Cells.

    Science.gov (United States)

    1983-11-30

    guanine as substrates and without the xanthine oxidase inhibitor allopurinol (5,8). DEAE-Cellulose Column Chromatography--Conditions utilized were similar...previously undocumented hypoxanthine incorporating activity has been assayed in rat liver and human leukemia cell cytosolic preparations, and it was resolved...the POD treated cells maintained levels 3 to 4-fold higher for most of the lifetime of the cultures. The specificity of the effect for an active tumor

  8. Diverse effects of naturally occurring base lesions on the structure and stability of the human telomere DNA quadruplex

    Czech Academy of Sciences Publication Activity Database

    Konvalinová, Helena; Dvořáková, Zuzana; Renčiuk, Daniel; Bednářová, Klára; Kejnovská, Iva; Trantírek, L.; Vorlíčková, Michaela; Sagi, J.

    2015-01-01

    Roč. 118, NOV 2015 (2015), s. 15-25 ISSN 0300-9084 R&D Projects: GA ČR(CZ) GAP205/12/0466; GA ČR(CZ) GA13-28310S; GA ČR(CZ) GA15-06785S Institutional support: RVO:68081707 Keywords : DNA secondary structure * Circular dichroism spectroscopy * Guanine quadruplex Subject RIV: BO - Biophysics Impact factor: 3.017, year: 2015

  9. G-quadruplex formation in the Oct4 promoter positively regulates Oct4 expression

    Czech Academy of Sciences Publication Activity Database

    Renčiuk, Daniel; Ryneš, J.; Kejnovská, Iva; Foldynova-Trantirkova, S.; Andaeng, M.; Trantírek, L.; Vorlíčková, Michaela

    2017-01-01

    Roč. 1860, č. 2 (2017), s. 175-183 ISSN 1874-9399 R&D Projects: GA ČR(CZ) GP14-33947P; GA ČR GAP205/12/0466; GA ČR(CZ) GA15-06785S Institutional support: RVO:68081707 Keywords : linked polymorphic region * guanine quadruplexes * transcription factor Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 5.018, year: 2016

  10. Benchmark RI-MP2 database of nucleic acid base trimers: performance of different density functional models for prediction of structures and binding energies

    Czech Academy of Sciences Publication Activity Database

    Kabeláč, Martin; Valdes, Haydee; Sherer, E. C.; Cramer, Ch. J.; Hobza, Pavel

    2007-01-01

    Roč. 9, č. 36 (2007), s. 5000-5008 ISSN 1463-9076 R&D Projects: GA MŠk LC512; GA ČR GA203/05/0009 Grant - others:NSF(US) CHE-0615401 Institutional research plan: CEZ:AV0Z40550506 Keywords : IR spectrum * guanine * deoxyguanosine Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.343, year: 2007

  11. Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A, G:G:A:A, and A:A:A:A tetrads

    Czech Academy of Sciences Publication Activity Database

    Sagi, J.; Renčiuk, Daniel; Tomaško, Martin; Vorlíčková, Michaela

    2010-01-01

    Roč. 93, č. 10 (2010), s. 880-886 ISSN 0006-3525 R&D Projects: GA AV ČR(CZ) IAA100040701; GA AV ČR(CZ) IAA500040903 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : human telomere quadruplex * adenine for guanine substitution * CD spectroscopy Subject RIV: BO - Biophysics Impact factor: 2.572, year: 2010

  12. Intraethnic variation in steroid-5-alpha-reductase polymorphisms in ...

    Indian Academy of Sciences (India)

    C/G. 2p13. 31805706. SRD5A2. C−2486459_10 rs9332975. C/T. 2p13. 31750417. SRD5A2. C−11160708_10 rs7594951. C/T. 2p13. 31791793. Chr., chromosome; C, cytosine; T, thymine; A, adenine; G, guanine. All the assays are commercially available at Applied Biosystems, except *Custom assays. Journal of Genetics ...

  13. Chemical Feasibility of the General Acid/Base Mechanism of glmS Ribozyme Self-Cleavage

    Czech Academy of Sciences Publication Activity Database

    Dubecký, M.; Walter, Nils G.; Šponer, Jiří; Otyepka, M.; Banáš, Pavel

    2015-01-01

    Roč. 103, č. 10 (2015), s. 550-562 ISSN 0006-3525 R&D Projects: GA MŠk(CZ) ED1.1.00/02.0068; GA ČR(CZ) GAP208/12/1878 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * ACTIVE-SITE GUANINE * HAIRPIN RIBOZYME Subject RIV: BO - Biophysics Impact factor: 2.248, year: 2015

  14. Thermal Inactivation of Bacillus Anthracis using Laser Irradiation of Micro-Etched Platforms

    Science.gov (United States)

    2009-03-01

    Al-Alami & Kiefer , 1983). Note that the average bond enthalpy of a C-C bond is 348 kJ/mol and that these reactions generate radicals, (ie: methyl...phosphate backbone of the DNA molecule ( Barbara Setlow, 1995). Guanine sites are slightly more reactive than adenine. Depurination occurs...Tool (SERPENT). SERPENT Overview Presentation . 4. Al-Alami, M. Z., & Kiefer , J. H. (1983). Shock-tube study of propane pyrolysis. Rate of

  15. Synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, an impurity of acyclovir.

    Science.gov (United States)

    Suárez, Rosa M; Matía, Maria Paz; Novella, José Luis; Molina, Andres; Cosme, Antonio; Vaquero, Juan José; Alvarez-Builla, Julio

    2012-07-25

    The synthesis of 9,9'-[1,2-ethanediylbis(oxymethylene)]bis-2-amino-1,9-dihydro-6H-purin-6-one, a minor impurity of acyclovir, is described. Starting with commercial N-(9-acetyl-6-oxo-1H-purin-2-yl)acetamide, the process uses an acid catalysed phase transfer catalysis (PTC) process to produce the selective alkylation at the 9 position of the guanine ring.

  16. Copper complexes as chemical nucleases

    Indian Academy of Sciences (India)

    Unknown

    hydrogen are as well expected to oxidize guanine or other nucleobases. Current efforts are on to ..... recording the absorption intensity at 260 nm with a molar extinction coefficient value. 6600 dm3 mol–1 cm–1 37. ... (H2A, 100 µM) followed by dilution with the Tris-HCl buffer to a total volume of 20 µL. The samples were ...

  17. Heat Shock-Induced Accumulation of Translation Elongation and Termination Factors Precedes Assembly of Stress Granules in S. cerevisiae

    Czech Academy of Sciences Publication Activity Database

    Groušl, Tomáš; Malcová, Ivana; Pompach, Petr; Frýdlová, Ivana; Slabá, Renata; Senohrábková, Lenka; Nováková, Lenka; Hašek, Jiří

    2013-01-01

    Roč. 8, č. 2 (2013), e57083 E-ISSN 1932-6203 R&D Projects: GA ČR GA204/09/1924; GA ČR GAP305/12/0480; GA ČR GPP305/10/P253 Institutional support: RVO:61388971 Keywords : CYTOPLASMIC PROCESSING BODIES * GUANINE-NUCLEOTIDE EXCHANGE * EUKARYOTIC MESSENGER-RNAS Subject RIV: EE - Microbiology, Virology Impact factor: 3.534, year: 2013

  18. Conformation of amine-modified DNA: 2-aminofluorene- and 2-(acetylamino)fluorene-modified deoxydinucleoside monophosphates with all possible nearest neighbors. A comparison of search and optimization methods.

    Science.gov (United States)

    Shapiro, R; Sidawi, D; Miao, Y S; Hingerty, B E; Schmidt, K E; Moskowitz, J; Broyde, S

    1994-01-01

    Although a significant part of the replication fork exists as single-stranded DNA, little is known about the effect of carcinogens and mutagens on single-strand conformation. Large-scale conformational searches with potential energy minimization, using the torsion angle space molecular mechanics program DUPLEX, were employed to explore the conformation of all 16 deoxydinucleoside monophosphates bearing 2-aminofluorene (AF) or 2-(acetylamino)fluorene (AAF) modification on guanine. We have thus examined the effect of 3' versus 5' modification, the presence or absence of the acetyl group, and the effect of four different neighbors in each case. The principal effect of the acetyl group appeared to be the destabilization of anti (and, to a lesser degree, borderline anti) conformations for modified guanine. This mattered little in the 5'-substituted dimers, where one conformational type predominated in the low-energy structures for the adducts of both AAF and AF: It was right-handed, with syn-guanine, imperfect base-base stacking, and fluorene to 3'-sugar contacts. Greater divergence was seen in the 3'-substituted series. The AAF-substituted 3'-adducts primarily displayed good base-fluorene stacking, with syn-guanine in contact with the 5'-sugar. The AF-substituted 3'-adducts displayed a variety of structures which included base-base and carcinogen-base stacked forms. Two novel forms were encountered [global minima for d(ApG-AF) and d(GpG-AF)], whose unusual structures suggest mutagenic capability. In order to address the multiple minimum problem, we conducted our searches of conformation space using two alternative optimization methods that also employ differing search strategies. We used the Powell algorithm, BOTM, with starting conformations that are selected combinations of rotamers, and the method of simulated annealing (SA), with random or arbitrary starting conformations. While both approaches were effective in defining the most important structures, SA was more

  19. Étude structurale de l'assemblage du complexe télomérique humain TRF2/RAP1

    OpenAIRE

    Gaullier , Guillaume

    2015-01-01

    Telomeres are the ends of eukaryotic linear chromosomes. They are made oftandem repeats of a short guanine-rich motif and bound by specific proteins.In vertebrates, these proteins form a complex called shelterin, theintegrity of which is critical to ensure proper replication of chromosomeends and to protect them against illicit targeting by DNA double-strandbreak repair pathways. Telomere dysfunctions lead to genome instability,which can ultimately cause senescence or cancer. Telomeres are a ...

  20. Synthesis of purine N9-[2-hydroxy-3-O-(phosphonomethoxy)propyl] derivatives and their side-chain modified analogues as potential antimalarial agents

    Czech Academy of Sciences Publication Activity Database

    Krečmerová, Marcela; Dračínský, Martin; Hocková, Dana; Holý, Antonín; Keough, D. T.; Guddat, L. W.

    2012-01-01

    Roč. 20, č. 3 (2012), s. 1222-1230 ISSN 0968-0896 R&D Projects: GA MŠk 1M0508; GA ČR GAP207/11/0108 Institutional research plan: CEZ:AV0Z40550506 Keywords : acyclic nucleoside phosphonates * Malaria * Plasmodium falciparum * Plasmodium vivax * hypoxanthine-guanine-(xanthine)-phosphoribosyltransferase Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

  1. Extent and patterns of MGMT promoter methylation in glioblastoma- and respective glioblastoma-derived spheres

    OpenAIRE

    Sciuscio D.; Diserens A. C.; van Dommelen K.; Martinet D.; Jones G.; Janzer R. C.; Pollo C.; Hamou M. F.; Kaina B.; Stupp R.; Levivier M.; Hegi M. E.

    2010-01-01

    PURPOSE: Quantitative methylation specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6 methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6 methyl guanine and has been shown ...

  2. Novel nucleotide analogues bearing (1H-1,2,3-triazol-4-yl)phosphonic acid moiety as inhibitors of Plasmodium and human 6-oxopurine phosphoribosyltransferases

    Czech Academy of Sciences Publication Activity Database

    Lukáč, Miloš; Hocková, Dana; Keough, D. T.; Guddat, L. W.; Janeba, Zlatko

    2017-01-01

    Roč. 73, č. 6 (2017), s. 692-702 ISSN 0040-4020 R&D Projects: GA ČR(CZ) GA16-06049S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * 6-oxopurine * hypoxanthine-guanine-(xanthine) phosphoribosyltransferase * copper(I)-catalyzed azide-alkyne cycloaddition Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 2.651, year: 2016

  3. Label-free detection of canonical DNA bases, uracil and 5-methylcytosine in DNA oligonucleotides using linear sweep voltammetry at a pyrolytic graphite electrode

    Czech Academy of Sciences Publication Activity Database

    Špaček, Jan; Daňhel, Aleš; Hasoň, Stanislav; Fojta, Miroslav

    2017-01-01

    Roč. 82, SEP2017 (2017), s. 34-38 ISSN 1388-2481 R&D Projects: GA ČR(CZ) GA16-01625S; GA MŠk EF15_003/0000477 Institutional support: RVO:68081707 Keywords : electrochemical oxidation * nucleic- acids * guanine * reduction Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.396, year: 2016

  4. Interactions of hydrated divalent metal cations with nucleic acid bases. How to relate the gas phase data to solution situation and binding selectivity in nucleic acids

    Czech Academy of Sciences Publication Activity Database

    Šponer, Judit E.; Sychrovský, Vladimír; Hobza, Pavel; Šponer, Jiří

    2004-01-01

    Roč. 6, č. 10 (2004), s. 2772-2780 ISSN 1463-9076 R&D Projects: GA MŠk LN00A016; GA MŠk LN00A032 Grant - others:Wellcome Trust(GB) GR067507MF Institutional research plan: CEZ:AV0Z5004920 Keywords : nucleic acids * gas phase * guanine Subject RIV: BO - Biophysics Impact factor: 2.076, year: 2004

  5. Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

    Science.gov (United States)

    Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

    2015-11-19

    Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

  6. Hippocampal Gαq/11 but not Gαo-coupled receptors are altered in aging

    Science.gov (United States)

    McQuail, Joseph A.; Davis, Kathleen N.; Miller, Frances; Hampson, Robert E.; Deadwyler, Samuel A.; Howlett, Allyn C.; Nicolle, Michelle M.

    2013-01-01

    Normal aging may limit the signaling efficacy of certain GPCRs by disturbing the function of specific Gα-subunits and leading to deficient modulation of intracellular functions that subserve synaptic plasticity, learning and memory. Evidence suggests that Gαq/11 is more sensitive to the effects of aging relative to other Gα-subunits, including Gαo. To test this hypothesis, the functionality of Gαq/11 and Gαo were compared in the hippocampus of young (6 months) and aged (24 months) F344×BNF1 hybrid rats assessed for spatial learning ability. Basal GTPγS-binding to Gαq/11 was significantly elevated in aged rats relative to young and but not reliably associated with spatial learning. mAChR stimulation of Gαq/11 with oxotremorine-M produced equivocal GTPγS-binding between age groups although values tended to be lower in the aged hippocampus and were inversely related to basal activity. Downstream Gαq/11 function was measured in hippocampal subregion CA1 by determining changes in [Ca2+]i after mAChR and mGluR (DHPG) stimulation. mAChR-stimulated peak change in [Ca2+]i was lower in aged CA1 relative to young while mGluR-mediated integrated [Ca2+]i responses tended to be larger in aged. GPCR modulation of [Ca2+]i was observed to depend on intracellular stores to a greater degree in aged than young. In contrast, measures of Gαo-mediated GTPγS-binding were stable across age, including basal, mAChR-, GABABR (baclofen)-stimulated levels. Overall, the data indicate that aging selectively modulates the activity of Gαq/11 within the hippocampus leading to deficient modulation of [Ca2+]i following stimulation of mAChRs but these changes are not related to spatial learning. PMID:23347951

  7. Neuron-astrocyte signaling is preserved in the aging brain.

    Science.gov (United States)

    Gómez-Gonzalo, Marta; Martin-Fernandez, Mario; Martínez-Murillo, Ricardo; Mederos, Sara; Hernández-Vivanco, Alicia; Jamison, Stephanie; Fernandez, Ana P; Serrano, Julia; Calero, Pilar; Futch, Hunter S; Corpas, Rubén; Sanfeliu, Coral; Perea, Gertrudis; Araque, Alfonso

    2017-04-01

    Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca 2+ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte-neuron signaling is derived from studies with young animals; however, the features of astrocyte-neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte-neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month-old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter-dependent intracellular Ca 2+ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG-induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte-neuron signaling in pathological conditions. Disruption of the astrocytic IP 3 R2 mediated-signaling, which is required for neurotransmitter-induced astrocyte Ca 2+ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte-neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca 2+ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569-580. © 2017 Wiley

  8. Proton- and ammonium- sensing by histaminergic neurons controlling wakefulness.

    Directory of Open Access Journals (Sweden)

    Yvgenij eYanovsky

    2012-04-01

    Full Text Available Orexinergic and histaminergic neurons in the posterior hypothalamus are involved in the control of arousal. Extracellular levels of acid /CO2 are fundamental physicochemical signals controlling wakefulness and breathing. Acidification excites orexinergic neurons like the chemosensory neurons in the brain stem. Hypercapnia induces c-Fos expression, a marker for increased neuronal activity, in the rat histaminergic tuberomamillary nucleus (TMN, but the mechanisms of this excitation are unknown. Acid-sensing ion channels (ASICs are gated by protons and also by ammonium. Recordings in rat brain slices revealed now that acidification within the physiological range (pH from 7.3 to 7.0 as well as ammonium chloride (5mM excite histaminergic neurons. We detected variable combinations of 4 known types of ASICs in single TMN neurons, along with the pharmacological properties of pH-induced current. At pH 7.0 however, activation of ASICs in TMN neurons was negligible. Block of type I metabotropic glutamate receptors abolished proton- but not ammonium- induced excitation. Mouse TMN neurons were identified within a novel HDC-Cre transgenic reporter mouse line. In contrast to the rat these lacked pH 7.0-induced excitation and showed only a minimal response to the mGluR I agonist DHPG (0.5µM. Ammonium-induced excitation was similar in mouse and rat. Thus glutamate, which is released by glial cells and orexinergic axons amplifies CO2/acid-induced arousal through the recruitment of the histaminergic system in rat but not in mouse. These results are relevant for the understanding of neuronal mechanisms controlling H+/CO2-induced arousal in hepatic encephalopathy and obstructive sleep apnoea. The new HDC-Cre mouse model will be a useful tool for studying the physiological and pathophysiological roles of the histaminergic system.

  9. Coupling of an indicator-free electrochemical DNA biosensor with polymerase chain reaction for the detection of DNA sequences related to the apolipoprotein E

    Energy Technology Data Exchange (ETDEWEB)

    Lucarelli, Fausto; Marrazza, Giovanna; Palchetti, Ilaria; Cesaretti, S.; Mascini, Marco

    2002-09-26

    This paper describes a disposable indicator-free electrochemical DNA biosensor applied to the detection of apolipoprotein E (apoE) sequences in PCR samples. In the indicator-free assays, the duplex formation was detected by measuring the electrochemical signal of the guanine base of nucleic acids. The biosensor format involved the immobilisation of an inosine-modified (guanine-free) probe onto a screen-printed electrode (SPE) transducer and the detection of the duplex formation in connection with the square-wave voltammetric measurement of the oxidation peak of the guanine of the target sequence. The indicator-free scheme has been characterised using 23-mer oligonucleotides as model: parameters affecting the hybridisation assay such as probe immobilisation conditions, hybridisation time, use of hybridisation accelerators were examined and optimised. The analysis of PCR samples (244 bp DNA fragments, obtained by amplification of DNA extracted from human blood) required a further optimisation of the experimental procedure. In particular, a lower steric hyndrance of the probe modified surface was essential to allow an efficient hybridisation of the target DNA fragment. Negative controls have been performed using the PCR blank and amplicons unrelated to the immobilised probe. A 10 min hybridisation time allowed a full characterisation of each sample.

  10. Kinetic and metabolic studies in HPRT deficiency

    International Nuclear Information System (INIS)

    Steyn, L.M.

    1983-12-01

    The patient T.K. presented with the clinical features, typical of purine overproduction and a partial hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency; uric acid lithiasis and gout. The variant enzyme activity in haemolysates showed substrate inhibition by the purine base substrates. This was responsible for the low HPRT activity observed in vitro. The substrate inhibition of the variant enzyme resulted from the formation of a dead-end complex between hypoxanthine and an enzyme-PP i intermediate. The HPRT activities were assayed using radioactive substrates. This assays depend on the detection of labelled products for the determination of the enzyme activity. Carbon 14 and Tritium have been used for labelling the compounds. A low HPRT activity was shown in cultured cells from the patient. The double label technique demonstrated a reduced flux through the hypoxanthine/guanine salvage pathway, relative to that through the thymidine or leucine pathways. The rate of purine synthesis de novo was higher in the transformed lymphoblasts compared with that of control cells. This was shown by the double label approach, and by the release of labelled hypoxanthine and guanine into the medium. The APRT activity in the HPRT-cells was similar to that in the control cells. The behaviour of cultured cells and cells known to be defective in HPRT acitivity was similar in selective media. Although these studies have shown this HPRT variant an interesting and unique enzyme, the molecular defect still has to be defined

  11. Electrochemical label-free and sensitive nanobiosensing of DNA hybridization by graphene oxide modified pencil graphite electrode.

    Science.gov (United States)

    Ahour, F; Shamsi, A

    2017-09-01

    Based on the strong interaction between single-stranded DNA (ss-DNA) and graphene material, we have constructed a novel label-free electrochemical biosensor for rapid and facile detection of short sequences ss-DNA molecules related to hepatitis C virus 1a using graphene oxide modified pencil graphite electrode. The sensing mechanism is based on the superior adsorption of single-stranded DNA to GO over double stranded DNA (ds-DNA). The intrinsic guanine oxidation signal measured by differential pulse voltammetry (DPV) has been used for duplex DNA formation detection. The probe ss-DNA adsorbs onto the surface of GO via the π- π* stacking interactions leading to a strong background guanine oxidation signal. In the presence of complementary target, formation of helix which has weak binding ability to GO induced ds-DNA to release from the electrode surface and significant variation in differential pulse voltammetric response of guanine bases. The results indicated that the oxidation peak current was proportional to the concentration of complementary strand in the range of 0.1 nM-0.5 μM with a detection limit of 4.3 × 10 -11  M. The simple fabricated electrochemical biosensor has high sensitivity, good selectivity, and could be applied as a new platform for a range of target molecules in future. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Preparation and characterization of radiolabeled magnetic nanoparticles as an imaging agent

    Science.gov (United States)

    İçhedef, Çiğdem; Teksöz, Serap; Ünak, Perihan; Medine, Emin İ.; Ertay, Türkan; Bekiş, Recep

    2012-08-01

    Magnetic nanoparticles were prepared by a reduction-precipitation method and coated with an amino silane coupling agent. Guanine (Gua) was conjugated to the magnetic nanoparticles (MNPs) using glutaraldehyde as a cross-linker. Common techniques (Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction, and vibrating electron microscopy) were used to assess the properties of the particles. Structural investigations showed that amino silane-coated MNPs had a particle size of about 40-60 nm in diameter with a spherical morphology. The guanine-conjugated MNPs were radiolabeled with 99mTc(I)-tricarbonyl core (99mTc(CO)3-MNP-Gua) with a labeling yield of 72 ± 4 %. Pure radiolabeled magnetic particles were obtained by washing them with saline solution, and the radiochemical purity of 99mTc(CO)3-MNP-Gua was 98 ± 2 % in the final solution. The biologic distribution of guanine MNPs was assessed in New Zealand rabbits using a gamma camera. In the in vivo experiment, a high level of radioactivity was observed in the lungs and liver soon after intravenous administration of 99mTc(CO)3-MNP-Gua.

  13. The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility.

    Science.gov (United States)

    Rapp, Micah; Schein, Jessica; Hunt, Kevin A; Nalam, Vamsi; Mourad, George S; Schultes, Neil P

    2016-03-01

    The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

  14. Determination of a complex crystal structure in the absence of single crystals: analysis of powder X-ray diffraction data, guided by solid-state NMR and periodic DFT calculations, reveals a new 2'-deoxyguanosine structural motif.

    Science.gov (United States)

    Hughes, Colan E; Reddy, G N Manjunatha; Masiero, Stefano; Brown, Steven P; Williams, P Andrew; Harris, Kenneth D M

    2017-05-01

    Derivatives of guanine exhibit diverse supramolecular chemistry, with a variety of distinct hydrogen-bonding motifs reported in the solid state, including ribbons and quartets, which resemble the G-quadruplex found in nucleic acids with sequences rich in guanine. Reflecting this diversity, the solid-state structural properties of 3',5'-bis- O -decanoyl-2'-deoxyguanosine, reported in this paper, reveal a hydrogen-bonded guanine ribbon motif that has not been observed previously for 2'-deoxyguanosine derivatives. In this case, structure determination was carried out directly from powder XRD data, representing one of the most challenging organic molecular structures (a 90-atom molecule) that has been solved to date by this technique. While specific challenges were encountered in the structure determination process, a successful outcome was achieved by augmenting the powder XRD analysis with information derived from solid-state NMR data and with dispersion-corrected periodic DFT calculations for structure optimization. The synergy of experimental and computational methodologies demonstrated in the present work is likely to be an essential feature of strategies to further expand the application of powder XRD as a technique for structure determination of organic molecular materials of even greater complexity in the future.

  15. Degenerative minimalism in the genome of a psyllid endosymbiont.

    Science.gov (United States)

    Clark, M A; Baumann, L; Thao, M L; Moran, N A; Baumann, P

    2001-03-01

    Psyllids, like aphids, feed on plant phloem sap and are obligately associated with prokaryotic endosymbionts acquired through vertical transmission from an ancestral infection. We have sequenced 37 kb of DNA of the genome of Carsonella ruddii, the endosymbiont of psyllids, and found that it has a number of unusual properties revealing a more extreme case of degeneration than was previously reported from studies of eubacterial genomes, including that of the aphid endosymbiont Buchnera aphidicola. Among the unusual properties are an exceptionally low guanine-plus-cytosine content (19.9%), almost complete absence of intergenic spaces, operon fusion, and lack of the usual promoter sequences upstream of 16S rDNA. These features suggest the synthesis of long mRNAs and translational coupling. The most extreme instances of base compositional bias occur in the genes encoding proteins that have less highly conserved amino acid sequences; the guanine-plus-cytosine content of some protein-coding sequences is as low as 10%. The shift in base composition has a large effect on proteins: in polypeptides of C. ruddii, half of the residues consist of five amino acids with codons low in guanine plus cytosine. Furthermore, the proteins of C. ruddii are reduced in size, with an average of about 9% fewer amino acids than in homologous proteins of related bacteria. These observations suggest that the C. ruddii genome is not subject to constraints that limit the evolution of other known eubacteria.

  16. Mechanism of Error-Free Bypass of the Environmental Carcinogen N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone Adduct by Human DNA Polymerase η.

    Science.gov (United States)

    Patra, Amritraj; Politica, Dustin A; Chatterjee, Arindom; Tokarsky, E John; Suo, Zucai; Basu, Ashis K; Stone, Michael P; Egli, Martin

    2016-11-03

    The environmental pollutant 3-nitrobenzanthrone produces bulky aminobenzanthrone (ABA) DNA adducts with both guanine and adenine nucleobases. A major product occurs at the C8 position of guanine (C8-dG-ABA). These adducts present a strong block to replicative polymerases but, remarkably, can be bypassed in a largely error-free manner by the human Y-family polymerase η (hPol η). Here, we report the crystal structure of a ternary Pol⋅DNA⋅dCTP complex between a C8-dG-ABA-containing template:primer duplex and hPol η. The complex was captured at the insertion stage and provides crucial insight into the mechanism of error-free bypass of this bulky lesion. Specifically, bypass involves accommodation of the ABA moiety inside a hydrophobic cleft to the side of the enzyme active site and formation of an intra-nucleotide hydrogen bond between the phosphate and ABA amino moiety, allowing the adducted guanine to form a standard Watson-Crick pair with the incoming dCTP. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Inhibition of epithelial Na+ transport by atriopeptin, protein kinase c, and pertussis toxin

    International Nuclear Information System (INIS)

    Mohrmann, M.; Cantiello, H.F.; Ausiello, D.A.

    1987-01-01

    The authors have recently shown the selective inhibition of an amiloride-sensitive, conductive pathway for Na + by atrial natriuretic peptide and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) in the renal epithelial cell line, LLC-PK i . Using 22 Na + fluxes, they further investigated the modulation of Na + transport by atrial natriuretic peptide and by agents that increase cGMP production, activate protein kinase c, or modulate guanine nucleotide regulatory protein function. Sodium nitroprusside increases intracellular cGMP concentrations without affecting cAMP concentrations and completely inhibits amiloride-sensitive Na + uptake in a time- and concentration-dependent manner. Oleoyl 2-acetylglycerol and phorbol 12-myristate 13-acetate, activators of protein kinase c, inhibit Na + uptake by 93 ± 13 and 51 ± 10%, respectively. Prolonged incubation with phorbol ester results in the downregulation of protein kinase c activity and reduces the inhibitory effect of atrial natriuretic peptide, suggesting that the action of this peptide involves stimulation of protein kinase c. Pertussis toxin, which induces the ADP-ribosylation of a 41-kDa guanine nucleotide regulatory protein in LLC-PK i cells, inhibits 22 Na + influx to the same extent as amiloride. Thus, increasing cGMP, activating protein kinase c, and ADP-ribosylating a guanine nucleotide regulatory protein all inhibit Na + uptake. These events may be sequentially involved in the action of atrial natriuretic peptide

  18. Migration and fate of charges initially produced in irradiated DNA: a review

    International Nuclear Information System (INIS)

    Faraggi, M.; Ferradini, C.; Jay-Gerin, J.P.

    1995-01-01

    It has been shown that the charges, electrons and 'holes', generated by the action of ionizing radiation on DNA, in the solid phase as well as in aqueous solution, can migrate along the DNA chain before being trapped. Electron migration distances are of the order of 100 base pairs (the inter-base pair distance being 3.4 A) in the first case, and about one order of magnitude smaller in the second case. Short-range hole migration processes seem to occur from the initial cation radicals to sites located predominantly at the purine bases [adenine (A) and guanine (G)], with guanine being the most favorable hole trapping center. This radical cation G +. may undergo charge recombination or lead to a reaction sequence susceptible to induce single-strand breaks. The electrons produced during the ionization processes seem to react preferentially with the pyrimidine bases [cytosine (C) and thymine (T)], with the formation of two anionic primary radicals, T -. . Based on thermodynamic and kinetic considerations about T -. , it seems that this latter is responsible for inducing single-strand breaks. When the electron is initially trapped by cytosine, a proton is transferred to C -. from the complementary guanine. The deprotonation of this guanine leads to a reduction potential decrease of 200 mV, creating an electron donor site G(-H) - . This site is susceptible to transfer an electron to a radical cation, or to its subsequent intermediates, located at a distance equal to, or less than the characteristic electron migration distance. A pair of coupled free radicals, derived from cytosine and guanine, namely, C(H) . and g(-H) . , may thus be produced. The possible reactions between these two radicals could lead to: (i) the reformation of C and G by recombination, (ii) the reaction of an interstrand covalent bond, and (iii) the induction of a double-strand break. These two latter processes may result in permanent damages of the DNA molecule. (authors). 97 refs., 6 figs., 3 tabs

  19. The 3'-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes.

    Science.gov (United States)

    Onel, Buket; Carver, Megan; Agrawal, Prashansa; Hurley, Laurence H; Yang, Danzhou

    2018-04-01

    While the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Recently, the 3'-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation. We use 1D and 2D high-field NMR, in combination with Dimethylsulfate Footprinting, Circular Dichroism Spectroscopy, and Electrophoretic Mobility Shift Assay. We determine that the PDGFR-β extended 3'-end NHE sequence forms two novel end-insertion intramolecular G-quadruplexes that co-exist in equilibrium under physiological salt conditions. One G-quadruplex has a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another has a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4). The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. Each end-insertion G-quadruplex has a low thermal stability as compared to the 5'-mid G-quadruplex, but the selective stabilization of GSA1129 shifts the equilibrium toward the 3'-end G-quadruplex in the PDGFR-β NHE. An equilibrium mixture of two unique end-insertion intramolecular G-quadruplexes forms in the PDGFR-β NHE 3'-end sequence that contains a GGA-run and non-adjacent guanines in both the 3'- and 5'- flanking segments; the novel end-insertion structures of the 3'-end G-quadruplex are selectively stabilized by GSA1129. We show for the first time that an equilibrium mixture of two unusual end-insertion G-quadruplexes forms in a native promoter sequence and appears to be the molecular recognition for PDGFR-β downregulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Potential energy surface of the reaction of imidazole with peroxynitrite: Density functional theory study

    Science.gov (United States)

    Gogonea, Valentin

    This article presents a theoretical investigation of the reaction mechanism of imidazole nitration by peroxynitrite using density functional theory calculations. Understanding this reaction mechanism will help in elucidating the mechanism of guanine nitration by peroxynitrite, which is one of the assumed chemical pathways for damaging DNA in cells. This work focuses on the analysis of the potential energy surface (PES) for this reaction in the gas phase. Calculations were carried out using Hartree-Fock (HF) and density functional theory (DFT) Hamiltonians with double-zeta basis sets ranging from 6-31G(d) to 6-31++G(d,p), and the triple-zeta basis set 6-311G(d). The computational results reveal that the reaction of imidazole with peroxynitrite in gas phase produces the following species: (i) hydroxide ion and 2-nitroimidazole, (ii) hydrogen superoxide ion and 2-nitrosoimidazole, and (iii) water and 2-nitroimidazolide. The rate-determining step is the formation of a short-lived intermediate in which the imidazole C2 carbon is covalently bonded to peroxynitrite nitrogen. Three short-lived intermediates were found in the reaction path. These intermediates are involved in a proton-hopping transport from C2 carbon to the terminal oxygen of the OO moiety of peroxynitrite via the nitroso (ON) oxygen. Both HF and DFT calculations (using the Becke3-Lee-Yang-Parr functional) lead to similar reaction paths for proton transport, but the landscape details of the PES for HF and DFT calculations differ. This investigation shows that the reaction of imidazole with peroxynitrite produces essentially the same types of products (nitro- and nitroso-) as observed experimentally in the reaction of guanine with peroxynitrite, which makes the former reaction a good model to study by computation the essential characteristics of the latter reaction. Nevertheless, the computationally determined activation energy for imidazole nitration by peroxynitrite in the gas phase is 84.1 kcal

  1. Insight into the Interaction between DNA Bases and Defective Graphenes: Covalent or Non-covalent

    Science.gov (United States)

    Xu, Zhenfeng; Meher, Biswa Ranjan; Eustache, Darnashley; Wang, Yixuan

    2013-01-01

    Although some metal clusters and molecules were found to more significantly bind to defective graphenes than to pristine graphenes, exhibiting chemisorptions on defective graphenes, the present investigation shows that the adsorption of DNA bases on mono- and di-vacant defective graphenes does not show much difference from that on pristine graphene, and is still dominantly driven by noncovalent interactions. In the present study the adsorptions of the nucleobases, adenine (A), cytosine (C), guanine, (G), and thymine (T) on pristine and defective graphenes, are fully optimized using a hybrid-meta GGA density functional theory (DFT), M06-2X/6-31G*, and the adsorption energies are then refined with both M06-2X and B97-D/6-311++G**. Graphene is modeled as nano-clusters of C72H24, C71H24, and C70H24 for pristine, mono- and divacant defective graphenes, respectively, supplemented by a few larger ones. The result shows that guanine has the maximum adsorption energy in all of the three adsorption systems; and the sequence of the adsorption strength is G>A>T>C on the pristine and di-vacant graphene and G>T>A>C on the mono-vacant graphene. In addition, the binding energies of the DNA bases with the pristine graphene are less than the corresponding ones with di-vacant defective graphene; however, they are greater than those of mono-vacant graphene with guanine and adenine, while it is dramatic that the binding energies of mono-vacant graphene with thymine and cytosine appear larger than those of pristine graphene. PMID:24215998

  2. Autroadiographic characterization of 125I-labeled 2,5-dimethoxy-4-iodophenylisopropylamine (DOI): A phenylisopropylamine derivative labeling both 5HT2 and 5HT1c receptors

    International Nuclear Information System (INIS)

    Appel, N.M.; Mitchell, W.M.; Garlick, R.K.; Glennon, R.A.; Titeler, M.; De Souza, E.B.

    1990-01-01

    The best-characterized 5HT 2 radioligands, such as [ 3 H]ketanserin and [ 3 H]spiperone, are antagonists that label both high- and low-affinity states of this receptor. Recently, the radiolabeled phenylisopropylamine hallucinogens DOB and DOI, which are agonists at 5HT 2 receptors, have been demonstrated to label selectively the high-affinity state of brain 5HT 2 receptors. In the present study, the authors determined optimum conditions for autoradiographic visualization of [ 125 I]DOI binding and characterized its pharmacology and guanine nucleotide sensitivity under those conditions. In slide-mounted tissue sections (rat forebrain; two 10 μm sections/slide), (±)[ 125 I]DOI binding was saturable, of high affinity (K D ∼4nM) and displayed a pharmacological profile [R(-)DOI > spiperone > DOB > (±)DOI > ketanserin > S(+)DOI > 5HT > DOM] comparable to that seen in homogenate assays. Consistent with coupling of 5HT 2 receptors to a guanine nucleotide regulatory protein, [ 125 I]DOI binding was inhibited by guanine nucleotides but not by ATP. In autoradiograms, high densities of [ 125 I]DOI binding sites were present in frontal cortex, olfactory tubercle, claustrum, caudate/putamen and mamillary nuclei with lower densities in trigeminal and solitary nuclei. The highest density of [ 125 l]DOI binding was observed in choroid plexus; these binding sites displayed a pharmacological profile characteristic of 5HT 1C receptors. These data suggest that [ 125 I]DOI labels both 5HT 2 and 5HT 1C receptors

  3. Chemical intervention of the NM23-H2 transcriptional programme on c-MYC via a novel small molecule.

    Science.gov (United States)

    Shan, Chan; Lin, Jing; Hou, Jin-Qiang; Liu, Hui-Yun; Chen, Shuo-Bin; Chen, Ai-Chun; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

    2015-08-18

    c-MYC is an important oncogene that is considered as an effective target for anticancer therapy. Regulation of this gene's transcription is one avenue for c-MYC-targeting drug design. Direct binding to a transcription factor and generating the intervention of a transcriptional programme appears to be an effective way to modulate gene transcription. NM23-H2 is a transcription factor for c-MYC and is proven to be related to the secondary structures in the promoter. Here, we first screened our small-molecule library for NM23-H2 binders and then sifted through the inhibitors that could target and interfere with the interaction process between NM23-H2 and the guanine-rich promoter sequence of c-MYC. As a result, a quinazolone derivative, SYSU-ID-01: , showed a significant interference effect towards NM23-H2 binding to the guanine-rich promoter DNA sequence. Further analyses of the compound-protein interaction and the protein-DNA interaction provided insight into the mode of action for SYSU-ID-01: . Cellular evaluation results showed that SYSU-ID-01: could abrogate NM23-H2 binding to the c-MYC promoter, resulting in downregulation of c-MYC transcription and dramatically suppressed HeLa cell growth. These findings provide a new way of c-MYC transcriptional control through interfering with NM23-H2 binding to guanine-rich promoter sequences by small molecules. © Oxford University Press OR Nucleic Acids Research 2015.

  4. Tiam1/Vav2-Rac1 axis: A tug-of-war between islet function and dysfunction.

    Science.gov (United States)

    Kowluru, Anjaneyulu

    2017-05-15

    Glucose-stimulated insulin secretion [GSIS] from the islet β-cell involves a well-orchestrated interplay between metabolic and cationic events. It is well established that intracellular generation of adenine and guanine nucleotide triphosphates [e.g., ATP and GTP] represents one of the requisite signaling steps in GSIS. The small molecular mass GTP-binding proteins [G-proteins; e.g., Rac1 and Cdc42] have been shown to regulate islet β-cell function including actin cytoskeletal remodeling and fusion of insulin granules with the plasma membrane for GSIS to occur. In this context, several regulatory factors for these G-proteins have been identified in the pancreatic β-cell; these include guanine nucleotide exchange factors [GEFs] and guanine nucleotide dissociation inhibitors [GDI]. Recent pharmacological and molecular biological evidence identified Tiam1 and Vav2 as GEFs for Rac1 in promoting physiological insulin secretion. Paradoxically, emerging evidence in multiple cell types, including the islet β-cell, suggests key roles for Rac1 in the onset of cellular dysfunction under conditions of metabolic stress and diabetes. Furthermore, functional inactivation of either Tiam1 or Vav2 appears to attenuate sustained activation of Rac1 and its downstream signaling events [activation of stress kinases] under conditions of metabolic stress. Together, these findings suggest both "friendly" and "non-friendly" roles for Tiam1/Vav2-Rac1 signaling axis in islet β-cell in health and diabetes. Our current understanding of the field and the knowledge gaps that exist in this area of islet biology are heighted herein. Furthermore, potential caveats in the specificity and selectivity of pharmacological inhibitors that are available currently are discussed in this Commentary. Published by Elsevier Inc.

  5. Asymmetric opening reaction mechanism of Z-DNA base pairs: a hydrogen exchange study

    International Nuclear Information System (INIS)

    Ramstein, J.; Vogt, N.; Leng, M.

    1985-01-01

    With the tritium-Sephadex method, the hydrogen-exchange kinetics of the five NH protons of guanine and cytosine residues in Z-form poly(dG-dC) X poly (dG-dC) were measured as a function of temperature and catalyst concentration. Over the measured temperature range from 0 to 34 degrees C, two classes of protons with constant amplitudes are found. The three protons of the fast class, which were assigned to the guanine amino and imino protons, have an exchange half-time in the minute time range (at 20 degrees C the half-time is 2.5 min) and an activation energy of 18 kcal mol-1. Since these two types of protons exchange at the same rate in spite of their grossly different pK values, the exchange of these protons must be limited by the same nucleic acid conformational change. The two cytosine amino protons of the slow class are especially slow with exchange half-times in the hour time range (at 20 degrees C the exchange half-time is 1 h) and the activation energy is 20 kcal mol-1. The exchange of these two protons is not limited by some nucleic acid conformational change as shown by the marked exchange acceleration of these protons upon addition of 0.2 M imidazole. In addition, we have also reexamined the hydrogen-deuterium exchange kinetics of the amino protons of guanosine cyclic 2',3'-monophosphate by a spectral difference method using a stopped-flow spectrophotometer. The measured kinetic process is monophasic with a rate constant of 3 s-1 at 20 degrees C, which is in the same range as the predicted rate constant of the guanine amino protons

  6. G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets.

    Science.gov (United States)

    Harris, Lynne M; Monsell, Katelyn R; Noulin, Florian; Famodimu, M Toyin; Smargiasso, Nicolas; Damblon, Christian; Horrocks, Paul; Merrick, Catherine J

    2018-03-01

    G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs. Copyright © 2018 American Society for Microbiology.

  7. Molecular Dynamics Insights into Polyamine-DNA Binding Modes: Implications for Cross-Link Selectivity.

    Science.gov (United States)

    Bignon, Emmanuelle; Chan, Chen-Hui; Morell, Christophe; Monari, Antonio; Ravanat, Jean-Luc; Dumont, Elise

    2017-09-18

    Biogenic polyamines, which play a role in DNA condensation and stabilization, are ubiquitous and are found at millimolar concentration in the nucleus of eukaryotic cells. The interaction modes of three polyamines-putrescine (Put), spermine (Spm), and spermidine (Spd)-with a self-complementary 16 base pair (bp) duplex, are investigated by all-atom explicit-solvent molecular dynamics. The length of the amine aliphatic chain leads to a change of the interaction mode from minor groove binding to major groove binding. Through all-atom dynamics, noncovalent interactions that stabilize the polyamine-DNA complex and prefigure the reactivity, leading to the low-barrier formation of deleterious DNA-polyamine cross-links, after one-electron oxidation of a guanine nucleobase, are unraveled. The binding strength is quantified from the obtained trajectories by molecular mechanics generalized Born surface area post-processing (MM-GBSA). The values of binding free energies provide the same affinity order, Putguanines and the ammonium group. These results imply that the formation of DNA-polyamine cross-links involves deprotonation of the guanine radical cation to attack the polyamines, which must be positively charged to lie in the vicinity of the B-helix. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Topoisomerase I plays a critical role in suppressing genome instability at a highly transcribed G-quadruplex-forming sequence.

    Directory of Open Access Journals (Sweden)

    Puja Yadav

    2014-12-01

    Full Text Available G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼ 20 kb or ∼ 100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

  9. Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells.

    Science.gov (United States)

    Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A; Nakamura, Jun

    2016-09-13

    Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS.

  10. Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts.

    Science.gov (United States)

    Thomas, D C; Veaute, X; Kunkel, T A; Fuchs, R P

    1994-08-02

    We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Replication product analyses reveal unique termination sites with each damaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct. We also observed a higher proportion of products representing replication of the undamaged strand compared to the damaged strand. This suggests that the undamaged strand is replicated more readily, either by uncoupling the first fork to encounter the lesion or by replication using the fork arriving from the other direction. Also included among replication products are covalently closed monomer-length molecules resistant to cleavage at the AAF-modified Nar I site. This resistance is characteristic of substrates containing the AAF adduct, suggesting that translesion bypass had occurred. Transformation of Escherichia coli cells with the replicated damaged DNA yielded lacZ alpha revertant frequencies significantly above values obtained with undamaged DNA or with damaged DNA not replicated in vitro. This increase was only seen with the substrate modified at the third guanine position. Analysis of mutant DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence. Generation of this position-dependent AAF-induced frameshift error in a human replication system is consistent with previous observations in E. coli suggesting that, after incorporation of dCMP opposite modified guanine in the third position, realignment of the template-primer occurs to form an intermediate with two

  11. Spectroscopic and immunochemical characterization of left-handed Z RNA under physiological conditions

    International Nuclear Information System (INIS)

    Hardin, C.C.; Zarling, D.A.; Tinoco, I. Jr.

    1986-01-01

    Limited chemical brominination of poly[r(C-G)] results in partial modification of guanine C8 and cytosine C5 producing a mixture of A and Z RNA forms in 110 mM NaCl buffer at 37 0 C. 1 H NMR of Br-poly[r(C-G)] shows a 1:1 mixture of A and Z RNAs. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1' and CH1 resonances in both the A and Z forms. Distance estimates obtained from GH8 to GH1' NOEs demonstrate that even unbrominated guanine residues in these polynucleotides undergo a transition to the syn conformation (Z form). The Z form of Br-poly[r(C-G)] in 110 mM NaCl has a guanine Raman scattering band at 643 cm -1 in agreement with the band in the Z form of poly[r(C-G)] stabilized by 6 M NaClO 4 . In A RNA the band is at 671 cm -1 . The corresponding shift in DNA is from 682 cm -1 (B DNA) to 625 cm -1 (Z DNA). This suggests different glycosidic torsion angles in Z RNA and Z DNA. Inoculating rabbits with Br-poly[r(C-G)] produces polyclonal antibodies specific for the Z forms of RNA and DNA (i.e., poly[d(br 5 C-G)] or poly[d(io 5 C-G)] as judged by radioimmunoassay in 100 mM NaCl buffers. Phosphate buffer or certain high ionic strength conditions affect the recognition of Z RNA and Z DNA by anti-Z RNA or anti-Z DNA IgGs suggesting that one determinant recognized by these antibodies is the zig-zag phosphodiester backbone

  12. Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

    Science.gov (United States)

    Nyakas, Adrien; Stucki, Silvan R.; Schürch, Stefan

    2011-05-01

    Therapeutic approaches for treatment of various diseases aim at the interruption of transcription or translation. Modified oligonucleotides, such as 2'- O-methyl- and methylphosphonate-derivatives, exhibit high resistance against cellular nucleases, thus rendering application for, e.g., antigene or antisense purposes possible. Other approaches are based on administration of cross-linking agents, such as cis-diamminedichloroplatinum(II) (cisplatin, DDP), which is still the most widely used anticancer drug worldwide. Due to the formation of 1,2-intrastrand cross links at adjacent guanines, replication of the double-strand is disturbed, thus resulting in significant cytotoxicity. Evidence for the gas-phase dissociation mechanism of platinated RNA is given, based on nano-electrospray ionization high-resolution multistage tandem mass spectrometry (MS n ). Confirmation was found by investigating the fragmentation pattern of platinated and unplatinated 2'-methoxy oligoribonucleotide hexamers and their corresponding methylphosphonate derivatives. Platinated 2'-methoxy oligoribonucleotides exhibit a similar gas-phase dissociation behavior as the corresponding DNA and RNA sequences, with the 3'-C-O bond adjacent to the vicinal guanines being cleaved preferentially, leading to wx-ion formation. By examination of the corresponding platinated methylphosphonate derivatives of the 2'-methoxy oligoribonucleotides, the key role of the negatively charged phosphate oxygen atoms in direct proximity to the guanines was proven. The significant alteration of fragmentation due to platination is demonstrated by comparison of the fragment ion patterns of unplatinated and platinated 2'- O-methyl- and 2'- O-methyl methylphosphonate oligoribonucleotides, and the results obtained by H/D exchange experiments.

  13. Direct fluorescence in situ hybridization on human metaphase chromosomes using quantum dot-platinum labeled DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Gyoyeon [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of); Lee, Hansol [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Lee, Jiyeon, E-mail: jylee@kist.re.kr [Chemical Kinomics Research Center, Future Convergence Research Division, Korea Institute of Science and Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791 (Korea, Republic of); Biological Chemistry, Korea University of Science and Technology, 217, Gajeong-ro, Yuseong-gu, Deajeon (Korea, Republic of)

    2015-11-13

    The telomere shortening in chromosomes implies the senescence, apoptosis, or oncogenic transformation of cells. Since detecting telomeres in aging and diseases like cancer, is important, the direct detection of telomeres has been a very useful biomarker. We propose a telomere detection method using a newly synthesized quantum dot (QD) based probe with oligonucleotide conjugation and direct fluorescence in situ hybridization (FISH). QD-oligonucleotides were prepared with metal coordination bonding based on platinum-guanine binding reported in our previous work. The QD-oligonucleotide conjugation method has an advantage where any sequence containing guanine at the end can be easily bound to the starting QD-Pt conjugate. A synthesized telomeric oligonucleotide was bound to the QD-Pt conjugate successfully and this probe hybridized specifically on the telomere of fabricated MV-4-11 and MOLT-4 chromosomes. Additionally, the QD-telomeric oligonucleotide probe successfully detected the telomeres on the CGH metaphase slide. Due to the excellent photostability and high quantum yield of QDs, the QD-oligonucleotide probe has high fluorescence intensity when compared to the organic dye-oligonucleotide probe. Our QD-oligonucleotide probe, conjugation method of this QD probe, and hybridization protocol with the chromosomes can be a useful tool for chromosome painting and FISH. - Highlights: • We prepared a probe linked between QD and telomeric oligonucleotide with platinum-guanine bonding. • Telomeres were detected by our new telomere probes successfully in three different human metaphase chromosomes. • QDPt-DNA probe has high fluorescence intensity in comparison with organic dye-DNA probe.

  14. Molecular investigation of evaporation of biodroplets containing single-strand DNA on graphene surface.

    Science.gov (United States)

    Akbari, Fahimeh; Foroutan, Masumeh

    2018-02-14

    In this study, the water droplet behaviour of four different types of single-strand DNA with homogeneous base sequence on a graphene substrate during evaporation of the droplet was investigated using molecular dynamics (MD) simulation. The simulation results indicated that the evaporation depended on the DNA sequence. The observed changes can be divided into four parts: (i) vaporization mode, (ii) evaporation flux, (iii) mechanism of single-strand placement on the surface, and (iv) consideration of remaining single strands after evaporation. Our simulation observations indicated different evaporation modes for thymine biodroplets as compared to those for other biodroplets. The evaporation of the thymine biodroplets occurred with an increase in the contact angle, while that of the other biodroplets occur in a constant contact angle mode. Moreover, thymine biodroplets generate the lowest contact line compared to other single strands, and it is always placed far away from the centre of the droplets during evaporation. Investigating variations in the evaporation flux shows that thymine has the highest evaporation flux and guanine has the lowest. Moreover, during initial evaporation, the flux of evaporation increases at the triple point of the biodroplets containing thymine single strands, while it decreases in the other biodroplets. The following observation was obtained from the study of the placement of single strands on the substrate: guanine and thymine interacted slower than other single strands during evaporation with graphene, adenine single strand had a higher folding during evaporation, and guanine single strand showed the lowest end-to-end distance. The investigation of single-strand DNA after evaporation shows that adenine produces the most stable structure at the end of evaporation. In addition, cytosine is the most stretched single-strand DNA due to its lack of internal π-π stacking and hydrogen bonding. Therefore, cytosine single strand is more

  15. Facile enzymatic synthesis of base J-containing oligodeoxyribonucleotides and an analysis of the impact of base J on DNA replication in cells.

    Directory of Open Access Journals (Sweden)

    Debin Ji

    Full Text Available We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs. We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.

  16. Mutagenic effects of 3-carbethoxypsoralen and 8-methoxypsoralen plus 365-nm irradiation in mammalian cells

    International Nuclear Information System (INIS)

    Papadopoulo, D.; Sagliocco, F.; Averbeck, D.

    1983-01-01

    Cell survival, i.e. colony-forming ability, and the induction of 6-thioguanine-resistant (6-TGsup(r)) mutants were determined in Chinese hamster V79 cells by using two photoreactive furocoumarins of photochemotherapeutic interest: the bifunctional compound 8-methoxypsoralen (8-MOP) and the monofunctional compound 3-carbethoxypsoralen (3-CPs). To quantify the mutation induction in V79 cells mutants deficient in the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) were selected with the purine analogue 6-thioguanine (6-TG). Both compounds exhibited lethal and mutagenic activities but the monofunctional compound 3-CPs was less lethal and mutagenic than the bifunctional compound 8-MOP. (Auth.)

  17. A Case of Juvenile Huntington Disease in a 6-Year-Old Boy

    Directory of Open Access Journals (Sweden)

    Jun-Sang Sunwoo

    2010-10-01

    Full Text Available Huntington disease is a neurodegenerative disorder distinguished by the triad of dominant inheritance, choreoathetosis and dementia, usually with onset in the fourth and fifth decades. It is caused by an unstable cytosine-adenine-guanine (CAG trinucleotide repeat expansion in the gene IT15 in locus 4p16.3. Juvenile HD that constitutes about 3% to 10% of all patients is clinically different from adult-onset form and characterized by a larger number of CAG repeats typically exceeding 60. We report a case of a 6-year-old boy with myoclonic seizure and 140 CAG repeats confirmed by molecular genetic analysis.

  18. Data on the effect of knockout of cytohesin-1 in myelination-related protein kinase signaling.

    Science.gov (United States)

    Tsuneishi, Ruri; Matsumoto, Naoto; Itaoka, Misa; Urai, Yuri; Kaneko, Minami; Watanabe, Natsumi; Takashima, Shou; Seki, Yoichi; Morimoto, Takako; Sakagami, Hiroyuki; Miyamoto, Yuki; Yamauchi, Junji

    2017-12-01

    Cytohesin-1 is the guanine-nucleotide exchange factor of Arf6, a small GTPase of Arf family, and participates in cellular morphological changes. Knockout mice of cytohesin-1 exhibit decreased myelination of neuronal axons in the peripheral nervous system (PNS) "Phosphorylation of cytohesin-1 by Fyn is required for initiation of myelination and the extent of myelination during development (Yamauchi et al., 2012) [1]". Herein we provide the data regarding decreased phosphorylation levels of protein kinases involved in two major myelination-related kinase cascades in cytohesin-1 knockout mice.

  19. DENN Domain Proteins: Regulators of Rab GTPases*

    Science.gov (United States)

    Marat, Andrea L.; Dokainish, Hatem; McPherson, Peter S.

    2011-01-01

    The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways. PMID:21330364

  20. DENN domain proteins: regulators of Rab GTPases.

    Science.gov (United States)

    Marat, Andrea L; Dokainish, Hatem; McPherson, Peter S

    2011-04-22

    The DENN domain is a common, evolutionarily ancient, and conserved protein module, yet it has gone largely unstudied; until recently, little was known regarding its functional roles. New studies reveal that various DENN domains interact directly with members of the Rab family of small GTPases and that DENN domains function enzymatically as Rab-specific guanine nucleotide exchange factors. Thus, DENN domain proteins appear to be generalized regulators of Rab function. Study of these proteins will provide new insights into Rab-mediated membrane trafficking pathways.