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Sample records for 1balpha guanine nucleotides

  1. Guanine nucleotides stimulate hydrolysis of phosphatidyl inositol bis phosphate in human myelin membranes

    SciTech Connect

    Boulias, C.; Moscarello, M.A. )

    1989-07-14

    Phosphodiesterase activity was stimulated in myelin membranes in the presence of guanine nucleotide analogues. This activity was reduced in myelin membranes which had been adenosine diphosphate ribosylated in the presence of cholera toxin which ADP-ribosylated three proteins of Mr 46,000, 43,000 and 18,500. Aluminum fluoride treatment of myelin had the same stimulatory effects on phosphodiesterase activity as did the guanine nucleotides.

  2. RasGRP Ras guanine nucleotide exchange factors in cancer

    PubMed Central

    Ksionda, Olga; Limnander, Andre

    2014-01-01

    Summary RasGRP proteins are activators of Ras and other related small GTPases by the virtue of functioning as guanine nucleotide exchange factors (GEFs). In vertebrates, four RasGRP family members have been described. RasGRP-1 through −4 share many structural domains but there are also subtle differences between each of the different family members. Whereas SOS RasGEFs are ubiquitously expressed, RasGRP proteins are expressed in distinct patterns, such as in different cells of the hematopoietic system and in the brain. Most studies have concentrated on the role of RasGRP proteins in the development and function of immune cell types because of the predominant RasGRP expression profiles in these cells and the immune phenotypes of mice deficient for Rasgrp genes. However, more recent studies demonstrate that RasGRPs also play an important role in tumorigenesis. Examples are skin- and hematological-cancers but also solid malignancies such as melanoma or prostate cancer. These novel studies bring up many new and unanswered questions related to the molecular mechanism of RasGRP-driven oncogenesis, such as new receptor systems that RasGRP appears to respond to as well as regulatory mechanism for RasGRP expression that appear to be perturbed in these cancers. Here we will review some of the known aspects of RasGRP biology in lymphocytes and will discuss the exciting new notion that RasGRP Ras exchange factors play a role in oncogenesis downstream of various growth factor receptors. PMID:24744772

  3. Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

    PubMed Central

    Glesne, D A; Collart, F R; Huberman, E

    1991-01-01

    To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides. Images PMID:1717828

  4. Transcription profiling of guanine nucleotide binding proteins during developmental regulation, and pesticide response in Solenopsis invicta (Hymenoptera: Formicidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Guanine nucleotide binding proteins (GNBP or G-protein) are glycoproteins anchored on the cytoplasmic cell membrane, and are mediators for many cellular processes. Complete cDNA of guanine nucleotide-binding protein gene ß-subunit (SiGNBP) was cloned and sequenced from S. invicta workers. To detect ...

  5. Human Sos1: A guanine nucleotide exchange factor for ras that binds to GRB2

    SciTech Connect

    Chardin, P. ); Camonis, J.; Gale, N.W.; Aelst, L. Van; Wigler, M.H.; Bar-Sagi, D. ); Schlessinger, J. )

    1993-05-28

    A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1. 42 refs., 5 figs.

  6. Guanine-nucleotide-dependent inhibition of adenylate cyclase of rabbit heart by glucagon.

    PubMed

    Kiss, Z; Tkachuk, V A

    1984-07-16

    The present study demonstrates an inhibitory effect of glucagon on the adenylate cyclase system of rabbit heart. Inhibition was maximal (22-40%) at 0.1-0.01 microM glucagon and required the presence of 0.01-0.1 mM GTP or guanosine 5'-[beta, gamma-imido]triphosphate (GuoPP[NH]P). Reduced or no inhibitor effect of glucagon was observed: (a) after limited proteolysis of plasma membrane proteins by trypsin, (b) in the presence of 1 mM Mn2+, (c) in the absence of Na+, and (d) during the first 10 min of incubation if GuoPP[NH]P was the activating ligand. With GTP as the activating ligand, inhibition of cyclase by glucagon occurred without delay. These data are consistent with a mediation of glucagon inhibition by a guanine-nucleotide-binding protein. In the presence of ethanol (0.2 M) or benzyl alcohol (0.05 M), agents which are known to increase the fluidity of biological membranes, glucagon increased the enzyme activity in a guanine-nucleotide-dependent manner. Activation of cyclase in the presence of alcohols was maximal (30-60%) at 0.1-1.0 microM glucagon and 0.01 mM guanine nucleotides. Data suggest that glucagon receptors can interact with both the activatory and inhibitory guanine-nucleotide-binding proteins and the physical state of membranes may play a role in determining which interaction will be preferential.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26558346

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

    NASA Astrophysics Data System (ADS)

    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.

  9. Binding of calcium ions to Ras promotes Ras guanine nucleotide exchange under emulated physiological conditions.

    PubMed

    Liao, Jun-Ming; Mo, Zhong-Ying; Wu, Ling-Jia; Chen, Jie; Liang, Yi

    2008-11-01

    Both Ras protein and calcium play significant roles in various cellular processes via complex signaling transduction networks. However, it is not well understood whether and how Ca(2+) can directly regulate Ras function. Here we demonstrate by isothermal titration calorimetry that Ca(2+) directly binds to the H-Ras.GDP.Mg(2+) complex with moderate affinity at the first binding site followed by two weak binding events. The results from limited proteinase degradation show that Ca(2+) protects the fragments of H-Ras from being further degraded by trypsin and by proteinase K. HPLC studies together with fluorescence spectroscopic measurements indicate that binding of Ca(2+) to the H-Ras.GDP.Mg(2+) complex remarkably promotes guanine nucleotide exchange on H-Ras under emulated physiological Ca(2+) concentration conditions. Addition of high concentrations of either of two macromolecular crowding agents, Ficoll 70 and dextran 70, dramatically enhances H-Ras guanine nucleotide exchange extent in the presence of Ca(2+) at emulated physiological concentrations, and the nucleotide exchange extent increases significantly with the concentrations of crowding agents. Together, these results indicate that binding of calcium ions to H-Ras remarkably promotes H-Ras guanine nucleotide exchange under emulated physiological conditions. We thus propose that Ca(2+) may activate Ras signaling pathway by interaction with Ras, providing clues to understand the role of calcium in regulating Ras function in physiological environments.

  10. Intermediates in the Guanine Nucleotide Exchange Reaction of Rab8 Protein Catalyzed by Guanine Nucleotide Exchange Factors Rabin8 and GRAB*

    PubMed Central

    Guo, Zhong; Hou, Xiaomin; Goody, Roger S.; Itzen, Aymelt

    2013-01-01

    Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange. PMID:24072714

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

    PubMed

    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. PMID:26986850

  12. Insights into the biological functions of Dock family guanine nucleotide exchange factors

    PubMed Central

    Laurin, Mélanie; Côté, Jean-François

    2014-01-01

    Rho GTPases play key regulatory roles in many aspects of embryonic development, regulating processes such as differentiation, proliferation, morphogenesis, and migration. Two families of guanine nucleotide exchange factors (GEFs) found in metazoans, Dbl and Dock, are responsible for the spatiotemporal activation of Rac and Cdc42 proteins and their downstream signaling pathways. This review focuses on the emerging roles of the mammalian DOCK family in development and disease. We also discuss, when possible, how recent discoveries concerning the biological functions of these GEFs might be exploited for the development of novel therapeutic strategies. PMID:24637113

  13. Rho Family Guanine Nucleotide Exchange Factor Brx Couples Extracellular Signals to the Glucocorticoid Signaling System*

    PubMed Central

    Kino, Tomoshige; Souvatzoglou, Emanuel; Charmandari, Evangelia; Ichijo, Takamasa; Driggers, Paul; Mayers, Chantal; Alatsatianos, Anton; Manoli, Irini; Westphal, Heiner; Chrousos, George P.; Segars, James H.

    2014-01-01

    Glucocorticoids regulate many crucial biologic functions through their cytoplasmic/nuclear glucocorticoid receptors (GR). Excess, deficiency, or alteration in tissue sensitivity to glucocorticoids has been associated with major causes of human morbidity and mortality. Brx, a cytoplasmic Rho family guanine nucleotide exchange factor, binds to and influences the activity of several nuclear hormone receptors. We examined the functional and molecular interactions between GR and Brx. The glucocorticoid sensitivity of lymphocytes obtained from mice haplo-insufficient for Brx was significantly decreased. Conversely, GR-mediated transcriptional activity of a glucocorticoid response element (GRE)-mediated glucocorticoid-responsive promoter was enhanced by Brx in a guanine nucleotide exchange factor domain-dependent fashion. Brx interacted with GR, forming a ternary complex with RhoA. In a chromatin immunoprecipitation assay, Brx and RhoA were co-precipitated with GREs only in the presence of ligand-activated GR. Extracellularly administered lyso-phosphatidic acid, which activates its signaling cascade through a specific membrane GTP-binding protein (G-protein)-coupled receptor in a G-protein α13-, Brx-, and RhoA-dependent fashion, enhanced GR transcriptional activity, whereas depletion of endogenous Brx attenuated this effect. These findings suggest that glucocorticoid signaling and, hence, the tissue sensitivity to glucocorticoids, may be coupled to extracellular signals via Brx and small G-proteins. Nuclear Brx might act as a local GRE-GR-transcripto-some activator by mediating the effect of small G-proteins on glucocorticoid-regulated genes. PMID:16469733

  14. The emerging role of guanine nucleotide exchange factors in ALS and other neurodegenerative diseases

    PubMed Central

    Droppelmann, Cristian A.; Campos-Melo, Danae; Volkening, Kathryn; Strong, Michael J.

    2014-01-01

    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 dedicator of cytokinesis proteins 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. 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 mRNA 3′ untranslated region 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. PMID:25309324

  15. Chromosomal localization of genes encoding guanine nucleotide-binding protein subunits in mouse and human.

    PubMed

    Blatt, C; Eversole-Cire, P; Cohn, V H; Zollman, S; Fournier, R E; Mohandas, L T; Nesbitt, M; Lugo, T; Jones, D T; Reed, R R

    1988-10-01

    A variety of genes have been identified that specify the synthesis of the components of guanine nucleotide-binding proteins (G proteins). Eight different guanine nucleotide-binding alpha-subunit proteins, two different beta subunits, and one gamma subunit have been described. Hybridization of cDNA clones with DNA from human-mouse somatic cell hybrids was used to assign many of these genes to human chromosomes. The retinal-specific transducin subunit genes GNAT1 and GNAT2 were on chromosomes 3 and 1; GNAI1, GNAI2, and GNAI3 were assigned to chromosomes 7, 3, and 1, respectively; GNAZ and GNAS were found on chromosomes 22 and 20. The beta subunits were also assigned--GNB1 to chromosome 1 and GNB2 to chromosome 7. Restriction fragment length polymorphisms were used to map the homologues of some of these genes in the mouse. GNAT1 and GNAI2 were found to map adjacent to each other on mouse chromosome 9 and GNAT2 was mapped on chromosome 17. The mouse GNB1 gene was assigned to chromosome 19. These mapping assignments will be useful in defining the extent of the G alpha gene family and may help in attempts to correlate specific genetic diseases with genes corresponding to G proteins. PMID:2902634

  16. Guanine-Centric Self-Assembly of Nucleotides in Water: An Important Consideration in Prebiotic Chemistry

    PubMed Central

    Cassidy, Lauren M.; Burcar, Bradley T.; Stevens, Wyatt; Moriarty, Elizabeth M.

    2014-01-01

    Abstract 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. Key Words: RNA world—Prebiotic chemistry—RNA polymerization

  17. Zizimin and Dock guanine nucleotide exchange factors in cell function and disease.

    PubMed

    Pakes, Nicholl K; Veltman, Douwe M; Williams, Robin S B

    2013-01-01

    Zizimin proteins belong to the Dock (Dedicator of Cytokinesis) superfamily of Guanine nucleotide Exchange Factor (GEF) proteins. This family of proteins plays a role in the regulation of Rho family small GTPases. Together the Rho family of small GTPases and the Dock/Zizimin proteins play a vital role in a number of cell processes including cell migration, apoptosis, cell division and cell adhesion. Our recent studies of Zizimin proteins, using a simple biomedical model, the eukaryotic social amoeba Dictyostelium discoideum, have helped to elucidate the cellular role of these proteins. In this article, we discuss the domain structure of Zizimin proteins from an evolutionary viewpoint. We also compare what is currently known about the mammalian Zizimin proteins to that of related Dock proteins. Understanding the cellular functions of these proteins will provide a better insight into their role in cell signaling, and may help in treating disease pathology associated with mutations in Dock/Zizimin proteins. PMID:23247359

  18. Solubilization and characterization of guanine nucleotide-sensitive muscarinic agonist binding sites from rat myocardium.

    PubMed Central

    Berrie, C. P.; Birdsall, N. J.; Hulme, E. C.; Keen, M.; Stockton, J. M.

    1984-01-01

    Muscarinic receptors from rat myocardial membranes may be solubilized by digitonin in good yield at low temperatures in the presence of Mg2+. Under these conditions, up to 60% of the soluble receptors show high affinity binding for the potent agonist [3H]-oxotremorine-M (KA = 10(9)M-1), which is inhibited by 5'-guanylylimidodiphosphate. The muscarinic binding site labelled with [3H]-oxotremorine-M has a higher sedimentation coefficient (13.4 s) than sites labelled with a 3H antagonist in the presence of guanylylimidodiphosphate (11.6 s) and probably represents a complex between the ligand binding subunit of the receptor and a guanine nucleotide binding protein. PMID:6478115

  19. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator.

  20. Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

    SciTech Connect

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

    1986-05-01

    The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.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 /sup 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/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..S 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/sub 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/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.

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

    PubMed

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

    2016-05-01

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

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

    PubMed

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

    2016-05-01

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

  3. Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras.

    PubMed

    Fam, N P; Fan, W T; Wang, Z; Zhang, L J; Chen, H; Moran, M F

    1997-03-01

    Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli. Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region. Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association with calmodulin in epithelial cells ectopically expressing Ras-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore-induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.

  4. The Guanine Nucleotide Exchange Factor ARNO mediates the activation of ARF and phospholipase D by insulin

    PubMed Central

    Li, Hai-Sheng; Shome, Kuntala; Rojas, Raúl; Rizzo, Mark A; Vasudevan, Chandrasekaran; Fluharty, Eric; Santy, Lorraine C; Casanova, James E; Romero, Guillermo

    2003-01-01

    Background Phospholipase D (PLD) is involved in many signaling pathways. In most systems, the activity of PLD is primarily regulated by the members of the ADP-Ribosylation Factor (ARF) family of GTPases, but the mechanism of activation of PLD and ARF by extracellular signals has not been fully established. Here we tested the hypothesis that ARF-guanine nucleotide exchange factors (ARF-GEFs) of the cytohesin/ARNO family mediate the activation of ARF and PLD by insulin. Results Wild type ARNO transiently transfected in HIRcB cells was translocated to the plasma membrane in an insulin-dependent manner and promoted the translocation of ARF to the membranes. ARNO mutants: ΔCC-ARNO and CC-ARNO were partially translocated to the membranes while ΔPH-ARNO and PH-ARNO could not be translocated to the membranes. Sec7 domain mutants of ARNO did not facilitate the ARF translocation. Overexpression of wild type ARNO significantly increased insulin-stimulated PLD activity, and mutations in the Sec7 and PH domains, or deletion of the PH or CC domains inhibited the effects of insulin. Conclusions Small ARF-GEFs of the cytohesin/ARNO family mediate the activation of ARF and PLD by the insulin receptor. PMID:12969509

  5. In vitro guanine nucleotide exchange activity of DHR-2/DOCKER/CZH2 domains.

    PubMed

    Côté, Jean-François; Vuori, Kristiina

    2006-01-01

    Rho family GTPases regulate a large variety of biological processes, including the reorganization of the actin cytoskeleton. Like other members of the Ras superfamily of small GTP-binding proteins, Rho GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and, when active, the GTPases relay extracellular signals to a large number of downstream effectors. Guanine nucleotide exchange factors (GEFs) promote the exchange of GDP for GTP on Rho GTPases, thereby activating them. Most Rho-GEFs mediate their effects through their signature domain known as the Dbl Homology-Pleckstrin Homology (DH-PH) module. Recently, we and others identified a family of evolutionarily conserved, DOCK180-related proteins that also display GEF activity toward Rho GTPases. The DOCK180-family of proteins lacks the canonical DH-PH module. Instead, they rely on a novel domain, termed DHR-2, DOCKER, or CZH2, to exchange GDP for GTP on Rho targets. In this chapter, the experimental approach that we used to uncover the exchange activity of the DHR-2 domain of DOCK180-related proteins will be described.

  6. Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1990-05-16

    Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin.

  7. Structure of the Rho-specific guanine nucleotide-exchange factor Xpln

    PubMed Central

    Murayama, Kazutaka; Kato-Murayama, Miyuki; Akasaka, Ryogo; Terada, Takaho; Yokoyama, Shigeyuki; Shirouzu, Mikako

    2012-01-01

    Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79 Å resolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the α4–α5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding. PMID:23192023

  8. Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease

    PubMed Central

    Cook, Danielle R.; Rossman, Kent L.; Der, Channing J.

    2016-01-01

    The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly by indirect mechanisms in disease. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflect the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2, Tiam1, Vav and P-Rex1/2. PMID:24037532

  9. The Guanine-Nucleotide Exchange Factor SGEF Plays a Crucial Role in the Formation of Atherosclerosis

    PubMed Central

    Kroon, Jeffrey; Welch, Christopher; Bakker, Erik N.; Matlung, Hanke L.; van den Berg, Timo K.; Sharek, Lisa; Doerschuk, Claire; Hahn, Klaus; Burridge, Keith

    2013-01-01

    The passage of leukocytes across the endothelium and into arterial walls is a critical step in the development of atherosclerosis. Previously, we showed in vitro that the RhoG guanine nucleotide exchange factor SGEF (Arhgef26) contributes to the formation of ICAM-1-induced endothelial docking structures that facilitate leukocyte transendothelial migration. To further explore the in vivo role of this protein during inflammation, we generated SGEF-deficient mice. When crossed with ApoE null mice and fed a Western diet, mice lacking SGEF showed a significant decrease in the formation of atherosclerosis in multiple aortic areas. A fluorescent biosensor revealed local activation of RhoG around bead-clustered ICAM-1 in mouse aortic endothelial cells. Notably, this activation was decreased in cells from SGEF-deficient aortas compared to wild type. In addition, scanning electron microscopy of intimal surfaces of SGEF−/− mouse aortas revealed reduced docking structures around beads that were coated with ICAM-1 antibody. Similarly, under conditions of flow, these beads adhered less stably to the luminal surface of carotid arteries from SGEF−/− mice. Taken together, these results show for the first time that a Rho-GEF, namely SGEF, contributes to the formation of atherosclerosis by promoting endothelial docking structures and thereby retention of leukocytes at athero-prone sites of inflammation experiencing high shear flow. SGEF may therefore provide a novel therapeutic target for inhibiting the development of atherosclerosis. PMID:23372835

  10. Proximal tubular epithelial cells possess a novel 42-kilodalton guanine nucleotide-binding regulatory protein.

    PubMed Central

    Zhou, J; Sims, C; Chang, C H; Berti-Mattera, L; Hopfer, U; Douglas, J

    1990-01-01

    The proximal tubule of the kidney represents an important location where adenylate cyclase regulates salt and water transport; yet a detailed characterization of the distribution and classification of guanine nucleotide-binding protein (G protein) and adenylate cyclase is lacking. We used purified brush border (20-fold) and basolateral membranes (14-fold) to characterize parathyroid hormone- and G protein-regulated adenylate cyclase and G-protein distribution. Adenylate cyclase was predominantly localized to basolateral membranes, while the 46-kDa alpha subunit of the stimulatory G protein (Gs) was 2-fold higher in brush border membranes than in basolateral membranes. The alpha subunit of the inhibitory G protein (Gi; 41 kDa) was equally distributed on immunoblotting but was 2-fold higher in brush border membranes than in basolateral membranes on radiolabeling with pertussis toxin. A 42-kDa cholera toxin substrate that cross-reacted with antisera to the common alpha subunit of G proteins and to Gs on immunoblotting and that was not immunoprecipitated with two Gi antisera was the most abundant alpha subunit and comprised approximately 1% of the total membrane proteins. These observations suggest that G proteins are important regulators of proximal tubular transport independent of adenylate cyclase. Images PMID:2120702

  11. Guanine nucleotide exchange factor H1 can be a new biomarker of melanoma

    PubMed Central

    Shi, Jie; Guo, Bingyu; Zhang, Yu; Hui, Qiang; Chang, Peng; Tao, Kai

    2016-01-01

    Guanine nucleotide exchange factor H1 (GEF-H1), which couples microtubule dynamics to RhoA activation, is a microtubule-regulated exchange factor. Studies have shown that GEF-H1 can be involved in various cancer pathways; however, the clinical significance of GEF-H1 expression and functions in melanoma has not been established. In this study, we investigated the relationship between clinical outcomes and GEF-H1 functions in melanoma. A total of 60 cases of different grades of melanoma samples were used to detect the expression of GEF-H1. Results showed that both messenger RNA and protein levels of GEF-H1 were significantly higher in high-grade melanomas. Furthermore, patients with high GEF-H1 expression had a shorter overall survival (22 months) than patients with low level of GEF-H1 expression (33.38 months). We also found that GEF-H1 can promote the proliferation and metastasis of melanoma cells. In summary, these results suggested that GEF-H1 may be a valuable biomarker for assessing the degree and prognosis of melanoma following surgery. PMID:27462139

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

    PubMed

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

    2015-05-01

    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.

  13. How not to do kinetics: examples involving GTPases and guanine nucleotide exchange factors.

    PubMed

    Goody, Roger S

    2014-01-01

    Guanine nucleotide exchange factors (GEFs) are crucial regulators of the action of GTPases in signal transduction and cellular regulation. Although their basic mechanism of action has been apparent for almost 20 years, there are still misconceptions concerning their properties, and these are confounded by superficial or incorrect interpretation of experimental results in individual cases. Here, an example is described in which an incorrect mechanism was derived because of an inadequate analysis of kinetic results. In a second example, a case is discussed where certain GTP analogs were erroneously described as being able to function as low molecular mass GEFs. In both cases, a lack of distinction between rates, rate constants, and apparent rate constants, together with a disregard of relative signal amplitudes, led to the misinterpretations. In a final example, it is shown how the lack of an appropriate kinetic investigation led to the false conclusion that a secreted protein from Legionella pneumophila can act not only as a GEF towards eukaryotic Rab1 but also as a factor that is able to actively dissociate the stable complex between Rab1 and GDP dissociation inhibitor. PMID:24112651

  14. Genetic interactions in yeast between Ypt GTPases and Arf guanine nucleotide exchangers.

    PubMed Central

    Jones, S; Jedd, G; Kahn, R A; Franzusoff, A; Bartolini, F; Segev, N

    1999-01-01

    Two families of GTPases, Arfs and Ypt/rabs, are key regulators of vesicular transport. While Arf proteins are implicated in vesicle budding from the donor compartment, Ypt/rab proteins are involved in the targeting of vesicles to the acceptor compartment. Recently, we have shown a role for Ypt31/32p in exit from the yeast trans-Golgi, suggesting a possible function for Ypt/rab proteins in vesicle budding as well. Here we report the identification of a new member of the Sec7-domain family, SYT1, as a high-copy suppressor of a ypt31/32 mutation. Several proteins that belong to the Sec7-domain family, including the yeast Gea1p, have recently been shown to stimulate nucleotide exchange by Arf GTPases. Nucleotide exchange by Arf GTPases, the switch from the GDP- to the GTP-bound form, is thought to be crucial for their function. Sec7p itself has an important role in the yeast secretory pathway. However, its mechanism of action is not yet understood. We show that all members of the Sec7-domain family exhibit distinct genetic interactions with the YPT genes. Biochemical assays demonstrate that, although the homology between the members of the Sec7-domain family is relatively low (20-35%) and limited to a small domain, they all can act as guanine nucleotide exchange factors (GEFs) for Arf proteins, but not for Ypt GTPases. The Sec7-domain of Sec7p is sufficient for this activity. Interestingly, the Sec7 domain activity is inhibited by brefeldin A (BFA), a fungal metabolite that inhibits some of the Arf-GEFs, indicating that this domain is a target for BFA. These results demonstrate that the ability to act as Arf-GEFs is a general property of all Sec7-domain proteins in yeast. The genetic interactions observed between Arf GEFs and Ypt GTPases suggest the existence of a Ypt-Arf GTPase cascade in the secretory pathway. PMID:10430582

  15. Catching Functional Modes and Structural Communication in Dbl Family Rho Guanine Nucleotide Exchange Factors.

    PubMed

    Raimondi, Francesco; Felline, Angelo; Fanelli, Francesca

    2015-09-28

    Computational approaches such as Principal Component Analysis (PCA) and Elastic Network Model-Normal Mode Analysis (ENM-NMA) are proving to be of great value in investigating relevant biological problems linked to slow motions with no demand in computer power. In this study, these approaches have been coupled to the graph theory-based Protein Structure Network (PSN) analysis to dissect functional dynamics and structural communication in the Dbl family of Rho Guanine Nucleotide Exchange Factors (RhoGEFs). They are multidomain proteins whose common structural feature is a DH-PH tandem domain deputed to the GEF activity that makes them play a central role in cell and cancer biology. While their common GEF action is accomplished by the DH domain, their regulatory mechanisms are highly variegate and depend on the PH and the additional domains as well as on interacting proteins. Major evolutionary-driven deformations as inferred from PCA concern the α6 helix of DH that dictates the orientation of the PH domain. Such deformations seem to depend on the mechanisms adopted by the GEF to prevent Rho binding, i.e. functional specialization linked to autoinhibition. In line with PCA, ENM-NMA indicates α6 and the linked PH domain as the portions of the tandem domain holding almost the totality of intrinsic and functional dynamics, with the α6/β1 junction acting as a hinge point for the collective motions of PH. In contrast, the DH domain holds a static scaffolding and hub behavior, with structural communication playing a central role in the regulatory actions by other domains/proteins. Possible allosteric communication pathways involving essentially DH were indeed found in those RhoGEFs acting as effectors of small or heterotrimeric RasGTPases. The employed methodology is suitable for deciphering structure/dynamics relationships in large sets of homologous or analogous proteins.

  16. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    SciTech Connect

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M. )

    1989-05-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increased the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.

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

    SciTech Connect

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

    1987-02-10

    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 ..cap alpha.. 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 ..beta.. subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the ..cap alpha.. 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 s..cap alpha../ relative to G/sub ichemically bond/ and G/sub ochemically bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with (/sup 125/I)protein. Immunohistochemical studies using an antiserum against the ..beta.. 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 ..cap alpha.. 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.

  18. The selective phosphorylation of a guanine nucleotide-binding regulatory protein

    SciTech Connect

    Carlson, K.E.

    1989-01-01

    Receptor-activated signal transduction pathways regulate the responsiveness of cells to external stimuli. These transduction pathways themselves are subject to regulation, most commonly by phosphorylation. Guanine nucleotide-binding regulatory proteins (G Proteins), as requisite signal transducing elements for many plasma membrane receptors, are considered likely targets for regulation by phosphorylation. Protein kinase C (PKC) has been shown to phosphorylate the {alpha} subunit of G{sub i} and other G proteins in solution. However, the occurrence of the phosphorylation of G{sub 1} within intact cells in response to activation of PKC has not been rigorously demonstrated. In this thesis, the extent to which the {alpha} subunits of G{sub i} undergo phosphorylation within human platelets in response to activation of PKC was examined by means of radiolabeling and immunoprecipitation. Incubation of platelets with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, promoted the phosphorylation of several proteins within saponin-permeabilized and intact platelets incubated with ({gamma}{sup 32}P)ATP and ({sup 32}P)H{sub 3}PO{sub 4}, respectively. None of the phosphoproteins, however, were precipitated by either of two antisera containing antibodies differing in specificities for epitopes within G{sub i{alpha}}-despite precipitation of a substantial fraction of the subunit itself. In contrast, other antisera, containing antibodies specific for the recently describe G{sub z{alpha}}, or antibodies for both G{sub z{alpha}} and G{sub i{alpha}}, precipitated a 40-kDa phosphoprotein.

  19. Activation of JNK by Epac is independent of its activity as a Rap guanine nucleotide exchanger.

    PubMed

    Hochbaum, Daniel; Tanos, Tamara; Ribeiro-Neto, Fernando; Altschuler, Daniel; Coso, Omar A

    2003-09-01

    Guanine nucleotide exchange factors (GEFs) and their associated GTP-binding proteins (G-proteins) are key regulatory elements in the signal transduction machinery that relays information from the extracellular environment into specific intracellular responses. Among them, the MAPK cascades represent ubiquitous downstream effector pathways. We have previously described that, analogous to the Ras-dependent activation of the Erk-1/2 pathway, members of the Rho family of small G-proteins activate the JNK cascade when GTP is loaded by their corresponding GEFs. Searching for novel regulators of JNK activity we have identified Epac (exchange protein activated by cAMP) as a strong activator of JNK-1. Epac is a member of a growing family of GEFs that specifically display exchange activity on the Rap subfamily of Ras small G-proteins. We report here that while Epac activates the JNK severalfold, a constitutively active (G12V) mutant of Rap1b does not, suggesting that Rap-GTP is not sufficient to transduce Epac-dependent JNK activation. Moreover, Epac signaling to the JNKs was not blocked by inactivation of endogenous Rap, suggesting that Rap activation is not necessary for this response. Consistent with these observations, domain deletion mutant analysis shows that the catalytic GEF domain is dispensable for Epac-mediated activation of JNK. These studies identified a region overlapping the Ras exchange motif domain as critical for JNK activation. Consistent with this, an isolated Ras exchange motif domain from Epac is sufficient to activate JNK. We conclude that Epac signals to the JNK cascade through a new mechanism that does not involve its canonical catalytic action, i.e. Rap-specific GDP/GTP exchange. This represents not only a novel way to activate the JNKs but also a yet undescribed mechanism of downstream signaling by Epac.

  20. Solution structures of oligonucleotides containing either a guanine or a cytosine in front of a gap of one nucleotide

    NASA Astrophysics Data System (ADS)

    Boulard, Y.; Faibis, V.; Fazakerley, G. V.

    1999-10-01

    We report NMR and molecular modelling studies on two DNA duplexes containing a gap of one nucleotides. The difference between the two oligonucleotides lies in the central base face to the gap, a guanine or a cytosine. For the gapG, we observed in solution a B-form conformation where the guanine stacks in the helix. For the gapC, we reveal the existence of two species, one majority where the cytosine is inside the helix and a second for which the cytosine is extrahelical. Nous présentons une étude par RMN et modélisation moléculaire sur deux duplexes d'ADN contenant une lacune de un nucléotide. La différence entre les deux oligonucléotides réside dans la base centrale en face de la lacune, une guanine ou une cytosine. Pour le duplex appelé gapG, nous observons en solution une hélice de type B dans laquelle la guanine est empilée à l'intérieur de l'hélice. Dans le cas du duplex gapC, nous montrons l'existence de deux formes, l'une où la cytosine est à l'intérieur de l'hélice; la seconde où la cytosine est extra hélicale.

  1. Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain.

    PubMed Central

    Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1994-01-01

    ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A. PMID:8159707

  2. The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial–mesenchymal transition

    PubMed Central

    Komiya, Y; Onodera, Y; Kuroiwa, M; Nomimura, S; Kubo, Y; Nam, J-M; Kajiwara, K; Nada, S; Oneyama, C; Sabe, H; Okada, M

    2016-01-01

    Epithelial tumor cells often acquire malignant properties, such as invasion/metastasis and uncontrolled cell growth, by undergoing epithelial–mesenchymal transition (EMT). However, the mechanisms by which EMT contributes to malignant progression remain elusive. Here we show that the Rho guanine nucleotide exchange factor (GEF) ARHGEF5 promotes tumor malignancy in a manner dependent on EMT status. We previously identified ARHGEF5, a member of the Dbl family of GEFs, as a multifunctional mediator of Src-induced cell invasion and tumor growth. In the present study, ARHGEF5 was upregulated during tumor growth factor-β-induced EMT in human epithelial MCF10A cells, and promoted cell migration by activating the Rho-ROCK pathway. ARHGEF5 was necessary for the invasive and in vivo metastatic activity of human colorectal cancer HCT116 cells. These findings underscore the crucial role of ARHGEF5 in cell migration and invasion/metastasis. An in vivo tumorigenesis assay revealed that ARHGEF5 had the potential to promote tumor growth via the phosphatidylinositol 3-kinase (PI3K) pathway. However, ARHGEF5 was not required for tumor growth in epithelial-like human colorectal cancer HCT116 and HT29 cells, whereas the growth of mesenchymal-like SW480 and SW620 cells depended on ARHGEF5. Induction of EMT by tumor necrosis factor-α or Slug in HCT116 cells resulted in the dependence of tumor growth on ARHGEF5. In these mesenchymal-like cells, Akt was activated via ARHGEF5 and its activity was required for tumor growth. Analysis of a transcriptome data set revealed that the combination of ARHGEF5 upregulation and E-cadherin downregulation or Snail upregulation was significantly correlated with poor prognosis in patients with colorectal cancers. Taken together, our findings suggest that EMT-induced ARHGEF5 activation contributes to the progression of tumor malignancy. ARHGEF5 may serve as a potential therapeutic target in a subset of malignant tumors that have undergone EMT. PMID

  3. The Rho guanine nucleotide exchange factor ARHGEF5 promotes tumor malignancy via epithelial-mesenchymal transition.

    PubMed

    Komiya, Y; Onodera, Y; Kuroiwa, M; Nomimura, S; Kubo, Y; Nam, J-M; Kajiwara, K; Nada, S; Oneyama, C; Sabe, H; Okada, M

    2016-01-01

    Epithelial tumor cells often acquire malignant properties, such as invasion/metastasis and uncontrolled cell growth, by undergoing epithelial-mesenchymal transition (EMT). However, the mechanisms by which EMT contributes to malignant progression remain elusive. Here we show that the Rho guanine nucleotide exchange factor (GEF) ARHGEF5 promotes tumor malignancy in a manner dependent on EMT status. We previously identified ARHGEF5, a member of the Dbl family of GEFs, as a multifunctional mediator of Src-induced cell invasion and tumor growth. In the present study, ARHGEF5 was upregulated during tumor growth factor-β-induced EMT in human epithelial MCF10A cells, and promoted cell migration by activating the Rho-ROCK pathway. ARHGEF5 was necessary for the invasive and in vivo metastatic activity of human colorectal cancer HCT116 cells. These findings underscore the crucial role of ARHGEF5 in cell migration and invasion/metastasis. An in vivo tumorigenesis assay revealed that ARHGEF5 had the potential to promote tumor growth via the phosphatidylinositol 3-kinase (PI3K) pathway. However, ARHGEF5 was not required for tumor growth in epithelial-like human colorectal cancer HCT116 and HT29 cells, whereas the growth of mesenchymal-like SW480 and SW620 cells depended on ARHGEF5. Induction of EMT by tumor necrosis factor-α or Slug in HCT116 cells resulted in the dependence of tumor growth on ARHGEF5. In these mesenchymal-like cells, Akt was activated via ARHGEF5 and its activity was required for tumor growth. Analysis of a transcriptome data set revealed that the combination of ARHGEF5 upregulation and E-cadherin downregulation or Snail upregulation was significantly correlated with poor prognosis in patients with colorectal cancers. Taken together, our findings suggest that EMT-induced ARHGEF5 activation contributes to the progression of tumor malignancy. ARHGEF5 may serve as a potential therapeutic target in a subset of malignant tumors that have undergone EMT. PMID

  4. A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus

    PubMed Central

    1994-01-01

    The Golgi apparatus is a dynamic organelle whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for rab1a, a GTPase previously associated with ER to Golgi and intra-Golgi transport, in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rab1a protein was microinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rab1a(S25N), a constitutively GDP-bound form (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225- 237), and mutant rab1a(N124I) defective in guanine nucleotide binding. Microinjection of wild-type rab1a protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of alpha-mannosidase II (Man II), used as a Golgi marker. In contrast, microinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rab1a mutants differs from BFA-induced disruption, since beta- COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rab1 mutants or GTP gamma S. We conclude that GTP exchange and hydrolysis by GTPases, specifically rab1a, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rab1a mutants to that occurring during

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

    PubMed Central

    Aoki, Takuji; Ueda, Shuji; Kataoka, Tohru; Satoh, Takaya

    2009-01-01

    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 the regulation of

  6. Guanine nucleotide is essential and Ca2+ is a modulator in the exocytotic reaction of permeabilized rat mast cells.

    PubMed Central

    Lillie, T H; Gomperts, B D

    1992-01-01

    Exocytosis from metabolically depleted permeabilized rat mast cells was measured in response to provision of Ca2+ and guanine nucleotide [GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S])]. For cells permeabilized in simple salt solutions (NaCl), both of these effectors were required to induce secretion. Exclusion of Mg2+ caused an increase in both the sensitivity of the system to GTP and the extent of secretion elicited, while having no such effects on secretion induced by GTP[S]. The effect of Mg2+ depletion on the ability of GTP to stimulate secretion is probably due to the dependence on Mg2+ of the GTPase activity of GE (a postulated GTP-binding protein which mediates exocytosis). This argues that a persistent stimulus to the G-protein is required to support secretion. Affinity for both GTP[S] and GTP is enhanced when the cells are permeabilized in zwitterionic electrolytes (glutamate, gamma-aminobutyric acid, glycine) instead of NaCl. Under these conditions, secretion occurs in response to provision of either GTP[S] [in the effective absence of Ca2+ (pCa 9)] or Ca2+ (in the absence of guanine nucleotide). Secretion induced by GTP[S] is strongly promoted by the presence of Mg2+ at concentrations in the millimolar range; this promotion by Mg2+ declines as the concentration of Ca2+ is elevated towards pCa 7. At pCa 6, Mg2+ is without effect. Ca(2+)-induced secretion requires the provision of MgATP. Since this is further enhanced by low concentrations (< 100 microM) and then inhibited by high concentrations of GDP, the essential role of ATP is likely to be in the maintenance of GTP via transphosphorylation by a nucleoside diphosphate kinase reaction. Thus, under conditions of high affinity (glutamate environment), GTP[S] alone is capable of inducing exocytosis. Ca2+ acts in concert with guanine nucleotides: it enhances the rate and extent of secretion and increases the affinity for Mg2+ and guanine nucleotides in the activation of the GTP-binding protein (GE

  7. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    PubMed Central

    Price, S R; Nightingale, M; Tsai, S C; Williamson, K C; Adamik, R; Chen, H C; Moss, J; Vaughan, M

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on the partial amino acid sequence of one of the soluble ARFs from bovine brain. Comparison of the deduced amino acid sequence of lambda ARF2B with sequences of peptides from the ARF protein (total of 60 amino acids) revealed only two differences. Whether these are cloning artifacts or reflect the existence of more than one ARF protein remains to be determined. Deduced amino acid sequences of ARF, Go alpha (the alpha subunit of a G protein that may be involved in regulation of ion fluxes), and c-Ha-ras gene product p21 show similarities in regions believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF apparently lacks a site analogous to that ADP-ribosylated by choleragen in G-protein alpha subunits. Although both the ARF proteins and the alpha subunits bind guanine nucleotides and serve as choleragen substrates, they must interact with the toxin A1 peptide in different ways. In addition to serving as an ADP-ribose acceptor, ARF interacts with the toxin in a manner that modifies its catalytic properties. PMID:3135549

  8. Gef1p, a New Guanine Nucleotide Exchange Factor for Cdc42p, Regulates Polarity in Schizosaccharomyces pombe

    PubMed Central

    Coll, Pedro M.; Trillo, Yadira; Ametzazurra, Amagoia; Perez, Pilar

    2003-01-01

    Schizosaccharomyces pombe cdc42+ regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1+ increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1+ causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1+ deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1Δ scd1Δ is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1+ or scd1+ causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed. PMID:12529446

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

    SciTech Connect

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

    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.

  10. Sestrins Function as Guanine Nucleotide Dissociation Inhibitors for Rag GTPases to Control mTORC1 Signaling

    PubMed Central

    Peng, Min; Yin, Na; Li, Ming O.

    2014-01-01

    SUMMARY Mechanistic target of rapamycin complex 1 (mTORC1) integrates diverse environmental signals to control cellular growth and organismal homeostasis. In response to nutrients, Rag GTPases recruit mTORC1 to the lysosome to be activated, but how Rags are regulated remains incompletely understood. Here we show that Sestrins bind to the heterodimeric RagA/B-RagC/D GTPases, and function as guanine nucleotide dissociation inhibitors (GDIs) for RagA/B. Sestrin overexpression inhibits amino acid-induced Rag guanine nucleotide exchange and mTORC1 translocation to the lysosome. Mutation of the conserved GDI motif creates a dominant-negative form of Sestrin that renders mTORC1 activation insensitive to amino acid deprivation, whereas a cell-permeable peptide containing the GDI motif inhibits mTORC1 signaling. Mice deficient in all Sestrins exhibit reduced postnatal survival associated with defective mTORC1 inactivation in multiple organs during neonatal fasting. These findings reveal a non-redundant mechanism by which the Sestrin family of GDIs regulates the nutrient-sensing Rag GTPases to control mTORC1 signaling. PMID:25259925

  11. Role of guanine nucleotide binding protein(s) in vasopressin-induced responses of a vascular smooth muscle cell line

    SciTech Connect

    Nambi, P.; Aiyar, N.; Whitman, M.; Stassen, F.L.; Crooke, S.T.

    1986-05-01

    Rat aortic smooth muscle cells (A-10) carry vascular V1 vasopressin receptors. In these cells, vasopressin inhibits isoproterenol-induced cAMP accumulation and stimulates phosphatidylinositol turnover and Ca/sup 2 +/ mobilization. Pretreatment of the cells with phorbol esters resulted in inhibition of the vasopressin-induced responses. The inactive phorbol ester aPDD was ineffective. These data suggested that phorbol ester might cause phosphorylation of the vasopressin receptor and/or coupling protein(s). Here, they studied the role of guanine nucleotide binding proteins by employing the novel radiolabeled vasopressin antagonist (/sup 3/H)-SKF 101926. In competition experiments with cell membranes, Gpp(NH)p shifted the vasopressin curve to the right indicating decreased agonist affinity. Phorbol ester pretreatment abolished the Gpp(NH)p effect. Pretreatment of the cells with N-ethylmaleimide (NEM) resulted in inhibition of vasopressin-induced phosphatidyinositol turnover. NEM also abolished the decrease in agonist affinity caused by Gpp(NH)p. These data showed that NEM and phorbol ester pretreatment of smooth muscle cells functionally uncoupled the vasopressin receptors and suggested that vasopressin V1 receptor responses are mediated through guanine nucleotide binding protein(s).

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

    PubMed Central

    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

  13. New insights into the dimerization of small GTPase Rac/ROP guanine nucleotide exchange factors in rice

    PubMed Central

    Akamatsu, Akira; Uno, Kazumi; Kato, Midori; Wong, Hann Ling; Shimamoto, Ko; Kawano, Yoji

    2015-01-01

    Molecular links between receptor-kinases and Rac/ROP family small GTPases mediated by activator guanine nucleotide exchange factors (GEFs) govern diverse biological processes. However, it is unclear how the Rac/ROP GTPases orchestrate such a wide variety of activities. Here, we show that rice OsRacGEF1 forms homodimers, and heterodimers with OsRacGEF2, at the plasma membrane (PM) and the endoplasmic reticulum (ER). OsRacGEF2 does not bind directly to the receptor-like kinase (RLK) OsCERK1, but forms a complex with OsCERK1 through OsRacGEF1 at the ER. This complex is transported from ER to the PM and there associates with OsRac1, resulting in the formation of a stable immune complex. Such RLK-GEF heterodimer complexes may explain the diversity of Rac/ROP family GTPase signalings. PMID:26251883

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

    PubMed Central

    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. Images PMID:2474826

  15. Guanine nucleotide exchange factors (GEFs) have a critical but not exclusive role in organelle localization of Rab GTPases.

    PubMed

    Cabrera, Margarita; Ungermann, Christian

    2013-10-01

    Membrane fusion at eukaryotic organelles is initiated by Rab GTPases and tethering factors. Rabs in their GDP-bound form are kept soluble in the cytoplasm by the GDP dissociation inhibitor (GDI) chaperone. Guanine nucleotide exchange factors (GEFs) are found at organelles and are critical for Rab function. Here, we surveyed the overall role of GEFs in Rab localization. We show that GEFs, but none of the proposed GDI displacement factors, are essential for the correct membrane localization of yeast Rabs. In the absence of the GEF, Rabs lost their primary localization to the target organelle. Several Rabs, such as vacuolar Ypt7, were found at the endoplasmic reticulum and thus were still membrane-bound. Surprisingly, a Ypt7 mutant that undergoes facilitated nucleotide exchange localized to vacuoles independently of its GEF Mon1-Ccz1 and rescued vacuole morphology. In contrast, wild-type Ypt7 required its GEF for localization and to counteract the extraction by GDI. Our data agree with the emerging model that GEFs are critical for Rab localization but raise the possibility that additional factors can contribute to this process.

  16. Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak.

    PubMed

    Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

    2014-01-01

    In this study, we compared the influence of GDP and GTP on isolated mitochondria respiring under conditions favoring oxidative phosphorylation (OXPHOS) and under conditions excluding this process, i.e., in the presence of carboxyatractyloside, an adenine nucleotide translocase inhibitor, and/or oligomycin, an FOF1-ATP synthase inhibitor. Using mitochondria isolated from rat kidney and human endothelial cells, we found that the action of GDP and GTP can differ diametrically depending on the conditions. Namely, under conditions favoring OXPHOS, both in the absence and presence of linoleic acid, an activator of uncoupling proteins (UCPs), the addition of 1 mM GDP resulted in the state 4 (non-phosphorylating respiration)-state 3 (phosphorylating respiration) transition, which is characteristic of ADP oxidative phosphorylation. In contrast, the addition of 1 mM GTP resulted in a decrease in the respiratory rate and an increase in the membrane potential, which is characteristic of UCP inhibition. The stimulatory effect of GDP, but not GTP, was also observed in inside-out submitochondrial particles prepared from rat kidney mitochondria. However, the effects of GDP and GTP were more similar in the presence of OXPHOS inhibitors. The importance of these observations in connection with the action of UCPs, adenine nucleotide translocase (or other carboxyatractyloside-sensitive carriers), carboxyatractyloside- and purine nucleotide-insensitive carriers, as well as nucleoside-diphosphate kinase (NDPK) are considered. Because the measurements favoring oxidative phosphorylation better reflect in vivo conditions, our study strongly supports the idea that GDP cannot be considered a significant physiological inhibitor of UCP. Moreover, it appears that, under native conditions, GTP functions as a more efficient UCP inhibitor than GDP and ATP.

  17. Guanine Nucleotide Exchange Factor OSG-1 Confers Functional Aging via Dysregulated Rho Signaling in Caenorhabditis elegans Neurons

    PubMed Central

    Duan, Zhibing; Sesti, Federico

    2015-01-01

    Rho signaling regulates a variety of biological processes, but whether it is implicated in aging remains an open question. Here we show that a guanine nucleotide exchange factor of the Dbl family, OSG-1, confers functional aging by dysregulating Rho GTPases activities in C. elegans. Thus, gene reporter analysis revealed widespread OSG-1 expression in muscle and neurons. Loss of OSG-1 gene function was not associated with developmental defects. In contrast, suppression of OSG-1 lessened loss of function (chemotaxis) in ASE sensory neurons subjected to conditions of oxidative stress generated during natural aging, by oxidative challenges, or by genetic mutations. RNAi analysis showed that OSG-1 was specific toward activation of RHO-1 GTPase signaling. RNAi further implicated actin-binding proteins ARX-3 and ARX-5, thus the actin cytoskeleton, as one of the targets of OSG-1/RHO-1 signaling. Taken together these data suggest that OSG-1 is recruited under conditions of oxidative stress, a hallmark of aging, and contributes to promote loss of neuronal function by affecting the actin cytoskeleton via altered RHO-1 activity. PMID:25527286

  18. The Garz Sec7 domain guanine nucleotide exchange factor for Arf regulates salivary gland development in Drosophila

    PubMed Central

    Szul, Tomasz; Burgess, Jason; Jeon, Mili; Zinn, Kai; Marques, Guillermo; Brill, Julie A

    2011-01-01

    Surface delivery of proteins involved in cell-cell and cell-matrix interactions in cultured mammalian cells requires the GBF1 guanine nucleotide exchange factor. However, the role of GBF1 in delivery of adhesion proteins during organogenesis in intact animals has not been characterized. Here, we report the function of the fly GBF1 homolog, Gartenzwerg (Garz) in the development of the salivary gland in Drosophila melanogaster. We used the GAL4/UAS system to selectively deplete Garz from salivary gland cells. We show that depletion of Garz disrupts the secretory pathway as evidenced by the collapse of Golgi-localized Lava lamp (Lva) and the TGN-localized γ subunit of the clathrin-adaptor protein complex (AP-1). Additionally, Garz depletion inhibits trafficking of cell-cell adhesion proteins cadherin (DE-cad) and Flamingo to the cell surface. Disregulation of trafficking correlates with mistargeting of the tumor suppressor protein Discs large involved in epithelial polarity determination. Garz-depleted salivary cells are smaller and lack well-defined plasma membrane domains. Garz depletion also inhibits normal elongation and positioning of epithelial cells, resulting in a disorganized salivary gland that lacks a well defined luminal duct. Our findings suggest that Garz is essential for establishment of epithelial structures and demonstrate an absolute requirement for Garz during Drosophila development. PMID:21686256

  19. Ccpg1, a Novel Scaffold Protein That Regulates the Activity of the Rho Guanine Nucleotide Exchange Factor Dbs▿

    PubMed Central

    Kostenko, Elena V.; Olabisi, Oyenike O.; Sahay, Sutapa; Rodriguez, Pedro L.; Whitehead, Ian P.

    2006-01-01

    Dbs is a Rho-specific guanine nucleotide exchange factor (RhoGEF) with in vitro exchange activity specific for RhoA and Cdc42. Like many RhoGEF family members, the in vivo exchange activity of Dbs is restricted in a cell-specific manner. Here we report the characterization of a novel scaffold protein (designated cell cycle progression protein 1 [Ccpg1]) that interacts with Dbs and modulates its in vivo exchange specificity. When coexpressed in mammalian cells, Ccpg1 binds to the Dbl homology/pleckstrin homology domain tandem motif of Dbs and inhibits its exchange activity toward RhoA, but not Cdc42. Expression of Ccpg1 correlates with the ability of Dbs to activate endogenous RhoA in cultured cells, and suppression of endogenous Ccpg1 expression potentiates Dbs exchange activity toward RhoA. The isolated Dbs binding domain of Ccpg1 is not sufficient to suppress Dbs exchange activity on RhoA, thus suggesting a regulatory interaction. Ccpg1 mediates recruitment of endogenous Src kinase into Dbs-containing complexes and interacts with the Rho family member Cdc42. Collectively, our studies suggest that Ccpg1 represents a new class of regulatory scaffold protein that can function as both an assembly platform for Rho protein signaling complexes and a regulatory protein which can restrict the substrate utilization of a promiscuous RhoGEF family member. PMID:17000758

  20. A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

    PubMed

    Reichman, Melvin; Schabdach, Amanda; Kumar, Meera; Zielinski, Tom; Donover, Preston S; Laury-Kleintop, Lisa D; Lowery, Robert G

    2015-12-01

    Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

  1. Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis.

    PubMed

    Okada, Keisuke; Miyake, Hideaki; Yamaguchi, Kohei; Chiba, Koji; Maeta, Kazuhiro; Bilasy, Shymaa E; Edamatsu, Hironori; Kataoka, Tohru; Fujisawa, Masato

    2014-02-28

    Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2(-)(/)(-) and wild-type mice. However, the testes of RA-GEF-2(-)(/)(-) male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2(-)(/)(-) males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2(-)(/)(-) mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2(-)(/)(-) testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice. PMID:24491570

  2. The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane Recycling in Osteoclasts*

    PubMed Central

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-01-01

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. PMID:24821726

  3. The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

    PubMed

    Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

    2014-06-27

    The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. PMID:24821726

  4. Alternative splicing of the guanine nucleotide-binding regulatory protein Go alpha generates four distinct mRNAs.

    PubMed Central

    Murtagh, J J; Moss, J; Vaughan, M

    1994-01-01

    Go alpha a guanine nucleotide-binding (G) protein abundant in brain and other neural tissues, has been implicated in ion channel regulation. Concerted efforts in several laboratories have revealed multiple Go alpha mRNAs and protein isoforms in different contexts. Go alpha is a single copy gene in mammalian species, although the structure, number and tissue localization of Go alpha mRNAs reported by investigators are inconsistent. To define the cell-specific expression of alternatively spliced variants of Go alpha mRNA, we employed several strategies, including Northern hybridizations with sequences-specific oligonucleotides, selective digestions of Go alpha mRNA using RNase H, and adaptations of the polymerase chain reaction. Four distinct alternatively spliced variants were identified, a 5.7-kb Go alpha 2 mRNA and three Go alpha 1 mRNAs with different 3' UTRs. The UTRs of the three Go alpha 1s are composed of different combinations of what have been referred to as UTR-A and UTR-B. The sequences of the spliced segments are well conserved among mammalian species, suggesting a functional role for these alternatively spliced 3' UTRs in post-transcriptional and/or tissue-specific regulation of Go alpha expression. The position of the intron-exon splice boundary at nucleotide 31 following T of the TGA stop codon is conserved in the Gi alpha 2 and Gi alpha 3 genes, consistent with the notion that similar alternative splicing of 3' UTRs occurs in products of these related genes. Images PMID:8139926

  5. The importance of interaction with membrane lipids through the pleckstrin homology domain of the guanine nucleotide exchange factor for rho family small guanosine triphosphatase, FLJ00018.

    PubMed

    Kimura, Shinji; Sato, Katsuya; Banno, Yoshiko; Nagase, Takahiro; Ueda, Hiroshi

    2013-01-01

    FLJ00018, a heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein (G protein) Gβγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, regulates cellular responses, including cell morphological changes and gene transcriptional regulation, and targets the cellular membranes. FLJ00018 contains a Dbl homology (DH) domain in addition to a pleckstrin homology (PH) domain. Here we show that the PH domain of FLJ00018 is required for FLJ00018-induced, serum response element-dependent gene transcription. Although the PH domain of KIAA1415/P-Rex1, another Gβγ subunit-activated guanine nucleotide exchange factor for Rho family small GTPases, binds to phosphatidylinositol 3,4,5-triphosphate and phosphatidylinositol 3,4-bisphosphate, the PH domain of FLJ00018 binds to polyphosphoinositides including phosphatidylinositol 4,5-bisphosphate, and phosphatidic acid. These results suggest that FLJ00018 is targeted via its PH domain to cellular membranes.

  6. Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation.

    PubMed

    Bobak, D A; Bliziotes, M M; Noda, M; Tsai, S C; Adamik, R; Moss, J

    1990-01-30

    Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF.

  7. Role of the guanine nucleotide exchange factor Ost in negative regulation of receptor endocytosis by the small GTPase Rac1.

    PubMed

    Ieguchi, Katsuaki; Ueda, Shuji; Kataoka, Tohru; Satoh, Takaya

    2007-08-10

    The Rho family of GTPases has been implicated in the regulation of intracellular vesicle trafficking. Here, we investigated the mechanism underlying the negative regulation of clathrin-mediated endocytosis of cell surface receptors mediated by the Rho family protein Rac1. Contrary to previous reports, only the activated mutant of Rac1, but not other Rho family members including RhoA and Cdc42, suppressed internalization of the transferrin receptor. On the other hand, down-regulation of Rac1 expression by RNA interference resulted in enhanced receptor internalization, suggesting that endogenous Rac1 in fact functions as a negative regulator. We identified a guanine nucleotide exchange factor splice variant designated Ost-III, which contains a unique C-terminal region including an Src homology 3 domain, as a regulator of Rac1 involved in the inhibition of receptor endocytosis. In contrast, other splice variants Ost-I and Ost-II exerted virtually no effect on receptor endocytosis. We also examined subcellular localization of synaptojanin 2, a putative Rac1 effector implicated in negative regulation of receptor endocytosis. Each Ost splice variant induced distinct subcellular localization of synaptojanin 2, depending on Rac1 activation. Furthermore, we isolated gamma-aminobutyric acid type A receptor-associated protein (GABARAP) as a protein that binds to the C-terminal region of Ost-III. When ectopically expressed, GABARAP was co-localized with Ost-III and potently suppressed the Ost-III-dependent Rac1 activation and the inhibition of receptor endocytosis. Lipid modification of GABARAP was necessary for the suppression of Ost-III. These results are discussed in terms of subcellular region-specific regulation of the Rac1-dependent signaling pathway that negatively regulates clathrin-mediated endocytosis.

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

    SciTech Connect

    Du, Yue; Meng, Jinyi; Huang, Yuhong; Wu, Jun; Wang, Bo; Ibrahim, Mohammed M.; Tang, Jianwu

    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.

  9. Oxytocin receptors on cultured astroglial cells. Regulation by a guanine-nucleotide-binding protein and effect of Mg2+.

    PubMed Central

    Di Scala-Guenot, D; Strosser, M T

    1992-01-01

    Specific binding sites for the radio-iodinated oxytocin (OT) antagonist d(CH2)5-[Tyr(Me)2,Thr4, Tyr-NH2(9)]OVT ([125I]OTA) have been characterized on cultured hypothalamic astroglial cell membranes. The rate of association of the ligand to OT-binding sites was identical in the presence and the absence of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (Gpp[NH]p, 0.1 mM), whereas the monophasic dissociation reaction became biphasic in the presence of Gpp[NH]p. Scatchard analysis of equilibrium binding of [125I]OTA resulted in a linear plot with a single class of binding sites (Kd 0.06 nM) which were insensitive to the addition of Gpp[NH]p. Unlabelled OT and [Arg8]vasopressin (AVP) bound to high- (H) and low- (L) affinity states with a dissociation constant ratio (KL/KH) of 100 for both hormones. Binding with both high and low affinity required the presence of Mg2+ in the incubation buffer, and the addition of Gpp[NH]p decreased the KL/KH ratio to 10 and increased the percentage of low-affinity binding sites. On the other hand, neither omission of Mg2+ from the buffer nor the addition of Gpp[NH]p altered the binding of either OT or V1 AVP antagonists to OT receptors. In the presence of a G-protein inactivator (N-ethylmaleimide; 3 mM) during OT competition studies the affinities of the two OT-binding sites were unchanged, but 90% of the high-affinity binding sites were converted into the low-affinity state. These results obtained with cultured hypothalamic astroglial cells provide further evidence for a coupling of OT receptors with a guanine-nucleotide-binding protein, with a requirement for Mg2+. PMID:1318032

  10. Interaction of brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 and kinesin motor protein KIF21A

    PubMed Central

    Shen, Xiaoyan; Meza-Carmen, Victor; Puxeddu, Ermanno; Wang, Guanghui; Moss, Joel; Vaughan, Martha

    2008-01-01

    Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates human ADP-ribosylation factor (ARF) 1 and 3 by accelerating the replacement of ARF-bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Liquid chromatography MS/MS analysis of peptides from proteins that co-precipitated with BIG1 antibodies identified “kinesin family member 21A” (KIF21A), a plus-end-directed motor protein that moves cargo on microtubules away from the microtubule-organizing center. Reciprocal immunoprecipitation (IP) of endogenous proteins and microscopically apparent overlap of immunoreactive BIG1 with overexpressed GFP-KIF21A in the perinuclear region were consistent with an interaction of KIF21A–BIG1. Overexpression of full-length KIF21A and BIG1 and their fragments in HEK293 cells followed by reciprocal IP revealed that the C-terminal tail of KIF21A, with seven WD-40 repeats, may interact with structure in the C-terminal region of BIG1. Interfering with cyclic activation and inactivation of ARF1 by overexpressing constitutively active ARF1(Q71L) or dominant inactive ARF1(T31N) altered the distribution of BIG1 as well as its interaction with KIF21A. A requirement for ARF1 was confirmed by its selective depletion with siRNA. Unlike disruption of microtubules with nocodazole, selective inhibition of transport by depletion of KIF21A with specific siRNA altered BIG1 distribution without changing that of intrinsic Golgi membrane proteins. These newly recognized interactions of BIG1 and KIF21A should enable us to understand better the mechanisms through which, acting together, they may integrate local events in membrane trafficking with longer-range transport processes and to relate those processes to the diverse signaling and scaffold functions of BIG1. PMID:19020088

  11. Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1*

    PubMed Central

    Pan, Dingxin; Barber, Mark A.; Hornigold, Kirsti; Baker, Martin J.; Toth, Judit M.; Oxley, David; Welch, Heidi C. E.

    2016-01-01

    P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates the small G protein (GTPase) Rac1 to control Rac1-dependent cytoskeletal dynamics, and thus cell morphology. Three mechanisms of P-Rex1 regulation are currently known: (i) binding of the phosphoinositide second messenger PIP3, (ii) binding of the Gβγ subunits of heterotrimeric G proteins, and (iii) phosphorylation of various serine residues. Using recombinant P-Rex1 protein to search for new binding partners, we isolated the G-protein-coupled receptor (GPCR)-adaptor protein Norbin (Neurochondrin, NCDN) from mouse brain fractions. Coimmunoprecipitation confirmed the interaction between overexpressed P-Rex1 and Norbin in COS-7 cells, as well as between endogenous P-Rex1 and Norbin in HEK-293 cells. Binding assays with purified recombinant proteins showed that their interaction is direct, and mutational analysis revealed that the pleckstrin homology domain of P-Rex1 is required. Rac-GEF activity assays with purified recombinant proteins showed that direct interaction with Norbin increases the basal, PIP3- and Gβγ-stimulated Rac-GEF activity of P-Rex1. Pak-CRIB pulldown assays demonstrated that Norbin promotes the P-Rex1-mediated activation of endogenous Rac1 upon stimulation of HEK-293 cells with lysophosphatidic acid. Finally, immunofluorescence microscopy and subcellular fractionation showed that coexpression of P-Rex1 and Norbin induces a robust translocation of both proteins from the cytosol to the plasma membrane, as well as promoting cell spreading, lamellipodia formation, and membrane ruffling, cell morphologies generated by active Rac1. In summary, we have identified a novel mechanism of P-Rex1 regulation through the GPCR-adaptor protein Norbin, a direct P-Rex1 interacting protein that promotes the Rac-GEF activity and membrane localization of P-Rex1. PMID:26792863

  12. The gall bladder cholecystokinin receptor exists in two guanine nucleotide-binding protein-regulated affinity states

    SciTech Connect

    Molero, X.; Miller, L.J. )

    1991-02-01

    To study proximal events in cholecystokinin (CCK) action on bovine gall bladder smooth muscle, we used the hormone analogue D-Tyr-Gly-((N1e28,31)CCK-26-32)-phenethyl ester (OPE), which has unique biological properties. This fully efficacious agonist differs from native CCK by not expressing supramaximal inhibition of cell shortening, yet it clearly interacts with the same receptor molecule. This was demonstrated in binding and affinity labeling studies, where both peptides label the same Mr 70,000-85,000 protein and both fully compete for binding of the other ligand. Further, its relatively high affinity for the low affinity CCK receptor permits the clear demonstration of two affinity states of a CCK receptor on a membrane preparation and makes possible evaluation of the molecular basis of these affinity states and their regulation. Analysis of homologous and heterologous binding curves performed with both CCK and OPE peptides and radioligands demonstrated the presence of two affinity states, with CCK being able to distinguish them (Kd1 = 0.48 +/- 0.04 nM and Kd2 = 56.5 +/- 7.4 nM) and OPE recognizing them equally (Kd1 = 0.94 +/- 0.31 nM and Kd2 = 0.96 +/- 0.23 nM). In the presence of nonhydrolyzable GTP analogues, there was a shift in distribution of receptors toward the low affinity state, with the total number of receptors and their absolute affinities for each peptide remaining constant. Thus, the gall bladder CCK receptor is a single molecule capable of assuming two interconvertible affinity states, regulated by a guanine nucleotide-binding protein. Two full agonists are capable of interacting with this molecule to yield different biological responses via different molecular events.

  13. A mammalian Rho-specific guanine-nucleotide exchange factor (p164-RhoGEF) without a pleckstrin homology domain.

    PubMed Central

    Rümenapp, Ulrich; Freichel-Blomquist, Andrea; Wittinghofer, Burkhard; Jakobs, Karl H; Wieland, Thomas

    2002-01-01

    Rho GTPases, which are activated by specific guanine-nucleotide exchange factors (GEFs), play pivotal roles in several cellular functions. We identified a recently cloned human cDNA, namely KIAA0337, encoding a protein containing 1510 amino acids (p164). It contains a RhoGEF-specific Dbl homology (DH) domain but lacks their typical pleckstrin homology domain. The expression of the mRNA encoding p164 was found to be at least 4-fold higher in the heart than in other tissues. Recombinant p164 interacted with and induced GDP/GTP exchange at RhoA but not at Rac1 or Cdc42. p164-DeltaC and p164-DeltaN are p164 mutants that are truncated at the C- and N-termini respectively but contain the DH domain. In contrast with the full-length p164, expression of p164-DeltaC and p164-DeltaN strongly induced actin stress fibre formation and activated serum response factor-mediated and Rho-dependent gene transcription. Interestingly, p164-DeltaN2, a mutant containing the C-terminus but having a defective DH domain, bound to p164-DeltaC and suppressed the p164-DeltaC-induced gene transcription. Overexpression of the full-length p164 inhibited M(3) muscarinic receptor-induced gene transcription, whereas co-expression with Gbeta(1)gamma(2) dimers induced transcriptional activity. It is concluded that p164-RhoGEF is a Rho-specific GEF with novel structural and regulatory properties and predominant expression in the heart. Apparently, its N- and C-termini interact with each other, thereby inhibiting its GEF activity. PMID:12071859

  14. Mutation Analysis of Inhibitory Guanine Nucleotide Binding Protein Alpha (GNAI) Loci in Young and Familial Pituitary Adenomas

    PubMed Central

    Demir, Hande; Donner, Iikki; Kivipelto, Leena; Kuismin, Outi; Schalin-Jäntti, Camilla; De Menis, Ernesto; Karhu, Auli

    2014-01-01

    Pituitary adenomas are neoplasms of the anterior pituitary lobe and account for 15–20% of all intracranial tumors. Although most pituitary tumors are benign they can cause severe symptoms related to tumor size as well as hypopituitarism and/or hypersecretion of one or more pituitary hormones. Most pituitary adenomas are sporadic, but it has been estimated that 5% of patients have a familial background. Germline mutations of the tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP) predispose to hereditary pituitary neoplasia. Recently, it has been demonstrated that AIP mutations predispose to pituitary tumorigenesis through defective inhibitory GTP binding protein (Gαi) signaling. This finding prompted us to examine whether germline loss-of-function mutations in inhibitory guanine nucleotide (GTP) binding protein alpha (GNAI) loci are involved in genetic predisposition of pituitary tumors. To our knowledge, this is the first time GNAI genes are sequenced in order to examine the occurrence of inactivating germline mutations. Thus far, only somatic gain-of-function hot-spot mutations have been studied in these loci. Here, we have analyzed the coding regions of GNAI1, GNAI2, and GNAI3 in a set of young sporadic somatotropinoma patients (n = 32; mean age of diagnosis 32 years) and familial index cases (n = 14), thus in patients with a disease phenotype similar to that observed in AIP mutation carriers. In addition, expression of Gαi proteins was studied in human growth hormone (GH), prolactin (PRL), adrenocorticotropic hormone (ACTH)-secreting and non-functional pituitary tumors. No pathogenic germline mutations affecting the Gαi proteins were detected. The result suggests that loss-of-function mutations of GNAI loci are rare or nonexistent in familial pituitary adenomas. PMID:25291362

  15. Leukemia-Associated Rho Guanine Nucleotide Exchange Factor Promotes Gαq-Coupled Activation of RhoA

    PubMed Central

    Booden, Michelle A.; Siderovski, David P.; Der, Channing J.

    2002-01-01

    Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein α subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of Gα12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific Gα subunits and couple Gα activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with Gα12 and Gα13 but also with Gαq. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of Gαq, Gα12, and Gα13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to Gαq and Gα13. Activated Gαq, as well as Gα12 and Gα13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three Gα subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways. PMID:12024019

  16. FAM123A Binds Microtubules and Inhibits the Guanine Nucleotide Exchange Factor ARHGEF2 to Decrease Actomyosin Contractility***

    PubMed Central

    Siesser, Priscila F.; Motolese, Marta; Walker, Matthew P.; Goldfarb, Dennis; Gewain, Kelly; Yan, Feng; Kulikauskas, Rima M.; Chien, Andy J.; Wordeman, Linda; Major, Michael B.

    2013-01-01

    The FAM123 gene family comprises three members, FAM123A, the tumor suppressor WTX(FAM123B) and FAM123C. WTX is required for normal development and causally contributes to human disease, in part through its regulation of β-catenin-dependent WNT signaling. The roles of FAM123A and FAM123C in signaling, cell behavior and human disease remain less understood. We defined and compared the protein-protein interaction networks for each member of the FAM123 family by affinity purification and mass spectrometry. Protein localization and functional studies suggest that the FAM123 family members have conserved and divergent cellular roles. In contrast to WTX and FAM123C, we found that microtubule-associated proteins were enriched in the FAM123A protein interaction network. FAM123A interacted with and tracked dynamic microtubules in a plus-end direction. Domain interaction experiments revealed a ‘SKIP’ amino acid motif in FAM123A that mediated interaction with the microtubule tip tracking proteins EB1 and EB3, and therefore with microtubules. Cells depleted of FAM123A showed compartment-specific effects on microtubule dynamics, increased actomyosin contractility, larger focal adhesions and decreased cell migration. These effects required binding of FAM123A to and inhibition of the guanine nucleotide exchange factor ARHGEF2, a microtubule-associated activator of RhoA. Together, these data suggest that the ‘family-unique’ SKIP motif enables FAM123A to bind EB proteins, localize to microtubules and coordinate microtubule dynamics and actomyosin contractility. PMID:22949735

  17. Guanine nucleotide regulation of muscarinic receptor-mediated inositol phosphate formation in permeabilized 1321N1 cells

    SciTech Connect

    Orellana, S.A.; Trilivas, I.; Brown, J.H.

    1986-03-05

    Carbachol and guanine nucleotides stimulate formation of the (/sup 3/H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with (/sup 3/H) inositol. Carbachol alone has little effect on formation of the (/sup 3/H) inositol phosphates (IPs), but GTP..gamma..S causes synergistic accumulation of (/sup 3/H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTP..gamma..S all support formation of the (/sup 3/H)IPs, with or without hormone, but GTP..gamma..S is the most effective. In the presence of GTP..gamma..S, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the (/sup 3/H)IPs occur at approx. 5 ..mu..M and approx. 100 ..mu..M carbachol, respectively; values close to those seen in intact cells. GTP..gamma..S alone stimulates formation of the (/sup 3/H)IPs after a brief lag time. The combination of GTP..gamma..S and carbachol both increases the rate of, and decreases the lag in, formation of the (/sup 3/H)IPs. LiCl increases (/sup 3/H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of (/sup 3/H)IP3. GTP..gamma..S and carbachol cause formation of (/sup 3/H)IPs in the absence of Ca/sup + +/, but formation induced by GTP..gamma..S with or without carbachol is Ca/sup + +/-sensitive over a range of physiological concentrations. Although carbachol, Ca/sup + +/, and GTP..gamma..S all have effects on formation of (/sup 3/H)IPs, GTP..gamma..S appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell.

  18. Defective Guanine Nucleotide Exchange in the Elongation Factor-like 1 (EFL1) GTPase by Mutations in the Shwachman-Diamond Syndrome Protein*

    PubMed Central

    García-Márquez, Adrián; Gijsbers, Abril; de la Mora, Eugenio; Sánchez-Puig, Nuria

    2015-01-01

    Ribosome biogenesis is orchestrated by the action of several accessory factors that provide time and directionality to the process. One such accessory factor is the GTPase EFL1 involved in the cytoplasmic maturation of the ribosomal 60S subunit. EFL1 and SBDS, the protein mutated in the Shwachman-Diamond syndrome (SBDS), release the anti-association factor eIF6 from the surface of the ribosomal subunit 60S. Here we report a kinetic analysis of fluorescent guanine nucleotides binding to EFL1 alone and in the presence of SBDS using fluorescence stopped-flow spectroscopy. Binding kinetics of EFL1 to both GDP and GTP suggests a two-step mechanism with an initial binding event followed by a conformational change of the complex. Furthermore, the same behavior was observed in the presence of the SBDS protein irrespective of the guanine nucleotide evaluated. The affinity of EFL1 for GTP is 10-fold lower than that calculated for GDP. Association of EFL1 to SBDS did not modify the affinity for GTP but dramatically decreased that for GDP by increasing the dissociation rate of the nucleotide. Thus, SBDS acts as a guanine nucleotide exchange factor (GEF) for EFL1 promoting its activation by the release of GDP. Finally, fluorescence anisotropy measurements showed that the S143L mutation present in the Shwachman-Diamond syndrome altered a surface epitope for EFL1 and largely decreased the affinity for it. These results suggest that loss of interaction between these proteins due to mutations in the disease consequently prevents the nucleotide exchange regulation the SBDS exerts on EFL1. PMID:25991726

  19. The mammalian guanine nucleotide exchange factor mSec12 is essential for activation of the Sar1 GTPase directing endoplasmic reticulum export.

    PubMed

    Weissman, J T; Plutner, H; Balch, W E

    2001-07-01

    The Sar1 GTPase is an essential component of COPII vesicle coats involved in export of cargo from the endoplasmic reticulum of mammalian cells. To begin to elucidate its mechanism of action, we now report the identity of the mammalian homolog to the yeast Sec12 guanine nucleotide exchange factor (18% identity) that promotes Sar1 activation. Mammalian Sec12 (mSec12) is a type II transmembrane protein with a large cytosolic domain, a fragment of which has previously been reported as the transcription factor prolactin regulatory element binding protein (PREB). mSec12 promotes efficient guanine nucleotide exchange on Sar1, but not Arf1 or Rab GTPases. mSec12 is localized to the endoplasmic reticulum and an antibody to the cytosolic domain of mSec12 potently inhibits Sar1 recruitment and the formation of COPII vesicles in vitro. The dominant negative GDP-restricted mutant Sar1[T39N] is shown to be a potent inhibitor of mSec12 activity, consistent with its role in preventing COPII vesicle formation in vitro and during transient expression in vivo. We propose that mSec12 is an evolutionarily distant guanine nucleotide exchange factor directing Sar1 GTPase activation in mammalian cells. Its divergence from yeast Sec12p may reflect the specialized needs of the mammalian endoplasmic reticulum involving the formation of Sar1-dependent transitional elements (Aridor M, et al. J Cell Biol 2001;152:213-229) and selection of cargo into prebudding complexes.

  20. Adenine-DNA adducts derived from the highly tumorigenic dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not

    PubMed Central

    Kropachev, Konstantin; Kolbanovskiy, Marina; Liu, Zhi; Cai, Yuqin; Zhang, Lu; Schwaid, Adam G.; Kolbanovskiy, Alexander; Ding, Shuang; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2013-01-01

    The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N2-dG) and adenine (N6-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N2-dG linkage site is ~ 35 times more susceptible to NER dual incisions than the stereochemically identical N6-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar, but somewhat smaller effect (factor of ~15) is observed. The striking resistance of the bulky N6-dA in contrast to the modest to good susceptibilities of the N2-dG adducts to NER are interpreted in terms of the balance between lesion-induced DNA-distorting and DNA-stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA. PMID:23570232

  1. Rho guanine nucleotide exchange factor is an NFL mRNA destabilizing factor that forms cytoplasmic inclusions in amyotrophic lateral sclerosis.

    PubMed

    Droppelmann, Cristian A; Keller, Brian A; Campos-Melo, Danae; Volkening, Kathryn; Strong, Michael J

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive disorder of unknown etiology characterized by the selective degeneration of motor neurons. Recent evidence supports the hypothesis that alterations in RNA metabolism in motor neurons can explain the development of protein inclusions, including neurofilamentous aggregates, observed in this pathology. In mice, p190RhoGEF, a guanine nucleotide exchange factor, is involved in neurofilament protein aggregation in an RNA-triggered transgenic model of motor neuron disease. Here, we observed that rho guanine nucleotide exchange factor (RGNEF), the human homologue of p190RhoGEF, binds low molecular weight neurofilament mRNA and affects its stability via 3' untranslated region destabilization. We observed that the overexpression of RGNEF in a stable cell line significantly decreased the level of low molecular weight neurofilament protein. Furthermore, we observed RGNEF cytoplasmic inclusions in ALS spinal motor neurons that colocalized with ubiquitin, p62/sequestosome-1, and TAR (trans-active regulatory) DNA-binding protein 43 (TDP-43). Our results provide further evidence that RNA metabolism pathways are integral to ALS pathology. This is also the first described link between ALS and an RNA binding protein with aggregate formation that is also a central cell signaling pathway molecule.

  2. The Structure of RalF, an ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

    SciTech Connect

    Amor,J.; Swails, J.; Zhu, X.; Roy, C.; Nagai, H.; Ingmundson, A.; Cheng, X.; Kahn, R.

    2005-01-01

    The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the 'glutamic finger,' conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.

  3. Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin.

    PubMed

    Tsai, S C; Adamik, R; Tsuchiya, M; Chang, P P; Moss, J; Vaughan, M

    1991-05-01

    Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.

  4. The effects of acute exposure to ethanol on neurotensin and guanine nucleotide-stimulation of phospholipase C activity in intact NIE-115 neuroblastoma cells

    SciTech Connect

    Smith, T.L. )

    1990-01-01

    Both ethanol and neurotensin produce sedation and hypothermia. When administered in combination the behavioral effects of these two substances are potentiated. In order to better understand the biochemical nature of this interaction, the direct effects of ethanol on neurotensin receptors and an associated signal transduction process were determined in NIE-115 neuroblastoma cells. Ethanol in physiologically relevant concentrations significantly reduced neurotensin stimulated ({sup 3}H)inositol phosphate production while having no effect on the specific binding of ({sup 3}H)neurotensin. In addition, ethanol up to 200 mM had no effect on GTPYS mediated ({sup 3}H)inositol phosphate production. The results indicate that acute exposure ethanol partially disrupts the normal coupling of activated neurotensin receptors to the guanine nucleotide binding protein associated with phospholipase C.

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

    SciTech Connect

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

    1990-06-01

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

  6. Molecular cloning and sequence determination of cDNAs for alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go from rat brain.

    PubMed Central

    Itoh, H; Kozasa, T; Nagata, S; Nakamura, S; Katada, T; Ui, M; Iwai, S; Ohtsuka, E; Kawasaki, H; Suzuki, K

    1986-01-01

    We have cloned cDNAs encoding alpha subunits of the guanine nucleotide-binding proteins Gs, Gi, and Go and determined their nucleotide sequences. Purified preparations of Gi and Go alpha subunits (Gi alpha and Go alpha) from rat brain were completely digested with trypsin, and peptides were subjected to amino acid sequence analysis. By screening of a cDNA library from rat C6 glioma cells with a synthetic probe corresponding to a 17 amino acid sequence, a clone encoding the sequence of Go alpha was obtained. Then, the library was rescreened with a Go alpha cDNA probe to isolate several strongly or weakly hybridizing clones. cDNAs encoding the complete sequences of Gi alpha and Gs alpha were thus obtained. From nucleotide sequence analysis, the amino acid sequences of Gs alpha and Gi alpha were deduced; they contain 394 and 355 amino acid residues (including the initiator methionine), respectively. The calculated molecular weights for Gs alpha and Gi alpha were 45,663 and 40,499, respectively. The Go alpha clone encoded a sequence of 310 amino acid residues that lacked the NH2 terminus. The homology of the alpha subunits of Gs, Gi, Go, transducin, and ras-encoded protein is discussed. PMID:3086867

  7. The auto-inhibitory state of Rho guanine nucleotide exchange factor ARHGEF5/TIM can be relieved by targeting its SH3 domain with rationally designed peptide aptamers.

    PubMed

    He, Ping; Tan, De-Li; Liu, Hong-Xiang; Lv, Feng-Lin; Wu, Wei

    2015-04-01

    The short isoform of Rho guanine nucleotide exchange factor ARHGEF5 is known as TIM, which plays diverse roles in, for example, tumorigenesis, neuronal development and Src-induced podosome formation through the activation of its substrates, the Rho family of GTPases. The activation is auto-inhibited by a putative helix N-terminal to the DH domain of TIM, which is stabilized by the intramolecular interaction of C-terminal SH3 domain with a poly-proline sequence between the putative helix and the DH domain. In this study, we systematically investigated the structural basis, energetic landscape and biological implication underlying TIM auto-inhibition by using atomistic molecular dynamics simulations and binding free energy analysis. The computational study revealed that the binding of SH3 domain to poly-proline sequence is the prerequisite for the stabilization of TIM auto-inhibition. Thus, it is suggested that targeting SH3 domain with competitors of the poly-proline sequence would be a promising strategy to relieve the auto-inhibitory state of TIM. In this consideration, we rationally designed a number of peptide aptamers for competitively inhibiting the SH3 domain based on modeled TIM structure and computationally generated data. Peptide binding test and guanine nucleotide exchange analysis solidified that these designed peptides can both bind to the SH3 domain potently and activate TIM-catalyzed RhoA exchange reaction effectively. Interestingly, a positive correlation between the peptide affinity and induced exchange activity was observed. In addition, separate mutation of three conserved residues Pro49, Pro52 and Lys54 - they are required for peptide recognition by SH3 domain -- in a designed peptide to Ala would completely abolish the capability of this peptide activating TIM. All these come together to suggest an intrinsic relationship between peptide binding to SH3 domain and the activation of TIM.

  8. Structure of Gαi1 bound to a GDP-selective peptide provides insight into guanine nucleotide exchange

    PubMed Central

    Johnston, Christopher A.; Willard, Francis S.; Jezyk, Mark R.; Fredericks, Zoey; Bodor, Erik T.; Jones, Miller B.; Blaesius, Rainer; Watts, Val J.; Harden, T. Kendall; Sondek, John; Ramer, J. Kevin; Siderovski, David P.

    2005-01-01

    Heterotrimeric G-proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP-bound and an active, GTP-bound state. Under basal conditions G-proteins exist in the inactive GDP-bound state, thus nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G-protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide-state-dependent Gα binding peptides. Herein, we report a GDP-selective Gα-binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Gαi subunits. Structural determination of the Gαi1/peptide complex reveals unique changes in the Gα switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Gα subunits adopt a conformation suitable for nucleotide exchange. PMID:16004878

  9. Rin-like, a novel regulator of endocytosis, acts as guanine nucleotide exchange factor for Rab5a and Rab22

    PubMed Central

    Woller, Barbara; Luiskandl, Susan; Popovic, Milica; Prieler, Barbara E.M.; Ikonge, Gloria; Mutzl, Michaela; Rehmann, Holger; Herbst, Ruth

    2011-01-01

    RIN proteins serve as guanine nucleotide exchange factors for Rab5a. They are characterized by the presence of a RIN homology domain and a C-terminal Vps9 domain. Currently three family members have been described and analyzed. Here we report the identification of a novel RIN family member, Rin-like (Rinl), that represents a new interaction partner of the receptor tyrosine kinase MuSK, which is an essential key regulator of neuromuscular synapse development. Rinl is localized to neuromuscular synapses but shows the highest expression in thymus and spleen. Rinl preferentially binds to nucleotide-free Rab5a and catalyzes the exchange of GDP for GTP. Moreover, Rinl also binds GDP-bound Rab22 and increases the GDP/GTP exchange implicating Rinl in endocytotic processes regulated by Rab5a and Rab22. Interestingly, Rinl shows a higher catalytic rate for Rab22 compared to Rab5a. Rinl is closely associated with the cytoskeleton and thus contributes to the spatial control of Rab5a and Rab22 signaling at actin-positive compartments. Most importantly, overexpression of Rinl affects fluid-phase as well as EGFR endocytosis. PMID:21419809

  10. Flow Cytometry for Real-Time Measurement of Guanine Nucleotide Binding and Exchange by Ras-like GTPases

    PubMed Central

    Schwartz, Samantha L.; Tessema, Mathewos; Buranda, Tione; Phlypenko, Olena; Rak, Alexey; Simons, Peter C.; Surviladze, Zurab; Sklar, Larry A.; Wandinger-Ness, Angela

    2008-01-01

    Ras-like small GTPases cycle between GTP-bound active and GDP-bound inactive conformational states to regulate diverse cellular processes. Despite their importance, detailed kinetic or comparative studies of family members are rarely undertaken due to the lack of real-time assays measuring nucleotide binding or exchange. Here, we report a bead-based, flow cytometric assay that quantitatively measures the nucleotide binding properties of GST-chimeras for prototypical Ras-family members Rab7 and Rho. Measurements are possible in the presence or absence of Mg2+, with magnesium cations principally increasing affinity and slowing nucleotide dissociation rate 8- to 10-fold. GST-Rab7 exhibited a 3-fold higher affinity for GDP relative to GTP that is consistent with a 3-fold slower dissociation rate of GDP. Strikingly, GST-Rab7 had a marked preference for GTP with ribose ring-conjugated BODIPY FL. The more commonly used γ-NH-conjugated BODIPY FL GTP analogue failed to bind to GST-Rab7. In contrast, both BODIPY analogues bound equally well to GST-RhoA and GST-RhoC. Comparisons of the GST-Rab7 and GST-RhoA GTP-binding pockets provide a structural basis for the observed binding differences. In sum, the flow cytometric assay can be used to measure nucleotide binding properties of GTPases in real-time and quantitatively assess differences between GTPases. PMID:18638444

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

    PubMed

    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. PMID:26797121

  12. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration-proliferation dichotomy.

    PubMed

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L; Garcia-Marcos, Mikel; Farquhar, Marilyn G; Ghosh, Pradipta

    2015-09-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development. PMID:26286990

  13. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration–proliferation dichotomy

    PubMed Central

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L.; Garcia-Marcos, Mikel; Farquhar, Marilyn G.; Ghosh, Pradipta

    2015-01-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously—a phenomenon called “migration–proliferation dichotomy.” We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration–proliferation dichotomy during cancer invasion, wound healing, and development. PMID:26286990

  14. Cyclin-dependent kinase 5 activates guanine nucleotide exchange factor GIV/Girdin to orchestrate migration-proliferation dichotomy.

    PubMed

    Bhandari, Deepali; Lopez-Sanchez, Inmaculada; To, Andrew; Lo, I-Chung; Aznar, Nicolas; Leyme, Anthony; Gupta, Vijay; Niesman, Ingrid; Maddox, Adam L; Garcia-Marcos, Mikel; Farquhar, Marilyn G; Ghosh, Pradipta

    2015-09-01

    Signals propagated by receptor tyrosine kinases (RTKs) can drive cell migration and proliferation, two cellular processes that do not occur simultaneously--a phenomenon called "migration-proliferation dichotomy." We previously showed that epidermal growth factor (EGF) signaling is skewed to favor migration over proliferation via noncanonical transactivation of Gαi proteins by the guanine exchange factor (GEF) GIV. However, what turns on GIV-GEF downstream of growth factor RTKs remained unknown. Here we reveal the molecular mechanism by which phosphorylation of GIV by cyclin-dependent kinase 5 (CDK5) triggers GIV's ability to bind and activate Gαi in response to growth factors and modulate downstream signals to establish a dichotomy between migration and proliferation. We show that CDK5 binds and phosphorylates GIV at Ser1674 near its GEF motif. When Ser1674 is phosphorylated, GIV activates Gαi and enhances promigratory Akt signals. Phosphorylated GIV also binds Gαs and enhances endosomal maturation, which shortens the transit time of EGFR through early endosomes, thereby limiting mitogenic MAPK signals. Consequently, this phosphoevent triggers cells to preferentially migrate during wound healing and transmigration of cancer cells. When Ser1674 cannot be phosphorylated, GIV cannot bind either Gαi or Gαs, Akt signaling is suppressed, mitogenic signals are enhanced due to delayed transit time of EGFR through early endosomes, and cells preferentially proliferate. These results illuminate how GIV-GEF is turned on upon receptor activation, adds GIV to the repertoire of CDK5 substrates, and defines a mechanism by which this unusual CDK orchestrates migration-proliferation dichotomy during cancer invasion, wound healing, and development.

  15. The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila.

    PubMed

    Chang, Ying-Ju; Zhou, Lily; Binari, Richard; Manoukian, Armen; Mak, Tak; McNeill, Helen; Stambolic, Vuk

    2016-01-01

    The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it's functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.

  16. Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge.

    PubMed

    Biersmith, Bridget; Wang, Zong-Heng; Geisbrecht, Erika R

    2015-06-01

    The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to engulfment and cell motility (ELMO) proteins, Dock-ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes, including cell motility, apoptotic cell clearance, and axon guidance. Overlapping expression patterns and functional redundancy among the 11 vertebrate Dock family members, which are subdivided into four families (Dock A, B, C, and D), complicate genetic analysis. In both vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism for understanding Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock A) and Sponge (Spg; Dock B). Here we show that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel. Moreover, we confirm the in vivo role of Mbc upstream of Rac and provide evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. Our findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis.

  17. The Rho-guanine nucleotide exchange factor PDZ-RhoGEF governs susceptibility to diet-induced obesity and type 2 diabetes

    PubMed Central

    Chang, Ying-Ju; Pownall, Scott; Jensen, Thomas E; Mouaaz, Samar; Foltz, Warren; Zhou, Lily; Liadis, Nicole; Woo, Minna; Hao, Zhenyue; Dutt, Previn; Bilan, Philip J; Klip, Amira; Mak, Tak; Stambolic, Vuk

    2015-01-01

    Adipose tissue is crucial for the maintenance of energy and metabolic homeostasis and its deregulation can lead to obesity and type II diabetes (T2D). Using gene disruption in the mouse, we discovered a function for a RhoA-specific guanine nucleotide exchange factor PDZ-RhoGEF (Arhgef11) in white adipose tissue biology. While PDZ-RhoGEF was dispensable for a number of RhoA signaling-mediated processes in mouse embryonic fibroblasts, including stress fiber formation and cell migration, it's deletion led to a reduction in their proliferative potential. On a whole organism level, PDZ-RhoGEF deletion resulted in an acute increase in energy expenditure, selectively impaired early adipose tissue development and decreased adiposity in adults. PDZ-RhoGEF-deficient mice were protected from diet-induced obesity and T2D. Mechanistically, PDZ-RhoGEF enhanced insulin/IGF-1 signaling in adipose tissue by controlling ROCK-dependent phosphorylation of the insulin receptor substrate-1 (IRS-1). Our results demonstrate that PDZ-RhoGEF acts as a key determinant of mammalian metabolism and obesity-associated pathologies. DOI: http://dx.doi.org/10.7554/eLife.06011.001 PMID:26512886

  18. Rac1 Controls the Subcellular Localization of the Rho Guanine Nucleotide Exchange Factor Net1A To Regulate Focal Adhesion Formation and Cell Spreading

    PubMed Central

    Carr, Heather S.; Morris, Christopher A.; Menon, Sarita; Song, Eun Hyeon

    2013-01-01

    RhoA is overexpressed in human cancer and contributes to aberrant cell motility and metastatic progression; however, regulatory mechanisms controlling RhoA activity in cancer are poorly understood. Neuroepithelial transforming gene 1 (Net1) is a RhoA guanine nucleotide exchange factor that is overexpressed in human cancer. It encodes two isoforms, Net1 and Net1A, which cycle between the nucleus and plasma membrane. Net1 proteins must leave the nucleus to activate RhoA, but mechanisms controlling the extranuclear localization of Net1 isoforms have not been described. Here, we show that Rac1 activation causes relocalization of Net1 isoforms outside the nucleus and stimulates Net1A catalytic activity. These effects do not require Net1A catalytic activity, its pleckstrin homology domain, or its regulatory C terminus. We also show that Rac1 activation protects Net1A from proteasome-mediated degradation. Replating cells on collagen stimulates endogenous Rac1 to relocalize Net1A, and inhibition of proteasome activity extends the duration and magnitude of Net1A relocalization. Importantly, we demonstrate that Net1A, but not Net1, is required for cell spreading on collagen, myosin light chain phosphorylation, and focal adhesion maturation. These data identify the first physiological mechanism controlling the extranuclear localization of Net1 isoforms. They also demonstrate a previously unrecognized role for Net1A in regulating cell adhesion. PMID:23184663

  19. The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila

    PubMed Central

    Chang, Ying-Ju; Zhou, Lily; Binari, Richard; Manoukian, Armen; Mak, Tak; McNeill, Helen; Stambolic, Vuk

    2016-01-01

    The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it’s functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila. PMID:27015411

  20. Expression and Distribution of the Guanine Nucleotide-binding Protein Subunit Alpha-s in Mice Skin Tissues and Its Association with White and Black Coat Colors

    PubMed Central

    Yin, Zhihong; Zhao, Xin; Wang, Zhun; Li, Zhen; Bai, Rui; Yang, Shanshan; Zhao, Min; Pang, Quanhai

    2016-01-01

    Guanine nucleotide-binding protein subunit alpha-s (Gnαs) is a small subunit of the G protein-couple signaling pathway, which is involved in the formation of coat color. The expression level and distribution of Gnαs were detected by quantitative real-time-polymerase chain reaction (qPCR), western blot, and immunohistochemistry to investigate the underlying mechanisms of coat color in white and black skin tissues of mice. qPCR and western blot results suggested that Gnαs was expressed at significantly higher levels in black mice compared with that of white mice, and transcripts and protein possessed the same expression in both colors. Immunohistochemistry demonstrated Gnαs staining in the root sheath and dermal papilla in hair follicle of mice skins. The results indicated that the Gnαs gene was expressed in both white and black skin tissues, and the expression level of Gnαs in the two types of color was different. Therefore, Gnαs may be involved in the coat color formation in mice. PMID:26954226

  1. The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila.

    PubMed

    Chang, Ying-Ju; Zhou, Lily; Binari, Richard; Manoukian, Armen; Mak, Tak; McNeill, Helen; Stambolic, Vuk

    2016-01-01

    The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it's functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila. PMID:27015411

  2. FgMon1, a guanine nucleotide exchange factor of FgRab7, is important for vacuole fusion, autophagy and plant infection in Fusarium graminearum.

    PubMed

    Li, Ying; Li, Bing; Liu, Luping; Chen, Huaigu; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2015-01-01

    The Ccz1-Mon1 protein complex, the guanine nucleotide exchange factor (GEF) of the late endosomal Rab7 homolog Ypt7, is required for the late step of multiple vacuole delivery pathways, such as cytoplasm-to-vacuole targeting (Cvt) pathway and autophagy processes. Here, we identified and characterized the yeast Mon1 homolog in Fusarium graminearum, named FgMon1. FgMON1 encodes a trafficking protein and is well conserved in filamentous fungi. Targeted gene deletion showed that the ∆Fgmon1 mutant was defective in vegetative growth, asexual/sexual development, conidial germination and morphology, plant infection and deoxynivalenol production. Cytological examination revealed that the ∆Fgmon1 mutant was also defective in vacuole fusion and autophagy, and delayed in endocytosis. Yeast two hybrid and in vitro GST-pull down assays approved that FgMon1 physically interacts with a Rab GTPase FgRab7 which is also important for the development, infection, membrane fusion and autophagy in F. graminearum. FgMon1 likely acts as a GEF of FgRab7 and constitutively activated FgRab7 was able to rescue the defects of the ∆Fgmon1 mutant. In summary, our study provides evidences that FgMon1 and FgRab7 are critical components that modulate vesicle trafficking, endocytosis and autophagy, and thereby affect the development, plant infection and DON production of F. graminearum.

  3. FgMon1, a guanine nucleotide exchange factor of FgRab7, is important for vacuole fusion, autophagy and plant infection in Fusarium graminearum

    PubMed Central

    Li, Ying; Li, Bing; Liu, Luping; Chen, Huaigu; Zhang, Haifeng; Zheng, Xiaobo; Zhang, Zhengguang

    2015-01-01

    The Ccz1-Mon1 protein complex, the guanine nucleotide exchange factor (GEF) of the late endosomal Rab7 homolog Ypt7, is required for the late step of multiple vacuole delivery pathways, such as cytoplasm-to-vacuole targeting (Cvt) pathway and autophagy processes. Here, we identified and characterized the yeast Mon1 homolog in Fusarium graminearum, named FgMon1. FgMON1 encodes a trafficking protein and is well conserved in filamentous fungi. Targeted gene deletion showed that the ∆Fgmon1 mutant was defective in vegetative growth, asexual/sexual development, conidial germination and morphology, plant infection and deoxynivalenol production. Cytological examination revealed that the ∆Fgmon1 mutant was also defective in vacuole fusion and autophagy, and delayed in endocytosis. Yeast two hybrid and in vitro GST-pull down assays approved that FgMon1 physically interacts with a Rab GTPase FgRab7 which is also important for the development, infection, membrane fusion and autophagy in F. graminearum. FgMon1 likely acts as a GEF of FgRab7 and constitutively activated FgRab7 was able to rescue the defects of the ∆Fgmon1 mutant. In summary, our study provides evidences that FgMon1 and FgRab7 are critical components that modulate vesicle trafficking, endocytosis and autophagy, and thereby affect the development, plant infection and DON production of F. graminearum. PMID:26657788

  4. Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

    PubMed Central

    Philip, Bevin; Levin, David E.

    2001-01-01

    Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2. PMID:11113201

  5. Crystallization and crystallographic analysis of yeast Sec2p, a guanine nucleotide-exchange factor for the yeast Rab GTPase Sec4p

    SciTech Connect

    Sato, Yusuke; Fukai, Shuya; Nureki, Osamu

    2007-07-01

    Crystal structure of a triple mutant (M115L, K121M, T142M) of SeMet-labelled Sec2p, a GEF for Rab GTPase, has been solved by SAD. Control of the selenium sites by mutagenesis much improved the resolution of the SeMet-labelled crystals. Sec2p is a guanine nucleotide-exchange factor (GEF) for the yeast Rab GTPase Sec4p. Sec2p accelerates GDP release from Sec4p and promotes GDP–GTP exchange for Sec4p activation. In order to elucidate this nucleotide-exchange mechanism using X-ray crystallography, three constructs of native Sec2p (Sec2{sub 1–160}p, Sec2{sub 18–160}p and Sec2{sub 31–160}p) and three constructs of selenomethionine-labelled Sec2p [Sec2{sub 31–160}p, Sec2{sub 31–160}p (M115L) and Sec2{sub 31–160}p (M115L, K121M, T142M)] were crystallized. These six crystals diffracted to 8.8, 4.8, 2.6, 4.0, 3.3 and 3.0 Å resolution, respectively. The data set from the SeMet-labelled Sec2{sub 31–160}p (M115L, K121M, T142M) crystal was processed for SAD phasing; the crystal belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 101.9, b = 176.6, c = 181.5 Å.

  6. Solubilization and functional reconstitution of polymorphonuclear leukocyte formyl-Methionyl-Leucyl-Phenylalanine receptors and guanine nucleotide binding proteins

    SciTech Connect

    Williamson, K.C.

    1987-01-01

    Formyl-Methionyl-Leucyl-Phenylalanine (fMLP) binds to specific polymorphonuclear leukocyte plasma membrane receptors stimulating chemotaxis and bactericidal responses. One of the initial events of the ligand receptor interaction is a rise in inositol trisphosphate, which triggers intracellular calcium release. The generation of inositol trisphosphate is mediated by the fMLP-activated phospholipase C via a GTP-binding protein (G-protein). In analogy to the adrenergic stimulation of adenylate cyclase, the following signal transduction model has been proposed: The fMLP receptor activates a G-protein which then stimulates phospholipase C to hydrolyse phosphatidylinositol bisphosphate to inositol trisphosphate and diacylglycerol. This work has focused on characterizing the structural and functional coupling fMLP receptor and G-proteins in native membranes, detergent micelles and reconstituted phospholipid vesicles. Tight coupling between the fMLP receptor and G-protein has been demonstrated in both native and solubilized membranes by assaying quanine nucleotide-induced inhibition of (/sup 3/H)fMLP binding and fMLP stimulated GTPase activity.

  7. Insights into the molecular activation mechanism of the RhoA-specific guanine nucleotide exchange factor, PDZRhoGEF.

    PubMed

    Bielnicki, Jakub A; Shkumatov, Alexander V; Derewenda, Urszula; Somlyo, Avril V; Svergun, Dmitri I; Derewenda, Zygmunt S

    2011-10-01

    PDZRhoGEF (PRG) belongs to a small family of RhoA-specific nucleotide exchange factors that mediates signaling through select G-protein-coupled receptors via Gα(12/13) and activates RhoA by catalyzing the exchange of GDP to GTP. PRG is a multidomain protein composed of PDZ, regulators of G-protein signaling-like (RGSL), Dbl-homology (DH), and pleckstrin-homology (PH) domains. It is autoinhibited in cytosol and is believed to undergo a conformational rearrangement and translocation to the membrane for full activation, although the molecular details of the regulation mechanism are not clear. It has been shown recently that the main autoregulatory elements of PDZRhoGEF, the autoinhibitory "activation box" and the "GEF switch," which is required for full activation, are located directly upstream of the catalytic DH domain and its RhoA binding surface, emphasizing the functional role of the RGSL-DH linker. Here, using a combination of biophysical and biochemical methods, we show that the mechanism of PRG regulation is yet more complex and may involve an additional autoinhibitory element in the form of a molten globule region within the linker between RGSL and DH domains. We propose a novel, two-tier model of autoinhibition where the activation box and the molten globule region act synergistically to impair the ability of RhoA to bind to the catalytic DH-PH tandem. The molten globule region and the activation box become less ordered in the PRG-RhoA complex and dissociate from the RhoA-binding site, which may constitute a critical step leading to PRG activation.

  8. Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

    SciTech Connect

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

    1987-11-01

    In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 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/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation of unidirectional /sup 45/Ca/sup 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 ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.

  9. Characterization of Synthetic-Lethal Mutants Reveals a Role for the Saccharomyces Cerevisiae Guanine-Nucleotide Exchange Factor Cdc24p in Vacuole Function and Na(+) Tolerance

    PubMed Central

    White, W. H.; Johnson, D. I.

    1997-01-01

    Cdc24p is the guanine-nucleotide exchange factor for the Cdc42p GTPase, which controls cell polarity in Saccharomyces cerevisiae. To identify new genes that may affect cell polarity, we characterized six UV-induced csl (CDC24 synthetic-lethal) mutants that exhibited synthetic-lethality with cdc24-4(ts) at 23°. Five mutants were not complemented by plasmid-borne CDC42, RSR1, BUD5, BEM1, BEM2, BEM3 or CLA4 genes, which are known to play a role in cell polarity. The csl3 mutant displayed phenotypes similar to those observed with calcium-sensitive, Pet(-) vma mutants defective in vacuole function. CSL5 was allelic to VMA5, the vacuolar H(+)-ATPase subunit C, and one third of csl5 cdc24-4(ts) cells were elongated or had misshapen buds. A cdc24-4(ts) Δvma5::LEU2 double mutant did not exhibit synthetic lethality, suggesting that the csl5/vma5 cdc24-4(ts) synthetic-lethality was not simply due to altered vacuole function. The cdc24-4(ts) mutant, like Δvma5::LEU2 and csl3 mutants, was sensitive to high levels of Ca(2+) as well as Na(+) in the growth media, which did not appear to be a result of a fragile cell wall because the phenotypes were not remedied by 1 M sorbitol. Our results indicated that Cdc24p was required in one V-ATPase mutant and another mutant affecting vacuole morphology, and also implicated Cdc24p in Na(+) tolerance. PMID:9286667

  10. Odontogenic Ameloblast-associated Protein (ODAM) Mediates Junctional Epithelium Attachment to Teeth via Integrin-ODAM-Rho Guanine Nucleotide Exchange Factor 5 (ARHGEF5)-RhoA Signaling.

    PubMed

    Lee, Hye-Kyung; Ji, Suk; Park, Su-Jin; Choung, Han-Wool; Choi, Youngnim; Lee, Hyo-Jung; Park, Shin-Young; Park, Joo-Cheol

    2015-06-01

    Adhesion of the junctional epithelium (JE) to the tooth surface is crucial for maintaining periodontal health. Although odontogenic ameloblast-associated protein (ODAM) is expressed in the JE, its molecular functions remain unknown. We investigated ODAM function during JE development and regeneration and its functional significance in the initiation and progression of periodontitis and peri-implantitis. ODAM was expressed in the normal JE of healthy teeth but absent in the pathologic pocket epithelium of diseased periodontium. In periodontitis and peri-implantitis, ODAM was extruded from the JE following onset with JE attachment loss and detected in gingival crevicular fluid. ODAM induced RhoA activity and the expression of downstream factors, including ROCK (Rho-associated kinase), by interacting with Rho guanine nucleotide exchange factor 5 (ARHGEF5). ODAM-mediated RhoA signaling resulted in actin filament rearrangement. Reduced ODAM and RhoA expression in integrin β3- and β6-knockout mice revealed that cytoskeleton reorganization in the JE occurred via integrin-ODAM-ARHGEF5-RhoA signaling. Fibronectin and laminin activated RhoA signaling via the integrin-ODAM pathway. Finally, ODAM was re-expressed with RhoA in regenerating JE after gingivectomy in vivo. These results suggest that ODAM expression in the JE reflects a healthy periodontium and that JE adhesion to the tooth surface is regulated via fibronectin/laminin-integrin-ODAM-ARHGEF5-RhoA signaling. We also propose that ODAM could be used as a biomarker of periodontitis and peri-implantitis.

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

    PubMed Central

    Takenaka, Nobuyuki; Nihata, Yuma; Satoh, Takaya

    2016-01-01

    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. PMID:27163697

  12. Measurement of thyroid stimulating immunoglobulins using a novel thyroid stimulating hormone receptor-guanine nucleotide-binding protein, (GNAS) fusion bioassay.

    PubMed

    Pierce, M; Sandrock, R; Gillespie, G; Meikle, A W

    2012-11-01

    Hyperthyroidism, defined by overproduction of thyroid hormones, has a 2-3% prevalence in the population. The most common form of hyperthyroidism is Graves' disease. A diagnostic biomarker for Graves' disease is the presence of immunoglobulins which bind to, and stimulate, the thyroid stimulating hormone receptor (TSHR), a G-protein coupled receptor (GPCR). We hypothesized that the ectopically expressed TSHR gene in a thyroid stimulating immunoglobulin (TSI) assay could be engineered to increase the accumulation of the GPCR pathway second messenger, cyclic AMP (cAMP), the molecule measured in the assay as a marker for pathway activation. An ectopically expressing TSHR-mutant guanine nucleotide-binding protein, (GNAS) Chinese hamster ovary (CHO) cell clone was constructed using standard molecular biology techniques. After incubation of the new clone with sera containing various levels of TSI, GPCR pathway activation was then quantified by measuring cAMP accumulation in the clone. The clone, together with a NaCl-free cell assay buffer containing 5% polyethylene glycol (PEG)6000, was tested against 56 Graves' patients, 27 toxic thyroid nodule patients and 119 normal patients. Using receiver operating characteristic analysis, when comparing normal with Graves' sera, the assay yielded a sensitivity of 93%, a specificity of 99% and an efficiency of 98%. Total complex precision (within-run, across runs and across days), presented as a percentage coefficient of variation, was found to be 7·8, 8·7 and 7·6% for low, medium and high TSI responding serum, respectively. We conclude that the performance of the new TSI assay provides sensitive detection of TSI, allowing for accurate, early detection of Graves' disease.

  13. Differential effect of sodium ions and guanine nucleotides on the binding of thioperamide and clobenpropit to histamine H3-receptors in rat cerebral cortical membranes.

    PubMed

    Clark, E A; Hill, S J

    1995-01-01

    1. Conflicting reports in the literature over heterogeneity (West et al., 1990) or homogeneity (Arrange et al., 1990) of histamine H3-receptor binding sites may be attributed to the use of different incubation conditions. In the present study we have investigated the extent to which the binding of H3-receptor ligands to rat cerebral cortical membranes can be modified by both sodium ions and guanine nucleotides. 2. The H3-selective antagonist, thioperamide, discriminated between two specific binding sites for [3H]-N alpha-methylhistamine (IC50 1 = 2.75 +/- 0.87 nM, IC50 2 101.6 +/- 12.0 nM, % site 1 = 24 +/- 2%) in 50 mM Tris HCl buffer, but showed homogeneity of binding in 50 mM Na/K phosphate buffer. 3. Sodium ions markedly altered the binding characteristics of thioperamide (i.e. heterogeneity was lost and IC50 value shifted towards the high affinity site). The competition curves for a second H3-antagonist, clobenpropit and the H3-agonist N alpha-methylhistamine however, were unaltered in the presence of sodium ions. 4. Guanylnucleotides displaced only 60% of specific [3H]-N alpha- methylhistamine binding and modulated thioperamide binding in the same way as sodium ions. 5. These data suggest that the H3-receptor can exist in different conformations for which thioperamide, but not N alpha-methylhistamine and clobenpropit, show differential affinity. 6. The potential nature of these sites, and the implications of this apparent receptor heterogeneity for H3-receptor antagonism by thioperamide, are discussed.

  14. Cdc42 and the Guanine Nucleotide Exchange Factors Ect2 and Trio Mediate Fn14-Induced Migration and Invasion of Glioblastoma Cells

    PubMed Central

    Fortin, Shannon P.; Ennis, Matthew J.; Schumacher, Cassie A.; Zylstra-Diegel, Cassandra R.; Williams, Bart O.; Ross, Julianna T.D.; Winkles, Jeffrey A.; Loftus, Joseph C.; Symons, Marc H.; Tran, Nhan L.

    2012-01-01

    Malignant glioblastomas are characterized by their ability to infiltrate into normal brain. We previously reported that binding of the multifunctional cytokine TNF-like weak inducer of apoptosis (TWEAK) to its receptor fibroblast growth factor–inducible 14 (Fn14) induces glioblastoma cell invasion via Rac1 activation. Here, we show that Cdc42 plays an essential role in Fn14-mediated activation of Rac1. TWEAK-treated glioma cells display an increased activation of Cdc42, and depletion of Cdc42 using siRNA abolishes TWEAK-induced Rac1 activation and abrogates glioma cell migration and invasion. In contrast, Rac1 depletion does not affect Cdc42 activation by Fn14, showing that Cdc42 mediates TWEAK-stimulated Rac1 activation. Furthermore, we identified two guanine nucleotide exchange factors (GEF), Ect2 and Trio, involved in TWEAK-induced activation of Cdc42 and Rac1, respectively. Depletion of Ect2 abrogates both TWEAK-induced Cdc42 and Rac1 activation, as well as subsequent TWEAK-Fn14–directed glioma cell migration and invasion. In contrast, Trio depletion inhibits TWEAK-induced Rac1 activation but not TWEAK-induced Cdc42 activation. Finally, inappropriate expression of Fn14 or Ect2 in mouse astrocytes in vivo using an RCAS vector system for glial-specific gene transfer in G-tva transgenic mice induces astrocyte migration within the brain, corroborating the in vitro importance of the TWEAK-Fn14 signaling cascade in glioblastoma invasion. Our results suggest that the TWEAK-Fn14 signaling axis stimulates glioma cell migration and invasion through two GEF-GTPase signaling units, Ect2-Cdc42 and Trio-Rac1. Components of the Fn14-Rho GEF-Rho GTPase signaling pathway present innovative drug targets for glioma therapy. PMID:22571869

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

    SciTech Connect

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

    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.

  16. Isolation and analysis of the faciogenital dysplasia (Aarskog-Scott syndrome) gene: A putative rho/rac guanine nucleotide exchange factor

    SciTech Connect

    Pasteris, N.G.; Cadle, A.; Glover, T.W.

    1994-09-01

    Faciogenital dysplasia (FGDY, MIM No. 305400) is an X-linked multisystemic growth disorder characterized by disproportionate short stature, diagnostic facial and skeletal anomalies, and urogenital malformations. Past genetic, cytologic and radiation hybrid analyses mapped FGDY to a 350 kb interval within Xp11.21 flanked by markers ALAS2 and DXS323. Using positional methods to clone the locus, YAC clones spanning an FGDY-specific t(X;8) breakpoint were isolated. Human fetal cDNA libraries were screened with regional DNA markers to isolate a transcript that was disrupted consequential to the breakpoint. SSCP analyses identified a confirming mutation co-segregating with FGDY in an affected family. The isolated 4156 bp cDNA, termed FGD1, detected a 4.4 kb transcript in most fetal and adult tissues. The FGD1 gene contained > 19 exons spanning 100 kb of Xp11.21. The predicted length of the FGD1 protein was 809 AA, with an 89 Kd mass. FGD1 had strong homology to ras-like rho/rac guanine nucleotide exchange factors (GEFs) including proto-oncogenes Dbl, ect2, Bcr, and Vav. FGD1 also contained a Pro-rich 5{prime} region including two potential Src-homology 3 (SH3) binding sites, suggesting that, like Sos (a ras GEF), FGD1 may be involved in intracellular signal transduction. These results strongly suggest that FGD1, a putative rho/rac GEF, is responsible for X-linked FGDY. Mutations in FGD1 may result in perturbed signal transduction and, consequently, developmental growth anomalies. An analysis of FGD1 should aid in understanding FGDY pathogenesis and may provide extra insight into signal transduction and mammalian development.

  17. Heparin inhibits pulmonary artery smooth muscle cell proliferation through guanine nucleotide exchange factor-H1/RhoA/Rho kinase/p27.

    PubMed

    Yu, Lunyin; Quinn, Deborah A; Garg, Hari G; Hales, Charles A

    2011-04-01

    Ras homolog gene family member A (RhoA) through Rho kinase kinase (ROCK), one of its downstream effectors, regulates a wide range of cell physiological functions, including vascular smooth muscle cell (SMC) proliferation, by degrading cyclin-dependent kinase inhibitor, p27. Our previous studies found that heparin inhibition of pulmonary artery SMC (PASMC) proliferation and pulmonary hypertension was dependent on p27 up-regulation. To investigate whether ROCK, a regulator of p27, is involved in regulation of heparin inhibition of PASMC proliferation, we analyzed ROCK expression in the lungs from mice and from human PASMCs exposed to hypoxia, and investigated the effect of ROCK expression in vitro by RhoA cDNA transfection. We also investigated the effect of guanine nucleotide exchange factor (GEF)-H1, an upstream regulator of RhoA, on heparin inhibition of PASMC proliferation by GEF-H1 cDNA transfection. We found that: (1) hypoxia increased ROCK expression in mice and PASMCs; (2) overexpression of RhoA diminished the inhibitory effect of heparin on PASMC proliferation and down-regulated p27 expression; and (3) overexpression of GEF-H1 negated heparin inhibition of PASMC proliferation, which was accompanied by increased GTP-RhoA and decreased p27. This study demonstrates that the RhoA/ROCK pathway plays an important role in heparin inhibition on PASMC proliferation, and reveals that heparin inhibits PASMC proliferation through GEF-H1/RhoA/ROCK/p27 signaling pathway, by down-regulating GEF-H1, RhoA, and ROCK, and then up-regulating p27.

  18. (3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

    SciTech Connect

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

    1985-12-01

    In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 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 (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 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 (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 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 (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 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 (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 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 (/sup 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 (/sup 3/H)WB4101 binding.

  19. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

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

    PubMed Central

    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

  1. 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

    SciTech Connect

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

    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.

  2. Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm

    PubMed Central

    Wen, Liuying; Fukuda, Masako; Sunada, Mariko; Ishino, Sonoko; Ishino, Yoshizumi; Okita, Thomas W.; Ogawa, Masahiro; Ueda, Takashi; Kumamaru, Toshihiro

    2015-01-01

    Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins. PMID:26136263

  3. Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm.

    PubMed

    Wen, Liuying; Fukuda, Masako; Sunada, Mariko; Ishino, Sonoko; Ishino, Yoshizumi; Okita, Thomas W; Ogawa, Masahiro; Ueda, Takashi; Kumamaru, Toshihiro

    2015-10-01

    Rice glutelin polypeptides are initially synthesized on the endoplasmic reticulum (ER) membrane as a proglutelin, which are then transported to the protein storage vacuole (PSV) via the Golgi apparatus. Rab5 and its cognate activator guanine nucleotide exchange factor (GEF) are essential for the intracellular transport of proglutelin from the Golgi apparatus to the PSV. Results from previous studies showed that the double recessive type of glup4/rab5a and glup6/gef mutant accumulated much higher amounts of proglutelin than either parent line. The present study demonstrates that the double recessive type of glup4/rab5a and glup6/gef mutant showed not only elevated proglutelin levels and much larger paramural bodies but also reduced the number and size of PSVs, indicating a synergistic mutation effect. These observations led us to the hypothesis that other isoforms of Rab5 and GEF also participate in the intracellular transport of rice glutelin. A database search identified a novel guanine nucleotide exchange factor, Rab5-GEF2. Like GLUP6/GEF, Rab5-GEF2 was capable of activating Rab5a and two other Rab5 isoforms in in vitro GTP/GDP exchange assays. GEF proteins consist of the helical bundle (HB) domain at the N-terminus, Vps9 domain, and a C-terminal region. By the deletion analysis of GEFs, the HB domain was found essential for the activation of Rab5 proteins. PMID:26136263

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

    PubMed

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

    2011-03-01

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

  5. Evidence for Natural Selection in Nucleotide Content Relationships Based on Complete Mitochondrial Genomes: Strong Effect of Guanine Content on Separation between Terrestrial and Aquatic Vertebrates

    PubMed Central

    Sorimachi, Kenji; Okayasu, Teiji

    2015-01-01

    The complete vertebrate mitochondrial genome consists of 13 coding genes. We used this genome to investigate the existence of natural selection in vertebrate evolution. From the complete mitochondrial genomes, we predicted nucleotide contents and then separated these values into coding and non-coding regions. When nucleotide contents of a coding or non-coding region were plotted against the nucleotide content of the complete mitochondrial genomes, we obtained linear regression lines only between homonucleotides and their analogs. On every plot using G or A content purine, G content in aquatic vertebrates was higher than that in terrestrial vertebrates, while A content in aquatic vertebrates was lower than that in terrestrial vertebrates. Based on these relationships, vertebrates were separated into two groups, terrestrial and aquatic. However, using C or T content pyrimidine, clear separation between these two groups was not obtained. The hagfish (Eptatretus burgeri) was further separated from both terrestrial and aquatic vertebrates. Based on these results, nucleotide content relationships predicted from the complete vertebrate mitochondrial genomes reveal the existence of natural selection based on evolutionary separation between terrestrial and aquatic vertebrate groups. In addition, we propose that separation of the two groups might be linked to ammonia detoxification based on high G and low A contents, which encode Glu rich and Lys poor proteins. PMID:25853054

  6. Evidence for Natural Selection in Nucleotide Content Relationships Based on Complete Mitochondrial Genomes: Strong Effect of Guanine Content on Separation between Terrestrial and Aquatic Vertebrates.

    PubMed

    Sorimachi, Kenji; Okayasu, Teiji

    2015-01-01

    The complete vertebrate mitochondrial genome consists of 13 coding genes. We used this genome to investigate the existence of natural selection in vertebrate evolution. From the complete mitochondrial genomes, we predicted nucleotide contents and then separated these values into coding and non-coding regions. When nucleotide contents of a coding or non-coding region were plotted against the nucleotide content of the complete mitochondrial genomes, we obtained linear regression lines only between homonucleotides and their analogs. On every plot using G or A content purine, G content in aquatic vertebrates was higher than that in terrestrial vertebrates, while A content in aquatic vertebrates was lower than that in terrestrial vertebrates. Based on these relationships, vertebrates were separated into two groups, terrestrial and aquatic. However, using C or T content pyrimidine, clear separation between these two groups was not obtained. The hagfish (Eptatretus burgeri) was further separated from both terrestrial and aquatic vertebrates. Based on these results, nucleotide content relationships predicted from the complete vertebrate mitochondrial genomes reveal the existence of natural selection based on evolutionary separation between terrestrial and aquatic vertebrate groups. In addition, we propose that separation of the two groups might be linked to ammonia detoxification based on high G and low A contents, which encode Glu rich and Lys poor proteins.

  7. Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

    PubMed Central

    Liu, Zhi; Ding, Shuang; Kropachev, Konstantin; Lei, Jia; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2015-01-01

    The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N2-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide

  8. Domain contributions to signaling specificity differences between Ras-guanine nucleotide releasing factor (Ras-GRF) 1 and Ras-GRF2.

    PubMed

    Jin, Shan-Xue; Bartolome, Christopher; Arai, Junko A; Hoffman, Laurel; Uzturk, B Gizem; Kumar-Singh, Rajendra; Waxham, M Neal; Feig, Larry A

    2014-06-01

    Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity.

  9. Yeast Ysl2p, Homologous to Sec7 Domain Guanine Nucleotide Exchange Factors, Functions in Endocytosis and Maintenance of Vacuole Integrity and Interacts with the Arf-Like Small GTPase Arl1p

    PubMed Central

    Jochum, Alexandra; Jackson, David; Schwarz, Heinz; Pipkorn, Rüdiger; Singer-Krüger, Birgit

    2002-01-01

    We previously described the isolation of ysl2-1 due to its genetic interaction with Δypt51/vps21, a mutant with a deletion of the coding sequence for the yeast Rab5 homolog, which regulates endocytic traffic between early and late endosomes. Here we report that Ysl2p is a novel 186.8-kDa peripheral membrane protein homologous to members of the Sec7 family. We provide multiple genetic and biochemical evidence for an interaction between Ysl12p and the Arf-like protein Arl1p, consistent with a potential function as an Arf guanine nucleotide exchange factor (GEF). The temperature-sensitive alleles ysl2-307 and ysl2-316 are specifically defective in ligand-induced degradation of Ste2p and α-factor and exhibit vacuole fragmentation directly upon a shift to 37°C. In living cells, green fluorescent protein (GFP)-Ysl2p colocalizes with endocytic elements that accumulate FM4-64. The GFP-Ysl2p staining is sensitive to a mutation in VPS27 resulting in the formation of an aberrant class E compartment, but it is not affected by a sec7 mutation. Consistent with the idea that Ysl2p and Arl1p have closely related functions, Δarl1 cells are defective in endocytic transport and in vacuolar protein sorting. PMID:12052896

  10. Threonine 680 Phosphorylation of FLJ00018/PLEKHG2, a Rho Family-specific Guanine Nucleotide Exchange Factor, by Epidermal Growth Factor Receptor Signaling Regulates Cell Morphology of Neuro-2a Cells*

    PubMed Central

    Sato, Katsuya; Sugiyama, Tsuyoshi; Nagase, Takahiro; Kitade, Yukio; Ueda, Hiroshi

    2014-01-01

    FLJ00018/PLEKHG2 is a guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. The function of FLJ00018 is regulated by the interaction of heterotrimeric GTP-binding protein Gβγ subunits or cytosolic actin. However, the details underlying the molecular mechanisms of FLJ00018 activation have yet to be elucidated. In the present study we show that FLJ00018 is phosphorylated and activated by β1-adrenergic receptor stimulation-induced EGF receptor (EGFR) transactivation in addition to Gβγ signaling. FLJ00018 is also phosphorylated and activated by direct EGFR stimulation. The phosphorylation of FLJ00018 by EGFR stimulation is mediated by the Ras/mitogen-activated protein kinase (MAPK) pathway. Through deletion and site-directed mutagenesis studies, we have identified Thr-680 as the major site of phosphorylation by EGFR stimulation. FLJ00018 T680A, in which the phosphorylation site is replaced by alanine, showed a limited response of the Neuro-2a cell morphology to EGF stimulation. Our results provide evidence that stimulation of the Ras/MAPK pathway by EGFR results in FLJ00018 phosphorylation at Thr-680, which in turn controls changes in cell shape. PMID:24554703

  11. Regulatory roles for Tiam1, a guanine nucleotide exchange factor for Rac1, in glucose-stimulated insulin secretion in pancreatic beta-cells.

    PubMed

    Veluthakal, Rajakrishnan; Madathilparambil, Suresh Vasu; McDonald, Phillip; Olson, Lawrence Karl; Kowluru, Anjaneyulu

    2009-01-01

    Using various biochemical, pharmacological and molecular biological approaches, we have recently reported regulatory roles for Rac1, a small G-protein, in glucose-stimulated insulin secretion (GSIS). However, little is understood with respect to localization of, and regulation by, specific regulatory factors of Rac1 in GSIS. Herein, we investigated regulatory roles for Tiam1, a specific nucleotide exchange factor (GEF) for Rac1, in GSIS in pancreatic beta-cells. Western blot analysis indicated that Tiam1 is predominantly cytosolic in distribution. NSC23766, a specific inhibitor of Tiam1-mediated activation of Rac1, markedly attenuated glucose-induced, but not KCl-induced insulin secretion in INS 832/13 cells and normal rat islets. Further, NSC23766 significantly reduced glucose-induced activation (i.e. GTP-bound form) and membrane association of Rac1 in INS 832/13 cells and rat islets. Moreover, siRNA-mediated knock-down of Tiam1 markedly inhibited glucose-induced membrane trafficking and activation of Rac1 in INS 832/13 cells. Interestingly, however, in contrast to the inhibitory effects of NSC23766, Tiam1 gene depletion potentiated GSIS in these cells; such a potentiation of GSIS was sensitive to extracellular calcium. Together, our studies present the first evidence for a regulatory role for Tiam1/Rac1-sensitive signaling step in GSIS. They also provide evidence for the existence of a potential Rac1/Tiam1-independent, but calcium-sensitive component for GSIS in these cells.

  12. Stimulatory and inhibitory effects of guanine nucleotides on arginine-vasotocin-sensitive adenylate cyclase in the epithelial cell membranes of the bullfrog bladder.

    PubMed

    Mishina, T; Shimada, H; Marumo, F

    1983-11-01

    The effects of arginine-vasotocin and nucleotides on the steady-state kinetics of the adenylate cyclase activity in the epithelial cell membranes of the bullfrog (Rana catesbiana) bladder were studied. Arginine-vasotocin stimulated adenylate cyclase more effectively than oxytocin or arginine-vasopressin, with respect to both the maximal hormonal activation ratio relative to basal, and the hormone concentration yielding a half-maximal response (apparent Km). Arginine-vasotocin, GTP and its analogue guanyl-5'-yl imidodiphosphate (Gpp(NH)p) increased the Vmax of the basal adenylate cyclase activity, but showed no effect of the apparent Km of the system for ATP. In addition, Gpp(NH)p enhanced the arginine-vasotocin-stimulated adenylate cyclase activity, further increasing the Vmax, while GTP showed no statistically significant effect. Dual effects of GDP were apparent: it was stimulatory at 1 x 10(-5) mol/l and inhibitory at 1 x 10(-3) mol/l, on both the basal and the arginine-vasotocin-stimulated adenylate cyclase activity. Guanosine 5'-monophosphate, CTP, UTP and ITP showed no apparent effect on the enzyme activity. Sodium fluoride acted in the same manner as GTP on the adenylate cyclase system, increasing only basal activity. Adenylate cyclase activities exhibited pH optima that were less distinct in the presence than in the absence of Gpp(NH)p. The Arrhenius plot of the temperature experiment showed that a high-energy step was involved for activation by Gpp(NH)p or arginine-vasotocin. When the relative activation ratios by arginine-vasotocin at different ATP concentrations were studied, a distinct activation optimum was shown at 2.5 x 10(-4) mol ATP/l, either in the absence or presence of Gpp(NH)p. The possibility that GTP, GDP nd ATP play a regulatory role in the epithelial cells of the bullfrog bladder by adjusting the responsiveness of the system to a natural hormone, arginine-vasotocin, is discussed. PMID:6606697

  13. An Adenine-DNA Adduct Derived from Nitroreduction of 6-Nitrochrysene is more Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts

    PubMed Central

    Krzeminski, Jacek; Kropachev, Konstantin; Reeves, Dara; Kolbanovskiy, Aleksandr; Kolbanovskiy, Marina; Chen, Kun-Ming; Sharma, Arun K.; Geacintov, Nicholas; Amin, Shantu; El-Bayoumy, Karam

    2013-01-01

    Previous studies in rats, mice and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: 1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts: N-(dG-8-yl)-6-AC, 5-(dG-N2-yl)-6-AC and N-(dA-8-yl)-6-AC, and 2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield: N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N2-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ~2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ~ 8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N2-yl)-6-AC ~ N-(dG-8-yl)-6-AC ~ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N2-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue, could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland. PMID:24112095

  14. Ras-Guanine Nucleotide-Releasing Factor 1 (Ras-GRF1) Controls Activation of Extracellular Signal-Regulated Kinase (ERK) Signaling in the Striatum and Long-Term Behavioral Responses to Cocaine

    PubMed Central

    Fasano, Stefania; D’Antoni, Angela; Orban, Paul C.; Valjent, Emmanuel; Putignano, Elena; Vara, Hugo; Pizzorusso, Tommaso; Giustetto, Maurizio; Yoon, Bongjune; Soloway, Paul; Maldonado, Rafael; Caboche, Jocelyne; Brambilla, Riccardo

    2010-01-01

    Background Ras-extracellular signal-regulated kinase (Ras-ERK) signaling is central to the molecular machinery underlying cognitive functions. In the striatum, ERK1/2 kinases are co-activated by glutamate and dopamine D1/5 receptors, but the mechanisms providing such signaling integration are still unknown. The Ras-guanine nucleotide-releasing factor 1 (Ras-GRF1), a neuronal specific activator of Ras-ERK signaling, is a likely candidate for coupling these neurotransmitter signals to ERK kinases in the striatonigral medium spiny neurons (MSN) and for modulating behavioral responses to drug abuse such as cocaine. Methods We used genetically modified mouse mutants for Ras-GRF1 as a source of primary MSN cultures and organotypic slices, to perform both immunoblot and immunofluorescence studies in response to glutamate and dopamine receptor agonists. Mice were also subjected to behavioral and immunohistochemical investigations upon treatment with cocaine. Results Phosphorylation of ERK1/2 in response to glutamate, dopamine D1 agonist, or both stimuli simultaneously is impaired in Ras-GRF1– deficient striatal cells and organotypic slices of the striatonigral MSN compartment. Consistently, behavioral responses to cocaine are also affected in mice deficient for Ras-GRF1 or overexpressing it. Both locomotor sensitization and conditioned place preference are significantly attenuated in Ras-GRF1– deficient mice, whereas a robust facilitation is observed in overexpressing transgenic animals. Finally, we found corresponding changes in ERK1/2 activation and in accumulation of FosB/ΔFosB, a well-characterized marker for long-term responses to cocaine, in MSN from these animals. Conclusions These results strongly implicate Ras-GRF1 in the integration of the two main neurotransmitter inputs to the striatum and in the maladaptive modulation of striatal networks in response to cocaine. PMID:19446794

  15. GS-9191 Is a Novel Topical Prodrug of the Nucleotide Analog 9-(2-Phosphonylmethoxyethyl)Guanine with Antiproliferative Activity and Possible Utility in the Treatment of Human Papillomavirus Lesions▿

    PubMed Central

    Wolfgang, Grushenka H. I.; Shibata, Riri; Wang, Jianying; Ray, Adrian S.; Wu, Sylvia; Doerrfler, Edward; Reiser, Hans; Lee, William A.; Birkus, Gabriel; Christensen, Neil D.; Andrei, Graciela; Snoeck, Robert

    2009-01-01

    GS-9191 is a novel double prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl)guanine (PMEG) designed as a topical agent to permeate skin and be metabolized to the active nucleoside triphosphate analog in the epithelial layer. The prodrug was shown to be metabolized intracellularly to 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6,diaminopurine (cPrPMEDAP) and subsequently deaminated to PMEG. The active form, PMEG diphosphate, was shown to be a potent inhibitor of DNA polymerase α and ß while showing weaker activity against mitochondrial DNA polymerase γ (50% enzyme inhibition observed at 2.5, 1.6, and 59.4 μM, respectively). GS-9191 was markedly more potent than PMEG or cPrPMEDAP in a series of human papillomavirus (HPV)-positive cell lines, with effective concentrations to inhibit 50% cell growth (EC50) as low as 0.03, 207, and 284 nM, respectively. In contrast, GS-9191 was generally less potent in non-HPV-infected cells and primary cells (EC50s between 1 and 15 nM). DNA synthesis was inhibited by GS-9191 within 24 h of treatment; cells were observed to be arrested in S phase by 48 h and to subsequently undergo apoptosis (between 3 and 7 days). In an animal model (cottontail rabbit papillomavirus), topical GS-9191 was shown to decrease the size of papillomas in a dose-related manner. At the highest dose (0.1%), cures were evident at the end of 5 weeks, and lesions did not recur in a 30-day follow-up period. These data suggest that GS-9191 may have utility in the treatment of HPV-induced lesions. PMID:19398642

  16. Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex123

    PubMed Central

    Maeta, Kazuhiro; Edamatsu, Hironori; Nishihara, Kaori; Ikutomo, Junji; Bilasy, Shymaa E.

    2016-01-01

    Abstract Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2flox/flox brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1G12V. These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells. PMID:27390776

  17. 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.

    PubMed

    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.

  18. 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

    SciTech Connect

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

    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.

  19. Changes in the phosphorylation state of the inhibitory guanine-nucleotide-binding protein Gi-2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats.

    PubMed

    Bushfield, M; Pyne, N J; Houslay, M D

    1990-09-11

    Treatment of intact, 32Pi-labelled hepatocytes from lean Zucker rats with a range of agents including 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the alpha-subunit of the inhibitory G protein of adenylate cyclase (alpha Gi-2). These agonist-induced phosphorylations of alpha Gi-2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5'-[beta,gamma imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist-induced phosphorylation of alpha Gi-2 and to p[NH]ppG-mediated inhibition of adenylate cyclase. The basal level of alpha Gi-2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 +/- 0.09 mol phosphate/mol alpha Gi-2 than in hepatocytes from lean animals which gave 0.54 +/- 0.09 mol phosphate/mol alpha Gi-2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of alpha Gi-2 in the hepatocytes from lean animals but had little effect on the phosphorylation of alpha Gi-2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of alpha Gi-2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of alpha Gi-2. The resistance to protein-kinase-C-mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine-nucleotide-mediated 'Gi function' seen in obese Zucker rats may be due to an inactivating phosphorylation of alpha Gi-2. PMID:2120055

  20. Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides.

    PubMed Central

    Seifert, R; Schultz, G; Richter-Freund, M; Metzger, J; Wiesmüller, K H; Jung, G; Bessler, W G; Hauschildt, S

    1990-01-01

    Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity. PMID:2160237

  1. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  2. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  3. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

  4. Rho-guanine nucleotide exchange factors during development

    PubMed Central

    Mulinari, Shai

    2010-01-01

    The development of multicellular organisms is associated with extensive rearrangements of tissues and cell sheets. The driving force for these rearrangements is generated mostly by the actin cytoskeleton. In order to permit the reproducible development of a specific body plan, dynamic reorganization of the actin cytoskeleton must be precisely coordinated in space and time. GTP-exchange factors that activate small GTPases of the Rho family play an important role in this process. Here we review the role of this class of cytoskeletal regulators during important developmental processes such as epithelial morphogenesis, cytokinesis, cell migration, cell polarity, neuronal growth cone extension and phagocytosis in different model systems. PMID:21686118

  5. 21 CFR 73.1329 - Guanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... eye, in amounts consistent with good manufacturing practice. (d) Labeling. The color additive and any... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1329 Guanine. (a) Identity. (1) The color additive guanine...

  6. 21 CFR 73.1329 - Guanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... eye, in amounts consistent with good manufacturing practice. (d) Labeling. The color additive and any... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1329 Guanine. (a) Identity. (1) The color additive guanine...

  7. 21 CFR 73.1329 - Guanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... eye, in amounts consistent with good manufacturing practice. (d) Labeling. The color additive and any... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1329 Guanine. (a) Identity. (1) The color additive guanine...

  8. 21 CFR 73.1329 - Guanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... eye, in amounts consistent with good manufacturing practice. (d) Labeling. The color additive and any... FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL LISTING OF COLOR ADDITIVES EXEMPT FROM CERTIFICATION Drugs § 73.1329 Guanine. (a) Identity. (1) The color additive guanine...

  9. Novel riboswitch ligand analogs as selective inhibitors of guanine-related metabolic pathways.

    PubMed

    Mulhbacher, Jérôme; Brouillette, Eric; Allard, Marianne; Fortier, Louis-Charles; Malouin, François; Lafontaine, Daniel A

    2010-04-01

    Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5'-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge. PMID:20421948

  10. Rates of Chemical Cleavage of DNA and RNA Oligomers Containing Guanine Oxidation Products

    PubMed Central

    2016-01-01

    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. PMID:25853314

  11. Radical-radical interactions among oxidized guanine bases including guanine radical cation and dehydrogenated guanine radicals.

    PubMed

    Zhao, Jing; Wang, Mei; Yang, Hongfang; Zhang, Meng; Liu, Ping; Bu, Yuxiang

    2013-09-19

    We present here a theoretical investigation of the structural and electronic properties of di-ionized GG base pairs (G(•+)G(•+),G(-H1)(•)G(•+), and G(-H1)(•)G(-H1)(•)) consisting of the guanine cation radical (G(•+)) and/or dehydrogenated guanine radical (G(-H1)(•)) using density functional theory calculations. Different coupling modes (Watson-Crick/WC, Hoogsteen/Hoog, and minor groove/min hydrogen bonding, and π-π stacking modes) are considered. We infer that a series of G(•+)G(•+) complexes can be formed by the high-energy radiation. On the basis of density functional theory and complete active space self-consistent (CASSCF) calculations, we reveal that in the H-bonded and N-N cross-linked modes, (G(•+)G(•+))WC, (G(-H1)(•)G(-H1)(•))WC, (G(-H1)(•)G(-H1)(•))minI, and (G(-H1)(•)G(-H1)(•))minIII have the triplet ground states; (G(•+)G(•+))HoogI, (G(-H1)(•)G(•+))WC, (G(-H1)(•)G(•+))HoogI, (G(-H1)(•)G(•+))minI, (G(-H1)(•)G(•+))minII, and (G(-H1)(•)G(-H1)(•))minII possess open-shell broken-symmetry diradical-characterized singlet ground states; and (G(•+)G(•+))HoogII, (G(•+)G(•+))minI, (G(•+)G(•+))minII, (G(•+)G(•+))minIII, (G(•+)G(•+))HoHo, (G(-H1)(•)G(•+))minIII, (G(-H1)(•)G(•+))HoHo, and (G(-H1)(•)G(-H1)(•))HoHo are the closed-shell systems. For these H-bonded diradical complexes, the magnetic interactions are weak, especially in the diradical G(•+)G(•+) series and G(-H1)(•)G(-H1)(•) series. The magnetic coupling interactions of the diradical systems are controlled by intermolecular interactions (H-bond, electrostatic repulsion, and radical coupling). The radical-radical interaction in the π-π stacked di-ionized GG base pairs ((G(•+)G(•+))ππ, (G(-H1)(•)G(•+))ππ, and (G(-H1)(•)G(-H1)(•))ππ) are also considered, and the magnetic coupling interactions in these π-π stacked base pairs are large. This is the first theoretical prediction that some di

  12. 21 CFR 73.2329 - Guanine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose....

  13. 21 CFR 73.2329 - Guanine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose....

  14. 21 CFR 73.2329 - Guanine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose....

  15. 21 CFR 73.2329 - Guanine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose....

  16. 21 CFR 73.2329 - Guanine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... ADDITIVES EXEMPT FROM CERTIFICATION Cosmetics § 73.2329 Guanine. (a) Identity and specifications. (1) The... coloring cosmetics generally, only those diluents listed under § 73.1001(a)(1); (ii) For coloring externally applied cosmetics, only those diluents listed in § 73.1001(b) and, in addition, nitrocellulose....

  17. Formation of guanine ribonucleotidyl-(3'-5')-adenosine in a flavinogenic strain of Eremothecium ashbyii.

    PubMed

    Mitsuda, H; Nishikawa, Y; Nakajima, K

    1976-01-01

    The addition of caffeine caused the accumulation of a new nucleotide compound simultaneously with the rigid inhibition of ribofalvin production in non-growing cells of Eremothecium ashbyii. In the present study we tried to identify the structure of the nucleotide compound using non-growing cells of the mold. 1) It became possible to obtain a large amount of mycelia by masscultivation in a reagent tank. 2) A new nucleotide compound, referred to as compound A in the paper, was extracted with perchloric acid solution and purified by the following subsequent procedures: 1) Dowex 1 x 2 (HCOO-) column, 2) charcoal treatment, 3) DEAE-Sephadex A25 (CI-) column, 4) Dowex 1 x 2 (C1-) column, and 5) DEAE-Sephadex A25 (HCO3-) column. 3) The structure of the new nucleotide compound was proved to be guanine ribonucleotidyl-(3'-5')-adenosine (GpA) from the results of the following analyses: 1) alkaline degradation, 2) UV-spectra, IR-spectra and NMR-spectra, and 3) enzymatic treatments with RNase T2 and phosphodiesterase. 4) The roles of caffeine and guanine ribonucleotidyl-(3'-5')-adenosine in connection with flavinogenesis of this mold were discussed. PMID:182940

  18. Guanine-vacancy-bearing G-quadruplexes responsive to guanine derivatives.

    PubMed

    Li, Xin-min; Zheng, Ke-wei; Zhang, Jia-yu; Liu, Hong-he; He, Yi-de; Yuan, Bi-feng; Hao, Yu-hua; Tan, Zheng

    2015-11-24

    G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G2 and three G3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg(2+). A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy-bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes.

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

    SciTech Connect

    Yeh, Hsin-chih; Sharma, Jaswinder; Yoo, Hyojong; Martinez, Jennifer S

    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).

  20. Nucleotide Salvage Deficiencies, DNA Damage and Neurodegeneration

    PubMed Central

    Fasullo, Michael; Endres, Lauren

    2015-01-01

    Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases. PMID:25923076

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

    SciTech Connect

    Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.; Clinch, Keith; Crump, Douglas R.; Rosario Jr., Irving; Merino, Emilio F.; Almo, Steve C.; Tyler, Peter C.; Schramm, Vern L.

    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.

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

    PubMed

    Kondo, Jiro; Westhof, Eric

    2011-10-01

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

  3. Thymine and guanine base specificity of human myeloma proteins with anti-DNA activity.

    PubMed Central

    Zouali, M; Stollar, B D

    1986-01-01

    To further our understanding of the molecular basis of DNA-autoantibody interactions, we have characterized the specificities of three IgG human myeloma proteins that bind DNA. We measured their binding to synthetic single- and double-stranded homopolynucleotides, random and alternating copolymers, oligonucleotides, and nucleotides or nucleosides conjugated to non-nucleic acid carriers. All three antibodies bound single-stranded nucleic acids, including both polyribonucleotides and polydeoxyribonucleotides. They varied in relative affinities for polynucleotides of varying base composition. Polymers containing the purines guanine or hypoxanthine and/or the pyrimidine thymine were most reactive with all three proteins. A myeloma protein that reacted with poly(G), poly(I), or poly(dT) also bound to the corresponding nucleosides or nucleotides conjugated to bovine serum albumin. None of the antibodies reacted with base-paired double-helical polynucleotides (double-stranded RNA, RNA-DNA hybrid or double-stranded DNA). The results indicate that base specificity is prominent in their reactions and that the accessible epitopes in single-stranded polynucleotides become masked upon base pairing in double-stranded helices. These findings suggest a model in which positions N1 and O6 of guanine and hypoxanthine and N3 and O4 of thymine interact with amino acids of the antibody-combining site. PMID:3771789

  4. Chlorophyll fluorescence control in microalgae by biogenic guanine crystals

    NASA Astrophysics Data System (ADS)

    Miyashita, Yuito; Iwasaka, Masakazu; Endo, Hirotoshi

    2015-05-01

    Magnetic fields were applied to water suspensions of guanine crystals to induce changes in light scattering as a possible way to control photosynthesis in microalgae. The effect of guanine microcrystals with and without an applied magnetic field on the photosynthesis of a unicellular microalgae (plant), Pleurochrysis. carterae (P. carterae), was investigated by examining chlorophyll fluorescence. The fluorescence intensity at 600-700 nm of the photosynthetic cells increased remarkably when the concentration ratio of guanine microcrystals was 10 times larger than that of the cells. This increase in fluorescence occurred reproducibly and was proportional to the amount of guanine microcrystals added. It is speculated that the guanine microcrystals enhance the intensity of the excitation light on the cells by concentrating the excitation light or prolonging the time of light exposure to the cells. Moreover, applying a 500-mT magnetic field allowed modulation of the fluorescence intensity, depending on the direction of the fluorescence light.

  5. Guanine quadruplex structures localize to heterochromatin

    PubMed Central

    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.

    2016-01-01

    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. PMID:26384414

  6. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs.

    PubMed

    Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga; Polonskaia, Anna; Malinina, Lucy; Phan, Anh Tuân; Hobartner, Claudia; Micura, Ronald; Breaker, Ronald R; Patel, Dinshaw J

    2004-12-01

    Metabolite-sensing mRNAs, or "riboswitches," specifically interact with small ligands and direct expression of the genes involved in their metabolism. Riboswitches contain sensing "aptamer" modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uridine and cytosine, respectively. PMID:15610857

  7. Structural Basis for Discriminative Regulation of Gene Expression by Adenine- and Guanine-Sensing mRNAs

    PubMed Central

    Serganov, Alexander; Yuan, Yu-Ren; Pikovskaya, Olga; Polonskaia, Anna; Malinina, Lucy; Phan, Anh Tuân; Hobartner, Claudia; Micura, Ronald; Breaker, Ronald R.; Patel, Dinshaw J.

    2015-01-01

    Summary Metabolite-sensing mRNAs, or “riboswitches,” specifically interact with small ligands and direct expression of the genes involved in their metabolism. Ribo-switches contain sensing “aptamer” modules, capable of ligand-induced structural changes, and downstream regions, harboring expression-controlling elements. We report the crystal structures of the add A-riboswitch and xpt G-riboswitch aptamer modules that distinguish between bound adenine and guanine with exquisite specificity and modulate expression of two different sets of genes. The riboswitches form tuning fork-like architectures, in which the prongs are held in parallel through hairpin loop interactions, and the internal bubble zippers up to form the purine binding pocket. The bound purines are held by hydrogen bonding interactions involving conserved nucleotides along their entire periphery. Recognition specificity is associated with Watson-Crick pairing of the encapsulated adenine and guanine ligands with uri-dine and cytosine, respectively. PMID:15610857

  8. Experimental observation of guanine tautomers with VUV photoionization

    SciTech Connect

    Zhou, Jia; Kostko, Oleg; Nicolas, Christophe; Tang, Xiaonan; Belau, Leonid; de Vries, Mattanjah S.; Ahmed, Musahid

    2008-12-01

    Two methods of preparing guanine in the gas phase, thermal vaporization and laser desorption, have been investigated. The guanine generated by each method is entrained in a molecular beam, single photon ionized with tunable VUV synchrotron radiation, and analyzed using reflectron mass spectrometry. The recorded photoionization efficiency (PIE) curves show a dramatic difference for experiments performed via thermal vaporization compared to laser desorption. The calculated vertical and adiabatic ionization energies for the eight lowest lying tautomers of guanine suggest the experimental observations arise from different tautomers being populated in the two different experimental methods.

  9. Guanine- Formation During the Thermal Polymerization of Amino Acids

    NASA Technical Reports Server (NTRS)

    Mc Caw, B. K.; Munoz, E. F.; Ponnamperuma, C.; Young, R. S.

    1964-01-01

    The action of heat on a mixture of amino acids was studied as a possible abiological pathway for the synthesis of purines and pyrimidines. Guanine was detected. This result is significant in the context of chemical evolution.

  10. The Formation and Biological Significance of N7-Guanine Adducts

    PubMed Central

    Boysen, Gunnar; Pachkowski, Brian F.; Nakamura, Jun; Swenberg, James A

    2009-01-01

    DNA alkylation or adduct formation occurs at nucleophilic sites in DNA, mainly the N7-position of guanine. Ever since identification of the first N7-guanine adduct, several hundred studies on DNA adducts have been reported. Major issues addressed include the relationships between N7-guanine adducts and exposure, mutagenesis, and other biological endpoints. It became quickly apparent that N7-guanine adducts are frequently formed, but may have minimal biological relevance, since they are chemically unstable and do not participate in Watson Crick base pairing. However, N7-guanine adducts have been shown to be excellent biomarkers for internal exposure to direct acting and metabolically activated carcinogens. Questions arise, however, regarding the biological significance for N7-guanine adducts that are readily formed, do not persist, and are not likely to be mutagenic. Thus, we set out to review the current literature to evaluate their formation and the mechanistic evidence for the involvement of N7-guanine adducts in mutagenesis or other biological processes. It was concluded that there is insufficient evidence that N7-guanine adducts can be used beyond confirmation of exposure to the target tissue and demonstration of the molecular dose. There is little to no evidence that N7-guanine adducts or their depurination product, apurinic sites, are the cause of mutations in cells and tissues, since increases in AP sites have not been shown unless toxicity is extant. However, more research is needed to define the extent of chemical depurination versus removal by DNA repair proteins. Interestingly, N7-guanine adducts are clearly present as endogenous background adducts and the endogenous background amounts appear to increase with age. Furthermore, the N7-guanine adducts have been shown to convert to ring opened lesions (FAPy), which are much more persistent and have higher mutagenic potency. Studies in humans are limited in sample size and differences between controls and

  11. Hypoxanthine-guanine phosphoribosyl transferase deficiency.

    PubMed

    de Bruyn, C H

    1976-02-29

    In man congential lack of enzyme of the purine salvage system, hypoxanthineguanine phosphoribosyl transferase (HG-PRT E.C. 2.4.2.8), is mostly accompanied by a picture known as the Lesch-Nyhan snydrome. The degree of deficiency may vary from zero to a few percent of normal activity but a correlation between the severity of HG-PRT deficiency and the clinical picture has not been observed, no more than a correlation HG-PRT deficiency and neurological dysfunction. But individuals with undetectable HG-PRT activity but without the Lesch-Nyhan syndrome have been described. Patients with partial HG-PRT defiency have clinically distinctive findings. Sometimes mild neurological abnormalities are observed. Because of marked overproduction of ric acid severe gouty arthritis and renal dysfunction are often encountered in both complete and partial deficiency. There is considerable molecular heterogeneity in HG-PRT deficiency in man. Mutant ebnzymes may exhibit different kinetic and electrophoretic properties, indicating that hterwe might be a mutation on the structural gene coding for HG-PRT. Lack of HG-PRT disturbs purine interconversions profoundly. In addition to an important function of HG-PRT in the uptake of the purine hypoxantine and guanine into the cell, the effective uptake of inosine, guanosine and adenosine also seems to be dependent on HG-PRT...

  12. Nucleotide-metabolizing enzymes in Chlamydomonas flagella.

    PubMed

    Watanabe, T; Flavin, M

    1976-01-10

    Nucleotides have at least two functions in eukaryotic cilia and flagella. ATP, originating in the cells, is utilized for motility by energy-transducing protein(s) called dynein, and the binding of guanine nucleotides to tubulin, and probably certain transformations of the bound nucleotides, are prerequisites for the assembly of microtubules. Besides dynein, which can be solubulized from Chlamydomonas flagella as a heterogeneous, Mg2+ or Ca2+-activated ATPase, we have purified and characterized five other flagellar enzymes involved in nucleotide transformations. A homogeneous, low molecular weight, Ca2+-specific adenosine triphosphatase was isolated, which was inhibited by Mg2+ and was not specific for ATP. This enzyme was not formed by treating purified dynein with proteases. It was absent from extracts of Tetrahymena cilia. Its function might be an auxiliary energy transducer, or in steering or tactic responses. Two species of adenylate kinase were isolated, one of which was much elevated in regenerating flagella; the latter was also present in cell bodies. A large part of flagellar nucleoside diphosphokinase activity could not be solubilized. Two soluble enzyme species were identified, one of which was also present in cell bodies. Since these enzymes are of interest because they might function in microtubule assembly, we studied the extent to which brain nucleoside diphosphokinase co-polymerizes with tubulin purified by repeated cycles of polymerization. Arginine kinase was not detected in Chlamydomonas flagellar extracts. PMID:397

  13. Guanine Holes Are Prominent Targets for Mutation in Cancer and Inherited Disease

    PubMed Central

    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. PMID:24086153

  14. Juvenile Hormone Regulation of Drosophila Epac - A Guanine Nucleotide Exchange Factor for Rap1 Small GTPase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previously, we utilized a microchip array encompassing probes for 14,010 genes of Drosophila melanogaster to analyze the effect of (10R) juvenile hormone III (JH) on genome-wide gene expression in Drosophila S2 cells. Treatment with JH yielded a collection of 32 gene transcripts that demonstrated a ...

  15. Rho guanine nucleotide dissociation inhibitor protein (RhoGDI) inhibits exocytosis in mast cells.

    PubMed Central

    Mariot, P; O'Sullivan, A J; Brown, A M; Tatham, P E

    1996-01-01

    Introducing non-hydrolysable analogues of GTP into the cytosolic compartment of mast cells results in exocytotic secretion through the activation of GTP binding proteins. The identity and mechanism of action of these proteins are not established. We have investigated the effects of Rho GDP dissociation inhibitor (RhoGDI) on exocytosis induced by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) in rat mast cells, introducing the protein into cells by means of a patch pipette and recording the progress of exocytosis by monitoring cell capacitance. To allow time for the protein to enter the cells and find its correct location, stimulation was provided 5-10 min after patch rupture by photolysing caged GTP-gamma-S included in the pipette solution. When bovine RhoGDI was introduced into mast cells, exocytosis was inhibited at concentrations of 200-400 nM for native protein and 800 nM to 8 microM for the recombinant form. Protein denatured by heat or N-ethylmaleimide treatment did not inhibit. In permeabilized cells, recombinant RhoGDI increased the rate at which cells lose their ability to respond to GTP-gamma-S. These data demonstrate that one or more small GTP binding proteins of the Rho family has a central role in the exocytotic mechanism in mast cells. Images PMID:8978674

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

    SciTech Connect

    Lindeberg, M.; Jacobs, M. )

    1989-04-01

    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{prime}-({gamma}-thio) triphosphate (GTP-{gamma}-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 {sigma} subunit of platelet G{sub s} protein. GTP-{gamma}-S can increase specific naphthylphthalamic acid (NPA) binding in zucchini microsomal membrane preparations, with its stimulation increasing with large tissue age. Al{sup +3} and F{sup {minus}} 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{sup {minus}}{sub 4} strongly inhibited {sup 3}H-indoleacetic acid nor accumulation; GTP-{gamma}-S effects on this system will be discussed.

  17. Modeling and structural analysis of human Guanine nucleotide-binding protein-like 3,nucleostemin

    PubMed Central

    Nazmi, Farinaz; Moosavi, Mohammad Amin; Rahmati, Marveh; Hoessinpour-Feizi, Mohammad Ali

    2015-01-01

    Human GNL3 (nucleostemin) is a recently discovered nucleolar protein with pivotal functions in maintaining genomic integrity and determining cell fates of various normal and cancerous stem cells. Recent reports suggest that targeting this GTP-binding protein may have therapeutic value in cancer. Although, sequence analyzing revealed that nucleostemin (NS) comprises 5 permuted GTP-binding motifs, a crystal structure for this protein is missing at Protein Data Bank (PDB). Obviously, any attempt for predicting of NS structure can further our knowledge on its functional sites and subsequently designing molecular inhibitors. Herein, we used bioinformatics tools and could model 262 amino acids of NS (132-393 aa). Initial models were built by MODELLER, refined with Scwrl4 program, and validated with ProsA and Jcsc databases as well as PSVS software. Then, the best quality model was chosen for motif and domain analyzing by Pfam, PROSITE and PRINTS. The final model was visualized by vmd program. This predicted model may pave the way for next studies regarding ligand binding states and interaction sites as well as screening of databases for potential inhibitors. PMID:26339152

  18. A potential role for guanine nucleotide-binding protein in the regulation of endosomal proton transport.

    PubMed Central

    Gurich, R W; Codina, J; DuBose, T D

    1991-01-01

    The effects of guanosine 5'-triphosphate (GTP) and GTP-gamma-S, known activators of GTP binding proteins, on proton transport were investigated in endosome-enriched vesicles (endosomes). Endosomes were prepared from rabbit renal cortex following the intravenous injection of FITC-dextran. The rate of intravesicular acidification was determined by measuring changes in fluorescence of FITC-dextran. Both GTP and GTP-gamma-S stimulated significantly the initial rate of proton transport. In contrast, GDP-beta-S, which does not activate GTP binding proteins, inhibited proton transport. The rank order of stimulation was GTP-gamma-S greater than GTP greater than control greater than GDP-beta-S. GTP-gamma-S stimulation of proton transport was also observed under conditions in which chloride entry was eliminated, i.e., 0 mM external chloride concentration in the presence of potassium/valinomycin voltage clamping. GTP-gamma-S did not affect proton leak in endosomes as determined by collapse of H+ ATPase-generated pH gradients. ADP ribosylation by treatment of endosomal membranes with pertussis toxin revealed two substrates corresponding to the 39-41 kD region and comigrating with alpha i subunits. Pretreatment of the membranes with pertussis toxin had no effect on proton transport in the absence of GTP or GTP-gamma-S. However, pretreatment with pertussis toxin blocked the stimulation of proton transport by GTP. In contrast, as reported in other membranes by others previously, pertussis toxin did not prevent the stimulation of proton transport by GTP-gamma-S. These findings, taken together, indicate that GTP binding proteins are present in endosomal membranes derived from renal cortex and that activation of G protein by GTP and GTP-gamma-S stimulates proton transport in a rank order identical to that reported for other transport pathways modulated by Gi proteins. Therefore, these studies suggest that G proteins are capable of stimulating the vacuolar H ATPase of endosomes directly. Images PMID:1850757

  19. The X-ray structure of the PurR-guanine-purF operator complex reveals the contributions of complementary electrostatic surfaces and a water-mediated hydrogen bond to corepressor specificity and binding affinity.

    PubMed

    Schumacher, M A; Glasfeld, A; Zalkin, H; Brennan, R G

    1997-09-01

    The purine repressor, PurR, is the master regulatory protein of de novo purine nucleotide biosynthesis in Escherichia coli. This dimeric transcription factor is activated to bind to cognate DNA operator sites by initially binding either of its physiologically relevant, high affinity corepressors, hypoxanthine (Kd = 9.3 microM) or guanine (Kd = 1.5 microM). Here, we report the 2.5-A crystal structure of the PurR-guanine-purF operator ternary complex and complete the atomic description of 6-oxopurine-induced repression by PurR. As anticipated, the structure of the PurR-guanine-purF operator complex is isomorphous to the PurR-hypoxanthine-purF operator complex, and their protein-DNA and protein-corepressor interactions are nearly identical. The former finding confirms the use of an identical allosteric DNA-binding mechanism whereby corepressor binding 40 A from the DNA-binding domain juxtaposes the hinge regions of each monomer, thus favoring the formation and insertion of the critical minor groove-binding hinge helices. Strikingly, the higher binding affinity of guanine for PurR and the ability of PurR to discriminate against 2-oxopurines do not result from direct protein-ligand interactions, but rather from a water-mediated contact with the exocyclic N-2 of guanine, which dictates the presence of a donor group on the corepressor, and the better electrostatic complementarity of the guanine base and the corepressor-binding pocket.

  20. Lethal Accumulation of Guanylic Nucleotides in Saccharomyces cerevisiae HPT1-Deregulated Mutants

    PubMed Central

    Breton, Annick; Pinson, Benoît; Coulpier, Fanny; Giraud, Marie-France; Dautant, Alain; Daignan-Fornier, Bertrand

    2008-01-01

    Guanylic nucleotide biosynthesis is a conserved and highly regulated process. Drugs reducing GMP synthesis affect the immunological response and mutations enabling guanylic-derivative recycling lead to severe mental retardation. While the effects of decreased GMP synthesis have been well documented, the consequences of GMP overproduction in eukaryotes are poorly understood. In this work, we selected and characterized several mutations making yeast hypoxanthine–guanine phosphoribosyltransferase insensitive to feedback inhibition by GMP. In these mutants, accumulation of guanylic nucleotides can be triggered by addition of extracellular guanine. We show that such an accumulation is highly toxic for yeast cells and results in arrest of proliferation and massive cell death. This growth defect could be partially suppressed by overexpression of Rfx1p, a transcriptional repressor of the DNA damage response pathway. Importantly, neither guanylic nucleotide toxicity nor its suppression by Rfx1p was associated with an alteration of forward mutation frequency. PMID:18245832

  1. Impedimetric investigation of gold nanoparticles - guanine modified electrode

    NASA Astrophysics Data System (ADS)

    Vulcu, A.; Pruneanu, S.; Berghian-Grosan, C.; Olenic, L.; Muresan, L. M.; Barbu-Tudoran, L.

    2013-11-01

    In this paper we report the preparation of a modified electrode with gold nanoparticles and guanine. The colloidal suspension of gold nanoparticles was obtained by Turkevich method and was next analyzed by UV-Vis spectroscopy and Transmission Electron Microscopy (TEM). The gold electrode was modified by self-assembling the gold nanoparticles with guanine, the organic molecule playing also the role of linker. The electrochemical characteristics of the bare and modified electrode were investigated by Electrochemical Impedance Spectroscopy (EIS). A theoretical model was developed based on an electrical equivalent circuit which contain solution resistance (Rs), charge transfer resistance (Rct), Warburg impedance (ZW) and double layer capacitance (Cdl).

  2. Analysis of Electric Properties of DNA Nucleotides

    NASA Astrophysics Data System (ADS)

    Zikic, R.; Zhang, X.-G.; Krstic, P. S.; Wells, J. C.; Fuentes-Cabrera, M.

    2006-05-01

    Calculation of the quantum tunnelling conductance through the DNA nucleotides between gold nanoelectrodes and analysis of the corresponding molecular spectra reveals that the tunneling conductance at low electric bias can be separated into two simple and approximately independent factors. The first is an exponential factor due to the potential barrier between the molecule and the electrode. The second factor is different for each molecule, but follows a universal form that can be expressed in terms of the bending angle of the DNA base relative to the sugar-phosphate group. This factor is also oscillatory indicating interference and resonance effects inside the molecule. Distinguishable conductances of Adenine (A), Cytosine (C), Guanine (G), and Thymine (T) are correlated to their differences in geometric dimensions.

  3. Guanine base stacking in G-quadruplex nucleic acids.

    PubMed

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

    2013-02-01

    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

  4. Crystal structures and inhibition of Trypanosoma brucei hypoxanthine–guanine phosphoribosyltransferase

    PubMed Central

    Terán, David; Hocková, Dana; Česnek, Michal; Zíková, Alena; Naesens, Lieve; Keough, Dianne T.; Guddat, Luke W.

    2016-01-01

    Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 μM and 77 μM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 μM (with guanine as base) and 15.8 μM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed. PMID:27786284

  5. Camptothecins guanine interactions: mechanism of charge transfer reaction upon photoactivation

    NASA Astrophysics Data System (ADS)

    Steenkeste, K.; Guiot, E.; Tfibel, F.; Pernot, P.; Mérola, F.; Georges, P.; Fontaine-Aupart, M. P.

    2002-01-01

    The potent activity exhibited by the antitumoral camptothecin (CPT) and its analog irinotecan (CPT-11) is known to be related to a close contact between the drug and the nucleic acid base guanine. This specificity of interaction between these two chromophores was examined by following changes in the photophysical properties of the drug using steady-state as well as time-resolved absorption and fluorescence methods. The observed effects on absorption, fluorescence emission and singlet excited state lifetimes give evidence for the occurrence of a stacking complex formation restricted to the quinoline part of CPT or CPT-11 and the guanine base but also with the adenine base. The triplet excited state properties of the drugs have been also characterized in absence and in presence of guanosine monophosphate and reveal the occurrence of an electron transfer from the guanine base to the drug. Support for this conclusion was obtained from the studies of a set of biological targets of various oxido-reduction potentials, adenosine monophosphate, cytidine, cytosine, tryptophan, tyrosine and phenylalanine. This finding gives an interpretation of the CPT-induced guanine photolesions previously reported in the literature. These data taken together are discussed in connection with the drug activity. The stacking complex CPT/guanine is necessary but not sufficient to explain the role of the chirality and of the lactone structure in the function of the drug. A stereospecific interaction with the enzyme topoisomerase I seems necessary to stabilize the stacking complex. The first experiments using time-resolved fluorescence by two-photon excitation confirms that CPT does not bind to the isolated enzyme.

  6. Multiplexed DNA detection with a composite molecular beacon based on guanine-quenching.

    PubMed

    Xiang, Dong-Shan; Zhai, Kun; Wang, Lian-Zhi

    2013-09-21

    We developed a multiplexed DNA detection method with a composite molecular beacon (MB) probe based on guanine-quenching by synchronous fluorescence analysis. It is demonstrated by two types of tumor-suppressor genes namely exon segments of p16 (T1) and p53 (T2) genes. The composite MB probe includes two loops and two stems, and two fluorophores of 6-carboxyfluorescein group (FAM) and tetramethyl-6-carboxyrhodamine (TAMRA) are connected to the two ends of molecular beacon. Every stem portion of MB include four continuous nucleotides with guanine (G) base as quencher, every loop portion is a probe sequence that is complementary to a corresponding target sequence. In the absence of target DNA, the composite MBs are in the stem-closed form, the fluorescence of FAM and TAMRA are quenched by G bases. At this time, the fluorescence signals of FAM and TAMRA are all very low. In the presence of target DNA, the MBs hybridize with the target DNA and form double-strands, FAM and TAMRA are separated from G bases, and the fluorescence of FAM and TAMRA recovers simultaneously. Thus, the simultaneous detection of two targets of DNA can be realized by measuring fluorescence signals of FAM and TAMRA, respectively. Under the optimum conditions, the fluorescence intensities of FAM and TAMRA all exhibit good linear dependence on their target DNA concentration in the range from 5 × 10(-11) to 5.5 × 10(-9) M. The detection limit of T1 is 4 × 10(-11) M (3σ), and that of T2 is 3 × 10(-11) M. This composite MB can be applied to detect the real sample, and can be applied to detect two aleatoric sequences of DNA. Compared with previously reported methods of detecting multiplexed target DNA with MBs, the proposed method has some advantages including easy synthesis of composite MB probes, low detection cost and shorter analytical time.

  7. Effect of base pairing on the electrochemical oxidation of guanine.

    PubMed

    Costentin, Cyrille; Hajj, Viviane; Robert, Marc; Savéant, Jean-Michel; Tard, Cédric

    2010-07-28

    The effect of base pairing by cytosine on the electrochemical oxidation of guanine is examined by means of cyclic voltammetry on carefully purified reactants in a solvent, CHCl(3), which strongly favors the formation of an H-bonded pair. The thermodynamics and kinetics of the oxidation reaction are not strongly influenced by the formation of the pair. They are actually similar to those of the reaction in which 2,6-lutidine, an encumbered base that cannot form a pair with guanine, replaces cytosine. The reaction does not entail a concerted proton-electron mechanism, as attested by the absence of H/D isotope effect. It rather involves the rate-determining formation of the cation radical, followed by its deprotonation and dimerization of the resulting neutral radical in competition with its further oxidation.

  8. Impedimetric investigation of gold nanoparticles - guanine modified electrode

    SciTech Connect

    Vulcu, A.; Pruneanu, S.; Berghian-Grosan, C.; Olenic, L.; Muresan, L. M.; Barbu-Tudoran, L.

    2013-11-13

    In this paper we report the preparation of a modified electrode with gold nanoparticles and guanine. The colloidal suspension of gold nanoparticles was obtained by Turkevich method and was next analyzed by UV-Vis spectroscopy and Transmission Electron Microscopy (TEM). The gold electrode was modified by self-assembling the gold nanoparticles with guanine, the organic molecule playing also the role of linker. The electrochemical characteristics of the bare and modified electrode were investigated by Electrochemical Impedance Spectroscopy (EIS). A theoretical model was developed based on an electrical equivalent circuit which contain solution resistance (R{sub s}), charge transfer resistance (R{sub ct}), Warburg impedance (Z{sub W}) and double layer capacitance (C{sub dl})

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

    PubMed

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

    2015-05-01

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

  10. `Guanigma': the revised structure of biogenic anhydrous guanine

    NASA Astrophysics Data System (ADS)

    Hirsch, Anna; Gur, Dvir; Polishchuk, Iryna; Levy, Davide; Pokroy, Boaz; Cruz-Cabeza, Aurora J.; Addadi, Lia; Kronik, Leeor; Leiserowitz, Leslie

    Living organisms display a spectrum of colors, produced by pigmentation, structural coloration, or both. A relatively well-studied system, which produces colors via an array of alternating anhydrous guanine crystals and cytoplasm, is responsible for the metallic luster of many fish. The structure of biogenic anhydrous guanine was believed to be the same as that of the synthetic one - a monoclinic polymorph. Here we re-examine the structure of biogenic guanine, using experimental X-ray and electron diffraction (ED) data exposing troublesome inconsistencies - namely, a 'guanigma'. To address this, we sought alternative candidate polymorphs using symmetry and packing considerations, then used first principles calculations to determine whether the selected candidates could be energetically stable. We identified theoretically a different monoclinic polymorph, were able to synthesize it, and to confirm using X-ray diffraction that it is this polymorph that occurs in biogenic samples. However, the ED data were still not consistent with this polymorph, but rather with a theoretically generated orthorhombic polymorph. This apparent inconsistency was resolved by showing how the ED pattern could be affected by crystal structural faults composed of offset molecular layers.

  11. In vivo formation of N7-guanine DNA adduct by safrole 2',3'-oxide in mice.

    PubMed

    Shen, Li-Ching; Chiang, Su-Yin; Lin, Ming-Huan; Chung, Wen-Sheng; Wu, Kuen-Yuh

    2012-09-18

    Safrole, a naturally occurring product derived from spices and herbs, has been shown to be associated with the development of hepatocellular carcinoma in rodents. Safrole 2',3'-oxide (SFO), an electrophilic metabolite of safrole, was shown to react with DNA bases to form detectable DNA adducts in vitro, but not detected in vivo. Therefore, the objective of this study was to investigate the formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SFO-Gua) resulting from the reaction of SFO with the most nucleophilic site of guanine in vitro and in vivo with a newly developed isotope-dilution high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method. N7γ-SFO-Gua and [(15)N(5)]-N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine ([(15)N(5)]-N7γ-SFO-Gua) were first synthesized, purified, and characterized. The HPLC-ESI-MS/MS method was developed to measure N7γ-SFO-Gua in calf thymus DNA treated with 60 μmol of SFO for 72 h and in urine samples of mice treated with a single dose of SFO (30 mg/kg body weight, intraperitoneally). In calf thymus DNA, the level of N7γ-SFO-Gua was 2670 adducts per 10(6)nucleotides. In urine of SFO-treated mice, the levels of N7γ-SFO-Gua were 1.02±0.14 ng/mg creatinine (n=4) on day 1, 0.73±0.68 ng/mg creatinine (n=4) on day 2, and below the limit of quantitation on day 3. These results suggest that SFO can cause in vivo formation of N7γ-SFO-Gua, which may then be rapidly depurinated from the DNA backbone and excreted through urine.

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

    PubMed

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

    2015-12-14

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

  13. Specific replacement of Q base in the anticodon of tRNA by guanine catalyzed by a cell-free extract of rabbit reticulocytes.

    PubMed Central

    Okada, N; Harada, F; Nishimura, S

    1976-01-01

    Guanylation of tRNA by a lysate of rabbit reticulocytes was reported previously by Farkas and Singh. This reaction was investigated further using 18 purified E. coli tRNAs as acceptors.Results showed that only tRNATyr, tRNAHis, tRNAAsn and tRNAAsp which contain the modified nucleoside Q in the anticodon acted as acceptors. Analysis of the nucleotide sequences in the guanylated tRNA showed that guanine specifically replaced Q base in these tRNAs. Images PMID:792816

  14. Rho guanine exchange factors in blood vessels: fine-tuners of angiogenesis and vascular function.

    PubMed

    Kather, Jakob Nikolas; Kroll, Jens

    2013-05-15

    The angiogenic cascade is a multi-step process essential for embryogenesis and other physiological and pathological processes. Rho family GTPases are binary molecular switches and serve as master regulators of various basic cellular processes. Rho GTPases are known to exert important functions in angiogenesis and vascular physiology. These functions demand a tight and context-specific control of cellular processes requiring superordinate control by a multitude of guanine nucleotide exchange factors (GEFs). GEFs display various features enabling them to fine-tune the actions of Rho GTPases in the vasculature: (1) GEFs regulate specific steps of the angiogenic cascade; (2) GEFs show a spatio-temporally specific expression pattern; (3) GEFs differentially regulate endothelial function depending on their subcellular location; (4) GEFs mediate crosstalk between complex signaling cascades and (5) GEFs themselves are regulated by another layer of interacting proteins. The aim of this review is to provide an overview about the role of GEFs in regulating angiogenesis and vascular function and to point out current limitations as well as clinical perspectives.

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

    PubMed Central

    Dhapola, Parashar; Chowdhury, Shantanu

    2016-01-01

    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. PMID:27185890

  16. Crystal Structure of a Replicative DNA Polymerase Bound to the Oxidized Guanine Lesion Guanidinohydantoin

    SciTech Connect

    Aller, Pierre; Ye, Yu; Wallace, Susan S.; Burrows, Cynthia J.; Doubli, Sylvie

    2010-04-12

    The oxidation of guanine generates one of the most common DNA lesions, 8-oxo-7,8-dihydroguanine (8-oxoG). The further oxidation of 8-oxoG can produce either guanidinohydantoin (Gh) in duplex DNA or spiroiminodihydantoin (Sp) in nucleosides and ssDNA. Although Gh can be a strong block for replicative DNA polymerases such as RB69 DNA polymerase, this lesion is also mutagenic: DNA polymerases bypass Gh by preferentially incorporating a purine with a slight preference for adenine, which results in G {center_dot} C {yields} T {center_dot} A or G {center_dot} C {yields} C {center_dot} G transversions. The 2.15 {angstrom} crystal structure of the replicative RB69 DNA polymerase in complex with DNA containing Gh reveals that Gh is extrahelical and rotated toward the major groove. In this conformation Gh is no longer in position to serve as a templating base for the incorporation of an incoming nucleotide. This work also constitutes the first crystallographic structure of Gh, which is stabilized in the R configuration in the two polymerase/DNA complexes present in the crystal asymmetric unit. In contrast to 8-oxoG, Gh is found in a high syn conformation in the DNA duplex and therefore presents the same hydrogen bond donor and acceptor pattern as thymine, which explains the propensity of DNA polymerases to incorporate a purine opposite Gh when bypass occurs.

  17. Production of guanine from NH(4)CN polymerizations

    NASA Technical Reports Server (NTRS)

    Levy, M.; Miller, S. L.; Oro, J.

    1999-01-01

    The synthesis of adenine from the polymerization of concentrated ammonium cyanide solutions is well known. We show here that guanine is also produced by this reaction but at yields ranging from 10 to 40 times less than that of adenine. This synthesis is effective at both +80 and -20 degrees C. Since high concentrations of NH(4)CN are obtainable only by freezing, this prebiotic synthesis would be applicable to frozen regions of the primitive Earth, the Jovian satellite Europa and other icy satellites, and the parent body of the Murchison meteorite.

  18. Production of guanine from NH(4)CN polymerizations.

    PubMed

    Levy, M; Miller, S L; Oró, J

    1999-08-01

    The synthesis of adenine from the polymerization of concentrated ammonium cyanide solutions is well known. We show here that guanine is also produced by this reaction but at yields ranging from 10 to 40 times less than that of adenine. This synthesis is effective at both +80 and -20 degrees C. Since high concentrations of NH(4)CN are obtainable only by freezing, this prebiotic synthesis would be applicable to frozen regions of the primitive Earth, the Jovian satellite Europa and other icy satellites, and the parent body of the Murchison meteorite. PMID:10441668

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

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  20. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

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

  1. The guanine cap of human guanylate-binding protein 1 is responsible for dimerization and self-activation of GTP hydrolysis.

    PubMed

    Wehner, Mark; Kunzelmann, Simone; Herrmann, Christian

    2012-01-01

    Human guanylate-binding protein 1 (hGBP1) belongs to the superfamily of large, dynamin-related GTPases. The expression of hGBP1 is induced by stimulation with interferons (mainly interferon-γ), and it plays a role in different cellular responses to inflammatory cytokines, e.g. pathogen defence, control of proliferation, and angiogenesis. Although other members of the dynamin superfamily show a diversity of cellular functions, they share a common GTPase mechanism that relies on nucleotide-controlled oligomerization and self-activation of the GTPase. Previous structural studies on hGBP1 have suggested a mechanism of GTPase and GDPase activity that, as a critical step, involves dimerization of the large GTP-binding domains. In this study, we show that the guanine cap of hGBP1 is the key structural element responsible for dimerization, and is thereby essential for self-activation of the GTPase activity. Studies of concentration-dependent GTP hydrolysis showed that mutations of residues in the guanine cap, in particular Arg240 and Arg244, resulted in higher dissociation constants of the dimer, whereas the maximum hydrolytic activity was largely unaffected. Additionally, we identified an intramolecular polar contact (Lys62-Asp255) whose mutation leads to a loss of self-activation capability and controlled oligomer formation. We suggest that this contact structurally couples the guanine cap to the switch regions of the GTPase, translating the structural changes that occur upon nucleotide binding to a change in oligomerization and self-activation. PMID:22059445

  2. Mobility enhancement of organic field-effect transistor based on guanine trap-neutralizing layer

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    We introduced a nucleic acid component guanine as a trap-neutralizing layer between silicon dioxide gate dielectric and a pentacene semiconducting layer to obtain increased field-effect mobility in organic field-effect transistors (OFETs). A tripling of the field-effect mobility, from 0.13 to 0.42 cm2/V s, was achieved by introducing a 2 nm guanine layer. By characterizing the surface morphology of pentacene films grown on guanine, we found that the effect of guanine layer on the topography of pentacene film was not responsible for the mobility enhancement of the OFETs. The increased field-effect mobility was mainly attributed to the hydrogen bonding capacity of otherwise unassociated guanine molecules, which enabled them to neutralize trapping sites on the silicon dioxide surface.

  3. Ab initio study of guanine damage by hydroxyl radical.

    PubMed

    Chaban, Galina M; Wang, Dunyou; Huo, Winifred M

    2015-01-15

    Multiconfigurational ab initio methods are used in this study to examine two initial reactions that take place during the OH radical attack of the DNA base guanine: a ring opening reaction and a hydrogen transfer reaction. The same reactions are also studied in the presence of a single water molecule. The ring opening reaction has a moderate barrier height of ∼20-25 kcal/mol that is relatively insensitive to the presence of water. The barrier of the H-transfer reaction, on the other hand, is lowered from ∼50 to ∼22 kcal/mol when one water molecule is added, thus becoming comparable to the barrier height of the ring opening reaction. PMID:25517252

  4. Guanine modification of inhibitory oligonucleotides potentiates their suppressive function.

    PubMed

    Römmler, Franziska; Jurk, Marion; Uhlmann, Eugen; Hammel, Monika; Waldhuber, Anna; Pfeiffer, Lavinia; Wagner, Hermann; Vollmer, Jörg; Miethke, Thomas

    2013-09-15

    Inhibitory TLR7 and/or TLR9 oligonucleotides (inhibitory oligonucleotide [INH-ODN]) are characterized by a phosphorothioate backbone and a CC(T)XXX₃₋₅GGG motif, respectively. INH-ODN 2088 is a prototypic member of this class of INH-ODN and acts as a TLR7 and TLR9 antagonist. It contains a G quadruple that leads to higher order structures by the formation of G tetrads. These structures are unfavorable for the prediction of their pharmacological and immunological behavior. We show in this study that modification of Gs within the G quadruple by 7-deaza-guanine or 7-deaza-2'-O-methyl-guanine avoids higher order structures and improves their inhibitory potential. Whereas TLR9-induced TNF-α secretion of bone marrow-derived macrophages and conventional dendritic cells was equally inhibited by INH-ODN 2088 and G-modified INH-ODNs such as INH-ODN 24888, TLR7-induced TNF-α release and TLR7- and TLR9-induced IL-12p40 release were significantly more impaired by G-modified INH-ODNs. Similarly, the IL-6 release of B cells from wild-type and autoimmune MRL/Mp-lpr/lpr mice was more efficiently impaired by G-modified INH-ODNs. Surprisingly, INH-ODN 2088 stimulated B cells to proliferate when used in higher doses. Finally, in vivo, in wild-type and autoimmune MRL/Mp-lpr/lpr mice, G-modified INH-ODN 24888 was significantly more efficient than unmodified INH-ODN 2088. In summary, G modification allows the development of INH-ODNs with superior inhibitory potency for inflammatory diseases with high medical need such as systemic lupus erythematosus. PMID:23966630

  5. Analysis of difference spectra of protonated DNA: determination of degree of protonation of nitrogen bases and the fractions of disordered nucleotide pairs.

    PubMed Central

    Smol'janinova, T I; Zhidkov, V A; Sokolov, G V

    1982-01-01

    The titration curves of nitrogen bases and fractions of disordered nucleotide pairs are obtained during DNA protonation. It is shown that purine bases are the first sites of the DNA double helix protonation. The cytosine protonation is due to proton-induced conformational transition within GC pairs with the sequence proton transfer from (N-7) of guanine to (N-3) of cytosine. Within DNA with unwound regions the bases are protonated in the following order: cytosine, adenine, guanine. It is shown that GC pairs are the primary centres in which the unwinding of protonated DNAs occurs. PMID:7079177

  6. A non-catalytic N-terminal domain negatively influences the nucleotide exchange activity of translation elongation factor 1Bα.

    PubMed

    Trosiuk, Tetiana V; Shalak, Vyacheslav F; Szczepanowski, Roman H; Negrutskii, Boris S; El'skaya, Anna V

    2016-02-01

    Eukaryotic translation elongation factor 1Bα (eEF1Bα) is a functional homolog of the bacterial factor EF-Ts, and is a component of the macromolecular eEF1B complex. eEF1Bα functions as a catalyst of guanine nucleotide exchange on translation elongation factor 1A (eEF1A). The C-terminal domain of eEF1Bα is necessary and sufficient for its catalytic activity, whereas the N-terminal domain interacts with eukaryotic translation elongation factor 1Bγ (eEF1Bγ) to form a tight complex. However, eEF1Bγ has been shown to enhance the catalytic activity of eEF1Bα attributed to the C-terminal domain of eEF1Bα. This suggests that the N-terminal domain of eEF1Bα may in some way influence the guanine nucleotide exchange process. We have shown that full-length recombinant eEF1Bα and its truncated forms are non-globular proteins with elongated shapes. Truncation of the N-terminal domain of eEF1Bα, which is dispensable for catalytic activity, resulted in acceleration of the rate of guanine nucleotide exchange on eEF1A compared to full-length eEF1Bα. A similar effect on the catalytic activity of eEF1Bα was observed after its interaction with eEF1Bγ. We suggest that the non-catalytic N-terminal domain of eEF1Bα may interfere with eEF1A binding to the C-terminal catalytic domain, resulting in a decrease in the overall rate of the guanine nucleotide exchange reaction. Formation of a tight complex between the eEF1Bγ and eEF1Bα N-terminal domains abolishes this inhibitory effect. PMID:26587907

  7. Characterization of lysine-guanine cross-links upon one-electron oxidation of a guanine-containing oligonucleotide in the presence of a trilysine peptide.

    PubMed

    Perrier, Sandrine; Hau, Jörg; Gasparutto, Didier; Cadet, Jean; Favier, Alain; Ravanat, Jean-Luc

    2006-05-01

    Formation of DNA-protein cross-links involving the initial formation of a guanine radical cation was investigated. For this purpose, riboflavin-mediated photosensitization of a TGT oligonucleotide in aerated aqueous solution in the presence of the KKK tripeptide was performed. We have shown that the nucleophilic addition of the epsilon-amino group of the central lysine residue of KKK to the C8 atom of either the guanine radical cation or its deprotonated form gives rise to the efficient formation of a Nepsilon-(guanin-8-yl)-lysine cross-link. Interestingly, the time course of formation of the above-mentioned cross-link was found to be not linear with the time of irradiation, and its formation rapidly reached a plateau. This is explained by secondary decomposition of the initially generated cross-link which could be further oxidized more efficiently than starting TGT oligonucleotide. One-electron oxidation of the initially generated cross-link was found to produce mainly two diastereomeric cross-links exhibiting a spiroimino-trilysine-dihydantoin structure as inferred from enzymatic digestion, CD, UV, NMR and mass spectrometry measurements. In addition, other minor cross-links, for which formation was favored at acidic pH, were assigned as lysine-guanine adducts in which the modified guanine base exhibits a guanidino-trilysine-iminohydantoin structure. A proposed mechanism for the formation of the different detected oligonucleotide-peptide cross-links is given. The high yield of formation of the detected cross-links strongly suggests that a DNA-protein cross-link involving a lysine residue linked to the C8 position of guanine could be generated in cellular systems if a lysine is located in the close vicinity of a guanine radical cation.

  8. Structural and Functional Studies on Nucleotide Excision Repair From Recognition to Incision.

    SciTech Connect

    Caroline Kisker

    2001-01-01

    Maintenance of the correct genetic information is crucial for all living organisms because mutations are the primary cause of hereditary diseases, as well as cancer and may also be involved in aging. The importance of genomic integrity is underscored by the fact that 80 to 90% of all human cancers are ultimately due to DNA damage. Among the different repair mechanisms that have evolved to protect the genome, nucleotide excision repair (NER) is a universal pathway found in all organisms. NER removes a wide variety of bulky DNA adducts including the carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo(a)pyrene-guanine adducts caused by smoking and the guanine-cisplatin adducts induced by chemotherapy. The importance of this repair mechanism is reflected by three severe inherited diseases in humans, which are due to defects in NER: xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.

  9. Enhanced Molecular Recognition between Nucleobases and Guanine-5'-monophosphate-disodium (GMP) by Surfactant Aggregates in Aqueous Solution.

    PubMed

    Liu, Zhang; Wang, Dong; Cao, Meiwen; Han, Yuchun; Xu, Hai; Wang, Yilin

    2015-07-15

    Only specific base pairs on DNA can bind with each other through hydrogen bonds, which is called the Watson-Crick (W/C) pairing rule. However, without the constraint of DNA chains, the nucleobases in bulk aqueous solution usually do not follow the W/C pairing rule anymore because of the strong competitive effect of water and the multi-interaction edges of nucleobases. The present work applied surfactant aggregates noncovalently functionalized by nucleotide to enhance the recognition between nucleobases without DNA chains in aqueous solution, and it revealed the effects of their self-assembling ability and morphologies on the recognition. The cationic ammonium monomeric, dimeric, and trimeric surfactants DTAB, 12-3-12, and 12-3-12-3-12 were chosen. The surfactants with guanine-5'-monophosphate-disodium (GMP) form micelles, vesicles, and fingerprint-like and plate-like aggregates bearing the hydrogen-bonding sites of GMP, respectively. The binding parameters of these aggregates with adenine (A), uracil (U), guanine (G), and cytosine(C) indicate that the surfactants can promote W/C recognitions in aqueous solution when they form vesicles (GMP/DTAB) or plate-like aggregates (GMP/12-3-12) with proper molecular packing compactness, which not only provide hydrophobic environments but also shield non-W/C recognition edges. However, the GMP/12-3-12 micelles with loose molecular packing, the GMP/12-3-12 fingerprint-like aggregates where the hydrogen bond sites of GMP are occupied by itself, and the GMP/12-3-12-3-12 vesicles with too strong self-assembling ability cannot promote W/C recognition. This work provides insight into how to design self-assemblies with the performance of enhanced molecule recognition.

  10. Radioimmunoassay for cyclic nucleotides

    SciTech Connect

    Chiang, C.S.

    1984-02-21

    An improved radioimmunoassay for the determination of cyclic nucleotides in body fluids which comprises adding a source of divalent cation prior to assay minimizes the effects of both endogenous calcium ion and EDTA used as an anticoagulant in blood plasma samples.

  11. Nucleotide diversity in gorillas.

    PubMed Central

    Yu, Ning; Jensen-Seaman, Michael I; Chemnick, Leona; Ryder, Oliver; Li, Wen-Hsiung

    2004-01-01

    Comparison of the levels of nucleotide diversity in humans and apes may provide valuable information for inferring the demographic history of these species, the effect of social structure on genetic diversity, patterns of past migration, and signatures of past selection events. Previous DNA sequence data from both the mitochondrial and the nuclear genomes suggested a much higher level of nucleotide diversity in the African apes than in humans. Noting that the nuclear DNA data from the apes were very limited, we previously conducted a DNA polymorphism study in humans and another in chimpanzees and bonobos, using 50 DNA segments randomly chosen from the noncoding, nonrepetitive parts of the human genome. The data revealed that the nucleotide diversity (pi) in bonobos (0.077%) is actually lower than that in humans (0.087%) and that pi in chimpanzees (0.134%) is only 50% higher than that in humans. In the present study we sequenced the same 50 segments in 15 western lowland gorillas and estimated pi to be 0.158%. This is the highest value among the African apes but is only about two times higher than that in humans. Interestingly, available mtDNA sequence data also suggest a twofold higher nucleotide diversity in gorillas than in humans, but suggest a threefold higher nucleotide diversity in chimpanzees than in humans. The higher mtDNA diversity in chimpanzees might be due to the unique pattern in the evolution of chimpanzee mtDNA. From the nuclear DNA pi values, we estimated that the long-term effective population sizes of humans, bonobos, chimpanzees, and gorillas are, respectively, 10,400, 12,300, 21,300, and 25,200. PMID:15082556

  12. Statistical analysis of nucleotide runs in coding and noncoding DNA sequences.

    PubMed

    Sprizhitsky YuA; Nechipurenko YuD; Alexandrov, A A; Volkenstein, M V

    1988-10-01

    A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.

  13. Cytochrome b nucleotide sequence variation among the Atlantic Alcidae.

    PubMed

    Friesen, V L; Montevecchi, W A; Davidson, W S

    1993-01-01

    Analysis of cytochrome b nucleotide sequences of the six extant species of Atlantic alcids and a gull revealed an excess of adenines and cytosines and a deficit of guanines at silent sites on the coding strand. Phylogenetic analyses grouped the sequences of the common (Uria aalge) and Brünnich's (U. lomvia) guillemots, followed by the razorbill (Alca torda) and little auk (Alle alle). The black guillemot (Cepphus grylle) sequence formed a sister taxon, and the puffin (Fratercula arctica) fell outside the other alcids. Phylogenetic comparisons of substitutions indicated that mutabilities of bases did not differ, but that C was much more likely to be incorporated than was G. Imbalances in base composition appear to result from a strand bias in replication errors, which may result from selection on secondary RNA structure and/or the energetics of codon-anticodon interactions. PMID:7916741

  14. Juvenile Hormone Regulates the Expression of Drosophila Epac– a Guanine Nucleotide Exchange Factor for Rap1 Small GTPase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The juvenile hormones (JH) are a key group of insect hormones involved in regulating larval development and adult reproductive processes. Although well-studied from the physiological standpoint, the molecular actions of JH remain unclear. Using cDNA microchip array technology, we previously identifi...

  15. Role of the inhibitory guanine nucleotide regulatory protein in high affinity. cap alpha. /sub 2/ adrenergic agonist binding

    SciTech Connect

    Kim, M.H.

    1987-01-01

    The purpose of this study was to determine whether regulatory protein, N/sub i/ was required for high affinity agonist binding to the a/sub 2/ adrenergic receptor in human platelet membranes. Human platelet membranes treated under alkaline conditions (pH 11.5) exhibited a selective and complete loss of high affinity agonist binding as measured by the parital agonist (/sup 3/H)-p-aminoclonidine and full agonist (/sup 3/H)UK 14,304 in direct binding studies. The binding parameters for (/sup 3/H)UK 14,304 are as follows: for control platelet membranes, the K/sub d/ was 0.88 +/- 0.17 and nM and the B/sub max/ was 280 +/- 20 fmol/mg compared to 1.89 +/- 0.34 nM and 75 fmol/mg for pH 11.5 treated membranes. For (/sup 3/H)p-aminoclonidine, the data for pH 11.5 treated membranes is as follows: B/sub max/ = 100 +/- 20 fmol/mg, K/sub d/ = 3.4 +/- 0.1 nM, compared to control membranes: (best fit with a two site fit) K/sub d1/ = 0.7 nM, K/sub d2/ = 8 nM, B/sub max1/ = 76 fmol/mg, B/sub max2/ = 198 fmol/mg. The ..cap alpha../sub 2/ antagonists, (/sup 3/H)yohimbine, was used to assess the presence of the receptor.

  16. Pioneer Axon Navigation Is Controlled by AEX-3, a Guanine Nucleotide Exchange Factor for RAB-3 in Caenorhabditis elegans.

    PubMed

    Bhat, Jaffar M; Hutter, Harald

    2016-07-01

    Precise and accurate axon tract formation is an essential aspect of brain development. This is achieved by the migration of early outgrowing axons (pioneers) allowing later outgrowing axons (followers) to extend toward their targets in the embryo. In Caenorhabditis elegans the AVG neuron pioneers the right axon tract of the ventral nerve cord, the major longitudinal axon tract. AVG is essential for the guidance of follower axons and hence organization of the ventral nerve cord. In an enhancer screen for AVG axon guidance defects in a nid-1/Nidogen mutant background, we isolated an allele of aex-3 aex-3 mutant animals show highly penetrant AVG axon navigation defects. These defects are dependent on a mutation in nid-1/Nidogen, a basement membrane component. Our data suggest that AEX-3 activates RAB-3 in the context of AVG axon navigation. aex-3 genetically acts together with known players of vesicular exocytosis: unc-64/Syntaxin, unc-31/CAPS, and ida-1/IA-2. Furthermore our genetic interaction data suggest that AEX-3 and the UNC-6/Netrin receptor UNC-5 act in the same pathway, suggesting AEX-3 might regulate the trafficking and/or insertion of UNC-5 at the growth cone to mediate the proper guidance of the AVG axon.

  17. Activated RhoA is a positive feedback regulator of the Lbc family of Rho guanine nucleotide exchange factor proteins.

    PubMed

    Medina, Frank; Carter, Angela M; Dada, Olugbenga; Gutowski, Stephen; Hadas, Jana; Chen, Zhe; Sternweis, Paul C

    2013-04-19

    The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs.

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  19. Fluorescence resonance energy transfer in the studies of guanine quadruplexes.

    PubMed

    Juskowiak, Bernard; Takenaka, Shigeori

    2006-01-01

    A guanine (G)-quadruplex DNA motif has recently emerged as a biologically important structure that is believed to interfere with telomere maintenance by telomerase. G-quadruplexes exhibit four-stranded structures containing one or more nucleic acid strands with central channel able to accommodate metal cations. Coordination of certain metal cations stabilizes G-quadruplex as with some promising small organic molecules that promote the formation and/or stabilization of G-quadruplex. Among many techniques employed to explore properties of G-quadruplexes, the fluorescence resonance energy transfer (FRET) technique has been recognized as a powerful tool to study G-quadruplex formation. This review summarizes the current developments in the uses of FRET technique for the fundamental structural investigations and its practical applications. Applications include FRET-based selection of efficient quadruplex-binding ligands, design of a nanomolecular machine, and a molecular aptamer beacon for protein recognition. We also describe a technique for detection of potassium ions in aqueous solution with the use of quadruplex-based sensor (potassium-sensing oligonucleotide).

  20. Mechanisms involved in the antinociception induced by spinal administration of inosine or guanine in mice.

    PubMed

    de Oliveira, Enderson D; Schallenberger, Cristhine; Böhmer, Ana Elisa; Hansel, Gisele; Fagundes, Aécio C; Milman, Michael; Silva, Marcos D P; Oses, Jean P; Porciúncula, Lisiane O; Portela, Luís V; Elisabetsky, Elaine; Souza, Diogo O; Schmidt, André P

    2016-02-01

    It is well known that adenine-based purines exert multiple effects on pain transmission. Recently, we have demonstrated that guanine-based purines may produce some antinociceptive effects against chemical and thermal pain in mice. The present study was designed to investigate the antinociceptive effects of intrathecal (i.t.) administration of inosine or guanine in mice. Additionally, investigation into the mechanisms of action of these purines, their general toxicity and measurements of CSF purine levels were performed. Animals received an i.t. injection of vehicle (30mN NaOH), inosine or guanine (up to 600nmol) and submitted to several pain models and behavioural paradigms. Guanine and inosine produced dose-dependent antinociceptive effects in the tail-flick, hot-plate, intraplantar (i.pl.) glutamate, i.pl. capsaicin and acetic acid pain models. Additionally, i.t. inosine inhibited the biting behaviour induced by spinal injection of capsaicin and i.t. guanine reduced the biting behaviour induced by spinal injection of glutamate or AMPA. Intrathecal administration of inosine (200nmol) induced an approximately 115-fold increase on CSF inosine levels. This study provides new evidence on the mechanism of action of extracellular guanine and inosine presenting antinociceptive effects following spinal administration. These effects seem to be related, at least partially, to the modulation of A1 adenosine receptors. PMID:26712379

  1. Nucleotide cleaving agents and method

    DOEpatents

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

    2000-01-01

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

  2. Real-time NMR Study of Three Small GTPases Reveals That Fluorescent 2′(3′)-O-(N-Methylanthraniloyl)-tagged Nucleotides Alter Hydrolysis and Exchange Kinetics*

    PubMed Central

    Mazhab-Jafari, Mohammad T.; Marshall, Christopher B.; Smith, Matthew; Gasmi-Seabrook, Geneviève M. C.; Stambolic, Vuk; Rottapel, Robert; Neel, Benjamin G.; Ikura, Mitsuhiko

    2010-01-01

    The Ras family of small GTPases control diverse signaling pathways through a conserved “switch” mechanism, which is turned on by binding of GTP and turned off by GTP hydrolysis to GDP. Full understanding of GTPase switch functions requires reliable, quantitative assays for nucleotide binding and hydrolysis. Fluorescently labeled guanine nucleotides, such as 2′(3′)-O-(N-methylanthraniloyl) (mant)-substituted GTP and GDP analogs, have been widely used to investigate the molecular properties of small GTPases, including Ras and Rho. Using a recently developed NMR method, we show that the kinetics of nucleotide hydrolysis and exchange by three small GTPases, alone and in the presence of their cognate GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, are affected by the presence of the fluorescent mant moiety. Intrinsic hydrolysis of mantGTP by Ras homolog enriched in brain (Rheb) is ∼10 times faster than that of GTP, whereas it is 3.4 times slower with RhoA. On the other hand, the mant tag inhibits TSC2GAP-catalyzed GTP hydrolysis by Rheb but promotes p120 RasGAP-catalyzed GTP hydrolysis by H-Ras. Guanine nucleotide exchange factor-catalyzed nucleotide exchange for both H-Ras and RhoA was inhibited by mant-substituted nucleotides, and the degree of inhibition depends highly on the GTPase and whether the assay measures association of mantGTP with, or dissociation of mantGDP from the GTPase. These results indicate that the mant moiety has significant and unpredictable effects on GTPase reaction kinetics and underscore the importance of validating its use in each assay. PMID:20018863

  3. Guanosine nucleotide precursor for flavinogenesis of Eremothecium Ashbyii.

    PubMed

    Mitsuda, H; Nakajima, K

    1975-01-01

    The purine precursor in the riboflavin biosynthetic pathway in Eremothecium ashbyii was examined using a guanine analogue, 8-azaguanine, with non-growing cell systems. 1. Riboflavin formation in the culture filtrate was determined at 0, 5, 10 and 20 hr after start of the incubation of the non-growing cells in the presence of xanthine or 8-azaguanine (1 mM, respectively). At 20 hr of incubation, the addition of xanthine stimulated riboflavin formation by 36% and the addition of 8-azaguanine inhibited the formation by 57%. 2. Acid soluble nucleotide pools in the cells were followed at 0, 5, 10 and 20 hr of the incubation period in the presence of xanthine or 8-azaguanine by means of anion exchange column chromatography. The result showed that the GTP pool changed markedly despite the fact that the adenosine nucleotide pool was almost constant irrespective of the presence or absence of these purines till 10 hr of incubation. But, the decrease of the former was overcome in part by the addition of flavinogenic xanthine. Furthermore, the total amounts of GTP and guanosine accumulated in cells in the presence of 8-azaguanine reached the maximum already at 5 hr, attaining a level twice as much as the GTP contents of the control. 3. The role of guanosine nucleotide pool in riboflavin formation was further examined using 8-azaguanine. In this experiment the drug was added to the suspension of non-growing cells at 3 hr or 6 hr after the incubation was started and the reaction was continued till the 12th hr. A more clear-cut correlationship between riboflavin formation and guanosine nucleotide pool was oberved by this experiment. The guanosine nucleotide pool (consisting of GMP, GDP and GTP) increased simultaneously with the inhibition of riboflavin formation. Of the guanosine nucleotides pools, the GMP pool increased 2.7 times above normal upon the addition of 8-azaguanine during the incubation for 6 hr and 5.3 fold for 9 hr. While, the GTP pool increased 1.9 fold above

  4. The crystal structure of a parallel-stranded guanine tetraplex at 0.95 A resolution.

    PubMed

    Phillips, K; Dauter, Z; Murchie, A I; Lilley, D M; Luisi, B

    1997-10-17

    In both DNA and RNA, stretches of guanine bases can form stable four-stranded helices in the presence of sodium or potassium ions. Sequences with a propensity to form guanine tetraplexes have been found in chromosomal telomers, immunoglobulin switch regions, and recombination sites. We report the crystal structure at 0.95 A resolution of a parallel-stranded tetraplex formed by the hexanucleotide d(TG4T) in the presence of sodium ions. The four strands form a right-handed helix that is stabilized by hydrogen-bonding tetrads of co-planar guanine bases. Well-resolved sodium ions are found between and, at defined points, within tetrad planes and are coordinated with the guanine O6 groups. Nine calcium ions have been identified, each with a well-defined hepta-coordinate hydration shell. Hydrogen-bonding water patterns are observed within the tetraplex's helical grooves and clustered about the phosphate groups. Water molecules in the groove may form a hydrogen bond with the O4', and may affect the stacking behavior of guanine. Two distinct stacking arrangements are noted for the guanine tetrads. The thymine bases do not contribute to the four-stranded conformation, but instead stack to stabilize the crystal lattice. We present evidence that the sugar conformation is strained and propose that this originates from forces that optimize guanine base stacking. Discrete conformational disorder is observed at several places in the phosphodiester backbone, which results from a simple crankshaft rotation that requires no net change in the sugar conformation. PMID:9367755

  5. Experimental and first-principles study of guanine adsorption on ZnO clusters.

    PubMed

    Chandraboss, V L; Karthikeyan, B; Senthilvelan, S

    2014-11-14

    Theoretical investigation of guanine, DNA base adsorption on the ZnO model clusters, viz., Zn2O2, Zn3O3, Zn4O4 ring (R) and Zn4O4 wurtzite (W) in terms of geometry, binding site, binding energy (EB), energy gap (Eg), electronic and spectral properties were studied by a density functional theory (DFT) method. The guanine adsorption on the ZnO (G-ZnO) clusters is modeled by the B3LYP/LanL2DZ method. The calculated binding energy (EB) and energy gap (Eg) of the guanine molecule are highly dependent on the nature of the cluster size and vary with the size of the clusters. Physisorption proceeded via formation of the NZn bond between guanine and the active Zn(2+) site on ZnO. The HOMO-LUMO energies show that charge transfer occurs in the G-ZnO clusters, from ZnO to guanine to better understand the interaction. The Mulliken charges are computed. The electronic properties of ZnO and G-ZnO clusters were compared with different basis sets (B3LYP/6-31G, B3LYP/6-311G, MP2/6-31G and MP2/LanL2DZ). Experimental information like microscopic and spectroscopic evidence is also included for understanding the guanine-ZnO interactions. The G-ZnO composite was prepared by a precipitation method and characterized by SEM with EDX, FT-IR and FT-RAMAN analysis. The interaction of guanine with ZnO nanoparticles was observed by UV-vis spectroscopy. The experimental results are compared with the DFT results in the light of these new insights. PMID:25266048

  6. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  7. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements.

  8. A new rapid amplification of cDNA ends method for extremely guanine plus cytosine-rich genes.

    PubMed

    Shi, Xianzong; Jarvis, Donald L

    2006-09-15

    Rapid amplification of cDNA ends (RACE) is widely used to determine the 5'- and 3'-terminal nucleotide sequences of genes. Many different RACE methods have been developed to meet various requirements, but none addresses the difficult problems that arise when trying to isolate the ends of extremely guanine plus cytosine (GC)-rich genes. In this study, we found that we were unable to isolate the correct 5' or 3' end of an insect gene, which appeared to include extremely GC-rich sequences, using current RACE methods. Thus, we developed a new RACE method that can be used for this purpose. This new method entails first-strand cDNA synthesis at 70 degrees C with Thermo-X reverse transcriptase in the presence of homoectoine, followed by a polymerase chain reaction with 98 degrees C denaturation steps and Phusion DNA polymerase in the presence of 1M betaine and 5% dimethyl sulfoxide (DMSO). The use of these conditions yielded 5'- and 3'-RACE products that were approximately 80% GC over 213 and 162bp, respectively, and included shorter internal regions of 82 to 89% GC. PMID:16875657

  9. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  10. Highly sensitive and selective fluorescent assay for guanine based on the Cu2 +/eosin Y system

    NASA Astrophysics Data System (ADS)

    Shi, Huimin; Cui, Yi; Gong, Yijun; Feng, Suling

    2016-05-01

    A fluorescent probe has been developed for the determination of guanine based on the quenched fluorescence signal of Cu2 +/eosin Y. Cu2 + interacted with eosin Y, resulting in fluorescence quenching. Subsequently, with the addition of guanine to the Cu2 +/eosin Y system, guanine reacted with Cu2 + to form 1:1 chelate cation, which further combined with eosin Y to form a 1:1 ternary ion-association complex by electrostatic attraction and hydrophobic interaction, resulting in significant decrease of the fluorescence. Hence, a fluorescent system was constructed for rapid, sensitive and selective detection of guanine with a detection limit as low as 1.5 nmol L- 1 and a linear range of 3.3-116 nmol L- 1. The method has been applied satisfactorily to the determination of guanine in DNA and urine samples with the recoveries from 98.7% to 105%. This study significantly expands the realm of application of ternary ion-association complex in fluorescence probe.

  11. Theoretical Study of the Photophysics of 8-Vinylguanine, an Isomorphic Fluorescent Analogue of Guanine.

    PubMed

    Kochman, Michał A; Pola, Martina; Miller, R J Dwayne

    2016-08-11

    Paving the way for the application of the algebraic-diagrammatic construction scheme of second-order (ADC(2)) to systems based on the guanine chromophore, we demonstrate the this excited-state electronic structure method provides a realistic description of the photochemistry of 9H-guanine, in close agreement with the benchmark provided by the CASPT2 method. We then proceed to apply the ADC(2) method to the photochemistry of 8-vinylguanine (8vG), a minimally modified analogue of guanine which, unlike the naturally occurring nucleobase, displays intense fluorescence, indicative of a much longer-lived excited electronic state. The emissive electronic state of 8vG is identified as an ππ*-type intramolecular charge transfer (ICT) state, in which a charge of roughly -0.2 e is transferred from the guanine moiety onto the vinyl substituent. The main radiationless deactivation pathway competing with fluorescence is predicted to involve the molecule leaving the minimum on the ICT ππ* state, and reaching a region of the S1 adiabatic state where it resembles the La ππ* state of unmodified 9H-guanine. The topology of the La ππ* region of the S1 state favors subsequent internal conversion at a crossing seam with the ground electronic state. The sensitivity of this process to environment polarity may explain the experimentally observed fluorescence quenching of 8vG upon incorporation in single- and double-stranded DNA. PMID:27427772

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

    PubMed Central

    Torres, Rosa J; Puig, Juan G

    2007-01-01

    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 lack of precise

  13. Telomere structure and stability: covalency in hydrogen bonds, not resonance assistance, causes cooperativity in guanine quartets.

    PubMed

    Fonseca Guerra, Célia; Zijlstra, Hester; Paragi, Gábor; Bickelhaupt, F Matthias

    2011-11-01

    We show that the cooperative reinforcement between hydrogen bonds in guanine quartets is not caused by resonance-assisted hydrogen bonding (RAHB). This follows from extensive computational analyses of guanine quartets (G(4)) and xanthine quartets (X(4)) based on dispersion-corrected density functional theory (DFT-D). Our investigations cover the situation of quartets in the gas phase, in aqueous solution as well as in telomere-like stacks. A new mechanism for cooperativity between hydrogen bonds in guanine quartets emerges from our quantitative Kohn-Sham molecular orbital (MO) and corresponding energy decomposition analyses (EDA). Our analyses reveal that the intriguing cooperativity originates from the charge separation that goes with donor-acceptor orbital interactions in the σ-electron system, and not from the strengthening caused by resonance in the π-electron system. The cooperativity mechanism proposed here is argued to apply, beyond the present model systems, also to other hydrogen bonds that show cooperativity effects.

  14. Effect of intense magnetic fields on the convection of biogenic guanine crystals in aqueous solution

    NASA Astrophysics Data System (ADS)

    Iwasaka, M.; Mizukawa, Y.

    2015-05-01

    In this study, the basic magneto-optic properties of biogenic microcrystals in aqueous media were investigated. Microcrystals, mica plates, silica, and microcrystals from a diatom cell and biogenic guanine crystals from goldfish showed light scattering inhibition when the crystals were observed in water under a 5 T magnetic field and dark-field illumination. In particular, in 50% ethanol/water medium, convection of the biogenic guanine particle aggregates was reversibly inhibited when the microcrystal suspension was exposed to a 5 T magnetic field. Microscopic observation comparing the biogenic guanine crystals in water with 95% ethanol or 99% acetone revealed that light flickering on the surface of the crystals was affected by the surface interaction of the crystal with the surrounding medium. By considering both the magnetic orientation of the microcrystals and the possible interactions of crystals with the surrounding medium, a magnetically controllable fluidic tracer was suggested.

  15. Prokaryotic Nucleotide Composition Is Shaped by Both Phylogeny and the Environment

    PubMed Central

    Reichenberger, Erin R.; Rosen, Gail; Hershberg, Uri; Hershberg, Ruth

    2015-01-01

    The causes of the great variation in nucleotide composition of prokaryotic genomes have long been disputed. Here, we use extensive metagenomic and whole-genome data to demonstrate that both phylogeny and the environment shape prokaryotic nucleotide content. We show that across environments, various phyla are characterized by different mean guanine and cytosine (GC) values as well as by the extent of variation on that mean value. At the same time, we show that GC-content varies greatly as a function of environment, in a manner that cannot be entirely explained by disparities in phylogenetic composition. We find environmentally driven differences in nucleotide content not only between highly diverged environments (e.g., soil, vs. aquatic vs. human gut) but also within a single type of environment. More specifically, we demonstrate that some human guts are associated with a microbiome that is consistently more GC-rich across phyla, whereas others are associated with a more AT-rich microbiome. These differences appear to be driven both by variations in phylogenetic composition and by environmental differences—which are independent of these phylogenetic composition differences. Combined, our results demonstrate that both phylogeny and the environment significantly affect nucleotide composition and that the environmental differences affecting nucleotide composition are far subtler than previously appreciated. PMID:25861819

  16. Prokaryotic nucleotide composition is shaped by both phylogeny and the environment.

    PubMed

    Reichenberger, Erin R; Rosen, Gail; Hershberg, Uri; Hershberg, Ruth

    2015-04-09

    The causes of the great variation in nucleotide composition of prokaryotic genomes have long been disputed. Here, we use extensive metagenomic and whole-genome data to demonstrate that both phylogeny and the environment shape prokaryotic nucleotide content. We show that across environments, various phyla are characterized by different mean guanine and cytosine (GC) values as well as by the extent of variation on that mean value. At the same time, we show that GC-content varies greatly as a function of environment, in a manner that cannot be entirely explained by disparities in phylogenetic composition. We find environmentally driven differences in nucleotide content not only between highly diverged environments (e.g., soil, vs. aquatic vs. human gut) but also within a single type of environment. More specifically, we demonstrate that some human guts are associated with a microbiome that is consistently more GC-rich across phyla, whereas others are associated with a more AT-rich microbiome. These differences appear to be driven both by variations in phylogenetic composition and by environmental differences-which are independent of these phylogenetic composition differences. Combined, our results demonstrate that both phylogeny and the environment significantly affect nucleotide composition and that the environmental differences affecting nucleotide composition are far subtler than previously appreciated.

  17. Mechanisms of oxidation of guanine in DNA by carbonate radical anion, a decomposition product of nitrosoperoxycarbonate.

    PubMed

    Lee, Young Ae; Yun, Byeong Hwa; Kim, Seog K; Margolin, Yelena; Dedon, Peter C; Geacintov, Nicholas E; Shafirovich, Vladimir

    2007-01-01

    Peroxynitrite is produced during inflammation and combines rapidly with carbon dioxide to yield the unstable nitrosoperoxycarbonate, which decomposes (in part) to CO(3) (.-) and (.)NO(2) radicals. The CO(3) (.-) radicals oxidize guanine bases in DNA through a one-electron transfer reaction process that ultimately results in the formation of stable guanine oxidation products. Here we have explored these mechanisms, starting with a spectroscopic study of the kinetics of electron transfer from 20-22mer double-stranded oligonucleotides to CO(3) (.-) radicals, together with the effects of base sequence on the formation of the end-products in runs of one, two, or three contiguous guanines. The distributions of these alkali-labile lesions were determined by gel electrophoresis methods. The cascade of events was initiated through the use of 308 nm XeCl excimer laser pulses to generate CO(3) (.-) radicals by an established method based on the photodissociation of persulfate to sulfate radicals and the oxidation of bicarbonate. Although the Saito model (Saito et al., J. Am. Chem. Soc. 1995, 117, 6406-6407) predicts relative ease of one-electron oxidations in DNA, following the trend 5'-GGG > 5'-GG > 5'-G, we found that the rate constants for CO(3) (.-)-mediated oxidation of guanines in these sequence contexts (k(5)) showed only small variation within a narrow range [(1.5-3.0)x10(7) M(-1) s(-1)]. In contrast, the distributions of the end-products are dependent on the base sequence context and are higher at the 5'-G in 5'-GG sequences and at the first two 5'-guanines in the 5'-GGG sequences. These effects are attributed to a combination of initial hole distributions among the contiguous guanines and the subsequent differences in chemical reaction yields at each guanine. The lack of dependence of k(5) on sequence context indicates that the one-electron oxidation of guanine in DNA by CO(3) (.-) radicals occurs by an inner-sphere mechanism. PMID:17335089

  18. High information throughput analysis of nucleotides and their isotopically enriched isotopologues by direct-infusion FTICR-MS.

    PubMed

    Lorkiewicz, Pawel; Higashi, Richard M; Lane, Andrew N; Fan, Teresa W-M

    2012-01-01

    Fourier transform-ion cyclotron resonance-mass spectrometry (FTICR-MS) is capable of acquiring unmatched quality of isotopologue data for stable isotope resolved metabolomics (SIRM). This capability drives the need for a continuous ion introduction for obtaining optimal isotope ratios. Here we report the simultaneous analysis of mono and dinucleotides from crude polar extracts by FTICR-MS by adapting an ion-pairing sample preparation method for LC-MS analysis. This involves a rapid cleanup of extracted nucleotides on pipet tips containing a C(18) stationary phase, which enabled global analysis of nucleotides and their (13)C isotopologues at nanomolar concentrations by direct infusion nanoelectrospray FTICR-MS with 5 minutes of data acquisition. The resolution and mass accuracy enabled computer-assisted unambiguous assignment of most nucleotide species, including all phosphorylated forms of the adenine, guanine, uracil and cytosine nucleotides, NAD(+), NADH, NADP(+), NADPH, cyclic nucleotides, several UDP-hexoses, and all their (13)C isotopologues. The method was applied to a SIRM study on human lung adenocarcinoma A549 cells grown in [U-(13)C] glucose with or without the anti-cancer agent methylseleninic acid. At m/z resolving power of 400,000, (13)C-isotopologues of nucleotides were fully resolved from all other elemental isotopologues, thus allowing their (13)C fractional enrichment to be accurately determined. The method achieves both high sample and high information throughput analysis of nucleotides for metabolic pathway reconstruction in SIRM investigations.

  19. Interaction of cyclic cytosine-, guanine-, thymine-, uracil- and mixed guanine-cytosine base tetrads with K+, Na+ and Li+ ions -- a density functional study.

    PubMed

    Meyer, Michael; Sühnel, Jürgen

    2003-02-01

    We have carried out B3LYP hybrid density functional studies of complexes formed by cyclic cytosine-, guanine-, thymine-, uracil- and mixed guanine cytosine-tetrads with Li+, Na+ and K+ ions to determine their structures and interaction energies. The conformations studied have been restricted to a hydrogen bond pattern closely related to the tetrads observed in experimental nucleic acid structures. A comparison of the alkali metal ion/tetrad complexes with the tetrads without cations indicates that alkali metal ions modulate the tetrad structures significantly and that even the hydrogen bond pattern may change. Guanine-tetrad cation complexes show the strongest interaction energy compared to other tetrads that occur less frequently in experimental structures. The most stable G-tetrad/metal ion structure adopts a nearly planar geometry that is especially suitable for tetraplex formation, which requires approximately parallel tetrad planes. In the cytosine-tetrad there is a very large central cavity suitable for cation recognition, but the complexes adopt a non-planar structure unsuitable for stacking, except possibly for ions with very large radii. Uracil and thymine tetrads show a significant different characteristics which may contribute to the differences between DNA and RNA PMID:12529150

  20. Theoretical and Experimental Study of Valence-Shell Ionization Spectra of Guanine

    NASA Astrophysics Data System (ADS)

    Zaytseva, Irina L.; Trofimov, Alexander B.; Schirmer, Jochen; Plekan, Oksana; Feyer, Vitaliy; Richter, Robert; Coreno, Marcello; Prince, Kevin C.

    2009-10-01

    The full valence-shell ionization spectra of the four most stable guanine tautomers were studied theoretically. The third-order algebraic-diagrammatic construction (ADC(3)) method for the one-particle Green's function was used to calculate the energies and relative intensities of the vertical ionization transitions. For low-lying transitions, the influence of planar and nonplanar guanine configurations on the ionization energies, as well as the convergence of the results with respect to basis set was studied at the level of the outer-valence Green's function (OVGF) approximation scheme. The results of the calculations were used to interpret recent synchrotron radiation valence-shell photoionization spectra of guanine in the gas phase under thermal equilibrium conditions. The photoelectron spectrum was modeled by summing individual tautomer spectra weighted by Boltzmann population ratios (BPR) of tautomers from our previous high-level ab initio thermochemical calculations. The theoretical spectra are in good agreement with the experimental results, providing assignments of most observed structures and offering insight into tautomerism of guanine in the gas phase. The first six molecular orbitals give rise to single-hole states with a binding energy of about 7-12 eV. At higher binding energy the spectral features are mainly due to satellite states.

  1. Exploring the Use of a Guanine-Rich Catalytic DNA for Sulfoxide Preparation.

    PubMed

    Dellafiore, María A; Montserrat, Javier M; Iribarren, Adolfo M

    2015-01-01

    A guanine-rich DNA oligonucleotide complexed with hemin was used to catalyze controlled oxygen transfer reactions to different sulfides for sulfoxide preparation in the presence of H2O2. Comparable activities were obtained when using fully modified L-DNA. In addition, oligonucleotide immobilization led to an active catalyst which could be successfully recovered and reused without loss of activity.

  2. Exploring the Use of a Guanine-Rich Catalytic DNA for Sulfoxide Preparation

    PubMed Central

    Dellafiore, María A.; Montserrat, Javier M.; Iribarren, Adolfo M.

    2015-01-01

    A guanine-rich DNA oligonucleotide complexed with hemin was used to catalyze controlled oxygen transfer reactions to different sulfides for sulfoxide preparation in the presence of H2O2. Comparable activities were obtained when using fully modified L-DNA. In addition, oligonucleotide immobilization led to an active catalyst which could be successfully recovered and reused without loss of activity. PMID:26066510

  3. 1-ethynylpyrene-modified guanine and cytosine as optical labels for DNA hybridization.

    PubMed

    Wagner, Clemens; Rist, Manuela; Mayer-Enthart, Elke; Wagenknecht, Hans-Achim

    2005-06-01

    1-ethynylpyrene shows remarkable absorption changes upon DNA hybridization when it is covalently attached to the 8-position of guanine. An absorption band at approximately 420 nm is only present in the duplex, exhibits thermal melting behaviour and provides the basis for a molecular beacon together with 1-ethynylpyrene-modified cytosine.

  4. Labeled nucleotide phosphate (NP) probes

    SciTech Connect

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

    2009-02-03

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

  5. Guanine-containing copper(II) complexes: synthesis, X-ray structures and magnetic properties.

    PubMed

    Mastropietro, Teresa F; Armentano, Donatella; Grisolia, Ettore; Zanchini, Claudia; Lloret, Francesc; Julve, Miguel; De Munno, Giovanni

    2008-01-28

    Three new compounds of formula {[Cu(gua)(H(2)O)(3)](BF(4))(SiF(6))(1/2)}(n) (1), {[Cu(gua)(H(2)O)(3)](CF(3)SO(3))(2).H(2)O}(n) (2) and [Cu(gua)(2)(H(2)O)(HCOO)]ClO(4).H(2)O.1/2HCOOH] (3) [gua = 2-amino-1H-purin-6(9H)-one] showing the unprecedented coordination of neutral guanine, have been synthesised and structurally characterized. The structures of the compounds 1 and 2 contain uniform copper(II) chains of formula [Cu(gua)(H(2)O)(3)](n)(2n+), where the copper atoms are bridged by guanine ligands coordinated via N(3) and N(7). The electroneutrality is achieved by uncoordinated tetrafluoroborate and hexafluorosilicate (1) and triflate (2). Each copper atom in 1 and 2 is five-coordinated in a distorted square pyramidal environment: two water molecules in trans positions and the N(3) and N(7a) nitrogen atoms of two guanine ligands build the basal plane whereas a water molecule fills the axial position. The values of the copper-copper separation across the bridging guanine ligand are 7.183(1) (1) and 7.123(1) A (2). is an ionic salt whose structure is made up of mononuclear [Cu(gua)(2)(H(2)O)(HCOO)](+) cations and perchlorate anions plus water and formic acid as crystallization molecules. The two guanine ligands in the cation are coordinated to the copper centre through the N(9) atom. The copper atom in 3 is four-coordinated with two monodentate guanine molecules in the trans position, a water molecule and a monodenate formate ligand building a quasi square planar surrounding. Magnetic susceptibility measurements for 1 and 2 in the temperature range 1.9-300 K show the occurrence of significant intrachain antiferromagnetic interactions between the copper(ii) ions across the guanine bridge [J = -9.6(1) (1) and -10.3(1) cm(-1) (2) with H = -J summation operator(i)S(i).S(i+1)].

  6. Hydrogen-bonded proton transfer in the protonated guanine-cytosine (GC+H)+ base pair.

    PubMed

    Lin, Yuexia; Wang, Hongyan; Gao, Simin; Schaefer, Henry F

    2011-10-13

    The single proton transfer at the different sites of the Watson-Crick (WC) guanine-cytosine (GC) DNA base pair are studied here using density functional methods. The conventional protonated structures, transition state (TS) and proton-transferred product (PT) structures of every relevant species are optimized. Each transition state and proton-transferred product structure has been compared with the corresponding conventional protonated structure to demonstrate the process of proton transfer and the change of geometrical structures. The relative energies of the protonated tautomers and the proton-transfer energy profiles in gas and solvent are analyzed. The proton-transferred product structure G(+H(+))-H(+)C(N3)(-H(+))(PT) has the lowest relative energy for which only two hydrogen bonds exist. Almost all 14 isomers of the protonated GC base pair involve hydrogen-bonded proton transfer following the three pathways, with the exception of structure G-H(+)C(O2). When the positive charge is primarily "located" on the guanine moiety (H(+)G-C, G-H(+)C(C4), and G-H(+)C(C6)), the H(1) proton transfers from the N(1) site of guanine to the N(3) site of cytosine. The structures G-H(+)C(C5) and G-H(+)C(C4) involve H(4a) proton transfer from the N(4) of cytosine to the O(6) site of guanine. H(2a) proton transfer from the N(2) site of guanine to the O(2) site of cytosine is found only for the structure G-H(+)C(C4). The structures to which a proton is added on the six-centered sites adjoining the hydrogen bonds are more prone to proton transfer in the gas phase, whereas a proton added on the minor groove and the sites adjoining the hydrogen bonds is favorable to the proton transfer in energy in the aqueous phase.

  7. Alpha-tubulin influences nucleotide binding to beta-tubulin: an assay using picomoles of unpurified protein.

    PubMed Central

    Farr, G W; Yaffe, M B; Sternlicht, H

    1990-01-01

    Tubulin binds guanine nucleotides tightly within its beta subunit. Whether the alpha subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free beta subunit with those of the heterodimer. The free beta subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires approximately 100-fold less protein than conventional assays. This assay exploits the observation that the recovery of beta-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized beta subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the alpha subunit enhances GTP binding to the beta subunit approximately 4-fold. However, in the absence of magnesium the alpha subunit appears to specifically weaken GTP binding to the beta subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the beta subunit. PMID:2367522

  8. Alpha-tubulin influences nucleotide binding to beta-tubulin: an assay using picomoles of unpurified protein.

    PubMed

    Farr, G W; Yaffe, M B; Sternlicht, H

    1990-07-01

    Tubulin binds guanine nucleotides tightly within its beta subunit. Whether the alpha subunit influences binding to this site has been unknown. This question was addressed by comparing the nucleotide binding properties of the free beta subunit with those of the heterodimer. The free beta subunit was obtained from an in vitro expression system and its nucleotide binding properties were determined by an assay that requires approximately 100-fold less protein than conventional assays. This assay exploits the observation that the recovery of beta-tubulin from Mono Q anion-exchange columns is dependent on added nucleotide. Our results demonstrate that the newly synthesized beta subunit and the heterodimer bind nucleotides with similar specificity. We found that in the presence of magnesium the alpha subunit enhances GTP binding to the beta subunit approximately 4-fold. However, in the absence of magnesium the alpha subunit appears to specifically weaken GTP binding to the beta subunit. Thus, nucleotide binding to the E site in the heterodimer may not be solely defined by the beta subunit.

  9. Rapid magnetic wiper featuring biogenic guanine particles: Magnetic non-contact switching of opto-fluidic mirrors featuring biogenic guanine crystals

    NASA Astrophysics Data System (ADS)

    Iwasaka, M.; Mizukawa, Y.; Miyashita, Y.

    2014-01-01

    In the present study, we prepared a diamagnetic fluid with magnetically controlled visual transparency. Light scattering control by the magnetic orientation of organic and diamagnetic microcrystals was applied for developing a functional diamagnetic fluid, by controlling its turbidity and transparency. The light scattering fluid was prepared by guanine crystal particles suspension (GPS), which were collected from the scales of goldfish, in an aqueous solution. It is revealed that GPS can control light scattering with magnetic fields. The method we developed can be utilized for controlling the visibility of light-reflective objects by using magnetic fields on a millitesla scale.

  10. Requirement of nucleotide exchange factor for Ypt1 GTPase mediated protein transport.

    PubMed

    Jones, S; Litt, R J; Richardson, C J; Segev, N

    1995-09-01

    Small GTPases of the rab family are involved in the regulation of vesicular transport. It is believed that cycling between the GTP- and GDP-bound forms, and accessory factors regulating this cycling are crucial for rab function. However, an essential role for rab nucleotide exchange factors has not yet been demonstrated. In this report we show the requirement of nucleotide exchange factor activity for Ypt1 GTPase mediated protein transport. The Ypt1 protein, a member of the rab family, plays a role in targeting vesicles to the acceptor compartment and is essential for the first two steps of the yeast secretory pathway. We use two YPT1 dominant mutations that contain alterations in a highly conserved GTP-binding domain, N121I and D124N. YPT1-D124N is a novel mutation that encodes a protein with nucleotide specificity modified from guanine to xanthine. This provides a tool for the study of an individual rab GTPase in crude extracts: a xanthosine triphosphate (XTP)-dependent conditional dominant mutation. Both mutations confer growth inhibition and a block in protein secretion when expressed in vivo. The purified mutant proteins do not bind either GDP or GTP. Moreover, they completely inhibit the ability of the exchange factor to stimulate nucleotide exchange for wild type Ypt1 protein, and are potent inhibitors of ER to Golgi transport in vitro at the vesicle targeting step. The inhibitory effects of the Ypt1-D124N mutant protein on both nucleotide exchange activity and protein transport in vitro can be relieved by XTP, indicating that it is the nucleotide-free form of the mutant protein that is inhibitory. These results suggest that the dominant mutant proteins inhibit protein transport by sequestering the exchange factor from the wild type Ypt1 protein, and that this factor has an essential role in vesicular transport.

  11. Exploitation of the Low Fidelity of Human Immunodeficiency Virus Type 1 (HIV-1) Reverse Transcriptase and the Nucleotide Composition Bias in the HIV-1 Genome To Alter the Drug Resistance Development of HIV

    PubMed Central

    Balzarini, Jan; Camarasa, Maria-José; Pérez-Pérez, Maria-Jesus; San-Félix, Ana; Velázquez, Sonsoles; Perno, Carlo-Federico; De Clercq, Erik; Anderson, John N.; Karlsson, Anna

    2001-01-01

    The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G→A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering the endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G→A) to an adenine-to-guanine (A→G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development. PMID:11390579

  12. Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins.

    PubMed

    Gao, Zhong Feng; Huang, Yan Li; Ren, Wang; Luo, Hong Qun; Li, Nian Bing

    2016-04-15

    Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10 min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform.

  13. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    SciTech Connect

    Abelson, H.T.; Gorka, C.

    1983-09-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no (3H)hypoxanthine uptake into tumor or tissue culture cells, no conversion of (3H)hypoxanthine to (3H)IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol.

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

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

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

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

    PubMed

    Usha, S; Selvaraj, S

    2015-01-01

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

  16. Mechanistic study of the deamination reaction of guanine: a computational study.

    PubMed

    Uddin, Kabir M; Almatarneh, Mansour H; Shaw, Dawn M; Poirier, Raymond A

    2011-03-17

    The mechanism for the deamination of guanine with H(2)O, OH(-), H(2)O/OH(-) and for GuaH(+) with H(2)O has been investigated using ab initio calculations. Optimized geometries of the reactants, transition states, intermediates, and products were determined at RHF/6-31G(d), MP2/6-31G(d), B3LYP/6-31G(d), and B3LYP/6-31+G(d) levels of theory. Energies were also determined at G3MP2, G3MP2B3, G4MP2, and CBS-QB3 levels of theory. Intrinsic reaction coordinate (IRC) calculations were performed to characterize the transition states on the potential energy surface. Thermodynamic properties (ΔE, ΔH, and ΔG), activation energies, enthalpies, and Gibbs free energies of activation were also calculated for each reaction investigated. All pathways yield an initial tetrahedral intermediate and an intermediate in the last step that dissociates to products via a 1,3-proton shift. At the G3MP2 level of theory, deamination with OH(-) was found to have an activation energy barrier of 155 kJ mol(-1) compared to 187 kJ mol(-1) for the reaction with H(2)O and 243 kJ mol(-1) for GuaH(+) with H(2)O. The lowest overall activation energy, 144 kJ mol(-1) at the G3MP2 level, was obtained for the deamination of guanine with H(2)O/OH(-). Due to a lack of experimental results for guanine deamination, a comparison is made with those of cytosine, whose deamination reaction parallels that of guanine.

  17. ESI-MS Characterization of a Novel Pyrrole-Inosine Nucleoside that Interacts with Guanine Bases

    PubMed Central

    Pierce, Sarah E.; Sherman, Courtney L.; Jayawickramarajah, Janarthanan; Lawrence, Candace M.; Sessler, Jonathan L.; Brodbelt, Jennifer S.

    2008-01-01

    Based on binding studies undertaken by electrospray ionization-mass spectrometry, a synthetic pyrrole-inosine nucleoside, 1, capable of forming an extended three-point Hoogsteen-type hydrogen-bonding interaction with guanine, is shown to form specific complexes with two different quadruplex DNA structures [dTG4T]4 and d(T2G4)4 as well as guanine rich duplex DNA. The binding interactions of two other analogs were evaluated in order to unravel the structural features that contribute to specific DNA recognition. The importance of the Hoogsteen interactions was confirmed through the absence of specific binding when the pyrrole NH hydrogen-bonding site was blocked or removed. While 2, with a large blocking group, was not found to interact with virtually any form of DNA, 3, with the pyrrole functionality missing, was found to interact non-specifically with several types of DNA. The specific binding of 1 to guanine rich DNA emphasizes the necessity of careful ligand design for specific sequence recognition. PMID:18790136

  18. Non-covalent functionalization of hexagonal boron nitride nanosheets with guanine.

    PubMed

    Anota, E Chigo; Tlapale, Y; Villanueva, M Salazar; Márquez, J A Rivera

    2015-08-01

    Density functional theory (DFT) calculations were performed to analyze changes in the structural and electronic properties generated by the interaction of a single nucleobase group (guanine) with the surface of boron nitride nanosheets with hexagonal symmetry (hBNNs). Nanosheets in two contexts were tested: pristine sheets and with point defects (doped with carbon atoms). The criterion of energy minimum was used to find the ground state of the nine possible isomers generated by the hBNNs-guanine interaction. The phenomenon of physisorption is known to occur at values less than 1.0 eV; the adsorption energy results revealed that the preferential geometry was a parallel arrangement between the two partners, with van der Waals-type bonds generated for the hBNNs doped with two carbon atoms. This was the only energetically stable configuration, thus revealing a vibrational mode rather than imaginaries. Furthermore, the hBNNs/C-guanine system has a low value for work function, and therefore could be used in health applications such drug transport and delivery. The increased polarity values suggest that these nanosheets could be solubilized in common solvents used in experimental processes. PMID:26227065

  19. N7-(carboxymethyl)guanine-Lithium Crystalline Complex: A Bioinspired Solid Electrolyte

    PubMed Central

    Dutta, Dipak; Nagapradeep, N.; Zhu, Haijin; Forsyth, Maria; Verma, Sandeep; Bhattacharyya, Aninda J.

    2016-01-01

    Electrochemical device with components having direct significance to biological life processes is a potent futuristic strategy for the realization of all-round green and sustainable development. We present here synthesis design, structural analysis and ion transport of a novel solid organic electrolyte (G7Li), a compound reminiscent of ion channels, derived from regioisomeric N7-guanine-carboxylate conjugate and Li-ions. G7Li, with it’s in-built supply of Li+-ions, exhibited remarkably high lithium-ion transference number (= 0.75) and tunable room temperature ionic conductivity spanning three decades (≈10−7 to 10−3 Ω−1 cm−1) as a function of moisture content. The ionic conductivity show a distinct reversible transition around 80–100 °C, from a dual Li+ and H+ (<100 °C) to a pure Li+ conductor (>100 °C). Systematic studies reveal a transition from water-assisted Li-ion transport to Li hopping-like mechanism involving guanine-Li coordination. While as-synthesized G7Li has potential in humidity sensors, the anhydrous G7Li is attractive for rechargeable batteries. PMID:27091631

  20. Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

    PubMed Central

    Mantaj, Julia; Jackson, Paul J. M.; Karu, Kersti; Rahman, Khondaker M.; Thurston, David E.

    2016-01-01

    Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks. PMID:27055050

  1. Singlet Oxygen Attack on Guanine: Reactivity and Structural Signature within the B-DNA Helix.

    PubMed

    Dumont, Elise; Grüber, Raymond; Bignon, Emmanuelle; Morell, Christophe; Aranda, Juan; Ravanat, Jean-Luc; Tuñón, Iñaki

    2016-08-22

    Oxidatively generated DNA lesions are numerous and versatile, and have been the subject of intensive research since the discovery of 8-oxoguanine in 1984. Even for this prototypical lesion, the precise mechanism of formation remains elusive due to the inherent difficulties in characterizing high-energy intermediates. We have probed the stability of the guanine endoperoxide in B-DNA as a key intermediate and determined a unique activation free energy of around 6 kcal mol(-1) for the formation of the first C-O covalent bond upon the attack of singlet molecular oxygen ((1) O2 ) on the central guanine of a solvated 13 base-pair poly(dG-dC), described by means of quantum mechanics/molecular mechanics (QM/MM) simulations. The B-helix remains stable upon oxidation in spite of the bulky character of the guanine endoperoxide. Our modeling study has revealed the nature of the versatile (1) O2 attack in terms of free energy and shows a sensitivity to electrostatics and solvation as it involves a charge-separated intermediate. PMID:27440482

  2. Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani.

    PubMed

    Allen, T E; Hwang, H Y; Jardim, A; Olafson, R; Ullman, B

    1995-07-01

    The gene encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani has been cloned and sequenced. The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targeting signal. Northern blot analysis of L. donovani RNA revealed two hgprt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript. The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both augmented approx. 5-fold in parasites incubated in the absence of purines. Southern blots of genomic DNA indicated only a single hgprt locus within the L. donovani genome. Overexpression of L. donovani hgprt in E. coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzymatically active HGPRT. The recombinant HGPRT was purified to homogeneity and recognized hypoxanthine, guanine and allopurinol, but not adenine or xanthine, as substrates. The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic target for the treatment of leishmaniasis and other diseases of parasitic origin. PMID:8577321

  3. N7-(carboxymethyl)guanine-Lithium Crystalline Complex: A Bioinspired Solid Electrolyte

    NASA Astrophysics Data System (ADS)

    Dutta, Dipak; Nagapradeep, N.; Zhu, Haijin; Forsyth, Maria; Verma, Sandeep; Bhattacharyya, Aninda J.

    2016-04-01

    Electrochemical device with components having direct significance to biological life processes is a potent futuristic strategy for the realization of all-round green and sustainable development. We present here synthesis design, structural analysis and ion transport of a novel solid organic electrolyte (G7Li), a compound reminiscent of ion channels, derived from regioisomeric N7-guanine-carboxylate conjugate and Li-ions. G7Li, with it’s in-built supply of Li+-ions, exhibited remarkably high lithium-ion transference number (= 0.75) and tunable room temperature ionic conductivity spanning three decades (≈10‑7 to 10‑3 Ω‑1 cm‑1) as a function of moisture content. The ionic conductivity show a distinct reversible transition around 80–100 °C, from a dual Li+ and H+ (<100 °C) to a pure Li+ conductor (>100 °C). Systematic studies reveal a transition from water-assisted Li-ion transport to Li hopping-like mechanism involving guanine-Li coordination. While as-synthesized G7Li has potential in humidity sensors, the anhydrous G7Li is attractive for rechargeable batteries.

  4. Electron microscopic visualization of complementary labeled DNA with platinum-containing guanine derivative.

    PubMed

    Loukanov, Alexandre; Filipov, Chavdar; Mladenova, Polina; Toshev, Svetlin; Emin, Saim

    2016-04-01

    The object of the present report is to provide a method for a visualization of DNA in TEM by complementary labeling of cytosine with guanine derivative, which contains platinum as contrast-enhanced heavy element. The stretched single-chain DNA was obtained by modifying double-stranded DNA. The labeling method comprises the following steps: (i) stretching and adsorption of DNA on the support film of an electron microscope grid (the hydrophobic carbon film holding negative charged DNA); (ii) complementary labeling of the cytosine bases from the stretched single-stranded DNA pieces on the support film with platinum containing guanine derivative to form base-specific hydrogen bond; and (iii) producing a magnified image of the base-specific labeled DNA. Stretched single-stranded DNA on a support film is obtained by a rapid elongation of DNA pieces on the surface between air and aqueous buffer solution. The attached platinum-containing guanine derivative serves as a high-dense marker and it can be discriminated from the surrounding background of support carbon film and visualized by use of conventional TEM observation at 100 kV accelerated voltage. This method allows examination of specific nucleic macromolecules through atom-by-atom analysis and it is promising way toward future DNA-sequencing or molecular diagnostics of nucleic acids by electron microscopic observation. PMID:26805035

  5. N7-(carboxymethyl)guanine-Lithium Crystalline Complex: A Bioinspired Solid Electrolyte.

    PubMed

    Dutta, Dipak; Nagapradeep, N; Zhu, Haijin; Forsyth, Maria; Verma, Sandeep; Bhattacharyya, Aninda J

    2016-01-01

    Electrochemical device with components having direct significance to biological life processes is a potent futuristic strategy for the realization of all-round green and sustainable development. We present here synthesis design, structural analysis and ion transport of a novel solid organic electrolyte (G7Li), a compound reminiscent of ion channels, derived from regioisomeric N7-guanine-carboxylate conjugate and Li-ions. G7Li, with it's in-built supply of Li(+)-ions, exhibited remarkably high lithium-ion transference number (= 0.75) and tunable room temperature ionic conductivity spanning three decades (≈10(-7) to 10(-3) Ω(-1) cm(-1)) as a function of moisture content. The ionic conductivity show a distinct reversible transition around 80-100 °C, from a dual Li(+) and H(+) (<100 °C) to a pure Li(+) conductor (>100 °C). Systematic studies reveal a transition from water-assisted Li-ion transport to Li hopping-like mechanism involving guanine-Li coordination. While as-synthesized G7Li has potential in humidity sensors, the anhydrous G7Li is attractive for rechargeable batteries. PMID:27091631

  6. Aquifex aeolicus tRNA (N2,N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA*

    PubMed Central

    Awai, Takako; Kimura, Satoshi; Tomikawa, Chie; Ochi, Anna; Ihsanawati; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Ohno, Satoshi; Nishikawa, Kazuya; Yokogawa, Takashi; Suzuki, Tsutomu; Hori, Hiroyuki

    2009-01-01

    Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m22G26) in tRNA. In the reaction, N2-guanine at position 26 (m2G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNACys has an m22G26m2G27 or m22G26m22G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m2G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme. PMID:19491098

  7. Aquifex aeolicus tRNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes transfer of methyl groups not only to guanine 26 but also to guanine 27 in tRNA.

    PubMed

    Awai, Takako; Kimura, Satoshi; Tomikawa, Chie; Ochi, Anna; Ihsanawati; Bessho, Yoshitaka; Yokoyama, Shigeyuki; Ohno, Satoshi; Nishikawa, Kazuya; Yokogawa, Takashi; Suzuki, Tsutomu; Hori, Hiroyuki

    2009-07-31

    Transfer RNA (N2,N2-guanine)-dimethyltransferase (Trm1) catalyzes N2,N2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m2G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.

  8. Rapid and sensitive detection of potassium ion based on K(+)-induced G-quadruplex and guanine chemiluminescence.

    PubMed

    Dong, Jingjing; Zhao, Hengzhi; Zhou, Fulin; Li, Baoxin

    2016-03-01

    A simple and rapid method for detection of potassium ion (K(+)) based on a guanine chemiluminescence (CL) system is presented. In this system, one guanine-rich DNA molecule is used as the recognition element. K(+) can cause the guanine-rich DNA to form a G-quadruplex conformation, resulting in remarkable quenching of the guanine CL intensity of guanine-rich DNA. The CL intensity of this CL system decreased with increasing K(+) concentration, revealing a linear relationship in K(+) concentration range from 3 × 10(-5) to 1 × 10(-3) M. A complete detection process can be accomplished in about 5 min. Other common cations (such as Na(+), NH4 (+), Mg(2+), Ca(2+), Zn(2+), and Pb(2+)) did not notably interfere with K(+) detection. The mechanism of this strategy is also discussed. The sensing strategy is low cost and simple without the requirement of complex labeling of probe DNA. The scheme is applicable to the detection of other guanine-rich aptamer-binding chemicals or biomolecules. PMID:26781100

  9. Strikingly different effects of hydrogen bonding on the photodynamics of individual nucleobases in DNA: comparison of guanine and cytosine.

    PubMed

    Zelený, Tomáš; Ruckenbauer, Matthias; Aquino, Adelia J A; Müller, Thomas; Lankaš, Filip; Dršata, Tomáš; Hase, William L; Nachtigallova, Dana; Lischka, Hans

    2012-08-22

    Ab initio surface hopping dynamics calculations were performed to study the photophysical behavior of cytosine and guanine embedded in DNA using a quantum mechanical/molecular mechanics (QM/MM) approach. It was found that the decay rates of photo excited cytosine and guanine were affected in a completely different way by the hydrogen bonding to the DNA environment. In case of cytosine, the geometrical restrictions exerted by the hydrogen bonds did not influence the relaxation time of cytosine significantly due to the generally small cytosine ring puckering required to access the crossing region between excited and ground state. On the contrary, the presence of hydrogen bonds significantly altered the photodynamics of guanine. The analysis of the dynamics indicates that the major contribution to the lifetime changes comes from the interstrand hydrogen bonds. These bonds considerably restricted the out-of-plane motions of the NH(2) group of guanine which are necessary for the ultrafast decay to the ground state. As a result, only a negligible amount of trajectories decayed into the ground state for guanine embedded in DNA within the simulation time of 0.5 ps, while for comparison, the isolated guanine relaxed to the ground state with a lifetime of about 0.22 ps. These examples show that, in addition to phenomena related to electronic interactions between nucleobases, there also exist relatively simple mechanisms in DNA by which the lifetime of a nucleobase is significantly enhanced as compared to the gas phase. PMID:22845192

  10. Functional classification of cNMP-binding proteins and nucleotide cyclases with implications for novel regulatory pathways in Mycobacterium tuberculosis.

    PubMed

    McCue, L A; McDonough, K A; Lawrence, C E

    2000-02-01

    We have analyzed the cyclic nucleotide (cNMP)-binding protein and nucleotide cyclase superfamilies using Bayesian computational methods of protein family identification and classification. In addition to the known cNMP-binding proteins (cNMP-dependent kinases, cNMP-gated channels, cAMP-guanine nucleotide exchange factors, and bacterial cAMP-dependent transcription factors), new functional groups of cNMP-binding proteins were identified, including putative ABC-transporter subunits, translocases, and esterases. Classification of the nucleotide cyclases revealed subtle differences in sequence conservation of the active site that distinguish the five classes of cyclases: the multicellular eukaryotic adenylyl cyclases, the eukaryotic receptor-type guanylyl cyclases, the eukaryotic soluble guanylyl cyclases, the unicellular eukaryotic and prokaryotic adenylyl cyclases, and the putative prokaryotic guanylyl cyclases. Phylogenetic distribution of the cNMP-binding proteins and cyclases was analyzed, with particular attention to the 22 complete archaeal and eubacterial genome sequences. Mycobacterium tuberculosis H37Rv and Synechocystis PCC6803 were each found to encode several more putative cNMP-binding proteins than other prokaryotes; many of these proteins are of unknown function. M. tuberculosis also encodes several more putative nucleotide cyclases than other prokaryotic species. PMID:10673278

  11. The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts.

    PubMed

    Wyss, Laura A; Nilforoushan, Arman; Williams, David M; Marx, Andreas; Sturla, Shana J

    2016-08-19

    Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide BENZI: opposite adducts, with up to 150-fold higher catalytic efficiency for O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies. PMID:27378785

  12. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

    NASA Astrophysics Data System (ADS)

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi; Fujii, Kentaro; Yokoya, Akinari; Fukuda, Yoshihiro; Saitoh, Yuji

    2014-08-01

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5'-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5'-monophosphate, and adenosine 5'-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  13. A New Apparatus for Studies of Low Energy Electron Collisions with Nucleotide Molecules

    NASA Astrophysics Data System (ADS)

    Duron, Jessica; Hargreaves, Leigh

    Low-energy electrons, the most copiously produced by-product of radiation cancer therapy, have been shown to be a strong driver of DNA damage in living cells [1]. Quantitative data describing these collisions are presently rare due to technological challenges in performing electron scattering measurements from the nucleobases, e.g. uracil, thymine, guanine, etc. These challenges include the low-vapor pressure of commercial samples (which are powders at room temperature), and the difficulty in making accurate flow measurements from heated gas sources, required to establish the absolute scale of the measured data. Based on techniques pioneered in positron collision physics [2], a new apparatus is presently undergoing commissioning at the California State University Fullerton, which aims to address these issues. We will make the first cross-section measurements for slow (E0 < 30eV) electron collisions with nucleotides. We will report design parameters and ongoing progress in the commissioning of this new experiment.

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

    PubMed Central

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

    2016-01-01

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

  15. Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label.

    PubMed

    Zhang, Xiaojun; Xu, Cui-Xia; Di Felice, Rosa; Sponer, Jiri; Islam, Barira; Stadlbauer, Petr; Ding, Yuan; Mao, Lingling; Mao, Zong-Wan; Qin, Peter Z

    2016-01-19

    Guanine-rich oligonucleotides can form a unique G-quadruplex (GQ) structure with stacking units of four guanine bases organized in a plane through Hoogsteen bonding. GQ structures have been detected in vivo and shown to exert their roles in maintaining genome integrity and regulating gene expression. Understanding GQ conformation is important for understanding its inherent biological role and for devising strategies to control and manipulate functions based on targeting GQ. Although a number of biophysical methods have been used to investigate structure and dynamics of GQs, our understanding is far from complete. As such, this work explores the use of the site-directed spin labeling technique, complemented by molecular dynamics simulations, for investigating GQ conformations. A nucleotide-independent nitroxide label (R5), which has been previously applied for probing conformations of noncoding RNA and DNA duplexes, is attached to multiple sites in a 22-nucleotide DNA strand derived from the human telomeric sequence (hTel-22) that is known to form GQ. The R5 labels are shown to minimally impact GQ folding, and inter-R5 distances measured using double electron-electron resonance spectroscopy are shown to adequately distinguish the different topological conformations of hTel-22 and report variations in their occupancies in response to changes of the environment variables such as salt, crowding agent, and small molecule ligand. The work demonstrates that the R5 label is able to probe GQ conformation and establishes the base for using R5 to study more complex sequences, such as those that may potentially form multimeric GQs in long telomeric repeats. PMID:26678746

  16. The role of N7 protonation of guanine in determining the structure, stability and function of RNA base pairs.

    PubMed

    Halder, Antarip; Bhattacharya, Sohini; Datta, Ayan; Bhattacharyya, Dhananjay; Mitra, Abhijit

    2015-10-21

    The roles of protonated nucleobases in stabilizing different structural motifs and in facilitating catalytic functions of RNA are well known. Among different polar sites of all the nucleobases, N7 of guanine has the highest protonation propensity at physiological pH. However, unlike other easily protonable sites such as N1 and N3 of adenine or N3 of cytosine, N7 protonation of guanine does not lead to the stabilization of base pairs involving its protonated Hoogsteen edge. It also does not facilitate its participation in any acid-base catalysis process. To explore the possible roles of N7 protonated guanine, we have studied its base pairing potentials involving WatsonCrick and sugar edges, which undergo major charge redistribution upon N7 protonation. We have carried out quantum chemical geometry optimization at the M05-2X/6-311G+(2d,2p) level, followed by interaction energy calculation at the MP2/aug-cc-pVDZ level, along with the analysis of the context of occurrence for selected base pairs involving the sugar edge or the WatsonCrick edge of guanine within a non-redundant set of 167 RNA crystal structures. Our results suggest that, four base pairs - G:C W:W trans, G:rC W:S cis, G:G W:H cis and G:G S:H trans may involve N7 protonated guanine. These base pairs deviate significantly from their respective experimental geometries upon QM optimization, but they retain their experimental geometries if guanine N7 protonation is considered during optimization. Our study also reveals the role of guanine N7 protonation (i) in stabilizing important RNA structural motifs, (ii) in providing a framework for designing pH driven molecular motors and (iii) in providing an alternative strategy to mimic the effect of post-transcriptional changes. PMID:26382322

  17. The EMBL nucleotide sequence database.

    PubMed Central

    Stoesser, G; Moseley, M A; Sleep, J; McGowran, M; Garcia-Pastor, M; Sterk, P

    1998-01-01

    The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl. html ) constitutes Europe's primary nucleotide sequence resource. DNA and RNA sequences are directly submitted from researchers and genome sequencing groups and collected from the scientific literature and patent applications (Fig. 1). In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute. Database releases are produced quarterly and are distributed on CD-ROM. EBI's network services allow access to the most up-to-date data collection via Internet and World Wide Web interface, providing database searching and sequence similarity facilities plus access to a large number of additional databases. PMID:9399791

  18. The Single Nucleotide Polymorphism Consortium

    NASA Technical Reports Server (NTRS)

    Morgan, Michael

    2003-01-01

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

  19. Necessary relations for nucleotide frequencies.

    PubMed

    Sinclair, Robert

    2015-06-01

    Genome composition analysis of di-, tri- and tetra-nucleotide frequencies is known to be evolutionarily informative, and useful in metagenomic studies, where binning of raw sequence data is often an important first step. Patterns appearing in genome composition analysis may be due to evolutionary processes or purely mathematical relations. For example, the total number of dinucleotides in a sequence is equal to the sum of the individual totals of the sixteen types of dinucleotide, and this is entirely independent of any assumptions made regarding mutation or selection, or indeed any physical or chemical process. Before any statistical analysis can be attempted, a knowledge of all necessary mathematical relations is required. I show that 25% of di-, tri- and tetra-nucleotide frequencies can be written as simple sums and differences of the remainder. The vast majority of organisms have circular genomes, for which these relations are exact and necessary. In the case of linear molecules, the absolute error is very nearly zero, and does not grow with contiguous sequence length. As a result of the new, necessary relations presented here, the foundations of the statistical analysis of di-, tri- and tetra-nucleotide frequencies, and k-mer analysis in general, need to be revisited.

  20. Necessary relations for nucleotide frequencies.

    PubMed

    Sinclair, Robert

    2015-06-01

    Genome composition analysis of di-, tri- and tetra-nucleotide frequencies is known to be evolutionarily informative, and useful in metagenomic studies, where binning of raw sequence data is often an important first step. Patterns appearing in genome composition analysis may be due to evolutionary processes or purely mathematical relations. For example, the total number of dinucleotides in a sequence is equal to the sum of the individual totals of the sixteen types of dinucleotide, and this is entirely independent of any assumptions made regarding mutation or selection, or indeed any physical or chemical process. Before any statistical analysis can be attempted, a knowledge of all necessary mathematical relations is required. I show that 25% of di-, tri- and tetra-nucleotide frequencies can be written as simple sums and differences of the remainder. The vast majority of organisms have circular genomes, for which these relations are exact and necessary. In the case of linear molecules, the absolute error is very nearly zero, and does not grow with contiguous sequence length. As a result of the new, necessary relations presented here, the foundations of the statistical analysis of di-, tri- and tetra-nucleotide frequencies, and k-mer analysis in general, need to be revisited. PMID:25843217

  1. Applications of adenine nucleotide measurements in oceanography

    NASA Technical Reports Server (NTRS)

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

    1975-01-01

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

  2. Effects of acyclovir and its metabolites on hypoxanthine-guanine phosphoribosyltransferase.

    PubMed

    Tuttle, J V; Krenitsky, T A; Elion, G B

    1983-10-15

    Acyclovir [9-(2-hydroxyethoxymethyl)guanine], a clinically useful anti-herpesvirus agent, was a weak inhibitor (Ki = 190 microM) of hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) from human erythrocytes. Nevertheless, this acyclic nucleoside analog was a more effective inhibitor than were its natural counterparts, guanosine (Ki = 1400 microM) and deoxyguanosine (Ki = 570 microM). The two oxidized metabolites of acyclovir, 9-carboxymethoxymethylguanine (Ki = 720 microM) and 8-hydroxy-9-(2-hydroxyethoxymethyl)guanine (Ki greater than 2000 microM), were less inhibitory than was the parent drug. None of the phosphorylated metabolites of acyclovir was as potent an inhibitor of HGPRTase as was GMP (Ki = 4 microM). However, the Ki value for acyclovir monophosphate was similar to that of dGMP (12 microM). The Ki values for acyclovir diphosphate (8.3 microM) and triphosphate (30 microM) were less than those for dGDP (110 microM) and dGTP (140 microM). The levels of these phosphate esters of acyclovir in cultured monkey kidney (Vero) and human embryo fibroblast (WI38) cells exposed to therapeutic levels of the drug were well below the observed Ki values. However, in herpesvirus-infected WI38 cells the levels of the phosphate esters of acyclovir were high enough potentially to inhibit the enzyme. Although inhibition of this enzyme by the phosphorylated metabolites of acyclovir may occur in these infected cells, concentrations of the drug very much higher than the EC50 concentration were required to achieve inhibitory levels. It is, therefore, unlikely that this inhibition contributes significantly to the antiviral activity.

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    Secondary electron emission is the most important stage in the mechanism of radiation damage to DNA biopolymers induced by primary ionizing radiation. These secondary electrons ejected by the primary electron impacts can produce further ionizations, initiating an avalanche effect, leading to genome damage through the energy transfer from the primary objects to sensitive biomolecular targets, such as nitrogenous bases, saccharides, and other DNA and peptide components. In this work, the formation of positive and negative ions of purine bases of nucleic acids (adenine and guanine molecules) under the impact of slow electrons (from 0.1 till 200 eV) is studied by the crossed electron and molecular beams technique. The method used makes it possible to measure the molecular beam intensity and determine the total cross-sections for the formation of positive and negative ions of the studied molecules, their energy dependences, and absolute values. It is found that the maximum cross section for formation of the adenine and guanine positive ions is reached at about 90 eV energy of the electron beam and their absolute values are equal to 2.8 × 10-15 and 3.2 × 10-15 cm2, respectively. The total cross section for formation of the negative ions is 6.1 × 10-18 and 7.6 × 10-18 cm2 at the energy of 1.1 eV for adenine and guanine, respectively. The absolute cross-section values for the molecular ions are measured and the cross-sections of dissociative ionization are determined. Quantum chemical calculations are performed for the studied molecules, ions and fragments for interpretation of the crossed beams experiments.

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

    SciTech Connect

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

    2014-05-07

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

  5. In vitro antiviral activity of mycophenolic acid and its reversal by guanine-type compounds.

    PubMed

    Cline, J C; Nelson, J D; Gerzon, K; Williams, R H; Delong, D C

    1969-07-01

    With the agar diffusion test and BS-C-1 cells, mycophenolic acid was found to give a straight-line dose-response activity in inhibiting the cytopathic effects of vaccinia, herpes simplex, and measles viruses. Plaque tests have shown 100% reduction of virus plaques by mycophenolic acid over drug ranges of 10 to 50 mug/ml and virus input as high as 6,000 plaque-forming units (PFU) per flask. Back titration studies with measles virus inhibited by mycophenolic acid have indicated that extracellular virus titers were reduced by approximately 3 logs(10) and total virus was reduced by 1 log(10). The agar diffusion test system lends itself readily to drug reversal studies. Mycophenolic acid incorporated into agar at 10 mug/ml gave 100% protection to virus-infected cells. Filter paper discs impregnated with selected chemical agents at concentrations of 1,000 mug/ml (20 mug per filter paper disc) were placed on the agar surface. Reversal of the antiviral activity of mycophenolic acid was indicated by virus breakthrough in those cells in close proximity to the filter paper disc. Chemicals showing the best reversal of the antiviral activity of mycophenolic acid were guanine, guanosine, guanylic acid, deoxyguanylic acid, and 2,6-diaminopurine. The reversal of antiviral activity was confirmed by titrations of virus produced with various amounts of both mycophenolic acid and guanine present and by isotope tracer methods with uptakes of labeled uridine, guanine, leucine, and thymidine in treated and nontreated, infected and noninfected cells as parameters. All antiviral effects of mycophenolic acid at 10 mug/ml could be reversed to the range shown by untreated controls by the addition of 10 mug/ml of those chemicals exhibiting reversal activity.

  6. Topoisomerase IB of Deinococcus radiodurans resolves guanine quadruplex DNA structures in vitro.

    PubMed

    Kota, Swathi; Misra, Hari S

    2015-12-01

    Deinococcus radiodurans genome contains a large number of guanine repeats interrupted by a few non-guanine bases, termed G motifs. Some of these G motifs were shown forming guanine quadruplex (G4) DNA structure in vitro. How is the formation and relaxation of G4 DNA regulated in the genome of D. radiodurans is not known and is worth investigating. Here, we showed that the topoisomerase Ib of D. radiodurans (DraTopoIB) could change the electrophoretic mobility of fast migrating intramolecular recF-G4 DNA into the slow migrating species. DraTopoIB also reduced the positive ellipticity in circular diachroism (CD) spectra of intramolecular recF-G4 DNA structures stabilized by K+. On the contrary, when DraTopoIB is incubated with G-motifs annealed without K+, it showed neither any change in electrophoretic mobility nor was ellipticity of the CD spectra affected. DNA synthesis by Taq DNA polymerase through G4 DNA structure was attenuated in the presence of G4 DNA binding drugs, which was abrogated by DraTopoIB. This implies that DraTopoIB could destabilize the G4 DNA structure, which is required for G4 drugs binding and stabilization. Camptothecin treatment inhibited DraTopoIB activity on intramolecular G4 DNA structures. These results suggested that DraTopoIB can relax intramolecular G4 DNA structure in vitro and it may be one such protein that could resolve G4 DNA under normal growth conditions in D. radiodurans.

  7. Effect of 10-T magnetic fields on structural colors in guanine crystals of fish scales

    NASA Astrophysics Data System (ADS)

    Iwasaka, M.; Miyashita, Y.; Kudo, M.; Kurita, S.; Owada, N.

    2012-04-01

    This work reports the magnetically modulated structural colors in the chromatophore of goldfish scales under static magnetic fields up to 10 T. A fiber optic system for spectroscopy measurements and a CCD microscope were set in the horizontal bore of a 10-T superconducting magnet. One leaf of a fish scale was set in a glass chamber, exposed to visible light from its side direction, and then static magnetic fields were applied perpendicular to the surface of the scale. In addition, an optical fiber for spectroscopy was directed perpendicular to the surface. During the magnetic field sweep-up, the aggregate of guanine thin plates partially showed a rapid light quenching under 0.26 to 2 T; however, most of the thin plates continued to scatter the side-light and showed changing iridescence, which was displayed individually by each guanine plate. For example, an aggregate in the chromatophore exhibited a dynamic change in structural color from white-green to dark blue when the magnetic fields changed from 2 to 10 T. The spectrum profile, which was obtained by the fiber optic system, confirmed the image color changes under magnetic field exposure. Also, a linearly polarized light transmission was measured on fish scales by utilizing an optical polarizer and analyzer. The transmitted polarized light intensities increased in the range of 500-550 nm compared to the intensity at 700 nm during the magnetic field sweep-up. These results indicate that the multi-lamella structure of nano-mirror plates in guanine hexagonal micro-plates exhibit diamagnetically modulated structure changes, and its light interference is affected by strong magnetic fields.

  8. Fluorescent Sensing of Guanine and Guanosine Monophosphate with Conjugated Receptors Incorporating Aniline and Naphthyridine Moieties.

    PubMed

    Lu, Shao-Hung; Phang, Riping; Fang, Jim-Min

    2016-04-15

    Ethyne-linked naphthyridine-aniline conjugated molecules are selective sensors of decylguanine in dichloromethane and guanosine monophosphate in water (Kass = 16,000 M(-1)). The 2-acetamido-1,8-naphthyridine moiety binds with guanine in a DAA-ADD triply hydrogen-bonded motif. The aniline moiety enhances an electron-donating effect, and the substituent is tuned to attain extra hydrogen bonds, π-π stacking, and electrostatic interactions. The proposed binding modes are supported by a Job plot, ESI-MS, (1)H NMR, UV-vis, and fluorescence spectral analyses.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25973536

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

    PubMed

    Kalaivani, Arumugam; Narayanan, Sangilimuthu Sriman

    2015-06-01

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

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

    PubMed

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

    2015-01-01

    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 Hg(2+). 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. PMID:25973536

  13. Promotion of purine nucleotide binding to thymidylate synthase by a potent folate analogue inhibitor, 1843U89.

    PubMed Central

    Weichsel, A; Montfort, W R; Cieśla, J; Maley, F

    1995-01-01

    A folate analogue, 1843U89 (U89), with potential as a chemotherapeutic agent due to its potent and specific inhibition of thymidylate synthase (TS; EC 2.1.1.45), greatly enhances not only the binding of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and dUMP to Escherichia coli TS but also that of dGMP, GMP, dIMP, and IMP. Guanine nucleotide binding was first detected by CD analysis, which revealed a unique spectrum for the TS-dGMP-U89 ternary complex. The quantitative binding of dGMP relative to GMP, FdUMP, and dUMP was determined in the presence and absence of U89 by ultrafiltration analysis, which revealed that although the binding of GMP and dGMP could not be detected in the absence of U89 both were bound in its presence. The Kd for dGMP was about the same as that for dUMP and FdUMP, with binding of the latter two nucleotides being increased by two orders of magnitude by U89. An explanation for the binding of dGMP was provided by x-ray diffraction studies that revealed an extensive stacking interaction between the guanine of dGMP and the benzoquinazoline ring of U89 and hydrogen bonds similar to those involved in dUMP binding. In addition, binding energy was provided through a water molecule that formed hydrogen bonds to both N7 of dGMP and the hydroxyl of Tyr-94. Accommodation of the larger dGMP molecule was accomplished through a distortion of the active site and a shift of the deoxyribose moiety to a new position. These rearrangements also enabled the binding of GMP to occur by creating a pocket for the ribose 2' hydroxyl group, overcoming the normal TS discrimination against nucleotides containing the 2' hydroxyl. Images Fig. 3 Fig. 4 Fig. 5 PMID:7724588

  14. Biological activity of analogs of guanine and guanosine against American Trypanosoma and Leishmania spp.

    PubMed Central

    Avila, J L; Rojas, T; Avila, A; Polegre, M A; Robins, R K

    1987-01-01

    The growth inhibitory effects of six guanine and guanosine analogs, 3-deazaguanine (compound 1); 3-deazaguanosine (compound 2); 6-aminoallopurinol (compound 3); 9-beta-xylofuranosyl guanine (compound 4); a ribosylated derivative of compound 3, 6-aminopyrazolo(3,4-d)pyrimidin-4-one (compound 5); and 5-aminoformycin B (compound 6), were tested against some pathogenic members of the family of American Trypanosomatidae. Compounds 1 and 2 were highly active against Trypanosoma cruzi, Trypanosoma rangeli, and American Leishmania spp. in in vitro culture forms. Both compounds also showed antiprotozoal activity in T. cruzi-infected mice, with the optimal dose being about 30 mg/kg of body weight per day given as 10 consecutive doses. Compound 3 was the most active compound in vitro, inhibiting all of the American Trypanosomatidae culture forms tested. It was also highly inhibitory in mice that were acutely infected with T. cruzi, with the optimal dose being about 10 mg/kg of body weight per day. Ribosylation of compound 3 resulted in a derivative that showed decreased inhibitory activity on Trypanosomatidae multiplication. Compound 6 was highly inhibitory of in vitro multiplication of American Leishmania and T. rangeli but had no effect on T. cruzi epimastigotes and on mice that were acutely infected with T. cruzi. Compound 4 showed only a slight effect on T. cruzi epimastigotes. PMID:3107463

  15. Reactions of the OOH radical with guanine: Mechanisms of formation of 8-oxoguanine and other products

    NASA Astrophysics Data System (ADS)

    Kumar, Nagendra; Shukla, P. K.; Mishra, P. C.

    2010-09-01

    The mutagenic product 8-oxoguanine (8-oxoGua) is formed due to intermediacy of peroxyl (OOR) radicals in lipid peroxidation and protein oxidation-induced DNA damage. The mechanisms of these reactions are not yet understood properly. Therefore, in the present study, the mechanisms of formation of 8-oxoGua and other related products due to the reaction of the guanine base of DNA with the hydroperoxyl radical (OOH) were investigated theoretically employing the B3LYP and BHandHLYP hybrid functionals of density functional theory and the polarizable continuum model for solvation. It is found that the reaction of the OOH radical with guanine can occur following seven different mechanisms leading to the formation of various products including 8-oxoGua, its radicals, 5-hydroxy-8-oxoguanine and CO 2. The mechanism that yields 8-oxoGua as an intermediate and 5-hydroxy-8-oxoGua as the final product was found to be energetically most favorable.

  16. Assessment of the photosensitization properties of cationic porphyrins in interaction with DNA nucleotide pairs.

    PubMed

    Cárdenas-Jirón, Gloria I; Cortez, Luis

    2013-07-01

    We present a theoretical assessment of the photosensitization properties of meso-mono(N-methylpyridyl) triphenylporphyrin (1, MmPyP(+)), which interacts with DNA nucleotide pairs [adenine (A)-thymine (T); guanine (G)-cytosine (C)] via an external binding mode. The photosensitization properties of the arrangements 1A, 1T, 1G and 1C were investigated. A set of density functionals (B3LYP, PBE0, CAM-B3LYP, M06-2X, B97D) with the 6-31G(d) basis set was used to calculate the electronic absorption spectra in solution (water) following TD-DFT methodology. In all the arrangements, with the exception of 1C, the functional PBE0 produced the lowest deviation of the Soret band (0.1-0.2 eV). Using this functional, we show that the porphyrin-nucleotide interaction is stabilized, as reflected by a larger HOMO-LUMO gap than free porphyrin. A more important effect of the interaction corresponds to the red-shift of the Soret band of MmPyP(+), which is in agreement with experimental results. This behavior could be explained by the higher symmetry found in arrangements with a lower dipole moment, and by the more symmetrical distribution of electronic density along the molecular orbitals, which provokes electronic transitions of lower energy. The structural model allowed us to show that MmPyP(+) improves the characteristics as a photosensitizer when it interacts with nucleotide pairs due to the longer wavelength required for the Soret band. Results obtained for porphyrins with larger monocationic substituents (2, MmAP+; 3, MONPP+) do not lead to the same behavior. Although the structural model is insufficient to describe porphyrin photosensitization, it suggests that improvements in this property are produced by the inclusion of a cationic charge in the pyridyl ring and a smaller size of the substituent leading to a better communication in the porphyrin-nucleotide pair.

  17. Nucleotide release by airway epithelia.

    PubMed

    Lazarowski, Eduardo R; Sesma, Juliana I; Seminario, Lucia; Esther, Charles R; Kreda, Silvia M

    2011-01-01

    The purinergic events regulating the airways' innate defenses are initiated by the release of purines from the epithelium, which occurs constitutively and is enhanced by chemical or mechanical stimulation. While the external triggers have been reviewed exhaustively, this chapter focuses on current knowledge of the receptors and signaling cascades mediating nucleotide release. The list of secreted purines now includes ATP, ADP, AMP and nucleotide sugars, and involves at least three distinct mechanisms reflecting the complexity of airway epithelia. First, the constitutive mechanism involves ATP translocation to the ER/Golgi complex as energy source for protein folding, and fusion of Golgi-derived vesicles with the plasma membrane. Second, goblet cells package ATP with mucins into granules, which are discharged in response to P2Y(2)R activation and Ca(2+)-dependent signaling pathways. Finally, non-mucous cells support a regulated mechanism of ATP release involving protease activated receptor (PAR)-elicited G(12/13) activation, leading to the RhoGEF-mediated exchange of GDP for GTP on RhoA, and cytoskeleton rearrangement. Together, these pathways provide fine tuning of epithelial responses regulated by purinergic signaling events. PMID:21560042

  18. N7-glycidamide-guanine DNA adduct formation by orally ingested acrylamide in rats: a dose-response study encompassing human diet-related exposure levels.

    PubMed

    Watzek, Nico; Böhm, Nadine; Feld, Julia; Scherbl, Denise; Berger, Franz; Merz, Karl Heinz; Lampen, Alfonso; Reemtsma, Thorsten; Tannenbaum, Steven R; Skipper, Paul L; Baum, Matthias; Richling, Elke; Eisenbrand, Gerhard

    2012-02-20

    Acrylamide (AA) is formed during the heating of food and is classified as a genotoxic carcinogen. The margin of exposure (MOE), representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be of concern. After ingestion, AA is converted by P450 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). We performed a dose-response study with AA in female Sprague-Dawley (SD) rats. AA was given orally in a single dosage of 0.1-10 000 μg/kg bw. The formation of urinary mercapturic acids and of N7-GA-Gua DNA adducts in liver, kidney, and lung was measured 16 h after application. A mean of 37.0 ± 11.5% of a given AA dose was found as mercapturic acids (MAs) in urine. MA excretion in urine of untreated controls indicated some background exposure from endogenous AA. N7-GA-Gua adduct formation was not detectable in any organ tested at 0.1 μg AA/kg bw. At a dose of 1 μg/kg bw, adducts were found in kidney (around 1 adduct/10(8) nucleotides) and lung (below 1 adduct/10(8) nucleotides) but not in liver. At 10, respectively, 100 μg/kg bw, adducts were found in all three organs, at levels close to those found at 1 μg AA/kg, covering a range of about 1-2 adducts/10(8) nucleotides. As compared to DNA adduct levels from electrophilic genotoxic agents of various origin found in human tissues, N7-GA-Gua adduct levels within the dose range of 0.1-100 μg AA/kg bw were at the low end of this human background. We propose to take the background level of DNA lesions in humans more into consideration when doing risk assessment of food-borne genotoxic carcinogens.

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

    SciTech Connect

    Monaco, L.; Murtagh, J.J.; Newman, K.B.; Tsai, Su-Chen; Moss, J.; Vaughan, M. )

    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. Leukaemia-associated Rho guanine nucleotide exchange factor (LARG) plays an agonist specific role in platelet function through RhoA activation.

    PubMed

    Zou, Siying; Teixeira, Alexandra M; Yin, Mingzhu; Xiang, Yaozu; Xavier-Ferruccio, Juliana; Zhang, Ping-Xia; Hwa, John; Min, Wang; Krause, Diane S

    2016-08-30

    Leukemia-Associated RhoGEF (LARG) is highly expressed in platelets, which are essential for maintaining normal haemostasis. We studied the function of LARG in murine and human megakaryocytes and platelets with Larg knockout (KO), shRNA-mediated knockdown and small molecule-mediated inhibition. We found that LARG is important for human, but not murine, megakaryocyte maturation. Larg KO mice exhibit macrothrombocytopenia, internal bleeding in the ovaries and prolonged bleeding times. KO platelets have impaired aggregation, α-granule release and integrin α2bβ3 activation in response to thrombin and thromboxane, but not to ADP. The same agonist-specific reductions in platelet aggregation occur in human platelets treated with a LARG inhibitor. Larg KO platelets have reduced RhoA activation and myosin light chain phosphorylation, suggesting that Larg plays an agonist-specific role in platelet signal transduction. Using two different in vivo assays, Larg KO mice are protected from in vivo thrombus formation. Together, these results establish that LARG regulates human megakaryocyte maturation, and is critical for platelet function in both humans and mice. PMID:27345948

  1. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  2. Guanine Nucleotide-binding Protein (Gα) Endocytosis by a Cascade of Ubiquitin Binding Domain Proteins Is Required for Sustained Morphogenesis and Proper Mating in Yeast*

    PubMed Central

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

    2014-01-01

    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. PMID:24722989

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

    PubMed

    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.

  4. Phytoestrogens regulate mRNA and protein levels of guanine nucleotide-binding protein, beta-1 subunit (GNB1) in MCF-7 cells.

    PubMed

    Naragoni, Srivatcha; Sankella, Shireesha; Harris, Kinesha; Gray, Wesley G

    2009-06-01

    Phytoestrogens (PEs) are non-steroidal ligands, which regulate the expression of number of estrogen receptor-dependent genes responsible for a variety of biological processes. Deciphering the molecular mechanism of action of these compounds is of great importance because it would increase our understanding of the role(s) these bioactive chemicals play in prevention and treatment of estrogen-based diseases. In this study, we applied suppression subtractive hybridization (SSH) to identify genes that are regulated by PEs through either the classic nuclear-based estrogen receptor or membrane-based estrogen receptor pathways. SSH, using mRNA from genistein (GE) treated MCF-7 cells as testers, resulted in a significant increase in GNB1 mRNA expression levels as compared with 10 nM 17beta estradiol or the no treatment control. GNB1 mRNA expression was up regulated two- to fivefold following exposure to 100.0 nM GE. Similarly, GNB1 protein expression was up regulated 12- to 14-fold. GE regulation of GNB1 was estrogen receptor-dependent, in the presence of the anti-estrogen ICI-182,780, both GNB1 mRNA and protein expression were inhibited. Analysis of the GNB1 promoter using ChIP assay showed a PE-dependent association of estrogen receptor alpha (ERalpha) and beta (ERbeta) to the GNB1 promoter. This association was specific for ERalpha since association was not observed when the cells were co-incubated with GE and the ERalpha antagonist, ICI. Our data demonstrate that the levels of G-protein, beta-1 subunit are regulated by PEs through an estrogen receptor pathway and further suggest that PEs may control the ratio of alpha-subunit to beta/gamma-subunits of the G-protein complex in cells. J. Cell. Physiol. 219: 584-594, 2009. (c) 2009 Wiley-Liss, Inc. PMID:19170076

  5. SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses.

    PubMed

    Fukaya, Masahiro; Kamata, Akifumi; Hara, Yoshinobu; Tamaki, Hideaki; Katsumata, Osamu; Ito, Naoki; Takeda, Shin'ichi; Hata, Yutaka; Suzuki, Tatsuo; Watanabe, Masahiko; Harvey, Robert J; Sakagami, Hiroyuki

    2011-03-01

    SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both β-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.

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

    PubMed

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

    2004-05-01

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

  7. Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

    PubMed

    Freitas-Mesquita, Anita Leocadio; Gomes, Marta T; Vieira, Danielle P; Paes-Vieira, Lisvane; Nascimento, Michelle T C; Lopes, Angela H C S; Meyer-Fernandes, José Roberto

    2016-10-01

    The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target.

  8. Comparative Genomic Analysis Reveals a Critical Role of De Novo Nucleotide Biosynthesis for Saccharomyces cerevisiae Virulence

    PubMed Central

    Pérez-Torrado, Roberto; Llopis, Silvia; Perrone, Benedetta; Gómez-Pastor, Rocío; Hube, Bernhard; Querol, Amparo

    2015-01-01

    In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other ‘opportunistic’ strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host. PMID:25816288

  9. Inhibitory effects promoted by 5'-nucleotides on the ecto-3'-nucleotidase activity of Leishmania amazonensis.

    PubMed

    Freitas-Mesquita, Anita Leocadio; Gomes, Marta T; Vieira, Danielle P; Paes-Vieira, Lisvane; Nascimento, Michelle T C; Lopes, Angela H C S; Meyer-Fernandes, José Roberto

    2016-10-01

    The protozoan parasite Leishmania amazonensis is the etiological agent of cutaneous leishmaniasis. During its life cycle, the flagellated metacyclic promastigote forms are transmitted to vertebrate hosts by sandfly bites, and they develop into amastigotes inside macrophages, where they multiply. L. amazonensis possesses a bifunctional enzyme, called 3'-nucleotidase/nuclease (3'NT/NU), which is able to hydrolyze extracellular 3'-monophosphorylated nucleosides and nucleic acids. 3'NT/NU plays an important role in the generation of extracellular adenosine and has been described as a key enzyme in the acquisition of purines by trypanosomatids. Furthermore, it has been observed that 3'NT/NU also plays a valuable role in the establishment of parasitic infection. In this context, this study aimed to investigate the modulation of the 3'-nucleotidase (3'NT) activity of L. amazonensis by several nucleotides. It was observed that 3'NT activity is inhibited by micromolar concentrations of guanosine and guanine nucleotides. The inhibition promoted by 5'-GMP on the 3'NT activity of L. amazonensis is reversible and uncompetitive because the addition of the inhibitor decreased the kinetic parameters Km and Vmax. Finally, we found that the addition of 5'-GMP is able to reverse the stimulation promoted by 3'-AMP in a macrophage-parasite interaction assay. The determination of compounds that can inhibit the 3'NT activity of Leishmania is very important because this enzyme does not occur in mammals, making it a potential therapeutic target. PMID:27531705

  10. Hydroxyl Radical (OH•) Reaction with Guanine in an Aqueous Environment: A DFT Study

    PubMed Central

    Kumar, Anil; Pottiboyina, Venkata; Sevilla, Michael D.

    2011-01-01

    The reaction of hydroxyl radical (OH•) with DNA accounts for about half of radiation-induced DNA damage in living systems. Previous literature reports point out that the reaction of OH• with DNA proceeds mainly through the addition of OH• to the C=C bond of the DNA bases. However, recently it has been reported that the principal reaction of OH• with dGuo (deoxyguanosine) is the direct hydrogen atom abstraction from its exocyclic amine group rather than addition of OH• to the C=C bond. In the present work, these two reaction pathways of OH• attack on guanine (G) in the presence of water molecules (aqueous environment) are investigated using the density functional theory (DFT) B3LYP method with 6-31G* and 6-31++G** basis sets. The calculations show that the initial addition of the OH• at C4=C5 double bond of guanine is barrier free and the adduct radical (G-OH•) has only a small activation barrier of ca. 1 – 6 kcal/mol leading to the formation of a metastable ion-pair intermediate (G•+---OH−). The formation of ion-pair is a result of the highly oxidizing nature of the OH• in aqueous media. The resulting ion-pair (G•+---OH−) deprotonates to form H2O and neutral G radicals favoring G(N1-H)• with an activation barrier of ca. 5 kcal/mol. The overall process from the G(C4)-OH• (adduct) to G(N1-H)• and water is found to be exothermic in nature by more than 13 kcal/mol. (G-OH•), (G•+---OH−), and G(N1-H)• were further characterized by the CAM-B3LYP calculations of their UV-visible spectra and good agreement between theory and experiment is achieved. Our calculations for the direct hydrogen abstraction pathway from N1 and N2 sites of guanine by the OH• show that this is also a competitive route to produce G(N2-H)•, G(N1-H)• and H2O. PMID:22050033

  11. Hydroxyl radical (OH•) reaction with guanine in an aqueous environment: a DFT study.

    PubMed

    Kumar, Anil; Pottiboyina, Venkata; Sevilla, Michael D

    2011-12-22

    The reaction of hydroxyl radical (OH(•)) with DNA accounts for about half of radiation-induced DNA damage in living systems. Previous literature reports point out that the reaction of OH(•) with DNA proceeds mainly through the addition of OH(•) to the C═C bonds of the DNA bases. However, recently it has been reported that the principal reaction of OH(•) with dGuo (deoxyguanosine) is the direct hydrogen atom abstraction from its exocyclic amine group rather than addition of OH(•) to the C═C bonds. In the present work, these two reaction pathways of OH(•) attack on guanine (G) in the presence of water molecules (aqueous environment) are investigated using the density functional theory (DFT) B3LYP method with 6-31G* and 6-31++G** basis sets. The calculations show that the initial addition of the OH(•) at C(4)═C(5) double bond of guanine is barrier free and the adduct radical (G-OH(•)) has only a small activation barrier of ca. 1-6 kcal/mol leading to the formation of a metastable ion-pair intermediate (G(•+)---OH(-)). The formation of ion-pair is a result of the highly oxidizing nature of the OH(•) in aqueous media. The resulting ion-pair (G(•+)---OH(-)) deprotonates to form H(2)O and neutral G radicals favoring G(N(1)-H)(•) with an activation barrier of ca. 5 kcal/mol. The overall process from the G(C(4))-OH(•) (adduct) to G(N(1)-H)(•) and water is found to be exothermic in nature by more than 13 kcal/mol. (G-OH(•)), (G(•+)---OH(-)), and G(N(1)-H)(•) were further characterized by the CAM-B3LYP calculations of their UV-vis spectra and good agreement between theory and experiment is achieved. Our calculations for the direct hydrogen abstraction pathway from N(1) and N(2) sites of guanine by the OH(•) show that this is also a competitive route to produce G(N(2)-H)(•), G(N(1)-H)(•) and H(2)O.

  12. The guanine cation radical: investigation of deprotonation states by ESR and DFT.

    PubMed

    Adhikary, Amitava; Kumar, Anil; Becker, David; Sevilla, Michael D

    2006-11-30

    This work reports ESR studies that identify the favored site of deprotonation of the guanine cation radical (G*+) in an aqueous medium at 77 K. Using ESR and UV-visible spectroscopy, one-electron oxidized guanine is investigated in frozen aqueous D2O solutions of 2'-deoxyguanosine (dGuo) at low temperatures at various pHs at which the guanine cation radical, G*+ (pH 3-5), singly deprotonated species, G(-H)* (pH 7-9), and doubly deprotonated species, G(-2H)*- (pH > 11), are found. C-8-deuteration of dGuo to give 8-D-dGuo removes the major proton hyperfine coupling at C-8. This isolates the anisotropic nitrogen couplings for each of the three species and aids our analyses. These anisotropic nitrogen couplings were assigned to specific nitrogen sites by use of 15N-substituted derivatives at N1, N2, and N3 atoms in dGuo. Both ESR and UV-visible spectra are reported for each of the species: G*+, G(-H)*, and G(-2H)*-. The experimental anisotropic ESR hyperfine couplings are compared to those obtained from DFT calculations for the various tautomers of G(-H)*. Using the B3LYP/6-31G(d) method, the geometries and energies of G*+ and its singly deprotonated state in its two tautomeric forms, G(N1-H)* and G(N2-H)*, were investigated. In a nonhydrated state, G(N2-H)* is found to be more stable than G(N1-H)*, but on hydration with seven water molecules G(N1-H)* is found to be more stable than G(N2-H)*. The theoretically calculated hyperfine coupling constants (HFCCs) of G*+, G(N1-H)*, and G(-2H)*- match the experimentally observed HFCCs best on hydration with seven or more waters. For G(-2H)*-, the hyperfine coupling constant (HFCC) at the exocyclic nitrogen atom (N2) is especially sensitive to the number of hydrating water molecules; good agreement with experiment is not obtained until nine or 10 waters of hydration are included.

  13. The Guanine Cation Radical: Investigation of Deprotonation States by ESR and DFT

    PubMed Central

    Adhikary, Amitava; Kumar, Anil; Becker, David; Sevilla, Michael D.

    2008-01-01

    This work reports ESR studies that identify the favored site of deprotonation of the guanine cation radical (G•+) in an aqueous medium at 77 K. Using ESR and UV-visible spectroscopy, one-electron oxidized guanine is investigated in frozen aqueous D2O solutions of 2′-deoxyguanosine (dGuo) at low temperatures at various pHs at which the guanine cation, G•+ (pH 3–5), singly deprotonated species, G(-H)• (pH 7–9) and doubly deprotonated species, G(-2H)•− (pH>11) are found. C-8-deuteration of dGuo to give 8-D-dGuo removes the major proton hyperfine coupling at C-8. This isolates the anisotropic nitrogen couplings for each of the three species and aids our analyses. These anisotropic nitrogen couplings were assigned to specific nitrogen sites by use of 15N substituted derivatives at N1, N2 N3 atoms in dGuo. Both ESR and UV-visible spectra are reported for each of the species: G•+, G(-H)•, and G(-2H)•−. The experimental anisotropic ESR hyperfine couplings are compared to those obtained from DFT calculations for the various tautomers of G(-H)•. Using the B3LYP/6–31G(d) method, the geometries and energies of G•+ and its singly deprotonated state in its two tautomeric forms, G(N1-H)• and G(N2-H)•, were investigated. In a non-hydrated state G(N2-H)• is found to be more stable than G(N1-H)• but on hydration with 7 water molecules G(N1-H)• is found to be more stable than G(N2-H)•. The theoretically calculated hyperfine coupling constants (HFCC) of G•+, G(N1-H)• and G(-2H)•− match the experimentally observed HFCCs best on hydration with 7 or more waters. For G(-2H)•−, the hyperfine coupling constant (HFCC) at the exocyclic nitrogen atom (N2) is especially sensitive to the number of hydrating water molecules; good agreement with experiment is not obtained until 9 or 10 waters of hydration are included. PMID:17125389

  14. Mosaic organization of DNA nucleotides

    NASA Technical Reports Server (NTRS)

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

    1994-01-01

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

  15. Nucleotide excision repair in humans.

    PubMed

    Spivak, Graciela

    2015-12-01

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

  16. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

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

  17. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

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

  18. Collision-induced dissociation (CID) of guanine radical cation in the gas phase: an experimental and computational study.

    PubMed

    Cheng, Ping; Li, Yanni; Li, Shuqi; Zhang, Mingtao; Zhou, Zhen

    2010-05-14

    Gas-phase guanine (G) radical cations were generated by electrospraying a solution of guanosine (L) and Cu(NO(3))(2). Collision-induced dissociation (CID) for guanine radical cations yielded five competing dissociation channels, corresponding to the elimination neutral molecules of NH(3), HCN, H(2)NC[triple bond]N (HN=C=NH), HNCO and the neutral radical N=C=NH, respectively. The primary product ions were further characterized by their relevant fragmentions. Ab initio and density functional theory (DFT) calculations were employed to explain the experimental observations. Ten stable radical cation isomers were optimized and the potential energy surfaces (PESs) for the isomerization processes were explored in detail. Starting with the most stable isomer, the primary dissociation channels of guanine radical cations were theoretically investigated. DFT calculations show that the energy barriers for the eliminations of NH(3), HCN, H(2)NC[triple bond]N (HN=C=NH), HNCO and N=C=NH are 397 kJ mol(-1), 479 kJ mol(-1), 294 kJ mol(-1) (298 kJ mol(-1)), 306 kJ mol(-1), and 275 kJ mol(-1), respectively. The results are consistent with the energy-resolved CID of guanine radical cation, in which the eliminations of NH(3) and HCN are less abundant than the other channels. PMID:20428546

  19. Antiviral activity and its mechanism of guanine 7-N-oxide on DNA and RNA viruses derived from salmonid.

    PubMed

    Hasobe, M; Saneyoshi, M; Isono, K

    1985-11-01

    Guanine 7-N-oxide produced by Streptomyces sp. was found to inhibit in vitro the replication of herpes virus (Oncorhynchus masou virus, OMV), rhabdo virus (infectious hematopoietic necrosis virus, IHNV) and a bi-segmented double-strand virus (infectious pancreatic necrosis virus, IPNV) derived from salmonids with IC50 values of about 10 micrograms/ml, 20 micrograms/ml and 32 micrograms/ml, respectively. The agent was not toxic for the host cells (chinook salmon embryo, CHSE-214) at the IC50 concentrations. Labeling of IHNV viral RNA and host cellular DNA and RNA with [3H]uridine and [3H]thymidine during drug treatment showed that guanine 7-N-oxide did not reduce the incorporation of these precusors into RNA and DNA. The anti-IHNV activity of guanine 7-N-oxide was enhanced synergistically by neplanocin A, an inhibitor of RNA methylation. The mechanism of action of guanine 7-N-oxide is discussed, in regard to maturation of viral messenger RNA including capping. PMID:3841124

  20. Adaptive ligand binding by the purine riboswitch in the recognition of guanine and adenine analogs

    PubMed Central

    Gilbert, Sunny D.; Reyes, Francis E.; Edwards, Andrea L.; Batey, Robert T.

    2009-01-01

    SUMMARY Purine riboswitches discriminate between guanine and adenine by at least 10,000-fold based on the identity of a single pyrimidine (Y74) that forms a Watson-Crick base pair with the ligand. To understand how this high degree of specificity for closely related compounds is achieved through simple pairing, we investigated their interaction with purine analogs with varying functional groups at the 2- and 6-positions that have the potential to alter interactions with Y74. Using a combination of crystallographic and calorimetric approaches, we find that binding these purines is often facilitated by either small structural changes in the RNA or tautomeric changes in the ligand. This work also reveals that, along with base pairing, conformational restriction of Y74 significantly contributes to nucleobase selectivity. These results reveal that compounds that exploit the inherent local flexibility within riboswitch binding pockets can alter their ligand specificity. PMID:19523903

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    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 pz 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 px,y electrons form double peaks with respective sites at about 0.5 and 1.5 a.u., only three px,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.

  2. Circular dichroism anisotrophy of DNA with different modifications at N7 of guanine.

    PubMed

    Zavriev, S K; Minchenkova, L E; Vorlícková, M; Kolchinsky, A M; Volkenstein, M V; Ivanov, V I

    1979-09-27

    The complexex DNA-Ag1+, DNA-Cu1+, protonated DNA and DNA methylated at N7 of guanine were oriented by pumping the solutions through a multicapillary cell in the direction of a light beam. The CD components along the DNA axis, delta epsilon parallel, and normal to it, 2 delta epsilon perpendicular, were calculated from the CD spectra of the oriented samples by the method of Chung and Holzwarth, (1975) J. Mol. Biol. 92, 449--466. It was shown that in most cases, except that of the protonated DNA, the degree of orientation was only slightly less than that for pure DNA. This demonstrated the absence of aggregation and of appreciable denaturation. In all cases the modifications of DNA give rise to a negative component 2 delta epsilon perpendicular, whose magnitude increased as the extent of modification increased. From both the CD spectra of non-oriented samples and the absorption spectra, an inference is drawn that Ag1+ and Cu1+ are attached to the same site as CH3 groups i.e., to the N7 atom of guanine. Proton transfer along the H-bond from the N1 atom of G to the N3 atom of the complementary cytosine is suggested to be a result of the modifications, although the case of H+-DNA may differ from the others. Based on the CD spectra for the anisotropic components, delta epsilon parallel and 2 delta epsilon perpendicular, it is proposed that ligand binding is accompanied by winding of the DNA helix.

  3. A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

    PubMed Central

    Liao, Y D

    1992-01-01

    A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS. Images PMID:1373237

  4. Kinetic studies of guanine recognition and a phosphate group subsite on ribonuclease T1 using substitution mutants at Glu46 and Lys41.

    PubMed

    Jo Chitester, Betty; Walz, Frederick G

    2002-10-01

    pH-Dependent kinetic studies were performed with ribonuclease T1 (RNase T1) and its Glu46Ser, Lys41Met, and Lys41Thr mutants with GpC and polyinosinic acid (PolyI) as substrates. Plots of pH versus log(k(cat)/K(M)) for both substrates had ascending slopes that were significantly greater for RNase T1 compared with Glu46Ser-RNase T1, which indicated that the gamma-carboxyl group of conserved Glu46 must be deprotonated (anionic) for maximal interaction with N1H and N2H of the guanine moiety of GpC or the N1H of the hypoxanthine moiety of PolyI. The involvement of the epsilon -ammonium group of nonconserved Lys41 at the 2p subsite (i.e., for an RNA phosphate group two nucleotide positions 5'-upstream from the active site) was supported by comparisons of Lys41Met-RNase T1 and Lys41Thr-RNase T1 with wild-type. These mutants shared identical catalytic properties (i.e., k(cat) and K(M)) with wild-type using GpC as a substrate. However, k(cat)/K(M) for both were identical with each other but lower than those for wild-type when PolyI was the substrate (PolyI has a phosphate group that could interact at a putative 2p site). The pH dependence of this latter difference can be interpreted as reflecting the loss of the 2p subsite interaction with the wild-type enzyme upon deprotonation of the epsilon -ammonium group of Lys41. Subsite interactions for ribonucleases are shown to mainly increase k(cat) and result in an attenuated pH dependence of k(cat)/K(M). PMID:12234492

  5. NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

    PubMed Central

    Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2012-01-01

    The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

  6. Automated Identification of Nucleotide Sequences

    NASA Technical Reports Server (NTRS)

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

    2007-01-01

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

  7. Long-range correlations in nucleotide sequences

    NASA Astrophysics Data System (ADS)

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

    1992-03-01

    DNA SEQUENCES have been analysed using models, such as an it-step Markov chain, that incorporate the possibility of short-range nucleotide correlations1. We propose here a method for studying the stochastic properties of nucleotide sequences by constructing a 1:1 map of the nucleotide sequence onto a walk, which we term a 'DNA walk'. We then use the mapping to provide a quantitative measure of the correlation between nucleotides over long distances along the DNA chain. Thus we uncover in the nucleotide sequence a remarkably long-range power law correlation that implies a new scale-invariant property of DNA. We find such long-range correlations in intron-containing genes and in nontranscribed regulatory DNA sequences, but not in complementary DNA sequences or intron-less genes.

  8. Long-range correlations in nucleotide sequences

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

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

    PubMed Central

    Jayamohan, Harikrishnan; Gale, Bruce K.; Minson, Bj; Lambert, Christopher J.; Gordon, Neil; Sant, Himanshu J.

    2015-01-01

    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 108 guanine tags per secondary bead (7.5 × 106 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′-bipyridine)ruthenium(II) ( Ru(bpy)32+) 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. We also demonstrate the use of the

  10. β-Cyclodextrin-grafted on multiwalled carbon nanotubes as versatile nanoplatform for entrapment of guanine-based drugs.

    PubMed

    Iannazzo, Daniela; Mazzaglia, Antonino; Scala, Angela; Pistone, Alessandro; Galvagno, Signorino; Lanza, Maurizio; Riccucci, Cristina; Ingo, Gabriel Maria; Colao, Ivana; Sciortino, Maria Teresa; Valle, Francesco; Piperno, Anna; Grassi, Giovanni

    2014-11-01

    The design of β-cyclodextrin/multiwalled carbon nanotubes hybrid (β-CD-MWCNT) as nanoplatform for the entrapment and delivery of guanine based drugs is described here. The functionalized carbon nanomaterials have been characterized by XPS spectroscopy, electron microscopy (FEG-SEM and TEM), AFM, TGA, and FT-IR to achieve insights on structure, morphology and chemical composition. The drug binding abilities of nanocarrier towards the guanine (G) and Acyclovir (Acy) were proved by UV-vis and DSC experiments. Host-guest equilibrium association constants and drug loading have been evaluated for G/β-CD-MWCNT and Acy/β-CD-MWCNT complexes. The release studies showed a sustained delivery of Acy without initial burst effect confirming a strong interaction of drug with the nanoplatform sites. The preliminary antiviral data indicated that the Acyclovir loaded into the β-CD-MWCNT platform interferes with HSV-1 replication and the antireplicative effect was higher than the free drug.

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

    NASA Technical Reports Server (NTRS)

    Yang, C. C.; Oro, J.

    1971-01-01

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

  12. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    PubMed

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT.

  13. Single Nucleotide Polymorphisms and Osteoarthritis

    PubMed Central

    Wang, Ting; Liang, Yuting; Li, Hong; Li, Haibo; He, Quanze; Xue, Ying; Shen, Cong; Zhang, Chunhua; Xiang, Jingjing; Ding, Jie; Qiao, Longwei; Zheng, Qiping

    2016-01-01

    Abstract Osteoarthritis (OA) is a complex disorder characterized by degenerative articular cartilage and is largely attributed to genetic risk factors. Single nucleotide polymorphisms (SNPs) are common DNA variants that have shown promising and efficiency, compared with positional cloning, to map candidate genes of complex diseases, including OA. In this study, we aim to provide an overview of multiple SNPs from a number of genes that have recently been linked to OA susceptibility. We also performed a comprehensive meta-analysis to evaluate the association of SNP rs7639618 of double von Willebrand factor A domains (DVWA) gene with OA susceptibility. A systematic search of studies on the association of SNPs with susceptibility to OA was conducted in PubMed and Google scholar. Studies subjected to meta-analysis include human and case-control studies that met the Hardy–Weinberg equilibrium model and provide sufficient data to calculate an odds ratio (OR). A total of 9500 OA cases and 9365 controls in 7 case-control studies relating to SNP rs7639618 were included in this study and the ORs with 95% confidence intervals (CIs) were calculated. Over 50 SNPs from different genes have been shown to be associated with either hip (23), or knee (20), or both (13) OA. The ORs of these SNPs for OA and the subtypes are not consistent. As to SNP rs7639618 of DVWA, increased knee OA risk was observed in all genetic models analyzed. Specifically, people from Asian with G-allele showed significantly increased risk of knee OA (A versus G: OR = 1.28, 95% CI 1.13–1.46; AA versus GG: OR = 1.60, 95% CI 1.25–2.05; GA versus GG: OR = 1.31, 95% CI 1.18–1.44; AA versus GA+GG: OR = 1.34, 95% CI 1.12–1.61; AA+GA versus GG: OR = 1.40, 95% CI 1.19–1.64), but not in Caucasians or with hip OA. Our results suggest that multiple SNPs play different roles in the pathogenesis of OA and its subtypes; SNP rs7639618 of DVWA gene is associated with a significantly increased

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

    PubMed

    Mikulski, Damian; Szeląg, Małgorzata; Molski, Marcin

    2011-12-01

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

  15. Guanine deaminase functions as dihydropterin deaminase in the biosynthesis of aurodrosopterin, a minor red eye pigment of Drosophila.

    PubMed

    Kim, Jaekwang; Park, Sang Ick; Ahn, Chiyoung; Kim, Heuijong; Yim, Jeongbin

    2009-08-28

    Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 degrees C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (k(cat)/K(m)) for guanine (8.6 x 10(6) m(-1).s(-1)) was 860-fold higher than that for 7,8-dihydropterin (1.0 x 10(4) m(-1).s(-1)). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism. PMID:19567870

  16. Advances in targeting cyclic nucleotide phosphodiesterases.

    PubMed

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

    2014-04-01

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

  17. Extracellular nucleotides as negative modulators of immunity

    PubMed Central

    Di Virgilio, Francesco; Boeynaems, Jean-Marie; Robson, Simon C.

    2014-01-01

    Nucleotides are well known for being the universal currency of intracellular energy transactions, but over the last decade it has become clear that they are also ubiquitous extracellular messenger. In the immune system there is increasing awareness that nucleotides serve multiple roles as stimulants of lymphocyte proliferation, ROS generation, cytokine and chemokine secretion: in one word as pro-inflammatory mediators. However, although often neglected, extracellular nucleotides exert an additional more subtle function as negative modulators of immunity, or as immunedepressants. The more we understand the peculiar biochemical composition of the microenvironment generated at inflammatory sites, the more we appreciate how chronic exposure to low extracellular nucleotide levels affect immunity and inflammation. A deeper understanding of this complex network will no doubt help design more effective therapies for cancer and chronic inflammatory diseases. PMID:19628431

  18. Self-catalyzed site-specific depurination of guanine residues within gene sequences.

    PubMed

    Amosova, Olga; Coulter, Richard; Fresco, Jacques R

    2006-03-21

    A self-catalyzed, site-specific guanine-depurination activity has been found to occur in short gene sequences with a potential to form a stem-loop structure. The critical features of that catalytic intermediate are a 5'-G-T-G-G-3' loop and an adjacent 5'-T.A-3' base pair of a short duplex stem stable enough to fix the loop structure required for depurination of its 5'-G residue. That residue is uniquely depurinated with a rate some 5 orders of magnitude faster than that of random "spontaneous" depurination. In contrast, all other purine residues in the sequence depurinate at the spontaneous background rate. The reaction requires no divalent cations or other cofactors and occurs under essentially physiological conditions. Such stem-loops can form in duplex DNA under superhelical stress, and their critical sequence features have been found at numerous sites in the human genome. Self-catalyzed stem-loop-mediated depurination leading to flexible apurinic sites may therefore serve some important biological role, e.g., in nucleosome positioning, genetic recombination, or chromosome superfolding.

  19. Synthesis and biological properties of caffeic acid-PNA dimers containing guanine.

    PubMed

    Gaglione, Maria; Malgieri, Gaetano; Pacifico, Severina; Severino, Valeria; D'Abrosca, Brigida; Russo, Luigi; Fiorentino, Antonio; Messere, Anna

    2013-01-01

    Caffeic acid (CA; 3,4-dihydroxycinnamic acid) is endowed with high antioxidant activity. CA derivatives (such as amides) have gained a lot of attention due to their antioxidative, antitumor and antimicrobial properties as well as stable characteristics. Caffeoyl-peptide derivatives showed different antioxidant activity depending on the type and the sequence of amino acid used. For these reasons, we decided to combine CA with Peptide Nucleic Acid (PNA) to test whether the new PNA-CA amide derivatives would result in an improvement or gain of CA's biological (i.e., antioxidant, cytotoxic, cytoprotective) properties. We performed the synthesis and characterization of seven dimer conjugates with various combinations of nucleic acid bases and focused NMR studies on the model compound ga-CA dimer. We demonstrate that PNA dimers containing guanine conjugated to CA exhibited different biological activities depending on composition and sequence of the nucleobases. The dimer ag-CA protected HepG2, SK-B-NE(2), and C6 cells from a cytotoxic dose of hydrogen peroxide (H₂O₂). PMID:23912270

  20. Mutation at a distance caused by homopolymeric guanine repeats in Saccharomyces cerevisiae.

    PubMed

    McDonald, Michael J; Yu, Yen-Hsin; Guo, Jheng-Fen; Chong, Shin Yen; Kao, Cheng-Fu; Leu, Jun-Yi

    2016-05-01

    Mutation provides the raw material from which natural selection shapes adaptations. The rate at which new mutations arise is therefore a key factor that determines the tempo and mode of evolution. However, an accurate assessment of the mutation rate of a given organism is difficult because mutation rate varies on a fine scale within a genome. A central challenge of evolutionary genetics is to determine the underlying causes of this variation. In earlier work, we had shown that repeat sequences not only are prone to a high rate of expansion and contraction but also can cause an increase in mutation rate (on the order of kilobases) of the sequence surrounding the repeat. We perform experiments that show that simple guanine repeats 13 bp (base pairs) in length or longer (G 13+ ) increase the substitution rate 4- to 18-fold in the downstream DNA sequence, and this correlates with DNA replication timing (R = 0.89). We show that G 13+ mutagenicity results from the interplay of both error-prone translesion synthesis and homologous recombination repair pathways. The mutagenic repeats that we study have the potential to be exploited for the artificial elevation of mutation rate in systems biology and synthetic biology applications.

  1. Mutation at a distance caused by homopolymeric guanine repeats in Saccharomyces cerevisiae.

    PubMed

    McDonald, Michael J; Yu, Yen-Hsin; Guo, Jheng-Fen; Chong, Shin Yen; Kao, Cheng-Fu; Leu, Jun-Yi

    2016-05-01

    Mutation provides the raw material from which natural selection shapes adaptations. The rate at which new mutations arise is therefore a key factor that determines the tempo and mode of evolution. However, an accurate assessment of the mutation rate of a given organism is difficult because mutation rate varies on a fine scale within a genome. A central challenge of evolutionary genetics is to determine the underlying causes of this variation. In earlier work, we had shown that repeat sequences not only are prone to a high rate of expansion and contraction but also can cause an increase in mutation rate (on the order of kilobases) of the sequence surrounding the repeat. We perform experiments that show that simple guanine repeats 13 bp (base pairs) in length or longer (G 13+ ) increase the substitution rate 4- to 18-fold in the downstream DNA sequence, and this correlates with DNA replication timing (R = 0.89). We show that G 13+ mutagenicity results from the interplay of both error-prone translesion synthesis and homologous recombination repair pathways. The mutagenic repeats that we study have the potential to be exploited for the artificial elevation of mutation rate in systems biology and synthetic biology applications. PMID:27386516

  2. Electron correlation effects and density analysis of the first-order hyperpolarizability of neutral guanine tautomers.

    PubMed

    Alparone, Andrea

    2013-08-01

    Dipole moments (μ), charge distributions, and static electronic first-order hyperpolarizabilities (β(μ)) of the two lowest-energy keto tautomers of guanine (7H and 9H) were determined in the gas phase using Hartree-Fock, Møller-Plesset perturbation theory (MP2 and MP4), and DFT (PBE1PBE, B97-1, B3LYP, CAM-B3LYP) methods with Dunning's correlation-consistent aug-cc-pVDZ and d-aug-cc-pVDZ basis sets. The most stable isomer 7H exhibits a μ value smaller than that of the 9H form by a factor of ca. 3.5. The β μ value of the 9H tautomer is strongly dependent on the computational method employed, as it dramatically influences the β(μ) (9H)/β(μ) (7H) ratio, which at the highest correlated MP4/aug-cc-pVDZ level is predicted to be ca. 5. The Coulomb-attenuating hybrid exchange-correlation CAM-B3LYP method is superior to the conventional PBE1PBE, B3LYP, and B97-1 functionals in predicting the β(μ) values. Differences between the largest diagonal hyperpolarizability components were clarified through hyperpolarizability density analyses. Dipole moment and first-order hyperpolarizability are molecular properties that are potentially useful for distinguishing the 7H from the 9H tautomer.

  3. Mutation at a distance caused by homopolymeric guanine repeats in Saccharomyces cerevisiae

    PubMed Central

    McDonald, Michael J.; Yu, Yen-Hsin; Guo, Jheng-Fen; Chong, Shin Yen; Kao, Cheng-Fu; Leu, Jun-Yi

    2016-01-01

    Mutation provides the raw material from which natural selection shapes adaptations. The rate at which new mutations arise is therefore a key factor that determines the tempo and mode of evolution. However, an accurate assessment of the mutation rate of a given organism is difficult because mutation rate varies on a fine scale within a genome. A central challenge of evolutionary genetics is to determine the underlying causes of this variation. In earlier work, we had shown that repeat sequences not only are prone to a high rate of expansion and contraction but also can cause an increase in mutation rate (on the order of kilobases) of the sequence surrounding the repeat. We perform experiments that show that simple guanine repeats 13 bp (base pairs) in length or longer (G13+) increase the substitution rate 4- to 18-fold in the downstream DNA sequence, and this correlates with DNA replication timing (R = 0.89). We show that G13+ mutagenicity results from the interplay of both error-prone translesion synthesis and homologous recombination repair pathways. The mutagenic repeats that we study have the potential to be exploited for the artificial elevation of mutation rate in systems biology and synthetic biology applications. PMID:27386516

  4. The Guanine-Based Purinergic System: The Tale of An Orphan Neuromodulation

    PubMed Central

    Garozzo, Roberta; Frinchi, Monica; Fernandez-Dueñas, Víctor; Di Iorio, Patrizia; Ciccarelli, Renata; Caciagli, Francesco; Condorelli, Daniele F.; Ciruela, Francisco; Belluardo, Natale

    2016-01-01

    Guanine-based purines (GBPs) have been recently proposed to be not only metabolic agents but also extracellular signaling molecules that regulate important functions in the central nervous system. In such way, GBPs-mediated neuroprotection, behavioral responses and neuronal plasticity have been broadly described in the literature. However, while a number of these functions (i.e., GBPs neurothophic effects) have been well-established, the molecular mechanisms behind these GBPs-dependent effects are still unknown. Furthermore, no plasma membrane receptors for GBPs have been described so far, thus GBPs are still considered orphan neuromodulators. Interestingly, an intricate and controversial functional interplay between GBPs effects and adenosine receptors activity has been recently described, thus triggering the hypothesis that GBPs mechanism of action might somehow involve adenosine receptors. Here, we review recent data describing the GBPs role in the brain. We focus on the involvement of GBPs regulating neuronal plasticity, and on the new hypothesis based on putative GBPs receptors. Overall, we expect to shed some light on the GBPs world since although these molecules might represent excellent candidates for certain neurological diseases management, the lack of putative GBPs receptors precludes any high throughput screening intent for the search of effective GBPs-based drugs. PMID:27378923

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-02-01

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

  7. In vitro incorporation of LNA nucleotides.

    PubMed

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    An LNA modified nucleoside triphosphate 1 was synthesized in order to investigate its potential to act as substrate for DNA strand synthesis by polymerases. Primer extension assays for the incorporation experiments revealed that Phusion High Fidelity DNA polymerase is an efficient enzyme for incorporation of the LNA nucleotide and for extending strand to full length. It was also observed that pfu DNA polymerase could incorporate the LNA nucleotide but it failed to extend the strand to a full length product. PMID:18058567

  8. Regulation of mammalian nucleotide metabolism and biosynthesis.

    PubMed

    Lane, Andrew N; Fan, Teresa W-M

    2015-02-27

    Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer.

  9. Regulation of mammalian nucleotide metabolism and biosynthesis

    PubMed Central

    Lane, Andrew N.; Fan, Teresa W.-M.

    2015-01-01

    Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer. PMID:25628363

  10. Probing adenosine nucleotide-binding proteins with an affinity-labeled nucleotide probe and mass spectrometry.

    PubMed

    Qiu, Haibo; Wang, Yinsheng

    2007-08-01

    Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli recombinase A (RecA) and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analogue with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification, and LC-MS/MS analysis, was relatively simple, fast, and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole-cell lysates of HeLa-S3 and WM-266-4 cells.

  11. Probing adenosine nucleotide-binding proteins with an affinity labeled-nucleotide probe and mass spectrometry

    PubMed Central

    Qiu, Haibo; Wang, Yinsheng

    2008-01-01

    Mass spectrometry combined with chemical labeling strategies has become very important in biological analysis. Herein, we described the application of a biotin-conjugated acyl nucleotide for probing adenosine nucleotide-binding proteins. We demonstrated that the probe reacted specifically with the lysine residue at the nucleotide-binding site of two purified adenosine nucleotide-binding proteins, Escherichia coli RecA and Saccharomyces cerevisiae alcohol dehydrogenase-I (YADH-I). A single conjugate peptide with a specifically labeled lysine residue was identified, by using LC-MS/MS, from the tryptic digestion mixture of the reaction products of the nucleotide analog with RecA or YADH-I. The strategy, which involved labeling reaction, enzymatic digestion, affinity purification and LC-MS/MS analysis, was relatively simple, fast and straightforward. The method should be generally applicable for the identification of lysine residues at the nucleotide-binding site of other proteins. The biotin-conjugated acyl nucleotide probe also allowed for the enrichment and identification of nucleotide-binding proteins from complex protein mixtures; we showed that more than 50 adenosine nucleotide-binding proteins could be identified from the whole cell lysates of HeLa-S3 and WM-266-4 cells. PMID:17602667

  12. A homogeneous quenching resonance energy transfer assay for the kinetic analysis of the GTPase nucleotide exchange reaction.

    PubMed

    Kopra, Kari; Ligabue, Alessio; Wang, Qi; Syrjänpää, Markku; Blaževitš, Olga; Veltel, Stefan; van Adrichem, Arjan J; Hänninen, Pekka; Abankwa, Daniel; Härmä, Harri

    2014-07-01

    A quenching resonance energy transfer (QRET) assay for small GTPase nucleotide exchange kinetic monitoring is demonstrated using nanomolar protein concentrations. Small GTPases are central signaling proteins in all eukaryotic cells acting as a "molecular switches" that are active in the GTP-state and inactive in the GDP-state. GTP-loading is highly regulated by guanine nucleotide exchange factors (GEFs). In several diseases, most prominently cancer, this process in misregulated. The kinetics of the nucleotide exchange reaction reports on the enzymatic activity of the GEF reaction system and is, therefore, of special interest. We determined the nucleotide exchange kinetics using europium-labeled GTP (Eu-GTP) in the QRET assay for small GTPases. After GEF catalyzed GTP-loading of a GTPase, a high time-resolved luminescence signal was found to be associated with GTPase bound Eu-GTP, whereas the non-bound Eu-GTP fraction was quenched by soluble quencher. The association kinetics of the Eu-GTP was measured after GEF addition, whereas the dissociation kinetics could be determined after addition of unlabeled GTP. The resulting association and dissociation rates were in agreement with previously published values for H-Ras(Wt), H-Ras(Q61G), and K-Ras(Wt), respectively. The broader applicability of the QRET assay for small GTPases was demonstrated by determining the kinetics of the Ect2 catalyzed RhoA(Wt) GTP-loading. The QRET assay allows the use of nanomolar protein concentrations, as more than 3-fold signal-to-background ratio was achieved with 50 nM GTPase and GEF proteins. Thus, small GTPase exchange kinetics can be efficiently determined in a HTS compatible 384-well plate format.

  13. Comparison of the acid-base properties of ribose and 2'-deoxyribose nucleotides.

    PubMed

    Mucha, Ariel; Knobloch, Bernd; Jezowska-Bojczuk, Małgorzata; Kozłowski, Henryk; Sigel, Roland K O

    2008-01-01

    The extent to which the replacement of a ribose unit by a 2'-deoxyribose unit influences the acid-base properties of nucleotides has not hitherto been determined in detail. In this study, by potentiometric pH titrations in aqueous solution, we have measured the acidity constants of the 5'-di- and 5'-triphosphates of 2'-deoxyguanosine [i.e., of H(2)(dGDP)(-) and H(2)(dGTP)(2-)] as well as of the 5'-mono-, 5'-di-, and 5'-triphosphates of 2'-deoxyadenosine [i.e., of H(2)(dAMP)(+/-), H(2)(dADP)(-), and H(2)(dATP)(2-)]. These 12 acidity constants (of the 56 that are listed) are compared with those of the corresponding ribose derivatives (published data) measured under the same experimental conditions. The results show that all protonation sites in the 2'-deoxynucleotides are more basic than those in their ribose counterparts. The influence of the 2'-OH group is dependent on the number of 5'-phosphate groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is most significantly enhanced (by about 0.2 pK units), while the effect on the phosphate groups and the N1H or N1H(+) sites is less pronounced but clearly present. In addition, (1)H NMR chemical shift change studies in dependence on pD in D(2)O have been carried out for the dAMP, dADP, and dATP systems, which confirmed the results from the potentiometric pH titrations and showed the nucleotides to be in their anti conformations. Overall, our results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact basis for the calculation, determination, and understanding of perturbed pK(a) values in DNAzymes and ribozymes, as needed for the delineation of acid-base mechanisms in catalysis.

  14. Role of human hypoxanthine guanine phosphoribosyltransferase in activation of the antiviral agent T-705 (favipiravir).

    PubMed

    Naesens, Lieve; Guddat, Luke W; Keough, Dianne T; van Kuilenburg, André B P; Meijer, Judith; Vande Voorde, Johan; Balzarini, Jan

    2013-10-01

    6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

  15. Deciphering the photochemical mechanisms describing the UV-induced processes occurring in solvated guanine monophosphate

    NASA Astrophysics Data System (ADS)

    Altavilla, Salvatore; Segarra-Martí, Javier; Nenov, Artur; Conti, Irene; Rivalta, Ivan; Garavelli, Marco

    2015-04-01

    The photophysics and photochemistry of water-solvated guanine monophosphate (GMP) are here characterized by means of a multireference quantum-chemical/molecular mechanics theoretical approach (CASPT2//CASSCF/AMBER) in order to elucidate the main photo-processes occurring upon UV-light irradiation. The effect of the solvent and of the phosphate group on the energetics and structural features of this system are evaluated for the first time employing high-level ab initio methods and thoroughly compared to those in vacuo previously reported in the literature and to the experimental evidence to assess to which extent they influence the photoinduced mechanisms. Solvated electronic excitation energies of solvated GMP at the Franck-Condon (FC) region show a red shift for the ππ* La and Lb states, whereas the energy of the oxygen lone-pair nπ* state is blue-shifted. The main photoinduced decay route is promoted through a ring-puckering motion along the bright lowest-lying La state towards a conical intersection (CI) with the ground state, involving a very shallow stationary point along the minimum energy pathway in contrast to the barrierless profile found in gas-phase, the point being placed at the end of the minimum energy path (MEP) thus endorsing its ultrafast deactivation in accordance with time-resolved transient and photoelectron spectroscopy experiments. The role of the nπ* state in the solvated system is severely diminished as the crossings with the initially populated La state and also with the Lb state are placed too high energetically to partake prominently in the deactivation photo-process. The proposed mechanism present in solvated and in vacuo DNA/RNA chromophores validates the intrinsic photostability mechanism through CI-mediated non-radiative processes accompanying the bright excited-state population towards the ground state and subsequent relaxation back to the FC region.

  16. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    SciTech Connect

    Jett, J.

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  17. Structural Landscape of the Proline-Rich Domain of Sos1 Nucleotide Exchange Factor

    PubMed Central

    McDonald, Caleb B.; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C.; Deegan, Brian J.; Seldeen, Kenneth L.; Lednev, Igor K.; Farooq, Amjad

    2013-01-01

    Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. PMID:23528987

  18. Structural landscape of the proline-rich domain of Sos1 nucleotide exchange factor.

    PubMed

    McDonald, Caleb B; Bhat, Vikas; Kurouski, Dmitry; Mikles, David C; Deegan, Brian J; Seldeen, Kenneth L; Lednev, Igor K; Farooq, Amjad

    2013-01-01

    Despite its key role in mediating a plethora of cellular signaling cascades pertinent to health and disease, little is known about the structural landscape of the proline-rich (PR) domain of Sos1 guanine nucleotide exchange factor. Herein, using a battery of biophysical tools, we provide evidence that the PR domain of Sos1 is structurally disordered and adopts an extended random coil-like conformation in solution. Of particular interest is the observation that while chemical denaturation of PR domain results in the formation of a significant amount of polyproline II (PPII) helices, it has little or negligible effect on its overall size as measured by its hydrodynamic radius. Our data also show that the PR domain displays a highly dynamic conformational basin in agreement with the knowledge that the intrinsically unstructured proteins rapidly interconvert between an ensemble of conformations. Collectively, our study provides new insights into the conformational equilibrium of a key signaling molecule with important consequences on its physiological function. PMID:23528987

  19. Echinacoside induces apoptotic cancer cell death by inhibiting the nucleotide pool sanitizing enzyme MTH1

    PubMed Central

    Dong, Liwei; Wang, Hongge; Niu, Jiajing; Zou, Mingwei; Wu, Nuoting; Yu, Debin; Wang, Ye; Zou, Zhihua

    2015-01-01

    Inhibition of the nucleotide pool sanitizing enzyme MTH1 causes extensive oxidative DNA damages and apoptosis in cancer cells and hence may be used as an anticancer strategy. As natural products have been a rich source of medicinal chemicals, in the present study, we used the MTH1-catalyzed enzymatic reaction as a high-throughput in vitro screening assay to search for natural compounds capable of inhibiting MTH1. Echinacoside, a compound derived from the medicinal plants Cistanche and Echinacea, effectively inhibited the catalytic activity of MTH1 in an in vitro assay. Treatment of various human cancer cell lines with Echinacoside resulted in a significant increase in the cellular level of oxidized guanine (8-oxoguanine), while cellular reactive oxygen species level remained unchanged, indicating that Echinacoside also inhibited the activity of cellular MTH1. Consequently, Echinacoside treatment induced an immediate and dramatic increase in DNA damage markers and upregulation of the G1/S-CDK inhibitor p21, which were followed by marked apoptotic cell death and cell cycle arrest in cancer but not in noncancer cells. Taken together, these studies identified a natural compound as an MTH1 inhibitor and suggest that natural products can be an important source of anticancer agents. PMID:26677335

  20. Nucleotide Binding Preference of the Monofunctional Platinum Anticancer-Agent Phenanthriplatin.

    PubMed

    Riddell, Imogen A; Johnstone, Timothy C; Park, Ga Young; Lippard, Stephen J

    2016-05-23

    The monofunctional platinum anticancer agent phenanthriplatin generates covalent adducts with the purine bases guanine and adenine. Preferential nucleotide binding was investigated by using a polymerase stop assay and linear DNA amplification with a 163-base pair DNA double helix. Similarly to cisplatin, phenanthriplatin forms the majority of adducts at guanosine residues, but significant differences in both the number and position of platination sites emerge when comparing results for the two complexes. Notably, the monofunctional complex generates a greater number of polymerase-halting lesions at adenosine residues than does cisplatin. Studies with 9-methyladenine reveal that, under abiological conditions, phenanthriplatin binds to the N(1) or N(7) position of 9-methyladenine in approximately equimolar amounts. By contrast, comparable reactions with 9-methylguanine afforded only the N(7) -bound species. Both of the 9-methyladenine linkage isomers (N(1) and N(7) ) exist as two diastereomeric species, arising from hindered rotation of the aromatic ligands about their respective platinum-nitrogen bonds. Eyring analysis of rate constants extracted from variable-temperature NMR spectroscopic data revealed that the activation energies for ligand rotation in the N(1) -bound platinum complex and the N(7) -linkage isomers are comparable. Finally, a kinetic analysis indicated that phenanthriplatin reacts more rapidly, by a factor of eight, with 9-methylguanine than with 9-methyladenine, suggesting that the distribution of lesions formed on double-stranded DNA is kinetically controlled. In addition, implications for the potent anticancer activity of phenanthriplatin are discussed herein.

  1. Insights into nucleotide recognition by cell division protein FtsZ from a mant-GTP competition assay and molecular dynamics.

    PubMed

    Schaffner-Barbero, Claudia; Gil-Redondo, Rubén; Ruiz-Avila, Laura B; Huecas, Sonia; Läppchen, Tilman; den Blaauwen, Tanneke; Diaz, J Fernando; Morreale, Antonio; Andreu, Jose M

    2010-12-14

    Essential cell division protein FtsZ forms the bacterial cytokinetic ring and is a target for new antibiotics. FtsZ monomers bind GTP and assemble into filaments. Hydrolysis to GDP at the association interface between monomers leads to filament disassembly. We have developed a homogeneous competition assay, employing the fluorescence anisotropy change of mant-GTP upon binding to nucleotide-free FtsZ, which detects compounds binding to the nucleotide site in FtsZ monomers and measures their affinities within the millimolar to 10 nM range. We have employed this method to determine the apparent contributions of the guanine, ribose, and the α-, β-, and γ-phosphates to the free energy change of nucleotide binding. Similar relative contributions have also been estimated through molecular dynamics and binding free energy calculations, employing the crystal structures of FtsZ-nucleotide complexes. We find an energetically dominant contribution of the β-phosphate, comparable to the whole guanosine moiety. GTP and GDP bind with similar observed affinity to FtsZ monomers. Loss of the regulatory γ-phosphate results in a predicted accommodation of GDP which has not been observed in the crystal structures. The binding affinities of a series of C8-substituted GTP analogues, known to inhibit FtsZ but not eukaryotic tubulin assembly, correlate with their inhibitory capacity on FtsZ polymerization. Our methods permit testing of FtsZ inhibitors targeting its nucleotide site, as well as compounds from virtual screening of large synthetic libraries. Our results give insight into the FtsZ-nucleotide interactions, which could be useful in the rational design of new inhibitors, especially GTP phosphate mimetics.

  2. Exploration of cone cyclic nucleotide-gated channel-interacting proteins using affinity purification and mass spectrometry.

    PubMed

    Ding, Xi-Qin; Matveev, Alexander; Singh, Anil; Komori, Naoka; Matsumoto, Hiroyuki

    2014-01-01

    Photopic (cone) vision essential for color sensation, central vision, and visual acuity is mediated by the activation of photoreceptor cyclic nucleotide-gated (CNG) channels. Naturally occurring mutations in the cone channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. This work investigated the functional modulation of cone CNG channel by exploring the channel-interacting proteins. Retinal protein extracts prepared from cone-dominant Nrl (- / -) mice were used in CNGA3 antibody affinity purification, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. The peptide mass fingerprinting of the tryptic digests and database search identified a number of proteins including spectrin alpha-2, ATPase (Na(+)/K(+) transporting) alpha-3, alpha and beta subunits of ATP synthase (H(+) transporting, mitochondrial F1 complex), and alpha-2 subunit of the guanine nucleotide-binding protein. In addition, the affinity-binding assays demonstrated an interaction between cone CNG channel and calmodulin but not cone Na(+)/Ca(2+)-K(+) exchanger in the mouse retina. Results of this study provide insight into our understanding of cone CNG channel-interacting proteins and the functional modulations.

  3. Role of cysteine residues in the redox-regulated oligomerization and nucleotide binding to EhRabX3.

    PubMed

    Chandra, Mintu; Datta, Sunando

    2016-08-01

    The enteric protozoan parasite, Entamoeba histolytica, an etiological agent of amebiasis, is involved in the adhesion and destruction of human tissues. Worldwide, the parasite causes about 50 million cases of amebiasis and 100,000 deaths annually. EhRabX3, a unique amoebic Rab GTPase with tandem G-domains, possesses an unusually large number of cysteine residues in its N-terminal domain. Crystal structure of EhRabX3 revealed an intra-molecular disulfide bond between C39 and C163 which is critical for maintaining the 3-dimensional architecture and biochemical function of this protein. The remaining six cysteine residues were found to be surface exposed and predicted to be involved in inter-molecular disulfide bonds. In the current study, using biophysical and mutational approaches, we have investigated the role of the cysteine residues in the assembly of EhRabX3 oligomer. The self-association of EhRabX3 is found to be redox sensitive, in vitro. Furthermore, the oligomeric conformation of EhRabX3 failed to bind and exchange the guanine nucleotide, indicating structural re-organization of the active site. Altogether, our results provide valuable insights into the redox-dependent oligomerization of EhRabX3 and its implication on nucleotide binding. PMID:27485554

  4. Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

    PubMed Central

    Suh, Hye-Young; Lee, Dong-Won; Lee, Kwang-Hoon; Ku, Bonsu; Choi, Sung-Jin; Woo, Jae-Sung; Kim, Yeon-Gil; Oh, Byung-Ha

    2010-01-01

    GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein. PMID:19942850

  5. Nucleotide `maps' of digests of deoxyribonucleic acid

    PubMed Central

    Murray, K.

    1970-01-01

    Various digests of 32P-labelled DNA were examined by two-dimensional ionophoresis on cellulose acetate and DEAE-cellulose paper. The products from digestion with pancreatic deoxyribonuclease and Neurospora crassa endonuclease were qualitatively closely similar, but very complex, and were used to investigate the mapping behaviour of nucleotides in various ionophoretic systems. Ionophoresis on DEAE-cellulose paper in triethylamine carbonate, pH 9.7, followed by ionophoresis in the second dimension at pH1.9 gave high resolution of nucleotides in very complex mixtures and permitted the fractionation of larger quantities than is possible on cellulose acetate. High resolution of nucleotides in compact spots was obtained with two-dimensional ionophoresis on cellulose acetate and AE-cellulose paper, a system that is a useful supplement to those based on DEAE-cellulose paper. ImagesPLATE 7PLATE 1PLATE 2PLATE 3PLATE 4PLATE 5PLATE 6 PMID:5476726

  6. Regulation of Ion Channels by Pyridine Nucleotides

    PubMed Central

    Kilfoil, Peter J.; Tipparaju, Srinivas M.; Barski, Oleg A.; Bhatnagar, Aruni

    2014-01-01

    Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion–transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide–binding β-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP+ to Kvβ removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K+ transporters. These nucleotides also have been shown to modify the activity of the plasma membrane KATP channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit—the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP+ metabolite, NAADP+, regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias. PMID:23410881

  7. Multisite-specific archaeosine tRNA-guanine transglycosylase (ArcTGT) from Thermoplasma acidophilum, a thermo-acidophilic archaeon.

    PubMed

    Kawamura, Takuya; Hirata, Akira; Ohno, Satoshi; Nomura, Yuichiro; Nagano, Tomoko; Nameki, Nobukazu; Yokogawa, Takashi; Hori, Hiroyuki

    2016-02-29

    Archaeosine (G(+)), which is found only at position 15 in many archaeal tRNA, is formed by two steps, the replacement of the guanine base with preQ0 by archaeosine tRNA-guanine transglycosylase (ArcTGT) and the subsequent modification of preQ0 to G(+) by archaeosine synthase. However, tRNA(Leu) from Thermoplasma acidophilum, a thermo-acidophilic archaeon, exceptionally has two G(+)13 and G(+)15 modifications. In this study, we focused on the biosynthesis mechanism of G(+)13 and G(+)15 modifications in this tRNA(Leu). Purified ArcTGT from Pyrococcus horikoshii, for which the tRNA recognition mechanism and structure were previously characterized, exchanged only the G15 base in a tRNA(Leu) transcript with (14)C-guanine. In contrast, T. acidophilum cell extract exchanged both G13 and G15 bases. Because T. acidophilum ArcTGT could not be expressed as a soluble protein in Escherichia coli, we employed an expression system using another thermophilic archaeon, Thermococcus kodakarensis. The arcTGT gene in T. kodakarensis was disrupted, complemented with the T. acidophilum arcTGT gene, and tRNA(Leu) variants were expressed. Mass spectrometry analysis of purified tRNA(Leu) variants revealed the modifications of G(+)13 and G(+)15 in the wild-type tRNA(Leu). Thus, T. acidophilum ArcTGT has a multisite specificity and is responsible for the formation of both G(+)13 and G(+)15 modifications.

  8. Combined Monte Carlo and quantum mechanics study of the hydration of the guanine-cytosine base pair.

    PubMed

    Coutinho, Kaline; Ludwig, Valdemir; Canuto, Sylvio

    2004-06-01

    We present a computer simulation study of the hydration of the guanine-cytosine (GC) hydrogen-bonded complex. Using first principles density-functional theory, with gradient-corrected exchange-correlation and Monte Carlo simulation, we include thermal contribution, structural effects, solvent polarization, and the water-water and water-GC hydrogen bond interaction to show that the GC interaction in an aqueous environment is weakened to about 70% of the value obtained for an isolated complex. We also analyze in detail the preferred hydration sites of the GC pair and show that on the average it makes around five hydrogen bonds with water.

  9. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells.

    PubMed

    Park, Joonghoon; Park, Jong Woo; Oh, Hawmok; Maria, Fernanda S; Kang, Jaeku; Tian, Xiuchun

    2016-01-01

    Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed

  10. Gene-Specific Assessment of Guanine Oxidation as an Epigenetic Modulator for Cardiac Specification of Mouse Embryonic Stem Cells

    PubMed Central

    Park, Joonghoon; Park, Jong Woo; Oh, Hawmok; Maria, Fernanda S.; Kang, Jaeku; Tian, Xiuchun

    2016-01-01

    Epigenetics have essential roles in development and human diseases. Compared to the complex histone modifications, epigenetic changes on mammalian DNA are as simple as methylation on cytosine. Guanine, however, can be oxidized as an epigenetic change which can undergo base-pair transversion, causing a genetic difference. Accumulating evidence indicates that reactive oxygen species (ROS) are important signaling molecules for embryonic stem cell (ESC) differentiation, possibly through transient changes on genomic DNA such as 7,8-dihydro-8-oxoguanine (8-oxoG). Technical limitations on detecting such DNA modifications, however, restrict the investigation of the role of 8-oxoG in ESC differentiation. Here, we developed a Hoogsteen base pairing-mediated PCR-sequencing assay to detect 8-oxoG lesions that can subsequently cause G to T transversions during PCR. We then used this assay to assess the epigenetic and transient 8-oxoG formation in the Tbx5 gene of R1 mouse ESCs subjected to oxidative stress by removing 2-mercaptoethanol (2ME) from the culture media. To our surprise, significantly higher numbers of 8-oxoG-mediated G∙C to C∙G transversion, not G∙C to T∙A, were detected at 7th and 9th base position from the transcription start site of exon 1 of Tbx5 in ESCs in the (-)2ME than (+)2ME group (p < 0.05). This was consistent with the decrease in the amount of amplifiable of DNA harboring the 8-oxoG lesions at the Tbx5 promoter region in the oxidative stressed ESCs. The ESCs responded to oxidative stress, possibly through the epigenetic effects of guanine oxidation with decreased proliferation (p < 0.05) and increased formation of beating embryoid bodies (EBs; p < 0.001). Additionally, the epigenetic changes of guanine induced up-regulation of Ogg1 and PolB, two base excision repairing genes for 8-oxoG, in ESCs treated with (-)2ME (p < 0.01). Together, we developed a gene-specific and direct quantification assay for guanine oxidation. Using oxidative stressed

  11. The role of topoisomerase I in suppressing genome instability associated with a highly transcribed guanine-rich sequence is not restricted to preventing RNA:DNA hybrid accumulation

    PubMed Central

    Yadav, Puja; Owiti, Norah; Kim, Nayun

    2016-01-01

    Highly transcribed guanine-run containing sequences, in Saccharomyces cerevisiae, become unstable when topoisomerase I (Top1) is disrupted. Topological changes, such as the formation of extended RNA:DNA hybrids or R-loops or non-canonical DNA structures including G-quadruplexes has been proposed as the major underlying cause of the transcription-linked genome instability. Here, we report that R-loop accumulation at a guanine-rich sequence, which is capable of assembling into the four-stranded G4 DNA structure, is dependent on the level and the orientation of transcription. In the absence of Top1 or RNase Hs, R-loops accumulated to substantially higher extent when guanine-runs were located on the non-transcribed strand. This coincides with the orientation where higher genome instability was observed. However, we further report that there are significant differences between the disruption of RNase Hs and Top1 in regards to the orientation-specific elevation in genome instability at the guanine-rich sequence. Additionally, genome instability in Top1-deficient yeasts is not completely suppressed by removal of negative supercoils and further aggravated by expression of mutant Top1. Together, our data provide a strong support for a function of Top1 in suppressing genome instability at the guanine-run containing sequence that goes beyond preventing the transcription-associated RNA:DNA hybrid formation. PMID:26527723

  12. Exploring the correlation between the sequence composition of the nucleotide binding G5 loop of the FeoB GTPase domain (NFeoB) and intrinsic rate of GDP release.

    PubMed

    Guilfoyle, Amy P; Deshpande, Chandrika N; Schenk, Gerhard; Maher, Megan J; Jormakka, Mika

    2014-01-01

    GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

  13. A syn–anti conformational difference allows SRSF2 to recognize guanines and cytosines equally well

    PubMed Central

    Daubner, Gerrit M; Cléry, Antoine; Jayne, Sandrine; Stevenin, James; Allain, Frédéric H-T

    2012-01-01

    SRSF2 (SC35) is a key player in the regulation of alternative splicing events and binds degenerated RNA sequences with similar affinity in nanomolar range. We have determined the solution structure of the SRSF2 RRM bound to the 5′-UCCAGU-3′ and 5′-UGGAGU-3′ RNA, both identified as SRSF2 binding sites in the HIV-1 tat exon 2. RNA recognition is achieved through a novel sandwich-like structure with both termini forming a positively charged cavity to accommodate the four central nucleotides. To bind both RNA sequences equally well, SRSF2 forms a nearly identical network of intermolecular interactions by simply flipping the bases of the two consecutive C or G nucleotides into either anti or syn conformation. We validate this unusual mode of RNA recognition functionally by in-vitro and in-vivo splicing assays and propose a 5′-SSNG-3′ (S=C/G) high-affinity binding consensus sequence for SRSF2. In conclusion, in addition to describe for the first time the RNA recognition mode of SRSF2, we provide the precise consensus sequence to identify new putative binding sites for this splicing factor. PMID:22002536

  14. Speciation of oxaliplatin adducts with DNA nucleotides.

    PubMed

    Zayed, Aref; Jones, George D D; Reid, Helen J; Shoeib, Tamer; Taylor, Sarah E; Thomas, Anne L; Wood, Joanna P; Sharp, Barry L

    2011-10-01

    This paper describes a set of fast and selective high performance liquid chromatography (HPLC) methods coupled to electro-spray ionisation linear ion trap mass spectrometry (ESI-MS), sector-field inductively coupled plasma mass spectrometry (SF-ICP-MS) and UV detection for in vitro studies of the bifunctional adducts of oxaliplatin with mono-nucleotides, di-nucleotides and cellular DNA. The stationary phases and the optimised conditions used for each separation are discussed. Interaction of oxaliplatin with A and G mono-nucleotides resulted in the formation of five bifunctional platinum diaminocyclohexane (DACHPt) adducts. These were two isomers of the A-DACHPt-A and A-DACHPt-G adducts, and one G-DACHPt-G adduct, as confirmed by MS/MS spectra obtained by collision induced dissociation. These adducts were also characterised by UV absorption data and SF-ICP-MS elemental (195)Pt and (31)P signals. Further, interaction of oxaliplatin with AG and GG di-nucleotides resulted in the formation of three adducts: DACHPt-GG and two isomers of the DACHPt-AG adduct, as confirmed by ESI-MS and the complementary data obtained by UV and SF-ICP-MS. Finally, a very sensitive LC-ICP-MS method for the quantification of oxaliplatin GG intra-strand adducts (DACHPt-GG) was developed and used for monitoring the in vitro formation and repair of these adducts in human colorectal cancer cells. The method detection limit was 0.14 ppb Pt which was equivalent to 0.22 Pt adduct per 10(6) nucleotides based on a 10 μg DNA sample. This detection limit makes this method suitable for in vivo assessment of DACHPt-GG adducts in patients undergoing oxaliplatin chemotherapy.

  15. The neurotrophin-3 receptor TrkC directly phosphorylates and activates the nucleotide exchange factor Dbs to enhance Schwann cell migration

    PubMed Central

    Yamauchi, Junji; Chan, Jonah R.; Miyamoto, Yuki; Tsujimoto, Gozoh; Shooter, Eric M.

    2005-01-01

    During the development of the peripheral nervous system, Schwann cells, the myelin-forming glia, migrate along axons before initiating myelination. We previously demonstrated that endogenous neurotrophin-3 (NT3) acting through the TrkC tyrosine kinase receptor enhances migration of premyelinating Schwann cells. This signaling pathway is mediated by the c-Jun N-terminal kinase (JNK) cascade regulated by the Rho GTPases Rac1 and Cdc42. However, missing is the link between TrkC and the GTPases. Here, we show that a guanine-nucleotide exchange factor (GEF), Dbl's big sister (Dbs), couples with TrkC to activate Cdc42 in Schwann cells. Furthermore, TrkC directly phosphorylates Dbs, thereby inducing the Cdc42-GEF activity. Taken together, activation of TrkC triggers Schwann cell migration by regulating Dbs upon direct tyrosine phosphorylation, providing a mechanism whereby a membrane receptor tyrosine kinase can induce the activation of Rho GTPase-GEFs. PMID:15758069

  16. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    SciTech Connect

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; Grimwood, Jane; Grigoriev, Igor V.; Van Etten, James L.; Keeling, Patrick J.

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.

  17. Measurements of single nucleotide electronic states as nanoelectronic fingerprints for identification of DNA nucleobases, their protonated and unprotonated states, isomers, and tautomers.

    PubMed

    Ribot, Josep Casamada; Chatterjee, Anushree; Nagpal, Prashant

    2015-04-16

    Several nanoelectronic techniques have been explored to distinguish the sequence of nucleic acids in DNA macromolecules. Identification of unique electronic signatures using nanopore conductance, tunneling spectroscopy, or other nanoelectronic techniques depends on electronic states of the DNA nucleotides. While several experimental and computational studies have focused on interaction of nucleobases with different substrates, the effect of nucleic acid biochemistry on its electronic properties has been largely unexplored. Here, we present correlated measurements of frontier molecular orbitals and higher-order electronic states for four DNA nucleobases (adenine, cytosine, thymine, and guanine), and first-principle quantum chemical density functional theoretical (DFT) computations. Using different pH conditions in our experiments, we show that small changes in the biochemical state of these nucleic acids strongly affect the intrinsic electronic structure, measured using scanning tunneling spectroscopy (STS). In our experimental measurements and computations, significant differences were observed between the position of frontier orbitals and higher-energy states between protonated and unprotonated nucleic acids, isomers, and different keto-enol tautomer's formed in these nucleotides, leading to their facile identification. Furthermore, we show unique "electronic fingerprints" for all nucleotides (A, G, T, C) using STS, with most distinct states identified at acidic pH. These results can have important implications for identification of nucleic acid sequences in DNA molecules using a high-throughput nanoelectronic identification technique.

  18. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence*

    PubMed Central

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L.; Embrey, Kevin J.; Golovanov, Alexander P.

    2016-01-01

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly 15N-labeled Ras as well as [13C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions. PMID:26565026

  19. Nitrogen K-edge X-ray absorption near edge structure (XANES) spectra of purine-containing nucleotides in aqueous solution

    SciTech Connect

    Shimada, Hiroyuki; Fukao, Taishi; Minami, Hirotake; Ukai, Masatoshi; Fujii, Kentaro; Yokoya, Akinari; Fukuda, Yoshihiro; Saitoh, Yuji

    2014-08-07

    The N K-edge X-ray absorption near edge structure (XANES) spectra of the purine-containing nucleotide, guanosine 5{sup ′}-monophosphate (GMP), in aqueous solution are measured under various pH conditions. The spectra show characteristic peaks, which originate from resonant excitations of N 1s electrons to π* orbitals inside the guanine moiety of GMP. The relative intensities of these peaks depend on the pH values of the solution. The pH dependence is explained by the core-level shift of N atoms at specific sites caused by protonation and deprotonation. The experimental spectra are compared with theoretical spectra calculated by using density functional theory for GMP and the other purine-containing nucleotides, adenosine 5{sup ′}-monophosphate, and adenosine 5{sup ′}-triphosphate. The N K-edge XANES spectra for all of these nucleotides are classified by the numbers of N atoms with particular chemical bonding characteristics in the purine moiety.

  20. Monitoring Ras Interactions with the Nucleotide Exchange Factor Son of Sevenless (Sos) Using Site-specific NMR Reporter Signals and Intrinsic Fluorescence.

    PubMed

    Vo, Uybach; Vajpai, Navratna; Flavell, Liz; Bobby, Romel; Breeze, Alexander L; Embrey, Kevin J; Golovanov, Alexander P

    2016-01-22

    The activity of Ras is controlled by the interconversion between GTP- and GDP-bound forms partly regulated by the binding of the guanine nucleotide exchange factor Son of Sevenless (Sos). The details of Sos binding, leading to nucleotide exchange and subsequent dissociation of the complex, are not completely understood. Here, we used uniformly (15)N-labeled Ras as well as [(13)C]methyl-Met,Ile-labeled Sos for observing site-specific details of Ras-Sos interactions in solution. Binding of various forms of Ras (loaded with GDP and mimics of GTP or nucleotide-free) at the allosteric and catalytic sites of Sos was comprehensively characterized by monitoring signal perturbations in the NMR spectra. The overall affinity of binding between these protein variants as well as their selected functional mutants was also investigated using intrinsic fluorescence. The data support a positive feedback activation of Sos by Ras·GTP with Ras·GTP binding as a substrate for the catalytic site of activated Sos more weakly than Ras·GDP, suggesting that Sos should actively promote unidirectional GDP → GTP exchange on Ras in preference of passive homonucleotide exchange. Ras·GDP weakly binds to the catalytic but not to the allosteric site of Sos. This confirms that Ras·GDP cannot properly activate Sos at the allosteric site. The novel site-specific assay described may be useful for design of drugs aimed at perturbing Ras-Sos interactions.

  1. Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between the GTP and the GDP states

    PubMed Central

    Yatime, Laure; Mechulam, Yves; Blanquet, Sylvain; Schmitt, Emmanuelle

    2007-01-01

    Initiation of translation in eukaryotes and in archaea involves eukaryotic/archaeal initiation factor (e/aIF)1 and the heterotrimeric initiation factor e/aIF2. In its GTP-bound form, e/aIF2 provides the initiation complex with Met–tRNAiMet. After recognition of the start codon by initiator tRNA, e/aIF1 leaves the complex. Finally, e/aIF2, now in a GDP-bound form, loses affinity for Met–tRNAiMet and dissociates from the ribosome. Here, we report a 3D structure of an aIF2 heterotrimer from the archeon Sulfolobus solfataricus obtained in the presence of GDP. Our report highlights how the two-switch regions involved in formation of the tRNA-binding site on subunit γ exchange conformational information with α and β. The zinc-binding domain of β lies close to the guanine nucleotide and directly contacts the switch 1 region. As a result, switch 1 adopts a not yet described conformation. Moreover, unexpectedly for a GDP-bound state, switch 2 has the “ON” conformation. The stability of these conformations is accounted for by a ligand, most probably a phosphate ion, bound near the nucleotide binding site. The structure suggests that this GDP–inorganic phosphate (Pi) bound state of aIF2 may be proficient for tRNA binding. Recently, it has been proposed that dissociation of eIF2 from the initiation complex is closely coupled to that of Pi from eIF2γ upon start codon recognition. The nucleotide state of aIF2 shown here is indicative of a similar mechanism in archaea. Finally, we consider the possibility that release of Pi takes place after e/aIF2γ has been informed of e/aIF1 dissociation by e/aIF2β. PMID:18000047

  2. Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa.

    PubMed

    Goupil, Serge; Maréchal, Loïze; El Hajj, Hassan; Tremblay, Marie-Ève; Richard, François J; Leclerc, Pierre

    2016-01-01

    In mammals, adenosine 3', 5'-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE) whose role is to degrade cAMP into 5'-AMP. The PDE superfamily is subdivided into 11 families (PDE1 to 11), which act on either cAMP or cGMP, or on both cAMP and cGMP although with different enzymatic properties. PDE10, which is more effective on cAMP than cGMP, has been known for almost 15 years and is mostly studied in the brain where it is associated with neurological disorders. Although a high level of PDE10A gene expression is observed in the testis, information on the identity of the isoforms or on the cell type that express the PDE10 protein is lacking. The objective of this study was to identify the PDE10A isoforms expressed in the testis and germ cells, and to determine the presence and localization of PDE10A in mature spermatozoa. As a sub-objective, since PDE10A transcript variants were reported strictly through analyses of bovine genomic sequence, we also wanted to determine the nucleotide and amino acid sequences by experimental evidence. Using RT-PCR, 5'- and 3'-RACE approaches we clearly show that PDE10A transcript variants X3 and X5 are expressed in bovine testis as well as in primary spermatocytes and spermatids. We also reveal using a combination of immunological techniques and proteomics analytical tools that the PDE10A isoform X4 is present in the area of the developing acrosome of spermatids and of the acrosome of mature spermatozoa. PMID:27548062

  3. Identification and Localization of the Cyclic Nucleotide Phosphodiesterase 10A in Bovine Testis and Mature Spermatozoa

    PubMed Central

    Goupil, Serge; Maréchal, Loïze; El Hajj, Hassan; Tremblay, Marie-Ève; Richard, François J.

    2016-01-01

    In mammals, adenosine 3’, 5’-cyclic monophosphate (cAMP) is known to play highly important roles in sperm motility and acrosomal exocytosis. It is known to act through protein phosphorylation via PRKA and through the activation of guanine nucleotide exchange factors like EPAC. Sperm intracellular cAMP levels depend on the activity of adenylyl cyclases, mostly SACY, though transmembrane-containing adenylyl cyclases are also present, and on the activity of cyclic nucleotide phosphodiesterases (PDE) whose role is to degrade cAMP into 5’-AMP. The PDE superfamily is subdivided into 11 families (PDE1 to 11), which act on either cAMP or cGMP, or on both cAMP and cGMP although with different enzymatic properties. PDE10, which is more effective on cAMP than cGMP, has been known for almost 15 years and is mostly studied in the brain where it is associated with neurological disorders. Although a high level of PDE10A gene expression is observed in the testis, information on the identity of the isoforms or on the cell type that express the PDE10 protein is lacking. The objective of this study was to identify the PDE10A isoforms expressed in the testis and germ cells, and to determine the presence and localization of PDE10A in mature spermatozoa. As a sub-objective, since PDE10A transcript variants were reported strictly through analyses of bovine genomic sequence, we also wanted to determine the nucleotide and amino acid sequences by experimental evidence. Using RT-PCR, 5’- and 3’-RACE approaches we clearly show that PDE10A transcript variants X3 and X5 are expressed in bovine testis as well as in primary spermatocytes and spermatids. We also reveal using a combination of immunological techniques and proteomics analytical tools that the PDE10A isoform X4 is present in the area of the developing acrosome of spermatids and of the acrosome of mature spermatozoa. PMID:27548062

  4. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    PubMed

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  5. Formation Pathways of a Guanine-Quadruplex DNA Revealed by Molecular Dynamics and Thermodynamic Analysis of the Substates

    PubMed Central

    Štefl, Richard; Cheatham, Thomas E.; Špačková, Nad'a; Fadrná, Eva; Berger, Imre; Koča, Jaroslav; Šponer, Jiří

    2003-01-01

    The formation of a cation-stabilized guanine quadruplex (G-DNA) stem is an exceptionally slow process involving complex kinetics that has not yet been characterized at atomic resolution. Here, we investigate the formation of a parallel stranded G-DNA stem consisting of four strands of d(GGGG) using molecular dynamics simulations with explicit inclusion of counterions and solvent. Due to the limitations imposed by the nanosecond timescale of the simulations, rather than watching for the spontaneous formation of G-DNA, our approach probes the stability of possible supramolecular intermediates (including two-, three-, and four-stranded assemblies with out-of-register basepairing between guanines) on the formation pathway. The simulations suggest that “cross-like” two-stranded assemblies may serve as nucleation centers in the initial formation of parallel stranded G-DNA quadruplexes, proceeding through a series of rearrangements involving trapping of cations, association of additional strands, and progressive slippage of strands toward the full stem. To supplement the analysis, approximate free energies of the models are obtained with explicit consideration of the integral cations. The approach applied here serves as a prototype for qualitatively investigating other G-DNA molecules using molecular dynamics simulation and free-energy analysis. PMID:12944293

  6. Quadruplexes of human telomere dG{sub 3}(TTAG{sub 3}){sub 3} sequences containing guanine abasic sites

    SciTech Connect

    Skolakova, Petra; Bednarova, Klara; Vorlickova, Michaela; Sagi, Janos

    2010-08-20

    Research highlights: {yields} Loss of a guanine base does not hinder the formation of G-quadruplex of human telomere sequence. {yields} Each depurination strongly destabilizes the quadruplex of dG{sub 3}(TTAG{sub 3}){sub 3} in NaCl and KCl. {yields} Conformational change of the abasic analogs of dG{sub 3}(TTAG{sub 3}){sub 3} is inhibited in KCl. {yields} The effects abasic sites may affect telomere-end structures in vivo. -- Abstract: This study was performed to evaluate how the loss of a guanine base affects the structure and stability of the three-tetrad G-quadruplex of 5'-dG{sub 3}(TTAG{sub 3}){sub 3}, the basic quadruplex-forming unit of the human telomere DNA. None of the 12 possible abasic sites hindered the formation of quadruplexes, but all reduced the thermodynamic stability of the parent quadruplex in both NaCl and KCl. The base loss did not change the Na{sup +}-stabilized intramolecular antiparallel architecture, based on CD spectra, but held up the conformational change induced in dG{sub 3}(TTAG{sub 3}){sub 3} in physiological concentration of KCl. The reduced stability and the inhibited conformational transitions observed here in vitro for the first time may predict that unrepaired abasic sites in G-quadruplexes could lead to changes in the chromosome's terminal protection in vivo.

  7. Mutagenic effects induced by the attack of NO2 radical to the guanine-cytosine base pair

    PubMed Central

    Cerón-Carrasco, José P.; Requena, Alberto; Zúñiga, José; Jacquemin, Denis

    2015-01-01

    We investigate the attack of the nitrogen dioxide radical (NO•2) to the guanine—cytosine (GC) base pair and the subsequent tautomeric reactions able to induce mutations, by means of density functional theory (DFT) calculations. The conducted simulations allow us to identify the most reactive sites of the GC base pair. Indeed, the computed relative energies demonstrate that the addition of the NO•2 radical to the C8 position of the guanine base forms to the most stable adduct. Although the initial adducts might evolve to non-canonical structures via inter-base hydrogen bonds rearrangements, the probability for the proton exchange to occur lies in the same range as that observed for undamaged DNA. As a result, tautomeric errors in NO2-attacked DNA arises at the same rate as in canonical DNA, with no macroscopic impact on the overall stability of DNA. The potential mutagenic effects of the GC–NO•2 radical adducts likely involve side reactions, e.g., the GC deprotonation to the solvent, rather than proton exchange between guanine and cytosine basis. PMID:25798437

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

    PubMed

    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.

  9. Comparison of static and dynamic methods of treatment of anharmonicity for the vibrational study of isolated and aqueous forms of guanine

    NASA Astrophysics Data System (ADS)

    Thicoipe, Sandrine; Carbonniere, Philippe; Pouchan, Claude

    2014-01-01

    This theoretical study provides the anharmonic vibrational wavenumbers of isolated and aqueous guanine. They were performed at the DFT B3LYP/6-31+G(d,p) level of theory using two different ways for the treatment of anharmonicity: time-independent (VPT2) and time-dependent (molecular dynamics) approaches. The wavenumbers obtained are compared to experimental data for isolated and aqueous forms: the VPT2 approach is slightly better than MD, especially for the determination of stretching and wagging (NH) motions. Finally, the structural model of solvatation used for aqueous guanine which combines an explicit solvent model with a polarizable continuum model (PCM) was validated.

  10. Radiation and thermal stabilities of adenine nucleotides.

    PubMed

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

    1995-03-01

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

  11. A highly specific and sensitive electroanalytical strategy for microRNAs based on amplified silver deposition by the synergic TiO2 photocatalysis and guanine photoreduction using charge-neutral probes.

    PubMed

    Li, Rui; Li, Shuying; Dong, Minmin; Zhang, Liyan; Qiao, Yuchun; Jiang, Yao; Qi, Wei; Wang, Hua

    2015-11-18

    TiO2 photocatalysis and guanine photoreduction were synergically combined for amplifying silver deposition for the electroanalysis of short-chain microRNAs with guanine bases using charge-neutral probes. It could allow for the highly specific and sensitive detection of microRNAs in the blood as well as the identification of their mutant levels.

  12. Nucleotide Sequence-Based Multitarget Identification

    PubMed Central

    Vinayagamoorthy, T.; Mulatz, Kirk; Hodkinson, Roger

    2003-01-01

    MULTIGEN technology (T. Vinayagamoorthy, U.S. patent 6,197,510, March 2001) is a modification of conventional sequencing technology that generates a single electropherogram consisting of short nucleotide sequences from a mixture of known DNA targets. The target sequences may be present on the same or different nucleic acid molecules. For example, when two DNA targets are sequenced, the first and second sequencing primers are annealed to their respective target sequences, and then a polymerase causes chain extension by the addition of new deoxyribose nucleotides. Since the electrophoretic separation depends on the relative molecular weights of the truncated molecules, the molecular weight of the second sequencing primer was specifically designed to be higher than the combined molecular weight of the first sequencing primer plus the molecular weight of the largest truncated molecule generated from the first target sequence. Thus, the series of truncated molecules produced by the second sequencing primer will have higher molecular weights than those produced by the first sequencing primer. Hence, the truncated molecules produced by these two sequencing primers can be effectively separated in a single lane by standard gel electrophoresis in a single electropherogram without any overlapping of the nucleotide sequences. By using sequencing primers with progressively higher molecular weights, multiple short DNA sequences from a variety of targets can be determined simultaneously. We describe here the basic concept of MULTIGEN technology and three applications: detection of sexually transmitted pathogens (Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum), detection of contaminants in meat samples (coliforms, fecal coliforms, and Escherichia coli O157:H7), and detection of single-nucleotide polymorphisms in the human N-acetyltransferase (NAT1) gene (S. Fronhoffs et al., Carcinogenesis 22:1405-1412, 2001). PMID:12843076

  13. Vacuum ultraviolet photoionization of carbohydrates and nucleotides.

    PubMed

    Shin, Joong-Won; Bernstein, Elliot R

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5(')-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results. PMID:25669546

  14. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    SciTech Connect

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5{sup ′}-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C–C and C–O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  15. Vacuum ultraviolet photoionization of carbohydrates and nucleotides.

    PubMed

    Shin, Joong-Won; Bernstein, Elliot R

    2014-01-28

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5(')-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  16. Vacuum ultraviolet photoionization of carbohydrates and nucleotides

    NASA Astrophysics Data System (ADS)

    Shin, Joong-Won; Bernstein, Elliot R.

    2014-01-01

    Carbohydrates (2-deoxyribose, ribose, and xylose) and nucleotides (adenosine-, cytidine-, guanosine-, and uridine-5'-monophosphate) are generated in the gas phase, and ionized with vacuum ultraviolet photons (VUV, 118.2 nm). The observed time of flight mass spectra of the carbohydrate fragmentation are similar to those observed [J.-W. Shin, F. Dong, M. Grisham, J. J. Rocca, and E. R. Bernstein, Chem. Phys. Lett. 506, 161 (2011)] for 46.9 nm photon ionization, but with more intensity in higher mass fragment ions. The tendency of carbohydrate ions to fragment extensively following ionization seemingly suggests that nucleic acids might undergo radiation damage as a result of carbohydrate, rather than nucleobase fragmentation. VUV photoionization of nucleotides (monophosphate-carbohydrate-nucleobase), however, shows that the carbohydrate-nucleobase bond is the primary fragmentation site for these species. Density functional theory (DFT) calculations indicate that the removed carbohydrate electrons by the 118.2 nm photons are associated with endocyclic C-C and C-O ring centered orbitals: loss of electron density in the ring bonds of the nascent ion can thus account for the observed fragmentation patterns following carbohydrate ionization. DFT calculations also indicate that electrons removed from nucleotides under these same conditions are associated with orbitals involved with the nucleobase-saccharide linkage electron density. The calculations give a general mechanism and explanation of the experimental results.

  17. p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

    PubMed

    Lloyd, Daniel R; Hanawalt, Philip C

    2002-09-15

    Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the

  18. Effects of nucleotides on [3H]bradykinin binding in guinea pig: further evidence for multiple B2 receptor subtypes.

    PubMed

    Seguin, L; Widdowson, P S

    1993-02-01

    We have suggested recently the existence of three subtypes of B2 bradykinin receptors in tissues of guinea pigs. We have classified these B2 bradykinin receptors into B2a, B2b, and B2c subtypes depending on their affinity for various bradykinin antagonists. Because the actions of bradykinin in different cell systems appear to be both dependent on and independent of G proteins, we sought to determine whether the binding of [3H]bradykinin to the B2 subtypes is sensitive to guanine nucleotides and, therefore, possibly coupled to G proteins. In the ileum, where we have demonstrated B2a and B2b subtypes, specific [3H]bradykinin binding was reduced with GDP (100 microM) and the nonmetabolized analogue of GTP, guanyl-5'-yl-imidodiphosphate (GppNHp; 100 microM). Competition studies with bradykinin and with [Hyp3]bradykinin, which shows approximately 20-fold greater selectivity for the B2a subtype than bradykinin, were performed in the presence or absence of GppNHp (100 microM). The competition experiments demonstrated that binding to the B2a subtype, which has higher affinity for [Hyp3]bradykinin and bradykinin than the B2b subtype, was lost in the presence of GppNHp, whereas binding to the B2b subtype was unaffected. In contrast, GppNHp (100 microM) and GDP (100 microM) failed to alter specific [3H]bradykinin binding to B2b and B2c subtypes in lung. [3H]Bradykinin binding was unaffected by AMP, ADP, ATP, and GMP (100 microM each). Based on this evidence, we suggest that the B2a bradykinin subtype is coupled to G proteins. The B2b and B2c subtypes are either not coupled to G proteins, or may be coupled to the Go-type GTP binding proteins, which have been suggested to be less sensitive to guanine nucleotides.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Molecular dynamics simulations reveal the balance of forces governing the formation of a guanine tetrad-a common structural unit of G-quadruplex DNA.

    PubMed

    Kogut, Mateusz; Kleist, Cyprian; Czub, Jacek

    2016-04-20

    G-quadruplexes (G4) are nucleic acid conformations of guanine-rich sequences, in which guanines are arranged in the square-planar G-tetrads, stacked on one another. G4 motifs formin vivoand are implicated in regulation of such processes as gene expression and chromosome maintenance. The structure and stability of various G4 topologies were determined experimentally; however, the driving forces for their formation are not fully understood at the molecular level. Here, we used all-atom molecular dynamics to probe the microscopic origin of the G4 motif stability. By computing the free energy profiles governing the dissociation of the 3'-terminal G-tetrad in the telomeric parallel-stranded G4, we examined the thermodynamic and kinetic stability of a single G-tetrad, as a common structural unit of G4 DNA. Our results indicate that the energetics of guanine association alone does not explain the overall stability of the G-tetrad and that interactions involving sugar-phosphate backbone, in particular, the constrained minimization of the phosphate-phosphate repulsion energy, are crucial in providing the observed enthalpic stabilization. This enthalpic gain is largely compensated by the unfavorable entropy change due to guanine association and optimization of the backbone topology. PMID:26980278

  20. Nitrosamine-induced carcinogenesis. The alkylation of N-7 of guanine of nucleic acids of the rat by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate

    PubMed Central

    Swann, P. F.; Magee, P. N.

    1971-01-01

    1. The extent of ethylation of N-7 of guanine in the nucleic acids of rat tissue in vivo by diethylnitrosamine, N-ethyl-N-nitrosourea and ethyl methanesulphonate was measured. 2. All compounds produced measurable amounts of 7-ethyl-guanine. 3. A single dose of diethylnitrosamine or N-ethyl-N-nitrosourea produced tumours of the kidney in the rat. Three doses of ethyl methanesulphonate produced kidney tumours, but a single dose did not. 4. A single dose of diethylnitrosamine produced twice as much ethylation of N-7 of guanine in DNA of kidney as did N-ethyl-N-nitrosourea. A single dose of both compounds induced kidney tumours, although of a different histological type. 5. A single dose of ethyl methanesulphonate produced ten times as much ethylation of N-7 of guanine in kidney DNA as did N-ethyl-N-nitrosourea without producing tumours. 6. The relevance of these findings to the hypothesis that alkylation of a cellular component is the mechanism of induction of tumours by nitroso compounds is discussed. PMID:5145908

  1. Highly sensitive sensor for picomolar detection of insulin at physiological pH, using GC electrode modified with guanine and electrodeposited nickel oxide nanoparticles.

    PubMed

    Salimi, Abdollah; Noorbakhash, Abdollah; Sharifi, Ensieh; Semnani, Abolfazl

    2008-12-01

    The electrochemical behavior of insulin at glassy carbon (GC) electrode modified with nickel oxide nanoparticles and guanine was investigated. Cyclic voltammetry technique has been used for electrodeposition of nickel oxide nanoparticles (NiOx) and immobilization of guanine on the surface GC electrode. In comparison to glassy carbon electrode modified with nickel oxide nanoparticles and bare GC electrode modified with adsorbed guanine, the guanine/nickel oxide nanoparticles/modified GC electrode exhibited excellent catalytic activity for the oxidation of insulin in physiological pH solutions at reduced overpotential. The modified electrode was applied for insulin detection using cyclic voltammetry or hydrodynamic amperometry techniques. It was found that the calibration curve was linear up to 4muM with a detection limit of 22pM and sensitivity of 100.9pA/pM under the optimized condition for hydrodynamic amperometry using a rotating disk modified electrode. In comparison to other electrochemical insulin sensors, this sensor shows many advantages such as simple preparation method without using any special electron transfer mediator or specific reagent, high sensitivity, excellent catalytic activity at physiological pH values, short response time, long-term stability and remarkable antifouling property toward insulin and its oxidation product. Additionally, it is promising for the monitoring of insulin in chromatographic effluents.

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

    SciTech Connect

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

    1995-01-01

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

  3. Molecular dynamics simulations reveal the balance of forces governing the formation of a guanine tetrad—a common structural unit of G-quadruplex DNA

    PubMed Central

    Kogut, Mateusz; Kleist, Cyprian; Czub, Jacek

    2016-01-01

    G-quadruplexes (G4) are nucleic acid conformations of guanine-rich sequences, in which guanines are arranged in the square-planar G-tetrads, stacked on one another. G4 motifs form in vivo and are implicated in regulation of such processes as gene expression and chromosome maintenance. The structure and stability of various G4 topologies were determined experimentally; however, the driving forces for their formation are not fully understood at the molecular level. Here, we used all-atom molecular dynamics to probe the microscopic origin of the G4 motif stability. By computing the free energy profiles governing the dissociation of the 3′-terminal G-tetrad in the telomeric parallel-stranded G4, we examined the thermodynamic and kinetic stability of a single G-tetrad, as a common structural unit of G4 DNA. Our results indicate that the energetics of guanine association alone does not explain the overall stability of the G-tetrad and that interactions involving sugar–phosphate backbone, in particular, the constrained minimization of the phosphate–phosphate repulsion energy, are crucial in providing the observed enthalpic stabilization. This enthalpic gain is largely compensated by the unfavorable entropy change due to guanine association and optimization of the backbone topology. PMID:26980278

  4. DNA-base guanine as hydrogen getter and charge-trapping layer embedded in oxide dielectrics for inorganic and organic field-effect transistors.

    PubMed

    Lee, Junyeong; Park, Ji Hoon; Lee, Young Tack; Jeon, Pyo Jin; Lee, Hee Sung; Nam, Seung Hee; Yi, Yeonjin; Lee, Younjoo; Im, Seongil

    2014-04-01

    DNA-base small molecules of guanine, cytosine, adenine, and thymine construct the DNA double helix structure with hydrogen bonding, and they possess such a variety of intrinsic benefits as natural plentitude, biodegradability, biofunctionality, low cost, and low toxicity. On the basis of these advantages, here, we report on unprecedented useful applications of guanine layer as hydrogen getter and charge trapping layer when it is embedded into a dielectric oxide of n-channel inorganic InGaZnO and p-channel organic heptazole field effect transistors (FETs). The embedded guanine layer much improved the gate stability of inorganic FETs gettering many hydrogen atoms in the gate dielectric layer of FET, and it also played as charge trapping layer to which the voltage pulse-driven charges might be injected from channel, resulting in a threshold voltage (Vth) shift of FETs. Such shift state is very ambient-stable and almost irrevocable even under a high electric-field at room temperature. So, Boolean logics are nicely demonstrated by using our FETs with the guanine-embedded dielectric. The original Vth is recovered only under high energy blue photons by opposite voltage pulse (charge-ejection), which indicates that our device is also applicable to nonvolatile photo memory.

  5. Quantum processes in 8-Oxo-Guanine-Ru(bipyridine)32+ photosynthetic systems of artificial minimal cells

    NASA Astrophysics Data System (ADS)

    Tamulis, Arvydas; Grigalavicius, Mantas; Krisciukaitis, Sarunas; Medzevicius, Giedrius

    2011-06-01

    Density functional theory methods were used to investigate various self-assembled photoactive bioorganic systems of interest for artificial minimal cells. The cell systems studied are based on nucleotides or their compounds and consisted of up to 123 atoms (not including the associated water or methanol solvent shells) and are up to 2.5 nm in diameter. The electron correlation interactions responsible for the weak hydrogen and Van derWaals chemical bonds increase due to the addition of a polar solvent (water or methanol). The precursor fatty acid molecules of the system also play a critical role in the quantum mechanical interaction based self-assembly of the photosynthetic center and the functioning of the photosynthetic processes of the artificial minimal cells. The distances between the separated sensitizer, fatty acid precursor, and methanol molecules are comparable to Van derWaals and hydrogen bonding radii. As a result the associated electron correlation interactions compress the overall system, resulting in an even smaller gap between the highest occupied molecular orbital (HOMO), and lowest unoccupied molecular orbital (LUMO) electron energy levels and photoexcited electron tunnelling occurs from the sensitizer (either Ru(bpy)32+ or [Ru(bpy)2(4-Bu-4'-Me-2,2'-bpy)]2++ derivatives) to the precursor fatty acid molecules (notation used: Me = methyl; Bu = butyl; bpy = bipyridine). The shift of the absorption spectrum to the red for the artificial protocell photosynthetic centers might be considered as the measure of the complexity of these systems.

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

    PubMed Central

    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. PMID:26999445

  7. Stereoselective synthesis of 9-beta-d-arabianofuranosyl guanine and 2-amino-9-(beta-d-arabianofuranosyl)purine.

    PubMed

    Yu, Xue-Jun; Li, Gai-Xia; Qi, Xiou-Xiang; Deng, You-Quan

    2005-02-01

    9-beta-d-Arabianofuranosyl guanine (6) and 2-amino-9-(beta-d-arabianofuranosyl)purine (8) were prepared from 2-amino-6-chloro-9-(2,3,5-triphenylmethoxyl-beta-d-arabianofuranosyl)purine (4), a key intermediate which was stereoselectively prepared from 2,3,5-triphenylmethoxyl-d-arabianofuranose and 2-amino-6-chloro-purine. The yield of the intermediate was obviously improved and only beta-isomer was formed by using the activated molecular sieve as environmental friendly catalyst, overcoming the defect that a 1:1 mixture of alpha- and beta-isomers was formed, which was difficult to separate, when toxic mercury cyanide was previously used as catalyst.

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

    PubMed

    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.

  9. Guanine-Specific Oxidation of Double-Stranded DNA by Cr(VI) and Ascorbic Acid Forms Spiroiminodihydantoin and 8-Oxo-2′-deoxyguanosine

    PubMed Central

    Slade, Peter G.; Hailer, M. Katie; Martin, Brooke D.; Sugden, Kent D.

    2005-01-01

    7,8-dihydro-8-oxoguanine (8-oxoG) is thought to be a major lesion formed in DNA by oxidative attack at the nucleobase guanine. Recent studies have shown that 8-oxoG has a lower reduction potential than the parent guanine and is a hot spot for further oxidation. Spiroiminodihydantoin (Sp) has been identified as one of these further oxidation products. Chromium(VI) is a human carcinogen that, when reduced by a cellular reductant such as ascorbate, can oxidize DNA. In this study, duplex DNA was reacted with Cr(VI) and ascorbate to identify and quantify the base lesions formed. Guanine bases were observed to be preferentially oxidized with 5′ guanines within purine repeats showing enhanced oxidation. Trapping of the guanine lesions by the base excision repair enzymes hOGG1 and mNEIL2 showed nearly exclusive trapping by mNEIL2, suggesting that 8-oxoG was not the major lesion but rather a lesion recognized by mNEIL2 such as Sp. Formation of the Sp lesion in the Cr(VI)/Asc oxidation reaction with DNA was confirmed by LC–ESI-MS detection. HPLC-ECD was used to identify and quantify any 8-oxoG arising from Cr(VI)/Asc oxidation of DNA. Concentrations of Cr(VI) (3.1–50 μM) with a corresponding 1:10 ratio of Asc oxidized between 0.3% and 1.5% of all guanines within the duplex DNA strand to Sp. 8-oxoG was also identified but with the highest Cr(VI) concentration converting ~0.1% of all guanines to 8-oxoG. These results show that Sp was present in concentrations ~20 times greater than that of 8-oxoG in this system. The results indicate that 8-oxoG, while present, was not the major product of Cr(VI)/Asc oxidation of DNA and that Sp predominates under these conditions. These results further imply that Sp may be the lesion that accounts for the carcinogenicity of this metal in cellular systems. PMID:16022506

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

    PubMed Central

    Kumar, Anil

    2011-01-01

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

  11. Formation of amino acids and nucleotide bases in a Titan atmosphere simulation experiment.

    PubMed

    Hörst, S M; Yelle, R V; Buch, A; Carrasco, N; Cernogora, G; Dutuit, O; Quirico, E; Sciamma-O'Brien, E; Smith, M A; Somogyi, A; Szopa, C; Thissen, R; Vuitton, V

    2012-09-01

    The discovery of large (>100 u) molecules in Titan's upper atmosphere has heightened astrobiological interest in this unique satellite. In particular, complex organic aerosols produced in atmospheres containing C, N, O, and H, like that of Titan, could be a source of prebiotic molecules. In this work, aerosols produced in a Titan atmosphere simulation experiment with enhanced CO (N(2)/CH(4)/CO gas mixtures of 96.2%/2.0%/1.8% and 93.2%/5.0%/1.8%) were found to contain 18 molecules with molecular formulae that correspond to biological amino acids and nucleotide bases. Very high-resolution mass spectrometry of isotopically labeled samples confirmed that C(4)H(5)N(3)O, C(4)H(4)N(2)O(2), C(5)H(6)N(2)O(2), C(5)H(5)N(5), and C(6)H(9)N(3)O(2) are produced by chemistry in the simulation chamber. Gas chromatography-mass spectrometry (GC-MS) analyses of the non-isotopic samples confirmed the presence of cytosine (C(4)H(5)N(3)O), uracil (C(5)H(4)N(2)O(2)), thymine (C(5)H(6)N(2)O(2)), guanine (C(5)H(5)N(5)O), glycine (C(2)H(5)NO(2)), and alanine (C(3)H(7)NO(2)). Adenine (C(5)H(5)N(5)) was detected by GC-MS in isotopically labeled samples. The remaining prebiotic molecules were detected in unlabeled samples only and may have been affected by contamination in the chamber. These results demonstrate that prebiotic molecules can be formed by the high-energy chemistry similar to that which occurs in planetary upper atmospheres and therefore identifies a new source of prebiotic material, potentially increasing the range of planets where life could begin.

  12. Formation of Amino Acids and Nucleotide Bases in a Titan Atmosphere Simulation Experiment

    PubMed Central

    Yelle, R.V.; Buch, A.; Carrasco, N.; Cernogora, G.; Dutuit, O.; Quirico, E.; Sciamma-O'Brien, E.; Smith, M.A.; Somogyi, Á.; Szopa, C.; Thissen, R.; Vuitton, V.

    2012-01-01

    Abstract The discovery of large (>100 u) molecules in Titan's upper atmosphere has heightened astrobiological interest in this unique satellite. In particular, complex organic aerosols produced in atmospheres containing C, N, O, and H, like that of Titan, could be a source of prebiotic molecules. In this work, aerosols produced in a Titan atmosphere simulation experiment with enhanced CO (N2/CH4/CO gas mixtures of 96.2%/2.0%/1.8% and 93.2%/5.0%/1.8%) were found to contain 18 molecules with molecular formulae that correspond to biological amino acids and nucleotide bases. Very high-resolution mass spectrometry of isotopically labeled samples confirmed that C4H5N3O, C4H4N2O2, C5H6N2O2, C5H5N5, and C6H9N3O2 are produced by chemistry in the simulation chamber. Gas chromatography–mass spectrometry (GC-MS) analyses of the non-isotopic samples confirmed the presence of cytosine (C4H5N3O), uracil (C5H4N2O2), thymine (C5H6N2O2), guanine (C5H5N5O), glycine (C2H5NO2), and alanine (C3H7NO2). Adenine (C5H5N5) was detected by GC-MS in isotopically labeled samples. The remaining prebiotic molecules were detected in unlabeled samples only and may have been affected by contamination in the chamber. These results demonstrate that prebiotic molecules can be formed by the high-energy chemistry similar to that which occurs in planetary upper atmospheres and therefore identifies a new source of prebiotic material, potentially increasing the range of planets where life could begin. Key Words: Astrochemistry—Planetary atmospheres—Titan—Astrobiology. Astrobiology 12, 809–817. PMID:22917035

  13. A single nucleotide polymorphism in NEUROD1 is associated with production traits in Nelore beef cattle.

    PubMed

    de Oliveira, P S N; Tizioto, P C; Malago, W; do Nascimento, M L; Cesar, A S M; Diniz, W J S; de Souza, M M; Lanna, D P D; Tullio, R R; Mourão, G B; de A Mudadu, M; Coutinho, L L; de A Regitano, L C

    2016-01-01

    Feed efficiency and carcass characteristics are late-measured traits. The detection of molecular markers associated with them can help breeding programs to select animals early in life, and to predict breeding values with high accuracy. The objective of this study was to identify polymorphisms in the functional and positional candidate gene NEUROD1 (neurogenic differentiation 1), and investigate their associations with production traits in reference families of Nelore cattle. A total of 585 steers were used, from 34 sires chosen to represent the variability of this breed. By sequencing 14 animals with extreme residual feed intake (RFI) values, seven single nucleotide polymorphisms (SNPs) in NEUROD1 were identified. The investigation of marker effects on the target traits RFI, backfat thickness (BFT), ribeye area (REA), average body weight (ABW), and metabolic body weight (MBW) was performed with a mixed model using the restricted maximum likelihood method. SNP1062, which changes cytosine for guanine, had no significant association with RFI or REA. However, we found an additive effect on ABW (P ≤ 0.05) and MBW (P ≤ 0.05), with an estimated allele substitution effect of -1.59 and -0.93 kg0.75, respectively. A dominant effect of this SNP for BFT was also found (P ≤ 0.010). Our results are the first that identify NEUROD1 as a candidate that affects BFT, ABW, and MBW. Once confirmed, the inclusion of this SNP in dense panels may improve the accuracy of genomic selection for these traits in Nelore beef cattle as this SNP is not currently represented on SNP chips. PMID:27420997

  14. Formation of amino acids and nucleotide bases in a Titan atmosphere simulation experiment.

    PubMed

    Hörst, S M; Yelle, R V; Buch, A; Carrasco, N; Cernogora, G; Dutuit, O; Quirico, E; Sciamma-O'Brien, E; Smith, M A; Somogyi, A; Szopa, C; Thissen, R; Vuitton, V

    2012-09-01

    The discovery of large (>100 u) molecules in Titan's upper atmosphere has heightened astrobiological interest in this unique satellite. In particular, complex organic aerosols produced in atmospheres containing C, N, O, and H, like that of Titan, could be a source of prebiotic molecules. In this work, aerosols produced in a Titan atmosphere simulation experiment with enhanced CO (N(2)/CH(4)/CO gas mixtures of 96.2%/2.0%/1.8% and 93.2%/5.0%/1.8%) were found to contain 18 molecules with molecular formulae that correspond to biological amino acids and nucleotide bases. Very high-resolution mass spectrometry of isotopically labeled samples confirmed that C(4)H(5)N(3)O, C(4)H(4)N(2)O(2), C(5)H(6)N(2)O(2), C(5)H(5)N(5), and C(6)H(9)N(3)O(2) are produced by chemistry in the simulation chamber. Gas chromatography-mass spectrometry (GC-MS) analyses of the non-isotopic samples confirmed the presence of cytosine (C(4)H(5)N(3)O), uracil (C(5)H(4)N(2)O(2)), thymine (C(5)H(6)N(2)O(2)), guanine (C(5)H(5)N(5)O), glycine (C(2)H(5)NO(2)), and alanine (C(3)H(7)NO(2)). Adenine (C(5)H(5)N(5)) was detected by GC-MS in isotopically labeled samples. The remaining prebiotic molecules were detected in unlabeled samples only and may have been affected by contamination in the chamber. These results demonstrate that prebiotic molecules can be formed by the high-energy chemistry similar to that which occurs in planetary upper atmospheres and therefore identifies a new source of prebiotic material, potentially increasing the range of planets where life could begin. PMID:22917035

  15. Pyrrolidine nucleotide analogs with a tunable conformation

    PubMed Central

    Poštová Slavětínská, Lenka; Rejman, Dominik

    2014-01-01

    Summary Conformational preferences of the pyrrolidine ring in nucleotide analogs 7–14 were investigated by means of NMR and molecular modeling. The effect of the relative configuration of hydroxy and nucleobase substituents as well as the effect of the alkylation or acylation of the pyrrolidine nitrogen atom on the conformation of the pyrrolidine ring were studied. The results of a conformational analysis show that the alkylation/acylation can be effectively used for tuning the pyrrolidine conformation over the whole pseudorotation cycle. PMID:25246956

  16. Complete Nucleotide Sequence of Tn10

    PubMed Central

    Chalmers, Ronald; Sewitz, Sven; Lipkow, Karen; Crellin, Paul

    2000-01-01

    The complete nucleotide sequence of Tn10 has been determined. The dinucleotide signature and percent G+C of the sequence had no discontinuities, indicating that Tn10 constitutes a homogeneous unit. The new sequence contained three new open reading frames corresponding to a glutamate permease, repressors of heavy metal resistance operons, and a hypothetical protein in Bacillus subtilis. The glutamate permease was fully functional when expressed, but Tn10 did not protect Escherichia coli from the toxic effects of various metals. PMID:10781570

  17. Performance characteristics of guanine incorporated PVDF-HFP/PEO polymer blend electrolytes with binary iodide salts for dye-sensitized solar cells

    NASA Astrophysics Data System (ADS)

    Senthil, R. A.; Theerthagiri, J.; Madhavan, J.; Arof, A. K.

    2016-08-01

    In this work, we have investigated the influence of guanine as an organic dopant in dye-sensitized solar cell (DSSC) based on poly(vinylidinefluoride-co-hexafluoropropylene) (PVDF-HFP)/polyethylene oxide (PEO) polymer blend electrolyte along with binary iodide salts (potassium iodide (KI) and tetrabutylammonium iodide (TBAI)) and iodine (I2). The PVDF-HFP/KI + TBAI/I2, PVDF-HFP/PEO/KI + TBAI/I2 and guanine incorporated PVDF-HFP/PEO/KI + TBAI/I2 electrolytes were prepared by solution casting technique using DMF as solvent. The PVDF-HFP/KI + TBAI/I2 electrolyte showed an ionic conductivity value of 9.99 × 10-5 Scm-1, whereas, it was found to be increased to 4.53 × 10-5 Scm-1 when PEO was blended with PVDF-HFP/KI + TBAI/I2 electrolyte. However, a maximum ionic conductivity value of 3.67 × 10-4 Scm-1 was obtained for guanine incorporated PVDF-HFP/PEO/KI + TBAI/I2 blend electrolyte. The photovoltaic properties of all these polymer electrolytes in DSSCs were characterized. As a consequence, the power conversion efficiency of the guanine incorporated PVDF-HFP/PEO/KI + TBAI/I2 electrolyte based DSSC was significantly improved to 4.98% compared with PVDF-HFP/PEO/KI + TBAI/I2 electrolyte based DSSC (2.46%). These results revealed that the guanine can be an effective organic dopant to enhance the performance of DSSCs.

  18. Microhydration of guanine...cytosine base pairs, a theoretical Study on the role of water in stability, structure and tautomeric equilibrium.

    PubMed

    Zelený, Tomás; Hobza, Pavel; Kabelác, Martin

    2009-05-14

    The potential energy surfaces of guanine...cytosine complexes and microhydrated guanine...cytosine (one and two water molecules) were investigated by the molecular dynamics/quenching method (MD/Q), using the empirical potential Parm94 force field, implemented in the Amber program package. The calculations were conducted for all the possible combinations of the four most stable tautomers of guanine and three of cytosine (covering the canonical forms in both cases). The obtained structures were sorted by their structural motifs into three main groups: planar hydrogen-bonded; stacked; and T-shaped structures. The most stable structures found at the empirical potential energy surfaces were fully reoptimised at the second-order Møller-Plesset perturbation theory as well as using the density functional method with an empirical dispersion term (DFT-D). A combination of the canonical form of guanine and cytosine and canonical cytosine with a guanine tautomer where the hydrogen is switched from position N9 to N7 are energetically preferred in microsolvated systems as well as those without the presence of a solvent. The rising number of water molecules leads to smaller differences between the stability of the various combinations of the tautomers of bases in the base pairs. For some of the tautomer combinations (mainly the enol-enol combination), two water molecules are sufficient for the preference of stacked structures over the H-bonded ones. The interaction energies and geometries obtained by the second-order Møller-Plesset perturbation theory method and the much less computationally demanding DFT-D method are comparable, except for stacked complexes, where the interaction energies are overestimated on average by 3 kcal mol(-1) at the MP2 level. PMID:19421545

  19. The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters.

    PubMed

    Di Noia, Maria Antonietta; Todisco, Simona; Cirigliano, Angela; Rinaldi, Teresa; Agrimi, Gennaro; Iacobazzi, Vito; Palmieri, Ferdinando

    2014-11-28

    The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport inorganic anions, amino acids, carboxylates, nucleotides, and coenzymes across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. Here two members of this family, SLC25A33 and SLC25A36, have been thoroughly characterized biochemically. These proteins were overexpressed in bacteria and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that SLC25A33 transports uracil, thymine, and cytosine (deoxy)nucleoside di- and triphosphates by an antiport mechanism and SLC25A36 cytosine and uracil (deoxy)nucleoside mono-, di-, and triphosphates by uniport and antiport. Both carriers also transported guanine but not adenine (deoxy)nucleotides. Transport catalyzed by both carriers was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. In confirmation of their identity (i) SLC25A33 and SLC25A36 were found to be targeted to mitochondria and (ii) the phenotypes of Saccharomyces cerevisiae cells lacking RIM2, the gene encoding the well characterized yeast mitochondrial pyrimidine nucleotide carrier, were overcome by expressing SLC25A33 or SLC25A36 in these cells. The main physiological role of SLC25A33 and SLC25A36 is to import/export pyrimidine nucleotides into and from mitochondria, i.e. to accomplish transport steps essential for mitochondrial DNA and RNA synthesis and breakdown.

  20. Oxidation of guanine in cellular DNA by solar UV radiation: biological role.

    PubMed

    Douki, T; Perdiz, D; Gróf, P; Kuluncsics, Z; Moustacchi, E; Cadet, J; Sage, E

    1999-08-01

    The formation of cyclobutane pyrimidine dimers (CPD) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) was investigated in Chinese hamster ovary cells upon exposure to either UVC, UVB, UVA or simulated sunlight (SSL). Two cell lines were used, namely AT3-2 and UVL9, the latter being deficient in nucleotide excision repair and consequently UV sensitive. For all types of radiation, including UVA, CPD were found to be the predominant lesions quantitatively. At the biologically relevant doses used, UVC, UVB and SSL irradiation yielded 8-oxodGuo at a rather low level, whereas UVA radiation produced relatively higher amounts. The formation of CPD was 10(2) and 10(5) more effective upon UVC than UVB and UVA exposure. These yields of formation followed DNA absorption, even in the UVA range. The calculated relative spectral effectiveness in the production of the two lesions showed that efficient induction of 8-oxodGuo upon UVA irradiation was shifted toward longer wavelengths, in comparison with those for CPD formation, in agreement with a photosensitization mechanism. In addition, after exposure to SSL, about 19% and 20% of 8-oxodGuo were produced between 290-320 nm and 320-340 nm, respectively, whereas CPD were essentially (90%) induced in the UVB region. However, the ratio of CPD to 8-oxodGuo greatly differed from one source of light to the other: it was over 100 for UVB but only a few units for UVA source. The extent of 8-oxodGuo and CPD was also compared to the lethality for the different types of radiation. The involvement of 8-oxodGuo in cell killing by solar UV radiation was clearly ruled out. In addition, our previously reported mutation spectra demonstrated that the contribution of 8-oxodGuo in the overall solar UV mutagenic process is very minor.

  1. Variance estimation for nucleotide substitution models.

    PubMed

    Chen, Weishan; Wang, Hsiuying

    2015-09-01

    The current variance estimators for most evolutionary models were derived when a nucleotide substitution number estimator was approximated with a simple first order Taylor expansion. In this study, we derive three variance estimators for the F81, F84, HKY85 and TN93 nucleotide substitution models, respectively. They are obtained using the second order Taylor expansion of the substitution number estimator, the first order Taylor expansion of a squared deviation and the second order Taylor expansion of a squared deviation, respectively. These variance estimators are compared with the existing variance estimator in terms of a simulation study. It shows that the variance estimator, which is derived using the second order Taylor expansion of a squared deviation, is more accurate than the other three estimators. In addition, we also compare these estimators with an estimator derived by the bootstrap method. The simulation shows that the performance of this bootstrap estimator is similar to the estimator derived by the second order Taylor expansion of a squared deviation. Since the latter one has an explicit form, it is more efficient than the bootstrap estimator.

  2. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  3. A Steric-inhibition model for regulation of nucleotide exchange via the Dock180 family of GEFs.

    PubMed

    Lu, Mingjian; Kinchen, Jason M; Rossman, Kent L; Grimsley, Cynthia; Hall, Matthew; Sondek, John; Hengartner, Michael O; Yajnik, Vijay; Ravichandran, Kodi S

    2005-02-22

    CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

  4. DNA lesion can facilitate base ionization: vertical ionization energies of aqueous 8-oxoguanine and its nucleoside and nucleotide.

    PubMed

    Palivec, Vladimír; Pluhařová, Eva; Unger, Isaak; Winter, Bernd; Jungwirth, Pavel

    2014-12-01

    8-Oxoguanine is one of the key products of indirect radiation damage to DNA by reactive oxygen species. Here, we describe ionization of this damaged nucleobase and the corresponding nucleoside and nucleotide in aqueous phase, modeled by the nonequilibrium polarizable continuum model, establishing their lowest vertical ionization energies of 6.8-7.0 eV. We thus confirm that 8-oxoguanine has even lower ionization energy than the parental guanine, which is the canonical nucleobase with the lowest ionization energy. Therefore, it can act as a trap for the cationic hole formed by ionizing radiation and thus protect DNA from further radiation damage. We also model using time-dependent density functional theory and measure by liquid jet photoelectron spectroscopy the valence photoelectron spectrum of 8-oxoguanine in water. We show that the calculated higher lying ionization states match well the experiment which, however, is not sensitive enough to capture the electron signal corresponding to the lowest ionization process due to the low solubility of 8-oxoguanine in water.

  5. A novel nuclear role for the Vav3 nucleotide exchange factor in androgen receptor coactivation in prostate cancer.

    PubMed

    Rao, S; Lyons, L S; Fahrenholtz, C D; Wu, F; Farooq, A; Balkan, W; Burnstein, K L

    2012-02-01

    Increased androgen receptor (AR) transcriptional activity mediated by coactivator proteins may drive castration-resistant prostate cancer (CRPC) growth. Vav3, a Rho GTPase guanine nucleotide exchange factor (GEF), is overexpressed in human prostate cancers, particularly in models of CRPC progression. Vav3 coactivates AR in a Vav3 pleckstrin homology (PH) domain-dependent but GEF-independent manner. Ectopic expression of Vav3 in androgen-dependent human prostate cancer cells conferred robust castration-resistant xenograft tumor growth. Vav3 but not a Vav3 PH mutant greatly stimulated interaction between the AR amino and carboxyl termini (N-C interaction), which is required for maximal receptor transcriptional activity. Vav3 was distributed between the cytoplasm and nucleus with nuclear localization-dependent on the Vav3 PH domain. Membrane targeting of Vav3 abolished Vav3 potentiation of AR activity, whereas nuclear targeting of a Vav3 PH mutant rescued AR coactivation, suggesting that nuclear localization is an important function of the Vav3 PH domain. A nuclear role for Vav3 was further demonstrated by sequential chromatin immunoprecipitation assays, which revealed that Vav3 and AR were recruited to the same transcriptional complexes of an AR target gene enhancer. These data demonstrate the importance of Vav3 in CRPC and define a novel nuclear function of Vav3 in regulating AR activity.

  6. Polyamine/Nucleotide Coacervates Provide Strong Compartmentalization of Mg²⁺, Nucleotides, and RNA.

    PubMed

    Frankel, Erica A; Bevilacqua, Philip C; Keating, Christine D

    2016-03-01

    Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5'-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg(2+) and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg(2+) concentration of 10 mM had 1-5 M Mg(2+) in the coacervates, and RNA random sequence (N54) partitioned ∼10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg(2+), and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg(2+) concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments. PMID:26844692

  7. Crystal structure of Escherichia coli cytidine triphosphate synthetase, a nucleotide-regulated glutamine amidotransferase/ATP-dependent amidoligase fusion protein and homologue of anticancer and antiparasitic drug targets.

    PubMed

    Endrizzi, James A; Kim, Hanseong; Anderson, Paul M; Baldwin, Enoch P

    2004-06-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 A between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

  8. Fast and reliable screening of mutations in human tumors: use of multiple fluorescence-based long linker arm nucleotides assay (mf-LLA).

    PubMed

    Marcelino, L A; Galvin, M; Martins, G M; Proença, M J; Mayrand, E; Rueff, J A; Monteiro, C J

    1999-06-01

    Human tumor samples were screened for point mutations by adapting a mobility-shift assay to automated DNA sizing. This screen identifies the type of point mutation and relative amount of mutated DNA sequences present in a sample. Test samples having known hypoxanthine-guanine phosphoribosyl transferase (hprt)/exon-3 sequence mutations were characterized by: (i) PCR amplification, (ii) fluorescent dye-primer extension with 36-atom linker derived deoxycytosine or deoxyuridine triphosphate and the remaining three natural nucleotides and (iii) sizing of the resulting fluorescently labeled modified strands, using an automated DNA sequencer. Routinely, a range of sizes is observed among the sequence variants of a single DNA target sequence. This is because nucleotide analogs are incorporated into DNA strands in a sequence-dependent manner, resulting in composition-dependent electrophoretic mobility. Thus, point mutations are identified as shifts in mobility between the fluorescently labeled modified strands of the control and test samples. The twenty different hprt/exon-3 single-base substitution mutations tested were easily identified, even at fourfold dilution with control DNA.

  9. Nicotinamide nucleotide synthesis in regenerating rat liver

    PubMed Central

    Ferris, G. M.; Clark, J. B.

    1971-01-01

    1. The concentrations and total content of the nicotinamide nucleotides were measured in the livers of rats at various times after partial hepatectomy and laparotomy (sham hepatectomy) and correlated with other events in the regeneration process. 2. The NAD content and concentration in rat liver were relatively unaffected by laparotomy, but fell to a minimum, 25 and 33% below control values respectively, 24h after partial hepatectomy. NADP content and concentration were affected similarly by both laparotomy and partial hepatectomy, falling rapidly and remaining depressed for up to 48h. 3. The effect of injecting various doses of nicotinamide on the liver DNA and NAD 18h after partial hepatectomy was studied and revealed an inverse correlation between NAD content and DNA content. 4. Injections of nicotinamide at various times after partial hepatectomy revealed that the ability to synthesize NAD from nicotinamide was impaired during the first 12h, rose to a peak at 26h and fell again by 48h after partial hepatectomy. 5. The total liver activity of NAD pyrophosphorylase (EC 2.7.7.1) remained at or slightly above the initial value for 12h after partial hepatectomy and then rose continuously until 48h after operation. The activity of NMN pyrophosphorylase (EC 2.4.2.12) showed a similar pattern of change after partial hepatectomy, but was at no time greater than 5% of the activity of NAD pyrophosphorylase. 6. The results are discussed with reference to the control of NAD synthesis in rapidly dividing tissue. It is suggested that the availability of cofactors and substrates for NAD synthesis is more important as a controlling factor than the maximum enzyme activities. It is concluded that the low concentrations of nicotinamide nucleotides in rapidly dividing tissues are the result of competition between NAD synthesis and nucleic acid synthesis for common precursor and cofactors. PMID:4398891

  10. Correlated Evolution of Nucleotide Positions within Splice Sites in Mammals.

    PubMed

    Denisov, Stepan; Bazykin, Georgii; Favorov, Alexander; Mironov, Andrey; Gelfand, Mikhail

    2015-01-01

    Splice sites (SSs)--short nucleotide sequences flanking introns--are under selection for spliceosome binding, and adhere to consensus sequences. However, non-consensus nucleotides, many of which probably reduce SS performance, are frequent. Little is known about the mechanisms maintaining such apparently suboptimal SSs. Here, we study the correlations between strengths of nucleotides occupying different positions of the same SS. Such correlations may arise due to epistatic interactions between positions (i.e., a situation when the fitness effect of a nucleotide in one position depends on the nucleotide in another position), their evolutionary history, or to other reasons. Within both the intronic and the exonic parts of donor SSs, nucleotides that increase (decrease) SS strength tend to co-occur with other nucleotides increasing (respectively, decreasing) it, consistent with positive epistasis. Between the intronic and exonic parts of donor SSs, the correlations of nucleotide strengths tend to be negative, consistent with negative epistasis. In the course of evolution, substitutions at a donor SS tend to decrease the strength of its exonic part, and either increase or do not change the strength of its intronic part. In acceptor SSs, the situation is more complicated; the correlations between adjacent positions appear to be driven mainly by avoidance of the AG dinucleotide which may cause aberrant splicing. In summary, both the content and the evolution of SSs is shaped by a complex network of interdependences between adjacent nucleotides that respond to a range of sometimes conflicting selective constraints. PMID:26642327

  11. Frequency and Correlation of Nearest Neighboring Nucleotides in Human Genome

    NASA Astrophysics Data System (ADS)

    Jin, Neng-zhi; Liu, Zi-xian; Qiu, Wen-yuan

    2009-02-01

    Zipf's approach in linguistics is utilized to analyze the statistical features of frequency and correlation of 16 nearest neighboring nucleotides (AA, AC, AG, ..., TT) in 12 human chromosomes (Y, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, and 12). It is found that these statistical features of nearest neighboring nucleotides in human genome: (i) the frequency distribution is a linear function, and (ii) the correlation distribution is an inverse function. The coefficients of the linear function and inverse function depend on the GC content. It proposes the correlation distribution of nearest neighboring nucleotides for the first time and extends the descriptor about nearest neighboring nucleotides.

  12. Time-resolved probes based on guanine/thymine-rich DNA-sensitized luminescence of terbium(III).

    PubMed

    Zhang, Min; Le, Huynh-Nhu; Jiang, Xiao-Qin; Yin, Bin-Cheng; Ye, Bang-Ce

    2013-12-01

    In this study, we have developed a novel strategy to highly sensitize the luminescence of terbium(III) (Tb(3+)) using a designed guanine/thymine-rich DNA (5'-[G3T]5-3') as an antenna ligand, in which [G3T]5 improved the luminescence of Tb(3+) by 3 orders of magnitude due to energy transfer from nucleic acids to Tb(3+) (i.e., antenna effect). Furthermore, label-free probes for the luminescent detection of biothiols, Ag(+), and sequence-specific DNA in an inexpensive, simple, and mix-and-read format are presented based on the [G3T]5-sensitized luminescence of Tb(3+) (GTSLT). The long luminescence lifetime of the probes readily enables time-resolved luminescence (TRL) experiments. Hg(2+) can efficiently quench the luminescence of Tb(3+) sensitized by [G3T]5 (Tb(3+)/[G3T]5); however, biothiols are readily applicable to selectively grab Hg(2+) for restoration of the luminescence of Tb(3+)/[G3T]5 initially quenched by Hg(2+), which can be used for "turn on" detection of biothiols. With the use of cytosine (C)-rich oligonucleotide c[G3T]5 complementary to [G3T]5, the formed [G3T]5/c[G3T]5 duplex cannot sensitize the luminescence of Tb(3+). However, in the presence of Ag(+), Ag(+) can combine the C base of c[G3T]5 to form C-Ag(+)-C complexes, leading to the split of the [G3T]5/c[G3T]5 duplex and then release of [G3T]5. The released [G3T]5 acts as an antenna ligand for sensitizing the luminescence of Tb(3+). Therefore, the Tb(3+)/[G3T]5/c[G3T]5 probe can be applied to detect Ag(+) in a "turn on" format. Moreover, recognition of target DNA via hybridization to a molecular beacon (MB)-like probe (MB-[G3T]5) can unfold the MB-[G3T]5 to release the [G3T]5 for sensitizing the luminescence of Tb(3+), producing a detectable signal directly proportional to the amount of target DNA of interest. This allows the development of a fascinating label-free MB probe for DNA sensing based on the luminescence of Tb(3+). Results and methods reported here suggest that a guanine/thymine-rich DNA

  13. Time-resolved probes based on guanine/thymine-rich DNA-sensitized luminescence of terbium(III).

    PubMed

    Zhang, Min; Le, Huynh-Nhu; Jiang, Xiao-Qin; Yin, Bin-Cheng; Ye, Bang-Ce

    2013-12-01

    In this study, we have developed a novel strategy to highly sensitize the luminescence of terbium(III) (Tb(3+)) using a designed guanine/thymine-rich DNA (5'-[G3T]5-3') as an antenna ligand, in which [G3T]5 improved the luminescence of Tb(3+) by 3 orders of magnitude due to energy transfer from nucleic acids to Tb(3+) (i.e., antenna effect). Furthermore, label-free probes for the luminescent detection of biothiols, Ag(+), and sequence-specific DNA in an inexpensive, simple, and mix-and-read format are presented based on the [G3T]5-sensitized luminescence of Tb(3+) (GTSLT). The long luminescence lifetime of the probes readily enables time-resolved luminescence (TRL) experiments. Hg(2+) can efficiently quench the luminescence of Tb(3+) sensitized by [G3T]5 (Tb(3+)/[G3T]5); however, biothiols are readily applicable to selectively grab Hg(2+) for restoration of the luminescence of Tb(3+)/[G3T]5 initially quenched by Hg(2+), which can be used for "turn on" detection of biothiols. With the use of cytosine (C)-rich oligonucleotide c[G3T]5 complementary to [G3T]5, the formed [G3T]5/c[G3T]5 duplex cannot sensitize the luminescence of Tb(3+). However, in the presence of Ag(+), Ag(+) can combine the C base of c[G3T]5 to form C-Ag(+)-C complexes, leading to the split of the [G3T]5/c[G3T]5 duplex and then release of [G3T]5. The released [G3T]5 acts as an antenna ligand for sensitizing the luminescence of Tb(3+). Therefore, the Tb(3+)/[G3T]5/c[G3T]5 probe can be applied to detect Ag(+) in a "turn on" format. Moreover, recognition of target DNA via hybridization to a molecular beacon (MB)-like probe (MB-[G3T]5) can unfold the MB-[G3T]5 to release the [G3T]5 for sensitizing the luminescence of Tb(3+), producing a detectable signal directly proportional to the amount of target DNA of interest. This allows the development of a fascinating label-free MB probe for DNA sensing based on the luminescence of Tb(3+). Results and methods reported here suggest that a guanine/thymine-rich DNA

  14. Pyridine nucleotide coenzymes: Chemical, biological, and medical aspects. Vol. 2, Pt. A

    SciTech Connect

    Dolphin, D.; Poulson, R.; Avramovic, O.

    1987-01-01

    This text contains the following: History of the Pyridine Nucleotides Nomenclature; Evolution of Pyridine Nucleotide; Relationship Between Biosynthesis and Evolution; Crystal Structure; Coenzyme Conformations; Protein Interactions; Optical Spectroscopy of the Pyridine Nucleotides; Excited States of Pyridine Nucleotide Coenzymes; Fluorescence and Phosphorescence; Nuclear Magnetic Resonance Spectroscopy of Pyridine Nucleotides; Mass Spectrometry of Pyridine Nucleotides; Mechanism of Action of the Pyridine Nucleotides; Chemical Stability and Reactivity of Pyridine Nucleotide Coenzymes; Stereochemistry of Fatty Acid Biosynthesis and Metabolism; Kinetics of Pyridine Nucleotide-Utilizing Enzymes; Preparation and Properties of NAD and NADP Analogs; Model Studies and Biological Activity of Analogs; and Spin-Labeled Pyridine Nucleotide Derivatives.

  15. VUV and mid-UV photoabsorption cross sections of thin films of guanine and uracil: application on their photochemistry in the solar system.

    PubMed

    Saïagh, Kafila; Cottin, Hervé; Aleian, Aicha; Fray, Nicolas

    2015-04-01

    We present a photostability study of two nucleobases, guanine and uracil. For the first time, the photoabsorption cross-section spectra of these molecules in the solid phase were measured in the VUV and mid-UV domain (115≤λ≤300 nm). They show a quite similar absorption level throughout this wavelength range, highlighting the importance of considering the whole VUV and UV domain during photolysis experiments in the laboratory. Their photolysis constant (J) can be estimated from those measurements as follows: 2.2×10(-2) s(-1)±11% for guanine and 5.3×10(-2) s(-1)±14% for uracil. This work shows that (i) measuring kinetic constants from a direct and "traditional" photolysis of a thin sample in the laboratory suffers strong limitations and (ii) achieving this measurement requires comprehensive modeling of the radiative transfer that occurs in any sample not optically thin (i.e.,≤2 nm). Moreover, this work has provided other data of interest: the refractive index of solid guanine and of uracil at 650 nm are 1.52 (±0.01) and 1.39 (±0.02), respectively, and the integrated IR band strengths (A) of solid guanine between 3700 and 2120 cm(-1) (3.4×10(-16) cm·molecule(-1)±13%) and of solid uracil between 3400 and 1890 cm(-1) (2.1×10(-16) cm·molecule(-1)±21%).

  16. Fourier transform infrared spectroscopy study on order-disorder transition in Langmuir-Blodgett films of 7-(2-octadecyloxycarbonylethyl)guanine before and after recognition to cytidine.

    PubMed

    Miao, Wangen; Luo, Xuzhong; Wu, Sanxie; Liang, Yingqiu

    2004-01-01

    Order-disorder transitions of 9-monolayer Langmuir-Blodgett (LB) films of 7-(2-octadecyloxycarbonylethyl)guanine (ODCG) before and after recognition to cytidine were investigated by Fourier transform infrared (FTIR) spectroscopy. The different order-disorder transitions suggest that molecular recognition between ODCG and cytidine influence these two LB films on the order-disorder process of alkyl tailchain. Cleavage of the multi-hydrogen bonds was also observed by the infrared spectroscopy at elevated temperature.

  17. VUV and mid-UV photoabsorption cross sections of thin films of guanine and uracil: application on their photochemistry in the solar system.

    PubMed

    Saïagh, Kafila; Cottin, Hervé; Aleian, Aicha; Fray, Nicolas

    2015-04-01

    We present a photostability study of two nucleobases, guanine and uracil. For the first time, the photoabsorption cross-section spectra of these molecules in the solid phase were measured in the VUV and mid-UV domain (115≤λ≤300 nm). They show a quite similar absorption level throughout this wavelength range, highlighting the importance of considering the whole VUV and UV domain during photolysis experiments in the laboratory. Their photolysis constant (J) can be estimated from those measurements as follows: 2.2×10(-2) s(-1)±11% for guanine and 5.3×10(-2) s(-1)±14% for uracil. This work shows that (i) measuring kinetic constants from a direct and "traditional" photolysis of a thin sample in the laboratory suffers strong limitations and (ii) achieving this measurement requires comprehensive modeling of the radiative transfer that occurs in any sample not optically thin (i.e.,≤2 nm). Moreover, this work has provided other data of interest: the refractive index of solid guanine and of uracil at 650 nm are 1.52 (±0.01) and 1.39 (±0.02), respectively, and the integrated IR band strengths (A) of solid guanine between 3700 and 2120 cm(-1) (3.4×10(-16) cm·molecule(-1)±13%) and of solid uracil between 3400 and 1890 cm(-1) (2.1×10(-16) cm·molecule(-1)±21%). PMID:25836367

  18. Experimental and first-principles investigation of the adsorption and entrapping of guanine with SiO2 clusters of sol-gel silicate material for understanding DNA photodamage.

    PubMed

    Chandraboss, V L; Karthikeyan, B; Senthilvelan, S

    2015-05-14

    We report a first principles density functional theoretical (DFT) investigation of guanine (G) adsorption onto SiO2 clusters, viz., Si2O4, Si3O6, Si4O8 and Si5O10 in terms of geometry, binding energy (EB), binding site, energy gap (Eg), and electronic and spectral properties. Guanine entrapped within a Si9O18 cluster was also studied in terms of geometry, energy gap (Eg), electronic and spectral properties. We observed that the most stable forms of the cluster were Si5O10 and Si9O18. Guanine adsorbed onto SiO2 (G-SiO2) and guanine entrapped within SiO2 (GE-SiO2) were analyzed by the B3LYP/LanL2DZ method. The HOMO-LUMO energies illustrate that charge transfer from ligand to metal (L → M) occurs in G-SiO2 clusters as guanine to SiO2. The composite of guanine with nanostructured silica material was prepared by simple precipitation and chemical sol-gel processes. The prepared G-SiO2 and GE-SiO2 composites were characterized by FT-IR and FE-SEM with EDX analysis. The resulting experimental evidence is included for better understanding the guanine adsorption and entrapment. The adsorption and entrapping of G-SiO2 and GE-SiO2 was also confirmed by UV-vis spectroscopy. Experimental results are compared with the DFT results. Furthermore, the sol-gel silicate material used to protect the DNA base (guanine) from UVA-irradiation has been highlighted. PMID:25875309

  19. Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types.

    PubMed

    Momose, Fumiyasu; Seo, Naohiro; Akahori, Yasushi; Sawada, Shin-Ichi; Harada, Naozumi; Ogura, Toru; Akiyoshi, Kazunari; Shiku, Hiroshi

    2016-01-01

    Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases. PMID:27101102

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

    PubMed

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

    2015-02-20

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  2. Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis.

    PubMed

    Choi, Jungkweon; Yang, Cheolhee; Fujitsuka, Mamoru; Tojo, Sachiko; Ihee, Hyocherl; Majima, Tetsuro

    2015-12-17

    The oxidation of guanine (G) is studied by using transient absorption and time-resolved resonance Raman spectroscopies combined with pulse radiolysis. The transient absorption spectral change demonstrates that the neutral radical of G (G(•)(-H(+))), generated by the deprotonation of G radical cation (G(•+)), is rapidly converted to other G radical species. The formation of this species shows the pH dependence, suggesting that it is the G radical cation (G(•+))' formed from the protonation at the N7 of G(•)(-H(+)). On one hand, most Raman bands of (G(•+))' are up-shifted relative to those of G, indicating the increase in the bonding order of pyrimidine (Pyr) and imidazole rings. The (G(•+))' exhibits the characteristic CO stretching mode at ∼1266 cm(-1) corresponding to a C-O single bond, indicating that the unpaired electron in (G(•+))' is localized on the oxygen of the Pyr ring. PMID:26632994

  3. Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamster cells using the polymerase chain reaction

    SciTech Connect

    Xu, Z.D.; Yu, Y.J.; Hsie, A.W.; Caskey, C.T.; Rossiter, B.; Gibbs, R.A. )

    1989-01-01

    We have developed a rapid screening method using the polymerase chain reaction (PCR) for detecting deletion mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in Chinese hamster cells. DNA was extracted from spontaneous and ultraviolet (UV) light- and X-ray-induced hprt-deficient mutants. Two primer sets were used to amplify 276 bp and 344 bp fragments containing the entire exon 3 and exon 9 coding sequence, respectively. The PCR was performed using Taq DNA polymerase for 40 cycles, and the PCR product was directly analyzed for the presence of the respective amplified DNA using electrophoresis on agarose gels stained with ethidium bromide. With this assay, we have analyzed 39 independently derived hprt-deficient mutants. Four of ten spontaneous mutants were found to have deletions in exon 9. UV light produced mutants with predominantly wild-type amplification patterns (10/14). X-ray induced mostly deletion patterns (11/15); six of these occurred only in exon 9, and five occurred in both exons 3 and 9. These observations are consistent with the classical notion that UV light induces predominantly missense mutations and X-ray produces a high proportion of deletion mutations. Deletion mutations occurred most frequently at the 3' end of the hprt gene, suggesting the possible existence of hot spots for deletions in this region. The PCR assay for deletion detection has the advantage that it can be completed in less than 4 hr without using radioisotopes. This assay should be useful for routine deletion screening.

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

    PubMed

    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. PMID:25181345

  5. High level expression in Escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.

    PubMed Central

    Craig, S P; Yuan, L; Kuntz, D A; McKerrow, J H; Wang, C C

    1991-01-01

    The bacterial alkaline phosphatase (phoA) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in Escherichia coli. Other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (HPRT; hypoxanthine phosphoribosyltransferase; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), which carries part of the signal peptide but remains in the cytosol of the bacteria. Herein, the phoA promoter without its associated signal peptide is used to regulate expression of the HPRT of Schistosoma mansoni and the ornithine decarboxylase (ODC; L-ornithine carboxy-lyase, EC 4.1.1.17) of Trypanosoma brucei, two enzymes that have been identified as potential targets for antiparasitic chemotherapy. The levels of recombinant expression range from 20% to 60% of the total bacterial protein, and the majority of both recombinant enzymes was soluble. The specific activity for the recombinant trypanosomal ODC was one-third to two-thirds that of the authentic native enzyme and yields were predicted to be 15-30 mg of active enzyme per liter of bacterial culture. The specific activity for the recombinant schistosomal HPRT was equivalent to that for the native enzyme purified from schistosomes and up to 10 mg of enzymatically active HPRT has been purified from a 0.5-liter culture of treated bacteria. These results represent a break-through in recombinant expression of HPRT and ODC. Images PMID:2006185

  6. Guanine-Rich Sequences Are a Dominant Feature of Exosomal microRNAs across the Mammalian Species and Cell Types

    PubMed Central

    Momose, Fumiyasu; Seo, Naohiro; Akahori, Yasushi; Sawada, Shin-ichi; Harada, Naozumi; Ogura, Toru; Akiyoshi, Kazunari; Shiku, Hiroshi

    2016-01-01

    Exosome is an extracellular vesicle released from multivesicular endosomes and contains micro (mi) RNAs and functional proteins derived from the donor cells. Exosomal miRNAs act as an effector during communication with appropriate recipient cells, this can aid in the utilization of the exosomes in a drug delivery system for various disorders including malignancies. Differences in the miRNA distribution pattern between exosomes and donor cells indicate the active translocation of miRNAs into the exosome cargos in a miRNA sequence-dependent manner, although the molecular mechanism is little known. In this study, we statistically analyzed the miRNA microarray data and revealed that the guanine (G)-rich sequence is a dominant feature of exosome-dominant miRNAs, across the mammalian species-specificity and the cell types. Our results provide important information regarding the potential use of exosome cargos to develop miRNA-based drugs for the treatment of human diseases. PMID:27101102

  7. Enforced Presentation of an Extrahelical Guanine to the Lesion Recognition Pocket of Human 8-Oxoguanine Glycosylase, hOGG1

    SciTech Connect

    Crenshaw, Charisse M.; Nam, Kwangho; Oo, Kimberly; Kutchukian, Peter S.; Bowman, Brian R.; Karplus, Martin; Verdine, Gregory L.

    2012-09-05

    A poorly understood aspect of DNA repair proteins is their ability to identify exceedingly rare sites of damage embedded in a large excess of nearly identical undamaged DNA, while catalyzing repair only at the damaged sites. Progress toward understanding this problem has been made by comparing the structures and biochemical behavior of these enzymes when they are presented with either a target lesion or a corresponding undamaged nucleobase. Trapping and analyzing such DNA-protein complexes is particularly difficult in the case of base extrusion DNA repair proteins because of the complexity of the repair reaction, which involves extrusion of the target base from DNA followed by its insertion into the active site where glycosidic bond cleavage is catalyzed. Here we report the structure of a human 8-oxoguanine (oxoG) DNA glycosylase, hOGG1, in which a normal guanine from DNA has been forcibly inserted into the enzyme active site. Although the interactions of the nucleobase with the active site are only subtly different for G versus oxoG, hOGG1 fails to catalyze excision of the normal nucleobase. This study demonstrates that even if hOGG1 mistakenly inserts a normal base into its active site, the enzyme can still reject it on the basis of catalytic incompatibility.

  8. DNA-directed aniline mustards with high selectivity for adenine or guanine bases: mutagenesis in a variety of Salmonella typhimurium strains differing in DNA-repair capability.

    PubMed

    Ferguson, L R; Denny, W A; Boritzki, T J

    1994-04-01

    Two closely-related aniline monomustards (1 and 2), linked to a DNA-targeting acridine chromophore by a linker chain of different length, show high selectivity for alkylation of polymer DNA. The shorter-chain derivative (2) alkylates mainly at guanine N7 sites, while the longer-chain analogue (1) reacts almost exclusively at adenine N1. The biological effects of these compounds have been studied in standard Ames Salmonella typhimurium strains in order to determine the mutagenic consequences of such well-defined DNA lesions, and the effect of DNA-repair systems on them. Both compounds caused detectable mutations in strains TA1537, TA98 or TA100 and some related strains. Mutation rates were greatly enhanced in strains carrying either a uvrB deletion or the plasmid pKM101. Frameshift mutagenesis by both compounds was completely eliminated by recA deletion, in both the presence or absence of the plasmid. The adenine-selective compound (1) appeared more sensitive to the DNA-repair defects than the guanine-selective derivative (2). Additionally, only the adenine-selective compound (1) caused statistically significant levels of detectable mutation in the repair-proficient strains TA102, TA4001 or TA4006. The bacterial mutagenesis evidence suggests that a bulky, major groove-residing adenine lesion may be more readily recognised by DNA-repair systems, and more likely to lead to a wider range of mutagenic events, than a similar guanine lesion.

  9. Microbial metabolism of thiopurines: A method to measure thioguanine nucleotides.

    PubMed

    Movva, Ramya; Lobb, Michael; Ó Cuív, Páraic; Florin, Timothy H J; Duley, John A; Oancea, Iulia

    2016-09-01

    Thiopurines are anti-inflammatory prodrugs. We hypothesised that bacteria may contribute to conversion to active drug. Escherichia coli strain DH5α was evaluated to determine whether it could metabolise the thiopurine drugs, thioguanine or mercaptopurine, to thioguanine nucleotides. A rapid and reliable high performance liquid chromatography (ultraviolet detection) method was developed to quantify indirectly thioguanine nucleotides, by measuring thioguanine nucleoside. PMID:27444548

  10. From Single Nucleotide Polymorphism to Transcriptional Mechanism

    PubMed Central

    Martini, Sebastian; Nair, Viji; Patel, Sanjeevkumar R.; Eichinger, Felix; Nelson, Robert G.; Weil, E. Jennifer; Pezzolesi, Marcus G.; Krolewski, Andrzej S.; Randolph, Ann; Keller, Benjamin J.; Werner, Thomas; Kretzler, Matthias

    2013-01-01

    Genome-wide association studies have proven to be highly effective at defining relationships between single nucleotide polymorphisms (SNPs) and clinical phenotypes in complex diseases. Establishing a mechanistic link between a noncoding SNP and the clinical outcome is a significant hurdle in translating associations into biological insight. We demonstrate an approach to assess the functional context of a diabetic nephropathy (DN)-associated SNP located in the promoter region of the gene FRMD3. The approach integrates pathway analyses with transcriptional regulatory pattern-based promoter modeling and allows the identification of a transcriptional framework affected by the DN-associated SNP in the FRMD3 promoter. This framework provides a testable hypothesis for mechanisms of genomic variation and transcriptional regulation in the context of DN. Our model proposes a possible transcriptional link through which the polymorphism in the FRMD3 promoter could influence transcriptional regulation within the bone morphogenetic protein (BMP)-signaling pathway. These findings provide the rationale to interrogate the biological link between FRMD3 and the BMP pathway and serve as an example of functional genomics-based hypothesis generation. PMID:23434934

  11. Human molecular cytogenetics: From cells to nucleotides.

    PubMed

    Riegel, Mariluce

    2014-03-01

    The field of cytogenetics has focused on studying the number, structure, function and origin of chromosomal abnormalities and the evolution of chromosomes. The development of fluorescent molecules that either directly or via an intermediate molecule bind to DNA has led to the development of fluorescent in situ hybridization (FISH), a technology linking cytogenetics to molecular genetics. This technique has a wide range of applications that increased the dimension of chromosome analysis. The field of cytogenetics is particularly important for medical diagnostics and research as well as for gene ordering and mapping. Furthermore, the increased application of molecular biology techniques, such as array-based technologies, has led to improved resolution, extending the recognized range of microdeletion/microduplication syndromes and genomic disorders. In adopting these newly expanded methods, cytogeneticists have used a range of technologies to study the association between visible chromosome rearrangements and defects at the single nucleotide level. Overall, molecular cytogenetic techniques offer a remarkable number of potential applications, ranging from physical mapping to clinical and evolutionary studies, making a powerful and informative complement to other molecular and genomic approaches. This manuscript does not present a detailed history of the development of molecular cytogenetics; however, references to historical reviews and experiments have been provided whenever possible. Herein, the basic principles of molecular cytogenetics, the technologies used to identify chromosomal rearrangements and copy number changes, and the applications for cytogenetics in biomedical diagnosis and research are presented and discussed.

  12. Genetic epidemiology of single-nucleotide polymorphisms.

    PubMed

    Collins, A; Lonjou, C; Morton, N E

    1999-12-21

    On the causal hypothesis, most genetic determinants of disease are single-nucleotide polymorphisms (SNPs) that are likely to be selected as markers for positional cloning. On the proximity hypothesis, most disease determinants will not be included among markers but may be detected through linkage disequilibrium with other SNPs. In that event, allelic association among SNPs is an essential factor in positional cloning. Recent simulation based on monotonic population expansion suggests that useful association does not usually extend beyond 3 kb. This is contradicted by significant disequilibrium at much greater distances, with corresponding reduction in the number of SNPs required for a cost-effective genome scan. A plausible explanation is that cyclical expansions follow population bottlenecks that establish new disequilibria. Data on more than 1,000 locus pairs indicate that most disequilibria trace to the Neolithic, with no apparent difference between haplotypes that are random or selected through a major disease gene. Short duration may be characteristic of alleles contributing to disease susceptibility and haplotypes characteristic of particular ethnic groups. Alleles that are highly polymorphic in all ethnic groups may be older, neutral, or advantageous, in weak disequilibrium with nearby markers, and therefore less useful for positional cloning of disease genes. Significant disequilibrium at large distance makes the number of suitably chosen SNPs required for genome screening as small as 30,000, or 1 per 100 kb, with greater density (including less common SNPs) reserved for candidate regions.

  13. Nucleotide Excision Repair in Caenorhabditis elegans

    PubMed Central

    Lans, Hannes; Vermeulen, Wim

    2011-01-01

    Nucleotide excision repair (NER) plays an essential role in many organisms across life domains to preserve and faithfully transmit DNA to the next generation. In humans, NER is essential to prevent DNA damage-induced mutation accumulation and cell death leading to cancer and aging. NER is a versatile DNA repair pathway that repairs many types of DNA damage which distort the DNA helix, such as those induced by solar UV light. A detailed molecular model of the NER pathway has emerged from in vitro and live cell experiments, particularly using model systems such as bacteria, yeast, and mammalian cell cultures. In recent years, the versatility of the nematode C. elegans to study DNA damage response (DDR) mechanisms including NER has become increasingly clear. In particular, C. elegans seems to be a convenient tool to study NER during the UV response in vivo, to analyze this process in the context of a developing and multicellular organism, and to perform genetic screening. Here, we will discuss current knowledge gained from the use of C. elegans to study NER and the response to UV-induced DNA damage. PMID:22091407

  14. The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1).

    PubMed

    Seye, Cheikh I; Yu, Ningpu; González, Fernando A; Erb, Laurie; Weisman, Gary A

    2004-08-20

    UTP stimulates the expression of pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells through activation of the P2Y(2) nucleotide receptor P2Y(2)R. Here, we demonstrated that activation of the P2Y(2)R induced rapid tyrosine phosphorylation of vascular endothelial growth factor receptor (VEGFR)-2 in human coronary artery endothelial cells (HCAEC). RNA interference targeting VEGFR-2 or inhibition of VEGFR-2 tyrosine kinase activity abolishes P2Y(2)R-mediated VCAM-1 expression. Furthermore, VEGFR-2 and the P2Y(2)R co-localize upon UTP stimulation. Deletion or mutation of two Src homology-3-binding sites in the C-terminal tail of the P2Y(2)R or inhibition of Src kinase activity abolished the P2Y(2)R-mediated transactivation of VEGFR-2 and subsequently inhibited UTP-induced VCAM-1 expression. Moreover, activation of VEGFR-2 by UTP leads to the phosphorylation of Vav2, a guanine nucleotide exchange factor for Rho family GTPases. Using a binding assay to measure the activity of the small GTPases Rho, we found that stimulation of HCAEC by UTP increased the activity of RhoA and Rac1 (but not Cdc42). Significantly, a dominant negative form of RhoA inhibited P2Y(2)R-mediated VCAM-1 expression, whereas expression of dominant negative forms of Cdc42 and Rac1 had no effect. These data indicate a novel mechanism whereby a nucleotide receptor transactivates a receptor tyrosine kinase to generate an inflammatory response associated with atherosclerosis.

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

    PubMed

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

    2011-04-01

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

  16. Oxidative DNA Damage and Nucleotide Excision Repair

    PubMed Central

    Melis, Joost P.M.; Luijten, Mirjam

    2013-01-01

    Abstract Significance: Oxidative DNA damage is repaired by multiple, overlapping DNA repair pathways. Accumulating evidence supports the hypothesis that nucleotide excision repair (NER), besides base excision repair (BER), is also involved in neutralizing oxidative DNA damage. Recent Advances: NER includes two distinct sub-pathways: transcription-coupled NER (TC-NER) and global genome repair (GG-NER). The CSA and CSB proteins initiate the onset of TC-NER. Recent findings show that not only CSB, but also CSA is involved in the repair of oxidative DNA lesions, in the nucleus as well as in mitochondria. The XPG protein is also of importance for the removal of oxidative DNA lesions, as it may enhance the initial step of BER. Substantial evidence exists that support a role for XPC in NER and BER. XPC deficiency not only results in decreased repair of oxidative lesions, but has also been linked to disturbed redox homeostasis. Critical Issues: The role of NER proteins in the regulation of the cellular response to oxidative (mitochondrial and nuclear) DNA damage may be the underlying mechanism of the pathology of accelerated aging in Cockayne syndrome patients, a driving force for internal cancer development in XP-A and XP-C patients, and a contributor to the mixed exhibited phenotypes of XP-G patients. Future Directions: Accumulating evidence indicates that DNA repair factors can be involved in multiple DNA repair pathways. However, the distinct detailed mechanism and consequences of these additional functions remain to be elucidated and can possibly shine a light on clinically related issues. Antioxid. Redox Signal. 18, 2409–2419. PMID:23216312

  17. The role of dietary nucleotides in single-stomached animals.

    PubMed

    Sauer, Nadja; Mosenthin, Rainer; Bauer, Eva

    2011-06-01

    The transition from liquid to solid feed during weaning results in morphological, histological and microbial changes in the young animal's intestinal tract and often is associated with diarrhoea. The ban of in-feed antibiotics in pig production in the European Union has led to increasing interest in alternatives to overcome weaning-associated problems. Among others, nucleotides may have the potential to alleviate health impairments due to weaning. Nucleotides are natural components of the non-protein fraction of milk and have important effects on the maintenance of health in young animals. Nucleotides and their related metabolic products play key roles in many biological processes and become essential dietary components when endogenous supply is insufficient for normal function. The present review summarises nucleotide composition of milk from different species, the biology of nucleotides and possible effects of dietary nucleotides on intestinal morphology and function, intestinal microbiota, immune function, nutrient metabolism, hepatic morphology and function as well as growth performance. Special attention is given to data available for pigs, and suggestions are made for inclusion of nucleotides in the diet to benefit piglets' health and reduce the consequences accompanying early weaning. PMID:21226977

  18. Levels of N7-(2-hydroxyethyl)guanine as a molecular dosimeter of drug delivery to human brain tumors.

    PubMed Central

    Bodell, W. J.; Giannini, D. D.; Hassenbusch, S.; Levin, V. A.

    2001-01-01

    The level of N7-(2-hydroxyethyl)guanine (N7-HOEtG), one of the DNA alkylation products formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment, was measured in human brain tumor samples by high performance liquid chromatography with electrochemical detection. The tumors from 6 recurrent chemotherapy-naive patients with recurrent glioblastoma multiforme were analyzed as controls. The mean level of N7-HOEtG in DNA of these specimens was 0.42 pmol/mg DNA. Samples were also obtained from a patient with a recurrent glioblastoma multiforme after direct intratumoral therapy with BCNU in ethanol (DTI-015). The levels of N7-HOEtG in the samples distal, medial, and adjacent to the site of injection were 0.8, 2.6, and 369.5 pmol/mg DNA, respectively. Comparison of the level of N7-HOEtG detected in the distal sample after injection with BCNU in ethanol with the mean level of the untreated samples indicated that it was not sufficiently different to be ruled out as a chance occurrence. Comparison of the levels of N7-HOEtG in the medial and adjacent brain tumor samples with the mean level of the control samples showed values that were approximately 6- and 879-fold higher. These results demonstrate that intratumoral administration of BCNU in ethanol produces significant levels of DNA alkylation and suggest that DNA adduct measurements provide a unique molecular dosimeter to evaluate delivery of alkylating agents to brain tumors. PMID:11584893

  19. Role of Bacillus subtilis Error Prevention Oxidized Guanine System in Counteracting Hexavalent Chromium-Promoted Oxidative DNA Damage

    PubMed Central

    Santos-Escobar, Fernando; Gutiérrez-Corona, J. Félix

    2014-01-01

    Chromium pollution is potentially detrimental to bacterial soil communities, compromising carbon and nitrogen cycles that are essential for life on earth. It has been proposed that intracellular reduction of hexavalent chromium [Cr(VI)] to trivalent chromium [Cr(III)] may cause bacterial death by a mechanism that involves reactive oxygen species (ROS)-induced DNA damage; the molecular basis of the phenomenon was investigated in this work. Here, we report that Bacillus subtilis cells lacking a functional error prevention oxidized guanine (GO) system were significantly more sensitive to Cr(VI) treatment than cells of the wild-type (WT) strain, suggesting that oxidative damage to DNA is involved in the deleterious effects of the oxyanion. In agreement with this suggestion, Cr(VI) dramatically increased the ROS concentration and induced mutagenesis in a GO-deficient B. subtilis strain. Alkaline gel electrophoresis (AGE) analysis of chromosomal DNA of WT and ΔGO mutant strains subjected to Cr(VI) treatment revealed that the DNA of the ΔGO strain was more susceptible to DNA glycosylase Fpg attack, suggesting that chromium genotoxicity is associated with 7,8-dihydro-8-oxodeoxyguanosine (8-oxo-G) lesions. In support of this notion, specific monoclonal antibodies detected the accumulation of 8-oxo-G lesions in the chromosomes of B. subtilis cells subjected to Cr(VI) treatment. We conclude that Cr(VI) promotes mutagenesis and cell death in B. subtilis by a mechanism that involves radical oxygen attack of DNA, generating 8-oxo-G, and that such effects are counteracted by the prevention and repair GO system. PMID:24973075

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

    PubMed Central

    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. PMID:27458535

  1. Nucleic acid analysis using terminal-phosphate-labeled nucleotides

    DOEpatents

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

    2008-04-22

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

  2. (/sup 3/H)dihydroergotamine as a high-affinity, slowly dissociating radioligand for 5-HT1B binding sites in rat brain membranes: evidence for guanine nucleotide regulation of agonist affinity states

    SciTech Connect

    Hamblin, M.W.; Ariani, K.; Adriaenssens, P.I.; Ciaranello, R.D.

    1987-12-01

    (/sup 3/H)Dihydroergotamine (DE) labels a population of binding sites in rat brain membranes with an affinity of approximately 70 pM in both hippocampus (maximal binding at saturation (Bmax) = 340 fmol/mg of protein) and cerebral cortex (Bmax = 250 fmol/mg of protein). Specific binding typically comprises about 97% of total binding at the Kd of the radioligand when nonspecific binding is determined in the presence of 100 nM unlabeled DE. Association kinetics at 37 degrees C are consistent with a uniform association rate constant for all sites labeled. Specific binding is completely reversible with addition of excess unlabeled DE, but dissociation does not proceed with simple first-order kinetics, suggesting the presence of more than one discrete binding site. Competition studies with selective drugs reveal alpha adrenergic, 5-HT1A and 5-HT1B components of (/sup 3/H)DE specific binding. When phentolamine (500 nM) is included to block alpha receptors and DPAT (100 nM) or spiroxatrine (500 nM) is included to block 5-HT1A receptors, specific binding is exclusively to sites with drug affinities characteristic of 5-HT1B receptors. Under these 5-HT1B-selective conditions, (/sup 3/H)DE binding is about 90% specific, with a Kd of about 50 to 60 pM and a Bmax of 96 fmol/mg of protein in hippocampus and 77 fmol/mg of protein in cortex. (/sup 3/H)DE binding to 5-HT1B sites is very slowly dissociable, with a T1/2 of greater than 2 h at 37 degrees C. 5-HT1B antagonists and DE itself yield competition curves at (/sup 3/H)DE-labeled 5-HT1B sites that are adequately fit assuming a single site in nonlinear regression analysis. Addition of 100 microM guanylyl 5'-imidodiphosphate appears to convert nearly all 5-HT1B sites to those having low affinity for agonists while having a much smaller effect on the binding of (/sup 3/H)DE.

  3. Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function.

    PubMed

    Houslay, M D; Gawler, D J; Milligan, G; Wilson, A

    1989-01-01

    Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats. PMID:2561940

  4. Stimulation of human thyroid growth via the inhibitory guanine nucleotide binding (G) protein Gi: constitutive expression of the G-protein alpha subunit Gi alpha-1 in autonomous adenoma.

    PubMed Central

    Selzer, E; Wilfing, A; Schiferer, A; Hermann, M; Grubeck-Loebenstein, B; Freissmuth, M

    1993-01-01

    The alpha subunits of the stimulatory and inhibitory G proteins, Gs alpha and Gi alpha, activate transmembrane-signaling systems involved in the control of cell proliferation. We have investigated the pattern of expression of Gi alpha subtypes and Gi alpha-mediated proliferative responses in the human thyroid. Human thyroid membranes contain two subtypes of Gi alpha, Gi alpha-1 and Gi alpha-2, as assessed by using specific antibodies. The expression of Gi alpha-1 is under tight control by thyrotropin in vivo and in primary cultures of thyroid epithelial cells. In contrast, Gi alpha-1 is expressed in the absence of thyrotropin in thyroid autonomous adenoma, an endocrine-active tumor, and its levels are not regulated by thyrotropin in thyroid epithelial cells prepared from these tumors. If thyroid epithelial cells are treated with pertussis toxin to block signal transduction via Gi, the mitogenic response to serum factors is reduced. These observations demonstrate that Gi subtypes transmit growth stimuli in the human thyroid. The constitutive expression of Gi alpha-1 in autonomous adenoma may allow for the unregulated stimulation of thyroid cell proliferation by a yet unidentified signaling pathway and, thus, be causally related to autonomous growth of thyroid cells. Images PMID:8434024

  5. Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter.

    PubMed Central

    Horisberger, M A; McMaster, G K; Zeller, H; Wathelet, M G; Dellis, J; Content, J

    1990-01-01

    The human protein p78 is induced and accumulated in cells treated with type I interferon or with some viruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-length cDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein. The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2 terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-alpha) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cells treated with IFN-alpha. In contrast, the p78-related protein was not expressed at detectable levels. The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-alpha was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins. Images PMID:2154602

  6. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions.

    PubMed

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao

    2016-06-01

    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation.

  7. Identifying 2'-O-methylationation sites by integrating nucleotide chemical properties and nucleotide compositions.

    PubMed

    Chen, Wei; Feng, Pengmian; Tang, Hua; Ding, Hui; Lin, Hao

    2016-06-01

    2'-O-methylationation is an important post-transcriptional modification and plays important roles in many biological processes. Although experimental technologies have been proposed to detect 2'-O-methylationation sites, they are cost-ineffective. As complements to experimental techniques, computational methods will facilitate the identification of 2'-O-methylationation sites. In the present study, we proposed a support vector machine-based method to identify 2'-O-methylationation sites. In this method, RNA sequences were formulated by nucleotide chemical properties and nucleotide compositions. In the jackknife cross-validation test, the proposed method obtained an accuracy of 95.58% for identifying 2'-O-methylationation sites in the human genome. Moreover, the model was also validated by identifying 2'-O-methylation sites in the Mus musculus and Saccharomyces cerevisiae genomes, and the obtained accuracies are also satisfactory. These results indicate that the proposed method will become a useful tool for the research on 2'-O-methylation. PMID:27191866

  8. Receptor binding of somatostatin-14 and somatostatin-28 in rat brain: differential modulation by nucleotides and ions.

    PubMed

    Srikant, C B; Dahan, A; Craig, C

    1990-02-01

    The tissue-selective binding of the two principal bioactive forms of somatostatin, somatostatin-14 (SS-14) and somatostatin-28 (SS-28), their ability to modulate cAMP-dependent and -independent regulation of post-receptor events to different degrees and the documentation of specific labelling of SS receptor subtypes with SS-28 but not SS-14 in discrete regions of rat brain suggest the existence of distinct SS-14 and SS-28 binding sites. Receptor binding of SS-14 ligands has been shown to be modulated by nucleotides and ions, but the effect of these agents on SS-28 binding has not been studied. In the present study we investigated the effects of adenine and guanine nucleotides as well as monovalent and divalent cations on rat brain SS receptors quantitated with radioiodinated analogs of SS-14 ([125I-Tyr11]SS14, referred to in this paper as SS-14) and SS-28 ([Leu8, D-Trp22, 125I-Tyr25] SS-28, referred to as LTT* SS-28) in order to determine if distinct receptor sites for SS-14 and SS-28 could be distinguished on the basis of their modulation by nucleotides and ions. GTP as well as ATP exerted a dose-dependent inhibition (over a concentration range of 10(-7)-10(-3) M) of the binding of the two radioligands. The nucleotide inhibition of binding resulted in a decrease the Bmax of the SS receptors, the binding affinity remaining unaltered. GTP (10(-4) M) decreased the Bmax of LTT* SS-28 binding sites to a greater extent than ATP (145 +/- 10 and 228 +/- 16 respectively, compared to control value of 320 +/- 20 pmol mg-1). Under identical conditions GTP was less effective than ATP in reducing the number of T* SS-14 binding sites (Bmax = 227 +/- 8 and 182 +/- 15, respectively, compared to 340 +/- 15 pmol mg-1 in the absence of nucleotides). Monovalent cations inhibited the binding of both radioligands, Li+ and Na+ inhibited the binding of T* SS-14 to a greater extent than K+. The effect of divalent cations on the other hand was varied. At low concentration (2 mM) Mg2+, Ba2

  9. Nucleotide excision repair of DNA: The very early history.

    PubMed

    Friedberg, Errol C

    2011-07-15

    This article, taken largely from the book Correcting the Blueprint of Life: An Historical Account of the Discovery of DNA Repair Mechanisms, summarizes the very early history of the discovery of nucleotide excision repair.

  10. ATP-Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling.

    PubMed

    Ji, Debin; Mohsen, Michael G; Harcourt, Emily M; Kool, Eric T

    2016-02-01

    A new strategy is reported for the production of luminescence signals from DNA synthesis through the use of chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. ATP-releasing nucleotides (ARNs) were synthesized as derivatives of the four canonical nucleotides. All four derivatives are good substrates for DNA polymerase, with Km values averaging 13-fold higher than those of natural dNTPs, and kcat values within 1.5-fold of those of native nucleotides. Importantly, ARNs were found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. When using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, thereby allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs.

  11. 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

    PubMed Central

    2015-01-01

    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 14Rtrans-anti-DB[a,l]P-N2-dG and the stereochemically analogous 10S- and 10R-B[a]P-N2-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N2-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N2-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. PMID

  12. Reducing nontemplated 3' nucleotide addition to polynucleotide transcripts

    DOEpatents

    Kao, C. Cheng

    2000-01-01

    Non-template 3' nucleotide addition to a transcript is reduced by transcribing a transcript from a template comprising an ultimate and/or penultimate 5' ribose having a C'2 substituent such as methoxy, which reduces non-template 3' nucleotide addition to the transcript. The methods are shown to be applicable to a wide variety of polymerases, including Taq, T7 RNA polymerase, etc.

  13. Mechanism of repair of acrolein- and malondialdehyde-derived exocyclic guanine adducts by the α-ketoglutarate/Fe(II) dioxygenase AlkB.

    PubMed

    Singh, Vipender; Fedeles, Bogdan I; Li, Deyu; Delaney, James C; Kozekov, Ivan D; Kozekova, Albena; Marnett, Lawrence J; Rizzo, Carmelo J; Essigmann, John M

    2014-09-15

    The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA.

  14. Nucleotide diversity analysis highlights functionally important genomic regions

    PubMed Central

    Tatarinova, Tatiana V.; Chekalin, Evgeny; Nikolsky, Yuri; Bruskin, Sergey; Chebotarov, Dmitry; McNally, Kenneth L.; Alexandrov, Nickolai

    2016-01-01

    We analyzed functionality and relative distribution of genetic variants across the complete Oryza sativa genome, using the 40 million single nucleotide polymorphisms (SNPs) dataset from the 3,000 Rice Genomes Project (http://snp-seek.irri.org), the largest and highest density SNP collection for any higher plant. We have shown that the DNA-binding transcription factors (TFs) are the most conserved group of genes, whereas kinases and membrane-localized transporters are the most variable ones. TFs may be conserved because they belong to some of the most connected regulatory hubs that modulate transcription of vast downstream gene networks, whereas signaling kinases and transporters need to adapt rapidly to changing environmental conditions. In general, the observed profound patterns of nucleotide variability reveal functionally important genomic regions. As expected, nucleotide diversity is much higher in intergenic regions than within gene bodies (regions spanning gene models), and protein-coding sequences are more conserved than untranslated gene regions. We have observed a sharp decline in nucleotide diversity that begins at about 250 nucleotides upstream of the transcription start and reaches minimal diversity exactly at the transcription start. We found the transcription termination sites to have remarkably symmetrical patterns of SNP density, implying presence of functional sites near transcription termination. Also, nucleotide diversity was significantly lower near 3′ UTRs, the area rich with regulatory regions. PMID:27774999

  15. Single nucleotide polymorphisms in nucleotide excision repair genes, cancer treatment, and head and neck cancer survival

    PubMed Central

    Wyss, Annah B.; Weissler, Mark C.; Avery, Christy L.; Herring, Amy H.; Bensen, Jeannette T.; Barnholtz-Sloan, Jill S.; Funkhouser, William K.

    2014-01-01

    Purpose Head and neck cancers (HNC) are commonly treated with radiation and platinum-based chemotherapy, which produce bulky DNA adducts to eradicate cancerous cells. Because nucleotide excision repair (NER) enzymes remove adducts, variants in NER genes may be associated with survival among HNC cases both independently and jointly with treatment. Methods Cox proportional hazards models were used to estimate race-stratified (White, African American) hazard ratios (HRs) and 95 % confidence intervals for overall (OS) and disease-specific (DS) survival based on treatment (combinations of surgery, radiation, and chemotherapy) and 84 single nucleotide polymorphisms (SNPs) in 15 NER genes among 1,227 HNC cases from the Carolina Head and Neck Cancer Epidemiology Study. Results None of the NER variants evaluated were associated with survival at a Bonferroni-corrected alpha of 0.0006. However, rs3136038 [OS HR = 0.79 (0.65, 0.97), DS HR = 0.69 (0.51, 0.93)] and rs3136130 [OS HR = 0.78 (0.64, 0.96), DS HR = 0.68 (0.50, 0.92)] of ERCC4 and rs50871 [OS HR = 0.80 (0.64, 1.00), DS HR = 0.67 (0.48, 0.92)] of ERCC2 among Whites, and rs2607755 [OS HR = 0.62 (0.45, 0.86), DS HR = 0.51 (0.30, 0.86)] of XPC among African Americans were suggestively associated with survival at an uncorrected alpha of 0.05. Three SNP-treatment joint effects showed possible departures from additivity among Whites. Conclusions Our study, a large and extensive evaluation of SNPs in NER genes and HNC survival, identified mostly null associations, though a few variants were suggestively associated with survival and potentially interacted additively with treatment. PMID:24487794

  16. Structure of Low-Lying Excited States of Guanine in DNA and Solution: Combined Molecular Mechanics and High-Level Coupled Cluster Studies

    DOE PAGES

    Kowalski, Karol; Valiev, Marat

    2007-01-01

    High-level ab-initio equation-of-motion coupled-cluster methods with singles, doubles, and noniterative triples are used, in conjunction with the combined quantum mechanical molecular mechanics approach, to investigate the structure of low-lying excited states of the guanine base in DNA and solvated environments. Our results indicate that while the excitation energy of the first excited state is barely changed compared to its gas-phase counterpart, the excitation energy of the second excited state is blue-shifted by 0.24 eV.

  17. An ab initio study of electronic structure and spectra of 8-bromoguanine: a comparative study with guanine.

    PubMed

    Mishra, S K; Mishra, P C

    2001-10-01

    Ground state geometries of the four tautomeric forms keto-N9H, keto-N7H, enol-N9H and enol-N7H of 8-bromoguanine (8BG) were optimized using the ab initio RHF procedure employing a mixed basis set consisting of the 6-311 + G* basis set for the nitrogen atom of the amino group and the bromine atom, and the 4-31G basis set for all other atoms. These calculations were followed by correlation correction of the total energy at the MP2 level using the same basis set. The different tautomeric forms of 8BG in the ground state were solvated using the isodensity surface polarized continuum model (IPCM) of the SCRF theory both at the RHF and MP2 levels. Excited states were generated employing configuration interaction among singly excited configurations (CIS) obtained using a limited window of filled and empty molecular orbitals. Formation of hydrogen-bonded complexes between 8BG and three water molecules in the ground and excited states was considered in order to account for solvent effects approximately. Excited state geometry was optimized in each case for the lowest singlet excited state which was found to be of pi-pi* type. Vibrational frequency analysis was performed in order to ensure that the stationary points located on the potential energy surfaces by geometry optimization were minima. It is found that 8BG would occur in the ground state dominantly in the keto-N7H form both at the aqueous solution-air interface and inside the bulk liquid. The observed absorption and fluorescence spectra of 8BG can be explained satisfactorily considering only the keto-N7H form of the molecule. The enol tautomers of 8BG do not appear to be important from the point of view of ground state properties or electronic spectra. The observed differences between the behaviors of guanine and 8BG can be easily explained on the basis of the results obtained.

  18. Ras activation in response to phorbol ester proceeds independently of the EGFR via an unconventional nucleotide-exchange factor system in COS-7 cells.

    PubMed

    Rubio, Ignacio; Rennert, Knut; Wittig, Ute; Beer, Katrin; Dürst, Matthias; Stang, Stacey L; Stone, Jim; Wetzker, Reinhard

    2006-09-01

    Ras is a major mediator of PE (phorbol ester) effects in mammalian cells. Various mechanisms for PE activation of Ras have been reported [Downward, Graves, Warne, Rayter and Cantrell (1990) Nature (London) 346, 719-723; Shu, Wu, Mosteller and Broek (2002) Mol. Cell. Biol. 22, 7758-7768; Roose, Mollenauer, Gupta, Stone and Weiss (2005) Mol. Cell. Biol. 25, 4426-4441; Grosse, Roelle, Herrlich, Höhn and Gudermann (2000) J. Biol. Chem. 275, 12251-12260], including pathways that target GAPs (GTPase-activating proteins) for inactivation and those that result in activation of GEFs (guanine nucleotide-exchange factors) Sos (son of sevenless homologue) or RasGRP (RAS guanyl releasing protein). However, a biochemical link between PE and GAP inactivation is missing and GEF stimulation is hard to reconcile with the observation that dominant-negative S17N-Ras does not compromise Ras-dependent ERK (extracellular-signal-regulated kinase) activation by PE. We have addressed this controversy and carried out an in-depth biochemical study of PE-induced Ras activation in COS-7 cells. Using a cell-permeabilization approach to monitor nucleotide exchange on Ras, we demonstrate that PE-induced Ras-GTP accumulation results from GEF stimulation. Nucleotide exchange stimulation by PE is prevented by PKC (protein kinase C) inhibition but not by EGFR [EGF (epidermal growth factor) receptor] blockade, despite the fact that EGFR inhibition aborts basal and PE-induced Shc (Src homology and collagen homology) phosphorylation and Shc-Grb2 (growth-factor-receptor-bound protein 2) association. In fact, EGFR inhibition ablates basal nucleotide exchange on Ras in growth-arrested COS-7 cells. These data disclose the existence of two separate GEF systems that operate independently from each other to accomplish PE-dependent formation of Ras-GTP and to maintain resting Ras-GTP levels respectively. We document that COS-7 cells do not express RasGRP and present evidence that the PE-responsive GEF system

  19. Prolonged nonhydrolytic interaction of nucleotide with CFTR's NH2-terminal nucleotide binding domain and its role in channel gating.

    PubMed

    Basso, Claudia; Vergani, Paola; Nairn, Angus C; Gadsby, David C

    2003-09-01

    CFTR, the protein defective in cystic fibrosis, functions as a Cl- channel regulated by cAMP-dependent protein kinase (PKA). CFTR is also an ATPase, comprising two nucleotide-binding domains (NBDs) thought to bind and hydrolyze ATP. In hydrolyzable nucleoside triphosphates, PKA-phosphorylated CFTR channels open into bursts, lasting on the order of a second, from closed (interburst) intervals of a second or more. To investigate nucleotide interactions underlying channel gating, we examined photolabeling by [alpha32P]8-N3ATP or [gamma32P]8-N3ATP of intact CFTR channels expressed in HEK293T cells or Xenopus oocytes. We also exploited split CFTR channels to distinguish photolabeling at NBD1 from that at NBD2. To examine simple binding of nucleotide in the absence of hydrolysis and gating reactions, we photolabeled after incubation at 0 degrees C with no washing. Nucleotide interactions under gating conditions were probed by photolabeling after incubation at 30 degrees C, with extensive washing, also at 30 degrees C. Phosphorylation of CFTR by PKA only slightly influenced photolabeling after either protocol. Strikingly, at 30 degrees C nucleotide remained tightly bound at NBD1 for many minutes, in the form of nonhydrolyzed nucleoside triphosphate. As nucleotide-dependent gating of CFTR channels occurred on the time scale of seconds under comparable conditions, this suggests that the nucleotide interactions, including hydrolysis, that time CFTR channel opening and closing occur predominantly at NBD2. Vanadate also appeared to act at NBD2, presumably interrupting its hydrolytic cycle, and markedly delayed termination of channel open bursts. Vanadate somewhat increased the magnitude, but did not alter the rate, of the slow loss of nucleotide tightly bound at NBD1. Kinetic analysis of channel gating in Mg8-N3ATP or MgATP reveals that the rate-limiting step for CFTR channel opening at saturating [nucleotide] follows nucleotide binding to both NBDs. We propose that ATP

  20. The tail domain of myosin M catalyses nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions.

    PubMed

    Geissler, H; Ullmann, R; Soldati, T

    2000-05-01

    Members of the myosin superfamily play crucial roles in cellular processes including management of the cortical cytoskeleton, organelle transport and signal transduction. GTPases of the Rho family act as key control elements in the reorganization of the actin cytoskeleton in response to growth factors, and other functions such as membrane trafficking, transcriptional regulation, growth control and development. Here, we describe a novel unconventional myosin from Dictyostelium discoideum, MyoM. Primary sequence analysis revealed that it has the appearance of a natural chimera between a myosin motor domain and a guanine nucleotide exchange factor (GEF) domain for Rho GTPases. The functionality of both domains was established. Binding of the motor domain to F-actin was ATP-dependent and potentially regulated by phosphorylation. The GEF domain displayed selective activity on Rac1-related GTPases. Overexpression, rather than absence of MyoM, affected the cell morphology and viability. Particularly in response to hypo-osmotic stress, cells overexpressing the MyoM tail domain extended massive actin-driven protrusions. The GEF was enriched at the tip of growing protuberances, probably through its pleckstrin homology domain. MyoM is the first unconventional myosin containing an active Rac-GEF domain, suggesting a role at the interface of Rac-mediated signal transduction and remodeling of the actin cytoskeleton. PMID:11208126

  1. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

  2. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

  3. Axonal transport of muscarinic cholinergic receptors in rat vagus nerve: high and low affinity agonist receptors move in opposite directions and differ in nucleotide sensitivity

    SciTech Connect

    Zarbin, M.A.; Wamsley, J.K.; Kuhar, M.J.

    1982-07-01

    The presence and transport of muscarinic cholinergic binding sites have been detected in the rat vagus nerve. These binding sites accumulate both proximal and distal to ligatures in a time-dependent manner. The results of double ligature and colchicine experiments are compatible with the notion that the anterogradely transported binding sites move by fast transport. Most of the sites accumulating proximal to ligatures bind the agonist carbachol with high affinity, while most of the sites accumulating distally bind carbachol with a low affinity. Also, the receptors transported in the anterograde direction are affected by a guanine nucleotide analogue (GppNHp), while those transported in the retrograde direction are less, or not, affected. The bulk of the sites along the unligated nerve trunk bind carbachol with a low affinity and are less sensitive to GppNHp modulation than the anterogradely transported sites. These results suggest that some receptors in the vagus may undergo axonal transport in association with regulatory proteins and that receptor molecules undergo changes in their binding and regulatory properties during their life cycle. These data also support the notion that the high and low affinity agonist form of the muscarinic receptor represent different modulated forms of a single receptor molecule.

  4. Universal 1/f noise, crossovers of scaling exponents, and chromosome-specific patterns of guanine-cytosine content in DNA sequences of the human genome

    NASA Astrophysics Data System (ADS)

    Li, Wentian; Holste, Dirk

    2005-04-01

    Spatial fluctuations of guanine and cytosine base content (GC%) are studied by spectral analysis for the complete set of human genomic DNA sequences. We find that (i) 1/fα decay is universally observed in the power spectra of all 24 chromosomes, and (ii) the exponent α≈1 extends to about 107 bases, one order of magnitude longer than has previously been observed. We further find that (iii) almost all human chromosomes exhibit a crossover from α1≈1 (1/fα1) at lower frequency to α2<1 (1/fα2) at higher frequency, typically occurring at around 30 000-100 000 bases, while (iv) the crossover in this frequency range is virtually absent in human chromosome 22. In addition to the universal 1/fα noise in power spectra, we find (v) several lines of evidence for chromosome-specific correlation structures, including a 500 000 base long oscillation in human chromosome 21. The universal 1/fα spectrum in the human genome is further substantiated by a resistance to reduction in variance of guanine and cytosine content when the window size is increased.

  5. Moss Phylogeny Reconstruction Using Nucleotide Pangenome of Complete Mitogenome Sequences.

    PubMed

    Goryunov, D V; Nagaev, B E; Nikolaev, M Yu; Alexeevski, A V; Troitsky, A V

    2015-11-01

    Stability of composition and sequence of genes was shown earlier in 13 mitochondrial genomes of mosses (Rensing, S. A., et al. (2008) Science, 319, 64-69). It is of interest to study the evolution of mitochondrial genomes not only at the gene level, but also on the level of nucleotide sequences. To do this, we have constructed a "nucleotide pangenome" for mitochondrial genomes of 24 moss species. The nucleotide pangenome is a set of aligned nucleotide sequences of orthologous genome fragments covering the totality of all genomes. The nucleotide pangenome was constructed using specially developed new software, NPG-explorer (NPGe). The stable part of the mitochondrial genome (232 stable blocks) is shown to be, on average, 45% of its length. In the joint alignment of stable blocks, 82% of positions are conserved. The phylogenetic tree constructed with the NPGe program is in good correlation with other phylogenetic reconstructions. With the NPGe program, 30 blocks have been identified with repeats no shorter than 50 bp. The maximal length of a block with repeats is 140 bp. Duplications in the mitochondrial genomes of mosses are rare. On average, the genome contains about 500 bp in large duplications. The total length of insertions and deletions was determined in each genome. The losses and gains of DNA regions are rather active in mitochondrial genomes of mosses, and such rearrangements presumably can be used as additional markers in the reconstruction of phylogeny. PMID:26615445