Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Nucleotides

  • adenine nucleotide
  • cyclic nucleotide
  • dietary nucleotide
  • extracellular nucleotide
  • other nucleotide
  • purine nucleotide
  • single nucleotide
  • sugar nucleotide

  • Terms modified by Nucleotides

  • nucleotide analogue
  • nucleotide base
  • nucleotide binding
  • nucleotide binding protein
  • nucleotide binding site
  • nucleotide biosynthesis
  • nucleotide change
  • nucleotide deletion
  • nucleotide difference
  • nucleotide divergence
  • nucleotide diversity
  • nucleotide exchange
  • nucleotide exchange factor
  • nucleotide exchanges
  • nucleotide excision repair
  • nucleotide identity
  • nucleotide insertion
  • nucleotide level
  • nucleotide long
  • nucleotide metabolism
  • nucleotide mutation
  • nucleotide phosphodiesterase
  • nucleotide polymorphism
  • nucleotide polymorphism genotyping
  • nucleotide polymorphism marker
  • nucleotide pool
  • nucleotide position
  • nucleotide receptor
  • nucleotide sequence
  • nucleotide sequence analysis
  • nucleotide sequence comparison
  • nucleotide sequence identity
  • nucleotide sequencing
  • nucleotide substitution
  • nucleotide synthesis
  • nucleotide variants
  • nucleotide variation

  • Selected Abstracts

    Biogeographical patterns of genetic differentiation in dung beetles of the genus Trypocopris (Coleoptera, Geotrupidae) inferred from mtDNA and AFLP analyses

    Loredana Carisio
    Abstract Aim, To examine the phylogeography and population structure of three dung beetle species of the genus Trypocopris (Coleoptera, Geotrupidae). We wanted to test whether genetic differences and genealogies among populations were in accordance with morphologically described subspecies and we aimed to establish times of divergence among subspecies to depict the appropriate temporal framework of their phylogeographical differentiation. We also wished to investigate the historical demographic events and the relative influences of gene flow and drift on the distribution of genetic variability of the different populations. Location, Europe (mostly Italy). Methods, We collected adult males from dung pats from 15 Italian localities over the period 2000,2002. For sequence analysis, some dried specimens from Albania, Croatia, Slovakia and Spain were also used. We applied cytochrome oxidase I mitochondrial DNA sequencing and the amplified fragment length polymorphism (AFLP) technique to determine whether phylogeographical patterns within the three species support the proposed hypotheses of subspecies designations, and to detect further structure among populations that might mediate diversification. Results and main conclusions, The results show a high concordance between the distribution of mtDNA variation and the main morphological groups recognized as subspecies, which thus may represent independent evolutionary units. The degree of mitochondrial divergence suggests that speciation events occurred during the Pliocene, while diversification of the main subspecific lineages took place in the Pleistocene, from c. 0.3 to 1.5 Ma. Mitochondrial and nuclear data also reveal that there is phylogeographical structuring among populations within each of the main groups and that both contemporary and historical processes determined this pattern of genetic structure. Geographical populations form monophyletic clades in both phylogenetic and network reconstructions. Despite the high levels of intrapopulational diversity, FST values indicate moderate but significant genetic differentiation among populations, and a Bayesian clustering analysis of the AFLP data clearly separates the geographical populations. Nucleotide and gene diversity estimates reveal interspecific differences in the degree of diversification among populations that may be related to the different ecological requirements of the three species. [source]

    Transcriptional Regulation of 2,,3,-Cyclic Nucleotide 3,-Phosphodiesterase Gene Expression by Cyclic AMP in C6 Cells

    M. Gravel
    Abstract: It was recently shown that the two transcripts encoding the isoforms of 2,,3,-cyclic nucleotide 3,-phosphodiesterase (CNP1 and CNP2) are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only CNP2 mRNA is present, whereas in differentiating oligodendrocytes, both CNP1 and CNP2 mRNAs are expressed. This pattern of CNP expression is likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Here, we report the influence of increased intracellular cyclic AMP (cAMP) levels on the transcription of both CNP1 and CNP2 mRNAs in rat C6 glioma cells. We found that the transcription of CNP1 mRNA was significantly increased in comparison with that of CNP2 mRNA in cells treated with cAMP analogues to elevate intracellular cAMP levels. This up-regulation of CNP1 expression (a) is due to an increase of transcription, (b) requires de novo protein synthesis, and (c) requires the activity of protein kinase A. These results are physiologically significant and support the idea that a cAMP-mediated pathway is part of the molecular mechanisms regulating the expression of CNP1 in oligodendrocytes. The regulation of CNP1 promoter activity by cAMP was then investigated in stably transfected C6 cell lines containing various deletions of the CNP promoter directing the bacterial chloramphenicol acetyltransferase gene. We showed that the sequence between nucleotides -126 and -102 was essential for the cAMP-dependent induction of CNP1 expression. Gel retardation analysis showed that two protein-DNA complexes are formed between this sequence and nuclear factors from C6 cells treated or not treated with cAMP. This suggests that the induction of CNP1 mRNA transcription is not mediated by changes in binding of nuclear factors that interact directly with the -126/-102 sequence. Sequence analysis of this region revealed the presence of a putative activator protein-2 (AP-2) binding site. It is interesting that mutagenesis of this region resulted in a significant reduction in transcriptional responses to cAMP, implying a possible role for the AP-2 factor in the expression of CNP1. In addition, we have shown that putative binding sites for activator protein-4 and nuclear factor-1 adjacent to the AP-2 site are required for efficient induction of CNP1 expression by cAMP. Taken together, our results show that the cAMP-dependent accumulation of CNP1 mRNA appears to depend on the synergistic interaction of several regulatory elements. [source]

    Demystifying Online Genetic Databases

    Carolyn Driscoll
    There has been an explosion of genetic information and keeping current can be difficult. Traditional methods for obtaining information may be obsolete. Many sources for genetic information are now found on the internet although they may be confusing to navigate and interpret. The purpose of this presentation is to outline commonly used genetic databases, and demonstrate how they may be accessed and used to interpret genetic data. The mission of the National Center for Biotechnology Information (NCBI), a resource for molecular biology information, is to develop new information technologies to support understanding of molecular and genetic processes related to health and disease. NCBI services include PubMed, Nucleotide, and the BLAST algorithm for sequence comparison. In this presentation, several genetic databases will be explored. Each database will be defined, the available genetic information described, database access demonstrated, and website information displayed. This presentation will provide education related to several genetic databases as a means of facilitating and promoting access to this information by a larger audience of nurses and health care providers involved with genetic health care. [source]

    Complete Nucleotide Sequence of a Coxsackievirus B4 Strain that Establishes Infection in ICR Mice Pancreas and Induces Glucose Intolerance

    Mi Zhou
    Abstract Some coxsackievirus B serotypes are potentially diabetogenic. Previous studies revealed that the virulence and the tissue damage varied with the genetics of the virus strain as well as with the genetics of the mice. A single amino acid variation can alter virulence and tropism in both murine and in vitro models. However, the genetic determinants of this phenomenon have not been determined. In this study, infections with a laboratory strain of coxsackievirus B4 resulted in a diabetes-like syndrome in ICR mice, characterized by chronic pancreatic inflammation together with dysregulation in glucose metabolism, loss of pancreatic acinar tissue and persistent infection in islets. To characterize the genetic determinants involved in the mouse pancreas adaptation, the laboratory strain of coxsackievirus B4 was cloned for molecular characterization. Comparing the whole genome sequence of this virus strain with the other coxsackievirus B4 strains revealed some differences. Altogether 15 nucleotides were changed, resulting in 10 amino acid substitutions, which might be responsible for the pathogenic phenotype of this strain in mice. Anat Rec, 291:601,609, 2008. © 2008 Wiley-Liss, Inc. [source]

    Die Entstehung erster Zellen , von der Nährstoffaufnahme hin zur Verlängerung eingeschlossener Nucleotide

    ANGEWANDTE CHEMIE, Issue 22 2010

    Abstract Ein neues Verständnis des Ursprungs des Lebens auf der Erde gründet sich auf jüngsten, bedeutenden Entdeckungen zur Biophysik von Membranen. Es wurde gezeigt, dass Vesikel aus Membran-Doppelschichten eine facettenreiche Mikroumgebung zur Verfügung stellen, in der sich erste Stoffwechselreaktionen entwickelt haben können. Zellmembran-ähnliche Aggregate amphiphiler Moleküle, die Oligonucleotide einschließen können, wurden erfolgreich im Labor hergestellt. Im Rahmen von Laborstudien zur Entstehung des Lebens gelang die Verlängerung eines DNA-Primers im Inneren von Fettsäurevesikeln, sobald das extravesikuläre Medium mit aktivierten Nucleotiden versetzt wurde. Diese Studien zeigten, dass zellähnliche Vesikel durchlässig genug sein können, um geladene Moleküle wie aktivierte Nucleotide aufzunehmen, die sich wiederum im Protozellinneren am Kopieren von DNA-Matrizen beteiligen. Wir fassen in diesem Aufsatz jüngste Experimente in diesem Bereich zusammen und beschreiben ein mögliches Szenario zum Ursprung erster primitiver Zellen, wobei wir der Verlängerung eingeschlossener Nucleotide besondere Aufmerksamkeit widmen. [source]

    Nucleotide,amino acid interactions in the l -His,IMP·MeOH·H2O complex

    Katarzyna, lepokura
    In the crystal structure of the methanol-solvated monohydrated complex of l -histidine (His) with inosine 5,-monophosphate (IMP), namely l -histidinium inosine-5,-phosphate methanol solvate monohydrate, C6H10N3O2+·C10H12N4O8P,·CH3OH·H2O, most of the interactions between IMP anions (anti/C3,- endo/gauche,gauche conformers) are realized between the riboses and hypoxanthine bases in a trans sugar-edge/sugar-edge geometry, and between the phosphate groups. The base Watson,Crick edge is involved in additional methanol-mediated IMP...MeOH...IMP contacts. Specific and nonspecific nucleotide,amino acid (IMP...His) interactions engage the Hoogsteen edges of the base and phosphate group, respectively. Additional stabilization of His...IMP contacts is provided by ,,, stacking between the imidazolium ring of His and the hypoxanthine base of IMP. The results may indicate the possible recognition mechanism between His and IMP. [source]

    Direct Polymerase Synthesis of Reactive Aldehyde-Functionalized DNA and Its Conjugation and Staining with Hydrazines,

    ANGEWANDTE CHEMIE, Issue 6 2010
    Veronika Raindlová
    In zwei Stufen zu reaktiver Aldehyd-modifizierter DNA: durch Suzuki-Kreuzkupplung halogenierter Nucleosidtriphosphate (dNTPs) mit 4-Formylthiophen-2-boronsäure und Polymerase- vermittelten Einbau der modifizierten Nucleotide in DNA (siehe Schema; PEX=Primerverlängerung, PCR=Polymerasekettenreaktion). Die Bildung von Hydrazonen mit Arylhydrazinen unter wässrigen Bedingungen wurde zum Anfärben der DNA verwendet. [source]

    Mitochondrial DNA diversity and origins of South and Central American goats

    ANIMAL GENETICS, Issue 3 2009
    M. Amills
    Summary We have analysed the genetic diversity of South and Central American (SCA) goats by partially sequencing the mitochondrial control region of 93 individuals with a wide geographical distribution. Nucleotide and haplotype diversities reached values of 0.020 ± 0.00081 and 0.963 ± 0.0012 respectively. We have also observed a rather weak phylogeographic structure, with almost 69% of genetic variation included in the within-breed variance component. The topology of a median-joining network analysis including 286 European, Iberian, Atlantic and SCA mitochondrial sequences was very complex, with most of the haplotypes forming part of independent small clusters. SCA sequences showed a scattered distribution throughout the network, and clustering with Spanish and Portuguese sequences occurred only occasionally, not allowing the distinguishing of a clear Iberian signature. Conversely, we found a prominent cluster including Canarian, Chilean, Argentinian and Bolivian mitochondrial haplotypes. This result was independently confirmed by constructing a Bayesian phylogenetic tree (posterior probability of 0.97). Sharing of mitochondrial haplotypes by SCA and Canarian goats suggests that goat populations from the Atlantic archipelagos, where Spanish and Portuguese ships en route to the New World used to stow food and supplies, participated in the foundation of SCA caprine breeds. [source]

    2,- N -(Pyren-1-yl)acetyl-2,-Amino-,-L-LNA: Synthesis and Detection of Single Nucleotide Mismatches in DNA and RNA Targets

    CHEMBIOCHEM, Issue 10 2007
    T. Santhosh Kumar
    Precise positioning of intercalators furnishes a SNP-detection tool. The conformationally locked 2-oxo-5-azabicyclo-[2.2.1]heptane skeleton of 2,-amino-,-L-LNA monomer X directs the N2,-linked pyrene moiety into nucleic acid duplex cores to give highly stabilized duplexes. We have used this precise positioning of pyrene moieties to develop probes that signal the presence of single-nucleotide mismatches in DNA/RNA targets by excimer signal formation. [source]

    Theoretical Study on Proton-Transfer Reaction of Intracellular Second-messenger 3,,5,-Cyclic Nucleotide

    Ai-Hua ZHANG
    Abstract The gas-phase proton-transfer reaction mechanism of intracellular second-messenger 3,,5,-cyclic nucleotide (cAMPm) has been theoretically investigated at the B3LYP/6-31G, , level. One or two H2O molecules have been used to simulate the catalyst. It is found that H shift reaction between conformation Bm and conformation Dm of cAMPm involves a cyclic transition state with one or two water molecules as a shuttle. Furthermore, H shift reaction proceeds easily with the participation of two water molecules. The results provide evidence in theory to study proton-transfer reaction mechanism of related phosphodiesters. Our present calculations have rationalized all the possible reaction channels. [source]

    Nucleotides and epidermal growth factor induce parallel cytoskeletal rearrangements and migration in cultured adult murine neural stem cells

    ACTA PHYSIOLOGICA, Issue 2 2010
    I. Grimm
    Abstract Aim:, The adult subventricular zone (SVZ) contains neural stem cells that generate neuroblasts migrating to the olfactory bulb (OB) and differentiating into interneurones. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, functional integration and cell survival are poorly understood. We have previously shown that cultured adult neural stem cells express a considerable variety of nucleotide receptors and that nucleotides and epidermal growth factor (EGF) induce converging intracellular signalling pathways that carry potential for synergism in the control of neural stem cell proliferation and cell survival. Here we investigate the role of EGF and the nucleotides ATP, ADP,S and UTP in neural stem cell migration. Methods:, Neural stem cells were prepared from adult mice and subjected to adherent culture. Labelling of F-actin was performed with tetramethylrhodamine isothiocyanate-phalloidin. Images were processed for quantitative evaluation of fluorescence labelling. Agonist-induced phosphorylation of AKT and focal adhesion kinase was analysed by quantitative Western blotting. Agonist-dependent cell migration was assayed using 48-well microchemotaxis chambers. Results:, Nucleotides and EGF induce the formation of stress fibres, an increase in the cortical actin cytoskeleton and in cell spreading. This is associated with increased phosphorylation of AKT and focal adhesion kinase. Using microchemotaxis chambers we demonstrate a parallel increase in cell migration. Conclusion:, Our results suggest that nucleotides and EGF acting as paracrine or autocrine signalling substances can be of relevance for structuring and maintaining the cytoarchitecture of the SVZ and the stream of neuroblasts migrating to the OB. [source]

    ATP activates both receptor and sustentacular supporting cells in the olfactory epithelium of Xenopus laevis tadpoles

    Dirk Czesnik
    Abstract Nucleotides and amino acids are acknowledged categories of water-borne olfactory stimuli. In previous studies it has been shown that larvae of Xenopus laevis are able to sense amino acids. Here we report on the effect of ATP in the olfactory epithelium (OE) of Xenopus laevis tadpoles. First, ATP activates a subpopulation of cells in the OE. The ATP-sensitive subset of cells is almost perfectly disjoint from the subset of amino acid-activated cells. Both responses are not mediated by the well-described cAMP transduction pathway as the two subpopulations of cells do not overlap with a third, forskolin-activated subpopulation. We further show that, in contrast to amino acids, which act exclusively as olfactory stimuli, ATP appears to feature a second role. Surprisingly it activated a large number of sustentacular supporting cells (SCs) and, to a much lower extent, olfactory receptor neurons. The cells of the amino acid- and ATP-responding subsets featured differences in shape, size and position in the OE. The latencies to activation upon stimulus application differed markedly in these subsets. To obtain these results two technical points were important. We used a novel dextran-tetramethylrhodamine-backfilled slice preparation of the OE and we found out that an antibody to calnexin, a known molecular chaperone, also labels SCs. Our findings thus show a strong effect of ATP in the OE and we discuss some of the possible physiological functions of nucleotides in the OE. [source]

    Synthesis of 3,-BODIPY-Labeled Active Esters of Nucleotides and a Chemical Primer Extension Assay on Beads

    Kerstin Gießler
    Abstract A solution-phase synthesis of active esters of 3,-fluorophore-labeled deoxynucleoside 5,-monophosphates was developed for thymine and cytosine as nucleobases by using two different BODIPY dyes. Starting from the respective 2,-amino-2,,3,-dideoxynucleoside-5,-monophosphate, the fluorescent oxyazabenzotriazolides can be prepared in one-pot procedures involving labeling and activation. Screening of a range of supports led to a chemical primer extension assay on beads with in situ detection of nucleobases in target DNA through optical read-out. [source]

    Synthesis and Properties of Fluorescent cycloSal Nucleotides Based on the Pyrimidine Nucleoside m5K and Its 2,,3,-Dideoxy Analog dm5K

    Henning J. Jessen
    Abstract The synthesis of the fluorescent thymidine nucleoside analog 5-methyl-pyrimidin-2-one nucleoside 1 (m5K) and that of its 2,,3,-dideoxy derivative 2 (dm5K) are described. Moreover, the conversion of 1 and 2 into the corresponding 3-methyl- cycloSal-phosphate triesters 11 and 12 or an enzyme-cleavable prodrug 5-acetoxymethylpropionate- cycloSal-phosphate triester 13, and the fluorescence and hydrolysis properties of these new lipophilic triesters are reported. Finally, the suitability of these cycloSal pronucleotides as probes is demonstrated in a cell-extract hydrolysis experiment and a model study for cell uptake. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006) [source]

    P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca2+ rise and change in mitochondrial potential

    Stefan Kahlert
    Abstract With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca2+ level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y1/2/4 receptor agonist in rat, caused a large cytosolic Ca2+ increase in astrocytes and a considerably smaller Ca2+ response in neighboring neurons. The P2Y1 receptor antagonist MRS2179 completely blocked the ATP-induced Ca2+ response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca2+ level. Stimulation with UDP, a P2Y6 receptor agonist; UTP, a P2Y2/4 receptor agonist; 2MeSATP, a P2Y1 receptor agonist; or 2MeSADP, a P2Y1/12/13 receptor agonist, evoked significant Ca2+ responses in astrocytes but small Ca2+ responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca2+ peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca2+ level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain. © 2007 Wiley-Liss, Inc. [source]

    The Neurospora circadian clock regulates a transcription factor that controls rhythmic expression of the output eas(ccg-2) gene

    Deborah Bell-Pedersen
    The circadian clock provides a link between an organism's environment and its behaviour, temporally phasing the expression of genes in anticipation of daily environmental changes. Input pathways sense environmental information and interact with the clock to synchronize it to external cycles, and output pathways read out from the clock to impart temporal control on downstream targets. Very little is known about the regulation of outputs from the clock. In Neurospora crassa, the circadian clock transcriptionally regulates expression of the clock-controlled genes, including the well-characterized eas(ccg-2) gene. Dissection of the eas(ccg-2) gene promoter previously localized a 68 bp sequence containing an activating clock element (ACE) that is both necessary and sufficient for rhythmic activation of transcription by the circadian clock. Using electrophoretic mobility shift assays (EMSAs), we have identified light-regulated nuclear protein factors that bind specifically to the ACE in a time-of-day-dependent fashion, consistent with their role in circadian regulation of expression of eas(ccg-2). Nucleotides in the ACE that interact with the protein factors were determined using interference binding assays, and deletion of the core interacting sequences affected, but did not completely eliminate, rhythmic accumulation of eas(ccg-2) mRNA in vivo, whereas deletion of the entire ACE abolished the rhythm. These data indicate that redundant binding sites for the protein factors that promote eas(ccg-2) rhythms exist within the 68 bp ACE. The ACE binding complexes formed using protein extracts from cells with lesions in central components of the Neurospora circadian clock were identical to those formed with extracts from wild-type cells, indicating that other proteins directly control eas(ccg-2) rhythmic expression. These data suggest that the Neurospora crassa circadian clock regulates an unknown transcription factor, which in turn activates the expression of eas(ccg-2) at specific times of the day. [source]

    Incorporation of Thymine Nucleotides by DNA Polymerases through T,HgII,T Base Pairing

    ANGEWANDTE CHEMIE, Issue 37 2010
    Hidehito Urata Prof.
    DNA in schlechter Gesellschaft: In Gegenwart von HgII -Ionen führten DNA-Polymerasen Thymidin-5,-triphosphat (TTP) gegenüber einem Thyminrest im Templatstrang ein bildeten zur Verlängerung des Primerstrangs eine Phosphodiesterbindung. DNA-Polymerasen erkannten dieses ungewöhnliche, durch ein Metallion verknüpfte Basenpaar und synthetisierten darüber hinweg das Volllängenprodukt (siehe Bild). [source]

    In-Stem Molecular Beacon Containing a Pseudo Base Pair of Threoninol Nucleotides for the Removal of Background Emission,

    ANGEWANDTE CHEMIE, Issue 38 2009
    Hiromu Kashida Dr.
    Aus heißt auch aus: Ein neuartiger molekularer Beacon wurde entwickelt, der in der Stamm-Region Pseudobasenpaare aus dem Fluorophor (Perylen) und dem Fluoreszenzlöscher (Anthrachinon) enthält, wobei D -Threoninol-Einheiten als Verbindungsstück dienen (in-stem molecular beacon, ISMB). Der ISMB war in der Lage, eine Einzeldeletionsmutante von der Wildtypsequenz zu unterscheiden, ohne dass Hintergrundemission auftrat (siehe Bild). [source]

    Novel 3,-O-Fluorescently Modified Nucleotides for Reversible Termination of DNA Synthesis

    CHEMBIOCHEM, Issue 1 2010
    Taek-Soo Kim Dr.
    Serving a double purpose: 3,- O -fluorophore-labeled dTTPs were synthesized and used as reversible terminators of DNA polymerization. Successful incorporation of nucleotides with a bulky 3,- O -fluorophore and detection of fluorescence for base calling can potentially be used to improve sequencing-by-synthesis technology. [source]

    Reaction of Cytidine Nucleotides with Cyanoacetylene: Support for the Intermediacy of Nucleoside-2,,3,-cyclic Phosphates in the Prebiotic Synthesis of RNA

    CHEMBIOCHEM, Issue 6 2006
    Michael A. Crowe
    Abstract A robust and prebiotically plausible synthesis of RNA is a key requirement of the "RNA World" hypothesis, but, to date, no such synthesis has been demonstrated. Monomer synthesis strategies involving attachment of preformed nucleobases to sugars have failed, and, even if activated 5,-nucleotides could be made, the hydrolysis of these intermediates in water makes their efficient oligomerisation appear unlikely. We recently reported a synthesis of cytidine-2,,3,-cyclic phosphate 1 (C>p) in which the nucleobase was assembled in stages on a sugar-phosphate template. However, 2,,3,-cyclic nucleotides (N>p's) also undergo hydrolysis, in this case giving a mixture of the 2,- and 3,-monophosphates. This hydrolysis has previously been seen as making the, otherwise promising, oligomerisation of N>p's seem as unlikely as that of the 5,-activated nucleotides. We now find that cyanoacetylene, the reagent used for the second stage of nucleobase assembly in the synthesis of C>p, also reverses the effect of the hydrolysis by driving efficient cyclisation of C2,p and C3,p back to C>p. Excess cyanoacetylene also derivatises the nucleobase, but this modification is reversible at neutral pH. These findings significantly strengthen the case for N>p's in a prebiotic synthesis of RNA. [source]

    Analysis of Platinum Adducts with DNA Nucleotides and Nucleosides by Capillary Electrophoresis Coupled to ESI-MS: Indications of Guanosine 5,-Monophosphate O6,N7 Chelation

    CHEMBIOCHEM, Issue 11 2004
    Ulrich Warnke Dr.
    Abstract DNA is the ultimate target of platinum-based anticancer therapy. Since the N7 of guanine is known to be the major binding site of cisplatin and its analogues, adduct formation with model nucleotides, especially 2,-deoxyguanosine 5,-monophosphate (dGMP), has been studied in detail. During the last few years a coupled capillary eletrophoresis/electrospray-ionization mass spectrometry (CE/ESI-MS) method has been advantageously used in order to separate and identify platinum adducts with nucleotides in submillimolar concentrations in aqueous solutions. Beside the bisadduct, [Pt(NH3)2(dNMP)2]2,(NMP=2,-deoxynucleoside 5,-monophosphate), and the well-known monochloro and monohydroxo adducts, [Pt(NH3)2Cl(dNMP)],and [Pt(NH3)2(dNMP)OH],, respectively, a third kind of monoadduct species with a composition of [Pt(NH3)2(dNMP)],can be separated by CE and detected through the m/z values measured with ESI-MS. Different experimental setups indicate the existence of an O6,N7 chelate, whereas the formation of N7,,PO4macrochelates or dinuclear species is unlikely. Additionally, offline MS experiments with 2,-deoxyguanosine (dG) and stabilization of the controversially discussed O6,N7 chelate by oxidation with hydrogen peroxide support the assumption of the existence of O6,N7 chelation. [source]

    2262: Study of the role of P2Y receptors in the development of experimental autoimmune uveitis

    3Article first published online: 23 SEP 2010, L JUDICE DE MENEZES RELVAS
    Purpose During autoimmune uveitis (AU), retinal specific auto-reactive T lymphocytes (TL) are activated, alter blood retinal barrier(BRB) and penetrate the eye where they start an inflammatory reaction. Nucleotides, normally present at low concentration in extracellular media, can act as a danger signal, through P2 receptors activation and might be implicated in AU. In this work we would like to investigate if the expression of the nucleotide receptor P2Y2 has any role in experimental autoimmune uveitis (EAU). Methods EAU will be induced in WT and P2Y2 KO mice by IRBP peptide 1-20 injection. 12 days later, TL from spleen and lymph nodes will be purified and restimulated by IRBP. TL proliferation will be measured by thymidine incorporation and cytokines secretion by ELISA. TL from the 2 groups of mice will also be adoptively transfered in WT mice. Similarly, TL from WT mice will be adoptively transfered in WT and P2Y2 KO mice. EAU development will be graded by clinical and histological scores. Results TL generated in KO mice proliferate and produce less IFN, and IL-17, after IRBP restimulation than TL generated in WT mice. Accordingly, adoptive transfer of TL generated in KO mice induce significantly a lower grade of disease than those generated in WT mice. Finally, adoptive transfer of TL generated in WT mice induce disease of significantly lower grade in KO mice recipient than in WT mice recipient. Conclusion Our preliminary data shows that P2Y2 KO mice have a defective immune response after immunisation and develop lower intraocular inflammation following adoptive transfer with TL. Altogether this suggests that P2Y2, as danger receptor signals, play an important role in the development of uveitis. [source]

    Hydrogen-Bond-Guided Self-Assembly of Nucleotides on a Receptor-Array Surface

    Dr. Dmitry
    Abstract The hydrogen-bond-guided self-assembly of 5,-ribonucleotides bearing adenine(A), cytosine (C), uracil (U), or guanine (G) bases from aqueous solution on a lipid-like surface decorated with synthetic bis(ZnII,cyclen) (cyclen=1,4,7,10-tetraazacyclodododecane) metal,complex receptor sites is described. The process was studied by using surface plasmon resonance spectroscopy. The data show that the mechanism of nucleotide binding to the 2D template is influenced by the chemistry of the bases and the pH,value of the solution. In a neutral solution of pH,7.5, the process is cooperative and selective with respect to Watson,Crick pairs (A,U and C,G), which form stable double planes in accordance with the Chargaff rule. In a more acidic solution at pH,6.0, the interactions between complementary partners become non-cooperative and the surface also stabilizes mismatched and wobble pairs due to the pH-induced changes in the receptor coordination state. The results suggest that hydrogen bonding plays a key role in the self-assembly of complementary nucleotides at the lipid-like interface, and the cooperative character of the process stems from the ideal matching of the orientation and chemistry of all the interacting components with respect to each other in neutral solution. [source]

    Fluorescent and Electrochemical Sensing of Polyphosphate Nucleotides by Ferrocene Functionalised with Two ZnII(TACN)(pyrene) Complexes

    Zhanghua Zeng Dr.
    Abstract The [Fcbis{ZnII(TACN)(Py)}] complex, comprising two ZnII(TACN) ligands (Fc=ferrocene; Py=pyrene; TACN=1,4,7-triazacyclononane) bearing fluorescent pyrene chromophores linked by an electrochemically active ferrocene molecule has been synthesised in high yield through a multistep procedure. In the absence of the polyphosphate guest molecules, very weak excimer emission was observed, indicating that the two pyrene-bearing ZnII(TACN) units are arranged in a trans -like configuration with respect to the ferrocene bridging unit. Binding of a variety of polyphosphate anionic guests (PPi and nucleotides di- and triphosphate) promotes the interaction between pyrene units and results in an enhancement in excimer emission. Investigations of phosphate binding by 31P,NMR spectroscopy, fluorescence and electrochemical techniques confirmed a 1:1 stoichiometry for the binding of PPi and nucleotide polyphosphate anions to the bis(ZnII(TACN)) moiety of [Fcbis{ZnII(TACN)(Py)}] and indicated that binding induces a trans to cis configuration rearrangement of the bis(ZnII(TACN)) complexes that is responsible for the enhancement of the pyrene excimer emission. Pyrophosphate was concluded to have the strongest affinity to [Fcbis{ZnII(TACN)(Py)}] among the anions tested based on a six-fold fluorescence enhancement and 0.1,V negative shift in the potential of the ferrocene/ferrocenium couple. The binding constant for a variety of polyphosphate anions was determined from the change in the intensity of pyrene excimer emission with polyphosphate concentration, measured at 475,nm in CH3CN/Tris-HCl (1:9) buffer solution (10.0,mM, pH,7.4). These measurements confirmed that pyrophosphate binds more strongly (Kb=(4.45±0.41)×106,M,1) than the other nucleotide di- and triphosphates (Kb=1,50×105,M,1) tested. [source]

    Polymerase-Catalysed Incorporation of Glucose Nucleotides into a DNA Duplex

    Marleen Renders
    Abstract Active but unselective: Nucleoside triphosphates possessing glucose moieties (such as those depicted) instead of the natural furanose rings are recognised by the active sites of polymerases. Polymerases therefore seem to be very unspecific in their recognition patterns. The enzymatic recognition of six-membered ring nucleoside triphosphates,in particular the 6,-triphosphates of (,- D -glucopyranosyl)thymine, (2,,3,-dideoxy-,- D -glucopyranosyl)thymine, (3,,4,-dideoxy-,- D -glucopyranosyl)thymine and (2,,3,-dideoxy-,- D -glucopyranosyl)adenine,was investigated. Despite the facts that the pyranose nucleic acids obtained by polymerisation of these monomers do not hybridise in solution with DNA and that the geometry of a DNA strand in a natural duplex differs from that of a pyranose nucleic acid, elongation of the DNA duplex with all four nucleotide analogues by Vent,(exo,) polymerase was observed. Modelling experiments showed that hydrogen bonds are formed when 2,,3,-dideoxy-,-homo-T building blocks or ,- D - gluco -T building blocks are incorporated opposite adenosine residues in the template but not when they are incorporated opposite thymine residues in the template. The model shows a near perfect alignment of a secondary hydroxy group at the end of the primer and the ,-phosphate group of the incoming triphosphate. The results of these experiments provide new information on the role of the active site of the enzyme in the polymerisation reaction. [source]

    The Search for a Potentially Prebiotic Synthesis of Nucleotides via Arabinose-3-phosphate and Its Cyanamide Derivative

    Carole Anastasi Dr.
    Abstract For the RNA world hypothesis to be accepted, the constitutional self-assembly of RNA will have to be demonstrated. Conceptually, the simplest route to RNA involves nucleotide polymerisation. Activated pyrimidine nucleotides can be derived from arabinose-3-phosphate under potentially prebiotic conditions, but the prebiotic synthesis of this sugar phosphate has not hitherto been investigated. The results of synthetic approaches involving phosphorylation, phosphate migration and 2,3-CC bond construction are described herein. [source]

    Changes in expression and activity levels of ecto-5,-nucleotidase/CD73 along the mouse female estrous cycle

    ACTA PHYSIOLOGICA, Issue 2 2010
    E. Aliagas
    Abstract Aim:, Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, hormone synthesis and maintenance of fluid composition. Moreover, adenosine is a key molecule for sperm capacitation. Extracellular nucleotide and nucleoside levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family is the most abundant and effective to hydrolyse ATP and ADP to AMP. In the female reproductive tract three members of this family have been recently identified: NTPDase1, NTPDase2 and NTPDase3 (Histochem. Cell Biol.131, 2009, 615). The purpose of the present study was to characterize in this system the expression profile of ecto-5,-nucleotidase (CD73), the enzyme generating adenosine from AMP. Methods:, Immunological techniques and in situ enzymatic assays were used to characterize the ecto-5,-nucleotidase expression in the mouse female reproductive tract along the four stages of the estrous cycle, that were determined by vaginal smear examination. Results:, Ecto-5,-nucleotidase was abundantly detected in the corpora lutea of the ovaries, as well as in several epithelia, such as that of oviducts, uterus and endometrial glands. Marked changes in endometrial ecto-5,-nucleotidase expression and activity along the estrous cycle are described, these being maximum at estrus phase, coinciding with optimal female sexual receptivity. Conclusion:, The adenosine generated thereby, besides other functions, might contribute to sperm capacitation, thus significantly influencing fertility. [source]

    Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy

    ELECTROPHORESIS, Issue 7 2009
    Chun-Chi Wang
    Abstract We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease. [source]

    High-sensitive determination of human ,-thrombin by its 29-mer aptamer in affinity probe capillary electrophoresis

    ELECTROPHORESIS, Issue 12 2008
    Yilin Li
    Abstract ACE technique provides an effective tool for the separation and identification of disease-related biomarkers in clinical analysis. In recent years, a couple of synthetic DNA or RNA oligonucleotides, known as aptamers, rival the specificity and affinity for targets to antibodies and are employed as one kind of powerful affinity probe in ACE. In this work, based on high affinity between antithrombin aptamer and thrombin (their dissociation constant is 0.5,nM), a carboxyfluorescein-labeled 29-nucleotide (nt) aptamer (F29-mer) was used and an aptamer-based affinity probe CE (aptamer-based APCE) method was successfully established for high-sensitive detection and quantitative analysis of thrombin. Experimental conditions including incubation temperature and time, buffer composition, and concentration of cations were investigated and optimized. Under the optimized condition, the linear range was from 0 to 400,nM and the LOD was 2,nM (74,ng/mL, S/N,=,3), i.e., 40,amol, both in running buffer and in 5% v/v human serum. This LOD is the lowest one than those achieved by the previous APCE methods but based on a 15-mer aptamer. This approach offers a promising method for the rapid, selective, and sensitive detection of thrombin in practical utility. Further binding experiments using one carboxyfluorescein-labeled aptamer and the other nonlabeled aptamer or vice versa were carried out to deduce the formation of ternary complex when these two aptamers coexisted in the free solution with thrombin. [source]

    Cloning and Characterization of the cDNA Encoding the Masquerade-like Serine Proteinase Homologue Gene of the Silkworm, Bombyx mori

    Doo-Sang PARK
    ABSTRACT From Bombyx mori larvae, RT-PCR and cDNA library screening isolated masquerade-like serine proteinase homologue cDNA gene, proposed to be related to insect immunity and its characteristics were examined. The isolated gene is composed of 1.3 kb of nucleotide and 420 amino acid residues were encoded. According to the results of database search, the isolated gene showed high sequence homology with Holotrichia and Tenebrio's 45 kDa protein, Drosophila CG5390 gene. Moreover, it is composed of regulatory domain and catalytic domain, which is characteristic of serine proteinase that can be found in the insect immune reaction and embryonic development processes. Enzyme activation site by proteolytic cleavage and the sequence of three amino acids participate in the catalytic triad of enzyme and 14 cystein residues used in disulfide bridges are well conserved with the compared genes. The mRNA expression was increased following E. coli injection and constitutive expression was also observed before injection by Northern blot analysis. [source]