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Journal of Biomolecular NMR May 2003Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine...
Triple resonance experiments for the simultaneous correlation of H6/H5 and exchangeable protons of pyrimidine nucleotides in 13C,15N-labeled RNA applicable to larger RNA molecules.
Triple-resonance two-dimensional H6/H5(C4N)H and C6/C5(C4N)H experiments are described that provide through-bond H6/H5 or C6/C5 to imino/amino correlations in pyrimidine bases in (13)C,(15)N-labeled RNA. The experiments simultaneously transfer H6/H5 magnetization by an INEPT step to the C6/C5 nuclei and by homonuclear CC- and heteronuclear CN-TOCSY steps via the intervening C4 nucleus to the N3/N4 nuclei and then by a reverse INEPT step to the imino/amino hydrogens. The sensitivity of these experiments is high as demonstrated using a 30-nucleotide pyrimidine rich RNA at a concentration of 0.9 mM at temperatures of 10 degrees C and 25 degrees C. This indicates the general applicability of the experiments and the possibility to obtain correlations for imino resonances in non-canonical regions of the target RNA.
Topics: Base Sequence; Carbon Isotopes; Isotope Labeling; Magnetic Resonance Spectroscopy; Models, Molecular; Nitrogen Isotopes; Nucleic Acid Conformation; Pyrimidine Nucleotides; RNA
PubMed: 12766404
DOI: 10.1023/a:1023040520291 -
Archives Internationales de Physiologie... Nov 1987These experiments were designed to determine through the study of uridine and cytidine kinase activity, the precise mechanisms of plasma nucleoside salvage leading to...
These experiments were designed to determine through the study of uridine and cytidine kinase activity, the precise mechanisms of plasma nucleoside salvage leading to pyrimidine nucleotide synthesis in the rat heart. The kinetic parameters were: Km = 10 microM, V = 4 nmol g-1 min-1 for cytidine kinase activity and Km = 43 microM and V = 18 nmol g-1 min-1 for uridine kinase activity. Competing activity as concerns the two nucleosides was shown to occur, suggesting that in the rat myocardium as in other cells, one and the same enzyme phosphorylates both uridine and cytidine. UTP and CTP were shown to exert a potent inhibitory action on nucleoside phosphorylation; two factors thus exert a joint influence on the control of pyrimidine nucleotide synthesis in the rat heart: the extracellular concentration of precursor and the intracellular level of UTP and CTP. The kinetic parameters for kinase activities are discussed, taking into account the actual concentration of plasmatic nucleosides. Comparison of these data with respectively those for incorporation of nucleosides into the pyrimidine nucleotides of isolated rat heart and with nucleotide turnover rates in vivo suggests that, under physiological conditions, the utilization of plasma cytidine is crucial to the synthesis of myocardial pyrimidine synthesis.
Topics: Animals; Binding, Competitive; Cytidine Triphosphate; Female; Kinetics; Male; Myocardium; Phosphotransferases; Pyrimidine Nucleotides; Rats; Uridine Kinase; Uridine Triphosphate
PubMed: 2453171
DOI: No ID Found -
Biochimica Et Biophysica Acta Jan 1960
Topics: Alkalies; Nucleosides; Nucleotides; Pyrimidine Nucleotides; Pyrimidines
PubMed: 13797596
DOI: 10.1016/0006-3002(60)90521-7 -
British Journal of Cancer Nov 1994In the present study we have used flow cytometric DNA measurements on synchronised human NHIK 3025 cells to measure cell cycle progression under various conditions of...
In the present study we have used flow cytometric DNA measurements on synchronised human NHIK 3025 cells to measure cell cycle progression under various conditions of reduced oxygenation. Our data indicate that addition of 0.1 mM deoxycytidine or uridine has no effect on the oxygen-dependent arrest in late G1 or on the inhibition of cell proliferation through S-phase under extremely hypoxic conditions. Following reoxygenation of cells exposed to extremely hypoxic conditions in G2 initiation of DNA synthesis in the subsequent cell cycle is delayed by several hours. This G2-induced delay is completely abolished for approximately 60% of the cell population by addition of deoxycytidine to hypoxic G2 cells. This finding supports our previous proposal that important steps in the preparation for DNA synthesis occur during G2 of the previous cell cycle, and it indicates that this preparation is connected to the de novo synthesis of pyrimidine deoxynucleotide precursors. The results show that cells are able to enter S-phase in the presence of 100-1,300 p.p.m. (0.01-0.13%) oxygen (here denoted 'moderate hypoxia'), but they are not able to complete DNA synthesis under such conditions. However, the cell cycle inhibition induced under moderate hypoxia is partially reversed in the presence of exogenously added deoxycytidine and uridine, while no such reversal is seen in the presence of purine deoxynucleosides (deoxyadenosine and deoxyguanosine). Thus, both deoxycytidine and uridine could replace reoxygenation under these conditions. These results indicate that the reduction of CDP to dCTP by ribonucleotide reductase, an enzyme which requires oxygen as an essential factor for the formation of tyrosyl radicals for its catalytic activity, does not seem to be the limiting step responsible for the reduced dCTP pool observed under moderate hypoxia. We conclude that the oxygen-dependent catalytic activity of the M2 subunit of ribonucleotide reductase is still intact and functional in NHIK 3025 cells even at oxygen concentration as low as 100 p.p.m. Therefore the cell cycle inhibition observed is probably due to inhibition of the respiratory chain-dependent UMP synthesis at the stage of dihydroorotate dehydrogenase.
Topics: Carcinoma in Situ; Cell Cycle; Cell Division; Cell Hypoxia; Cell Survival; Female; Humans; Kinetics; Oxygen; Pyrimidine Nucleotides; Tumor Cells, Cultured; Uterine Cervical Neoplasms
PubMed: 7947090
DOI: 10.1038/bjc.1994.411 -
Japanese Journal of Ophthalmology 1990Effects of nucleotide phosphates on the sialyltransferase activity in the neurosensory retina of the bovine eye were studied. Enzyme activity was assayed using cytidine...
Effects of nucleotide phosphates on the sialyltransferase activity in the neurosensory retina of the bovine eye were studied. Enzyme activity was assayed using cytidine monophosphate-[14C]-N acetylneuraminic acid as a substrate and desialylated fetuin as an exogenous acceptor. Cytidine-5'-diphosphate and adenosine triphosphate inhibited the enzyme activity. Uridine diphosphate and guanosine diphosphate increased the enzyme activity at low concentrations and decreased the activity at high concentrations. Cyclic adenosine monophosphate and cyclic guanosine monophosphate increased the enzyme activity at concentrations up to 8 mM. It is thus concluded that sialyltransferase activity of the neural retina may be affected by various nucleotides, its alteration depending on either the type of nucleotides or their concentration.
Topics: Animals; Cattle; Enzyme Stability; Purine Nucleotides; Pyrimidine Nucleotides; Retina; Sialyltransferases; Substrate Specificity; beta-D-Galactoside alpha 2-6-Sialyltransferase
PubMed: 2082061
DOI: No ID Found -
Cells Jan 2022The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of...
The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.
Topics: Head and Neck Neoplasms; Humans; Metabolomics; Pyrimidine Nucleotides; Saliva; Sjogren's Syndrome; Xerostomia
PubMed: 35159133
DOI: 10.3390/cells11030323 -
Origins of Life and Evolution of the... Jun 2011Selective adsorption of D, L-ImpA with D, L-ImpU on the platelets of montmorillonite demonstrates an important reaction pathway for the origin of homochirality in RNA...
Selective adsorption of D, L-ImpA with D, L-ImpU on the platelets of montmorillonite demonstrates an important reaction pathway for the origin of homochirality in RNA synthesis. Our earlier studies have shown that the individual reactions of D, L-ImpA or D, L-ImpU on montmorillonite catalyst produced oligomers which were only partially inhibited by the incorporation of both D- and L-enantiomers. Homochirality in these reactions was largely due to the formation of cyclic dimers that cannot elongate. We investigated the quaternary reactions of D, L-ImpA with D, L-ImpU on montmorillonite. The chain length of these oligomers increased from 9-mer to 11-mer as observed by HPLC, with a concomitant increase in the yield of linear dimers and higher oligomers in the reactions involving D, L-ImpA with D, L-ImpU as compared to the similar reactions carried out with D-enantiomers only. The formation of cyclic dimers of U was completely inhibited in the quaternary reactions. The yield of cyclic dimers of A was reduced from 60% to 10% within the dimer fraction. 12 linear dimers and 3 cyclic dimers were isolated and characterized from the quaternary reaction. The homochirality and regioselectivity of dimers were 64.1% and 71.7%, respectively. Their sequence selectivity was shown by the formation of purine-pyrimidine (54-59%) linkages, followed by purine-purine (29-32%) linkages and pyrimidine-pyrimidine (9-13%) linkages. Of the 16 trimers detected, 10 were homochiral with an overall homochirality of 73-76%. In view of the greater homochirality, sequence- and regio- selectivity, the quaternary reactions on montmorillonite demonstrate an unexpectedly favorable route for the prebiotic synthesis of homochiral RNA compared with the separate reactions of enantiomeric activated mononucleotides.
Topics: Bentonite; Catalysis; Chromatography, High Pressure Liquid; Hydrolysis; Isomerism; Models, Molecular; Phosphorylation; Purine Nucleotides; Pyrimidine Nucleotides; RNA
PubMed: 20725859
DOI: 10.1007/s11084-010-9222-1 -
Journal of Inorganic Biochemistry Nov 2006The interactions of Cu(II) ions with adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP) and 1,12-diamino-4,9-dioxadodecane (OSpm) were studied. A...
Interactions of 1,12-diamino-4,9-dioxadodecane (OSpm) and Cu(II) ions with pyrimidine and purine nucleotides: adenosine-5'-monophosphate (AMP) and cytidine-5'-monophosphate (CMP).
The interactions of Cu(II) ions with adenosine-5'-monophosphate (AMP), cytidine-5'-monophosphate (CMP) and 1,12-diamino-4,9-dioxadodecane (OSpm) were studied. A potentiometric method was applied to determine the composition and stability constants of complexes formed, while the mode of interactions was analysed by spectral methods (ultraviolet and visible spectroscopy (UV-Vis), electron paramagnetic resonance (EPR), (13)C NMR, (31)P NMR). In metal-free systems, molecular complexes nucleotide-polyamine (NMP)H(x)(OSpm) were formed. The endocyclic nitrogen atoms of the purine ring N(1), N(7), the nitrogen atom of the pyrimidine ring N(3), the oxygen atoms of the phosphate group of the nucleotide and the protonated nitrogen atoms of the polyamine were the reaction centres. The mode of interaction of the metal ion with OSpm and the nucleotides (AMP or CMP) in the coordination compounds was established. In the system Cu(II)/OSpm the dinuclear complex Cu(2)(OSpm) forms, while in the ternary systems Cu(II)/nucleotide/OSpm the species type MH(x)LL' and MLL' appear. In the MH(x)LL' type species, the main centres of copper (II) ion binding in the nucleotide are the phosphate groups. The protonated amino groups of OSpm are involved in non-covalent interaction with the nitrogen atoms N(1), N(7) or N(3) of the purine or pyrimidine ring, whereas at higher pH, deprotonated nitrogen atoms of polyamine are engaged in metallation in MLL' species.
Topics: Adenosine Monophosphate; Copper; Cytidine Monophosphate; Drug Interactions; Electron Spin Resonance Spectroscopy; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Molecular Structure; Purine Nucleotides; Pyrimidine Nucleotides; Spermine
PubMed: 16899296
DOI: 10.1016/j.jinorgbio.2006.06.009 -
Nucleic Acids Research 2005Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are...
Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3'-->P5' phosphoramidate (amidate), 5-(1-propynyl)-2'-deoxyuridine (pdU), 2'-O-methyl-ribose (2'-O-Me), 2'-O-(2-aminoethyl)-ribose, or 2'-O, 4'-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.
Topics: Animals; Binding Sites; CHO Cells; COS Cells; Chlorocebus aethiops; Cricetinae; Cricetulus; DNA; DNA Repair; Deoxyribonucleases; HeLa Cells; Humans; Oligonucleotides; Pyrimidine Nucleotides; Recombination, Genetic
PubMed: 15961731
DOI: 10.1093/nar/gki659 -
Bioorganic & Medicinal Chemistry Letters Jan 2016Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically...
Recently, 7-substituted 7-deazapurine nucleoside triphosphates and 5-substituted pyrimidine nucleoside triphosphates (dN(am)TPs) were synthesized to extend enzymatically using commercially available polymerase. However, extension was limited when we attempted to incorporate the substrates consecutively. To address this, we have produced a mutant polymerase that can efficiently accept the modified nucleotide with amphiphilic groups as substrates. Here we show that the KOD polymerase mutant, KOD exo(-)/A485L, had the ability to incorporate dN(am)TP continuously over 50nt, indicating that the mutant is sufficient for generating functional nucleic acid molecules.
Topics: DNA-Directed DNA Polymerase; Oligodeoxyribonucleotides; Point Mutation; Polyethylene Glycols; Purine Nucleotides; Pyrimidine Nucleotides; Temperature
PubMed: 26627581
DOI: 10.1016/j.bmcl.2015.11.079