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The Journal of Biological Chemistry Jul 1955
Topics: Nucleotides; Phosphates; Pyrimidine Nucleotides; Uracil Nucleotides; Uridine
PubMed: 14392174
DOI: No ID Found -
Biochemical Pharmacology Aug 1999Transformed cells are characterized by imbalances in metabolic routes. In particular, different key enzymes of nucleotide metabolism and DNA biosynthesis, such as CTP... (Review)
Review
Transformed cells are characterized by imbalances in metabolic routes. In particular, different key enzymes of nucleotide metabolism and DNA biosynthesis, such as CTP synthetase, thymidylate synthase, dihydrofolate reductase, IMP dehydrogenase, ribonucleotide reductase, DNA polymerase, and DNA methyltransferase, are markedly up-regulated in certain tumor cells. Together with the concomitant down-modulation of the purine and pyrimidine degradation enzymes, the increased anabolic propensity supports the excessive proliferation of transformed cells. However, many types of cancer cells have maintained the ability to differentiate terminally into mature, non-proliferating cells not only in response to physiological receptor ligands, such as retinoic acid, vitamin D metabolites, and cytokines, but also following exposure to a wide variety of non-physiological agents such as antimetabolites. Interestingly, induction of tumor cell differentiation is often associated with reversal of the transformation-related enzyme deregulations. An important class of differentiating compounds comprises the antimetabolites of purine and pyrimidine nucleotide metabolism and nucleic acid synthesis, the majority being structural analogs of natural nucleosides. The CTP synthetase inhibitors cyclopentenylcytosine and 3-deazauridine, the thymidylate synthase inhibitor 5-fluoro-2'-deoxyuridine, the dihydrofolate reductase inhibitor methotrexate, the IMP dehydrogenase inhibitors tiazofurin, ribavirin, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) and mycophenolic acid, the ribonucleotide reductase inhibitors hydroxyurea and deferoxamine, and the DNA polymerase inhibitors ara-C, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and aphidicolin, as well as several nucleoside analogs perturbing the DNA methylation pattern, have been found to induce tumor cell differentiation through impairment of DNA synthesis and/or function. Thus, by selectively targeting those anabolic enzymes that contribute to the neoplastic behavior of cancer cells, the normal cellular differentiation program may be reactivated and the malignant phenotype suppressed.
Topics: Animals; Antimetabolites; Antineoplastic Agents; Cell Differentiation; Cell Transformation, Neoplastic; Humans; Neoplasms; Purine Nucleotides; Pyrimidine Nucleotides
PubMed: 10413291
DOI: 10.1016/s0006-2952(99)00035-0 -
Archives of Biochemistry and Biophysics Jan 1998HOCl-induced chlorination of pyrimidine nucleotides, PyNH, strikingly depends on the nature of the available chlorine acceptor group. For CMP, with an -NH2 group as...
HOCl-induced chlorination of pyrimidine nucleotides, PyNH, strikingly depends on the nature of the available chlorine acceptor group. For CMP, with an -NH2 group as acceptor, the reaction is slow and involves predominantly the acid [k(CMP + HOCl) approximately 100 M-1 s-1 at pH 6]; apparent rate constants of the reaction decrease around the pK alpha (HOCl), to 0 in alkaline solution. For TMP and UMP, with a heterocyclic > NH group (at 3N) as acceptor, the reaction is faster and involves mainly the conjugated CIO- anion [e.g., k(UMP + ClO-) approximately 3 x 10(4) M-1 s-1 and k(UMP + HOCl) approximately 200 M-1 s-1]. The 3-N-methylthymidine derivative is inert toward HOCl. Reactions of ClO- with TMP, UMP, and poly(U) are shown to be reversible, PyNH + ClO- = PyNCl + OH-; an increase in pH due to this reaction was confirmed, and equilibrium constants have been estimated. The chlorinated derivatives of TMP and UMP are very reactive toward GSH, disulfide, aliphatic amines, and NADH. In contrast, the PyNCl derivative of CMP is unreactive, except with GSH. Rate constants of reactions of PyNCl species with various substrates are presented. Oxidation of NADH, by both HOCl and PyNCl derivatives, leads to a stable product (not NAD+) which is irreversibly degraded by reaction with excess HOCl, but inert toward acsorbate, GSH, and H2O2. Thiols (GSH) and disulfides (DTPA) were previously found capable of scavenging up to four HOCl molecules (Prütz, W. A., Arch. Biochem. Biophys. 332, 110-120, 1996). In the present study it was established that reactions of GSH or DTPA with excess HOCl give rise to a rapid drop in the pH by release of up to four HCl molecules per GSH or DTPA, as expected for a sequence of consecutive sulfoxidations. Reactions of GSH and DTPA with PyNCl efficiently regenerate PyNH, namely up to four molecules per GSH or DTPA in the case of TMP and UMP, but only one molecule per GSH in the case of CMP. The PyNCl derivatives of TMP and UMP transfer chlorine slowly but completely to CMP or AMP. Such chlorine transfer between nucleic acid bases is likely to occur also in DNA; it is shown that HOCl in fact induces a complex series of reactions on interaction with native DNA.
Topics: Animals; Glutathione; Hypochlorous Acid; NAD; Pyrimidine Nucleotides
PubMed: 9439597
DOI: 10.1006/abbi.1997.0440 -
Bioscience, Biotechnology, and... Jun 1997Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a...
Enzymatic production of pyrimidine nucleotides using Corynebacterium ammoniagenes cells and recombinant Escherichia coli cells: enzymatic production of CDP-choline from orotic acid and choline chloride (Part I).
Enzymatic production of cytidine diphosphate choline (CDP-choline) using orotic acid and choline chloride as substrates was investigated using a 200-ml beaker as a reaction vessel. When Cornybacterium ammoniagenes KY13505 cells were used as the enzyme source, UMP was accumulated up to 28.6 g/liter (77.6 mM) from orotic acid after 26 h of reaction. In this reaction, UDP and UTP were also accumulated, but CTP, a direct precursor of CDP-choline, was not accumulated sufficiently. Escherichia coli JF646/pMW6 cells, which overproduce CTP synthetase by selfcloning of the pyrG gene, were used together with cells of KY12505 for the enzymatic reaction using orotic acid as a substrate. CTP was produced at 8.95 g/liter (15.1 mM) after 23 h of this reaction. To produce CDP-choline, two additional enzyme activities were needed. E. coli MM294/pUCK3 and MM294/pCC41 cells, which express a choline kinase from Saccharomyces cerevisiae (CKIase; encoded by the CKI gene) and a cholinephosphate cytidylyltransferase from S. cerevisiae (CCTase; encoded by the CCT gene) respectively, were added to this CTP-producing reaction system. After 23 h of the reaction using orotic acid and choline chloride as substrates, 7.7 g/liter (15.1 mM) of CDP-choline was accumulated without addition of ATP or phosphoribosylpyrophosphate (PRPP). ATP and PRPP required in the CDP-choline forming reaction system are biosynthesized by those cells using glucose as a substrate.
Topics: Carbon-Nitrogen Ligases; Choline; Chromatography, High Pressure Liquid; Corynebacterium; Cytidine Diphosphate Choline; Cytidine Triphosphate; Escherichia coli; Gene Expression Regulation, Enzymologic; Ligases; Orotic Acid; Phosphoribosyl Pyrophosphate; Plasmids; Pyrimidine Nucleotides; Saccharomyces cerevisiae; Substrate Specificity; Uridine Diphosphate; Uridine Monophosphate; Uridine Triphosphate
PubMed: 9214753
DOI: 10.1271/bbb.61.956 -
Chemistry, An Asian Journal Mar 2009The enzymatic incorporation of a series of emissive pyrimidine analogues into RNA oligonucleotides is explored. T7 RNA polymerase is challenged with accepting three...
The enzymatic incorporation of a series of emissive pyrimidine analogues into RNA oligonucleotides is explored. T7 RNA polymerase is challenged with accepting three non-natural, yet related, triphosphates as substrates and incorporating them into diverse RNA transcripts. The three ribonucleoside triphosphates differ only in the modification of their uracil nucleus and include a thieno[3,2-d]pyrimidine nucleoside, a thieno[3,4-d]pyrimidine derivative, and a uridine containing a thiophene ring conjugated at its 5-position. All thiophene-containing uridine triphosphates (UTPs) get incorporated into RNA oligonucleotides at positions that are remote to the promoter, although the yields of the transcripts vary compared with the transcript obtained with only native triphosphates. Among the three derivatives, the 5-modified UTP is found to be the most "polymerase-friendly" and is well accommodated by T7 RNA polymerase. Although the fused thiophene analogues cannot be incorporated next to the promoter region, the 5-modified non-natural UTP gets incorporated near the promoter (albeit in relatively low yields) and even in multiple copies. Labeling experiments shed light on the mediocre incorporation of the fused analogues, suggesting the enzyme frequently pauses at the incorporation position. When incorporation does take place, the enzyme fails to elongate the modified oligonucleotide and yields aborted transcripts. Taken together, these results highlight the versatility and robustness, as well as the scope and limitation, of T7 RNA polymerase in accepting and incorporating reporter nucleotides into modified RNA transcripts.
Topics: Base Sequence; DNA-Directed RNA Polymerases; Fluorescent Dyes; Isotope Labeling; Oligonucleotides; Pyrimidine Nucleotides; Ribonucleotides; Substrate Specificity; Transcription, Genetic; Uridine Triphosphate; Viral Proteins
PubMed: 19072942
DOI: 10.1002/asia.200800370 -
The Journal of Endocrinology Nov 1973
Topics: Animals; Carbon Radioisotopes; Castration; Cytidine; Cytosine Nucleotides; DNA; Decidua; Estradiol; Female; Mice; Organ Size; Orotic Acid; Pregnancy; Progesterone; Pyrimidine Nucleotides; RNA; Sesame Oil; Time Factors; Tritium; Uracil Nucleotides; Uridine; Uterus
PubMed: 4759593
DOI: 10.1677/joe.0.0590275 -
Journal of the American Chemical Society Nov 2006The sequential addition of water molecules to protonated and deprotonated forms of the four mononucleotides dAMP, dCMP, dGMP, and dTMP was studied experimentally by...
The sequential addition of water molecules to protonated and deprotonated forms of the four mononucleotides dAMP, dCMP, dGMP, and dTMP was studied experimentally by equilibrium measurements using an electrospray mass spectrometer equipped with a drift cell and theoretically by computational methods including molecular modeling and density functional theory calculations. Experiments were carried out in positive and negative ion mode, and calculations included the protonated and deprotonated forms of the four nucleotides. For deprotonated anionic nucleotides the experimental enthalpies of hydration (DeltaH degrees n) were found to be similar for all four systems and varied between -10.1 and -11.5 kcal mol-1 for the first water molecule (n = 1) and -8.3 and -9.6 kcal mol-1 for additional water molecules (n = 2-4). Theory indicated that the first water molecule binds to the charge-carrying phosphate group. Simulations of deprotonated mononucleotides with four water molecules yielded a large number of structures with similar energies. In some of the structures all four water molecules cluster around the phosphate group, and in other structures the four water molecules each hydrate a different functional group of the nucleotide. These include the phosphate group, the deoxyribose hydroxyl group, and various functional groups on the nucleobases. Experimental DeltaH degrees 1 values for the protonated cationic mononucleotides ranged from -10.5 to -13.5 kcal mol-1 with more negative values (< or =-12 kcal mol-1) for dCMP, dGMP, and dTMP and the least negative value for dAMP. For n = 2-4 DeltaH degrees n values varied from -6.9 to -9.7 kcal/mol and were similar in value to the deprotonated nucleotides except for dAMP. Theory on the protonated nucleotides indicated that the first water molecule binds to the charge-carrying group for dCMP, dGMP, and dTMP. For protonated dAMP, on the other hand, the charge-carrying N3 group is well self-solvated by the phosphate group and not readily available for a hydrogen bond with the water molecule. The insight gained on nucleotide stabilization by individual water molecules is used to discuss the competition between hydration of individual nucleotides and Watson-Crick base pairing.
Topics: DNA; Mass Spectrometry; Models, Molecular; Nucleic Acid Conformation; Purine Nucleotides; Pyrimidine Nucleotides; Thermodynamics; Water
PubMed: 17117867
DOI: 10.1021/ja062418o -
The Journal of Pharmacology and... Jan 2011Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3',5'-diguanosine monophosphate (c-di-GMP), which is degraded by specific... (Comparative Study)
Comparative Study
Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3',5'-diguanosine monophosphate (c-di-GMP), which is degraded by specific phosphodiesterases. c-di-GMP levels control the transition of bacteria from a motile to a biofilm-forming lifestyle. These bacterial communities are highly resistant to antibiotic treatment and represent the predominant lifestyle in most chronic infections. Hence, DGCs serve as starting point for the development of novel therapeutics interfering with the second messenger-signaling network in bacteria. In previous studies, we showed that 2'(3')-O-(N-methylanthraniloyl) (MANT)- and 2',3'-O-(2,4,6-trinitrophenyl) (TNP)-substituted nucleotides are potent adenylyl and guanylyl cyclase inhibitors. The catalytic domain of DGCs is homologous to the mammalian adenylyl cyclase catalytic domain. Therefore, we investigated the interaction of various MANT purine and pyrimidine nucleotides with the model DGC YdeH from Escherichia coli. We observed strong fluorescence resonance energy transfer between tryptophan and tyrosine residues of YdeH and the MANT group of MANT-NTPs (MANT-ATP, -CTP, -GTP, -ITP, -UTP, and -XTP) and an enhanced direct MANT fluorescence upon interaction with YdeH. We assessed the affinity of MANT-NTPs to YdeH by performing competition assays with NTPs. We conducted an amino acid alignment of YdeH with the earlier crystallized Caulobacter crescentus DGC PleD and found high similarities in the nucleotide-binding site of PleD. In vitro mass-spectrometric activity assays with YdeH resulted in the identification of new MANT/TNP nucleotide-based inhibitors of DGC activity. Together, the analysis of interactions between MANT/TNP nucleotides and YdeH provides a new basis for the identification and development of DGC inhibitors and allows insights into nucleotide-protein interactions.
Topics: Amino Acid Sequence; Escherichia coli; Escherichia coli Proteins; Molecular Sequence Data; Phosphorus-Oxygen Lyases; Protein Binding; Purine Nucleotides; Pyrimidine Nucleotides
PubMed: 20947637
DOI: 10.1124/jpet.110.170993 -
The Journal of Biological Chemistry Jan 2002The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around...
The present study describes the distribution and properties of enzymes of the catabolic pathway of pyrimidine nucleotides in Riftia pachyptila, a tubeworm living around deep-sea hydrothermal vents and known to be involved in a highly specialized symbiotic association with a bacterium. The catabolic enzymes, 5'-nucleotidase, uridine phosphorylase, and uracil reductase, are present in all tissues of the worm, whereas none of these enzymatic activities were found in the symbiotic bacteria. The 5'-nucleotidase activity was particularly high in the trophosome, the symbiont-harboring tissue. These results suggest that the production of nucleosides in the trophosome may represent an alternative source of carbon and nitrogen for R. pachyptila, because these nucleosides can be delivered to other parts of the worm. This process would complement the source of carbon and nitrogen from organic metabolites provided by the bacterial assimilatory pathways. The localization of the enzymes participating in catabolism, 5'-nucleotidase and uridine phosphorylase, and of the enzymes involved in the biosynthesis of pyrimidine nucleotides, aspartate transcarbamylase and dihydroorotase, shows a non-homogeneous distribution of these enzymes in the trophosome. The catabolic enzymes 5'-nucleotidase and uridine phosphorylase activities increase from the center of the trophosome to its periphery. In contrast, the anabolic enzymes aspartate transcarbamylase and dihydroorotase activities decrease from the center toward the periphery of the trophosome. We propose a general scheme of anatomical and physiological organization of the metabolic pathways of the pyrimidine nucleotides in R. pachyptila and its bacterial endosymbiont.
Topics: 5'-Nucleotidase; Animals; Chromatography, Ion Exchange; Cytidine Triphosphate; Hydrogen-Ion Concentration; Polychaeta; Pyrimidine Nucleotides; Substrate Specificity; Symbiosis
PubMed: 11591717
DOI: 10.1074/jbc.M108035200 -
The Journal of Chemical Physics May 2015X-ray absorption near edge structure (XANES) was measured at energies around the N K-edge of the pyrimidine-containing nucleotides, cytidine 5'-monophosphate (CMP),... (Comparative Study)
Comparative Study
X-ray absorption near edge structure (XANES) was measured at energies around the N K-edge of the pyrimidine-containing nucleotides, cytidine 5'-monophosphate (CMP), 2'-deoxythymidine 5'-monophosphate (dTMP), and uridine 5'-monophosphate (UMP), in aqueous solutions and in dried films under various pH conditions. The features of resonant excitations below the N K-edge in the XANES spectra for CMP, dTMP, and UMP changed depending on the pH of the solutions. The spectral change thus observed is systematically explained by the chemical shift of the core-levels of N atoms in the nucleobase moieties caused by structural changes due to protonation or deprotonation at different proton concentrations. This interpretation is supported by the results of theoretical calculations using density functional theory for the corresponding nucleobases in the neutral and protonated or deprotonated forms.
Topics: Electrons; Hydrogen-Ion Concentration; Hydroxyl Radical; Models, Chemical; Molecular Structure; Nitrogen; Protons; Pyrimidine Nucleotides; Solutions; Water; X-Ray Absorption Spectroscopy
PubMed: 25956126
DOI: 10.1063/1.4919744