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At least 307 records · Page 17Linked to original sources
PubMed2000

Constitutive release of ATP and evidence for major contribution of ecto-nucleotide pyrophosphatase and nucleoside diphosphokinase to extracellular nucleotide concentrations.

Nucleotides are important extracellular signaling molecules. At least five mammalian P2Y receptors exist that are specifically activated by ATP, UTP, ADP, or UDP. Although the existence of ectoenzymes that metabolize extracellular nucleotides is well established, the relative flux of ATP and UTP through their extracellular metabolic products remains undefined. Therefore, we have studied the kinetics of accumulation and metabolism of endogenous ATP in the extracellular medium of four different cell lines. ATP concentrations reached a maximum immediately after change of medium and decreased thereafter with a single exponential decay (t(1/2);1 approximately;230-40 min). ATP levels did not fall to zero but attained a base-line concentration that was independent of the medium volume and of the initial ATP concentration. Although the base-line concentration of ATP remained stable for up to 12 h, [gamma-(32)P]ATP added to resting cells as a radiotracer was completely degraded within 120 min, indicating that steady state reflected a basal rate of ATP release balanced by ATP hydrolysis (20-200 fmol x min(-)(1) x cell(-)(6)). High performance liquid chromatography analysis revealed that the gamma-phosphate of ATP was rapidly, although transiently, transferred during steady state to species subsequently identified as UTP and GTP, indicating the existence of both ecto-nucleoside diphosphokinase activity and the accumulation of endogenous UDP and GDP. Conversely, addition of [gamma-(32)P]UTP to resting cells resulted in transient formation of [gamma-(32)P]ATP, indicating phosphorylation of endogenous ADP by nucleoside diphosphokinase. The final (32)P-products of [gamma-(32)P]ATP metabolism were [(32)P]orthophosphoric acid and a (32)P-labeled species that was further purified and identified as [(32)P]inorganic pyrophosphate. In C6 cells, the formation of [(32)P]pyrophosphate from [gamma-(32)P]ATP at steady state exceeded by 3-fold that of [(32)P]orthophosphate. These results illustrate for the first time a constitutive release of ATP and other nucleotides and reveal the existence of a complex extracellular metabolic pathway for released nucleotides. In addition to the existence of an ecto-ATPase activity, our results suggest a major scavenger role of ecto-ATP pyrophosphatase and a transphosphorylating activity of nucleoside diphosphokinase.

Adenosine Triphosphate↗
PubMed1978

Purine nucleoside and nucleotide interactions on normal and subsensitive alpha adrenoreceptor responsiveness in guinea-pig vas deferens.

Adenosine and adenosine 5'-monophosphate (AMP) augment contractile responses to norepinephrine (NE) in isolated guinea-pig vas deferens. Dipyridamole slightly enhances, while theophylline antagonizes, adenosine effects on responses to NE. Adenosine triphosphate (ATP) and the nonhydrolyzable analog, adenosine 5'-(beta,gamma-imido)triphosphate (AppNp) depress responses to NE. Adenine and adenosine diphosphate (ADP) are ineffective in influencing alpha adrenoreceptor responsiveness. Repetitive stimulation of isolated vas deferens with maximal concentrations of NE markedly reduce the contractile response to test concentrations of 6 micron NE. Spontaneous resensitization of responses to NE to control levels occurs within 25 to 35 minutes after the end of desensitization treatment. Adenine nucleosides and nucleotides promote a more rapid rate of alpha adrenoreceptor resensitization, with a potency order: AMP greater than adenosine greater than ADP. Adenine and ATP did not influence the rate of alpha adrenoreceptor resensitization. The adenine nucleotides ADP, ATP and the analog AppNp elicit concentration-dependent contractions of guinea-pig vas deferens. Theophylline antagonizes this contractile activity to adenine nucleotides. AMP, adenosine and adenine are devoid of agonistic activity. In the presence of NE, however, AMP and adenosine produce contractile responses of isolated vas deferens strips, and the agonistic activity of ADP, ATP and AppNp is profoundly enhanced. Agonistic actions of purines in the presence of NE are antagonized by phentolamine much more effectively than by theophylline. The results suggest the existence of a purinergic receptor mediating excitatory responses of guinea-pig vas deferens. Furthermore, there appears to be mutual interaction between purinergic and alpha adrenoreceptor mechanisms. That adenyl derivatives are capable of augmenting subsensitive alpha adrenoreceptor responsiveness suggests that adenine nucleosides or nucleotides, released during sympathetic transmission, may be required for maintenance of normal alpha adrenoreceptor sensitivity.

Adenine Nucleotides↗
PubMed2002

HPLC determination of thiopurine nucleosides and nucleotides in vivo in lymphoblasts following mercaptopurine therapy.

BACKGROUND: Mercaptopurine is a prodrug requiring intracellular activation to thiopurine nucleotides to exert antileukemic effect. We developed a reversed-phase liquid chromatographic assay for the quantification of mercaptopurine, thioguanine, and methylmercaptopurine nucleoside and nucleotide concentrations in the target tissue, the leukemic lymphoblast. METHODS: Leukemic blasts were isolated from peripheral blood and bone marrow by a standard Ficoll-hypaque procedure. Proteins were removed by ultrafiltration in the presence of dithiothreitol. Thiopurine ribonucleotides were converted into their respective ribonucleosides by treatment of ultrafiltrate with acid phosphatase. Thiopurine nucleosides and bases were measured by direct injection of ultrafiltrate into the chromatographic system. Thiopurine nucleotide concentrations were calculated by subtracting the thiopurine nucleoside concentrations measured after treatment with acid phosphatase from those measured after direct injection of ultrafiltrate in the chromatographic system. Analytes were separated on a C18 Supelco column with ammonium phosphate-methanol eluent coupled with ultraviolet detection. RESULTS: CVs for intra- and interday precision were 1.1-14% (median, 4.9%), and recovery of added analyte was 89-126% (median, 105%) at low and high concentrations of analytes, except for mercaptopurine riboside. The median signal for each of the five metabolites in lymphoblast samples was 98% (range, 80-106%) of that in water. Detection limits for thiopurine bases and nucleosides ranged from 0.5 to 4.5 pmol/5 x 10(6) cells. CONCLUSIONS: This method is suitable for measurement of thiopurine metabolite concentrations in lymphoblasts in children with acute lymphoblastic leukemia following a single dose of intravenous mercaptopurine.

Antimetabolites, Antineoplastic↗
PubMed2001

NDP kinase reactivity towards 3TC nucleotides.

Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.

Animals↗
PubMed2004

The role of nucleoside and nucleotide reverse transcriptase inhibitor backbones in antiretroviral therapy.

Several dual nucleoside reverse transcriptase inhibitor (NRTI) combinations provide efficacy when combined with a 3rd agent. However, there are a number of issues with current NRTI and nucleotide reverse transcriptase inhibitor (NtRTI) combinations that often lead to treatment failure and limited treatment options. These issues include suboptimal potency, drug interactions, toxicities, tolerability issues, and selection of resistance mutations that confer cross-resistance. Options for simplified NRTI backbones include fixed-dose combinations and agents that allow once-daily dosing; however, once-daily treatment choices are currently limited because of a lack of data on potential combinations. This article provides an historical perspective on the use of NRTI backbones in the treatment of HIV infection and outlines the advantages and disadvantages of currently available backbone combinations. In addition, it provides a brief introduction to backbone combinations under investigation as potential options for initial therapy. In an environment where several NRTI/NtRTI backbones offer comparable efficacy, treatment decisions will increasingly be made based on toxicity, resistance, and convenience considerations.

Anti-HIV Agents↗
PubMed1992

Interactions of praseodymium and neodymium with nucleosides and nucleotides: absorption difference and comparative absorption spectral study.

The interactions of praseodymium(III) and neodymium(III) with nucleosides and nucleotides have been studied in different stoichiometry in water and water-DMF mixtures by employing absorption difference and comparative absorption spectrophotometry. The 4f-4f bands were analysed by linear curve analysis followed by gaussian curve analysis, and various spectral parameters were computed, using partial and multiple regression method. The magnitude of changes in both energy interaction and intensity were used to explore the degree of outer and inner sphere coordination, incidence of covalency and the extent of metal 4f-orbital involvement in chemical bonding. Crystalline complexes of the type [Ln(nucleotide)2(H2O)2]- (where nucleotide--GMP or IMP) were characterized by IR, 1H NMR, 31P NMR data. These studies indicated that the binding of the nucleotide is through phosphate oxygen in a bidentate manner and the complexes undergo substantial ionisation in aqueous medium, thereby supporting the observed weak 4f-4f bands and lower values for nephelauxetic effect (1-beta), bonding (b) and covalency (delta) parameters derived from coulombic and spin orbit interaction parameters.

Guanosine Monophosphate↗
PubMed2000

Structure, energetics, vibrational frequencies and charge transfer of base pairs, nucleoside pairs, nucleotide pairs and B-DNA pairs of trinucleotides: ab initio HF/MINI-1 and empirical force field study.

Geometries, interaction energies and vibrational frequencies of base pairs, nucleoside pairs and nucleotide pairs were studied by ab initio Hartree-Fock (HF) method using MINI-1 basis set and empirical Cornell et al. force field (AMBER 4.1). A good agreement was found between HF/MINI-1 and AMBER results. In addition, both methods provide reasonable agreement with available high-level ab initio data. Finally, AMBER potential was used to determine the structure, energetics and vibrational frequencies of B-DNA pairs of trinucleotides. Stabilization energies of clusters are lowered when passing from base pairs to nucleoside pairs, nucleotide pairs and to pairs of trinucleotides. The lowest vibrations of base pairs and nucleoside pairs correspond to intermolecular motions of bases, specifically to buckle and propeller motions. In the case of pairs of larger subunits the lowest vibrations are of intramolecular nature (rotation around glycosidic bond, sugar and phosphate vibration). The spectra of these clusters became more complicated and quasi-degenerate. Intermolecular charge transfer between bases in H-bonded and stacked pairs is negligible, while a significant intramolecular charge transfer was observed.

Base Pairing↗
PubMed2001

Mutational patterns in the HIV genome and cross-resistance following nucleoside and nucleotide analogue drug exposure.

A variety of key mutations in HIV reverse transcriptase (RT) have been associated with nucleoside reverse transcriptase inhibitor (NRTI) exposure, which give rise to a diverse range of effects in terms of altered drug susceptibilities, viral replicative capacity and RT biochemistry. There are three basic mechanisms of resistance conferred by specific mutations in the coding region of RT. The first is drug discrimination, whereby a particular drug or drugs are either selectively excluded from uptake or from the RT-primer-template catalytic complex. Drug discrimination is, for the most part, relatively specific for individual drugs. Repositioning of the template-primer to prevent a catalytically competent complex in the presence of a bound drug molecule has also been observed in some instances, and forms a second mechanism. The third, and potentially most significant for long-term efficacy of the NRTIs, is pyrophosphorolysis, the primary mode of resistance to zidovudine. Mutations selected by this drug or stavudine serve to elevate the natural rate of the reverse reaction for RT. Pyrophosphorolysis uncouples the last nucleoside monophosphate added to the proviral transcript, and attaches it to either a free pyrophosphate (regenerating a deoxynucleoside triphosphate) or to a nucleoside di- or triphosphate (usually ATP). Uncoupling a chain-terminating NRTI residue therefore rescues reverse transcription and reduces drug susceptibility across the class, since the process is not specific for the selecting drug. Of all the nucleoside-associated mutations, the best known and most studied are the six associated with thymidine analogue exposure. These six mutations (M41L, D67N, K70R, L210W, T215Y/F, K219Q) enhance RT pyrophosphorolysis to confer high-level viral resistance to zidovudine, and clinically significant loss of response to stavudine and didanosine. They have also been found to confer reduced susceptibility to lamivudine and abacavir, particularly when present alongside other NRTI-induced changes. Other key mutations generally confer more limited resistance to specific agents, although the primary lamivudine- and abacavir-associated M184V substitution generates a broad spectrum of drug-dependent phenotypes, and uncommon mutational complexes conferring resistance across the entire class are well known. In addition to 'classical' multi-nucleoside-resistant genotypes, database-driven 'virtual phenotyping' for accumulations of NRTI-associated mutations around a core of thymidine analogue-induced changes predicts drug susceptibilities below wild-type across the entire NRTI class, even in the absence of key mutations associated with individual agents. When the natural range of drug susceptibilities for treatment-naive isolates is used as the basis for defining resistance, retrospective analysis of clinical isolates in the Virco database shows a significantly increased incidence of reduced susceptibility for the dideoxy NRTIs (didanosine, stavudine and zalcitabine) that was undetected in previous assays. These data imply a cumulative degradation of response to

Anti-HIV Agents↗
PubMed2004

Biodegradation of DNA and nucleotides to nucleosides and free bases.

Thirty-two different microorganisms were examined in order to check their ability to degrade an exogenous DNA. Bacteria from species: Stenotrophomonas maltophilia, Brevundimonas diminuta, Bacillus subtilis, Mycobacterium butyricum and fungus Fusarium moniliforme were capable to degrade DNA to nucleic bases or their derivatives. Degradation of DNA by S. maltophilia resulted in formation of free bases, such as hypoxanthine, thymine, uracil and xanthine. The optimum concentration of DNA seemed to be 3 mg ml(-1). The mode of degradation of DNA nucleotides depended on the type of nucleotide and its concentration, but nucleic bases or their derivatives were always formed at the end of the reaction process.

Biodegradation, Environmental↗
PubMedDate not supplied

2-substituted thioadenine nucleoside and nucleotide analogues: synthesis and receptor subtype binding affinities (1).

The design, synthesis, and receptor subtype binding affinities of several 2-substituted thioadenosine nucleoside and nucleotide analogues are described. Alkylation of 2-thioadenosine (1) with iodopentenylboronic acid followed by iododeboronation gave 2-((E)-1-iodo-1-penten-5-yl) thioadenosine (9). Compound 1 on treatment with 4-nitrobenzyl bromide and propargyl bromide furnished compounds 3 and 5, respectively. The 5'-monophosphate analogues of compounds 3, 5, 7, and 9 were prepared similarly using 2-thioadenosine 5'-monophosphate (2). Treatment of 1 with bromoethylamine hydrobromide provided 2-[(aminoethyl)thio]adenosine (11) which on coupling with N-succinimidyl 3-(4-hydroxyphenyl)propionate gave 2-[[[3-(4-hydroxyphenyl)propionamido]ethyl]thio]adenosine (12). Iodination of 12 gave 2-[[[3-(4-hydroxy-3-iodophenyl)propionamido]ethyl]thio]adenosine (13). Compounds 3-13 were evaluated for their affinities toward A1 and A2 adenosine receptors in rat brain cortex and striatum, respectively using [3H]DPCPX and [3H]CGS21680 as ligands. The nucleotide analogues 4, 6, 8, and 10 inhibited binding of [3H]DPCPX by 10-20% and of [3H]CGS21680 by 40-50% at a concentration of 100 microM suggesting weak affinity toward adenosine receptors. The nucleoside analogues 3, 5, 7, 9, 12, and 13 inhibited the A2 receptor binding of [3H]CGS21680 with Ki values of 1.2-3.67 microM, while A1 receptor binding of [3H]DPCPX was inhibited with Ki values 10-17 microM. The A1/A2 ratios suggest 4-8-fold A2 receptor selectivity.

Adenine Nucleotides↗
PubMedDate not supplied

Ribose modified nucleosides and nucleotides as ligands for purine receptors.

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.

Animals↗
PubMed1991

The modification of guanine nucleosides and nucleotides by the borderline arylamine carcinogens 4-methyl- and 4-methoxyaniline: chemistry and structural characterization.

We report here on the conformational analysis of C8-arylamine nucleoside and nucleotide adducts of the borderline carcinogens 4-methylaniline and 4-methoxyaniline. The non-phosphorylated adducts show anti conformation of the glycosidic link, while the corresponding 5'-phosphorylated adducts have a syn conformation. All adducts exhibit a predominant C2'-endo conformation of the sugar ring and a gg conformation of the exocyclic bond.

Aniline Compounds↗