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At least 91 records · Page 5Linked to original sources
PubMed2003

Two nucleoside uptake systems in Lactococcus lactis: competition between purine nucleosides and cytidine allows for modulation of intracellular nucleotide pools.

A method for measuring internal nucleoside triphosphate pools of lactococci was optimized and validated. This method is based on extraction of (33)P-labeled nucleotides with formic acid and evaluation by two-dimensional chromatography with a phosphate buffer system for the first dimension and with an H(3)BO(3)-LiOH buffer for separation in the second dimension. We report here the sizes of the ribo- and deoxyribonucleotide pools in laboratory strain MG1363 during growth in a defined medium. We found that purine- and pyrimidine-requiring strains may be used to establish physiological conditions in batch fermentations with altered nucleotide pools and growth rates by addition of nucleosides in different combinations. Addition of cytidine together with inosine to a purine-requiring strain leads to a reduction in the internal purine nucleotide pools and a decreased growth rate. This effect was not seen if cytidine was replaced by uridine. A similar effect was observed if cytidine and inosine were added to a pyrimidine-requiring strain; the UTP pool size was significantly decreased, and the growth rate was reduced. To explain the observed inhibition, the nucleoside transport systems in Lactococcus lactis were investigated by measuring the uptake of radioactively labeled nucleosides. The K(m) for for inosine, cytidine, and uridine was determined to be in the micromolar range. Furthermore, it was found that cytidine and inosine are competitive inhibitors of each other, whereas no competition was found between uridine and either cytidine or inosine. These findings suggest that there are two different high-affinity nucleoside transporters, one system responsible for uridine uptake and another system responsible for the uptake of all purine nucleosides and cytidine.

Adenosine Triphosphate↗
PubMed2000

Design of anti-HIV compounds: from nucleoside to nucleoside 5'-triphosphate analogs. Problems and perspectives.

To date, human immunodeficiency virus infection remains incurable although a variety of antiviral agents have been identified and characterized. Even though nucleoside analogs have been the most successful prodrugs, there remains the need to develop new compounds that exhibit a more favorable toxicity profile, less susceptible to cross-resistance, and greater efficacy. As prodrugs, the nucleoside analogs should be sequentially phosphorylated by cellular kinases to yield triphosphate form before they can inhibit HIV replication at the reverse transcriptase level. The efficiency of phosphorylation of nucleoside analogs is a key factor in their antiviral activity and strongly depends on nucleoside structure and cell type. In recent years, several attempts have been made to improve therapeutic potential of nucleoside analogs by the use of nucleotide prodrugs (pronucleotides), that can avoid the first step of phosphorylation. This review focuses on problems of intracellular phosphorylation of nucleoside analogs and perspectives of developing of a new class of nucleotide analogs modified at phosphate group as a form for the delivery of nucleotide analogs into the cell.

Animals↗
PubMed1991

Effect of nucleoside transport inhibitors on thymidine salvage and the toxicity of nucleoside analogs in mouse bone marrow granulocyte-macrophage progenitor cells.

In an attempt to elucidate the types of nucleoside transporters present in bone marrow stem cells, this study examined the effect of nucleoside transport inhibitors on the toxicity of nucleoside analogs and on the salvage of thymidine by mouse bone marrow granulocyte and macrophage progenitor cells using the CFU-GM assay. Concentrations of NBMPR (nitrobenzylmercaptopurine riboside) as low as 10 nM protected these cells from the toxicity of the adenosine analog tubercidin and provided a partial block of thymidine-rescue of the granulocyte-macrophage progenitor cells from methotrexate toxicity. Dipyridamole had similar effects but generally required higher concentrations. These results suggested that the major nucleoside transporter in these cells is the NBMPR-sensitive equilibrative carrier, es. In contrast to the results with tubercidin, the toxicity of 2-chlorodeoxyadenosine was increased 8- to 10-fold by 1 microM NBMPR. These results suggested that the bone marrow granulocyte-macrophage progenitor cells also have a concentrative nucleoside transporter that is capable of pumping 2-chlorodeoxyadenosine into the cells while efflux of the nucleoside via es is blocked by NBMPR.

Animals↗
PubMed1988

Thiamine pyrophosphatase (nucleoside diphosphatase) in the Golgi apparatus is distinct from microsomal nucleoside diphosphatase.

The properties of thiamine pyrophosphatase in the Golgi apparatus of rat liver were studied. Thiamine pyrophosphatase in an extract of the Golgi apparatus was separated into 6 bands of between pH 5.4 and 6.3 by isoelectric focusing on polyacrylamide gel. On the gels all these subforms catalyzed the hydrolyses of GDP, IDP, UDP, and CDP as well as that of thiamine pyrophosphate. The characteristics resembled those of Type B nucleoside diphosphatase of rat brain, though the enzyme did not have 3 subforms of Type B nucleoside diphosphatase in the higher pH region on isoelectric focusing. Thiamine pyrophosphatase of the Golgi apparatus was separated from microsomal nucleoside diphosphatase by DEAE-cellulose column chromatography. The properties of the enzyme were quite similar to those of Type B nucleoside diphosphatase with respect to its substrate specificity, optimum pH for activity, and inhibition by ATP. These findings suggest that thiamine pyrophosphatase in the Golgi apparatus is different from microsomal nucleoside diphosphatase and that it might be basically the same enzyme as Type B nucleoside diphosphatase except for different extents of modification.

Acid Anhydride Hydrolases↗
PubMed2000

Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family.

We have isolated and characterized the cDNA encoding nucleoside triphosphate diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside triphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an open reading frame of 1365 bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971 Da and a predicted N-terminal hydrophobic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% and 51% identity respectively with sequences of the two related human and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transcripts with differing abundances. PCR analysis suggests a single open reading frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary (CHO) and PC12 cells for immunological analysis and protein isolation. The protein was contained in membrane fractions of transfected CHO cells and occurred in a soluble form in the cell culture supernatants. NTPDase6 preferentially hydrolysed nucleoside 5'-diphosphates. With different substrates the order of activity was GDP>IDP>>UDP,CDP>>ADP. Nucleoside 5'-triphosphates were hydrolysed only to a minor extent and no hydrolysis of nucleoside 5'-monophosphates was observed. The enzyme was strongly and equally activated by Ca(2+) and Mg(2+) and had a K(m) for GDP of 211 microM. The immunohistochemical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is associated with the Golgi apparatus and to a small extent also with the plasma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of extracellular nucleotides.

Amino Acid Sequence↗
PubMed1988

Genetic control of red-cell nucleoside transport and its association with the B blood-group locus and nucleoside phosphorylase activity in sheep.

Nucleoside transport in sheep red cells is controlled by two allelomorphic genes, the gene for nucleoside transport deficiency (NuI) being dominant to that for the functional presence of carrier-mediated nucleoside transport activity (Nui). Sheep are also polymorphic with respect to their red-cell nucleoside phosphorylase (NP) activity, some having high activities and others low activities of this enzyme. The gene for high activity (NPH) is incompletely dominant to that for low activity (NPL). Inheritance data indicate that the Nu locus is genetically linked to that for the B blood-group system and, in addition, exerts a pleiotropic effect on NP activity, Nu permeability stabilizing the heat-labile NPL gene product. Nu-permeable cells have a higher ATP content than Nu-impermeable red cells, and within the Nu-impermeable subgroup, NP deficiency causes a further reduction in red cell ATP concentration. It is concluded that the nucleoside inosine supplements glucose as a physiological energy substrate in sheep red cells.

ABO Blood-Group System↗
PubMed1996

Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase.

An extensive study has been made of the fluorescence emission properties of the neutral and ionic forms in aqueous medium of the azapurine nucleosides, 8-azaadenosine (8-azaAdo), 8-azainosine (8-azaIno), 8-azaguanosine (8-azaGuo), and their aglycons. The fluorescence of 8-azaGuo at pH 7 originates from its anionic species (pKa = 8.05, phi= 0.55), as is also the case for 8-azaIno (pKa = 8.0, phi = 0.02), whereas 8-azaAdo is a strong emitter (phi = 0.06) as the neutral species. By contrast the corresponding free 8-azapurines are only weakly fluorescent in aqueous medium, with the exception of 8-azaguanine (8-azaG). Examination of the emission properties of N-substituted 8-azaguanines demonstrated that the observed blue emission of the neutral form of 8-azaG (phi = 0.05 to 0.33, dependent on lambda exc) originates from a minor tautomer of the compound, the N(8)-H form, present to the extent of 10-15%; while the principal N(9)-H tautomer is virtually nonfluorescent. The 8-azapurines are substrates of purine nucleoside phosphorylase (PNP), leading to their irreversible conversion to the corresponding nucleosides in the synthetic pathway of this enzyme. The fluorescent properties of these compounds, together with spectrophotometric methods, were applied to determine the basic kinetic parameters for synthesis of 8-azapurine nucleosides by PNP from mammalian (calf spleen) and bacterial (Escherichia coli) sources. The fluorimetric method was also used to determine the kinetic parameters for the second substrate, alpha-D-ribose 1-phosphate, and for the analytical titration of the latter in solution. The pH optimum of the reverse synthetic PNP reaction with 8-azapurines as substrates is below pH 7, due to their enhanced acidity in comparison with natural purines. The 8-azapurine nucleosides, but not their aglycons, are reasonably good inhibitors of phosphorolysis of Ino and Guo by E. coli PNP. The most effective is 8-azaIno (Ki approximately 20 microM), also the only one to inhibit phosphorolysis by the calf spleen enzyme (Ki approximately 40 microM). The nature of this inhibition is apparently uncompetitive.

Hydrogen-Ion Concentration↗
PubMed1981

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases.

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.

Escherichia coli↗
PubMed2002

Characterization of imidazo[4,5-d]pyridazine nucleosides as modulators of unwinding reaction mediated by West Nile virus nucleoside triphosphatase/helicase: evidence for activity on the level of substrate and/or enzyme.

Compounds that interact with DNA or RNA generally act as inhibitors of enzymes that unwind DNA or RNA. In the present study we describe the synthesis and properties of some nucleoside analogues that interact with double-stranded DNA but that, in contrast, facilitate the unwinding reaction mediated by West Nile (WN) virus nucleoside triphosphatase (NTPase)/helicase. The nucleoside analogues described, 1-(2'-O-methyl-beta-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)-dione (HMC-HO4), 1-(beta-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)-dione, and 1-(2'-deoxy-alpha-D-ribofuranosyl)imidazo[4,5-d]pyridazine-4,7(5H,6H)dione, all contain the imidazo[4,5-d]pyridazine ring system. The extent of the enhancing effect on helicase activity was found to be dependent on the time of exposure of the DNA substrate to the compounds and their concentrations. The nucleoside analogues were nevertheless found to be capable of uncoupling the ATPase and helicase activities of the enzyme by a mechanism operating on the level of the enzyme. Thus, in the case of HMC-HO4, the direct interaction with the enzyme caused inhibition of its helicase activity, with a half-maximal inhibitory concentration of 30 microM. The similar potency of the compound against replication of WN virus in cell culture suggests that inhibition of the helicase activity of the viral enzyme is responsible for the observed antiviral activity of HMC-HO4 and may indeed represent an important mode of action of antiviral drugs in general. Comparative studies performed with the related NTPase/helicase from hepatitis C virus revealed that the extent of the effects mediated by imidazo[4,5-d]pyridazine nucleosides is enzyme specific. The substances described may represent a starting point for the development of a new class of helicase-specific antivirals.

Acid Anhydride Hydrolases↗
PubMed1977

[Escherichia coli K-12 mutants capable of catabolizing purine nucleosides in the absence of purine nucleoside phosphorylase].

Strains of Escherichia coli K-12 defective in purine nucleoside phosphorylase (pup gene) formed on the medium with inosine as the source of carbon and energy phenotypical reversions for the ability of utilizing inosine as source of carbon or purines. The phenotypical suppression of the purine nucleoside phosphorylase deficiency is the result of the mutations (called pnd), which are mapped on the chromosome of E. coli beyond the region of the structural pup-gene location and have phenotypic manifestation distinct from that of pup+ allele: a) pnd mutants divide into some groups for the ability of utilizing several purine nucleosides, including xantosine that cannot be metabolized by pnd+ strains of E. coli; b) pnd mutations do not restore the ability of purine auxotrophs (pur) defective in purine nucleoside phosphorylase (pup) and adenine phosphoribosyltransferase (apt) to grow on the medium with adenine as the sole source of purines. Cell-free extracts of pnd mutants fail to degrade the guanine nucleosides in the absence of phosphate or arsenate ions. These data (and also the ability of pnd mutants to utilize both purine ribonucleosides and deoxyribonucleosides) seem to indicate that the activities induced by pnd mutations are phosphorylase activities.

Chromosome Mapping↗
PubMed2005

Synthesis and antiviral evaluation of novel exomethylene acyclic nucleosides and phosphonic acid nucleosides.

This paper describes a very simple synthesis route of novel acyclic nucleosides and phosphonic acid nucleosides. The condensation of the mesylates 6 and 17 with the natural nucleosidic bases (A, C, U, T) under nucleophilic substitution (K(2)CO(3), 18-Crown-6, DMF) and deprotection afforded the target nucleosides (11, 12, 13, 14) and phosphonic acid nucleosides (22, 23, 24, 25). In addition, these compounds were evaluated for their antiviral properties against various viruses. Uracil derivative 24 shows significant anti-HCMV activity (EC(50) = 10.24 microM).

Antiviral Agents↗
PubMed2006

Analysis of urinary nucleosides. V. Identification of urinary pyrimidine nucleosides by liquid chromatography/electrospray mass spectrometry.

Modified urinary nucleosides are potentially invaluable in cancer diagnosis, as they reflect altered RNA turnovers. High-performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, tandem mass spectrometry, MS(n) analysis and accurate mass measurements in order to identify pyrimidine nucleosides purified from urine. Potential nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the various mass spectrometric techniques. In this manner numerous pyrimidine nucleosides were identified in the urine samples from cancer patients including pseudouridine, cytidine, two methylcytidines and an acetylcytidine. Furthermore, a number of novel modified pyrimidine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data.

Biomarkers, Tumor↗