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PubMed2003

Nucleoside transporter profiles in human pancreatic cancer cells: role of hCNT1 in 2',2'-difluorodeoxycytidine- induced cytotoxicity.

PURPOSE: Concentrative nucleoside transporter (CNT) 1, CNT3, equilibrative nucleoside transporter (ENT) 1, and, to a lesser extent, ENT2, appear to be the transporters responsible for 2',2'-difluorodeoxycytidine (gemcitabine; Gemzar) uptake into cells. Gemcitabine is used currently in the treatment of pancreatic cancer, but the role of specific nucleoside carrier proteins in gemcitabine cytotoxicity has not been elucidated. Indeed, it is not known which nucleoside transporters are expressed in human pancreas. EXPERIMENTAL DESIGN: In this study we have used four cell lines [pancreatic neoplasia (NP)9, NP18, NP29, and NP31] derived from human pancreatic adenocarcinomas to monitor the pattern of nucleoside transporter expression, and we have heterologously expressed the high-affinity gemcitabine transporter human orthologue (h) CNT1 to monitor its role in drug responsiveness. RESULTS: All of the cell lines take up gemcitabine mostly via the hENT1 transporter, which is expressed at high levels. Reverse transcription-PCR analysis of the other four cloned plasma membrane transporter mRNAs revealed very different expression patterns among NP cell lines, with apparent selective loss or decrease of hCNT mRNAs. NP cells transiently express hCNT1-type Na(+)-dependent nucleoside transport activity at low/medium cell density but not in confluent cultures. Cells expressing hCNT1 in a more constitutive manner were cloned after stable transfection of hCNT1. Despite high constitutive hENT1 activity, this increased sensitivity to gemcitabine. CONCLUSION: In summary, human pancreatic adenocarcinoma cells overexpress hENT1, although they retain the ability to express a functional hCNT1 transporter, an isoform that confers sensitivity to gemcitabine.

Antimetabolites, Antineoplastic↗
PubMedDate not supplied

Clinical implications of HIV drug resistance to nucleoside and nucleotide reverse transcriptase inhibitors.

This review focuses on issues pertinent to the epidemiology and clinical interpretation of resistance to nucleos(t)ide reverse transcriptase inhibitors. Nucleoside reverse transcriptase inhibitor resistance mutations, and especially thymidine analog mutations, remain the most common form of resistance detected in drug-naive patients. At the same time, improved treatment strategies, changes in prescribing policies, and prompt management of treatment failure are changing the prevalence and patterns of nucleoside reverse transcriptase inhibitor resistance in treatment-experienced patients. The clinical interpretation of nucleoside reverse transcriptase inhibitor resistance is being increasingly refined, helped by improved understanding of resistance pathways, development of sophisticated methods of analysis of genotypic resistance patterns, and introduction of clinically relevant cutoffs. Correlation of genotypic and phenotypic resistance data to clinical outcomes is essential to allow appropriate interpretation. In some cases, phenotypic data, either obtained directly by phenotypic tests or extrapolated from genotypic results, provide the most immediate predictors of virologic response. In other cases, genotypic analyses identify mutations that impact on responses without showing a marked effect on the phenotype, by either acting as "sentinel" markers for the presence of resistance undetectable by standard methods, or by lowering the genetic barrier to the evolution of resistance. The potential benefits of nucleoside reverse transcriptase inhibitor resistance, through hypersusceptibility and fitness effects, are also increasingly understood and exploited in clinical practice. Although progress has been significant, there remain many challenges. It is often questioned whether genotypic scores and clinical cutoffs obtained by various methods and from frequently small datasets can be reliably extrapolated to the general population of treated patients. At the same time, there is a need to define the role of reverse transcriptase mutations that are identified by statistical analyses as being associated with nucleoside reverse transcriptase inhibitor exposure, but have unknown effects on virus phenotype and clinical outcome. Novel mechanisms have also been proposed to play a role in nucleoside reverse transcriptase inhibitor resistance, including changes in RNaseH that are not targeted by routine testing at present. One additional, currently unresolved issue is the clinical relevance of minority resistant species and the feasibility of introducing ultra-sensitive resistance tests in routine diagnostic settings. The most appropriate viral-load cutoff for performing resistance tests and the reliability of results obtained at low copy numbers are similarly controversial. In spite of these limitations, resistance testing with appropriate interpretation provides an important guide to successful treatment outcomes and necessary support to the introduction of new treatment strategies.

Drug Resistance, Multiple, Viral↗
PubMed1976

Transport mechanisms in isolated plasma membranes. Nucleoside processing by membrane vesicles from mouse fibroblast cells grown in defined medium.

Plasma membrane vesicles were isolated from a subline of L929 mouse fibroblasts grown on defined medium in the absence of serum. These vesicles were not significantly contaminated by mitochondria or endoplasmic reticulum. The isolation procedure, a modification of that originally developed by McKeel and Jarett (McKeel, D.W., and Jarett, L. (1970) J. Cell Biol. 44, 417-432) employs mechanical homogenization in isotonic medium followed by differential centrifugation. The resultant plasma membrane vesicles take up radioactivity when exposed to uniformly labeled nucleosides. Two subfractions of the plasma membrane were isolated, distinguished by their differing activity of 5'-nucleotidase and (Na+,K+)-stimulated ATPase, two well known plasma membrane enzyme markers. Uptake of nucleoside radioactivity was extensively studied in one subfraction; it was linear with time and membrane concentration over ranges used for the studies. Apparent Km values for uptake of radioactivity from adenosine, inosine, and uridine were 7.1 +/- 26 muM, respectively. Uptake of radioactivity from all three nucleosides exhibits a broad pH optimum from pH 7 to pH 9, but falls off rapidly at lower pH. N-Ethylmaleimide was an effective inhibitor of uptake of radioactivity from all three nucleosides; uptake of radioactivity from uridine is more sensitive than uptake of radioactivity from the purine nucleosides. Adenosine inhibited uptake of radioactivity from inosine more than from uridine. Inosine inhibited the uptake of radioactivity from adenosine, but uridine did not. Caffeine and 6-methylaminopurine riboside (6-N-methyladenosine differentially inhibit uptake of radioactivity from adenosine and inosine, and thus the vesicles apparently possess seperate transport systems for uptake of radioactivity from purine nucleosides and from uridine.

Adenosine Triphosphatases↗
PubMed1989

Effects of adenosine A2 receptor agonists on nucleoside transport.

A series of adenosine A2 receptor agonists were examined for their ability to activate adenosine A2 receptors and inhibit nucleoside transport. A2 receptor activation was measured by the ability of these adenosine agonists to relax porcine coronary smooth muscle, where the compounds varied in their EC50 values from 4.5 nM (CGS 21680A (2-[p-(2-carboxyethyl) phenylethylamino]-5'-N-ethylcarboxamidoadenosine)] to 3.6 microM (CGS 23321 [2 alpha,3 alpha-dihydroxy-1 beta-hydroxymethyl-4 beta-(2-phenylamino-9- adenyl)-cyclopentane]). Nucleoside transport was measured as the nitrobenzylthioinosine-sensitive cellular accumulation of [3H]uridine into guinea pig erythrocytes at 22 degrees C. The initial velocity of transport was dependent on substrate concentration and a substrate-velocity curve yielded a Km of 78 +/- 16 microM and a Vmax of 0.31 +/- 0.049 mmol/l of cell water per hr (mean +/- S.D., n = 4). Dipyridamole, a known potent inhibitor of nucleoside transport, blocked cellular [3H]uridine accumulation with an EC50 of 29.4 nM. Whereas a number of the adenosine agonists tested showed little or no inhibition of nucleoside transport, CV 1808 (2-phenylaminoadenosine) inhibited transport with an EC50 of 140 nM. In addition, two carbocyclic derivatives of CV 1808, CGS 23321 and CGS 23302 [(-)2S,3R-dihydroxy-4R-hydroxymethyl-1R-[2-(p-ethoxycarbonyl)- phenylamino-9-adenyl]-cyclopentane) inhibited nucleoside transport with respective EC50 values of 366 and 168 nM. The data suggest that these compounds have a different structure-activity relationship for adenosine A2 receptors and for the site mediating nucleoside transport inhibition.

Adenosine↗
PubMed1979

Patterns of urinary excretion of modified nucleosides.

A recently developed high-performance liquid chromatography method permits quantitative measurement of low levels of modified nucleosides in urine. We report on the patterns of excretion of seven modified nucleosides by normal subjects and cancer patients. It was found that the excretion of these nucleosides expressed as a function of creatinine concentration was constant, not episodic nor related to diet. Thus, randomly collected samples of urine are satisfactory sources for measurement of nucleosides, and the level of nucleosides is of significance when related to creatinine excretion. The constancy of the excretion of the modified nucleosides in normal subjects is quite remarkable. It implies strict metabolic control of transfer RNA turnover. The values for the individual nucleoside/creatinine ratios were found to be significantly elevated in the urine of colon cancer patients.

Adult↗
PubMed1982

Axonal transport and transcellular transfer of nucleosides and polyamines in intact and regenerating optic nerves of goldfish: speculation on the axonal regulation of periaxonal cell metabolism.

The axonal transport, metabolism, and transcellular transfer of uridine, adenosine, putrescine, and spermidine have been examined in intact and regenerating optic nerves of goldfish. Following intraocular injection of labeled nucleosides, axonal transport was determined by comparing left-right differences in tectal radioactivity, and transcellular transfer was indicated by light autoradiographic analysis. The results demonstrated axonal transport, transcellular transfer, and periaxonal cell utilization of both nucleosides in intact axons and severalfold increases of all of these processes in regenerating axons. Experiments in which the metabolism of the nucleosides was studied resulted in data which suggested that uridine and adenosine, when delivered to the tectum by axonal transport, are protected from degradation and thus are relatively more available for periaxonal cell utilization than nucleosides reaching these cells via the blood. In intact axons, the majority of the nonmetabolized radioactivity was present as UMP, UDP, and UTP following [3H]uridine injections, whereas the majority of the radioactivity following [3H]adenosine injections was present as adenosine, with the phosphorylated derivatives constituting a smaller proportion. During nerve regeneration, the relative proportion of nucleosides to nucleotides was reversed, with uridine being the principal labeled compound in the first case, and AMP, ADP, and ATP being the major labeled compounds in the latter case. The nucleosides also were found to be different from each other in that adenosine, but not uridine, can be taken up by optic axons and transported retrogradely from the tectum to retinal ganglion cell bodies in the eye. Following intraocular injection of [3H]spermidine, radioactivity was transported to the optic tectum and transferred to tectal cells in the vicinity of the regenerating axons. Following [3H]putrescine injections, silver grains were found over periaxonal glia, but preliminary findings suggest that they are not present over tectal neurons nor over radial glial cells in the periependymal layers. Analysis of tectal radioactivity showed in each case that it was composed primarily of the injected compounds. These studies indicate that, following axonal transport, the polyamines do not remain within regenerating axons but are transferred to cells surrounding the axon. On the basis of these and previous findings, we speculate that the axonal transport and transcellular transfer of uridine, adenosine, polyamines, and perhaps other small molecules are means of communication between axons and periaxonal cells; that the axon can affect RNA and protein synthesis in periaxonal cells by regulating the availability of these small molecules; and that, during nerve regeneration, the increased metabolic needs of periaxonal cells are met by an increased axonal supply of precursors (adenosine and uridine) and other molecules (polyamines) critical for protein synthesis.

Adenosine↗
PubMed1983

Relationship of urinary excretion of modified nucleosides to disease status in childhood acute lymphoblastic leukemia.

The levels of urinary excretion of five modified nucleosides were quantitated by high-performance liquid chromatography for 15 normal children and 24 children with acute lymphoblastic leukemia (ALL). Excretion of each nucleoside decreased linearly with age when quantitation was based on urine creatinine content. Patients with childhood ALL at initial diagnosis or in relapse had significantly higher concentrations of 1-methylinosine, N2,N2-dimethylguanosine, 1-methylguanosine, and pseudouridine in their urine when compared to the concentrations in either patients in remission (P less than 0.001, P less than 0.001, P less than 0.01, and P less than 0.05, respectively) or normal controls (P less than 0.001, P less than 0.02, P less than 0.01, and P less than 0.001, respectively). Excretion of 2-pyridone-5-carboxamide-N'-ribofuranoside did not show significant differences. Urinary excretion of 1-methylinosine demonstrated a positive linear relationship with the percentage of blast cells in the bone marrow [correlation coefficient (r) = 0.90]; the other nucleosides had lower degrees of correlation. In comparison, the absolute blast cell count in the peripheral blood showed less correlation to the percentage of blast cells in the bone marrow (r = 0.47) than did four of the five nucleosides. The data demonstrate that excretion of modified nucleosides reflects disease activity in childhood ALL and that the urinary nucleosides could be useful clinical markers for this disease.

Adolescent↗
PubMed2003

Functional characterization of a H+/nucleoside co-transporter (CaCNT) from Candida albicans, a fungal member of the concentrative nucleoside transporter (CNT) family of membrane proteins.

Human and other mammalian concentrative (Na(+)-linked) nucleoside transport proteins belong to a membrane protein family (CNT, TC 2.A.41) that also includes Escherichia coli H(+)-dependent nucleoside transport protein NupC. Here, we report the cDNA cloning and functional characterization of a CNT family member from the pathogenic yeast Candida albicans. This 608 amino acid residue H(+)/nucleoside symporter, designated CaCNT, contains 13 predicted transmembrane domains (TMs), but lacks the exofacial, glycosylated carboxyl-terminus of its mammalian counterparts. When produced in Xenopus oocytes, CaCNT exhibited transport activity for adenosine, uridine, inosine and guanosine but not cytidine, thymidine or the nucleobase hypoxanthine. Apparent K(m) values were in the range 16-64 micro M, with V(max) : K(m) ratios of 0.58-1.31. CaCNT also accepted purine and uridine analogue nucleoside drugs as permeants, including cordycepin (3'-deoxyadenosine), a nucleoside analogue with anti-fungal activity. Electrophysiological measurements under voltage clamp conditions gave a H(+) to [(14)C]uridine coupling ratio of 1 : 1. CaCNT, obtained from logarithmically growing cells, is the first described cation-coupled nucleoside transporter in yeast, and the first member of the CNT family of proteins to be characterized from a unicellular eukaryotic organism.

Amino Acid Sequence↗
PubMed2004

Allelic isoforms of the H+/nucleoside co-transporter (CaCNT) from Candida albicans reveal separate high- and low-affinity transport systems for nucleosides.

Contigs 19-10196 and 19-20196 of the Stanford Candida albicans genome sequence databank encode two putative allelic isoforms of C. albicans CaCNT, a recently characterized 608 amino acid residue H+-coupled fungal member of the CNT family of concentrative nucleoside transport proteins. The single Ser/Gly difference between CaCNT/19-20196 and CaCNT occurs at position 328 in putative TM 7, and corresponds to a Ser/Gly substitution previously shown to contribute to the contrasting pyrimidine and purine nucleoside selectivities of human (h) and rat (r) Na+-dependent CNT1 and CNT2. CaCNT/19-10196 differs from CaCNT by four amino acids, but has Gly at position 328. These new proteins were recreated by site-directed mutagenesis of CaCNT and characterized functionally by heterologous expression in Xenopus laevis oocytes. In marked contrast to h/rCNT1/2, both CaCNT/19-10196 and CaCNT/19-20196 exhibited permeant selectivities for purine nucleosides (adenosine, guanosine and inosine) and uridine similar to that of CaCNT. However, although H+-coupled, CaCNT/19-20196 exhibited a approximately 10-fold higher apparent Km for uridine than either CaCNT or CaCNT/19-10196. CaCNT/19-20196 also exhibited a low apparent affinity for inosine. We conclude that the three proteins correspond to high-affinity (CaCNT, CaCNT/19-10196) and low-affinity (CaCNT/19-20196) allelic isoforms of the C. albicans CNT nucleoside transporter. This is the first example of a single amino acid residue substitution altering a CNT protein's overall apparent affinity for nucleosides.

Amino Acid Sequence↗
PubMed1992

Nucleoside transport-deficient mutants of PK-15 pig kidney cell line.

Previous studies indicated that PK-15 pig kidney cells express solely a nitrobenzylthioinosine-sensitive, equilibrative nucleoside transporter. In the present study, PK-15 cells were mutagenized by treatment with ICR-170 and nucleoside transport-deficient mutants selected in a single step in growth medium containing tubercidin and cytosine arabinoside at a frequency of about 2 x 10(6). The mutants were simultaneously at least 100-times more resistant to tubercidin, cytosine arabinoside and 5-fluorodeoxyuridine than the wild-type parent cells. The mutants failed to transport thymidine and uridine and had lost all high affinity nitrobenzylthioinosine binding sites. Residual low level uptake of thymidine by the mutants was shown to be due to nonmediated permeation (passive diffusion), which explains the sensitivity of the mutants to growth inhibition by high concentrations of the nucleoside drugs. Passive diffusion of thymidine at a concentration of 16 microM was not rapid enough to support the growth of nucleoside transport-deficient mutant cells that had been made thymidine-dependent by treatment with methotrexate, whereas wild-type cells grew normally under these conditions. The nucleoside transport-deficient mutants exhibited about the same growth rate and plating efficiency (60-80%) as wild-type cells, but formed larger colonies than wild-type cells because of a more extensive spread of the cells on the surface of culture dishes. PK-15 cells adhere very strongly to the surface of culture dishes and have been transformed with high efficiency with plasmid DNA either via lipofection or electroporation.

Animals↗
PubMed1990

Mapping the binding site of the nucleoside transporter protein: a 3D-OSAR study.

The nucleoside transporter is an intrinsic membrane protein that mediates salvage of nucleosides from the extracellular medium. In this report, its binding sites have been characterized by a 3D-QSAR (three-dimensional structure-directed quantitative structure-activity relationships) receptor mapping technique. REMOTEDISC. The algorithm is applied to a set of 19 nucleoside analogues, each of which binds to the transporter. The methodology includes: (i) conformational analysis of each ligand; (ii) estimation of physicochemical properties of each ligand at the atomic level; (iii) structural comparison of the low energy conformation of each ligand in the series with a reference structure on the basis of physicochemical property matching; (iv) construction of a predicted binding site cavity from the alignments of step (iii); and (v) multiple regression analysis of the binding data with respect to the 3-dimensional physicochemical descriptors in different 'site-pockets' of the binding cavity. The pharmacophore model that emerges consists of the geometry of the binding site cavity and the relative weights of various properties in different pockets for each of the ligands considered. The study suggests that binding free energy is sensitive to the composition, size and hydrophobicity of the heterocyclic base in the ligand. Though both syn and anti conformations are tried as active forms, the anti conformation gives a better solution and is chosen for modeling the binding site cavity. The best model obtained divides the binding site into six pockets and uses nine independent variables, fitting the observed data with a correlation coefficient of 0.94, a standard deviation of 0.22 and an explained variance of 0.80. Results of our model are consistent with a hypothesis that the 5'-OH group hydrogen bonds with the receptor. This model provides tentative design criteria for development of new nucleoside drugs and transport inhibitors. The model will undoubtedly continue to evolve (i) as the 3D-QSAR algorithm is further refined, and (ii) as data on additional nucleoside analogues become available.

Algorithms↗
PubMed1994

A plant nucleoside diphosphate kinase homologous to the human Nm23 gene product: purification and characterization.

Nucleoside diphosphate kinases (NDPKs) catalyze the transfer of high-energy phosphates from nucleoside triphosphates to nucleoside diphosphates and may be involved in the regulation of growth, development, and signal transduction processes. We report here the purification and characterization of NDPK from detergent-solubilized extracts of dark-grown oat (Avena) tissue. The purification was achieved primarily through adsorption to GTP-agarose, followed by elution with ATP. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicated that the purified protein is composed of six 18 kDa subunits. Substrate specificity experiments indicated that the purified kinase is capable of using all tested nucleosides as substrates. N-terminal sequencing of the Avena protein revealed that 87% of the 23 amino acids sequenced were identical to the human Nm23 protein, a nucleoside diphosphate kinase identified as a possible tumor metastasis suppressor and transcriptional activator of the myc oncogene.

Amino Acid Sequence↗
PubMed2005

Taxonomic distribution and quantitative analysis of free purine and pyrimidine nucleosides in snake venoms.

The nucleoside content of 32 elapid and viperid venoms was examined. Free purines, principally adenosine (ADO), inosine (INO), and guanosine (GUA), comprised as much as 8.7% of the solid components of some venoms. Thus, purines are far more abundant in some venoms than many proteinaceous toxins. Hypoxanthine (HYP) was found in about half of elapid and viperine venoms, in which it is a relatively minor constituent (<60 microg/g). Adenosine monophosphate (AMP) was tentatively identified in only three elapid and two viperid venoms. The pyrimidines, uridine (URI) and cytidine (CYT), were also found in most elapid and viperine venoms. In most of these, the amount of uridine was substantially greater than that of cytidine. Thymidine (THY) was not found in any venom, indicating that DNA from disintegration of glandular cells is not the source of venom nucleosides. In contrast to elapid and viperine venoms, most crotaline venoms are devoid of free nucleosides. Elapid and viperine venoms also contained other minor, low molecular weight constituents that could not be positively identified. Some had spectra identical to those of adenosine, nicotinamide adenine dinucleotide (NAD), inosine, xanthosine (XAN), and guanosine, while others had unique spectra. There is no apparent correlation between quantities of venom nucleosides and literature values for the three dominant venom enzymes that release endogenous nucleosides, 5'-nucleotidase (5NUC), phosphodiesterase (PDE), and alkaline phosphomonoesterase (PME).

5'-Nucleotidase↗
PubMed2003

Biosynthesis of the 7-deazaguanosine hypermodified nucleosides of transfer RNA.

Transfer RNA (tRNA) is structurally unique among nucleic acids in harboring an astonishing diversity of post-transcriptionally modified nucleoside. Two of the most radically modified nucleosides known to occur in tRNA are queuosine and archaeosine, both of which are characterized by a 7-deazaguanosine core structure. In spite of the phylogenetic segregation observed for these nucleosides (queuosine is present in Eukarya and Bacteria, while archaeosine is present only in Archaea), their structural similarity suggested a common biosynthetic origin, and recent biochemical and genetic studies have provided compelling evidence that a significant portion of their biosynthesis may in fact be identical. This review covers current understanding of the physiology and biosynthesis of these remarkable nucleosides, with particular emphasis on the only two enzymes that have been discovered in the pathways: tRNA-guanine transglycosylase (TGT), which catalyzes the insertion of a modified base into the polynucleotide with the concomitant elimination of the genetically encoded guanine in the biosynthesis of both nucleosides, and S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA), which catalyzes the penultimate step in the biosynthesis of queuosine, the construction of the carbocyclic side chain.

Catalysis↗
PubMed2000

Sustained depolarisation induces changes in the extracellular concentrations of purine and pyrimidine nucleosides in the rat thalamus.

ATP and adenosine are well-known neuroactive compounds. Other nucleotides and nucleosides may also be involved in different brain functions. This paper reports on extracellular concentrations of nucleobases and nucleosides (uracil, hypoxanthine, xanthine, uridine, 2'-deoxycytidine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenosine) in response to sustained depolarisation, using in vivo brain microdialysis technique in the rat thalamus. High-potassium solution, the glutamate agonist kainate, and the Na(+)/K(+) ATPase blocker ouabain were applied in the perfusate of microdialysis probes and induced release of various purine and pyrimidine nucleosides. All three types of depolarisation increased the level of hypoxanthine, uridine, inosine, guanosine and adenosine. The levels of measured deoxynucleosides (2'-deoxycytidine, 2'-deoxyuridine and thymidine) decreased or did not change, depending on the type of depolarisation. Kainate-induced changes were TTX insensitive, and ouabain-induced changes for inosine, guanosine, 2'-deoxycytidine and 2'-deoxyuridine were TTX sensitive. In contrast, TTX application without depolarisation decreased the extracellular concentrations of hypoxanthine, uridine, inosine, guanosine and adenosine. Our data suggest that various nucleosides may be released from cells exposed to excessive activity and, thus, support several different lines of research concerning the regulatory roles of nucleosides.

Animals↗
PubMed1999

Nucleoside transporters: molecular biology and implications for therapeutic development.

The uptake of nucleosides (or nucleobases) is essential for nucleic acid synthesis in many human cell types and in parasitic organisms that cannot synthesize nucleotides de novo. The transporters responsible are also the route of entry for many cytotoxic nucleoside analogues used in cancer and viral chemotherapy. Moreover, by regulating adenosine concentrations in the vicinity of its cell-surface receptors, nucleoside transporters profoundly affect neurotransmission, vascular tone and other processes. The recent molecular characterization of two families of human nucleoside transporters has provided new insights into the mechanisms of natural nucleoside and drug uptake and into future developments of improved therapies.

Animals↗
PubMed1998

One-third-the-sites transition-state inhibitors for purine nucleoside phosphorylase.

Genetic defects in human purine nucleoside phosphorylase cause T-cell deficiency as the major phenotype. It has been proposed that efficient inhibitors of the enzyme might intervene in disorders of T-cell function. Compounds with features of the transition-state structure of purine nucleoside phosphorylase were synthesized and tested as inhibitors. The transition-state structure for purine nucleoside phosphorylase is characterized by (1) an elevated pKa at N7 of the purine ring for protonation or favorable H-bond interaction with the enzyme and (2) oxocarbenium ion formation in the ribosyl ring (Kline, P. C., and Schramm, V. L. (1995) Biochemistry 34, 1153-1162). Both features have been incorporated into the stable transition-state analogues, (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol (immucillin-H) and (1S)-1-(9-deazaguanin-9-yl)-1,4-dideoxy-1, 4-imino-D-ribitol (immucillin-G). Both inhibitors exhibit slow-onset tight-binding inhibition of calf spleen and human erythrocyte purine nucleoside phosphorylase. The inhibitors exhibit equilibrium dissociation constants (Ki) from 23 to 72 pM and are the most powerful inhibitors reported for the enzyme. Complete inhibition of the homotrimeric enzyme occurs at one mole of inhibitor per mole of enzymic trimer. Binding of the transition-state inhibitor at one site per trimer prevents inhibitor binding at the remaining two sites of the homotrimer. A mechanism of sequential catalysis at each subunit, similar to that of F1 ATPase, is supported by these results. Slow inhibitor dissociation (e.g., t1/2 of 4.8 h) suggests that these compounds will have favorable pharmacologic properties. Interaction of transition-state inhibitors with purine nucleoside phosphorylase is different from reactant-state (substrate and product analogue) inhibitors of the enzyme which bind equally to all subunits of the homotrimer.

Animals↗
PubMed1999

Modified nucleosides in the first positions of the anticodons of tRNA(Leu)4 and tRNA(Leu)5 from Escherichia coli.

Minor leucine tRNA species, tRNA(Leu)4 and tRNA(Leu)5, from Escherichia coli B have been reported to recognize leucine codons UUA and UUG [Goldman, E., Holmes, W. M., and Hatfield, G. W. (1979) J. Mol. Biol. 129, 567-585]. In the present study, these two tRNA(Leu) species were purified from E. coli A19, and the nucleotide sequences were determined by a post-labeling method. tRNA(Leu)5 was found to correspond to the tRNA gene reported as su degrees6 tRNA [Yoshimura, M., Inokuchi, H., and Ozeki, H. (1984) J. Mol. Biol. 177, 627-644]. The first letter of the anticodon was identified to be 2'-O-methylcytidine (Cm). tRNA(Leu)4 was identified as the minor leucine tRNA that has been sequenced previously (tRNA(Leu)UUR) [Yamaizumi, Z., Kuchino, Y., Harada, F., Nishimura, S., and McCloskey, J. A. (1980) J. Biol. Chem. 255, 2220-2225]. There was an unidentified modified nucleoside (N*) in the first position of the anticodon of tRNA(Leu)4. Nucleoside N* was isolated to homogeneity (1 A260 unit). By 1H NMR spectroscopy, nucleoside N was found to be a 2'-O-methyluridine derivative with a substituent having a -CH2NH2+CH2COO- moiety in position 5 of the uracil ring. On the basis of these NMR analyses together with mass spectrometry, the chemical structure of nucleoside N* was determined as 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um). Nucleoside N* was thus found to be a novel type of naturally occurring modified uridine. Because of the conformational rigidity of Cm and cmnm5Um in the first position of the anticodon, these tRNA(Leu) species recognize the leucine codons UUA++ and UUG correctly, but never recognize the phenylalanine codons UUU and UUC.

Anticodon↗