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Nucleoside transporters (NTs) are integral membrane proteins necessary for the cellular entry of nucleoside analog drugs used in chemotherapeutic treatment of conditions such as cancer and viral or parasitic infections. NTs are also the targets of certain drugs used in the treatment of various cardiovascular conditions. Because of the importance of NTs in drug uptake, determination of the three-dimensional structure of these proteins, particularly hENT1, has the potential to improve these treatments through structure-based design of more specifically targeted and transported drugs. In this paper, we use NMR spectroscopy to investigate the structure of the large intracellular loop between transmembrane domains 6 and 7 and we also describe a method for the successful overexpression of full-length hENT1 in a bacterial system. Recombinant tandem histidine-affinity (HAT) and 3 x FLAG tagged hENT1 was overexpressed in E. coli, affinity purified, and functionally characterized by nitrobenzylthioinosine (NBTI) binding. Anti-3xFLAG immunodetection confirmed the expression of N-HAT-3 xFLAG-hENT1, while increased NBTI binding (3.2-fold compared with controls) confirmed the conformational integrity of the recombinant hENT1 within the bacterial inner membrane. Yields of recombinant hENT1 using this approach were ~ 15 µg/L of bacterial culture and this approach provides a basis for large-scale production of protein for a variety of purposes. Key words: hENT1, structure, recombinant, overexpression, bacteria. Les transporteurs de nucleosides (TN) consistent en proteines membranaires integrales neecessaires a l'entree dans les cellules d'analogues des nucleosides utilises en chimiotheerapie pour traiter des maladies ou des situations comme le cancer ou les infections virales/parasitaires. Les TN sont aussi les cibles de certains medicaments utilises dans le traitement de certaines maladies cardiovasculaires. A cause de l'importance des TN dans la capture de medicaments, la determination de la structure tridimensionnelle de ces proteines, particulierement hENTl, pourrait permettre d'ameliorer ces traitements par la conception, d'apres cette structure, de medicaments cibles et transportes de maniere plus specifique. Dans cet article, nous utilisons la spectroscopie RMN afin d'examiner la structure de la grande boucle intracellulaire situee entre les domaines 6 et 7, et nous decrivons aussi une methode efficace de surexpression de hENTl de pleine longueur dans un systeme bacterien. Un transporteur hENTl recombinant portant des etiquettes d'affinite d'histidine en tandem (HAT) et 3 x FLAG a ete surexprime; chez E. coli, purifie; par affinite et caracterise fonctionnellement par la liaison de nitrobenzylthioionsine (NBTI). La detection immunologique a l'aide d'un anti-3xFLAG a confirme l'expression de N-HAT3xFLAG-hENTl, alors que l'augmentation de la liaison de NBTI (3,2 fois comparativement au controle) a confirmee l'integrite conformationnelle du hENTl recombinant au sein de la membrane interne bacterienne. La recolte de hENTl recombinant etait d'environ 15 µg/L de culture bacterienne en utilisant cette approche, celle-ci constituant une base a partir de laquelle la production a grande eechelle de la proteine sera envisageee a differentes fins. Mots-cles: hENTl, structure, recombinant, surexpression, bacterie. [Traduit par la Redaction], Introduction Structural characterization of proteins, especially those involved in cellular drug processing, has greatly increased in recent years, allowing more advanced structure-based drug design (Congreve et al. 2005; Blundell et [...]

To better understand the role of equilibrative nucleoside transporters (ENT) in purine nucleoside-dependent physiology of the cardiovascular system, we investigated whether the ENTI-null mouse heart was cardioprotected in response to ischemia (coronary occlusion for 30 min followed by reperfusion for 2 h). We observed that ENTI-null mouse hearts showed significantly less myocardial infarction compared with wild-type littermates. We confirmed that isolated wild-type adult mouse cardiomyocytes express predominantly ENT1, which is primarily responsible for purine nucleoside uptake in these cells. However, ENT1-null cardiomyocytes exhibit severely impaired nucleoside transport and lack ENT1 transcript and protein expression. Adenosine receptor expression profiles and expression levels of ENT2, ENT3, and ENT4 were similar in cardiomyocytes isolated from ENT1-null adult mice compared with cardiomyocytes isolated from wild-type littermates. Moreover, small interfering RNA knockdown of ENT1 in the cardiomyocyte cell line, HL-1, mimics findings in ENTl-null cardiomyocytes. Taken together, our data demonstrate that ENT1 plays an essential role in cardioprotection, most likely due to its effects in modulating purine nucleoside-dependent signaling and that the ENT1-null mouse is a powerful model system for the study of the role of ENTs in the physiology of the cardiomyocyte. cardiovascular; hypoxia; adenosine; cardiomyocytes doi:10.1152/ajpheart.00711.2009

This study examined the roles of human nucleoside transporters (hNTs) in mediating transepithelial fluxes of adenosine, 2'-deoxyadenosine, and three purine nucleoside anti-cancer drugs across polarized monolayers of human renal proximal tubule cells (hRPTCs), which were shown in previous studies to have human equilibrative NT 1 (hENT1) and 2 (hENT2) and human concentrative NT 3 (hCNT3) activities (11). Early passage hRPTCs were cultured on transwell inserts under conditions that induced formation of polarized monolayers with experimentally accessible apical and basolateral domains. Polarized hRPTC cultures were monitored for inhibitor sensitivities and sodium-dependence of the following: 1) transepithelial fluxes of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine (9-[beta]-D-arabinosyl-2-fluoroadenine), cladribine (2-chloro-2'-deoxyadenosine), and clofarabine (2-chloro-2'-fluoro-deoxy-9-[beta]-D-arabinofuranosyladenine); 2) mediated uptake of radiolabeled adenosine, 2'-deoxyadenosine, fludarabine, cladribine, and clofarabine from either apical or basolateral surfaces; and 3) relative apical cell surface hCNT3 protein levels. Transepithelial fluxes of adenosine were mediated from apical-to-basolateral sides by apical hCNT3 and basolateral bENT2, whereas transepithelial fluxes of 2'-deoxyadenosine were mediated from basolateral-to-apical sides by apical hENT1 and basolateral human organic anion transporters (hOATs). The transepithelial fluxes of adenosine, hCNT3-mediated cellular uptake of adenosine, and relative apical cell surface hCNT3 protein levels correlated positively in polarized hRPTCs. The purine nucleoside anti-cancer drugs fludarabine, cladribine, and clofarabine, like adenosine exhibited apical-to-basolateral fluxes. Collectively, this evidence suggested that apical hCNT3 and basolateral hENT2 are involved in proximal tubular reabsorption of adenosine and some nucleoside drugs and that apical hENT1 and basolateral hOATs are involved in proximal tubular secretion of 2'-deoxyadenosine. kidney; fludarabine; cladribine; clofarabine

Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified an Na-independent purineselective nucleobase/nucleoside transport system in the nucleoside transporter-deficient PK15NTD cells. This transport system has 1,000-fold higher affinity for nucleobases than nucleosides with [K.sub.m] values of 2.5 [+ or -] 0.7 [micro]M for [[sup.3]H]adenine, 6.4 [+ or -] 0.5 [micro]M for [[sup.3]H]guanine, 1.1 [+ or -] 0.1 mM for [[sup.3]H]guanosine, and 4.2 [+ or -] 0.5 mM [[sup.3]H]adenosine. The uptake of [[sup.3]H]guanine (0.05 [micro]M) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1 mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2' and/or 5 positions (all at 1 mM) were more potent than adenosine in competing the uptake of [[sup.3]H]guanine: 2-chloro-2'-deoxyadenosine > 2-chloroadenosine > 2'3'-dideoxyadenosine = 2'-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-Chloro-2'-deoxyadenosine and 2-chloroadenosine inhibited [[sup.3]H]guanine uptake with ICso values of 68 [+ or -] 5 and 99 [+ or -] 10 [micro]M, respectively. The nucleobase/nucleoside transporter was resistant to nitrobenzylthioinosine {6-[(4-nitrobenzyl) thiol]-9-[beta]-D-ribofuranosylpurine}, dipyridamole, and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor decynium-22 ([IC.sub.50] of ~1 [micro]M), and by acidic pH (pH = 5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells. [[sup.3]H]guanine; adenine; cladribine; adenosine

The development of a flexible multicomponent synthesis of a range of differently functionalized triazinane diones is reported. The process could be used to synthesize unnatural nucleoside analogues.

The purine nucleoside phosphorylase (PNP) from Anopheles gambiae (AgPNP) is expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP's rapid pre-steady-state turnover number for guanosine supports high catalytic potential.

Nucleoside transporters in kidney mediate renal reabsorption and secretion of nucleosides. Using RT-PCR, we demonstrated mRNAs encoding hENT1, hENT2, hCNT1, hCNT2, and hCNT3 in both cortex and medulla. Immunoblotting with crude membrane preparations revealed abundant hENT1 and hCNT3 in both cortex and medulla, and little, if any, hENT2, hCNT1, or hCNT2, indicating that the latter were either absent or below limits of detection of immunoassays, hENT1 immunostaining was observed on apical surfaces of proximal tubules and on both apical and basal surfaces of thick ascending loops of Henle and collecting ducts. Prominent hCNT3 immunostaining was observed on apical surfaces of proximal tubules and thick ascending loops of Henle in addition to some cytoplasmic staining. Equilibrium binding of [[sup.3]H]nitrobenzylmercaptopurine ribonucleoside (NBMPR), a high-affinity inhibitor of hENT1, to brush-border membrane vesicles from cortex confirmed the presence of hENT1 on apical surfaces of proximal tubules. Uptake of [[sup.3]H]uridine by polarized renal proximal tubule cells exhibited a sodium-dependent component that was inhibited by thymidine and inosine as well as a sodium-independent component that was partially inhibited by NBMPR and completely inhibited by dilazep, indicating high levels of hENT1 and hCNT3 and low levels of hENT2 activities. The presence of 1) transcripts for hENT1/2 and hCNT1/2/3 and the hENT1 and hCNT3 proteins in human kidneys and 2) hENT1, hENT2, and hCNT3 activities in cultured proximal tubule cells suggest involvement of hENT1, hCNT3, and possibly also hENT2 in renal handling of nucleosides and nucleoside drugs. renal transport of nucleosides: tissue distribution of nucleoside transporters doi:10.1152/ajprenal.00007.2007

A neoceptor approach is employed to analyze the binding site of the [A.sub.1] adenosine receptor, which is modified by tailored nucleosides for orthogonal recognition. The His residue is found to induce conformational changes in a ligand to activate the full spectrum of functional effects.

Bis- and trisuracil nucleosides with nucleobases anchored to isoxazoline ring-fused 11-, 12-, and 16-membered macrooxacycles are synthesized by nucelosidation of 1,2-isopropylidenefuranose ring-fused macrocycles. The nucleosides have displayed spherical and fiber-like morphologies in water and in one case, the morphology is changed by complementary base pairing.

Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaceharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-l-phosphate uridylyltransferase mediates the reversible conversion of glucose-l-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-l-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-l-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.

Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent [K.sub.m] values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ ATPDase. The partial N[H.sub.2]-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage. ecto-ATPDase; P1/P2 receptors; ATP; adenosine; nucleoside transporters

Expression of the Streptomyces exfoliatus lipA gene, which encodes an extracellular lipase, depends on LipR, a transcriptional activator that belongs to the STAND class of P-loop nucleoside triphosphatases. LipR is closely related to activators present in some antibiotic biosynthesis clusters of actinomycetes, forming the LipR/TchG family of regulators. In this work we showed that purified LipR protein is essential for activation of lipA transcription in vitro and that this transcription depends on the presence of a conserved inverted repeat, the LipR box, located upstream of the lipA promoter. Mutagenesis of the lipA promoter region indicated that most transcription depends on LipR binding to the proximal half-site of the LipR box in close proximity to the -35 region of the promoter. Our experiments also indicated that LipR establishes contact with the RNA polymerase on both sides of the LipR box, since some activation was observed when only the distal half-site was present or when the entire LipR box was moved further upstream. We also showed that the LipR proteins of S. exfoliatus and Streptomyces coelicolor are functionally interchangeable both in vitro and in vivo, revealing the functional conservation of the regulatory elements in these two species.

The multifunctional cell-surface protein dipeptidyl peptidase IV (DPPIV/CD26) is aberrantly expressed in many cancers and plays a key role in tumorigenesis and metastasis. Its diverse cellular roles include modulation of chemokine activity by cleaving dipeptides from the chemokine N[H.sub.2]-terminus, perturbation of extracellular nucleoside metabolism by binding the ecto-enzyme adenosine deaminase, and interaction with the extracellular matrix by binding proteins such as collagen and fibronectin. We have recently shown that DPPIV can be downregulated from the cell surface of HT-29 colorectal carcinoma cells by adenosine, which is a metabolite that becomes concentrated in the extracellular fluid of hypoxic solid tumors. Most of the known responses to adenosine are mediated through four different subtypes of G protein-coupled adenosine receptors: [A.sub.1], [A.sub.2A], [A.sub.2B], and [A.sub.3]. We report here that adenosine downregulation of DPPIV from the surface of HT-29 cells occurs independently of these classic receptor subtypes, and is mediated by a novel cell-surface mechanism that induces an increase in protein tyrosine phosphatase activity. The increase in protein tyrosine phosphatase activity leads to a decrease in the tyrosine phosphorylation of ERK1/2 MAP kinase that in turn links to the decline in DPPIV mRNA and protein. The downregulation of DPPIV occurs independently of changes in the activities of protein kinases A or C, phosphatidylinositol 3-kinase, other serine/threonine phosphatases, or the p38 or JNK MAP kinases. This novel action of adenosine has implications for our ability to manipulate adenosine-dependent events within the solid tumor microenvironment. CD26; deaminase binding protein; mitogen-activated protein kinases; nucleosides; tumor microenvironment doi:10.1152/ajpcell.00238.2005.

The Salmonella enterica tsx gene encodes a nucleoside-specific outer membrane channel. The Tsx porin is essential for the prototrophic growth of S. enterica sv. Typhi in the absence of nucleosides. RT-PCR analysis shows that the tsx gene is cotranscribed with an open reading frame unique to S. enterica, impX (STY0450), which encodes an inner membrane protein 108 amino acids in length, which is predicted to have only two transmembrane [alpha]-helices. Fusions of the lacZ gene to both tsx and impX reveal that the transcription of both genes is induced in the presence of adenosine. A null mutation in the S. Typhi impX gene suppresses the induced auxotrophy for adenosine or thymidine resulting from a tsx mutation and confers sensitivity to high concentrations of adenosine or thymidine. The ImpX protein, when tagged with a 3xFLAG epitope, is functional and associates with the inner membrane; impX mutants are defective in the export of [sup.3]Hradio-labeled thymidine. Taken together, these and other results suggest that the S. Typhi Tsx porin and ImpX inner membrane protein facilitate competing mechanisms of thymidine influx and efflux, respectively, to maintain the steady-state levels of internal nucleoside pools.

In the testis, nucleosides and nucleobases are important substrates of the salvage pathway for nucleotide biosynthesis, and one of the roles of Sertoli cells is to provide nutrients and metabolic precursors to spermatogenic cells located within the blood-testis barrier (BTB). We have already shown that concentrative and equilibrative nucleoside transporters are expressed and are functional in primary-cultured rat Sertoli cells as a BTB model, but little is known about nucleobase transport at the BTB or about the genes encoding specific nucleobase transporters in mammalian cells. In the present study, we examined the uptake of purine ([[sup.3]H]guanine) and pyrimidine ([[sup.3]H]uracil) nucleobases by primary-cultured rat Sertoli cells. The uptake of both nucleobases was time and concentration dependent. Kinetic analysis showed the involvement of three different transport systems in guanine uptake. In contrast, uracil uptake was mediated by a single [Na.sup.+]-dependent high-affinity transport system. Guanine uptake was inhibited by other purine nucleobases but not by pyrimidine nucleobases, whereas uracil uptake was inhibited only by pyrimidine nucleobases. In conclusion, it was suggested that there might be purine- or pyrimidine-selective nucleobase transporters in rat Sertoli cells. nucleoside; Sertoli cells; transporter

The fluorescent probe 3-[4-(3-phenyl-2-pyrazolin-1-yl)benzene-1-sulfonyl amido]phenylboronic acid (PPBA)-binding site in the complexes, enzymatic and fluorescence properties of the S1PPBA nucleotide complexes were investigated. Docking simulation of PPBA on S1 nucleotide complex revealed that Phe472 interacts with PPBA.

The internal dynamics of triosephosphate isomerase is investigated with elastic networks, with and without a substrate bound. It is shown that elastic network computations on protein system combine atoms for functional parts of the structure with coarse-grained representations of the remainder of the structure in several different ways.

A description of a novel methodology for real-time visualization of nucleoside transport processes in living cells is presented. Quantitative analysis of fluorescence signals revealed two stages of FuPmR uptake, a fast first stage that represents the initial uptake rate, followed by a slow long-lasting second stage.

Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Thus, synthetic entry into a new class of these analogues, 2'-C-difluoromethylribonucleosides, is described.

The preparation of two complete series of RNA nucleoside phosphoramidities, each having an appropriately protected amine or thiol functional group is presented. The new phosphoramidites would be particularly valuable for enabling site-specific introduction of biophysical probes and constraints into RNA.

The bifunctional protein regulators of G protein signaling 14 (RGS proteins) are both a guanosine triphosphatase activating protein for Gi(alpha) and Go(alpha) and a guanine nucleotide dissociation inhibitor (GDI) for Gi(alpha). This GDI activity is isolated to a region of the protein distinct from the RGS domain that contains an additional G-protein binding domain.

A study was conducted to develop two new strategies for the introduction of a boranophosphate moiety into amino acids to obtain nucleoside amino acid boranophosphoramidates. Nucleoside boranophosphates and nucleoside amino acid phosphoramidates are shown to be potent antiviral and anticancer agents with a potential to act as nucleoside prodrugs.

Directed syntheses of spirocyclic nucleosides featuring adenine and guanine as the nucleobases is successfully developed. The configurational assignments are based on the thermal interconversions and supporting MM3 steric energy calculations.

Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3-300 [micro]M), dilazep (1-100 [micro]M), NBTI (10-500 nM), and adenosine (50-250 [micro]M) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 [micro]M). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a close-dependent inhibition of glucose transport by dipyridamole (basal, I[C.sub.50] = 12.2 [micro]M, insulin stimulated, I[C.sub.50] = 13.09 [micro]M) and dilazep (basal, I[C.sub.50] = 5.7 [micro]M, insulin stimulated, I[C.sub.50] = 19 [micro]M) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses. adenosine; equilibrative nucleoside transporters; regulation

Nucleoside transporters are important in the disposition of nucleosides and nucleoside analogs in the kidney. Two human equilibrative nucleoside transporters have been cloned and characterized, hENT1 and hENT2. The primary goal of this study was to localize these transporters in polarized renal epithelia, hENT1 and hENT2 were tagged with green fluorescence protein, stably expressed in renal epithelial cells, and localized by immunofluorescence and functiona] analysis. Our data demonstrated that both transporters are expressed on the basolateral membrane, hENT1 is also present on the apical membrane. Additionally, we examined the importance to basolateral targeting of two COOH-terminal targeting motifs: a RXXV motif for hENT1 and a dileucine repeat for hENT2. Neither motif appeared to affect targeting, but the dileucine repeat was implicated in surface expression of hENT2. In addition, a splice variant of hENT2 was identified that is predicted to result in a 156-residue COOH-terminal truncation. This variant had a tissue distribution similar to wild-type hENT2 but was retained intracellularly. These data suggest that hENT1 and hENT2 on the basolateral membrane function with concentrative nucleoside transporters on the apical membrane to mediate active reabsorption of nucleosides within the kidney. equilibrative nucleoside transporter; Madin-Darby canine kidney; dileucine; splice variant

Research shows that bioactive nucleosides bearing thiocyanate function such as thiocyanates can be derived by cleavage of 2-Trimethylsilylethyl sulfides with cyanogen bromide in methanol. Results indicate that thiocyanates are precursors for nucleoside diphosphates which interfere with the radical reactions at the active site of ribonucleoside diphosphate reductase.

The stereodefined rearangement is suitable for oxygen labeling of a variety of pyrimidine nucleosides. Inactivation of ribonucleoside diphosphate reductase gives rise to electron paramagnetic resonance signals representing nitrogen-centered radical derived fromazide.

Research has been conducted on the purine nucleoside phosphorylases (PNPs) encoded by Mycobacterium tuberculosis. The crystal structure of M. tuberculosis PNP has been investigated in complex with the transition-state analogue immucillin-H and inorganic phosphate to confirm its trimeric structure.

Research has been conducted on the effect of Mycobacterium tuberculosis genome sequence on purine salvage pathways. The results indicate that M. tuberculosis deoD gene exhibit purine nucleoside activity after having been cloned, expressed and purified.

Research has been conducted on the carbocyclic C-nucleosides. The synthesis of these nucleosides that gave spiranic carbocyclic C-nucleoside and enantiomerically pure forms of unsaturated nucleosides is discussed.

Research has been conducted on the pyrazine C-nucleosides. A sequential dilithiation-addition method developed for the synthesis of these nucleosides is discussed.

Research has been conducted on the pyrrolo[2,3-d][1,2,3]triazines. The study initiated to provide additional members of this ring system is discussed.

Research has been conducted on the rat equilibrative nucleoside transporters belonging to the integral membrane protein family. The creation of chimeras between nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive nucleoside transporters has been carried out.

Research has been conducted on the GTP cyclohydrolase I which catalyzes complex ring expansion affording dihydroneopterin triphosphate from GTP. The slow enzyme reaction under single turnover conditions has been investigated.

A synthesis of carcocyclic and hexanopyranosyl nucleosides is developed. The strategy combines three key transfotmations: an asymmetric aldol addition to establish the relative and absolute configuration of the pseudosugar, a ring-closing metathesis to construct the pseudosugar ring, and a trost-type palladium(0)-mediated substitution to assemble the pseudosugar and the aromatic base.

Results of a study conducted suggest that the two C-nucleosides d2APy and d2APm, although similar structure, exhibit two different selectives based upon their pK(sub a) values, 6.8 and 3.3, respectively. The d2APy residue with a greater pK(sub a) value, even higher than dC(pK(sub a)=4.3) is more effective than dC in targeting dG-dC base particularly at slight basis pH value.

Research has been conducted on the modified nucleosides in the anticodon domain of Escherichia coli tRNA (super)Lys. The NMR solution structures for the modified 17-nucleotide E. coli tRNA (super)Lys stemloop domain have been investigated and the results are presented.

Research reveals that the key step in the synthetic strategy for the synthesis of deoxyribonucleosides is the selective deoxygenation of a benzoyl or 3-trifluromethyl)benzoyl group of the respective nucleosides via a photoinduced electron-transfer. Data indicate that the method is scalable to yield gram quantities of desired deoxynucleosides.