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Mixtures of 13 normal and modified nucleosides in a 70 mM phosphate buffer solution (pH 6.7) supplemented with 100 mM of sodium dodecyl sulfate were separated by micellar electrokinetic chromatography in 25 min. Electrophoretic profiles of urine are obtained; it is demonstrated that 1-methylinosine and 1-methylguanosine nucleosides can be used as markers of precancerous states (mastopathy) and benign tumors (fibroadenoma). Keywords: tumor markers, diagnosis of cancer, nucleosides, urine, micellar electrokinetic chromatography, Determination of tumor markers in biological fluids (blood serum, urine) is an important component of the diagnostic complex in oncology. The term 'tumor markers' denotes compounds found in biological fluids [...]

The binding motifs for many RNA-binding proteins are normally buried within structured regions; now, the N.sup.6-methyladenosine modification is shown to act as a switch to remodel these regions, expose the motif, and thereby facilitate binding of RNA-binding proteins. N.sup.6-methyladenosine influences RNA-protein interactions N.sup.6-methyladenosine (m.sup.6A) is a common internal modification found in many eukaryotic mRNAs and long non-coding RNAs. It is involved in the control of various cellular functions including circadian rhythm, meiosis and stem cell development. This study uncovers a previously unknown mechanism by which m.sup.6A regulates RNA-protein interactions. Tao Pan and colleagues show that the binding motifs for many RNA binding proteins (RBPs) are normally buried within structured regions. The m.sup.6A modification acts as a switch to reorganize these regions, inducing structural changes that expose the motif and thereby facilitate RBP binding. This finding implies that m.sup.6A functions as an RNA structure remodeller, for example, to affect mRNA maturation through interference with post-transcriptional regulator binding activities. RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs).sup.1,2,3. However, RBMs can be buried within their local RNA structures.sup.4,5,6,7, thus inhibiting RNA-protein interactions. N.sup.6-methyladenosine (m.sup.6A), the most abundant and dynamic internal modification in eukaryotic messenger RNA.sup.8,9,10,11,12,13,14,15,16,17,18,19, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs.sup.15, but how m.sup.6A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m.sup.6A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m.sup.6A-switch'. We found that m.sup.6A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing.sup.20,21,22,23,24. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m.sup.6A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m.sup.6A-switches among HNRNPC-binding sites; and global m.sup.6A reduction decreased HNRNPC binding at 2,798 high-confidence m.sup.6A-switches. We determined that these m.sup.6A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m.sup.6A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m.sup.6A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology., Author(s): Nian Liu [sup.1] , Qing Dai [sup.1] , Guanqun Zheng [sup.2] , Chuan He [sup.1] [sup.2] [sup.3] [sup.4] , Marc Parisien [sup.2] [sup.5] , Tao Pan [sup.2] [sup.3] Author [...]

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes (1,2). Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes (3-5). A wide range of nucleoside-derived drugs, including anticancer drugs (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes (4,6-13). Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 Å. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes., Humans have three isoforms of concentrative nucleoside transporter (hCNT), and the substrate specificities and tissue distributions of these isoforms are different (14-16). Knowledge of the mechanism of these transporters would [...]

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.

A comprehensive ab initio conformational analysis of thymidine, DNA canonical nucleoside was performed. It was confirmed that dozens of energetically representative conformers are necessary to reproduce satisfactorily the T low temperature matrix isolation IR spectrum.

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.

A new series of viologen linked pyrene conjugates with the general formula [PYL.sub.n][V.sup.2} having a different number of methylene spacer units (Ln) was synthesized and investigated with the objective of developing efficient DNA oxidizing agents. The results show that the pyrene moiety in these molecules constitutes an interesting variation and plays a major role in controlling the electron-transfer pathways, which lead to the efficient photooxidation of DNA.

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 hydrolysis of nucleoside triphosphates by enzymes is used as a regulation mechanism in key biological processes. Here, the GTP hydrolysis of the protein complex of Ras with its GTPase-activating protein is monitored at atomic resolution in a noncrystalline state by time-resolved FTIR spectroscopy. At 900 ms, after the attack of water at the [gamma]-phosphate, there appears a [H.sub.2]P[O.sup.-.sub.4] intermediate that is shown to be hydrogen-bonded in an eclipsed conformation to the [beta]-phosphate of GDP. The [H.sub.2]P[O.sup.-.sub.4] intermediate is in a position where it can either reform GTP or be released from the protein in 7 s in the rate-limiting step of the GTPase reaction. We propose that such an intermediate also occurs in other GTPases and ATPases. proteins | reaction mechanism | FTIR spectroscopy | signal transduction

The preliminary efficacy of four different short-course nucleoside analogue regimens, stavudine (d4T), didanosine (ddI), d4T+ddI, and zidovudine are reported for the prevention of mother-to-child transmission of HIV-1 in a response-limited setting. The use of monotherapy in the study afforded the opportunity to demonstrate the relative intrinsic activity of several nucleoside analogues, while the results suggested that of the three nucleoside analogues evaluated in monotherapy, ddI was the most potent.

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.

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.

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.

A study is made to compare the efficacy and safety of a nucleoside sparing approach with a conventional highly active antiretroviral therapy (HAART) regimen in antiretroviral-experienced patients with prolonged viral suppression. The result showed that all patients maintained viral suppression after 48 weeks and no subject discontinued therapy because of adverse events.

The relation between clinical lipoatrophy (LA) and mitochondrial abnormalities occurring in the muscle of HIV-infected patients on highly active antiretroviral therapy (HAART) is investigated. The investigation confirmed that LA in long-term nucleoside reverse transcriptase inhibitors treated patients was associated with muscular mitochondrial dysfunction as shown by rapid lactic academia increase and mitochondrial histoenzymatic abnormalities.

The wider range of choices available for NRTI/NtRTI backbones has increased the complexity of HIV resistance considerations in clinical practice. Ongoing monitoring of the prevalence of resistance and the resistance outcomes from long-term clinical trials will continue to play an important role in guiding selection of the NRTI/NtRTI backbone in combination ART.

The selection of the nucleoside/nucleotide reverse transcriptase inhibitor (NRTI/NtRTI) backbone is an important step in constructing a regimen for the treatment of HIV infection. Data published since 2000 that address NRTI/NtRTI backbone performances and safety when included in HAART regimen is reviewed.

Mitochondrial toxicity has been implicated in the development of a variety of nucleoside reverse transcriptase inhibitors-associated syndromes. Clinical syndromes mediated by mitochondrial toxicity include hyperlactatemia and lactic acidosis, lipoatrophy, peripheral nephropathy, HANWS, and hepatic steatosis.

The nucleoside/nucleotide reverse inhibitor (NRTI/NtRTI) class continues to serve as an important component of the standard of care for HIV infection. Recent efficacy and safety data on emerging NRTI/NtRTI combination backbone is also summarized.

A historical perspective on the use of NRTI backbones in the treatment of HIV infection and the advantages and disadvantages of currently available backbone combinations is described. In an environment where several NRTI/NtRTI backbones offer comparable efficacy, treatment decisions will increasingly be made based on toxicity, resistance, and convenience considerations.

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) remain the backbone of highly active antiretroviral therapy (HAART). The pharmacokinetic properties, particularly those of the various phosphorylation steps, of the NRTIs/NtRTIs are reviewed.

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.

The long-term immunologic and virologic efficacy of the dual- and triple-nucleoside therapy for HIV infection was compared in a retrospective analysis of two randomized clinical trials in antiretroviral-naive patients in Thailand. The triple-nucleoside therapy proved more effective in inducing a sustained virologic response than the dual-nucleoside therapy as stated.

Suicide gene therapy with the herpes simplex virus type-1 thymidine kinase gene (HSV-1 tk) is considered to be a promising approach to the treatment of cancer. Making use of the lower specificity of the viral enzyme compared to human thymidine kinase, the therapy involves the administration of antiviral agents (e.g., ganciclovir) as prodrugs to induce enzymatic cell death in those cells that express the transferred gene. [sup.18]F-labelled derivatives have been described for monitoring location, duration, and magnitude of the viral kinase enzyme activity by positron emission tomography (PET). Since an optimal radiotracer has not been developed, novel substances were synthesized for monitoring gene expression. A group of 13 nucleoside analogues were synthesized, among them [N.sup.1]-methyl-9-[(l,3-dihydroxy-2-propoxy)methyl]guanine (5) and [N.sup.1]-methyl-9-[(4-hydroxy)-3-hydroxymethylbutyl]guanine (7) as methyl analogues of ganciclovir and penciclovir and their related fluoro compounds (6, 8). Further novel derivatives include [N.sup.6]-methyl-9-[(l,3-dihydroxy-2-propoxy)methyl]-, [N.sup.6]-methyl-9-[(4-hydroxy)-3-hydroxymethylbutyl]adenine (9, 10), as well as the uracil derivatives 5-hydroxy-1-[(1,3-dihydroxy-2-propoxy)methyl]uracil (11), 6-methyl-1-[(l,3-dihydroxy-2-propoxy)-methyl]uracil (12), and its 3-fluoro-derivative (13). Key words: fluorinated nucleoside analogues, gene therapy, PET, thymidine kinase.

A study was conducted, by using the time-resolved infrared spectroscopy (TRIR), laser flash photolysis with UV-vis detection, fluorescence quenching, absorption spectroscopy, and density functional theory calculations, to investigate the photoreaction between riboflavin tetraacetate and nucleosides. The TRIR spectroscopy demonstrates that triplet riboflavin tetraacetate reacts with G' to form a neutral hydroflavin radical by an electron transfer-proton transfer mechanism.

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 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.

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 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.

Research describes two methodologies for the syntheses of unsaturated analogues of nucleosides, consisting of four- and six-membered rings, using the same 2-benzyloxycycloalkanones as the starting material. The synthetic approach is based on the alkylation-elimination procedure.

Research reveals that the synthesis of the diastereoisomeric locked nucleic acid nucleosides involve efficient reaction cascades from di-O-tosyl and tri-O-mesyl nucleoside derivatives. However, the stereochemical inversion at the 2'-carbon atoms limits the utilization of the reaction in the preparation of pyrimidine nucleosides.

Improved methods for the synthesis of thio-substituted nucleosides, 4-thiouridine, 6-thioinosine, and 6-thioguanosine 3'-5'-diphosphates and their derivatives are presented. Further, syntheses of three types of pNp donors for RNA ligation are described. Data indicate that the donors facilitate incorporation of thionucleotides onto the 3-terminal site of the TMG-cped RNA tetramer.

Research has been conducted on the nucleoside diphosphate kinase (Ndk). The localization of the Ndk secretion motif has been carried out.

Research has been conducted on the nucleoside phosphorylation process. The investigation of this process that uses food additive phosphate as the phosphate source is described.