Search

NADP is essential for biosynthetic pathways, energy, and signal transduction. In living organisms, NADP biosynthesis proceeds through the phosphorylation of NAD with a reaction catalyzed by NAD kinase. We expressed, purified, and characterized Bacillus subtilis NAD kinase. This enzyme represents a new member of the inorganic polyphosphate [poly(P)]/ATP NAD kinase subfamily, as it can use poly(P), ATP, or other nucleoside triphosphates as phosphoryl donors. NAD kinase showed marked positive cooperativity for the substrates ATP and poly(P) and was inhibited by its product, NADP, suggesting that the enzyme plays a major regulatory role in NADP biosynthesis. We discovered that quinolinic acid, a central metabolite in NAD(P) biosynthesis, behaved like a strong allosteric activator for the enzyme. Therefore, we propose that NAD kinase is a key enzyme for both NADP metabolism and quinolinic acid metabolism.

Research has been conducted on Escherichia coli 2-thiouridine biosynthesis. The enzymatic pathway of this biosynthesis has been characterized via the study of the cloned nmnA from E. coli and via the ability of purified protein to bind tRNA, and the results demonstrate that MnmA binds to its substrate tRNA and catalyzes 2-thiouridine synthesis in vivo by utilizing sulfur.

Research has been conducted on Fapy.dA which is produced in DNA as an oxidative stress result. The study of interactions between DNA containing Fapy.dA with repair enzyme MutY and bifunctional enzyme Fpg is described.

Research has been conducted on natural nucleotides. The synthesis of these nucleotides at the position 1915 of Escherichia coli rRNA, 3-methylpseudouridine, has been carried out and their conversion into corresponding phosphoramidite and incorporation into 19-nucleotide helix 69 RNA are described.

Research has been conducted on Salmonella enterica serovar Typhimurium strain TR970. The importance of the active IscS protein for the synthesis of thiolated nucleosides present in S. enterica tRNA is discussed.

Research has been conducted on Trichomonas vaginalis purine nucleoside phosphorylase. The identification and characterization of this phosphorylase have been carried out and the results suggest that it will favor the anabolic reaction for the purine nucleoside synthesis.

Research has been conducted on the ribonucleoside analogue 1-beta-D-ribofuranosyl-3-nitropyrrole. The results of the analogue's chemical synthesis, biochemical analysis, antiviral evaluation and structural characterization are presented.

Research has been conducted on the encoded by herpes simplex virus type 1 thymidine kinase. Results indicate that the single A167Y mutation can convert this thymidine kinase into the exclusive purine (guanine) nucleoside analogue kinase.

Research has been conducted on the human equilibrative nucleoside transporter hENT1, sensitive to the nitrobenzylthioinosine inhibition. The expression of this transporter in different tissues has been carried out and the results indicate possible involvement of the transmembrane domains of hENT1 in the nucleoside binding.

All parasitic protozoa studied to date are incapable of purine biosynthesis and must therefore salvage purine nucleobases or nucleosides from their hosts. This salvage process is initiated by purine transporters on the parasite cell surface. We have used a mutant line (TUBA5) of Leishmania donovani that is deficient in adenosine/pyrimidine nucleoside transport activity (LdNT1) to clone genes encoding these nucleoside transporters by functional rescue. Two such genes, LdNT1.1 and LdNT1.2, have been sequenced and shown to encode deduced polypeptides with significant sequence identity to the human facilitative nucleoside transporter hENT1. Hydrophobicity analysis of the LdNT1.1 and LdNT1.2 proteins predicted 11 transmembrane domains. Transfection of the adenosine/pyrimidine nucleoside transport-deficient TUBA5 parasites with vectors containing the LdNT1.1 and LdNT1.2 genes confers sensitivity to the cytotoxic adenosine analog tubercidin and concurrently restores the ability of this mutant line to take up [3H]adenosine and [3H]uridine. Moreover, expression of the LdNT1.2 ORF in Xenopus oocytes significantly increases their ability to take up [3H]adenosine, confirming that this single protein is sufficient to mediate nucleoside transport. These results establish genetically and biochemically that both LdNT1 genes encode functional adenosine/pyrimidine nucleoside transporters.

Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates. Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 were tested for in vivo function by complementation of a [Delta]prp16 null strain. In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373-377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable. The S505A and T507A mutants in motif III (SAT) were viable. In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth. Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan. Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells. Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo. These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors.

Research shows that sodium-dependent purine nucleoside transporter hSPNT1 exists within the human kidney. Experiments using reverse transcriptase-polymerase chain reactions, northern analysis and homology cloning strategies are described. Genetic analysis demonstrates hSPNT1 gene location at chromosome 15. Genetic sequences representing hSPNT1 are described.

A study has been conducted to examine the mode of action of amicetin, a nucleoside antibiotic that induces the expression of chloramphenicol resistance gene cat-86. Assays of ribosomes from wild-type cells and mutant cells for amicetin indicated that amicetin inhibits bacterial peptidyl transferase by interacting with the peptidyl transferase region of bacterial ribosomes.

Studies have classified gene nm23 and the awd gene from Drosophila as nucleoside diphosphate kinases, a group of enzymes which play a role in cellular regulation and metastatic control of tumors. Gene nm23, which has been proven to be responsible for mammalian tumor malignancy, was discovered through the differential screening of the cDNA. It is often correlated with high metastatic potential.

Research has been conducted on Fapy.dG which is produced in DNA after the oxidative stress. The authors have investigated the inetarctions of DNA containing Fapy.dG with Fpg and MutY, and they report the results.

Research has been conducted on nucleoside excision repair. The authors demonstrate that UV response of the DDB2 and CSA complexes affect nucleoside excision repair mechanism.

The author discusses the studies on human and mouse genomes. The topics of interest include the comparison of these genomes, and the pattern of nucleoside substitution between human and mouse genes.

Research has been conducted on bacterial endospores. Results suggest that the addition of exogenous adenosine and L-alanine is required for Bacillus anthracis in vivo germination.

Research has been conducted on VirB4 family of proteins encoded by conjugative plasmids and type IV secretion system. The hypothesis that these proteins are similar in their structure and that they hydrolyze nucleotides has been tested, and the results are presented.

Characterization of nucleoside transport systems in cultured rat epididymal epithelium. Am J Physiol Cell Physiol 280: C1076-C1082, 2001.--The nucleoside transport systems in cultured epididymal epithelium were characterized and found to be similar between the proximal (caput and corpus) and distal (cauda) regions of the epididymis. Functional studies revealed that 70% of the total nucleoside uptake was [Na.sup.+] dependent, while 30% was [Na.sup.+] independent. The [Na.sup.+]-independent nucleoside transport was mediated by both the equilibrative nitrobenzylthioinosine (NBMPR)-sensitive system (40%) and the NBMPR-insensitive system (60%), which was supported by a biphasic dose response to NBMPR inhibition. The [Na.sup.+]-dependent [[sup.3]H]uridine uptake was selectively inhibited 80% by purine nucleosides, indicating that the purine nucleoside-selective N1 system is predominant. Since [Na.sup.+]-dependent [[sup.3]H]guanosine uptake was inhibited by thymidine by 20% and [Na.sup.+]-dependent [[sup.3]H]thymidine uptake was broadly inhibited by purine and pyrimidine nucleosides, this suggested the presence of the broadly selective N3 system accounting for 20% of [Na.sup.+]-dependent nucleoside uptake. Results of RT-PCR confirmed the presence of mRNA for equilibrative nucleoside transporter (ENT) 1, ENT2, and concentrative nucleoside transporter (CNT) 2 and the absence of CNT1. It is suggested that the nucleoside transporters in epididymis may be important for sperm maturation by regulating the extracellular concentration of adenosine in epididymal plasma. sperm maturation