Problems of uridine protection

Various routes to synthesize 5'-O-dimethoxytrityl-O4-p-nitrophenylethyl- 2'-O-p-nitrophenylethylsulfonyluridine (7) as a useful intermediate in oligoribonucleotide synthesis have been investigated. The direct sulfonylation of 5'-dimethoxytrityl-O4-p-nitrophenylethyluridine (4) gave the best results despite the fact that 7 is formed in almost equal amounts with its 3'-NPES isomer (8) and the 2',3'-di-O-NPES derivative (6).     

Phosphorylation of uridine with inorganic phosphates

Phosphorylation of uridine by heating with inorganic orthophosphates can take place through reaction of nucleoside with thermally produced polyphosphate or direct reaction of nucleoside with orthophosphate or a combination of both. The relative importance of the two pathways varies with the orthophosphate and temperature.     

Homeostatic control of uridine and the role of uridine phosphorylase: a biological and clinical update

Both a 25-hydroxylation and a 1alpha-hydroxylation are necessary for the conversion of vitamin D(3) into the calcium-regulating hormone 1alpha,25-dihydroxyvitamin D(3). According to current knowledge, the hepatic mitochondrial cytochrome P450 (CYP) 27A and microsomal CYP2D25 are able to catalyze the former bioactivation step. Substantial 25-hydroxylase activity has also been demonstrated in kidney. This paper describes the molecular cloning and characterization of a microsomal vitamin D(3) 25- and 1alpha-hydroxylase in kidney. The enzyme purified from pig kidney and the recombinant enzyme expressed in COS cells catalyzed 25-hydroxylation of vitamin D(3) and 1alpha-hydroxyvitamin D(3) and, in addition, 1alpha-hydroxylation of 25-hydroxyvitamin D(3). The cDNA encodes a protein of 500 amino acids. Both the DNA sequence and the deduced peptide sequence of the renal enzyme are homologous with those of the hepatic vitamin D(3) 25-hydroxylase CYP2D25. Genomic Southern blot analysis suggested the presence of a single gene for CYP2D25 in the pig. Immunohistochemistry experiments indicated that CYP2D25 is expressed almost exclusively in the cells of cortical proximal tubules. The expression of CYP2D25 in kidney, but not in liver, was much higher in the adult pig than in the newborn. These findings indicate a tissue-specific developmental regulation of CYP2D25. The results from the current and previous studies on renal vitamin D hydroxylations imply that CYP2D25 has a biological role in kidney.     

Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine

UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc. The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc. Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi. The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively. Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis. No other nucleosides caused inhibition of the enzyme activity except uridine. Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity. But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.     

Enzymatic analysis of uridine diphosphate N-acetyl-D-glucosamine

The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD⁺-dependent oxidation of the 3′′ position of uridine diphosphate N-acetyl-d-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation, forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae, and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations in time series experiments or inhibitor screens.     

尿苷对线粒体功能的影响

尿苷是生物体内必需的营养物之一,需将尿苷浓度维持在一定浓度水平才能维持胞内正常的生长代谢。近年来,有研究发现尿苷可通过多种机制缓解生物体炎症反应,并能参与胞内糖酵解等代谢途径,且可调节胞内糖基化、乙酰化等蛋白修饰作用。此外,其还能通过减轻胞内氧化应激、促进高能化合物合成等方式保护细胞免受缺氧性损伤。研究表明,这些保护作用与尿苷对线粒体功能的调节作用息息相关。因此,本文主要综述了尿苷及其代谢反应(物)对线粒体功能的研究进展。