A acid-stable analogue of the 3-beta-D-ribofuranoside of Y-base.
A cyclonucleoside analogue of Y(TU) riboside has been prepared and shown to be relatively stable in M-hydrochloric acid solution at room temperature. (+info)
Inhibition of vaccinia virus growth by the nucleoside analogue 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (virazole, ribavirin).
Virazole or Ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) inhibits the growth of vaccinia virus at a concentration ode to a certain extent in the presence of Virazole, the DNA fails to acquire resistance to deoxyribonuclease and virus particles are not formed. Reversibility of the antiviral effect occurs when the drug is washed out from the infected cultures or when guanosine at an equimolar concentration is added. (+info)
Human high-Km 5'-nucleotidase effects of overexpression of the cloned cDNA in cultured human cells.
5'-Nucleotidases participate, together with nucleoside kinases, in substrate cycles involved in the regulation of deoxyribonucleotide metabolism. Three major classes of nucleotidases are known, one on the plasma membrane and two in the cytosol. The two cytosolic classes have been named high-Km nucleotidases and 5'(3')-nucleotidases. Starting from two plasmids with partial sequences (Oka, J., Matsumoto, A., Hosokawa, Y. & Inoue, S. (1994) Biochem. Biophys. Res. Commun. 205, 917-922) we cloned the complete cDNA of the human high-Km nucleotidase into vectors suitable for transfection of Escherichia coli or mammalian cells. After transfection, E. coli overproduced large amounts of the enzyme. Most of the enzyme was present in inclusion bodies that also contained many partially degraded products of the protein. Part of the enzyme, corresponding to approximately 2% of the soluble proteins, was in a soluble active form. Stably transfected human 293 cells were obtained with a vector where the 3'-end of the nucleotidase coding sequence is linked to the 5'-end of the green fluorescent protein coding sequence. Several green clones overproduced both mRNA and fusion protein. Two clones with 10-fold higher enzyme activity were analyzed further. The nucleotidase activity of cell extracts showed the same substrate specificity and allosteric regulation as the high-Km enzyme. The growth rate of the two clones did not differ from the controls. The cells were not resistant to deoxyguanosine or deoxyadenosine, and did not show an increased ability to phosphorylate dideoxyinosine. Both ribonucleoside and deoxyribonucleoside triphosphate pools were decreased slightly, suggesting participation of the enzyme in their regulation. (+info)
Functional characterization of a human purine-selective, Na+-dependent nucleoside transporter (hSPNT1) in a mammalian expression system.
Nucleosides and nucleoside analogs are actively transported in the human kidney. With the recent cloning of a purine-selective, Na+-dependent, nucleoside transporter (hSPNT1, also termed hCNT2) from human kidney, it is now possible to study the interaction of nucleosides and nucleoside analogs with this transport protein and gain a more detailed knowledge of the underlying mechanisms of nucleoside transport in the human kidney. In this study we examined the substrate selectivity of hSPNT1 for nucleosides and nucleoside analogs. We determined that the naturally occurring nucleosides adenosine, inosine, and uridine are substrates for this carrier, whereas thymidine is not. The nucleoside analogs (0.5 mM) 2', 3'-dideoxyadenosine; 2',3'-dideoxyinosine; and 2-chloro-2'deoxyadenosine (2CdA), significantly inhibited the uptake of [3H]inosine in HeLa cells transiently transfected with hSPNT1. However, there was no significant Na+-dependent uptake of [3H]2', 3'-dideoxyinosine or [3H]2CdA in the transfected cells, suggesting that these nucleoside analogs are not permeants of hSPNT1. Interestingly, 2CdA was considerably less potent in inhibiting [3H]inosine uptake in HeLa cells expressing hSPNT1 than in cells expressing the rat homolog rSPNT (IC50 = 371 microM versus 13.8 microM), suggesting that there may be notable species differences in the kinetic interactions of some nucleoside analogs with purine- selective nucleoside transporters. (+info)
Isolation and characterization of mutations in the Escherichia coli regulatory protein XapR.
In this work, the LysR-type protein XapR has been subjected to a mutational analysis. XapR regulates the expression of xanthosine phosphorylase (XapA), a purine nucleoside phosphorylase in Escherichia coli. In the wild type, full expression of XapA requires both a functional XapR protein and the inducer xanthosine. Here we show that deoxyinosine can also function as an inducer in the wild type, although not to the same extent as xanthosine. We have isolated and characterized in detail the mutants that can be induced by other nucleosides as well as xanthosine. Sequencing of the mutants has revealed that two regions in XapR are important for correct interactions between the inducer and XapR. One region is defined by amino acids 104 and 132, and the other region, containing most of the isolated mutations, is found between amino acids 203 and 210. These regions, when modelled into the three-dimensional structure of CysB from Klebsiella aerogenes, are placed close together and are most probably directly involved in binding the inducer xanthosine. (+info)
Phosphoinositide-dependent activation of Rho A involves partial opening of the RhoA/Rho-GDI complex.
Rho GTPases have two interconvertible forms and two cellular localizations. In their GTP-bound conformation, they bind to the cell membrane and are activated. In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI). We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides. We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex. In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides. In both cases, phosphoinositides partially opened, but did not fully dissociate the complex. Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced. Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with [alpha32P]GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled. We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP. The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins. (+info)
5' AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle.
It has previously been reported that exercise causes an increase in glucose uptake in skeletal muscle and also an increase in 5' AMP-activated protein kinase (AMPK) activity. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA-riboside), an analog of adenosine, is taken up into cells and phosphorylated to form AICA-riboside monophosphate (ZMP), which can also activate AMPK. This study was designed to determine whether the increase in glucose uptake observed with AMPK activation by AICA-riboside is due to GLUT4 translocation from an intracellular location to the plasma membranes, similar to that seen in response to contraction. Rat hindlimbs were perfused with Krebs-Henseleit bicarbonate containing 4% bovine serum albumin, washed bovine erythrocytes, 8 mmol/l glucose, and +/-2 mmol/AICA-riboside or +/-60 nmol/l insulin. Perfusion medium containing AICA-riboside was found to significantly increase AMPK activity, glucose uptake, and GLUT4 translocation in skeletal muscle above basal levels. Insulin-perfused muscles showed significant increases in glucose uptake and GLUT4 translocation, but AMPK activation was not significantly changed from basal levels. These results provide evidence that the increased glucose uptake observed with AMPK activation by AICA-riboside in perfused rat hindlimb muscles is due to an increase in the translocation of GLUT4 to surface membranes. (+info)
Inhibition of Epstein-Barr virus replication by a benzimidazole L-riboside: novel antiviral mechanism of 5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole.
Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. D-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5, 6-dichloro-2-bromo-1-beta-D-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to the L-riboside 1263W94 [5, 6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1. 1 microM, compared with 10 microM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D. (+info)