Role of UDP-Sugar Receptor P2Y14 in Murine Osteoblasts.

The purinergic (P2) receptor P2Y14 is the only P2 receptor that is stimulated by uridine diphosphate (UDP)-sugars and its role in bone formation is unknown. We confirmed P2Y14 expression in primary murine osteoblasts (CB-Ob) and the C2C12-BMP2 osteoblastic cell line (C2-Ob). UDP-glucose (UDPG) had undiscernible effects on cAMP levels, however, induced dose-dependent elevations in the cytosolic free calcium concentration ([Ca2+]i) in CB-Ob, but not C2-Ob cells. To antagonize the P2Y14 function, we used the P2Y14 inhibitor PPTN or generated CRISPR-Cas9-mediated P2Y14 knockout C2-Ob clones (Y14KO). P2Y14 inhibition facilitated calcium signalling and altered basal cAMP levels in both models of osteoblasts. Importantly, P2Y14 inhibition augmented Ca2+ signalling in response to ATP, ADP and mechanical stimulation. P2Y14 knockout or inhibition reduced osteoblast proliferation and decreased ERK1/2 phosphorylation and increased AMPKα phosphorylation. During in vitro osteogenic differentiation, P2Y14 inhibition modulated the timing of osteogenic gene expression, collagen deposition, and mineralization, but did not significantly affect differentiation status by day 28. Of interest, while P2ry14-/- mice from the International Mouse Phenotyping Consortium were similar to wild-type controls in bone mineral density, their tibia length was significantly increased. We conclude that P2Y14 in osteoblasts reduces cell responsiveness to mechanical stimulation and mechanotransductive signalling and modulates osteoblast differentiation.

The effects of UDP-sugars, UDP and Mg2+on uridine diphosphate glucuronosyltransferase activity in human liver microsomes.

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation. 2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg2+ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation. 3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by including Mg2+ in the assay. The Mg2+ effect can be offset using EDTA, and is dependent on the concentration of UDP-GlcA in the assay. 4. We propose that formation of a Mg2+-UDP complex prevents UDP from affecting the enzyme. Our results suggest that 5 mM UDP-GlcA and 10 mM Mg2+ be used for UGT assays in fully disrupted HLM.

Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1ß-stimulated human monocytes.

P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell-cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the ß-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1: ) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1ß (IL-1ß). Between 1 and 24 hours after IL-1ß stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1ß-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1ß-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1: also affects the cell surface levels of adhesion molecule CD11b in IL-1ß-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1: , which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1ß-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development.

UDP-glucose sensing P2Y14R: A novel target for inflammation.

Uridine 5'-diphosphoglucose (UDP-G) as a preferential agonist, but also other UDP-sugars, such as UDP galactose, function as extracellular signaling molecules under conditions of cell injury and apoptosis. Consequently, UDP-G is regarded to function as a damage-associated molecular pattern (DAMP), regulating immune responses. UDP-G promotes neutrophil recruitment, leading to the release of pro-inflammatory chemokines. As a potent endogenous agonist with the highest affinity for the P2Y14 receptor (R), it accomplishes an exclusive relationship between P2Y14Rs in regulating inflammation via cyclic adenosine monophosphate (cAMP), nod-like receptor protein 3 (NLRP3) inflammasome, mitogen-activated protein kinases (MAPKs), and signal transducer and activator of transcription 1 (STAT1) pathways. In this review, we initially present a brief introduction into the expression and function of P2Y14Rs in combination with UDP-G. Subsequently, we summarize emerging roles of UDP-G/P2Y14R signaling pathways that modulate inflammatory responses in diverse systems, and discuss the underlying mechanisms of P2Y14R activation in inflammation-related diseases. Moreover, we also refer to the applications as well as effects of novel agonists/antagonists of P2Y14Rs in inflammatory conditions. In conclusion, due to the role of the P2Y14R in the immune system and inflammatory pathways, it may represent a novel target for anti-inflammatory therapy.

A glucurono(arabino)xylan synthase complex from wheat contains members of the GT43, GT47, and GT75 families and functions cooperatively.

Glucuronoarabinoxylans (GAXs) are the major hemicelluloses in grass cell walls, but the proteins that synthesize them have previously been uncharacterized. The biosynthesis of GAXs would require at least three glycosyltransferases (GTs): xylosyltransferase (XylT), arabinosyltransferase (AraT), and glucuronosyltransferase (GlcAT). A combination of proteomics and transcriptomics analyses revealed three wheat (Triticum aestivum) glycosyltransferase (TaGT) proteins from the GT43, GT47, and GT75 families as promising candidates involved in GAX synthesis in wheat, namely TaGT43-4, TaGT47-13, and TaGT75-4. Coimmunoprecipitation experiments using specific antibodies produced against TaGT43-4 allowed the immunopurification of a complex containing these three GT proteins. The affinity-purified complex also showed GAX-XylT, GAX-AraT, and GAX-GlcAT activities that work in a cooperative manner. UDP Xyl strongly enhanced both AraT and GlcAT activities. However, while UDP arabinopyranose stimulated the XylT activity, it had only limited effect on GlcAT activity. Similarly, UDP GlcUA stimulated the XylT activity but had only limited effect on AraT activity. The [(14)C]GAX polymer synthesized by the affinity-purified complex contained Xyl, Ara, and GlcUA in a ratio of 45:12:1, respectively. When this product was digested with purified endoxylanase III and analyzed by high-pH anion-exchange chromatography, only two oligosaccharides were obtained, suggesting a regular structure. One of the two oligosaccharides has six Xyls and two Aras, and the second oligosaccharide contains Xyl, Ara, and GlcUA in a ratio of 40:8:1, respectively. Our results provide a direct link of the involvement of TaGT43-4, TaGT47-13, and TaGT75-4 proteins (as a core complex) in the synthesis of GAX polymer in wheat.

Gi-dependent cell signaling responses of the human P2Y14 receptor in model cell systems.

Eight G protein-coupled receptors comprise the P2Y receptor family of cell signaling proteins. The goal of the current study was to define native cell signaling pathways regulated by the uridine nucleotide sugar-activated P2Y(14) receptor (P2Y(14)-R). The P2Y(14)-R was stably expressed in human embryonic kidney (HEK) 293 and C6 rat glioma cells by retroviral infection. Nucleotide sugar-dependent P2Y(14)-R activation was examined by measuring inhibition of forskolin-stimulated cAMP accumulation. The effect of P2Y(14)-R activation on mitogen-activated protein kinase signaling also was studied in P2Y(14)-HEK293 cells and in differentiated HL-60 human myeloid leukemia cells. UDP-Glc, UDP-galactose, UDP-glucuronic acid, and UDP-N-acetylglucosamine promoted inhibition of forskolin-stimulated cAMP accumulation in P2Y(14)-HEK293 and P2Y(14)-C6 cells, and this signaling effect was abolished by pretreatment of cells with pertussis toxin. Inhibition of cAMP formation by nucleotide sugars also was observed in direct assays of adenylyl cyclase activity in membranes prepared from P2Y(14)-C6 cells. UDP-Glc promoted concentration-dependent and pertussis toxin-sensitive extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y(14)-HEK293 cells. P2Y(14)-R mRNA was not observed in wild-type HL-60 cells but was readily detected in dimethyl sulfoxide-differentiated cells. Consistent with this observation, no effect of UDP-Glc was observed in wild-type HL-60 cells, but UDP-Glc-promoted pertussis toxin-sensitive activation of ERK1/2 occurred after differentiation. These results illustrate that the human P2Y(14)-R signals through G(i) to inhibit adenylyl cyclase, and P2Y(14)-R activation also leads to ERK1/2 activation. This work also identifies two stable P2Y(14)-R-expressing cell lines and differentiated HL-60 cells as model systems for the study of P2Y(14)-R-dependent signal transduction.

Functional expression of the P2Y14 receptor in human neutrophils.

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y(14) receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in human neutrophils. UDP-glucose (IC(50)=1 microM) induced a small but significant inhibition (circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 microM) had no significant effect, whereas 100 microM UDP-N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 microM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y(14) receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y(14) receptor expressed in human neutrophils.

Modification of benzo(a)pyrene metabolism with microsomes by addition of uridine 5'-diphosphoglucuronic acid.

The metabolism of benzo(a)pyrene (BaP) by rat liver microsomes was examined in the presence and absence of uridine 5'-diphosphoglucuronic acid (UDPGA). BaP metabolites were separated by high-pressure liquid chromatography. The normal chromatographic patterns of the metabolities were altered by the addition of UDPGA. At concentrations of UDPGA at which the elutions of dihydrodiol components were unchanged, phenol and quinone elution profiles were selectively decreased. The decreased activities of microsomal mixed-function oxidases by UDPGA were also observed with the use of the aryl hydrocarbon hydroxylase assay. The decrease may not be due to inhibition of those enzymes, but rather to formation of glucuronide conjugates with oxygenated BaP metabolites. These results suggest that glucuronidation may be important in the detoxification of BaP.

An inhibitor of chondroitin sulfate proteoglycan synthesis promotes central nervous system remyelination.

Remyelination is the generation of new myelin sheaths after injury facilitated by processes of differentiating oligodendrocyte precursor cells (OPCs). Although this repair phenomenon occurs in lesions of multiple sclerosis patients, many lesions fail to completely remyelinate. A number of factors have been identified that contribute to remyelination failure, including the upregulated chondroitin sulfate proteoglycans (CSPGs) that comprise part of the astrogliotic scar. We show that in vitro, OPCs have dramatically reduced process outgrowth in the presence of CSPGs, and a medication library that includes a number of recently reported OPC differentiation drugs failed to rescue this inhibitory phenotype on CSPGs. We introduce a novel CSPG synthesis inhibitor to reduce CSPG content and find rescued process outgrowth from OPCs in vitro and accelerated remyelination following focal demyelination in mice. Preventing CSPG deposition into the lesion microenvironment may be a useful strategy to promote repair in multiple sclerosis and other neurological disorders.

Evidence indicating that UDP-N-acetylglucosamine does not appear to stimulate hepatic microsomal UDP-glucuronosyltransferase by interaction with the catalytic unit of the enzyme.

The binding studies in this paper indicate that the catalytic unit(s) of microsomal UDP glucuronosyltransferase(s) is not accessible to N-ethylmaleimide or UDP-N-acetylglucosamine, when the enzyme is in its membrane environment. Thus a separate regulatory factor may exist within the endoplasmic reticulum membrane that mediates the stimulation of UDPglucuronosyltransferase(s) by UDP-N-acetylglucosamine. The possible role and the mode of interaction of the putative regulatory factor with the multiple forms of UDPglucuronosyltransferase are discussed.

Special effects of UDP-sugar binding to bovine liver uridine diphosphoglucose dehydrogenase.

The binding of NADH to uridine diphosphate glucose dehydrogenase has been examined by equilibrium dialysis. There is an absolute requirement for the presence of UDP-glucose for the binding of NADH. Other analogs such as UDPxylose, UDPgalactose and UDPglucuronic acid cannot replace UDPglucose as an effector of NADH binding. UDPxylose competes with UDPglucose for the UDP-sugar-binding site, and in so doing releases the bound NADH. The binding of NADH to UDPglucose dehydrogenase in the presence of UDPglucose reaches a saturation limit of 3 mol NADH bound per enzyme hexamer, and displays positive cooperativity, Hill number = 1.34. The effects of UDP-sugars on the fluorescence of UDPglucose dehydrogenase derivatized at the catalytic sites with a fluorophore have also been studied. Two classes of UDPxylose-binding site have been detected. One class has high affinity (Kdiss = 3 microM, determined by equilibrium dialysis) but does not affect fluorophore fluorescence, and the other has lower affinity (Kdiss = 120 microM) and leads to red-shifted fluorescence quenching, presumably by effecting exposure of the fluorophore to solvent. The high-affinity sites are identified as the UDP-sugar subsites of the underivatized catalytic sites, and the low-affinity sites as UDP-sugar subsites of the fluorophore-labeled catalytic sites.

Effect of phosphoenolpyruvate analogs on the activity of enoylpyruvate transferase and the effect of UDP-N-acetylglucosamine on the reactivity of the active site SH group.

Effect of several phosphoenolpyruvate analogs on the activity of enoylpyruvate (phosphoenolpyruvate: UDP-2-acetamido-2-deoxy-D-glucose 2-enoyl-1-carboxyethyltransferase, EC 2.5.1.7) transferase was examined. The results suggest that the phosphoenolpyruvate binding site of the transferase is very similar to that of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40). Evidence is presented to show that the binding of UDP-GlcNAc to the transferase enhances the reactivity of the active site SH group.

Kinetic properties of the rat intestinal microsomal 1-naphthol:UDP-glucuronosyl transferase. Inhibition by UDP and UDP-N-acetylglucosamine.

The kinetic properties of the rat intestinal microsomal 1-naphthol:UDPglucuronosyltransferase (EC 2.4.1.17) were investigated in fully activated microsomes prepared from isolated mucosal cells. The enzyme appeared to follow an ordered sequential bireactant mechanism in which 1-naphthol and UDP-glucuronic acid (UDPGlcUA) are the first and second binding substrates and UDP and 1-naphthol glucuronide the first and second products, respectively. Bisubstrate kinetic analysis yielded the following kinetic constants: Vmax = 102 +/- 6 nmol/min per mg microsomal protein, Km (UDPGlcUA) = 1.26 +/- 0.10 mM, Km (1-naphthol) = 96 +/- 10 microM and Ki (1-naphthol) = 25 +/- 7 microM. The rapid equilibrium random or ordered bireactant mechanisms, as well as the iso-Theorell-Chance mechanism, could be excluded by endproduct inhibition studies with UDP.UDP-N-acetylglucosamine (UDPGlcNAc), usually found to be an activator of UDP glucuronosyltransferase in liver microsomes, acted as a full competitive inhibitor towards UDPGlcUA in rat intestinal microsomes. With regard to 1-naphthol UDPGlcNAc exhibited a dual effect: both inhibition and activation was observed. The effect of activation by MgCl2 and Triton X-100 on the kinetic constants and the inhibition patterns of UDP and UDPGlcNAc were investigated. The results obtained suggest that latency in rat intestinal microsomes may be due to endproduct inhibition by UDP. This endproduct inhibition could be abolished by in vitro treatment with MgCl2 and Triton X-100.

Functional expression of the P2Y14 receptor in murine T-lymphocytes.

Quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis has previously shown that the P2Y(14) receptor is expressed in peripheral immune cells including lymphocytes. Although in transfected cells the P2Y(14) receptor couples to pertussis toxin-sensitive G(i/o) protein, the functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity has not been reported. Therefore, the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in murine spleen-derived T- and B-lymphocyte-enriched populations. RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in whole spleen and isolated T- and B-lymphocytes. In T cells, UDP-glucose (EC(50) = 335 nM) induced a small but significant inhibition (circa 20%) of forskolin-stimulated cAMP accumulation, suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine had no significant effect alone but behaved as partial agonists by blocking UDP-glucose responses. In B cells, UDP-glucose (100 microM) had no significant effect on forskolin-stimulated cAMP accumulation. Treatment of T cells with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. T-cell proliferation in response to anti-CD3 monoclonal antibody (1 microg ml(-1)) was significantly inhibited by UDP-glucose (59% inhibition; p[IC(50)] = 5.9 +/- 0.3), UDP-N-acetylglucosamine (37%; 6.1 +/- 0.3), UDP-galactose (56%; 8.2 +/- 0.2) and UDP-glucuronic acid (49%; 6.3 +/- 0.2). Interleukin-2- (5 ng ml(-1)) induced T-cell proliferation was also significantly inhibited by all four agonists. In summary, we have shown that the P2Y(14) receptor appears to be functionally expressed in murine spleen-derived T-lymphocytes. These observations suggest that UDP-glucose and related sugar nucleotides presumably via the P2Y(14) receptor may play an important role in modulating immune function.

Uridine 5'-diphosphate glucose analogues. Inhibitors of protein glycosylation that show antiviral activity.

A series of analogues of uridine 5'-diphosphate glucose and uridine 5'-diphosphate glucosamine have been synthesized by reaction of 2,3,4,6-tetra-O-benzyl-, 2,3,4,6-tetra-O-benzoyl-, 2,3,4,6-tetra-O-acetyl-, and 2,3,4,6-tetra-O-palmitoyl-alpha-D-glucopyranose and 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranose with chlorosulfonyl isocyanate and 2',3'-O-isopropylideneuridine. Isopropylidene and acetyl groups of the resulting 5'-O-[[[[(alpha-D-glucopyranosyl)oxy]carbonyl]amino]sulfonyl] -2',3'-O-isopropylideneuridine derivatives were removed by reaction with a TFA/water (5:1) mixture and methanolic ammonia, respectively. The 5'-O-[[[[(2",3",4",6"-tetra-O-benzyl-and 2",3",4",6"-tetra-O-benzoyl-alpha-D-glucopyranosyl)oxy]carbonyl] amino]sulfonyl]-2',3'-O-isopropylideneuridine (13 and 19) and the corresponding deisopropylidenated derivatives showed antiviral activity as determined by the inhibition of the cytopathic effect induced by HSV-1 replication and by the plaque assay method. Compound 13 inhibited glycosylation of proteins in HSV-1 infected HeLa cells.

Inhibition of UDP-N-acetylglucosamine import into Golgi membranes by nucleoside monophosphates.

The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5'-phosphate. The enzyme is extremely tolerant to changes at the 3'-position, and the presence of 3'-F actually increases binding of the NMP to the enzyme. At the 2'-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.

Effects of uridine diphosphoglucose (UDPG) infusion on 5-phosphoribosyl pyrophosphate (PRPP) levels of mouse tissues.

Uridine diphosphoglucose (UDPG) has been shown to have tissue-specific effects that have proved to be of clinical value in the treatment of some liver ailments. In an effort to determine something about the mechanism of action, we investigated the effect of UDPG on the levels of 5-phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase in mouse liver, spleen and transplanted tumors. Three strains of mice were studied with and without tumors under various experimental conditions. Balb/c mice were infused with UDPG intraperitoneally at levels of 0.16 g/kg/day (0.28 mmole) to 1.6 g/kg/day (2.8 mmoles) for 5 days. At the low dose rate the PRPP level in the liver was found to increase 3-fold. A slight increase was noted in the activity of PRPP synthetase. However, when the UDPG was infused at a level of 2.8 mmoles/kg/day, the increases in both the synthetase and PRPP were inhibited. Both CRF1 and CD8 mice were less sensitive to the effects of UDPG per se. However, the high level of PRPP in the tumors they carried was greatly affected by the UDPG infusion. The tumor-specific inhibition of PRPP suggests that this action might prove to be useful combination therapy with inhibitors of purine and pyrimidine nucleotide synthesis in various rescue regimens. UDPG was found to enter cells intact before it was cleaved into glucose phosphate and UMP. The fact that UDPG was also found in the membrane fraction suggests that either there is a specific transport mechanism or UDPG exerts its action via interaction with the cell membrane.

Antiviral activity of uridine 5'-diphosphate glucose analogues against some enveloped viruses in cell culture.

Twenty five analogues of uridine 5'-diphosphate glucose were screened against herpes simplex type 2, vaccinia virus, Sindbis virus and African swine fever virus. After screening, the compound 5'-[[[[(2",3",4",6"-tetra-O-benzoyl-alpha-D- glucopyranosyl)oxi]carbonyl]amino]sulfonyl]uridine (2), the synthesis of which has been reported (Camarasa et al., J. Med. Chem. 28, 40-46, 1985), was selected for further study. This compound showed in vitro activity against all viruses tested. The replication of herpes virus type 2 and African swine fever virus was completely inhibited at 100 micrograms/ml and 150 micrograms/ml respectively; vaccinia virus and Sindbis virus were inhibited to a lesser extent. The compound may inhibit several steps in the viral replication process.

Uridine sugar nucleotides modified in their nucleoside moieties and their effect on lipid-linked saccharide formation in vitro.

Uridine diphospho glucose (UDP-Glc) and uridine diphospho N-acetylglucosamine (UDP-GlcNAc), modified in the uridine moiety by either periodate oxidation of the ribose ring or substitution at position 5 of the uracil ring with fluorine, have been tested as potential inhibitors of glucosyl monophosphoryl dolichol (Glc-P-Dol) or N,N-diacetylchitobiosyl pyrophosphoryl dolichol [GlcNAc)2-PP-Dol) assembly in chick embryo cell membranes. The periodate oxidised sugar nucleotides inhibited glycosyl transfer from their respective natural counterparts by 50% at 230 micron periodate oxidised UDP-Glc and 70 micron periodate oxidised UDP-GlcNAc respectively. Inhibition in both cases was irreversible and addition of exogenous Dol-P stimulated only the residual non-inhibited reaction. Periodate oxidised UDP-GlcNAc preferentially inhibited the transfer of GlcNAc to GlcNac-PP-Dol. The sugar nucleotide containing 5-fluorouridine were, on the other hand, alternative substrates for Glc-P-Dol or (GlcNAc)2-PP-Dol synthesis. FUDP-Glc was a good substrate for Glc-P-Dol formation; having Km and Vmax values equal to those of UDP-Glc, whereas FUDP-GlcNAc was a less efficient substrate for the formation of (GlcNAc)2-PP-Dol; having Km and Vmax values one half and one third respectively of those of UDP-GlcNAc.

Effect of uridine diphospho-N-acetylglucosamine and sodium salicylate on L-glutamine-D-fructose-6-phosphate aminotransferase activity from rat gastric mucosa.

Mechanisms of inhibitory action of sodium salicylate on L-glutamine-D-fructose-6-phosphate aminotransferase, prepared from rat gastric mucosa, were studied. Sodium salicylate at lower concentrations (10-20 mM) inhibited reversibly aminotransferase activity by competing with fructose-6-phosphate. At higher concentrations, sodium salicylate inactivated the enzyme irreversibly, with an inactivation rate following first-order kinetics with respect to the enzyme concentration. Uridine-5'-diphospho-N-acetylglucosamine is an endogenous feedback inhibitor. It inhibited the aminotransferase-catalyzed reaction also by competing with fructose-6-phosphate but with an inhibiting activity 1000 times that of sodium salicylate. Uridine-5'-diphospho-N-acetylglucosamine reduced the salicylate inhibition of the enzymic reaction and protected the enzyme from salicylate-induced irreversible inactivation. At a fixed concentration of uridine-5'-diphospho-N-acetylglucosamine (7x10(-6)M), an increase of salicylate concentration produced an increase in enzyme activity as compared with the control.