Caenorhabditis elegans sqv mutants are defective in vulval epithelial invagination and have a severe reduction in hermaphrodite fertility. The gene sqv-7 encodes a multitransmembrane hydrophobic protein resembling nucleotide sugar transporters of the Golgi membrane. A Golgi vesicle enriched fraction of Saccharomyces cerevisiae expressing SQV-7 transported UDP-glucuronic acid, UDP-N-acetylgalactosamine, and UDP-galactose (Gal) in a temperature-dependent and saturable manner. These nucleotide sugars are competitive, alternate, noncooperative substrates. The two mutant sqv-7 missense alleles resulted in a severe reduction of these three transport activities. SQV-7 did not transport CMP-sialic acid, GDP-fucose, UDP-N-acetylglucosamine, UDP-glucose, or GDPmannose. SQV-7 is able to transport UDP-Gal in vivo, as shown by its ability to complement the phenotype of Madin-Darby canine kidney ricin resistant cells, a mammalian cell line deficient in UDP-Gal transport into the Golgi. These results demonstrate that unlike most nucleotide sugar transporters, SQV-7 can transport multiple distinct nucleotide sugars. We propose that SQV-7 translocates multiple nucleotide sugars into the Golgi lumen for the biosynthesis of glycoconjugates that play a pivotal role in development.M ost cell surface and secreted proteins and some lipids undergo covalent modifications by the addition of carbohydrates (1, 2). These macromolecules play essential roles in multicellular organisms by participating in normal embryonic development and cell-cell and cell-matrix interactions. The carbohydrate-deficient glycoprotein syndromes, a group of autosomal recessive multisystemic diseases characterized by defective glycosylation of N-glycans, and studies of null mutations of N-glycan biosynthetic enzymes in mice provide strong evidence that the glycan moieties of glycoproteins play essential roles in the normal development and physiology of mammals and probably of all multicellular organisms (3-5). Disorders affecting the assembly of the glycosaminoglycan moieties of proteoglycans suggest the importance of these macromolecules in connective tissue, cartilage, and bone development (3). Heparan sulfate glycosaminoglycans (GAGs) are critical components of Wingless and fibroblast growth factor signaling in Drosophila melanogaster, and defects in heparan sulfate GAG assembly are associated with effects on Drosophila embryonic development, such as abnormalities in segment-polarity cuticle patterns and in mesodermal and tracheal cell migrations (6-9). Mutations of the EXT genes, a tumor suppressor family that includes glycosyltransferases involved in polymerization of heparan sulfates, have been associated with hereditary multiple exostoses, a skeletal dysplasia characterized by multiple cartilage-capped skeletal tumors (10).Nucleotide sugar transporters translocate nucleotide sugars from the cytosol, their site of synthesis, into the lumen of the Golgi apparatus, where they are used as sugar-donor substrates by glycosyltransferases (11). Yeast, Leishmania do...

Lumenal ecto-nucleoside tri-and di-phosphohydrolases (ENTPDases) of the secretory pathway of eukaryotes hydrolyze nucleoside diphosphates resulting from glycosyltransferase-mediated reactions, yielding nucleoside monophosphates. The latter are weaker inhibitors of glycosyltransferases than the former and are also antiporters for the transport of nucleotide sugars from the cytosol to the endoplasmic reticulum (ER) and Golgi apparatus (GA) lumen. Here we describe the presence of two cation-dependent nucleotide phosphohydrolase activities in membranes of Caenorhabditis elegans: one, UDA-1, is a UDP/GDPase encoded by the gene uda-1, whereas the other is an apyrase encoded by the gene ntp-1. UDA-1 shares significant amino acid sequence similarity to yeast GA Gda1p and mammalian UDP/GDPases and has a lumenal active site in vesicles displaying an intermediate density between those of the ER and GA when expressed in S. cerevisiae. NTP-1 expressed in COS-7 cells appeared to localize to the GA. The transcript of uda-1 but not those of two other C. elegans ENTPDase mRNAs (ntp-1 and mig-23) was induced up to 3.5-fold by high temperature, tunicamycin, and ethanol. The same effectors triggered the unfolded protein response as shown by the induction of expression of green fluorescent protein under the control of the BiP chaperone promoter and the UDP-glucose:glycoprotein glucosyltransferase. Up-regulation of uda-1 did not occur in ire-1-deficient mutants, demonstrating the role of this ER stress sensor in this event. We hypothesize that up-regulation of uda-1 favors hydrolysis of the glucosyltransferase inhibitory product UDP to UMP, and that the latter product then exits the lumen of the ER or pre-GA compartment in a coupled exchange with the entry of UDP-glucose, thereby further relieving ER stress by favoring protein re-glycosylation.The occurrence of the eukaryotic endomembrane system has brought forth a need for a mechanism to coordinate the metabolic flux of nucleotides, nucleotide sugars, and nucleotide sulfate between the cytosol and the lumen of secretory organelles. E-type ATPases play an important role in this function. They have been conserved through evolution with their catalytic site in the ecto-position, facing the outer surface of the plasma membrane or its topological equivalent, the lumen of intracellular organelles such as the endoplasmic reticulum (ER), 1 Golgi apparatus (GA), and lysosomes/vacuoles. These enzymes, members of the ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) family, hydrolyze nucleoside tri-and/or diphosphates in the presence of cations and share, at the amino acid sequence level, five conserved motifs called apyrase-conserved regions (ACRs) (1).Extracellular nucleotides such as ATP and ADP are intercellular signaling molecules in virtually every tissue where, modulated by ecto-apyrases, they participate in a broad range of biological processes such as the regulation of immune responses (2) and modulation of signaling by neuronal cells (3). Specifically, mammalian ENTPDase1/CD...

Indomethacin, a nonselective cyclooxygenase (COX) inhibitor, was modified in three distinct regions in an attempt both to increase cyclooxygenase-2 (COX-2) selectivity and to enhance drug safety by covalent attachment of an organic nitrate moiety as a nitric oxide donor. A human whole-blood COX assay shows the modifications on the 3-acetic acid part of the indomethacin yielding an amide-nitrate derivative 32 and a sulfonamide-nitrate derivative 61 conferred COX-2 selectivity. Along with their respective des-nitrate analogs, for example, 31 and 62, the nitrates 32 and 61 were effective antiinflammatory agents in the rat air-pouch model. After oral dosing, though, only 32 increased nitrate and nitrite levels in rat plasma, indicating that its nitrate tether served as a nitric oxide donor in vivo. In a rat gastric injury model, examples 31 and 32 both show a 98% reduction in gastric lesion score compared to that of indomethacin. In addition, the nitrated derivative 32 inducing 85% fewer gastric lesions when coadministered with aspirin as compared to the combination of aspirin and valdecoxib.

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Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat inflammation and to provide pain relief but suffer from a major liability concerning their propensity to cause gastric damage. As nitric oxide (NO) is known to be gastro-protective we have synthesized a NO-donating prodrug of naproxen named NMI-1182. We evaluated two cyclo-oxygenase (COX)-inhibiting nitric oxide donors (CINODs), NMI-1182 and AZD3582, for their ability to be gastro-protective compared to naproxen and for their anti-inflammatory activity. NMI-1182 and AZD3582 were found to produce similar inhibition of COX activity to that produced by naproxen. Both NMI-1182 and AZD3582 produced significantly less gastric lesions after oral administration than naproxen. All three compounds effectively inhibited paw swelling in the rat carrageenan paw edema model. In the carrageenan air pouch model all three compounds significantly reduced PGE2 levels in the pouch exudate but only NMI-1182 and naproxen inhibited leukocyte influx. These data demonstrate that NMI-1182 has comparable anti-inflammatory activity to naproxen but with a much reduced likelihood to cause gastric damage.

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