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(1/2075) Proteoglycan involvement in polyamine uptake.

We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki=0.16+/-0.04 microM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-beta-D-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by alpha-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or beta-D-xyloside, which might have future therapeutical implications.  (+info)

(2/2075) Determining anomericity of the glycosidic bond in Zn(II)-diethylenetriamine-disaccharide complexes using MSn in a quadrupole ion trap.

Zinc-diethylenetriamine (Zn-dien) N-glycoside complexes of four 1,4 and four 1,6 linked disaccharides are prepared. Each reaction mixture is ionized by electrospray and the resulting species [Zn(dien)(disaccharide)-H]+ is allowed to undergo collision-induced dissociation in a quadrupole ion trap. An MS3 analysis is used to differentiate alpha versus beta anomericity of the glycosidic bond in the disaccharide moiety. In addition, the MS2 and MS3 spectra can be used together to determine the linkage position of this glycosidic bond.  (+info)

(3/2075) Characterization of a novel unconjugated pteridine glycoside, cyanopterin, in Synechocystis sp. PCC 6803.

A new pteridine glycoside, called cyanopterin, was isolated from Synechocystis sp. PCC 6803 and its structure was elucidated as 6-[1-(4-O-methyl-(alpha-d-glucuronyl)-(1, 6)-(beta-d-galactosyloxy]methylpterin by chemical degradation and 1H- and 13C-NMR spectroscopic means. Cyanopterin is constitutively synthesized at a relatively high intracellular concentration that is comparable to that of chlorophyll a in a molar ratio of approximately 1 to 1.6. The in vivo oxidation state of cyanopterin is primarily the fully reduced 5,6,7,8-tetrahydro form. The cellular function is unknown at present. The findings have established a model system, using Synechocystis sp. PCC 6803, for studies of the physiological functions of unconjugated pteridine glycosides found mostly in cyanobacteria.  (+info)

(4/2075) Collagen synthesis and deposition in cartilage during disrupted proteoglycan production.

A simple system was developed to investigate in vitro the possible relationship between collagen and proteoglycan synthesis in cartilage. When production of complete proteoglycan molecules was effectively inhibited with 4-methylumbelliferyl beta-D-xyloside collagen synthesis and distribution were virtually unaffected.  (+info)

(5/2075) Stimulation of collagen galactosyltransferase and glucosyltransferase activities by lysophosphatidylcholine.

Lysophosphatidylcholine stimulated the activities of collagen galactosyl- and glucosyl-transferases in chick-embryo extract and its particulate fractions in vitro, whereas essentially no stimulation was noted in the high-speed supernatant, where the enzymes are soluble and membrane-free. The stimulatory effect of lysophosphatidylcholine was masked by 0.1% Triton X-100. In kinetic experiments lysophosphatidylcholine raised the maximum velocities with respect to the substrates and co-substrates, whereas no changes were observed in the apparant Km values. Phospholipase A preincubation of the chick-embryo extract resulted in stimulation of both transferase activities, probably gy generating lysophosphatides from endogenous phospholipids. No stimulation by lysophosphatidylcholine was found when tested with 500-fold-purified glycosyltransferase. The results suggest that collagen glycosyltransferases must be associated with the membrane structures of the cell in order to be stimulated by lysophosphatidylcholine. Lysophosphatidylcholine could have some regulatory significance in vivo, since its concentration in the cell is comparable with that which produced marked stimulation in vitro.  (+info)

(6/2075) Initiation of galactosaminoglycan biosynthesis. Separate galactosylation and dephosphorylation pathways for phosphoxylosylated decorin protein and exogenous xyloside.

By using various radiolabelled precursors, glycosylation and phosphorylation of decorin in a rat fibroblast cell line was investigated in the presence of increasing concentrations of p-nitrophenyl-O-beta-d-xylopyranoside. Decorin core protein glycanation was suppressed to approximately 25% of the normal level in the presence of 2 mm and 3 mm xyloside. Glycans/saccharides were released from the core protein and size-separated by gel chromatography. The intracellular decorin obtained from cells treated with 2 mm xyloside was substituted with Xyl and also with Gal-Xyl and Gal-Gal-Xyl, but not with longer saccharides. Only the trisaccharide contained an almost fully phosphorylated Xyl. We conclude that galactosylation of endogenous, xylosylated decorin and exogenous xyloside probably follow separate pathways or that xylosides and early decorin glycoforms are kept separated. At the addition of the first glucuronic acid the two pathways seem to merge and dephosphorylation of decorin takes place. Xyloside-primed and secreted galactosaminoglycan chains produced simultanously retained phosphorylated Xyl. Inadequate dephosphorylation could be due to excess substrate or to a short transit.time. As shown previously [Moses, J., Oldberg, A., Eklund, E. & Fransson, L.-A. (1997) Eur. J. Biochem. 248, 767-774], brefeldin A-arrested decorin is substituted with the linkage-region extended with an undersulphated and incomplete galactosaminoglycan chain. In cells treated with this drug, xylosides were unable to prime galactosaminoglycan synthesis and unable to inhibit glycosylation and phosporylation of decorin.  (+info)

(7/2075) Fusicoccin, 14-3-3 proteins, and defense responses in tomato plants.

Fusicoccin (FC) is a fungal toxin that activates the plant plasma membrane H+-ATPase by binding with 14-3-3 proteins, causing membrane hyperpolarization. Here we report on the effect of FC on a gene-for-gene pathogen-resistance response and show that FC application induces the expression of several genes involved in plant responses to pathogens. Ten members of the FC-binding 14-3-3 protein gene family were isolated from tomato (Lycopersicon esculentum) to characterize their role in defense responses. Sequence analysis is suggestive of common biochemical functions for these tomato 14-3-3 proteins, but their genes showed different expression patterns in leaves after challenges. Different specific subsets of 14-3-3 genes were induced after treatment with FC and during a gene-for-gene resistance response. Possible roles for the H+-ATPase and 14-3-3 proteins in responses to pathogens are discussed.  (+info)

(8/2075) Characterization of the UDP-glucuronosyltransferases involved in the glucuronidation of an antithrombotic thioxyloside in rat and humans.

To investigate the glucuronidation on the hydroxyl group of carbohydrate-containing drugs, the in vitro formation of glucuronides on the thioxyloside ring of the antithrombotic drug, LF 4.0212, was followed in rat and human liver microsomes and with recombinant UDP-glucuronosyltransferases (UGT). The reaction revealed a marked regioselectivity in rat and humans. Human liver microsomes glucuronidated the compound mainly on the 2-hydroxyl position of the thioxyloside ring, whereas rat was able to form glucuronide on either the 2-, 3-, or 4- hydroxyl group of the molecule, although to a lower extent. LF 4.0212 was a much better substrate of human UGT than the rat enzyme (Vmax/Km 30.0 and 0.06 microl/min/mg, respectively). Phenobarbital, 3-methylcholanthrene, and clofibrate enhanced the glucuronidation of LF 4.0212 on positions 2, 3, and 4 of the thioxyloside ring, thus indicating that several UGT isoforms were involved in this process. The biosynthesis of the 2-O-glucuronide isomer was catalyzed by the human UGT1A9 and 2B4, but not by UGT1A6 and 2B11. By contrast, the rat liver recombinant UGT1A6 and 2B1 failed to form the 2-O-glucuronide isomers. From all the recombinant UGTs tested, none catalyzed the formation of the 3-O-glucuronide isomer. Interestingly, glucuronidation on the 4-position was found in all the metabolic competent V79 cell lines considered, including the nontransfected V79 cells, suggesting the presence of an endogenous UGT in fibroblasts able to actively glucuronidate the drug. This activity, which was nonsensitive to the inhibitory effect of 7,7,7-triphenylheptanoic acid, a potent UGT inhibitor, could reflect the existence of a different enzyme.  (+info)