Swainsonine, an indolizidine alkaloid, inhibits the processing ofasparagine-linked glycoproteins in both cell-free extracts and animal cells in culture. Thus, in a liver particulate enzyme preparation, swainsonine at 0.1-1.0 FM inhibited the mannosidase that releases [3H]mannose from a high mannose glycopeptide but only slightly inhibited the release of glucose from a glucose-labeled glycopeptide. MDCK and Chinese hamster ovary cells in culture incorporate [2-3H]mannose and [6-3H]-glucosamine into both high mannose and complex types ofoligosaccharides. When these cells were incubated with swainsonine and then labeled with mannose or glucosamine, there was a dramatic decrease in the amount oflabel in the complex type ofglycopeptide and a substantial increase in the radioactivity in the high mannose type. This change was monitored by the increase in radioactivity that became susceptible to digestion by endoglucosaminidase H with increasing concentrations of swainosine. The endoglucosaminidase H-released oligosaccharide(s) from swainsonine-treated cells was larger and more homogenous than that from controls and eluted from Bio-Gel P4 at the position of Man9GlcNAc. Several tissue culture cell lines were grown in the presence of swainsonine to determine its effect on cell surface glycoproteins. Cells grown in the alkaloid showed an increased capacity to bind Escherichia coli B886, a bacterium that binds to high mannose glycoproteins. These cells also showed an increasing binding of [3H]concanavalinA.During the synthesis of the oligosaccharide portion of asparagine-linked glycoproteins, a Glc3Man9GlcNAc2 oligosaccharide is transferred from its dolichol derivative to the protein (1). This newly formed glycoprotein is apparently the precursor of both the high mannose and the complex types ofoligosaccharide and undergoes a number ofprocessing or trimming reactions to form these oligosaccharides. These reactions result in the removal of all three glucose residues and a number of mannose units (3). Several enzymes having glucosidase and mannosidase activity have been described in various membrane fractions (4-9). While the mannose residues are being removed, the sugars that characterize the complex oligosaccharides are added from the appropriate sugar nucleotide donors to form the trisaccharide structures, NeuNAc -* galactose --GlcNAc, linked to the mannose core (10).In this paper, we describe a new inhibitor, swainsonine, that blocks the processing of asparagine-linked glycoproteins. Swainsonine is an indolizidine alkaloid isolated from the plant Swainsona canescens (11). The compound has been shown to inhibit lysosomal a-mannosidase and to produce symptoms of a-mannosidosis in cattle that eat the plant (12). We also show that this alkaloid inhibits the a-mannosidase involved in glycoprotein processing and therefore leads to an increase in high mannose oligosaccharides and a decrease in complex types at the cell surface.EXPERIMENTAL PROCEDURES Studies in Cell-Free Systems. The effect of swainsonine on neutral a-m...

No abstract

No abstract

Treatment of mesangial cells with interleukin I p (IL-10) or tumour necrosis factor a (TNFa) has been shown to increase cGMP formation, most probably due to induction of nitric oxide synthase. Here we report that maximum stimulation of cGMP formation over a 24-h period required the presence of IL-1p or TNFa during the first 18 h of induction. N4-monomethyl-~-arginine (L-NMMA) was a potent inhibitor of cytokine-induced cGMP formation while N4-nitro-~-arginine (L-NNA) was less active. Formation of nitric oxide was detected in the cytosol of cytokine-treated mesangial cells by activation of purified soluble guanylate cyclase and was stimulated by tetrahydrobiopterin, but not by calcium calmodulin. Treatment of cells with IL-1p or TNFa markedly attenuated the contractile response to a subsequent challenge with angiotensin 11. Furthermore, conditioned medium from SLIb-treated cells increased cGMP in untreated control cells. Abbreviations. IL-1, interleukin 1 ; TNF, tumour necrosis factor; TGF, transforming growth factor; L-NMMA, N4-monomethyl-~-arginine; L-NNA, N4-nitro-~-arginine.suggestion gains support from our recent observations, demonstrating that anti-inflammatory steroids and transforming growth factor p2 (TGFp,) potently antagonize IL-lp-induced and TNFa-induced cGMP formation in mesangial cells [20,21]. The present data suggest that IL-1p and TNFa induce the macrophage-type of NO synthase in mesangial cells and thereby alter the contractile responsiveness of the cells. MATERIALS AND METHODS Chemicals[N-~'P]GTP (800 Cijmmol) was purchased from DuPont de Nemours (Dreieich, Germany); recombinant human IL-1 fl (5. lo7 Ujmg), recombinant human TGFB2 (> 2 . lo6 Ujmg) and indomethacin were prepared by Ciba-Geigy Ltd (Basel, Switzerland); recombinant human TNFa (> 2. lo7 Ujmg), superoxide dismutase (3000 Ujmg) and all cell culture nutrients were from Boehringer (Mannheim, Germany); angiotensin SI, 3-isobutyl-1-methylxanthine and L-NMMA were from Sigma (St Louis, MO); (6R)-5,6,7,8-tetrahydrobiopterin was from Dr Schircks Laboratories (Jona, Switzerland); L-arginine, NADPH, nitrate reductase from Aspergillus and L-NNA were from Serva (Heidelberg, Germany); all other chemicals were from Merck (Darmstadt, Germany). Cell cultureRat glomerular mesangial cells were cultivated as described previously [22]. In a second step, single cells were cloned by limited dilution using 96-n~icrowell plates. Clones with apparent mesangial cell morphology were used for further processing [20]. The cells exhibited the typical stellate morphology. Moreover, there was positive staining for the

Human peripheral blood neutrophils are primed, or enabled to respond to formyl peptide, by prior exposure to bacterial lipopolysaccharide (LPS). The activity of LPS and the size of its aggregates are altered by plasma constituents such as high density lipoprotein (HDL) and the recently discovered acute phase reactant lipopolysaccharide binding protein (LBP) Tobias et al.: J. Exp. Med. 164,777, 1986]. The ability of LPS, LPS-LBP, and LPS-HDL complexes to activate a number of cellular responses have been compared. LPS-LBP and LPS-HDL were prepared using LBP and HDL from rabbit serum. LPS from Salmonella minnesota Re595 and its LPS-LBP and LPS-HDL complexes differed in their ability to prime PMN O2- production in response to formyl peptide (f-Nle-Leu-Phe-Nle-Tyr-Leu [FNLPNTL]). Human PMN prepared under conditions in which O2- production is minimal (less than 1 nmol O2-/10(6) PMN/10 min) after exposure to 10(-7) M FNLPNTL can be primed with 0.1-100 ng/ml LPS in a dose- and time-dependent manner to produce up to 12 nmol O2-/10(6) PMN/10 min. LBP complexation accelerated the priming induced by LPS, whereas HDL complexation retarded it. Priming was accompanied by a parallel two- to threefold increase in formyl peptide receptor number as determined by FACS analysis of fluoresceinated FNLPNTL binding and SDS-PAGE autoradiographic analysis of photoaffinity ligand binding. Thus binding of LPS to plasma proteins changes the response of the PMS to LPS and may represent one way in which the response of the PMN is regulated during infection. Since LBP concentrations change during an acute phase response, complexation of LPS with LBP is a mechanism that may regulate neutrophil responses in vivo during inflammation.