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...

The antimicrobial activity of 5-chloro-2(2,4-dichlorophenoxy) phenol (triclosan, one of the active ingredients of Logamel®, Ciba-Geigy) was compared in vitro with that of other antimicrobials exclusively or occasionally used as topical agents in dermatology: hexachlorophene, clioquinol, chlorquinaldol. gentamicin, neomycin, nystatin, econazole, clotrimazole and salicylic acid. Upon determination of the MICs for 53 strains of aerobic and anaerobic bacteria, yeasts and fungi, triclosan was found to display a high degree of activity against most of the test organisms and to have the broadest spectrum of chemotherapeutically significant antimicrobial activity of the substances tested.

Sorangicin A, a macrolide polyether antibiotic and the ansamycin antibiotic rifampicin inhibit DNA-dependentRNApolymerase to a similar extent. Resistance to sorangicin A is due to a mutation in the RNApolymerase which renders the enzyme less sensitive. Parallel investigations with rifampicin revealed partial cross-resistance, which was more marked in sorangicin A-resistant mutants than in rifampicin-resistant mutants. Sorangicin A is a new type of macrolide polyether antibiotic isolated from the gliding bacterium Sorangium cellulosum. Its antimicrobial properties are similar to those of the ansamycin antibiotic rifampicin. Sorangicin A is highly active against Gram-positive bacteria (MIC<0.01~2^g/ml) and less so against Gram-negative bacteria (MIC 2~>32^g/ml). The drug also resembles rifampicin in its mechanism of action, since it specifically affects bacterial DNA-dependent RNApolymerase1~3). The chemical structures of the two drugs are, however, quite different. The aim of the present work was to analyze the mechanismof resistance of Escherichia coli to sorangicin A and to compare it to the resistance against rifampicin.

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase 11. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 pg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-IOI), 8-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100-500 ng/ml), these cells showed a 50-100% increase in their ability to bind [3H]concanavalin A, and a substantial decrease in the binding of [3H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 pglml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [3H]leucine or [3H]proline into protein.There was, however, a significant inhibition in the incorporation of [3H]fucose, [3H]glucosamine, and [3H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, gl...

CGP 47969A is a novel piperazine derivative that inhibits the synthesis of inflammatory cytokines, such as interleukin-1 alpha (IL-1), IL-1 beta and tumor necrosis factor alpha (TNF), in human monocytes stimulated with lipopolysaccharide (LPS), zymosan or IL-1 itself. IC50 values are in the range of 0.3-5 mumol/l. CGP 47969A does not inhibit total protein or RNA synthesis indicating selectivity for cytokine inhibition. CGP 47969A exerts its inhibitory effect at a post-transcriptional level, most probably by reducing translational efficiency of IL-beta mRNA, as steady-state levels of IL-1 beta mRNA are not inhibited while the primary translation product, the 31 kD IL-1 beta precursor molecule, is dose-dependently inhibited by CGP 47969A. The compound is devoid of cyclooxygenase and phospholipase A2 inhibitory activity but efficiently inhibits the generation of PGE2 and LTC4 in zymosan-stimulated mouse macrophages with an IC50 of 1.2 and 0.6 mumol/l, respectively. Antagonism of IL-1 and/or TNF is thought to have a beneficial effect on the course of inflammatory diseases. CGP 47969A may therefore represent a mechanistically new approach to the treatment of such diseases.