Enzymatic characterization of CMP-NeuAc:Galβ1-4GlcNAc-R α(2-3)-sialyltransferase from human placenta

In this report we present the enzymatic characterization of CMP-NeuAc:Gal1-4GlcNAc-R (2-3)-sialyltransferase from human placenta using placenta membranes as an enzyme preparation. This sialyltransferase is highly sensitive to detergents and prefers type 2 chain (Gal1-4GlcNAc) over type 1 chain (Gal1-3GlcNAc) acceptors. Oligosaccharides and glycopeptides were better acceptor substrates than glycoproteins. Of the branched oligosaccharides, those with a bisectedN-acetylglucosamine (GlcNAc) structure appeared to be poorer substrates, while triantennary structures containing a Gal1-4GlcNAc1-4Man1-3Man branch were preferred. Product characterization, using 400 MHz¹H-NMR spectroscopy, confirmed that sialic acid was introduced into the Gal1-4GlcNAc-R units of the acceptor substrates in an (2-3) linkage, and revealed that this sialytransferase does not prefer either of the two branches of a complex type diantennary glycopeptide acceptor for sialic acid attachment. These properties distinguish this enzyme from all other sialyltransferases characterized to date.Abbreviations NeuAc N-acetylneuraminic acid - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - GP-F2 and GP-F4 diantennary complex type glycopeptides from desialylated fibrinogen - GP-Trf diantennary complex type glycopeptide from desialylated transferrin - LNT Gal1-3GlcNAc1-3Gal1-4Glc (lacto-N-tetraose) - 6-sialytransferase CMP-NeuAc:Gal1-4GlcNAc-R (2-6)-sialytransferase - 3-sialytransferaseO CMP-NeuAc:Gal1-3GalNAc-R (2-3)-sialyltransferase - 3-sialytransferase I CMP-NeuAc:Gal1-3(4)GlcNAc-R (2-3)-sialyltransferase - 3-sialytransferase II CMP-NeuAc:Gal1-4GlcNAc-R (2-3)-sialytransferase     

Purification and characterization of α(2-6)-sialyltransferase from human liver

A Gal1-4GlcNAc (2-6)-sialyltransferase from human liver was purified 34 340-fold with 18% yield by dye chromatography on Cibacron Blue F3GA and cation exchange FPLC. The enzyme preparation was free of other sialyltransferases. It did not contain CMP-NeuAc hydrolase, protease, or sialidase activity, and was stable at –20°C for at least eight months. The donor substrate specificity was examined with CMP-NeuAc analogues modified at C-5 or C-9 of theN-acetylneuraminic acid moiety. Affinity of the human enzyme for parent CMP-NeuAc and each CMP-NeuAc analogue was substantially higher than the corresponding Gal1-4GlcNAc (2-6)-sialyltransferase from rat liver.Abbreviations FPLC fast protein liquid chromatography - NeuAc 5-N-acetyl-d-neuraminic acid - 9-amino-NeuAc 5-acetamido-9-amino-3,5,9-trideoxy-d-glycero-2-nonulosonic acid - 9-acetamido-NeuAc 5,9-diacetamido-3,5,9-trideoxy-d-glycero--d-2-nonulosonic acid - 9-benzamido-NeuAc 5-acetamido-9-benzamido-3,5,9-trideoxy-d-glycero--d-galacto-2-nonulosonic acid - 9-fluoresceinyl-NeuAc 9-fluoresceinylthioureido-NeuAc - 5-formyl-Neu 5-formyl--d-neuraminic acid - 5-aminoacetyl-Neu 5-aminoacetyl--d-neuraminic acid - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - G_M1 Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc-ceramide - ST sialyltransferase - DTE 1,4-dithioerythritol Enzyme: Gal1-4GlcNAc (2-6)-sialyltransferase, EC 2.4.99.1.     

Benzyl-N-acetyl-α-d-galactosaminide inhibits the sialylation and the secretion of mucins by a mucin secreting HT-29 cell subpopulation

We have analysed the mucins synthesized by the HT-29 MTX cell subpopulation, derived from the HT-29 human colon carcinoma cells through a selective pressure with methotrexate (Lesuffleuret al., 1990,Cancer Res 50: 6334–43), in the presence of benzyl-N-acetyl--galactosaminide (GalNAc-O-benzyl), which is a potential competitive inhibitor of the 1,3-galactosyltransferase that synthesizes the T-antigen. The main observation was a 13-fold decrease in the sialic acid content of mucins after 24 h of exposure to 5mm GalNAc-O-benzyl. This effect was accompanied by an increased reactivity of these mucins to peanut lectin, testifying to the higher amount of T-antigen. The second observation was a decrease in the secretion of the mucins by GalNAc-O-benzyl treated cells. The decrease in mucin sialyation was achieved through thein situ -galactosylation of GalNAc-O-benzyl into Gal1–3GalNAc-O-benzyl, which acts as a competitive substrate of Gal1–3GalNAc 2,3-sialyltransferase, as shown by the intracellular accumulation of NeuAc2–3Gal1–3GalNAc-O-benzyl in treated cells.Abbreviations BSM bovine submaxillary mucin - MTX methotrexate - PBS sodium phosphate 10mm, NaCl 0.15m, pH 7.4 buffer - pNp p-nitrophenol - TBS Tris/HCl 10mm, NaCl 0.15m, pH 7.4 buffer Enzymes: CMP-NeuAc: Gal1–3/4GlcNAc 2,3-sialyltransferase, ST3(N), EC 2.4.99.6; CMP-NeuAc: Gal1–4GlcNAc 2,6-sialyltransferase, ST6(N), EC 2.4.99.1; CMP-NeuAc: Gal1–3GalNAc 2,3-sialyltransferase, ST3(O), EC 2.4.99.4; CMP-NeuAc: R-GalNAc1-O-Ser 2,6-sialyltransferase, ST6(O)-I, EC 2.4.99.3; CMP-NeuAc: NeuAc2–3Gal1–3GalNAc 2,6-sialyltransferase, ST6(O)-II, EC 2.4.99.7; UDP-GlcNAc: Gal1–3GalNAc-R·(GlcNAc to GalNAc) 1,6-N-acetylglucosaminyltransferase, EC 2.4.1.102; UDP-GlcNAc: GalNAc-R 1,3-N-acetylglucosaminyltransferase, EC 2.4.1.147; UDP-Gal: GalNAc-R 1,3-galactosyltransferase, EC 2.4.1.122.     

Effect of α(2–6)-linked sialic acid and α(1–3)-linked fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA)

The effects of branching and substitution of branches by sialic acid and fucose on the interaction ofN-linked glycopeptides and related oligosaccharides with immobilizedPhaseolus vulgaris leukoagglutinating lectin (L-PHA) were examined. Asialo bi-, tri-and tetra-antennary glycans were all retarded but to different extents on a long column of L-PHA-agarose. Asialo tri- and tetra-antennary glycans containing the pentasaccharide unit Gal1-4GlcNAc1-2[Gal1-4GlcNAc1-6]Man were strongly retarded, whereas asialo bi- and tri-antennary glycans lacking the Gal1-4GlcNAc1-6 branch were only weakly retarded. In all instances the interaction with the lectin was completely abolished when either (2–6)-linkedN-acetylneuraminic acid or (1–3)-linked fucose was present at the galactose orN-acetylglucosamine residue of the Gal1-4GlcNAc1-6Man1-6 branch, respectively. The same substitutions on the Gal1-4GlcNAc1-6Man1-6 branch decreased but did not abolish the affinity of the lectin for the glycans. The presence of NeuAc2-6 and Fuc1-3 on the other two branches did not interfere with the binding of the glycans to L-PHA. Furthermore, it appeared that the presence of the Man1-4GlcNAc unit is requried for interaction with the lectin. In order to obtain reliable information on the relative occurrence of tri- and tetra-antennary glycopeptides, this study shows that it is essential to desialylate and to defucosylate the glycans prior to application to L-PHA-agarose.Abbreviations L-PHA leukoagglutinating phytohemagglutinin - CMP-NeuAc cytidine-5-monophospho-N-acetylneuraminic acid - GP glycopeptide - OS oligosaccharide - HPLC high-performance liquid chromatography - FNR fraction not retarded - FR fraction retarded suffixes MS, BS and TS indicate mono-, bi- and trisialyl derivatives respectively; suffix MF indicates monofucosyl derivatives.structures of the substratesOS2, OS3, OS3, OS4, GP2, GP3, GP4, GP4-MF, OS2(3) andOS2(-) are presented in Fig. 2     

Sialidase of swine influenza A viruses: variation of the recognition specificities for sialyl linkages and for the molecular species of sialic acid with the year of isolation

The sialidase of swine influenza A viruses of N1 and N2 subtypes, isolated from 1930 to 1992, was studied for substrate specificity with ganglio-series, lacto-series type II and GM3 gangliosides containing Neu5Ac2-3Gal, Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. All viral sialidases tested showed that the activity for hydrolysing substrates with Neu5Ac2-3Gal was higher than the activities with Neu5Gc2-3Gal and Neu5Ac2-6Gal linkages. When GM1b, GM3 and sialylparagloboside were used as substrates, the earliest strain (A/Wisconsin/15/30 H1N1, isolated in 1930) showed the activity ratio of Neu5Ac2-6Gal to Neu5Ac2-3Gal to be 0.13:0.2, and the ratio Neu5Gc2-3Gal/Neu5Ac2-3Gal to be 0.19:0.37, while those strains isolated from 1978 to 1992 exhibited ratios of 0.29:0.58 for Neu5Ac2-6Gal/Neu5Ac2-3Gal and 0.51:0.76 for Neu5Gc2-3Gal/Neu5Ac2-3Gal. The above results indicate that the substrate specificities of sialidases from swine influenza A viruses towards sialyl linkages and the molecular species of sialic acid are related to the year of isolation, i.e. strains isolated after 1978 exhibited higher activity towards Neu5Ac2-6Gal and Neu5Gc2-3Gal linkages when compared with strains isolated in an earlier year, 1930.Abbreviation Neu5Ac 5-N-acetylneuraminic acid - Neu5Gc 5-N-glycolyneuraminic acid - Gal d-galactose - Glc d-glucose - Cer Ceramide - II³(Neu5Ac)Lac Neu5Ac2-3Gal1-4Glc - GM3(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-4Glc1-Cer - GM3(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-4Glc1-Cer - GM1b(Neu5Ac2-3Gal) Neu5Ac2-3Gal1-3GalNac1-4Gal1-4Glc1-Cer - GMlb(Neu5Gc2-3Gal) Neu5Gc2-3Gal1-3GalNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Ac)nLc4Cer Neu5Ac2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV³(Neu5Gc)nLc4Cer Neu5Gc2-3Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - IV⁶(Neu5Ac)nLc4Cer Neu5Ac2-6Gal1-3GlcNAc1-4Gal1-4Glc1-Cer - TDC taurodeoxycholate.     

Sialyltransferase activity in FR3T3 cells transformed withras oncogene: decreased CMP-Neu5Ac:Galβ1-3GalNAc α-2,3-sialyltransferase

We have investigated the activity of CMP-Neu5Ac:Gal\1-3GalNAc -2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the -galactosidase -2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activatedras gene decreases the activity of this specific -2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of activeO-glycan -2,3-sialyltransferase polypeptides inras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface ofras-transformed FR3T3 suggesting that no change in the sialylation ofO-glycan core 1 appeared in these cells, although the activity of the -2,3-sialyltransferase was decreased.Abbreviations -2,3-ST(O) CMP-Neu5Ac:Gal1-3GalNAc-R -2,3-sialyltransferase - -2,3-ST(N/O) CMP-Neu5Ac:Gal1-3/4GlcNAc-R -2,3-sialyltransferase - -2,6-ST(N) CMP-Neu5Ac:Gal1-4GlcNAc-R -2,6-sialyltransferase - -2,6-ST(O)I CMP-Neu5Ac:R-GalNAc(1-O)Ser -2,6-sialyltransferase - -2,6-ST(O)II CMP-Neu5Ac:Neu5Ac2-3Gal1-3GalNAc-R -2,6-sialyltransferase - ASFet asialofetuin - FR3T3 Fisher rat fibroblast - FRras Ha-ras-transfected FR3T3 fibroblasts - NaCl/P_i sodium phosphate 10mm, NaCl 0.15m, pH 7.4, buffer - pNp p-nitrophenol     

The glycolipid specificity ofErythrina cristagalli agglutinin

The interaction of¹²⁵I-labeledErythrina cristagalli agglutinin (ECA) with neutral glycosphingolipids on thin layer chromatograms was examined by the overlay technique followed by radioautography. The lectin bound topara-globoside with a sensitivity about 10 times higher than to lactosylceramide or globoside, in agreement with the specificity of the lectin forN-acetyllactosamine. The lower limit of detection ofpara-globoside was about 0.66 nmol. The specific binding of ECA to this glycolipid was confirmed by a highly sensitive enzyme-linked lectin assay (ELLA), utilizing the horseradish peroxidase-avidin-biotin system for detection of bound lectin. Overlays of neutral glycosphingolipid extracts from human erythrocyte membranes and from human granulocytes with ECA demonstrated that the lectin can be employed for the detection of small amounts ofpara-globoside in biological materials also in the presence of excess globoside. No staining was obtained when thin layer chromatograms of neutral glycosphingolipid extracts from rabbit erythrocyte membranes were overlayed with¹²⁵I-ECA. Afterin situ treatment of the chromatograms with -galactosidase, the lectin bound to several components, one of which had a mobility corresponding to that of the pentahexosylceramide Gal3Gal4GlcNAc3Gal4Glc1Cer, the major neutral glycosphingolipid of rabbit erythrocytes, thus providing further evidence for the specificity of ECA forpara-globoside.Abbreviations GSL glycosphingolipid(s) - CDH lactosylceramide, Gal4Glc1Cer - CTH trihexosylceramide, Gal4Gal4Glc1Cer - GLOB globoside, GalNac3Gal4Gal4Glc1Cer - PG para-globoside, Gal4GlcNAc3Gal4Glc1Cer - AsG_M1 asialo-G_M1, Gal3GalNAc4Gal4Glc1Cer - FORS Forsmann antigen, GalNAc3GalNAc3Gal4Gal4Glc1Cer - CPH pentahexosylceramide, Gal3Gal4GlcNAc3Gal4Glc1Cer - ECA Erythrina cristagalli agglutinin - SBA soybean agglutinin - PBS phosphate-buffered saline - PVP-40 polyvinylpyrrolidone M.W. 40000 - BSA bovine serum albumin - HRP-avidin horseradish peroxidase conjugated to avidin - ELLA enzyme-linked lectin assay - ELISA enzyme-linked immunosorbent assay - PMNL polymorphonuclear leukocytes - HPTLC high performance thin layer chromatography     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

Specificity of human antibodies against galα1-3gal carbohydrate epitope and distinction from natural antibodies reacting with galα1-2gal or galα1-4 gal

Antibodies against galactosyl-1-3-galactose epitopes were characterized in normal and patient sera by radioimmunoassay binding to mouse laminin and oligosaccharide inhibition. Binding was strictly dependent on -linked galactose in a terminal position. Reduced affinities were observed for digalactoses with (1-2)-, (1-6)- and (1-4)-linkages and for the blood group B epitope, Gal1-3(Fuc1-2)Gal. Conformational models of various active and inactive oligosaccharides provided a clearer picture of the epitope requirements for the observed antibody specificity. Some antibody heterogeneity was detected by comparing individual sera and by hapten elution from a laminin adsorbent. New assays were developed with synthetic Gal1-3Gal-albumin conjugates and these were shown to be more sensitive than assays with mouse laminin. Two more ubiquitous human antibodies could be detected with Gal1-2Gal and Gal1-4Gal conjugates. They were distinct from Gal1-3Gal-specific antibodies as shown by carbohydrate inhibition. This demonstrates a considerable diversity in the recognition of -linked galactose epitopes by natural antibodies.     

N-andO-alkylation of glycoconjugates and polysaccharides by solid base in dimethyl sulphoxide/alkyl lodide

O-Methylation of simple neutral oligosaccharides is readily accomplished in dimethyl sulphoxide containing solid sodium hydroxide and methyl iodide [Cincanu I, Kerek F (1984) Carbohydr Res 131209-17]. This procedure has been extended to 2-acetamido-2-deoxy sugars and sialic acid-containing oligosaccharides. CompleteO-andN-methylation was in most cases achieved in 15 min. Esterification of carboxylic groups in uronic acids was fast and resulted in concomitant -elimination. The method is also suitable for methylation of glycoproteins and glycosphingolipids. Polysaccharides can also be methylated by this technique. Analysis of the products by gas-liquid chromatography and mass spectrometry showed no degradation products.Abbreviations lacto-N-tetraose LcOse₄, Gal3GlcNAc3Gal4Glc - lacto-N-fucopentaose III III³Fuc-nLcOse₄, Gal4[Fuc3]GlcNAc3Gal4Glc - trihexosylceramide GbOse₃Cer, Gal4Gal4Glc1-1Cer - globoside GbOse₄Cer, GalNAc3Gal4Glc1-1Cer - FAB-MS fas atom bombardment mass spectrometry     

Structure of the main ganglioside from the brain of Xenopus laevis

The main component of the ganglioside¹ mixture from the brain of the adult amphibian Xenopus laevis accounts for 35% of the total, as lipid bound sialic acid. This ganglioside has been purified and characterized by thin layer chromatography, partial and exhaustive enzymatic hydrolysis with sialidase, TLC-overlay procedures with anti-Gg₄Cer and anti-Neu5Ac6GalNAc specific monoclonal antibodies and mass spectrometry. All together the results suggest the following structure:Neu5Ac8Neu5Ac3Gal3(Neu5Ac8Neu5Ac6)GalNAc4Gal4Glc1Ceror, IV³--Neu5Ac₂,III⁶--Neu5Ac₂-Gg₄Cer.     

The effect of fumonisin B1 on developing chick embryos: correlation between de novo sphingolipid biosynthesis and gross morphological changes

Fumonisins, mycotoxins produced byFusarium moniliforme and a number of other fungi, are potent inhibitors of the sphinganine-N-acyltransferase, a key enzyme of sphingolipid biosynthesis, and cause neuronal degeneration, liver and renal toxicity, cancer and other injury to animals.In this study we investigated the effect of fumonisin B₁ on the sphingolipids of developing chick embryos. After yolk sac injection of fumonisin B₁ a concentration and time dependent increase of the sphinganine-over-sphingosine ratio of the embryos could be demonstrated. Studies were done to evaluate the effect of fumonisin B₁ on the glycosphingolipid pattern of the chick embryos. In the presence of 72 µg fumonisin B₁ per egg the incorporation of [¹⁴C]galactose and of [¹⁴C]serine into embryonic glycosphingolipids was reduced by about 70%, although the mass of glycosphingolipids was not affected by the toxin. However, a reduction of the wet weight of the treated embryos was observed. Additionally, histological examinations of whole embryo sections of control and fumonisin B₁ treated embryos are presented. Fumonisin B₁ caused haemorrhages under the skin as well as in the liver of treated embyros. A close correlation between disruption of sphingoid metabolism and light microscopic detectable tissue lesions could be observed.Abbreviations Cer ceramide (N-acylsphingosine) - FB₁ fumonisin B₁ - GM3 NeuAc23Gal14Glc11Cer - GD3 NeuAc28NeuAc23Gal14Glc11Cer - GD1a NeuAc23Gal13GalNAc14(NeuAc23)Gal14Glc11Cer - GT1b NeuAc23Gal13GalNAc14(NeuAc28NeuAc23) Gal14Glc11Cer - HPLC high pressure liquid chromatography - PBS phosphate buffered saline - PDMP 1-phenyl-2-dodecanoylamino-3-morpholino-1-propanol - Sa sphinganine - So sphingosine - Sa/So sphinganine-over-sphingosine - TLC thin layer chromatography - Tris Tris(hydroxymethyl)aminomethan Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Contribution to the knowledge of the enzymatic activity of yeasts of clinical interest

The determination of the enzymatic activity of the yeasts has been applied to the identification of species, specially that ofCandida albicans. In order to know its usefulness in species of clinical interest, we have tested the commercial system API ZYM (Bio Mérieux) on 500 isolated strains of different organic samples, belonging to eight genera and twenty species. All the strains showed positivity to Phosphatase alcaline, Esterase (C4), Esterase lipase (C8), Leucine arylamidase and Phosphatase acid, and negativity to Lipase (C14), Trypsin, Chymotrypsin, -galactosidase, -glucoronidase, -manosidase and -fucosidase. Fourteen enzymatic activity patterns were obtained considering the substrates with variable results for the whole of the strains: Valine arylamidase, Cystine arylamidase, Naphthol-AS-BI-phosphohydrolase, -galactosidase, -glucosidase, -glucosidase and N-acetyl--glucosaminidase. In the majority of the species, the enzymatic profile did not have very specific results since it is usually shared by more than one species.C. albicans is that which presents the greatest number of enzymatic variations, some of these are similar to those of other common clinical species, such asCandida krusei, Candida parapsilosis andCandida tropicalis. This system is proposed as a rapid method for identification and as an epidemiological marker of medically important yeasts.Abbreviations AGL -glucosidase - BGA -galactosidase - BGL -glucosidase - CAA Cystine arylamidase - NAG N.Acetyl--glucosaminidase - PHO Naphthol-AS-BI-phosphohydrolase - VAA Valine arylamidase     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Polyvalent GalNAcα1→Ser/Thr (Tn) and Galβ1→3GalNAcα1→Ser/Thr (T <Subscript>α</Subscript>) as the most potent recognition factors involved in <Emphasis Type="Italic">Maclura pomifera</Emphasis> agglutinin–glycan interactions

Summary The agglutinin isolated from the seeds of Maclura pomifera (MPA) recognizes a mucin-type disaccharide sequence, Gal13GalNAc (T) on a human erythrocyte membrane. We have utilized the enzyme-linked lectinosorbent assay (ELLSA) and inhibition assay to more systematically analyze the carbohydrate specificity of MPA with glyco-recognition factors and mammalian Gal/GalNAc structural units in lectin–glycoform interactions. From the results, it is concluded that the high densities of polyvalent GalNAc1Ser/Thr (Tn) and Gal13GalNAc1Ser/Thr (T) glycotopes in macromolecules are the most critical factors for MPA binding, being on a nanogram basis 2.0 × 10⁵, 4.6 × 10⁴ and 3.9 × 10⁴ more active than monovalent Gal, monomeric T and Tn glycotope, respectively. Other carbohydrate structural units in mammalian glycoconjugates, such as human blood group Sd (a⁺) related disaccharide (GalNAc14Gal) and P^k/P₁ active disaccharide (Gal14Gal) were inactive. These results demonstrate that the configurations of carbon-4 and carbon-2 are essential for MPA binding and establish the importance of affinity enhancement by high-density polyvalencies of Tn/T glycotopes in MPA–glycan interactions. The overall binding profile of MPA can be defined in decreasing order as high density of polyvalent Tn/T (M.W. > 4.0 × 10⁴) >> Tn-containing glycopeptides (M.W. < 3.0 × 10³) > monomeric T/Tn and P (GalNAc13Gal) > GalNAc > Gal >> Man, LAra, DFuc and Glc (inactive). Our findings should aid in the selection of this lectin for elucidating functions of carbohydrate chains in life processes and for applications in the biomedical sciences.