Isolation and properties of a sialidase fromTrypanosoma rangeli

In the culture supernatant ofTrypanosoma rangeli, strain El Salvador, a sialidase was present with an activity of 0.1 U/mg protein as determined with the 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid as substrate. This enzyme was purified about 700-fold almost to homogeneity by gel chromatography on Sephadex G-100 and Blue Sepharose, and affinity chromatographies on 2-deoxy-2,3-didehydroneuraminic acid and horse submandibular gland mucin, both immobilized on Sepharose. The pH optimum is at 5.4–5.6, and the molecular weight was determined by gel chromatography, high performance liquid chromatography and sodium dodecyl sulphate gel electrophoresis to be 70 000. The substrate specificity of the enzyme is comparable to bacterial, viral and mammalian sialidases with cleavage rates for the following substrates in decreasing order: N-acetylneuraminyl-(2–3)-lactose> N-glycoloylneuraminy-(2–3)-lactose> N-acetylneuraminyl-(2–6)-lactose >sialoglycoproteins>gangliosides>9-O-acetylated sialoglycoproteins.4-O-Acetylated derivatives are resistant towards the action of this sialidase. The enzyme activity can be inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid, Hg²⁺ ions, andp-nitrophenyloxamic acid; it is not dependent on the presence of Ca²⁺ Mn²⁺ or Mg²⁺ ions.Abbreviations BSA bovine serum albumin - BSM bovine submandibular gland mucin - CMP cytidine monophosphate - EDIA ethylenediaminetetraacetic acid - ESM equine submandibular gland mucin - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HPLC high performance liquid chromatography - Lac lactose - MU-Neu5Ac 4-methylumbelliferyl glycoside of -N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4Ac5Gc N-glycoloyl-4-O-acetylneuraminic acid - Neu2en 2-deoxy-2,3-didehydroneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - PMSF phenylmethylsulfonyl fluoride - PSM pig submandibular gland mucin - SDS sodium dodecyl sulfate - Tris tris-(hydroxymethyl)aminomethane Dedicated to Professor Dr. Heinz Mühlpfordt on the occasion of his 65th birthday.     

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.     

N-Acetyl-4-deoxy-d-neuraminic acid is activated and transferred on to asialoglycoprotein

The sialic acid analogue,N-acetyl-4-deoxy-neuraminic acid, is readily activated by CMP-sialic acid synthase from bovine brain. We also show that sialyl-transfer from CMP-N-acetyl-4-deoxy-neuraminic acid to asialo- ₁-acid glycoprotein is achieved at a high rate using Gal1-4GlcNAc (2.6)-sialyltransferase from rat liver.In contrast toVibrio cholerae sialidase, fowl plague virus sialidase liberates boundN-acetyl-4-deoxy-neuraminic acid from the glycoprotein. Thus, as opposed to the general view, the action of neither synthase nor transferase depends on the presence of the hydroxy group at C-4 ofN-acetylneuraminic acid.Abbrevations BSA bovine serum albumin - DTE dithioerythritol - HPLC high performance liquid chromatography - NeuAc N-acetyl-d-neuraminic acid - 4-deoxy-NeuAc N-acetyl-4-deoxy-d-neuraminic acid - 4-epi-NeuAc 4-acetamido-3,5-dideoxy-d-glycero-d-talononulosonic acid - CMP-NeuAc Cytidine-5-monophospho-N-acetylneuraminic acid - CMP-4-deoxy-NeuAc Cytidine-5-monophospho-N-acetyl-4-deoxy-neuraminic acid - FPV-sialidase Fowl plague virus sialidase - VCN Vibrio cholerae neuraminidase     

Synthesis of methyl α- and β-N-dansyl-d-galactosaminides,probes for the combining sites ofN-acetyl-d-galactosamine-specific lectins

The synthesis of the methyl - and -N-dansyl-d-galactosaminides is described using methyl ,-2-azido-2-deoxy-d-galactopyranoside as starting material. This was reduced to the corresponding methyl ,-2-amino-2-deoxy-d-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl ,-N-dansyl-d-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl -N-dansyl-d-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with theN-acetyl-d-galactosamine specific lectin fromErythrina corallodendron.Abbreviations ECorL Erythrina corallodendron lectin - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside - MeGalNDns methyl 2-deoxy-2-(5-dimethylamino-1-naphthalenesulfamido)--d-galactopyranoside Dedicated to Hilde De Boeck (1958–1991).     

Reassessment of the acceptor specificity and general properties of the Lewis blood-group gene associated α-3/4-fucosyltransferase purified from human milk

The acceptor specificity and general properties of a Lewis blood-group gene associated -3/4-L-fucosyltransferase isolated from human milk have been examined at the penultimate purification stage involving affinity chromatography on GDP-hexanolamine Sepharose, and after a subsequent gel filtration step on Sephacryl S-200. Both preparations transferred fucose to theO-4 position ofN-acetylglucosamine in Type 1 (Gal1-3GlcNAc-R) acceptors and theO-3 position of glucose in lactose-based (Gal1-4Glc) oligosaccharides, and both used Type 1 sialylated compounds when the terminalN-acetylneuraminic acid was present in -2,3 linkage. The striking difference between the two preparations was in their reactivity with Type 2 (Gal1-4GlcNAc-R) chains; after Sephacryl S-200 chromatography the apparentK _M values for the -3/4- preparation with unsubstituted low-molecular-weight Type 2 oligosaccharides were considerably increased. Substitution of the terminal galactose with sialic acid in -2,3 linkage decreased theK _M values for low-molecular-weight oligosaccharides but no detectable incorporation of fucose was observed intoN-acetyllactosamine end-groups of glycoproteins withN-linked oligosaccharide chains, irrespective of the presence of sialic acid in the terminal sequences.Deceased 25 June 1991.     

Spatiotemporal expression patterns of sialoglycoconjugates during nephron morphogenesis and their regional and cell type-specific distribution in adult rat kidney

The expression of 2,6- and 2,3-linked sialic acids on N-glycans was studied in embryonic, postnatal, and adult rat kidney. Histochemistry and blotting using Polyporus squamosus and Sambucus nigra lectins for 2,6-linked sialic acids and the Maackia amurensis lectin for 2,3-linked sialic acids were performed and sialyltransferase activity was assayed. N-glycans with 2,6- and 2,3-linked sialic acid were differently expressed in the two embryonic anlagen and early stages of nephron. Metanephrogenic mesenchyme was positive for 2,3-linked sialic acid but not for the 2,6-linked one, which became detectable initially in the proximal part of S-shaped bodies. Collecting ducts were positive for 2,6-linked sialic acid, whereas 2,3-linked sialic acid was restricted to their ampullae. Although positive in embryonic kidney, S1 and S2 of proximal tubules became unreactive for 2,3-linked sialic acid in postnatal and adult kidneys. In adult kidney, intercalated but not principal cells of collecting ducts were reactive for 2,3-linked sialic acid. In contrast, 2,6-linked sialic acids were detected in all cells of adult kidney nephron. Blot analysis revealed a different but steady pattern of bands reactive for 2,6- and 2,3-linked sialic acid in embryonic, postnatal, and adult kidney. Activity of 2,6 and 2,3 sialyltransferases was highest in embryonic kidney and decreased over postnatal to adult kidney with the activity of 2,6 sialyltransferase always being three to fourfold that of 2,3 sialyltransferase. Thus, 2,6- and 2,3-linked sialic acids are differently expressed in embryonic anlagen and mesenchyme-derived early stages of nephron and show regional and cell type-specific differences in adult kidney.     

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.     

Glycerol-, inositol-, and reducing end hexose-containing oligosaccharides in human urine

Ten previously unreported oligosaccharides have been purified from the urines of human subjects using a combination of gel filtration, ion exchange, and thin-layer chromatographies. Their structures were determined by direct probe mass spectrometry, methylation analysis, and proton NMR spectroscopy of the permethylated oligosaccharide alditols.On the basis of composition, the oligosaccharides could be divided into three groups. Five oligosaccharides containing glycerol were characterized as glucosyl1-1glycerol; glucosyl1-1glycerol; galactosyl1-1glycerol; glucosyl-1-1(fucosyl-1-2)glycerol and/or fucosyl-1-1(glucosyl-1-2)glycerol; and glucosyl-1-1(galactosyl-1-2)glycerol or galactosyl-1-1(glucosyl-1-2)glycerol. Four inositol-containing oligosaccharides were characterized as galactosyl1 (fucosyl1)inositol,N-acetylgalactosaminyl1 (fucosyl1)inositol, fucosyl1-2galactosyl1 (N-acetylgalactosaminyl1)inositol and fucosyl1-2galactosyl1-4-N-acetylglucosaminyl1(N-acetylgalactosaminyl1)inositol. Finally, galactosyl1-3(fucosyl1-2)galactosyl1-6galactosyl1-4(fucosyl1-3)glucose, an oligosaccharide with glucose at its reducing end, was tentatively identified. The significance and possible origins of the carbohydrate structures are discussed.     

Effect of 2-deoxyglucose, α-methylglucoside,and glucosamine on aflatoxin production by Aspergillus parasiticus

The effects of 2-deoxyglucose (2-DOG), -methylglucoside (-MG), and glucosamine (GA) on aflatoxin production by Aspergillus parasiticus were studied using conidia-initiated and replacement cultures. In conidia-initiated, 2-DOG, -MG, and GA supported varying amounts of growth when employed as sole carbon sources. In both conidia-initiated and replacement cultures, 2-DOG, but not -MG nor GA, as sole carbon sources support toxin formation. None of the compounds inhibited aflatoxin production when used in combination with glucose. It appears that neither 2-DOG, -MG, nor GA can be considered nonmetabolizable analogs of glucose in A. parasiticus.Abbreviations YES yeast extract sucrose - PMS peptone-mineral salts - 2-DOG L-deoxyglucose - -MG -methylglucoside - GA glucosamine     

Structure and properties of aCephalosporium acremonium α-galactosidase

The amino acid and sugar composition of the enzyme protein, the effect of urea, sodium dodecyl sulphate and Concanavalin A on the purified -galactosidase (EC 3.2.1.22) from the moldCephalosporium acremonium has been studied. The results obtained by gas liquid chromatography indicated the presence ofN-acetylglucosamine, mannose, galactose andN-acetylneuramic acid in the molar proportions 27311. The presence of two types of Asn-linked oligosaccharide structures in the enzyme molecule is assumed. The -galactosidase liberates (1–3), (1–4) and (1–6)-linkedd-galactose units from various synthetic and natural substrates which have been tested. The effects of pH, substrate concentration and temperature on the catalytic activity of the enzyme are described. The purified -galactosidase also exhibited a lectin activity with an affinity towards glucose, and to some extent mannose.Abbreviations p-NPG p-nitrophenyl--d-galactopyranoside - 4-MUG 4-methylumbelliferyl--d-galactopyranoside - HU hemagglutinin unit - PBS phosphate buffered saline - SDS sodium dodecyl sulphate - ConA Concanavalin A - WGA wheat germ agglutinin - LCA Lens culinaris agglutinin - PHA phytohemagglutinin fromPhaseolus vulgaris     

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     

Chemoenzymatic galactosialylation with integrated cofactor regeneration

As a precursor for the chemical synthesis of sialylated oligosaccharides, the trisaccharide glycoside Neu5Ac (2-8)Gal(1-4)GlcNAc(1-O)-pent-4-ene was synthesized starting from GlcNAc(1-O)-pent-4-ene, UDP-glucose andN-acetylneuraminic acid in a one pot reaction employing galactosyltransferase and (2-6)sialyl-transferase in a complete cofactor regeneration system.Abbreviations Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5-monophosphosialate - CMP cytidine 5-monophosphate - CDP cytidine 5-diphosphate - CTP cytidine 5-triphosphate - Gal galactose - GlcNAc N-acetylglucosamine - UDP uridine 5-diphosphate - UDP-Glc uridine-5-diphosphoglucose - UDP-Gal uridine-5-diphosphogalactose - PEP phosphoenolpyruvate     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant     

Evidence of regional differences in the lectin histochemistry along the ductus epididymis of the lizard,Podarcis sicula Raf

The regional difference in the carbohydrate components of the ductus epididymis epithelium of a lizard was delineated by means of 13 lectins. Basal cells expressed only N-acetylglucosamine (GlcNAc). Throughout the ductus, the secretory cells showed oligosaccharides with terminal N-acetylneuraminic acid (Neu5Ac)(2,6)galactose (Gal)/N-acetylgalactosamine (GalNAc) and internal mannose (Man) and/or glucose (Glc) in the whole cytoplasm, oligosaccharides terminating in Neu5Ac(2,6)Gal(1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, GalNAc, GlcNAc, and fucose (Fuc) in the supra-nuclear zone, and also glycans terminating in Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal(1,3)GalNAc, Gal (1,4)GlcNAc on the luminal surface. In the caput and corpus regions, the supra-nuclear cytoplasm was characterized by terminal Gal(1,4)GlcNAc and GalNAc, the luminal surface by GalNAc and Gal. The Golgi zone, showing oligosaccharides with terminal Neu5Ac(2,3)Gal (1,4)GlcNAc, Neu5Ac(2,6)Gal (1,3)GalNAc, Neu5Ac(2,6)Gal (1,4)GlcNAc, and internal GlcNAc, expressed terminal Gal (1,4)GlcNAc and GalNAc in the caput, and terminal GalNAc in the corpus. The granules showed all the investigated carbohydrates in their peripheral zone except terminal GalNAc and Fuc, whereas internal Man/Glc and terminal Gal were expressed in the central core, and Fuc throughout the ductus, terminal GlcNAc in the caput and corpus, and terminal GalNAc only in the corpus.     

Glycosylation with thioglycosides activated by dimethyl(methylthio)sulfonium tetrafluoroborate: synthesis of two trisaccharide glycosides corresponding to the blood group A and B determinants

Two trisaccharide glycosides,p-trifluoroacetamidophenylethyl 3-O-(2-acetamido-2-deoxy--d-galactopyranosyl)-2-O-(-l-fucopyranosyl)--d-galactopyranoside andp-trifluoroa-cetamidophenylethyl 2-O-(-l-fucopyranosyl)-3-O-(-d-galactopyranosyl)--d-galactopyranoside, corresponding to the human blood group A and B determinants, were synthesized. A key fucosylgalactosyl disaccharide derivative was glycosylated with galactosaminyl or galactosyl donors, respectively. Dimethyl (thiomethyl)sulfonium tetrafluoroborate was used for thioglycoside activation in coupling reactions.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Plant regeneration and genetic transformation of Lotus angustissimus

Culture conditions have been established for callus induction and growth from different explants in L. angustissimus L. Calli were obtained from hypocotyls, leaves, stems, cotyledons and roots cultured on media containing 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid with kinetin, N⁶ – ² or benzyladenine in different combinations and concentrations. Only those calli induced in presence of -naphthaleneacetic acid with benzyladenine or kinetin produced shoots. Calli induced from hypocotyl explants were the most efficient in regeneration of shoots. Transformation with an Agrobacterium rhizogenes binary vector carrying the plasmid pBI 121.1 is reported. The percentage of cotransformation was estimated by testing GUS activity in hairy roots. The integration of Ri T-DNA and the NPTII gene in transformed plants was confirmed by molecular analyses and in vitro culture of transgenic tissues in the presence of kanamycin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - 1AA indole-3-acetic acid - NAA -naphthaleneacetic acid - 2iP N⁶ – ² - PA proanthocyanidins - NOS nopaline synthase - NI TII neomycin phosphotransferase - GUS -glucuronidase - CaMV cauliflower mosaic virus     

O-Glycosidically linked oligosaccharides from peptidorhamnomannans ofSporothrix schenckii

-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases ofSporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine. Man-(1–2)Man-ol, Rha(1–3)Man(1–2)Man-ol, Rha(1–4)GlcA(1–2)Man(1–2)Man-ol, and Rha(1–4)[Rha(1–2)] GlcA(1–2)Man(1–2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry.Abbreviations FAB fast atom bombardment - GLC gas liquid chromatography - GlcA d-glucopyranosyluronic acid - Man d-mannopyranose - Man-ol d-mannitol - MS mass spectrometry - NMR nuclear magnetic resonance - Rha l-rhamnopyranose