Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Isolation and characterization of free glycans of the oligomannoside type from the extracellular medium of a plant cell suspension

The oligosaccharides Man₅GlcNAc and Man₃(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by¹H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc l-fucose - Man d-mannose - Xyl d-xylose - GlcNAc N-acetyl-d-glucosamine - Gal d-galactose - Glc d-glucose - FAB-MS fast atom bombardment mass spectrometry - NMR nuclear magnetic resonance     

Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin

Levels of basal chitin synthetase in cell-free extracts from Phycomyces blakesleeanus were reduced by breakage of cells in the presence of EDTA or EGTA. Addition of Ca²⁺ to these extracts activated chitin synthetase. Maximal activation was obtained after 2 h at a Ca²⁺ concentration of 2–5 mM. Activation by calcium was not reduced by any protease inhibitor tested but benzamidine, whereas the weak proteolytic activity of the extracts was inhibited by antipain. Larger levels of chitin synthetase activation were obtained by the simultaneous addition of calcium and calmodulin in most, but not all extracts. This further activation by calmodulin was prevented by TFP. ATP or cAMP did not stimulate activation by calcium or calcium-calmodulin.Abbreviations EGTA ethylene glycol-bis(B-aminoethylether)-N,NN-tetraacetic acid - GlcNAc N-acetyl-d-glucosamine - PMSF phenylmethylsulfonyl fluoride - SBTI soybean trypsin inhibitor - TFP trifluoperazine - TLCK N-p-tosyl-l-lysine choromethyl ketone - UDPGlcNAc uridine diphosphate N-acetyl-d-glucosamine     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Determination of the structure of sulfated tetra- and pentasaccharides obtained by alkaline borohydride degradation of hen ovomucin. A fast atom bombardment-mass spectrometric and1H-NMR spectroscopic study

Alkaline borohydride reductive cleavage of hen ovomucin resulted in the release of a series of neutral and acidic oligosaccharide-alditols.¹H-NMR spectroscopy in combination with fast ion bombardment-mass spectrometry in negative ion mode were used for investigation of the structures of three oligosaccharide-alditols. The following structures were established: Abbreviations NeuAc N-acetyl-d-neuraminic acid - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Gal-NAc-ol N-acetyl-d-galactosaminitol - NMR nuclear magnetic resonance - FAB-MS fast atom bombardmentmass spectrometry     

Specificity towards oligomannoside and hybrid type glycans of the endo-β-N-acetylglucosaminidase B from the basidiomy ceteSporotrichum dimorphosporum

We have previously shown that an endo--N-acetylglucosaminidase (EC 3.2.1.96) named Endo B, isolated from culture filtrates of the basidiomyceteSporotrichum dimorphosporum cleaves asialo-, and to some extent, monosialylated bi-antennary glycans of theN-acetyllactosamine type linked to the asparagine residue of peptide or protein moieties [Bouquelet S, Strecker G, Montreuil J, Spik G (1980) Biochimie 62:43–49]. In the present paper, the substrate specificity of the enzyme towards oligomannoside and hybrid type glycans has been analyzed. The results obtained indicate that ovalbumin glycopeptides containing four to seven mannose residues and bovine lactotransferrin glycopeptides containing four to nine mannose residues were completely hydrolyzed by the enzyme. The degree of cleavage was variable among hybrid type structures, since glycopeptides containing the following glycans: (Gal)₁(GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₅(GlcNAc)₂; (GlcNAc)₃(Man)₄(GlcNAc)₂ were not hydrolyzed by the enzyme while the percentage of hydrolysis of a glycopeptide containing (GlcNAc)₂(Man)₅(GlcNAc)₂ glycan reached 90%. The bovine lactotransferrin was partially deglycosylated (40%) in the absence of non-ionic detergent while native ovalbumin glycoprotein was not hydrolyzed by the enzyme.The oligomannoside-and theN-acetyllactosamine-type degrading activities present in the culture filtrates were not separated at any step of the purification procedure. Both activities were eluted as a single component with an apparent molecular mass of 89 kDa suggesting that they are located on the same enzyme molecule.Endo B represents a powerful tool for removing oligomannoside-andN-acetyllactosamine-type glycans fromN-glycopeptides andN-glycoproteins. Moreover, advantages in the use of Endo B in a soluble form as well as in an immobilized form result in its high activity and in its stability to heat denaturation and storage.Abbreviations Gal d-galactose - Man d-mannose - GlcNAc N-acetyl-d-glucosamine - Con A concanavalin A - Asn asparagine - GLC gas liquid chromatography - TLC thin layer chromatography - Endo endo--N-acetylglucosaminidase - Endo B endo--N-acetylglucosaminidase isolated fromSporotrichum dimorphosporum - PBE polybuffer exchanger - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Immunochemical characterization of antisera reactivities toN-acetyl-d-glucosamine oligosaccharides with theβ(1–4)-glycosidic linkage

The (1–4)-linked oligosaccharides ofN-acetyl-d-glucosamine (GlcNAc) isolated from chitin were used to prepare synthetic immunogens and antigens by reductive amination of (GicNAc)_n to bovine serum albumin (BSA). The rabbit antisera produced to the (GlcNAc)_n-BSA conjugates were characterized using an enzyme-linked immunosorbent assay (ELISA) system under conditions that, only the antibodies with carbohydrate specificity were reactive with the solid-phase adsorbed (GlcNAc)_n-BSA antigens. Inhibition assays using the (GlcNAc)_n-BSA, (GlcNAc)_n oligosaccharides, and the reduced oligosaccharides showed a relative specificity of the antisera for the chain length of the (GlcNAc)_n sequences. For example, the anti-(GlcNAc)₅-and anti-(GlcNAc)₄-sera were inhibited best by the longer chain (GlcNAc)_n ologosaccharides with the antibody combining sites directed mainly to the cyclic GlcNAc residues of the (GlcNAc)_n-BSA conjugates. The antibody combining sites were in part directed to the acyclic moiety of the reducing end of the oligosaccharides as shown by the increased inhibitory activities of the reduced (GlcNAc)_n oligosaccharides particularly, with the anti-(GlcNAc)₂-and anti-(GlcNAc)₃-sera. The best hapten inhibitors for the anti-(GlcNAc)₂-BSA and anti-(GlcNAc)₁-BSA sera were theN-butylamine derivatives of (GlcNAc)₂ and (GlcNAc)₁, respectively, indicating that the antibodies were also reactive with the secondary amine formed between the reducing end of the oligosaccharides and the -amino groups of lysine.Abbreviations ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - GicNAc N-acetyl-d-glucosamine - (GlcNAc)_n Oligosaccharides containing GlcNAc in 1–4 linkages - (GlcNAc)₂ DGlcNAc(1–4)-d-GlcNAc - (GlcNAc)₃ (GlcNAc)₄ and (GlcNAc)₅, the homologous oligosaccharides of (GlcNAc)₂ - PBS phosphate buffered saline (0.01 M sodium phosphate, pH73 containing 0.15%M (NaCl) - PBSA PBS containing 1% BSA and 0.1% Tween-20 - ONPG o-nitrophenyl--d-galactopyranoside     

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl--d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.Abbreviations MS minimal salts - GlcNAc N-acetyl--d-glucosamine - pNP-GlcNAc p-nitrophenyl-2-acetamido-2-deoxy--d-glucopyranoside - d.a. degree of N-acetylation - TLC thin-layer chromatography     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004     

Metabolic products of microorganisms

The effect of the nucleoside-peptide antibiotics nikkomycin Z, nikkomycin X, and polyoxin A was tested on chitosomal chitin synthetase from yeast cells of the dimorphic fungus Mucor rouxii. The K _i was 0.6 M for polyoxin A and 0.5 M for nikkomycin X; nikkomycin Z was slightly less inhibitory (K _i=3.5M). Whereas the minimum inhibitory concentrations of the nikkomycins for growth and germination were quite low (about 1M, or lower), polyoxin A displayed no antifungal activity against yeast cells and sporangiospores of the test organism, even when present in high concentrations. These results are discussed with respect to structure/activity relationships.Abbreviations MIC minimum inhibitory concentration (i.e. concentration required to completely suppress growth: cf. Drews, 1979) - GlcNAc N-acetyl-d-glucosamine - UDP-GlcNAc uridine 5-diphospho-N-acetyl-d-glucosamine Metabolic products of microorganisms. 202. H. P. Kaiser and W. Keller-Schierlein: Strukturaufklärung von Elaiophylin: Spektroskopische Untersuchungen und Abbau. Helv. Chim. Acta 64: 407–424 (1981)     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

Production of N-acetyl-β-d-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl--d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)₂ and (GlcNAc)₃, whereas above 50°C the major product was (GlcNAc)₂. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.     

Transfer of Man9GlcNAc tol-fucose by endo-β-N-acetylglucosaminidase fromArthrobacter protophormiae

We have reported that transglycosylation activity of endo--N-acetylglucosaminidase fromArthrobacter protophormiae (endo-A) can be enhanced to near completion using GlcNAc as an acceptor in a medium containing 30% acetone (Fan J-Q, Takegawa K, Iwahara S, Kondo A, Kato I, Abeygunawardana C, Lee YC (1995)J Biol Chem 270: 17723–29). In this paper, we found that the endo-A can also transfer an oligosaccharide, Man₉GlcNAc, tol-Fuc using Man₉GlcNAc₂Asn as donor substrate in a medium containing 35% acetone. The transglycosylation yield was greater than 25% when 0.2m l-Fuc was used as acceptor. The transglycosylation product was purified by high performance liquid chromatography on a graphitized carbon column and the presence ofl-Fuc was confirmed by sugar composition analysis and electrospray mass spectrometry. Sequential exo-glycosidase digestion of pyridyl-2-aminated transglycosylation product, Man₉GlcNAc-l-Fuc-PA, revealed that a -anomeric configuration linkage was formed between GlcNAc andl-Fuc. The GlcNAc was found to be 1,2-linked tol-Fuc by two methods; i) collision-induced decomposition on electrospray mass spectrometry after periodate oxidation, reduction and permethylation of Man₉GlcNAc-l-Fuc; and ii) preparation of Man₉GlcNAc-l-Fuc-PA, its periodate oxidation and reduction, followed by hydrolysis and HPLC analysis. Thus, the structure of the oligosaccharide synthesized by endo-A transglycosylation was determined to be Man₉GlcNAc(1,2)-l-Fuc. Methyl -l-fucopyranoside,l-Gal are also acceptors for the enzymic transglycosylation. However, transglycosylation failed when methyl -l-fucopyranoside,d-Fuc andd-Gal were used. These results indicate that the endo-A requires not only 3-OH and 4-OH to be equatorial but also a⁴C₁-conformation or equivalent conformation of the acceptor to perform transglycosylation.Abbreviations endo-A endo--N-acetylglucosaminidase fromArthrobacter protophormiae - PA pyridyl-2-amino- - AP aminopyridine - GlcNAc N-acetyl-d-glucosamine - Man mannose - Gal galactose - Fuc fucose - Glc glucose - PA-C₂ PA-glycolaldehyde - PA-C₃ PA-l-glyceraldehyde - PA-C₄ PA-d-threose - HPAEC-PAD high performance anion exchange chromatography with pulsed amperometric detector - HPLC high performance liquid chromatography - ODS octadecylsilyl - ES-MS electrospray mass spectrometry - CID collision-induced decomposition     

Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.)

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23000–28000 and isoelectric points 10.3–10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-d-glucosamine oligosaccharide (GlcNAc)₃. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8–100% acetylated chitosans and (GlcNAc)_4–6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)_2–6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (Ab_PLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.Mention of a trademark, warranty, propriety, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.Abbreviations Ab antibody - BCLVC basic chitinase/lysozyme cv. Valencia callus - BCVC basic chitinase cv. Valencia callus - CE capillary electrophoresis - CM-chitin-RBV carboxymethyl-chitin-remazol brilliant violet - GlcNAc N-acetyl-d-glucosamine - HEWL hen egg-white lysozyme - M_r relativemolecular mass - pI isoelectric point - PLC potato leaf chitinase - PR pathogenesis-related - SEC size exclusion chromatography We thank Mr. M. Burkhart, Ms. T.-T. Ho, and Ms. M. Doherty for their valuable technical assistance. A portion of the funding for this work was made available from the Citrus Production Research Marketing Order by the Division of Marketing and Development, Florida Department of Agriculture and Consumer Services, Bob Crawford, Commissioner.     

Coupled bioconversion for preparation of N-acetyl-d-neuraminic acid using immobilized N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic acid lyase

N-Acetyl-d-neuraminic acid (Neu5Ac) can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-d-manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-d-neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-d-glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was ～73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate.     

A full assignment of proton resonances for an α(1-3)-linked fucose residue in keratan sulphate from bovine articular cartilage

Full proton NMR assignments have been achieved for the (1-3)-linked fucose residues contained in alkaline borohydride reduced keratan sulphate chains derived from bovine articular cartilage. This involved 500 MHz spectroscopy at 60°C and included COSY and RELAYED-COSY determinations.Abbreviations KS keratan sulphate - TSP sodium 3-trimethylsilylpropionate - Fuc -l-fucose - Gal -d-galactose - GalNAc-ol N-acetylgalactosaminitol - GlcNAc -N-acetyl-d-glucosamine     

Beauveria bassiana submerged conidia production in a defined medium containing chitin,two hexosamines or glucose

Summary Beauveria bassiana in liquid culture can produce blastospores and occasionally submerged conidia. For use as a bioinsecticide, conidia have definite advantages. Numerous studies have investigated conidia production in liquid cultures using synthetic and industrial grade media supplemented with glucose. We have studied growth, development and sporulation in microcultures using growth media containing chitin monomers. For the production of submerged conidia growth media containing N-acetyl-d-glucosamine (GlcNAc) proved to be better than yeast extract-peptone-glucose (YPG), glucose plus ammonium salts (Glc+NH₄Cl) or N-acetyl-d-galactosamine (GalNAc). Sixty-one percent of the spores in the GlcNAc medium were submerged conidia with the remainder being blastospores. The concentration of submerged conidia reached 8.0 × 10⁵/ ml after two days in GlcNAc medium as compared to 8.9 × 10⁵/ml in YPG medium. Therefore, in terms of percentage of submerged conidia produced, GlcNAc medium generated more submerged conidia in spite of its lower cell yields. Growth in a medium containing chitin, a polymer of GlcNAc, resulted in 86.3% of the spores as submerged conidia exceeding 10⁶/ml after 48 h. Growth under phosphate limitation resulted in an increased percentage of submerged conidia for all media tested. Electron microscopy and spore protein analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that structural and compositional differences exist between the spore types.     

Biosynthesis of a blood group H1 antigen by α1,2-fucosyltransferase in PC12 cells

We have examined the expression of GDP-fucose: glycosphingolipid fucosyltransferase activity in PC12 cells and PC12 sublines in relation to the neuronal differentiation induced by nerve growth factor (NGF) or dexamethasone. Transfer of fucose to paragloboside (nLc₄Cer) yielded a product which was determined to be a blood group H₁ antigen (Fuc1-2Gal1-4GlcNAc1-3Gal1-4Glc-Cer) by gas chromatography/mass spectrometry analysis and enzymatic hydrolysis, suggesting that PC12 cells have an 1,2-fucosyltransferase. Lactosylceramide was also fucosylated at a reduced rate. When the differentiation of PC12 cells and PC12 subline cells, PC12D and MR31, was induced by exposure to either NGF or dexamethasone, the fucosyltransferase activity for nLc₄Cer was found to decrease in both cell lines, suggesting the association with cell differentiation. This is the first report of the presence of an 1,2-fucosyltransferase in cultured neuronal cell lines which catalyses thein vitro biosynthesis from nLc₄Cer of a type-2 chain glycosphingolipid having the blood group H₁ determinant. The disaccharides, -lactose andN-acetyllactosamine, were also fucosylated by PC12 cell enzyme, although the specificity for the carbohydrate structure was different from that for glycosphingolipids.Abbreviations Glc d-glucose - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - Cer ceramide - nLc₄Cer neolactotetraosylceramide (paragloboside) - GDP guanosine diphosphate - CDP cytidine diphosphate - CTP cytidine triphosphate - NGF nerve growth factor - DX dexamethasone - GC/MS gas chromatography/mass spectrometry     

Histochemical study of Hurler's disease by the use of peroxidase-labelled lectins

Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders.     

Isolation and Identification of ( – ) - Quinic Acid as an Unidentified Major Tea-component

A new enzyme, N-acetyl- d-hexosamine dehydrogenase (N-acety 1-α-d-hexosamine: NAD⁺ 1-oxidoreductase), was purified to homogeneity on polyacrylamide gel electrophoresis from a strain of Pseudomonas sp. about 900-fold with a yield of 12 %. The molecular weight of the enzyme was about 124,000 on gel filtration and 30,000 on SD S-polyacrylamide gel electrophoresis, respectively. Its isoelectric point was 4.7. The optimum pH was about 10.0. The enzyme was most stable between pH 8.0 and pH 10.5. The highest enzyme activity was observed with N-acetyl-d-glucosamine (Km = 5.3mm) and N-acetyl-d-galactosamine (Km = 0.8mm) as the sugar substrate. But it was not so active on N-acetyl-d-mannosamine. NAD⁺ was used specifically as the hydrogen acceptor. The anomeric requirement of the enzyme for N-acetyl-d-glucosamine was the α-pyranose form, and the reaction product was N-acetyl-d-glucosaminic acid. The enzyme activity was inhibited by Hg and SDS, but many divalent cations, metal-chelating reagents, and sulfhydryl reagents had no effect.