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     

Structures of polysaccharides and oligosaccharides of some Gram-negative marine Proteobacteria

The chemical structures of polysaccharides and LPS core oligosaccharides, isolated from various Gram-negative marine bacteria from the genera Pseudoalteromonas and Shewanella belonging to the Alteromonadaceae family and gamma-subclass of Proteobacteria, are reviewed. The polysaccharides are distinguished by the acidic character (e.g., due to the presence of hexuronic and aldulosonic acids and their derivatives) and the occurrence of unusual sugars, including N-acyl derivatives of 6-deoxyamino sugars, such as N-acetyl-D-quinovosamine, N-acetyl-L-fucosamine and N-acetyl-6-deoxy-L-talosamine, and higher sugars like 2,6-dideoxy-2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-D-galactopyranose (shewanellose). Many constituent sugars have various uncommon non-sugar substituents, such as alanine, formic, lactic and hydroxybutyric acids, sulfate, phosphate, and 2-aminopropane-1,3-diol.     

Endogenous ammonia production by Anacystis nidulans R-2 induced by methionine sulfoximine

Anacystis nidulans R-2 produced ammonia from endogenous sources for at least 6 h when illuminated without external nitrogen source but with CO₂ in the presence of 50 M methionine sulfoximine. The onset of ammonia release coinciding with complete inhibition of glutamine synthetase. The total quantity of ammonia which could be released exceeded the nitrogen content of small molecule pools, and suggested protein degradation as the most likely source of the nitrogen. Ammonia release was not accompanied by leakage of carbon compounds from the cells. Methionine sulfoximine-induced ammonia release was energy requiring, and was barely detectable under dark anaerobic conditions, or in the presence of 10 M carbonyl cyanide m-chlorophenylhydrazone in light. Phenyl methyl sulfonylfluoride, an inhibitor of serine proteases, eliminated ammonia release, and the rate of release was reduced to one-third of control values, after a lag, in the presence of 50–75 g/ml chloramphenicol. The rate of NH ₊ ⁴ release was maximal (1.4 nmol·min^-1·mg^-1 protein) if suspensions were bubbled with 100% O₂, but could not be reduced below 0.6 nmol·min^-1·mg^-1 protein in air: CO₂, suggesting that release was at most only partly due to photorespiration.Abbreviations used MSX L-methionine D,L-sulfoximine - PMSF phenylmethylsulfonylfluoride - CAP chloramphenicol - CCCP carbonyl cyanide-m-chlorophenyl hydrazone     

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.     

Pregnancy-associated changes in oligomannose oligosaccharides of human and bovine uromodulin (Tamm-Horsfall glycoprotein)

The urinary glycoprotein uromodulin (Tamm-Horsfall glycoprotein) exhibits a pregnancy-associated ability to inhibit antigen-specific T cell proliferation, and the activity is associated with a carbohydrate moiety [Muchmore and Decker (1985) Science 229:479–81; Hessionet al., (1987) Science 237:1479–84; Muchmore, Shifrin and Decker (1987) J Immunol 138:2547–53]. We report here that the Man₆₍₇₎GlcNAc₂-R glycopeptides derived from uromodulin inhibit antigen-specific T cell proliferation by 50% at 0.2–2 M, and further studies, reported elsewhere, confirm that oligomannose glycopeptides from other sources are also inhibitory, with Man₉GlcNAc₂-R the most inhibitory of those tested [Muchmoreet al., J Leukocyte Biol (in press)]. In this work, we have extended the observation of pregnancy-associated inhibitory activity to a second species, and have compared the oligomannose profile of Tamm-Horsfall glycoprotein (nonpregnant) with that of uromodulin (pregnant) derived from both human and bovine sources. Surprisingly, there was a pregnancy-associated decrease in the total content of oligomannose chains due predominantly to a reduction in Man₅GlcNAc₂-R and Man₆GlcNAc₂-R. Man₇GlcNAc₂-R, which did not decrease with pregnancy, comprised a significantly greater proportion of the total oligomannose chains in pregnant vs. nonpregnant samples from both species (human; 34.6% vs. 25.9%: bovine; 14.4% vs. 7.2%).     

Utilization of nitrogen compounds and ammonia assimilation by chromatiaceae

Chromatium vinosum strain D, Thiocapsa roseopersicina strain 6311 and Ectothiorhodospira mobilis strain 8112 were grown anaerobically in the light with various single nitrogen sources. When substituted for NH₄Cl only glutamine and casamino acids supported good growth of all strains tested. Peptone and urea were utilized by C. vinosum and T. roseopersicina, glutamate, asparagine and nitrate only by C. vinosum. The strains were able to grow with molecular nitrogen; complete inhibition of this growth was observed in the presence of alanine with E. mobilis, and of alanine or asparagine with T. roseopersicina.Glutamate dehydrogenase, requiring either NADH or NADPH, NADH-linked glutamate synthase, and glutamine synthetase were demonstrate in the above organisms grown on NH₄Cl.     

Discriminative behavioral responses by aphids to various plant matrix polysaccharides

Three species of aphids, Schizaphis graminum (Rondani) (the greenbug), Acyrthosiphon pisum (Harris) (the pea aphid) and Myzus persicae (Sulzer) (the green peach aphid), showed significantly different behavioral responses to various plant mono- or polysaccharides incorporated within thin (<200 m) films of agar overlaying artificial diets. Thirteen polysaccharides (i.e.: polygalacturonic acid, low methoxy pectin, high methoxy pectin, arabinogalactan, xylan, galactan, cellulose, starch, amylose, laminarin, dextran, 2,3-diacetyl pectin and trifluoroacetyl pectin) and 5 monosaccharides (arabinose, xylose, galactose, glucose and galacturonic acid) were tested in paired choices against a control overlay containing only agar. Glucose, dextran and laminarin were the only compounds that all three species of aphids responded to similarly (viz., glucose stimulated, dextran had no effect and laminarin deterred ingestion of underlying diets). None of the aphids exhibited negative responses to the monosaccharides tested. These monosaccharides either had no significant effect or were stimulatory. The polysaccharides tested induced a variation of unaltered, stimulatory or inhibitory behavioral responses in the 3 species of aphids. The behavioral responses of both greenbugs and pea aphids, which are oligophagous, were significantly affected by 6 out of 7 plant matrix polysaccharides tested. Feeding behavior by green peach aphids was affected by only one of these polysaccharides.Electronic monitoring of aphid probing showed that aphids salivated in and test probed the overlays prior to inserting their stylets into and ingesting from the underlying diets. Recorded responses of aphids probing overlays containing inhibitory polysaccharides resembled the recorded behavioral responses of aphids on resistant or nonhost plants (i.e., numerous short probes with either a lack of or a prolonged period prior to ingestion). The differential behavioral responses of the aphids to the various polysaccharides supports the view that plant matrix polysaccharides play a role in the chemical basis of aphid — plant interactions.

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Zusammenfassung Drei Blattlausarten, Schizaphis graminum, Acyrthosiphon pisum and Myzus persicae, zeigten deutliche Unterschiede in ihrem Probeverhalten auf künstlichen Nährmedien, die mit dünnen (<200 m), verschiedene Mono- und Polysaccharide enthaltenden Agarschichten bedeckt waren.Die in den Agarschichten enthaltenen Monosaccharide bewirkten bei allen drei Arten eine deutliche Steigerung des Probeverhaltens. Im Gegensatz dazu war der Einfluss der Polysaccharide von Art zu Art verschieden. Sechs von sieben getesteten pflanzlichen Matrixpolysacchariden beeinflussten deutlich das Probeverhalten von Schizaphis graminum und Acyrthosiphon pisum. Im Gegensatz dazu wurde das Probeverhalten von Myzus persicae nur von einem der untersuchten Polysaccharide beeinflusst. Die Zahl der Wirtspflanzen für Schizaphis graminum und Acyrthosiphon pisum ist relativ klein im Vergleich zu der von Myzus persicae. Dies ist möglicherweise darauf zurückzuführen, daß die Fähigkeit, chemosensorisch zwischen pflanzlichen Matrixpolysacchariden zu unterscheiden, bei Myzus persicae weniger stark ausgeprägt ist.Elektronische Registrierung des Probeverhaltens zeigte, daß Schizaphis graminum vor der Aufnahme der künstlichen Nährmedien zunächst die darüber liegenden Agarschichten testete. Dies geschah durch Einstechen des Stiletts in die Agarschicht, Absonderung von Speichel, Aufsaugen der verflüssigten Matrix und auschliessendes Wiederausstoßen.Das Probeverhalten von Schizaphis graminum auf Trifluoroacetylpektin, welches eine Aufnahme der darunter liegenden Nährstoffe verhinderte, war vergleichbar zu dem Probeverhalten von Schizaphis Graminum auf resistenten oder Nichtwirtspflanzen. Andererseits glich das Probeverhalten von Schizaphis graminum stark methyliertem Pektin, welche die Nährstoffaufnahme steigerte, dem auf natürlichen Wirtspflanzen. Die Ergebnisse dieser Untersuchung zeigen, daß pflanzliche Matrixpolysaccharide für die Wechselwirkung zwischen Blattläusen und Pflanzen keine chemisch inerten Komponenten darstellen. Der unterschiedliche Einfluss der pflanzlichen Matrixpolysaccharide auf die verschiedenen Blattlausarten zeigt, daß diese pflanzlichen Biopolymere eine entscheidene Rolle bei der Erkennung von Wirtspflanzen durch Blattläuse spielen könnten.Darüberhinaus zeigt das extrem unterschiedliche Probeverhalten von Schizaphis graminum auf Trifluoroacetyl- bzw. stark methyliertem Pektin, daß selbst geringe Änderungen in den chemischen Eigenschaften dieser Komponenten das Probeverhalten von Blattläusen deutlich beeinflussen können.

     

Derivatization procedures for reducing oligosaccharides,part 2: Chemical transformation of 1-deoxy-1-(4-trifluoroacetamidophenyl)aminoalditols

Three chemical transformations of oligosaccharide 1-deoxy-1-(4-trifluoroacetamidophenyl)aminoalditols are described. 1) Oxidation with hydrogen peroxide to give the corresponding reducing oligosaccharides. 2) Oxidation with cerium ammonium nitrate to give the corresponding 1-amino-1-deoxyalditols. 3) Treatment with acetic anhydride to give the correspondingN-acetylated derivatives, which are more stable towards oxidation.     

Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase

Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B. licheniformis SK-1 chitinase.     

Increased calcium in carrots by expression of an Arabidopsis H+/Ca2+ transporter

We demonstrate that carrots expressing the Arabidopsis H⁺/Ca²⁺ transporter CAX1 (Cation Exchanger 1) contained up to 50% more calcium (Ca) than plants transformed with control vectors. The CAX1-expressing carrots were fertile, and robust plant growth was seen in the majority of the transgenic plants. CAX1-expressing carrots were crossed to a commercial carrot variety to confirm that the increased Ca accumulation was mediated by CAX1-expression, and the increased Ca content was clearly correlated with the transgene. This study suggests that modulation of ion transporters could be an important means of increasing the Ca content of agriculturally important crops. To our knowledge, this study represents the first attempts to use biotechnology to increase the Ca content of an agriculturally important crop.     

Effect of galactoglucomannan-derived oligosaccharides on elongation growth of pea and spruce stem segments stimulated by auxin

Galactoglucomannan-derived oligosaccharides (GGMOs) (degree of polymerization 4–8) isolated from the wood of poplar (Populus monilifera Ait.) were shown to be inhibitors of the 2,4-dichlorophenoxyacetic acid-stimulated elongation growth of pea (Pisum sativum L. cv. Tyrkys) and spruce [Picea abies (L.) Karst] stem segments. A dependence on the concentration of GGMOs (between 10^-5-10^-10M) as well as plant species was ascertained. Pea stem segments were much more sensitive (10^-10M) than spruce (10^-8M). The GGMOs did not exhibit toxicity even at high concentrations and during long-term bioassays. The timing of the action of GGMOs and auxin in the growth process was also studied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - d.p degree of polymerization - GGMOs galactoglucomannan-derived oligosaccharides This research was supported by the Slovak Grant Agency for Science.     

灵芝液态发酵胞内外多糖结构特征及其活性研究进展

灵芝是一味传统的中药材,具有很高的药用价值,多糖是其主要的活性成分之一,可从其子实体、孢子粉、发酵菌丝体和胞外液中获得。近年来,从灵芝菌丝体和胞外液中发现的多糖越来越得到研究者们的关注。本文从发酵培养基成分及发酵条件对灵芝胞内外多糖的影响、灵芝胞内外多糖的结构、灵芝胞内外多糖生物活性3个方面进行综述,为发酵来源灵芝多糖的开发利用提供科学理论依据。     

Formate dependent nitrate and nitrite reduction to ammonia by Citrobacter freundii and competition with denitrifying bacteria

Citrobacter freundii, Paracoccus denitrificans and Pseudomonas stutzeri were grown either singly or in mixed culture in anaerobic nitrate or nitrite limited chemostats with formate and/or succinate as electron donors and carbon sources. C. freundii reduced nitrate or nitrite stoichiometrically to ammonia. Maximum molar growth yields for nitrate (nitrite) were 15.3 (9.9) g/mol for C. freundii on formate with succinate as carbon source, 15.3 (9.5) g/mol for Ps. stutzeri on succinate and 32.3 (20.4) g/mol for Pa. denitrificans on succinate. The almost identical growth yields indicate that the ATP output of the anaerobic processes in the nitrate (nitrite) ammonifying organism and Ps. stutzeri are nearly the same. In mixed cultures with either Ps. stutzeri or Pa. denitrificans, C. freundii was the best competitor for nitrate. These results show that in anaerobic environments C. freundii may compete successfully with denitrifying organisms.     

Heterogeneity in oligosaccharides from theO-polysaccharide chain of the lipopolysaccharide fromSalmonella typhi 253Ty determined by fast atom bombardment mass spectrometry

Oligosaccharides from theO-polysaccharide chain of theSalmonella typhi 253Ty lipopolysaccharide (LPS) were prepared from delipidated LPS by digestion with bacteriophage P22. The oligosaccharides were separated by gel chromatography into fractions representing monomers to polymers of the basic tetrasaccharide repeating unit. Fractions containing the dimer and the trimer were analysed by fast atom bombardment-mass spectrometry as methylated alditols. The mass spectra clearly showed heterogeneity in terms ofd-glucose substitution of thed-galactose residue in the tetrasaccharide repeating unit: dimers with none, one, or twod-glucosyl branches and trimers with none, one, two, or threed-glucosyl branches were found. This suggests a randomd-glucosylation of theO-polysaccharide chain ofS. typhi 253Ty.     

Assignment of the1H- and13C-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series

The assignment of the 13C- and 1H-NMR spectra of eight oligosaccharides of the lacto-N-tetraose and neotetraose series was obtained from homonuclear and heteronuclear correlation spectroscopy. These analyses were performed on the following compounds: 1. Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 2. NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc; 3. Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 4. NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 5. NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc; 6. Fuc alpha 1-2Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc; 7. Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc; 8. NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc.     

Glycosylation of dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside as saccharide primers in cells

Syntheses of oligosaccharides expressed on cells are indispensable for the improvement of the functional analyses of the oligosaccharides and their applications. We are developing saccharide primers for synthesizing oligosaccharides using living cells. In this study, dodecyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (GlcNAc-C12) and dodecyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside (LacNAc-C12) were examined for their abilities to prime the syntheses of neolacto-series oligosaccharides in HL60 cells. When GlcNAc-C12 was incubated with HL60 cells in serum-free medium for 2 days, 14 kinds of glycosylated products were collected from the culture medium. They were separated by high-performance liquid chromatography. The sequences of the products were determined to be neolacto-series oligosaccharides including Lewis(X), sialyl Lewis(X), polylactosamine, and sialylpolylactosamine by mass spectrometry. GlcNAc-C12 was also glycosylated by B16 cells and gave sialyllactosamine. Furthermore, LacNAc-C12 gave similar glycosylated products to GlcNAc-C12.     

Contribution of L-type Ca2+ channels to early afterdepolarizations induced by I Kr and I Ks channel suppression in guinea pig ventricular myocytes

Early afterdepolarizations (EADs) induced by suppression of cardiac delayed rectifier I (Kr) and/or I (Ks) channels cause fatal ventricular tachyarrhythmias. In guinea pig ventricular myocytes, partial block of one of the channels with complete block of the other reproducibly induced EADs. Complete block of both I (Kr) and I (Ks) channels depolarized the take-off potential and reduced the amplitude of EADs, which in some cases were not clearly separated from the preceding action potentials. A selective L-type Ca(2+) (I (Ca,L)) channel blocker, nifedipine, effectively suppressed EADs at submicromolar concentrations. As examined with the action potential-clamp method, I (Ca,L) channels mediated inward currents with a spike and dome shape during action potentials. I (Ca,L) currents decayed mainly due to inactivation in phase 2 and deactivation in phase 3 repolarization. When EADs were induced by complete block of I (Kr) channels with partial block of I (Ks) channels, repolarization of the action potential prior to EAD take-off failed to increase I (K1) currents and thus failed to completely deactivate I (Ca,L) channels, which reactivated and mediated inward currents during EADs. When both I (Kr) and I (Ks) channels were completely blocked, I (Ca,L) channels were not deactivated and mediated sustained inward currents until the end of EADs. Under this condition, the recovery and reactivation of I (Ca,L) channels were absent before EADs. Therefore, an essential mechanism underlying EADs caused by suppression of the delayed rectifiers is the failure to completely deactivate I (Ca,L) channels.     

Side products of glycosidation with selected 2-acetamido-2-deoxy-D-glucopyranosides

Allyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-formyl-alpha-D-glucopyranoside, N-acetyl-2,3,4-tri-O-acetyl-L-fucopyranosylamine and products of O-acetyl group migration were found as side products during glycosidation of selected 2-acetamido-2-deoxy-D-glucopyranosides.     

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.