Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Conformational analysis of the Sda determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations

The conformational behaviour of the spacer-linked synthetic Sd^a tetrasaccharide -d-GalpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (1) and the two mimics -d-Galp-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (2) and -d-GlcpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (3) were investigated by ¹H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D ¹H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1–3 are rather rigid, especially the -d-Hex(NAc)-(14)-[-Neu5Ac-(23)]--d-Galp fragments. The - Neu5-Ac-(23)--d-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1–3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

A pyrophosphate bridge links the pyruvate-containing secondary cell wall polymer of Paenibacillus alvei CCM 2051 to muramic acid

The peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed:[(Pyr4,6)--D-Manp NAc-(14)--D-Glcp NAc-(13)]_ñ11-(Pyr4,6)--D-Manp NAc-(14)--D-Glcp NAc-(1O)-PO₂-O-PO₂-(O6)-MurNAc-Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. ³¹P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex.In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Heteronuclear NMR studies of 13C-labeled yeast cell wall β-glucan oligosaccharides

Summary The structures of uniformly ¹³C-labeled -glucan octa- and undeca-oligosaccharides enzymatically prepared by the yeast cell wall glucanosyl transferase of Candida albicans were characterized by using a combination of HCCH-COSY, HCCH-TOCSY, and HMBC experiments. The oligosaccharide structures indicate that the cell wall glucanosyl transferase cleaves two glucosyl units from the reducing end of the initial linear (13) penta-oligosaccharide and subsequently transfers the remainder to another oligosaccharide at the nonreducing end via a (16) linkage. These results indicate that the combined action of cell wall glucanase and glucanosyl transferase activities could not only introduce intrachain (16) linkages within a single glucan strand, but also result in cross-linking of two initially separate glucan strands with concurrent introduction of intrachain (16) linkages. Since isolated fungal membranes only synthesize linear (13) glucan strands, wall-associated enzymes probably participate in the assembly of the final wall glucan structure during cell growth and division.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

On the action of hydroxylamine,hydrazine and their derivatives on the water-oxidizing complex

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S₀ S₁, S₁ S₂, S₂ S₃, S₃ (S₄) S₀. State S₁ is most stable in the dark. Oxygen is released during S₃ (S₄) S₀. Hydroxylamine and hydrazine interact with S₁. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S₁ S₂ : S₂ S₃ : S₃ (S₄) S₀ : S₀ S₁ :... in the absence of the agents, to S₁ ^* S₀ : S₀ S₁ : S₁ S₂ : S₂ S₃ :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H(S₃ S₀) : 1H⁺(S₀ S₁) :... to 2H⁺(S₁ ^* S₀) : 1H⁺(S₀ S₁) : OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H⁺(S₃ S₀) :... The 2H⁺ were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53–57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n3 molecules of hydroxylamine and of n2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl^- or OH^-, which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S₁.Abbreviations DNP-INT dinitrophenylether of iodonitrothymol - FWHM full width at half maximum - NR neutral red (3-amino-7-dimethylamino-2-methylphenazine-HCI) - PS II photosystem II - WOC or (in formulas:) W water-oxidizing complex Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Hemoglobin vancouver [α2β2 73(E17) Asp→ Tyr]: Its structure and function

Summary Hemoglobin Vancouver is a new abnormal hemoglobin with an amino acid substitution of the normal aspartyl residue 73 of the chain by a tyrosyl residue. It was discovered in a man of Chinese descent in association with thalassemia. It was subsequently detected in a sister in association with normal Hb A. The oxygen affinity of the abnormal hemoglobin is decreased but its subunit interaction is normal. The Bohr effect may be slightly increased.This is the fourth abnormal hemoglobin to be found with a substitution at73. The others are Hb C-Harlem ( _{2 2} 6GluVal and 73 AspAsn), Hb Korle-Bu ( _{2 2} 73 AspAsn), and Hb Mobile ( _{2 2} 73 AspVal). Although Hb Mobile was found in the present studies to have a decreased affinity for oxygen, Hbs C-Harlem and Korle-Bu have been reported to be normal. These observations of functional differences for variants of73 added to earlier observations of the role of the normal73 residue to the aggregation of sickle deoxyhemoglobin indicate that this position of the molecule may be important in intra as well as intermolecular interactions.     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Structural study of fucoidan from Cladosiphon okamuranus tokida

A structural study was carried out on a fucoidan isolated from the brown seaweed Cladosiphon okamuranus. The polysaccharide contained fucose, glucuronic acid and sulfate in a molar ratio of about 6.1 : 1.0 : 2.9. The results of Smith degradation showed that this polysaccharide has a linear backbone of 13-linked -fucopyranose with a half sulfate substitution at the 4-positions, and a portion of the fucose residues was O-acetylated. The data obtained from partial acid hydrolysis, a methylation analysis and NMR spectra indicated that the -glucuronic acid residue is linked to the 2-positions of the fucose residues, which were not substituted by a sulfate group. These results indicated that the average structure of this fucoidan is as follows: -[(3Fuc-4(±OSO₃-)1–)₅3[GlcA12]Fuc1–]_n–. (Half of each fucose residue was sulfated. One O-acetyl ester was present in every 6 fucose residues.)     

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo--galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fuc12Gal14(±SO–36)GlcNAc (type 2H), Gal14(Fuc13)(±SO–36)GlcNAc(Lex) and Fuc13 or 4(±SO–36)GlcNAc, are expressed in the repeated outer chain moieties.     

Carbohydrate Structural Units in Glycosphingolipids as Receptors for Gal and GalNAc Reactive Lectins

Glycosphingolipids (GSLs) contain many carbohydrate epitopes or crypto-glycotopes for Gal and GalNAc reactive lectins. Many of them are in the nervous system and function as important receptors in various life processes. During the past two decades, 11 mammalian structural units have been used to express the binding domain of applied lectins. They are: F, GalNAc1 3GalNAc; A, GalNAc1 3Gal; T, Gal1 3GalNAc; I, Gal1 3GlcNAc; II, Gal1 4GlcNAc; B, Gal1 3Gal; E, Gal1 4Gal; L, Gal1 4Glc; P, GalNAc1 3Gal; S, GalNAc1 4Gal, and Tn, GalNAc1 Ser(Thr). Although 10 of them occur in GSLs, only 3 (L , S , and T ) are found in human brain, and 2 (L and II ) are present in the inner structures of human blood group active GSLs. In the families of gangliosides, L and II represent 55% of the total structural units, while the other three units (T , P , and S ) constitute the rest. To facilitate the selection of lectins that could serve as structural probes, the carbohydrate binding specificities of Gal/GalNAc reactive lectins have been classified according to their highest affinity for the structural units and their binding properties expressed by decreasing order of reactivity. Hence, the binding relation between GSLs and Gal/GalNAc specific lectins can be established.     

The catalysis of nucleotide polymerization by compounds of divalent lead

Summary Soluble lead salts and a number of lead-containing minerals catalyze the formation of oligonucleotides from nucleoside 5-phosphorimidazolides. The effectiveness of lead compounds correlates strongly with their solubility. Under optimal conditions we were able to obtain 18% of pentamer and higher oligomers from ImpA. Reactions involving ImpU gave smaller yields.Abbreviations A adenosine - U uridine - Im imidazole - MeIm 1-methyl-imidazole - EDTA ethylenediaminetetraacetic acid - pA adenosine 5-phosphate - pU uridine 5-phosphate - Ap adenosine cyclic 2:3-phosphate - ATP adenosine 5-triphosphate - AppA P₁,P₂-diadenosine 5-diphosphate - pNp (N = A,U) nucleotide 2(3), 5-diphosphate - ImpA adenosine 5-phosphoreimidazolide - ImpU uridine 5-phosphorimidazolide - A ²pA adenylyl-[25]-adenosine - A ³pA adenylyl-[35]-adenosine - pA ²pA 5-phospho-adenylyl-[25]-adenosine - pA ³pA 5-phospho-adenylyl-[35]-adenosine - pUpU 5-phospho-uridylyl-uridine - pApU 5-phospho-adenylyl-uridine - pUpA 5-phospho-uridylyladenine - (pA)n (n, 2,3,4,) oligoadenylates with 5 terminal phosphate - ImpApA 5-phosphorimidazolide of adenylyl adenosine - (pA) ₅₊ pentamer and higher oligoadenylates with 5 terminal phosphate - (Ap)nA (n = 2,3,4) oligoadenylates without terminal phosphates In the following we do not specify the nature of the internucleotide linkageIn the following we do not specify the nature of the internucleotide linkage     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.