Preparative use of the analytical column of a sugar autoanalyzer for resolution of gluco-oligosaccharides of the same molecular weight.

A sugar autoanalyzer was used on a preparative scale to resolve a gluco-oligosaccharide mixture. In this way the components of the following mixtures were resolved: O-alpha-D-glucopyranosyl-(1-3)-O-[alpha-D-glucopyranosyl-(1-6)]-D-glucose (1), O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-3)-D-glucose (2) and O-alpha-D-glucopyranosyl-(1-3)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (3), O-alpha-D-glucopyranosyl-(1-3)-O-alpha-D-glucopyranosyl-(1-4)-D-glucose (4) and O-alpha-D-glucopyranosyl-(1-4)-O-alpha-D-glucopyranosyl-(1-3)-D-glucose (5), and O-alpha-D-glucopyranosyl-(1-2)-O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (6) and O-alpha-D-glucopyranosyl-(1-3)--O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-O-alpha-D-glucopyranosyl-(1-6)-D-glucose (7).     

Chemical characterization of milk oligosaccharides of the common brushtail possum (Trichosurus vulpecula)

Structural characterizations of marsupial milk oligosaccharides have been performed in only three species: the tammar wallaby, the red kangaroo and the koala. To clarify the homology and heterogeneity of milk oligosaccharides among marsupials, 21 oligosaccharides of the milk carbohydrate fraction of the common brushtail possum were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of mid-lactation milk and characterized by ¹H-nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The structures of the 7 neutral oligosaccharides were Gal(β1-3)Gal(β1-4)Glc (3’-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3”, 3’-digalactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I), Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl lacto-N-novopentaose I), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-3)Gal(β1-4)Glc (galactosyl lacto-N-novopentaose II). The structures of the 14 acidic oligosaccharides detected were Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3’-galactosyllactose), Gal(β1-3)(O-3-sulfate)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate a) Gal(β1-3)[Gal(β1-4)(O-3-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I sulfate b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)(?3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulfate)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(?3-O-sulphate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)(?3-O-sulphate)GlcNAc(β1-6)]Gal(β1-4)Glc and Gal(β1-3)Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (galactosyl sialyl lacto-N-novopentaose b). No fucosyl oligosaccharides were detected. Galactosyl lacto-N-novopentaose II, lacto-N-novopentaose I sulfate a, lacto-N-novopentaose I sulfate b and galactosyl sialyl lacto-N-novopentaose b are novel oligosaccharides. The results are compared with those of previous studies on marsupial milk oligosaccharides.     

Vaccination of lactating dairy cows for the prevention of aflatoxin B1 carry over in milk

The potential of anaflatoxin B(1) (AnAFB(1)) conjugated to keyhole limpet hemocyanin (KLH) as a vaccine (AnAFB(1)-KLH) in controlling the carry over of the aflatoxin B(1) (AFB(1)) metabolite aflatoxin M(1) (AFM(1)) in cow milk is reported. AFB(1) is the most carcinogenic compound in food and foodstuffs amongst aflatoxins (AFs). AnAFB(1) is AFB(1) chemically modified as AFB(1)-1(O-carboxymethyl) oxime. In comparison to AFB(1), AnAFB(1) has proven to be non-toxic in vitro to human hepatocarcinoma cells and non mutagenic to Salmonella typhimurium strains. AnAFB(1)-KLH was used for immunization of cows proving to induce a long lasting titer of anti-AFB(1) IgG antibodies (Abs) which were cross reactive with AFB(1), AFG(1), and AFG(2). The elicited anti-AFB(1) Abs were able to hinder the secretion of AFM(1) into the milk of cows continuously fed with AFB(1). Vaccination of lactating animals with conjugated AnAFB(1) may represent a solution to the public hazard constituted by milk and cheese contaminated with AFs.     

Light-harvesting function of carotenoids in photo-synthesis: the roles of the newly found 1(1)Bu- state

This minireview article highlights the energetics and the dynamics of the 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids discovered very recently. Those "hidden" covalent states have been revealed by measurements of resonance-Raman excitation profiles of crystalline carotenoids. The dependence of the energies of the low-lying singlet states, including the 1(1)B(u)(+), 3(1)A(g)(-), 1(1)B(u)(-), and 2(1)A(g)(-) states, on the number of conjugated double bonds (n) is in agreement with the extrapolation of those state energies calculated by Tavan and Schulten for shorter polyenes (P. Tavan and K. Schulten, Journal of Chemical Physics, 1986, vol. 85, pp. 6602-6609). It has also been shown that the internal-conversion processes among those singlet states take place in accord with the state ordering, i.e., 1(1)B(u)(+) --> 1(1)B(u)(-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) (the ground state) for carotenoids having n = 9 and 10, whereas 1(1)B(u)(+) --> 3(1)A(g)(-) --> 1(1)B(u) (-) --> 2(1)A(g)(-) --> 1(1)A(g)(-) for carotenoids having n = 11-13. Radiative transitions of 1(1)B(u)(+) --> 2(1)A(g)(-) and 1(1)B(u)(-) --> 2(1)A(g)(-) as well as a branching into the triplet manifold of 1(1)B(u)(-) --> 1(3)A(g) --> 1(3)B(u) have also been found. Those low-lying singlet states of all-trans carotenoids can facilitate multiple channels of singlet-energy transfer to bacteriochlorophyll in the LH2 antenna complexes of purple photosynthetic bacteria. Thus, the newly found 1(1)B(u)(-) and 3(1)A(g)(-) states of carotenoids need to be incorporated into the picture of carotenoid-to-bacteriochlorophyll singlet-energy transfer.     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by ¹H-NMR as follows: the saccharides from one animal: Gal(α1-3)Gal(β1-4)Glc (α3′-galactosyllactose), Fuc(α1-2)Gal(β1-4)Glc (2′-fucosyllactose), Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (B-tetrasaccharide), GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)Glc (A-tetrasaccharide), Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)[Fuc(α1-2)]Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)Gal(β1-4)GlcNAc(β1-3)[Gal(α1-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc; the saccharides from another animal: α3′-galactosyllactose, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]Glc, A-tetrasaccharide, GalNAc(α1-3)[Fuc(α1-2)]Gal(β1-4)[Fuc(α1-3)]Glc (A-pentasaccharide), Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc, Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)[Fuc(α1-3)]Glc (difucosylheptasaccharide) and Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3){Gal(α1-3)Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had α-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.     

Immunochemistry of I/i-active oligo- and polyglycosylceramides from rabbit erythrocyte membranes. Determination of branching patterns of a ceramide pentadecasaccharide by 1H nuclear magnetic resonance

1H NMR spectra of the ceramide hexasaccharide obtained after the removal of the terminal alpha-Gal and subterminal beta-Gal residues from the ceramide decasaccharide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (beta 1-6)]Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, showed that terminal and internal GlcNAc residues are differentiated by their chemical shifts. This finding enabled us to determine the primary structure of the title compound as Gal(alpha 1-3)Gal(beta 1-4)GlcNAc (beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-6)]Gal(beta 1-4)GlcNAc (beta 1-3)[Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-6)]Gal(beta 1-4)GlcNAc (beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer. Alternative branching of this oligosaccharide chain was excluded since the removal of all terminal alpha-Gal and penultimate beta-Gal residues yielded a ceramide nonasaccharide containing one terminal and two internal 1----3-linked GlcNAc residues, as well as two terminal 1----6-linked GlcNAc units. The intermediate degradation products of the ceramide deca- and pentadecasaccharides , viz. the ceramide octa- and dodecasaccharide , obtained by the removal of alpha-Gal residues only, as well as the linear ceramide heptasaccharide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3) Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, and ceramide hexasaccharide, Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)GlcNAc (beta 1-3)Gal(beta 1-4)Glc(beta 1-1)Cer, were also investigated. The usefulness of the glycosylation-induced chemical shifts is discussed.     

Chemical characterization of milk oligosaccharides of the polar bear, Ursus maritimus

Two trisaccharides, three tetrasaccharides, two pentasaccharides, one hexasaccharide, one heptasaccharide, one octasaccharide and one decasaccharide were isolated from polar bear milk samples by chloroform/methanol extraction, gel filtration, ion exchange chromatography and preparative thin-layer chromatography. The oligosaccharides were characterized by 1H-NMR as follows: the saccharides from one animal: Gal(alpha1-3)Gal(beta1-4)Glc (alpha3'-galactosyllactose), Fuc(alpha1-2)Gal(beta1-4)Glc (2'-fucosyllactose), Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (B-tetrasaccharide), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc (A-tetrasaccharide), Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc, Gal(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)GlcNAc(beta1-3)[Gal(alpha1-3)Gal(beta1-4)Glc NAc(beta1-6)]Gal(beta1-4)Glc; the saccharides from another animal: alpha3'-galactosyllactose, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, A-tetrasaccharide, GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)[Fuc(alpha1-3)]Glc (A-pentasaccharide), Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Gl c, Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[F uc(alpha1-3)]Glc (difucosylheptasaccharide) and Gal(alpha1-3)Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)?Gal(alpha1-3) Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-6)?Gal(beta1-4)Glc (difucosyldecasaccharide). Lactose was present only in small amounts. Some of the milk oligosaccharides of the polar bear had alpha-Gal epitopes similar to some oligosaccharides in milk from the Ezo brown bear and the Japanese black bear. Some milk oligosaccharides had human blood group A antigens as well as B antigens; these were different from the oligosaccharides in Ezo brown and Japanese black bears.     

Synthesis of structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B

O-alpha-d-Glucopyranosyl-(1----3)-alpha, beta-L-rhamnopyranose (15), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)-al pha, beta-L-rhamnopyranose (17), O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----3)-D-ribitol (23), and O-alpha-D-galactopyranosyl-(1----3)-O-alpha-D-glucopyranosyl-(1----3)- O-alpha-L-rhamnopyranosyl-(1----4)-D-ribitol (27), which are structural elements of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B ([----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap- (1----X)- D-Rib-ol-(5-P----]n; 6A X = 3, 6B X = 4), have been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside (3) was coupled with benzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside (4), and subsequent deallylation (----14) and debenzylation gave 15. Condensation of 14 with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside (2) followed by debenzylation gave 17. Acetylation of 17 followed by removal of AcO-1, conversion into the imidate, coupling with 1,2,4,5-tetra-O-benzyl-D-ribitol (11), deacetylation, and debenzylation gave 23. Coupling of the imidate with 1-O-allyloxycarbonyl-2,3,5-tri-O-benzyl-D-ribitol (12) followed by deallyloxycarbonylation, deacetylation, and debenzylation yielded 27.     

Synthesis of two heptasaccharide analogues of the lentinan repeating unit

beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (18) and the allyl glycoside of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)[-beta-D-Glcp-(1-->6)]-alpha-D-Glcp (29) were synthesized as the analogues of the lentinan repeating heptaose by building the pentasaccharide backbones first, followed by attaching the side chains. 4,6-O-benzylidenated mono-13 or disaccharide 8 were used as the acceptor to ensure the beta linkage in the synthesis of 18, while 4,6-O-benzylidenated disaccharides 21 and 23 were used as the donor and acceptor, respectively, to ensure the beta linkage in the synthesis of 29.     

Synthesis and in-vitro evaluation of novel low molecular weight thiocarbamates as inhibitors of human leukocyte elastase

A series of novel low molecular weight thiocarbamate esters (1e-6e) were synthesized and evaluated as inhibitors of human leukocyte elastase (HLE). The thiocarbamate esters studied consist of a substituted primary or secondary aliphatic or aromatic amine and a 1-phenyl-1H-tetrazole-5-thiol (Table I). The HLE catalyzed hydrolysis of N-methoxysuccinyl- L-Ala-L-Ala-L-Pro-L-Val-p-nitroanilide substrate was utilized as the measure of inhibition. N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (1e) exhibited the highest inhibitory activity (k(obs) /[I] = 2.1 x 10(5) M(-1). min(-1) ) and N-allyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (2e) (K(obs) /[I] = 6.1 x 10(4) M(-1). min(-1) ) exhibited the second highest inhibitory activity of all the thiocarbamates. The aromatic N-phenyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (4e) showed the lowest inhibitory activity (K(obs) /[I] = 1.9 x 10(2) M(-1). min(-1) ) among the N-monosubstituted derivatives, similar to that of N-ethyl-N-n-butyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (5e) (K(obs) /[I] = 1.8 x 10(2) M(-1).min(-1) ). The N-isopropyl, 1-phenyl-1H-tetrazole-5-thiocarbamate (3e) (K(obs) /[I] = 3.3 x 10(3) M(-1).min(-1) ) was about 10 fold more active than (4e) and N, N-diisopropyl, 1-phenyl-1H-tetrazole- 5-thiocarbamate (6e) showed no inhibitory activity against HLE. In the present work less than 3% of HLE specific activity was regained after 24 hours incubation with each of the tested N-monosubstituted thiocarbamates (1e-4e). The time-dependent inhibition of HLE by the thiocarbamate compounds (1e-5e) seems to involve the interaction and possible chemical modification of one enzyme residue. Straight chain nonpolar aliphatic substituents on the nitrogen of the thiocarbamate functionality may be essential for high inhibitory activity. As the degree of substitution (branching) on the nitrogen of the thiocarbamate functionality increases the inhibitory activity of the compounds decreases. The time-dependent inhibition of HLE and the slow deacylation rates by the N-monosubstituted thiocarbamates are consistent with irreversible inhibition.     

The structures of oligosaccharides excreted by sheep with swainsonine toxicosis

Eleven oligosaccharides were purified form the urine of sheep with swainsonine toxicosis induced by the feeding of Astragalus lentiginosus. Oligosaccharides were extracted by charcoal adsorption, chromatographed on Bio-Gel P-2, and partially fractionated by preparative-layer chromatography. Separation into individual compounds was completed by semi-preparative high pressure liquid chromatography. Structures were determined by a combination of high pressure liquid chromatography and exo- and endo- glycosidase action, methanolysis followed by gas-liquid chromatography, methylation analysis, and high resolution nuclear magnetic resonance spectroscopy. Two homologous series of oligosaccharides were identified: (a) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp+ ++-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----2)-alpha-D-Manp(1----3)-[alpha-D-Manp+ ++-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----2)-alpha-D-Manp-(1----2)-alpha-D-Manp+ ++-(1----3)-[alpha- D-Manp-(1----6)]-beta-D-Manp-(1----4)-D-GlcpNAc (minor series); (b) alpha-D-Manp-(1----6)-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp(1----3)-alpha-D-Manp-(1----6)-beta-D-Manp -(1----4)-beta-D-GlcpNAc- (1----4)-D-GlcpNAc, alpha-D-Manp-(1----6)-alpha-D-Manp-(1----6)-beta-D-Manp++ +-(1----4)-beta-D-GlcpNAc - (1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)-[alpha-D-Manp -(1----3)]-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)-beta-D- Manp-(1----4)-beta-D-GlcpNAc-(1----4)-D-GlcpNAc, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man p-(1----6)- [alpha-D-Manp-(1----3)]-beta-D-Manp-(1----4)-beta-D-GlcpNAc- (1----4)-D- GlcpNAc (major series).     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.     

Structural examination of antitumour, water-soluble glucans from Grifora umbellata by use of four types of glucanase.

Antitumour glucans (GU) from the fungus Grifora umbellata have been subjected to periodate oxidation, Smith degradation, methylation analysis, and treatment with endo-(1 leads to 6)-beta-D-, endo-(1 leads to 3)-beta-D-, and exo-(1 leads to 3)-beta-D-glucanases, and alpha-amylase; the following structural features were revealed. GU-2 contains a backbone involving (1 leads to 6)-beta- and () leads to 3)-beta linkages, and two kinds of branches involving (1 leads to 6)-beta and (1 leads to 4)-alpha linkages. GU-3 has a (1 leads to 3)-beta-linked backbone and branches involving (1 leads to 6)-beta linkages or (1 leads to 4)-alpha and (1 leads to 6)-beta linkages. GU-4 also contains a (1 leads to 3) beta-D-glucan backbone and a small number of (1 leads to 6)-beta-linked branches. Probable structural units of these glucans are proposed.     

Domain organization of XAF1 and the identification and characterization of XIAP(RING) -binding domain of XAF1

X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) has been implicated as a novel tumor suppressor, which was proposed to exert pro-apoptotic effect by antagonizing the anticaspase activity of XIAP. Here, we delineated the domain architecture of XAF1 by applying limited proteolysis and peptide mass fingerprinting analysis. Our results indicated that XAF1 has a distinct domain organization, with a highly compact N-terminal domain (XAF1(NTD) ) followed by a middle domain (XAF1(MD) ), a 42-residue unstructured linker and a C-terminal domain (XAF1(CTD) ). The search of XIAP binding region within XAF1 revealed that a modest affinity XIAP(RING) binding site (dissociation constant, K(d) , ～18 μM) is located at the C-terminal portion of XAF1. This C-terminal region, embracing XAF1(CTD) and a flexible tail at C-terminus (residue Thr251-Ser301), is functionally identified as XIAP(RING) -binding domain of XAF1 (XAF1(RBD) ) in the present study. We have also mapped the interaction sites for XAF1(RBD) on XIAP(RING) by using NMR spectroscopy. By applying in vitro ubiquitination assay, we observed that XAF1(RBD) /XIAP interaction is essential for the ubiquitination of GST-XAF1(RBD) fusion protein. In addition, the C-terminal XAF1 fragment harboring XAF1(RBD) was found to be substantially ubiquitinated by XIAP(RING) . Base on these observations, we speculate a possible role of XAF1(RBD) in targeting XAF1 for XIAP-mediated ubiquitination.     

Chemical characterization of acidic oligosaccharides in milk of the red kangaroo (Macropus rufus)

In the milk of marsupials, oligosaccharides usually predominate over lactose during early to mid lactation. Studies have shown that tammar wallaby milk contains a major series of neutral galactosyllactose oligosaccharides ranging in size from tri- to at least octasaccharides, as well as β(1-6) linked N-acetylglucosamine-containing oligosaccharides as a minor series. In this study, acidic oligosaccharides were purified from red kangaroo milk and characterized by (1)H-nuclear magnetic resonance spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to be as follows: Neu5Ac(α2-3)Gal(β1-4)Glc (3'-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Glc (sialyl 3'-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc, Gal(β1-3)(-3-O-sulfate)Gal(β1-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc. These acidic oligosaccharides were shown to be sialylated or sulfated in the non-reducing ends to the major linear and the minor branched series of neutral oligosaccharides of tammar wallaby milk.     

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Myosin subfragment 1 (S-1) can be fractionated into two isozymes, (A1)S-1 containing alkali light chain 1 and (A2)S-1 containing alkali light chain 2. The predominant difference in the behavior of the two isozymes of S-1 is that, at low ionic strength, the actin concentration required for half-maximal ATPase activity is considerably lower for (A1)S-1 than for (A2)S-1; that is, the apparent binding constant KATPase for (A1)S-1 is greater than KATPase for (A2)S-1 [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. This difference disappears at high ionic strength [Wagner, P. D., Slater, C. S., Pope, B., & Weeds, A.G. (1979) Eur. J. Biochem. 99, 385-394]. In the present study we investigated whether the difference in the KATPase values of (A1)S-1 and (A2)S-1 is due to a difference in the actual affinity of these S-1 isozymes for actin. Binding was measured in the presence of ATP and AMP-PNP and in the absence of nucleotide at varied ionic strengths. We found that at low ionic strength where KATPase is several times stronger for (A1)S-1 than for (A2)S-1, the binding of (A1)S-1 to actin is correspondingly stronger than that of (A2)S-1 irrespective of the nucleotide present. Furthermore, as the ionic strength is increased, just as the difference between the KATPase values for (A1)S-1 and (A2)S-1 disappears so too does the difference in the affinity of the two isozymes for actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural features of a polysaccharide from the mucin of water hyacinth

The mucin found in the nodal region of the weed, water hyacinth (Eichhornia crassipes), is a heteropolysaccharide composed of d-xylose, l-galactose and l-arabinose in the mol ratio of 1.3:1.2:1.0. Partial hydrolysis with acid gave four oligosaccharides which were characterized as: d-Xylp-(1 → 3)-l-Ara, l-Galp-(1 -→ 2)-l-Ara, d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Ara, and d-Xylp-(1 → 2)-d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Ara. These, together with the results of methylation analysis using GC and GC/MS and periodate oxidation, indicated that the trisaccharide repeating unit, → 4)-d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Araf-(1 →, constitutes the backbone of the polysaccharide. Further, all the d-xylopyranosyl residues of the backbone are substituted at O-2 and, in addition, one out of seven such residues is also substituted at O-3; the substituents being l-Araf-(1 →, d-Xylp-(1 →, l-Galp-(1 →, d-Xylp-(1 → 3)-l-Araf-(1 →, residues.     

Elongation of both branches of biantennary backbones of oligo-(N-acetyllactosamino)glycans by human serum (1----3)-N-acetyl-beta-D- glucosaminyltransferase.

Partial reactions catalyzed by a (1----3)-N-acetyl-beta-D- glucosaminyltransferase (EC2.4.1.149), known to be present in human serum, were studied by use of biantennary "backbone" saccharides of oligo-N-acetyllactosamine-type as acceptors. Incubation of the radiolabeled blood-group I-active hexasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp- (1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-GlcNAc (1) and UDP-GlcNAc with serum gave first a transient 1:1 mixture of two isomeric heptasaccharides, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D- Galp-(1----4)-D-GlcNAc (2) and beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----3)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-Glc NAc (3), showing that both branches of 1 react equally well. The two heptasaccharides reacted further in the incubation mixture to form the radiolabeled octasaccharide, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[be ta-D- GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Ga lp- (1----4)-D-GlcNAc (4); during this second reaction, the composition of the heptasaccharide mixture remained unchanged, indicating that 2 and 3 reacted at approximately equal rates. The heptasaccharides 2 and 3 could not be separated from each other, but they could be detected, identified, and quantitatively determined by stepwise enzymic degradations. Partial (1----3)-N-acetyl-beta-D-glucosaminylation reactions, carried out with another acceptor, the branched pentasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Gal (11), revealed that it reacted also equally well at both branches.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation analysis of modified tetrasaccharide sequences of the N-glycoprotein type--problem of the alpha-(1 to 6)-glycosidic bond

The conformational analysis of the recently synthesized tetrasaccharides alpha-D-Manp (1----3)-[alpha-D-Manp-(1----6)]-4-deoxy-beta-D-lyx-hexp+ ++-(1----4)-D-GlcNAc (2) and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)-D-GlcNAc (3) will be described. The preferred solution conformation of 2 and 3 is a gt-conformation, which is nearly identical with the preferred conformation of the naturally occurring tetrasaccharide alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-D-GlcNAc (1). The main structural feature is the backfolding of the alpha-(1----6)-linked D-Man to the reducing D-GlcNAc unit. Conformational analysis of the tetrasaccharides alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-1,6- anhydro-beta-D-GlcNAc (4), alpha-D-Manp-(1----3)-alpha-D-Manp-(1----6)]-4-deoxy-beta-D- lyx-hexp-(1----4)- 1,6-anhydro-beta-D-GlcNAc (5), and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Talp -(1----4)- 1,6-anhydro-beta-D-GlcNAc (6) gave additional proof for this backfolding. The substitution of the reducing unit leads to a smaller amount of gt- and a greater amount of gg-conformers. The method used for conformational analysis of 2-6 is a combination of n.m.r.-experiments and HSEA-calculations with the program GESA. Concerning the application of new 2D-techniques, the COLOC-experiment turned out to be extremely useful in sequencing oligosaccharides.     

Chemical characterization of milk oligosaccharides of the koala (Phascolarctos cinereus)

Previous structural characterizations of marsupial milk oligosaccharides had been performed in only two macropod species, the tammar wallaby and the red kangaroo. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of the koala milk carbohydrate fraction were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of milk of the koala, a non-macropod marsupial, and characterized by ¹H-nuclear magnetic resonance spectroscopy. The structures of the neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3′-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3′,3″-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I) and Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl lacto-N-novopentaose I), while those of the acidic saccharides were Neu5Ac(α2-3)Gal(β1-4)Glc (3′-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Gal (sialyl 3′-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), and Neu5Ac(α2-3)Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl sialyl lacto-N-novopentaose a). The neutral oligosaccharides, other than fucosyl lacto-N-novopentaose I, a novel hexasaccharide, had been found in milk of the tammar wallaby, a macropod marsupial, while the acidic oligosaccharides, other than fucosyl sialyl lacto-N-novopentaose a had been identified in milk carbohydrate of the red kangaroo. The presence of fucosyl oligosaccharides is a significant feature of koala milk, in which it differs from milk of the tammar wallaby and the red kangaroo.