A colorimetric minisequencing assay for the mutation in codon 506 of the coagulation factor V gene.

We describe a colorimetric screening method for the mutation in codon 506 of the coagulation factor V gene. The nucleotide at the site of the FV:Q506 mutation is identified in an immobilized amplified DNA template by extension of a primer with a hapten-labelled dNTP using a DNA polymerase. The incorporated hapten is detected by an antibody-alkaline phosphatase conjugate that catalyses the formation of a coloured end product. The assay is carried out in a microtiter plate format, and the procedure is identical to that of enzyme immunoassays. It unequivocally identifies the FV:Q506 mutation in heterozygous and homozygous form. The colorimetric minisequencing method gave the same result as a 3H-based minisequencing assay and restriction site analysis with Mn11 used as reference methods. Because of its simple format and numeric result, the novel colorimetric minisequencing method should be an attractive alternative for screening for the FV:Q506 mutation in clinical laboratories.

Structural studies of the O-antigen polysaccharides of Klebsiella O5 and Escherichia coli O8.

The O-antigen polysaccharides of Klebsiella serotype O5 and Escherichia coli serotype O8 are serologically very similar or identical. The structures of these two polysaccharides have now been re-investigated. N.m.r. spectroscopy, chromium trioxide oxidation, hydrolysis with a specific phage enzyme, and f.a.b. mass spectrometry were the principal methods used. It is concluded that the O-antigen has the following structure, in which D-Man3Me is 3-O-methyl-D-mannose and n is approximately 10. (Formula: see text) Biosynthetic studies indicate that these antigens are synthesised by addition of D-mannopyranosyl groups to the "non-reducing" end of the mannan chain, and it seems possible that addition of a 3-O-methyl-D-mannopyranosyl group involves termination.

Synthesis of a disaccharide component of the capsular polysaccharide antigen of Streptococcus pneumoniae type 1.

Methyl 2-acetamido-4-amino-2,4,6-trideoxy-3-O-(alpha-D-galactopyranosyl-uronic acid)-alpha-D-galactopyranoside has been synthesised. The parent disaccharide is a structural element of the capsular polysaccharide antigen of Streptococcus pneumoniae type 1.

Structural studies of the Escherichia coli O-antigen 6.

The structure of the Escherichia coli O-antigen 6 has been investigated using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure. (Formula: see text)

Carbohydrate binding studies on the lectin from Datura stramonium seeds.

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (Mr = 86,000) possesses two carbohydrate-binding sites for N,N'N",N"'- tetraacetylchitotetritol /mol protein, with an apparent Ka = 8.7 X 10(3) M-1 at 4 degrees C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N'- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N',N"'- triacetylchitotriose was over 6-fold more potent than the disaccharide (N,N'- diacetylchitobiose ) which, in turn, was 90 times more reactive than N-acetyl-D-glucosamine. N-Acetyllactosamine [beta-D-Gal-(1----4)-D-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N'- diacetylchitobiose . The requirement for an N-acetyl-D-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.

Capsular polysaccharides and lipopolysaccharides from two strains of Bacteroides fragilis.

The capsular polysaccharide from Bacteroides fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of B. fragilis strain ATCC 23745 in addition contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose, but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyltetradecanoic, 3-hydroxypentadecanoic as major constituents). The capsular polysaccharide of both strains promoted abscess formation, whereas the lipopolysaccharide failed to do so.

Synthesis of a nona- and an undeca-saccharide that form part of the complex type of carbohydrate moiety of glycoproteins.

Silver trifluoromethanesulfonate-promoted condensation of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-beta-D-glucopyranosyl bromide with benzyl 2,4,-di-O-benzyl-6-O-(3,4-di-O-benzyl-alpha-D-mannopyranosyl)-3-O-(3,6-di-O- benzyl-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside gave nonasaccharide and undecasaccharide derivatives. The nonasaccharide 2 and the undecasaccharide 3 were obtained by removal of the protecting groups followed by N-acetylation. (formula; see text).

Capsular polysaccharides and lipopolysaccharides from two Bacteroides fragilis reference strains: chemical and immunochemical characterization.

Fermentor growth of Bacteroides fragilis under controlled conditions in a complex medium containing 1% glucose and 10% fetal calf serum resulted in high yields of bacteria. After hot phenol-water extraction of the organisms, capsular polysaccharide was isolated from the aqueous phase and purified by Sephacryl S-300 chromatography in a buffer with 3% sodium deoxycholate. Lipopolysaccharide was isolated by phenol-chloroform-light petroleum ether extraction. The capsular polysaccharide from B. fragilis strain NCTC 9343 contained six sugars: L-fucose, D-galactose, D- and L-quinovosamine, D-glucosamine, and galacturonic acid. The capsule of strain ATCC 23745 also contained D-glucose, L-fucosamine, L-rhamnosamine, and a 3-amino-3,6-dideoxyhexose but lacked D-quinovosamine. The latter capsule also contained alanine (4%). The capsular polysaccharides were different immunochemically by ELISA inhibition. The lipopolysaccharide of both strains contained the same sugars (L-rhamnose, D-glucose, D-galactose, and D-glucosamine) and fatty acids (13-methyl-tetradecanoic and 3-hydroxy-hexadecanoic and 3-hydroxy-15 methyl-hexadecanoic as major constituents) and were identical by ELISA inhibition.

Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features.

A series of synthetic oligosaccharides, resembling natural N-acetyllactosamine type glycans, were tested for their ability to inhibit the binding of labeled ligand to the mammalian hepatic lectin on rabbit hepatocytes at 2 degrees C. A dramatic hierarchy of inhibitory potency (tetraantennary greater than triantennary much greater than biantennary much greater than monoantennary) could be demonstrated. The range of concentration required for 50% inhibition of labeled ligand binding extended from approximately 1 mM, for the monoantennary oligosaccharides, to approximately 1 nM for triantennary oligosaccharides, even though the absolute Gal concentration increased only 3-fold. It was found that the number of Gal residues/cluster and their branching mode are major determinants of binding affinity of ligands to the hepatic lectin on the surface of hepatocytes.

Synthesis of oligosaccharides that form parts of the complex type of carbohydrate moieties of glycoproteins. Three glycosides prepared for conjugation to proteins.

Condensation of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-2-deoxy-2- phthalimido-beta-D-glucopyranosyl bromide with p-nitrophenyl 3-O-benzoyl-4,6-di-O-benzylidene-alpha-D-mannopyranoside, p-nitrophenyl 3,6-di-O-benzyl-alpha-D-mannopyranoside and p-nitrophenyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave protected tri- and pentasaccharides. The oligosaccharide glycosides 1, 2 and 3 were obtained after de-protection of these condensation products. These oligosaccharides will, after suitable conversions, be conjugated to proteins for biological studies.

Mitogenic leukoagglutinin from Phaseolus vulgaris binds to a pentasaccharide unit in N-acetyllactosamine-type glycoprotein glycans.

The carbohydrate binding specificity of leukoagglutinin (La; Phaseolus vulgaris isolectin L4) was studied by using quantitative precipitation and precipitation-inhibition. A series of purified glycopeptides and synthetic oligosaccharides were used as inhibitors. The minimal structural unit required for La binding was the disaccharide GlcNac(1 leads to beta 2)Man. Additions for this basic unit of different sugar residues gave a positive or negative contribution to binding. The most complementary structure was the pentasaccharide (formula: see text). This pentasaccharide units occurs in tetraantennary N-acetyllactosamine-type glycoprotein glycans. Glycoproteins containing such structures were accordingly precipitated by La. Selected glycopeptides and oligosaccharides were also tested as inhibitors of La-induced DNA synthesis in human lymphocytes. The pattern of inhibition was essentially the same as that obtained by precipitation-inhibition, indicating that binding to lymphocytes via the carbohydrate binding site of the lectin is an essential step in the activation process.

Synthesis of three oligosaccharides that form part of the complex type of carbohydrate moiety of glycoproteins.

Silver trifluoromethanesulfonate-promoted condensation of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl bromide with benzyl 3,6-di-O-benzyl-alpha-D-mannopyranoside and benzyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave the protected 2,4- and 2,6-linked trisaccharides in yields of 54 and 32%, respectively. After exchanging the 2-deoxy-2-phthalimido groups for 2-acetamido-2-deoxy groups and de-blocking, the trisaccharides 2,4-di-O-(2-deoxy-beta-D-glucopyranosyl)-D-mannose and 2,6-di-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-D-mannose were obtained. Similar condensation of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranosyl bromide with benzyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave a pentasaccharide derivative in 52% yield. After transformations analogous to those applied to the trisaccharides, 2,6-di-O-[beta-D-galactopyranosyl-(1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)]-D-mannose was obtained.

Structural studies of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O 55.

The structure of the O-specific side-chains of the lipopolysaccharide from Escherichia coli O 55 has been investigated, methylation analysis, specific degradations, and n.m.r. spectroscopy being the principal methods used. It is concluded that the O-specific side-chains are composed of pentasaccharide repeating-units having the following structure [where Col stands for colitose (3,6-dideoxy-L-xylo-hexose)].(See formula in text).

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 1.

The capsular polysaccharide from Streptococcus pneumoniae type 1 is composed of D-galactopyranosyluronic acid residues and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranosyl residues. The latter sugar, previously unknown in Nature, was not isolated but was identified from the products obtained on deamination of the polymer. Using n.m.r. spectroscopy, methylation analysis, and Smith degradation as the principal methods of structural investigation, it is concluded that the polysaccharide is composed of trisaccharide repeating-units having the structure: leads to 3)-alpha-Sugp-(1 leads to 4)-alpha-D-GalpA-(1 leads to 3)-alpha-D-GalpA-(1 leads to, in which Sug denotes the new sugar.