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Mycobacterial polysaccharides. II. Comparison of polysaccharides from strains of four species of mycobacteria.

Evidence from chemical and serological studies indicates that a cellular heteropolysaccharide, also found in lipid extracts and culture filtrate, is present as a group antigen in Mycobacterium tuberculosis H37Ra and in other strains of mycobacteria representing M. kansasii, scotochromogenic and Battey strains. Polysaccharides from the four strains contain the same main sugars, arabinose, and galactose, as revealed by thin-layer chromatography and spectrophotometric studies. In Ouchterlony gel diffusions, bands of identity are produced between the polysaccharides by using rabbit antiserum prepared against any of the four mycobacteria. Immune adsorption studies also confirm the presence of identical antigenic determinant groups. In skin tests with tuberculopolysaccharide I, a skin reaction of about equal size was elicited in guinea pigs sensitized with either M. tuberculosis H37Ra or heterologous mycobacterial antigens in Freund's incomplete adjuvant. In animals sensitized with M. tuberculosis H37Ra, skin tests with both homologous and heterologous polysaccharides elicited similar responses.

Animals↗

Biosynthesis of the Escherichia coli K5 polysaccharide, a representative of group II capsular polysaccharides: polymerization in vitro and characterization of the product.

Biosynthesis of the capsular K5 polysaccharide of Escherichia coli, which has the structure 4)-beta GlcA-1,4-alpha GlcNAc-(1, was studied with membrane preparations from an E. coli K5 wild-type strain and from a recombinant K-12 strain expressing the K5 capsule. Polymerization occurs at the inner face of the cytoplasmic membrane without the participation of lipid-linked oligosaccharides. The serological K5 specificity of the in vitro product was determined with a K5-specific monoclonal antibody in an antigen-binding assay. The K5 polysaccharide, as obtained from the membranes after an in vitro incubation, has 2-keto-3-deoxyoctulosonic acid as the reducing sugar, which indicates that the polysaccharide grows by chain elongation at the nonreducing end.

Antigens, Bacterial↗

[Comparison of the immunomodulatory effects of spore polysaccharides and broken spore polysaccharides isolated from Ganoderma lucidum on murine splenic lymphocytes and peritoneal macrophages in vitro].

OBJECTIVE: To compare the immunomodulatory effects of spore polysaccharides (Gl-SP) and broken spore polysaccharides (Gl-BSP) isolated from Ganoderma lucidum(Leyss et Fr.) Karst. on murine splenic lymphocytes and peritoneal macrophages in vitro. METHODS: Mixed lymphocyte culture reaction (MLR), lymphocyte proliferation in the presence or absence of mitogen, and the cytotoxic activity of splenic natural killer (NK) cells were detected with MTT assay in vitro. The percentage of phagocytosis of neutral red (NR) by mouse peritoneal macrophages was detected by colorimetric assay. Splenic T-lymphocyte subpopulations were measured with flow cytometry(FCM). IL-2, IFN-gamma and TNF-alpha in the culture supernatants were detected by ELISA and biological assay. Nitric oxide (NO) production was examined by Griess reaction. RESULTS: At the concentration range of 0.2-12.8 mg/L, Gl-SP and Gl-BSP were shown to increase lymphocyte proliferation in the presence or absence of mitogen, enhance NK cytotoxic activity, augment the production of TNF-alpha and NO in Gl-SP- or Gl-BSP-activated macrophages, as well the percentage of phagocytosis of NR by macrophages in vitro. Both Gl-SP and Gl-BSP could promote MLR, however, at the dose of 12.8 mg/L, Gl-BSP showed higher activity than Gl-SP in the proliferation of lymphocytes. These two kinds of polysaccharide could significantly increase the secretion of IL-2 and IFN-gamma in doublejway MLR at the concentrations of 0.2-12.8 mg/L, but Gl-BSP had stronger effects than Gl-SP at the same concentrations. Both Gl-SP and Gl-BSP could increase the ratio of T-lymphocyte subpopulations in double-way MLR. At the concentrations of 0.2-12.8 mg/L or 3.2-12.8 mg/L, Gl-BSP demonstrated more significant activity in increasing the percentage of the CD4(+) or CD8(+) subset than Gl-SP. At the concentrations of 0.2-0.8 mg/L, the ratio of the CD4(+) and CD8(+) subset in the Gl-BSP treated group was higher than that of the Gl-SP treated group. CONCLUSION: Gl-SP and Gl-BSP have similar immunomodulatory effects in vitro, as though the immunomodulatory effects of Gl-BSP are stronger than that of Gl-SP.

Adjuvants, Immunologic↗

Application of two new methods for cleavage of polysaccharides into specific oligosaccharide fragments. Structure of the capsular and extracellular polysaccharides of Rhizobium japonicum that bind soybean lectin.

The extracellular polysaccharide produced by the bacterium Rhizobium japonicum has been implicated in the recognition between symbionts which takes place in the association of R. japonicum with soybean. The complete primary structure of the polysaccharide produced by R. japonicum strain 3I1b 138 has been determined by a combination of conventional and unconventional methods. The polymer contains glucose, mannose, and galacturonic acid in the molar ratio of 2:1:1 and contains a varying proportion of galactose and 4-O-methylgalactose, the sum of these two being equivalent to the amount of mannose (Mort, A. J., and Bauer, W. D. (1980) Plant Physiol, 66, 158-163). The polymer was specifically degraded and its galacturonic acid residues by treatment with lithium in ethylenediamine to a tetrasaccharide. By sequential specific glycosidase hydrolysis and methylation analysis the tetrasaccharide was shown to have Structure 1: (formula: see text). Anhydrous liquid hydrogen fluoride at -23 degrees C degraded the polymer to a trisaccharide and monosaccharides. The galactose and 4-O-methylgalactose released by the HF as monosaccharides were found to be partially acetylated. By 1H NMR spectrometry and methylation analysis the trisaccharide was found to have Structure 2: (formula: see text). From the composition and the structural analyses of the tetra- and trisaccharides produced in high yields by specific degradations of the polysaccharide, the polymer is deduced to have a pentasaccharide repeating unit with Structure 3: (formula: see text).

Carbohydrate Conformation↗

[Properties of polysaccharide complexes produced by Azospirillum brasilense and polysaccharides isolated from these complexes].

Azospirillum brasilense strains Sp7, Sp7 (S-form), Sp107, Sp245, Sp246, S17, and 15 when cultivated in a liquid synthetic malate medium up to the end of the logarithmic phase of growth were shown to produce at least two complex polysaccharide-containing components. The components were arbitrarily called lipopolysaccharide-protein complex (LPPC) and polysaccharide-lipid complex (PSLC). The LPPCs and PSLCs from the strains Sp7, Sp107, Sp245, Sp246, and S17 were shown to interact with a wheat germ agglutinin (WGA). From PSLCs, acidic polysaccharides (PS) were isolated and their specific rotation, molecular masses, affinity for WGA, and monosaccharide composition were determined. The PSs of all strains contained rhamnose, galacturonic acid, and galactose (except the strain Sp246), and glucosamine (except the strain Sp7 (S-form)), while the PSs of the strains Sp7 and S17 also contained fucose and mannose, respectively. It is suggested that LPPCs and PSLCs may be involved in the process of interaction of azospirilla with wheat root surfaces, and that PSsare probably active parts of the LPPCs and PSLCs. The ability of LPPCs and PSs to interact with Congo red in aqueous solutions was investigated.

Azospirillum brasilense↗

[Specific polysaccharides of alkali-forming enterobacteria. II. Separation of a hydrolysate of B. faecalis alcaligenes specific polysaccharide on a column with cellulose].

Paper and gas-liquid chromatography was applied to the study of monosaccharide composition of specific polysaccharide from B. faecalis alcaligenes. Lipophilic sugar of unknown chemical nature was revealed in the composition of this polysaccharide. Conditions for separation of acid hydrolyzate of the specific polysaccharide of this culture in a column with cellulose powder for the purpose of preparative obtaining of the unknown sugar were elaborated.

Alcaligenes↗

Oligosaccharide fragments of the type III group B streptococcal polysaccharide derived from S. pneumoniae type 14 capsular polysaccharide by a chemoenzymatic method.

Partial N-deacetylation fo the GlcNAc residues in S. pneumoniae type 14 capsular polysaccharide (Pn14-PS) backbone was achieved by treatment with base, and the product was subsequently enzymatically sialylated at the 3-O-positions of the terminal galactose residues. The resultant, partially N-deacetylated type III Group B streptococcus capsular polysaccharide (GBSIII-PS) was subjected to nitrous acid deamination, which resulted in the degradation of GBSIII-PS polysaccharide into oligosaccharides containing increasing numbers of the identical repeating units. The oligosaccharides were then separated by passage through a Superdex 30 column and characterized by ESIMS and NMR spectroscopic analysis.

Bacterial Capsules↗

Core-lipid A on the K40 polysaccharide of Escherichia coli O8:K40:H9, a representative of group I capsular polysaccharides.

From the capsular K40 polysaccharide of E. coli O8:K40:H9, a fraction was obtained by gel permeation chromatography which in SDS-PAGE exhibited a ladder-like pattern characteristic of lipopolysaccharides. In Western blots, this fraction reacted with a K40-specific antiserum but not with an O8-specific antiserum. It contained, in addition to the constituents of the K40 polysaccharide (glucuronic acid, glucosamine and serine), glucose, galactose, heptose, and KDO. Mild acid hydrolysis of this fraction liberated a lipid moiety which by chemical analysis was characterized as lipid A. From these results, we conclude that the capsular polysaccharide of E. coli O8:K40:H9 is in part bound to core lipid A. The significance of this finding is discussed.

Bacterial Capsules↗

[Experimental and theoretical conformation analysis of the O-antigenic polysaccharide from Pseudomonas cepacia strain 3181. II. The polysaccharide].

The spatial structure of Pseudomonas cepacia 3181 polysaccharide in aqueous solution is discussed basing on the data of nuclear Overhauser effect, observed with preirradiation of anomeric protons of all 3 D-rhamnose residues in the repeat unit, and theoretical conformational analysis. It is shown that conformational states of the free disaccharides and corresponding disaccharide units of the polysaccharide are similar. All conformations of the polysaccharide may be described by one shape representing an extended structure with characteristic turns in the D-Rha alpha 1-2-D-Rha beta 1-3-D-Rha units.

Antigens, Bacterial↗

[Bacterial antigenic polysaccharides. 20. The structure of the O-specific polysaccharide chain of Pseudomonas aeruginosa O(3a),3d,3f (Lányi) lipopolysaccharide].

Mild acid degradation of lipopolysaccharide from Pseudomonas aeruginosa O(3a), 3d, 3f (Lányi classification) afforded O-specific polysaccharide containing N-acetyl-D-fucosamine, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, 3-acetamidino-2-acetamido-2,3-dideoxy-L-guluronic and D-mannuronic acid as well as O-acetyl groups. On the basis of O-deacetylation, selective cleavage with anhydrous fluoride, chemical transformation of the oligosaccharides obtained (hydrolysis or reductive deamination of the acetamidino group into acetamido or ethylamino group, respectively) and analysis by 13C NMR spectroscopy, it was concluded that the polysaccharide is built up mainly by trisaccharide repeating units of types A and B in the ratio approximately 2:1: (Formula: see text). The units of both types most probably enter the same polymeric chain. If so, such a hybrid structure can be accounted for by incompleteness of epimerization at C5 of the acetamidino derivative of mannuronic acid at the polymer level in the course of biosynthesis of this polysaccharide.

Antigens, Bacterial↗

[Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide].

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.

Antigens, Bacterial↗

[Antigenic bacterial polysaccharides. 24. The structure of the O-specific polysaccharide chain of Salmonella arizonae 063 (Arizona 08) lipopolysaccharide].

The O-specific polysaccharide chain of the Salmonella arizonae O63 lipopolysaccharide is composed of D-glucose, D-galactose, N-acetyl-D-galactosamine, and 3-acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) residues in the ratio 1:1:2:1. On the basis of methylation analysis and calculations of 13C-NMR-spectra of the polysaccharide and of the product of its selective cleavage with anhydrous hydrogen fluoride, the linear polymer lacking 3-acetamido-3,6-dideoxygalactose, it was concluded that the polysaccharide has the following structure: (Formula: see text).

Antigens, Bacterial↗

[Antigenic bacterial polysaccharides. 29. The structure of the polysaccharide chain of Pseudomonas holci 8300 (serogroup I) lipopolysaccharide].

The Pseudomonas holci 8300 lipopolysaccharide has an O-specific polysaccharide chain, containing L-rhamnose and 3-acetamido-3-deoxy-D-fucose residues in the ratio 4:1. On the basis of methylation, Smith degradation, and 1H- and 13C-NMR spectroscopy data, it was concluded that the polysaccharide is built up of pentasaccharide units of A and B types in the ratio approximately 2.5:1. In some stretches of the polysaccharide, minor B units form rather long chains, and in the others they alternate with predominant A units. (formula; see text)

Antigens, Bacterial↗

[Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)].

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.

Antigens, Bacterial↗

[Antigenic bacterial polysaccharides. 36. A study of the structure of the O-specific polysaccharide chain of the lipopolysaccharide of Pseudomonas fluorescens IMV 2763 (biostrain G)].

Chemical and serological characterization of the Pseudomonas fluorescens IMV 2763 (biovar G) lipopolysaccharide was carried out. The O-specific polysaccharide chain of the lipopolysaccharide is composed of D-mannose, 6-deoxy-L-talose, N-acetyl-D-galactosamine and O-acetyl groups in the ratio of approximately 2:1:1:1. The polysaccharide is branched and a half of residues of 6-deoxytalose and monosubstituted mannose carry O-acetyl groups. On the basis of methylation, partial acid hydrolysis and 13C NMR analysis it was concluded that the repeating unit of the polysaccharide has the following structure: (formula; see text)

Antigens, Bacterial↗

[Analysis of polysaccharides used as food additives. V. Gaschromatographic identification of hydrolysis products in uronic acids containing polysaccharides].

Alduronic acids can be found among the monomeric units of polysaccharides, which are used as stabilisators in the food industry. Gaschromatographically they cannot be separated directly by the way of their aldonitrilacetates but it is possible by the procedure described. The 1-0-methylalduronic-acidmethylesters, obtained by the methanolysis of the polysaccharides, are reduced with boronhydrid to the corresponding methyl glycosides; there are split with acid to the aldoses, which are converted in pyridine with hydroxylamine to the aldoximes and than with acetic anhydride to the aldonitrilacetates, which can be separated by gaschromatography without difficulty.

Chromatography, Gas↗

The O-polysaccharide from the lipopolysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides.

The O-polysaccharide (O-antigen) of Providencia stuartii O44:H4 (strain 3768/51) was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure: [Formula: see text].

Carbohydrate Sequence↗

Immunization with meningococcal polysaccharide-tetanus toxoid conjugate induces polysaccharide-reactive T cells in mice.

T cell clones were generated from mice immunized with a meningococcal group C (alpha2 --> 9-sialic acid) polysaccharide-tetanus toxoid (MCPS-TT) conjugate. Many clones were found to be specific for tetanus toxoid (TT), however, clones reactive with MCPS-TT and polysaccharide (PS) were isolated. Two clones were specific for MCPS and two cross-reacted with Escherichia coli K1-PS (alpha2 --> 8-sialic acid). Both TT and PS reactive clones were CD4+ and CD8-. TT and MCPS-TT-specific T cell clones were major histocompatibility complex (MHC) restricted, however, the PS-reactive clones were not. Both MHC-restricted TT clones and non-restricted PS clones, however, were dependent on contact with antigen presenting cells (APC) for maximal stimulation. The data suggest that multivalent repeating epitopes on PS antigen (Ag) can overcome the need for MHC restricted interactions, but not the requirement for cell-cell contact.

Animals↗