Phenol-water Extraction Research Articles

Metabolic labeling to characterize the overall composition ofFrancisellalipid A and LPS grown in broth and in human phagocytes

A hallmark of Francisella tularensis, a highly virulent Gram-negative bacterium, is an unusual LPS that possesses both structural heterogeneity and characteristics that may contribute to innate immune evasion. However, none of the methods yet employed has been sufficient to determine the overall LPS composition of Francisella. We now demonstrate that metabolic labeling of francisellae with [(14)C]acetate, combined with fractionation of [(14)C]acetate-labeled lipids by ethanol precipitation rather than hot phenol-water extraction, permits a more sensitive and quantitative appraisal of overall compositional heterogeneity in lipid A and LPS. The majority of lipid A of different francisellae strains grown in diverse bacteriologic media and within human phagocytes accumulated as very hydrophobic species, including free lipid A, with <10% of the lipid A molecules substituted with O-Ag polysaccharides. The spectrum of lipid A and LPS species varied in a medium- and strain-dependent fashion, and growth in THP-1 cells yielded lipid A species that were not present in the same bacteria grown in brain heart infusion broth. In summary, metabolic labeling with [(14)C]acetate greatly facilitates assessment of the effect of genotypic and/or environmental variables on the synthesis and accumulation of lipid A and LPS by Francisella, including during growth within the cytosol of infected host cells.

Structural analysis of the O-polysaccharide of the lipopolysaccharide from Azospirillum brasilense Jm6B2 containing 3-O-methyl-d-rhamnose (d-acofriose)

Two types of neutral O-polysaccharides were obtained by mild acid degradation of the lipopolysaccharide isolated by phenol–water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum brasilense Jm6B2. The following structure of the major O-polysaccharide was established by composition and methylation (ethylation) analyses, Smith degradation, and 1D and 2D 1H and 13C NMR spectroscopy: [Display omitted] where a non-stoichiometric (∼60%) 3-O-methylation of d-rhamnose is indicated by italics.

Structure of a polysaccharide from Providencia rustigianii O11 containing a novel amide of 2-acetamido-2-deoxygalacturonic acid with l-glutamyl-l-alanine

An acidic polysaccharide was isolated from Providencia rustigianii O11 by the phenol–water extraction. The polysaccharide was cleaved by solvolysis with triflic acid to yield disaccharides with uronic acid derivatives at the non-reducing end. The polysaccharide and the disaccharides were studied by chemical analyses, high-resolution ESI MS, and 2D 1H and 13C NMR spectroscopy, and the following structure of the tetrasaccharide repeating unit of the polysaccharide was established:▪where GalNAcA stands for 2-acetamido-2-deoxygalacturonic acid, GalNAcA6GluAla for N-(2-acetamido-2-deoxygalacturonoyl)-l-glutam-1-yl-l-alanine, QuiNAc4NAcyl for 2-acetamido-4-[(S)-3-hydroxybutanoylamino]-2,4,6-trideoxyglucose (∼75%) or 2,4-diacetamido-2,4,6-trideoxyglucose (∼25%); the d configuration of GalNA and QuiN4N was ascribed tentatively. To the best of our knowledge, this is for the first time that an amide of uronic acid with a dipeptide is found in bacterial polysaccharides.

Aldobiouronic acid domains in Helicobacter pylori

Helicobacter pylori cell-surface glycans exert strong influences in host–microbe interplays and define the strain’s immunological signature. Envisaging the development of a carbohydrate-based vaccine against the gastroduodenal pathogen H. pylori, several clinical isolates are being screened for their cell-surface glycan profile. The present work concerns H. pylori clinical specimen PTAV79 that abundantly expressed amylose-like glycans. These polysaccharides were isolated in glycan-rich fractions resultant from phenol–water extractions and purified by Bio-Gel P2. Structural studies showed that the glycans are linked to glycerol and present aldobiouronic acid domains composed of [→3)-α- d-GlcA-(1→4)-α- d-Glc-(1→] repeating units. The amylose domains were constituted by an average of 19 Glc residues and the acidic moieties had an average number of 10 aldobiouronic acid repeating units. These polysaccharides were isolated in fractions that, although hydrophilic, were rich in stearic acid, strongly suggesting that they are present as glycerolipids anchored to cell-surface.

Suppressive effect of the antimicrobial peptide LL‐37 on expression of IL‐6, IL‐8 and CXCL10 induced by Porphyromonas gingivalis cells and extracts in human gingival fibroblasts

Porphyromonas gingivalis is a major periodontogenic bacterium and possesses immunostimulatory components, such as lipopolysaccharides (LPS) and fimbriae. The host antimicrobial peptide, LL-37, suppresses proinflammatory responses of immune cells but its effect on human gingival fibroblasts (HGFs) is not known. In this study, we assessed the effect of LL-37 on the proinflammatory responses of HGFs stimulated with P. gingivalis cells and their components. Live P. gingivalis cells did not induce proinflammatory responses of HGFs, and LL-37 did not alter these responses. However, LL-37 was able to suppress the killed P. gingivalis cell-induced secretion of interleukin (IL)-6 and IL-8. LL-37 also suppressed the expression of IL6, IL8, and CXCL10 genes that was induced by P. gingivalis components, including phenol-water extracts, lipid A, and fimbriae, and the induction of phosphorylation of p38 and extracellular signal-regulated kinase (ERK) by P. gingivalis lipopolysaccharide (LPS). CAMP was found to be expressed in oral epithelial cells but not in HGFs, despite stimulation with P. gingivalis components. Therefore, LL-37 can exert a suppressive effect on P. gingivalis-induced proinflammatory responses of HGFs in a paracrine manner, suggesting that excess inflammatory responses to P. gingivalis in the gingival tissue are suppressed by LL-37 in vivo.

Chemical composition of lipopolysaccharides isolated from various endophytic nitrogen-fixing bacteria of the genus Herbaspirillum

Bacteria from the genus Herbaspirillum are endophytes responsible for nitrogen fixation in gramineous plants of economic importance such as maize, sugarcane, sorghum, rice, and wheat. Some species are known to produce plant growth substances. In contrast, Herbaspirillum rubrisubalbicans strains are known to be mild plant pathogens. The molecular communication between the plant and the microbes might involve lipopolysaccharides present in the outer membrane of these gram-negative bacteria. Phenol-water extraction was used to obtain lipopolysaccharides from 7 strains of Herbaspirillum seropedicae (SmR1, Z67, Z78, ZA95, and M2) and H. rubrisubalbicans (M1 and M4). The electrophoretic profiles and chemical composition of the lipopolysaccharides obtained in the phenol and aqueous extracts were shown herein.

Affinity chromatography for purification of antibodies to Neisseria gonorrhoeae and Neisseria meningitidis lipopolysaccharides.

Lipopolysaccharides (LPSs) were prepared by phenol-water extraction of the gonococcal strain 8551 and the group B meningococcal strain 44/76, digested with pronase, and purified by ultracentrifugation and Sepharose CL-6B fractionation in the presence of 1.5 per cent sodium deoxycholate. On SDS-PAGE with 10 per cent acrylamide the purified 125I-labelled LPSs migrated as single, low-molecular-weight components. The LPSs were coupled to CNBr-activated Sepharose 4B for affinity purification of antibodies to the common antigenic factor 1 and the sero-type factor 5 of LPS 8551, and antibodies to LPS 44/76. The antibodies eluted showed ELISA activity against wells coated with LPS or whole cells of the bacteria, the antibody activity being inhibited by LPS. SDS-PAGE of whole cells of the strain 8551 and immunoblotting with the anti-factor 1 or -factor 5 antibodies resulted in single, broad bands corresponding to the low-molecular-weight LPS subunits.

Isolation, purification and antitumor activity of lipopolysaccharide from cow placenta

Lipopolysaccharide from cow placenta (LPS-CP-2) has been isolated and purified by hot phenol–water extraction, enzyme hydrolysis, chloroform–petroleum ether method, ion-exchange and gel-filtration chromatography. Also, LPS-PS-2 was evaluated for antitumor activity against Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. LPS-PS-2 caused significant ( P < 0.05) decrease in tumor volume, and viable cell count; and it prolonged the life span of EAC-tumor-bearing mice. Hematological profile indicates that LPS-CP-2 possessed protective action on the haemopoietic system. Further, administration of LPS-CP-2 reduced the tumor volume of both DLA and EAC cell lines in a dose-dependent way. The LPS-PS was found to be devoid of pyrogenic response in the rabbits. These results indicate that LPS-PS exhibited significant antitumor activity without pyrogenic response, suggesting its potential as antitumor agent.

An Indirect Enzyme-Linked Immunosorbent Assay for Detection of Antibodies to Actinobacillus Pleuropneumoniae Serovar 7 in Pig Serum

Lipopolysaccharide (LPS) antigen was purified from Actinobacillus pleuropneumoniae serovar 7 by phenol-water extraction and fractionated on a, S-100 Sephacryl column. High molecular weight fractions of LPS purified from the S-100 column were pooled and used as antigen in an indirect serovar 7 ELISA. The ELISA was evaluated with sera from pigs experimentally infected with 11 different A. pleuropneumoniae serovars of biotype 1. Estimation of sensitivity and specificity of the A. pleuropneumoniae serovar 7 ELISA was performed using pig sera from herds naturally infected with A. pleuropneumoniae serovar 7 as well as sera from herds free of infection with A. pleuropneumoniae serovar 7. When compared to the complement fixation test (CFT) as a reference test, the ELISA showed much higher sensitivity and statistically equivalent specificity.

Lipid A‐associated proteins from Porphyromonas gingivalis stimulate release of nitric oxide by inducing expression of inducible nitric oxide synthase

The purpose of this study was to examine the effects of lipid A-associated proteins from Porphyromonas gingivalis, a major cause of inflammatory periodontal disease, on the production of nitric oxide and expression of inducible nitric oxide synthase in the murine macrophage cell line, RAW264.7. We also attempted to throw light on the signaling mechanisms involved in P. gingivalis lipid A-associated protein-induced nitric oxide production. The lipid A-associated proteins from P. gingivalis 381 were prepared by standard hot phenol-water extraction of endotoxin isolated by the butanol method. Nitric oxide production was assayed by measuring the accumulation of nitrite in culture supernatants. Western blot analysis of inducible nitric oxide synthase and analysis of reverse transcription-polymerase chain reaction products were carried out. We found that P. gingivalis lipid A-associated proteins can induce inducible nitric oxide synthase expression and stimulate the release of nitric oxide without additional stimuli, and we demonstrated that multiple signaling pathways, such as nuclear factor-kappaB, microtubule polymerization, protein tyrosine kinase, protein kinase C, and mitogen-activated protein kinase cascades, are involved in P. gingivalis lipid A-associated protein-stimulated nitric oxide production. The production of nitric oxide required l-arginine. The present study clearly shows that P. gingivalis lipid A-associated proteins fully induced inducible nitric oxide synthase expression and nitric oxide production in RAW264.7 cells in the absence of other stimuli. The ability of P. gingivalis lipid A-associated proteins to promote the production of nitric oxide may be important in the pathogenesis of inflammatory periodontal disease.

Structure and heterogeneity of the O-antigen chain ofSalmonellaAgona lipopolysaccharide

Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.

Contribution of Interferon-β to the Murine Macrophage Response to the Toll-like Receptor 4 Agonist, Lipopolysaccharide

Interferon-beta (IFN-beta) has been identified as the signature cytokine induced via the Toll-like receptor (TLR) 4, "MyD88-independent" signaling pathway in macrophages stimulated by Gram-negative bacterial lipopolysaccharide (LPS). In this study, we analyzed the responses of macrophages derived from wild-type (IFN-beta(+/+)) mice or mice with a targeted mutation in IFN-beta (IFN-beta(-/-)) to the prototype TLR4 agonist, Escherichia coli LPS. A comparison of basal and LPS-induced gene expression (by reverse transcription-PCR, real-time PCR, and Affymetrix microarray analyses) resulted in the identification of four distinct patterns of gene expression affected by IFN-beta deficiency. Analysis of a subset of each group of differentially regulated genes by computer-assisted promoter analysis revealed putative IFN-responsive elements in all genes examined. LPS-induced activation of intracellular signaling molecules, STAT1 Tyr-701, STAT1 Ser-727, and Akt, but not p38, JNK, and ERK MAPK proteins, was significantly diminished in IFN-beta(-/-) versus IFN-beta(+/+) macrophages. "Priming" of IFN-beta(-/-) macrophages with exogenous recombinant IFN-beta significantly increased levels of LPS-induced gene expression for induction of monocyte chemotactic protein 5, inducible nitric-oxide synthase, IP-10, and IL-12 p40 mRNA, whereas no increase or relatively small increases were observed for IL-1beta, IL-6, monocyte chemotactic protein 1, and MyD88 mRNA. Finally, IFN-beta(-/-) mice challenged in vivo with LPS exhibited increased survival when compared with wild-type IFN-beta(+/+) controls, indicating that IFN-beta contributes to LPS-induced lethality; however, not to the extent that one observes in mice with more complete pathway deficiencies (e.g. TLR4(-/-) or TRIF(-/-) mice). Collectively, these findings reveal unanticipated regulatory roles for IFN-beta in response to LPS in vitro and in vivo.

Differentiation of Crude Lipopolysaccharides from Escherichia coli Strains Using Fourier Transform Infrared Spectroscopy and Chemometrics

ABSTRACTCrude phenol‐phase extracts containing bacterial lipopolysaccharides (LPS) from 5 strains of Escherichia coli were investigated to differentiate the strains using Fourier transform infrared (FTIR) spectroscopy and multivariate statistical analysis. The strains used were E. coli K12, E. coli DH5α, E. coli O157:H7, E. coli O157:H12, and E. coli O157:H19. LPS‐containing extracts were isolated from each E. coli strain using a hot phenol‐water extraction procedure. The extracts were 1st analyzed by deoxycholic acid‐polyacrylamide gel electrophoresis and visualized by silver‐staining. Analysis of the extracts from E. coli K12 and E. coli DH5α showed rough‐type LPS on the lower half of the gel, whereas E. coli O157:H7, E. coli O157:H12, and E. coli O157:H19 yielded abundant smooth LPS (high‐molecular‐weight LPS that include the O‐polysaccharides). Spectra (4000 cm‐1 to 700 cm‐1) of crude E. coli LPS extracts and intact cells were collected using a FTIR spectrometer. Spectral data were compressed by principle component analysis and analyzed using canonical variate analysis (CVA) of 4000 cm‐1 to 700 cm‐1 or 1200 cm‐1 to 900 cm‐1 spectral regions. CVA showed better separation between strains using LPS extracts than intact cells in the 1200 cm‐1 to 900 cm‐1 spectral region. The same separation trend was found using Mahalanobis distances that quantified spectral differences between the E. coli strains, providing 80% and &gt;95% correct classifications of intact cells and LPS extracts, respectively. This article is the first to report the successful differentiation of E. coli strains at a serotype level using FTIR spectra of bacterial phenol‐phase extracts (crude LPS preparations).

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 expresses the monofucosyl A type 1 histo-blood group epitope.

The lipopolysaccharide of Helicobacter mustelae type strain ATCC 43772 was obtained by phenol-water extraction of bacterial cells. Structural investigations were made on the lipid A free saccharide moiety released from the lipopolysaccharide by mild acetic acid hydrolysis. Nuclear magnetic resonance, gas liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry were employed in the characterization of products from chemical manipulations. A monoclonal antibody specific for blood group A reacted strongly with lipopolysaccharide of H. mustelae. Chemical and serological data showed that the outer core region of the lipopolysaccharide from H. mustelae ATCC 43772 expresses the monofucosyl A type 1, alpha-D-GalNAc-(1-->3)-[alpha-L-Fuc-(1-->2]-beta-D-Gal-(1-->3)- beta-D-GlcNAc, blood group determinant, a mimic of animal cell surface glycolipids and glycoproteins.

The structure of the lipopolysaccharide from a galU mutant of Pseudomonas aeruginosa serogroup-O11

The lipopolysaccharide (LPS) of a galU mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain, was sequentially extracted with phenol–chloroform–petroleum ether (PCP) followed by hot phenol–water extraction of the bacterial pellet remaining after PCP extraction. LPS was found in both the PCP extract as well as in the water phase of the hot phenol–water extract. Analysis of the carbohydrate portion released by mild acid hydrolysis of both LPS preparations, both before and after removal of all phosphate groups by treatment with aqueous HF, was performed by glycosyl composition and linkage analyses as well as by NMR and mass spectrometric analyses. The results showed that the carbohydrate portion of these two LPS extracts contained the same structure: namely, α-GalN(Ala)-(1→3)-α-(7-Cm)HepII-(1→3)-α-HepI-(1→5)-α-Kdo-(2→. The oligosaccharide preparation from PCP-extracted LPS consisted of a variety of structures containing up to six phosphate groups present as mono-, pyro-, and possibly triphosphate, primarily located on the HepI residue with some molecules having a monophosphate on HepII. The oligosaccharide preparation from the hot phenol–water-extracted LPS contained a similar variety of structures, but with an additional structure in which HepI contained a PPEA group at O-2. In addition, PAGE immunoblot analysis of the crude cellular extract with anti-A-antibodies revealed the presence of A-band material in both PA103 and the galU mutant. The A-band material was purified and characterized by glycosyl composition and linkage analyses, as well as by NMR spectroscopy, which confirmed that the A-band rhamnan polysaccharide was present but not as typical LPS since lipid-A or LPS core oligosaccharide components were not detected.

Development, characterization and diagnostic application of a monoclonal antibody specific for a proteinase K resistant Lawsonia intracellularis antigen

Proliferative enteropathy (PE) is one of the most important infections in pigs caused by Lawsonia intracellularis, an obligate intracellular bacterium. The purpose of the present investigation was to develop monoclonal antibodies with specificity to L. intracellularis useful both for diagnostic purposes (by immunohistochemistry) and for bacterial characterization. Several antibody producing hybridomas were established by fusion of mouse myeloma with spleen cells from BALB/c mice immunized with mucosa scrapings of the intestinal mucosa from a L. intracellularis infected pig. A monoclonal antibody (mAb), Law1-DK, isotyped as IgG2b was selected by indirect immunofluorescence antibody test (IFAT). Histological sections of the intestines from pigs affected by proliferative enteropathy and in vitro grown bacteria in cell culture were tested positive for the presence of L. intracellularis with the mAb. A molecule at 21 kDa was recognized by the mAb in a Western blotting analysis when a whole-cell preparation of L. intracellularis was run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This antigen was released from L. intracellularis by mild heat treatment and was resistant to proteinase K digestion, suggesting it to be non-protein, e.g., lipopolysaccharide (LPS). This suggestion was supported by its presence in the aqueous phase of a phenol–water extract. The inhibitory effect of periodate oxidation on the antigen–antibody binding confirmed the participation of a carbohydrate epitope. The new mAb was tested highly specific for L. intracellularis by applying in situ hybridization with a L. intracellularis specific probe targeting 16S ribosomal RNA simultaneously with the IFAT.

Purification and characterisation of lipoglycan macroamphiphiles from Propionibacterium acnes.

Lipidated macroamphiphiles such as the lipoteichoic acids and mycobacterial lipoarabinomannans are cell envelope components of Gram-positive bacteria that have been extensively associated with the pathogenesis of disease. In order to study such associations, purification of these macroamphiphiles is essential for resolving their structures and diverse biological effects. We describe herein a method for purification of lipoglycan components from Propionibacterium acnes. This method uses the existing phenol-water extraction, followed by hydrophobic interaction chromatography and an additional purification step that utilises preparative electrophoresis for the separation of two lipoglycan components. Analysis of these lipoglycans revealed evidence for a lipid anchor based on fatty acids whilst the polysaccharide moiety contained significant amounts of mannose, glucose and galactose, together with an amino sugar suspected of being a diaminohexuronic acid. These latter components have been previously identified as components of the P. acnes cell wall polysaccharide. Consequently, it is proposed that there may be a relationship between the structures of these distinctive cell envelope polymers.

The structure of the glycopeptides from the fish pathogen Flavobacterium columnare

Proteolytic digestion of the phenol–water extraction product of the fish pathogen Flavobacterium columnare afforded a mixture of glycopeptides in which the oligosaccharide moiety was an unusual hexasaccharide composed of 4- O-methyl-2-acetamido-2-deoxy- d-glucuronic acid (GlcNAcA), d-glucuronic acid ( d-GlcA), 2,3-di- O-acetyl- d-xylose ( d-Xyl), 2- O-methyl- d-glucuronic acid ( d-GlcA), d-mannose ( d-Man), and 2- O-methyl- l-rhamnose ( l-Rha). By the application of high-resolution 1D and 2D NMR, mass spectrometry, and chemical analysis, the hexasaccharide structure was determined to be: where all monosaccharides have the d-configuration except for 2- O-methyl- l-rhamnose; and were in the pyranose form. Only one carbohydrate structure was found. The peptide part was represented by tri- to hepta-peptides with a minimal common tripeptide fragment Asp-Ser-Ala, extended with Ala and Val.

Development and evaluation of a mixed long-chain lipopolysaccharide based ELISA for serological surveillance of infection with Actinobacillus pleuropneumoniae serotypes 2, 6 and 12 in pig herds

The objective was to develop an enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of antibodies against Actinobacillus pleuropneumoniae (Ap) serotypes 2, 6 and 12. The assay was designated MIX-ELISA. Lipopolysaccharide (LPS) from Ap serotypes 2, 6 and 12 was purified using hot phenol–water extraction followed by fractionation by size-exclusion chromatography. A mixture of fractions containing molecules with molecular weight above 50 kDa from all three serotypes was used as antigen. The MIX-ELISA was evaluated with sera from pigs experimentally infected with the serotypes 1, 2, 5b, 6, 7, 8, 10 and 12 of Ap biotype 1. In addition to reaction with sera from pigs inoculated with Ap serotypes 2, 6 and 12, reaction was observed with sera from pigs inoculated with serotype 8. Furthermore, the sensitivity and specificity of the test on a herd level were evaluated with sera from herds naturally infected with serotypes 2, 6 or 12 and with sera from herds free of infection with any Ap serotype of biotype 1. The ELISA showed a high herd sensitivity (0.98; 95% confidence interval: 0.89–1.00) and specificity (0.95; 0.88–0.99). The high diagnostic sensitivity and specificity of the assay indicate that screening of herds for Ap infection can be performed using this ELISA. Efficient serological surveillance can be achieved by using such mixed antigen ELISAs coated with size-selected LPS-antigens from the most prevalent serotypes.

Structures of two polysaccharides of Campylobacter jejuni 81116

Campylobacter jejuni 81116 has been extensively investigated in studies on genes associated with the synthesis of Campylobacter lipopoly/lipooligosaccharides (LPS/LOS). Despite these investigations, data on the chemical structure of polysaccharides from C. jejuni 81116 have been absent. The present study was undertaken to fill that void. Biomass was grown in large quantities on agar medium, harvested and extracted by hot phenol–water extraction. Subsequently, extracts were treated by DNase, RNase and proteinase K to remove contaminants. After mild acid treatment, followed by preparative gel-permeation and anion-exchange chromatography, fractions were isolated and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H, 13C HMQC and HMBC experiments. These advanced investigations revealed the occurrence of two different polysaccharides in the approximate ratio of 3:1, each having a tetrasaccharide repeating unit. Polysaccharide A contained glucose, glucuronic acid and mannose, and is O-acetylated. Polysaccharide B contained glucose, galactose and N-acetylglucosamine. Importantly, polysaccharide A is acidic, whereas polysaccharide B is neutral.