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PubMed1999

A dye binding assay for the quantification of soluble and cell-bound acidic polysaccharides produced by red algae.

Polysaccharides produced in technical scale from red algae serve in a variety of industrial applications. Ruthenium red (RR) is a cytochemical stain which has been used to visualize these mostly acidic polysaccharides in light microscopy. The binding of RR to algal polysaccharides is highly dependent on the ionic strength of the surrounding medium. The dye is firmly bound at low ionic strength, but is released at increasing salt concentrations. These properties were exploited to develop a new method to quantify the amount of cell wall material synthesized by algal cells under different physiological conditions. Soluble or extracted polysaccharides are quantified by a dot-blot procedure with subsequent image analysis, while cell-bound polysaccharides are determined in situ by a dye-binding assay followed by photometry. In comparison to the current methods of quantification, the new assay is quick, reliable, and avoids extensive use of hazardous chemicals.

Calibration↗
PubMed1996

Peptide libraries define the fine specificity of anti-polysaccharide antibodies to Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated fungus that causes a life-threatening meningoencephalitis in patients with AIDS. Monoclonal antibodies to the capsular glucuronoxylomannan can modulate the infection in mice, but the epitopes on this complex polysaccharide recognized by protective and non-protective antibodies have not been defined. We have used 2H1, one of our most protective antibodies, to screen phage display peptide libraries for peptide mimotopes that would allow us to explore the fine specificity of anti-cryptococcal polysaccharide antibodies. Hexa- and decapeptides have been identified with sequence homologies that define four motifs: 1, (E)TPXWM/LM/L; 2, W/YXWM/ LYE; 3, DWXDW; and 4, (Ar)WDGQ(Ar). Peptides representing these motifs compete with each other for a shared binding site that overlaps the polysaccharide binding site. Motifs 1 and 2 confer high affinity binding, and PA1, which displays a motif 1 peptide with the sequence LQYTPSWMLV, binds to 2H1 with a Kd of 295 nM. Analysis of the interaction between the 2H1 binding peptides and 24 structurally related anti-polysaccharide antibodies reveals a complex pattern of reactivity that strongly suggests binding to or close to the complementary determining regions. Furthermore, those antibodies that have been shown to have different specificity, and in some cases different protective potential, do not bind any of the peptides selected by the protective 2H1 antibody. This study shows that peptide mimotopes for a complex microbial polysaccharide can be identified by screening phage peptide libraries and demonstrates the usefulness of such peptides in analyzing closely related interactive sites of proteins in general and of antibodies in particular.

Amino Acid Sequence↗
PubMed1990

Pneumococcal C and type polysaccharide detection in the concentrated urine of patients with bacteremia.

The C polysaccharide of Streptococcus pneumoniae was detected in the concentrated urine of 23 of 33 patients with pneumococcal bacteremia using latex agglutination. Type-specific polysaccharides were detected in the urine of 17 of these 33 patients including 4 patients lacking C polysaccharide in their urine. These 4 with the 23 detected above gave a total sensitivity of 82% (27/33). The concentrated urine from an additional 11 patients with other bacteremias were tested by C polysaccharide and type-specific reagents and were negative. C polysaccharide detection in the concentrated urine of patients may be helpful in the diagnosis of pneumococcal infections.

Adolescent↗
PubMed1985

Isolation of an acidic polysaccharide antigen from Propionibacterium acnes.

An acidic polysaccharide antigen is released from Propionibacterium acnes I and II during growth. The molecular weight of the antigen was heterogeneous and when fractionated on a Sepharose CL-6B column, the antigen was detected at Kav values of between 0.1 and 1.0. The pI of the antigen was below 3.5. Rabbit antiserum raised against purified acidic-polysaccharide agglutinated P. acnes showing that the polysaccharide was a surface antigen. Human antibodies towards whole P. acnes-II organisms were quantitated by an agglutination technique, and antibodies towards purified acidic polysaccharide were quantitated by single radial immunodiffusion. A high prevalence of antibodies against whole bacteria and the acidic polysaccharide was found in sera from healthy individuals. The antibody titre in sera from acne patients was significantly higher than that in sera from blood donors.

Acne Vulgaris↗
PubMed1991

Effects of base-soluble proteins and methanol-soluble polysaccharides from corn on mycelial growth of Aspergillus flavus.

The antifungal activity of base-soluble proteins (BSP) and methanol-soluble polysaccharides (PS) from A. flavus resistant (Yellow Creole) and susceptible (Huffman) genotypes of corn were investigated by in vitro studies. Bioassays of fungal growth inhibition in agar media showed antifungal activity by proteins and polysaccharides only from the Huffman genotype. Microgramme quantities of protein and polysaccharides were required to retard fungal growth. The polysaccharides have molecular weights greater than 3.5 kilodaltons. Cathodic PAGE of native protein from the two genotypes showed six protein bands with differences in staining intensity of individual components. SDS-PAGE showed four distinct bands in Yellow Creole that were absent in Huffman. Both of the protein samples contained traces of carbohydrate. Analysis of hydrolyzed polysaccharide from the two genotypes showed different proportions of mannose and glucose.

Aspergillus flavus↗
PubMed1996

High-field NMR as a technique for the determination of polysaccharide structures.

NMR spectroscopy has played a developing role in the study of polysaccharide structures for over 30 years. Many new bacterial polysaccharide repeat unit structures have recently been published as a result of the application of modern NMR techniques. NMR can also be used to elucidate the structures of both regular and heterogeneous polysaccharides from fungal and plant sources, as well as complex glycosaminoglycans of animal origin. In addition to covalent structure, conformation and dynamics of polysaccharides are susceptible to NMR analysis, both in solution and in the solid state. Improvements in NMR technology with potential applications to polysaccharide studies hold promise for the future.

Animals↗
PubMed2000

Heterogeneity of the genus Myrothecium as revealed by cell wall polysaccharides.

The polysaccharides obtained from the alkali-extractable, water-soluble fraction (F1SS) from the cell wall of Myrothecium verrucaria and Myrothecium atroviride were shown to be composed of beta-(1-->6)-galactofuranose fully substituted at O-2 by terminal residues of alpha-glucopyranose and alpha-glucuronic acid. Glucuronic acid was substituted at O-4 by glucopyranose in the Myrothecium species M. inundatum, M. setiramosum, M. prestonii, M. tongaense and M. roridum. The acidic polysaccharides from Phaeostilbella atra (=Myrothecium atrum) and Myrothecium gramineum lacked the backbone of 2,6 di-O-substituted galactofuranose and contained a high amount of O-5-substituted beta-galactofuranose. The structures of the polysaccharides isolated from Myrothecium cinctum and Myrothecium penicilloides were unrelated to each other and to the polysaccharides from the other species analysed. The usefulness of these polysaccharides as a characteristic for delimitation of the genus Myrothecium is discussed.

Ascomycota↗
PubMed2001

Strains degrading polysaccharides produced by bacteria from paper machines.

Biofilm-degrading enzymes are potential agents for slime control in paper machines. In this work, extracellular polysaccharides were produced by bacteria isolated from paper machines and the isolated polysaccharides were used as substrates for the screening of polysaccharide-degrading microbes. Polysaccharide yields of 1.5-3.5 g/l were obtained by ethanol precipitation from cultures of strains of Klebsiella pneumoniae, Bacillus licheniformis and Pseudomonas fluorescens on sucrose medium. Two K. pneumoniae strains apparently produced an identical heteropolysaccharide containing galacturonic acid. Fructose-containing polysaccharides were the main products of B. licheniformis and P. fluorescens. Bacteria capable of hydrolyzing the fructose-containing polymers (levans) appeared to be relatively common among the strains selected for screening. None of the bacteria or mixed cultures screened were able to utilize the Klebsiella heteropolysaccharides.

Bacteria↗
PubMed2003

Localisation and characterisation of cell wall mannan polysaccharides in Arabidopsis thaliana.

Polysaccharides containing beta-1,4-mannosyl residues (mannans) are abundant in the lignified secondary cell walls of gymnosperms, and are also found as major seed storage polysaccharides in some plants, such as legume species. Although they have been found in a variety of angiosperm tissues, little is known about their presence and tissue localisation in the model angiosperm, Arabidopsis thaliana (L.) Heynh. In this study, antibodies that specifically recognised mannans in competitive ELISA experiments were raised in rabbits. Using these antibodies, we showed that Golgi-rich vesicles derived from Arabidopsis callus were able to synthesise mannan polysaccharides in vitro. Immunofluorescence light microscopy and immunogold electron microscopy of Arabidopsis inflorescence stem sections revealed that the mannan polysaccharide epitopes were localised in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfascicular fibres. Similarly, mannan epitopes were present in the xylem of the leaf vascular bundles. Surprisingly, the thickened epidermal cell walls of both leaves and stems also contained abundant mannan epitopes. Low levels were observed in most other cell types examined. Thus, mannans are widespread in Arabidopsis tissues, and may be of particular significance in both lignified and non-lignified thickened cell walls. Polysaccharide analysis using carbohydrate gel electrophoresis (PACE) of cell wall preparations digested with a specific mannanase showed that there is glucomannan in inflorescence stems. The findings show that Arabidopsis can be used as a model plant in studies of the synthesis and functions of mannans.

Arabidopsis↗
PubMed2007

Secretion marker proteins and cell-wall polysaccharides move through different secretory pathways.

The building up of the cell wall is tightly dependent on the functionality of the secretory pathway. Syntaxins as well as other SNARE proteins play important roles during vesicle secretion and fusion. We have compared the secretion of newly synthesised cell-wall polysaccharides to that of secretory marker proteins such as secreted green-fluorescent protein (sec-GFP) and secreted rat preputial beta-glucuronidase (secRGUS) in leaf protoplasts and roots of wild-type and transgenic Nicotiana tabacum plants, overexpressing a syntaxin homologue NtSyr1 (Sp1) and its soluble variant Sp2 that interferes specifically with Sp1 function, affecting post-Golgi transport. In protoplasts transiently transformed with secGFP and Sp1, no variation was observed in the pattern of fluorescence with respect to control; on the contrary, GFP fluorescence accumulate within the cells in protoplasts co-transformed with secGFP and Sp2. Sp2 reduced the percentage of marker protein secretion to 53% as quantified with secRGUS. In protoplasts obtained from leaves of wild-type and transformed tobacco plants expressing Sp1, Sp2 and Sp1 plus Sp2, no remarkable differences in the percentage of newly synthesised polysaccharides incorporated into the regenerating cell walls were observed. The same results were confirmed in roots of whole transformed seedlings. Tests with cytochalasin D (CD) showed a marked decrease in the amount of newly synthesised polysaccharides into the wall and a simultaneous sharp increase in membrane-associated polysaccharides. SecRGUS secretion was also inhibited by CD. The data indicate that marker proteins and matrix polysaccharides, as well as cellulose synthase complexes, are secreted through the involvement of different secretory machineries.

Carbon Radioisotopes↗
PubMed1983

A sensitive staining method for detecting acidic polysaccharides in cellulose acetate and agarose gels.

A sensitive staining method has been developed for the detection of acidic polysaccharides in cellulose acetate and agarose gels. The method is based on the precipitation of bovine serum albumin by acidic polysaccharides at acidic pH values and the subsequent staining of precipitated protein with amido black or Coomassie brilliant blue R-250 stains. The detection limit of acidic polysaccharides is 15-40 ng on cellulose acetate strips and 50-150 ng on agarose plates. The sensitivity of the described staining technique is of the same order for a wide range of acidic polysaccharides of different origin in contrast to Alcian blue and toluidine blue stains, which detect only mucopolysaccharides of animal origin at comparable levels. The method was also applied to the colorimetric quantitative determination of acidic polysaccharides after electrophoretic separation.

Colorimetry↗
PubMed1992

Carbohydrate composition analysis of bacterial polysaccharides: optimized acid hydrolysis conditions for HPAEC-PAD analysis.

The capsular polysaccharide from Haemophilus influenzae type b (polyribosyl ribitol-phosphate; PRP) and the capsular polysaccharides from Streptococcus pneumoniae types 6B, 14, 18C, and 23F (Pn6B, Pn14, Pn18C, and Pn23F) were subjected to acid hydrolysis using hydrofluoric (HF) and/or trifluoroacetic acid (TFA) and high-pH anion-exchange chromatography with pulsed amperometric detection in an effort to identify optimum hydrolysis conditions for composition analysis of their carbohydrate components. With the exception of PRP, composition analyses of polysaccharides containing a phosphate moiety in the repeating unit structure (Pn6B, Pn18C, and Pn23F) are significantly improved by subjecting the sample to HF hydrolysis (65 degrees C, 1 h) followed by TFA hydrolysis (98 degrees C, 16 h). This results in essentially quantitative hydrolysis of the phosphodiester bond to the carbohydrate components, which otherwise remained predominantly phosphorylated and poorly accounted for in the analysis. Optimum analysis of PRP was achieved following a 2-h hydrolysis with TFA at 80 degrees C, whereas Pn14 showed optimum results after a 16-h hydrolysis with TFA at 98 degrees C. These analyses also provide information about the relative susceptibility to acid hydrolysis of the various glycosidic and phosphodiester bonds in these polysaccharides, with evidence to suggest that the acid lability of a given bond can be dramatically different from one polysaccharide to another.

Bacteria↗
PubMed1992

Carbohydrate analysis of water-soluble uronic acid-containing polysaccharides with high-performance anion-exchange chromatography using methanolysis combined with TFA hydrolysis is superior to four other methods.

Sulfuric acid hydrolysis according to the Saeman procedure, TFA hydrolysis, and methanolysis combined with TFA hydrolysis were compared for the hydrolysis of water-soluble uronic acid-containing polysaccharides originating from fungi, plants, and animals. The constituent sugar residues released were subsequently analyzed by either conventional GLC analysis of alditol acetates or high-performance anion-exchange chromatography with pulsed-amperometric detection. It was shown that TFA hydrolysis alone is not sufficient for complete hydrolysis. Sulfuric acid hydrolysis of these polysaccharides resulted in low recoveries of 6-deoxy-sugar residues. Best results were obtained by methanolysis combined with TFA hydrolysis. Methanolysis with 2 M HCl prior to TFA hydrolysis resulted in complete liberation of monosaccharides from pectic material and from most fungal and animal polysaccharides tested. Any incomplete hydrolysis could be assessed easily by HPAEC, by the detection of characteristic oligomeric products, which is difficult using alternative methods currently in use. Methanolysis followed by TFA hydrolysis of 20 micrograms water-soluble uronic acid containing polysaccharides and subsequent analysis of the liberated sugar residues by HPAEC allowed us to determine the carbohydrate composition of these polysaccharides rapidly and accurately in one assay without the need for derivatization.

Animals↗
PubMed1985

Inhibition by cocoa extracts of biosynthesis of extracellular polysaccharide by human oral bacteria.

Extracts of defatted cocoa inhibited the biosynthesis of extracellular polysaccharide by both cell-free and cell-associated streptococcal glucosyltransferases, but did not affect growth or acid production. Both water-insoluble and water-soluble polysaccharide syntheses were inhibited, and the net result was a shift away from adherence-supportive polysaccharide. Inhibition was non-competitive with respect to sucrose. The inhibition of polysaccharide biosynthesis was observed also with Actinomyces species. The findings suggest that the inhibitory effects of cocoa on plaque accumulation and caries formation are due to inhibition of bacterial polysaccharide production.

Actinomyces↗
PubMed1983

Structural studies on an antitumor polysaccharide from Microellobosporia grisea.

The structure of the antitumor polysaccharide from the actinomycete Microellobosporia grisea has been investigated. By methylation and periodate-oxidation studies, the polysaccharide was shown to consist of (nonreducing) D-mannosyl groups, (1 leads to 4)-linked D-glucosyl residues, and 3,6-branched, (1 leads to 4)-linked D-glucosyl residues in the approximate molar ratios of 2:1:1. Periodate oxidation of the polysaccharide, followed by borohydride reduction and mild hydrolysis with acid yielded glycerol, erythritol, 2-O-beta-D-glucopyranosyl-D-erythritol, and 5-O-beta-D-glucopyranosyl-2,4-bis(hydroxymethyl)-1,3-dioxane, which were isolated in the molar ratios of 2.0:0.14:0.74:0.35. Partial hydrolysis of the polysaccharide gave alpha-D-Manp-(1 leads to 6)-D-Glcp, beta-D-Glcp-(1 leads to 4)-D-Glcp, alpha-D-Manp-(1 leads to 3)-D-Glcp, and beta-D-Glcp-(1 leads to 4)-[alpha-D-Manp-(1 leads to 3)-]-D-Glcp. From these results, it is proposed that the polysaccharide is mainly composed of tetrasaccharide repeating-units having the following structure. (formula: see text)

Actinomycetales Infections↗
PubMed1985

Bacteriophage degradation of Klebsiella K44 polysaccharide: an n.m.r. study and chemical proof of the position of the acetate group.

Bacteriophages (phi) have been used to degrade polysaccharides into oligosaccharides containing one or more of their repeating units. The capsular polysaccharide from Klebsiella K44 contains an acetate group, and n.m.r. spectroscopy and chemical methods have been employed to prove its linkage to O-6 of the 4-linked glucose residue. Phage phi 44 was shown to be an alpha-glucosidase not influenced by the acetate moiety and thus able to depolymerize the polysaccharide into pentasaccharide repeating units, some of which contained acetate on O-6 of the reducing glucose residue. The two oligosaccharides were studied by 1H- and 13C-n.m.r. spectroscopy, and their spectra were compared with those of the native and the deacetylated polysaccharide. 13C-n.m.r. was a useful tool for locating the 6-linked acetate, the position of which was confirmed by the method of temporary protection using methyl vinyl ether. The importance of using bacteriophages to obtain oligosaccharides is highlighted by the better results obtained with the oligosaccharide in comparison to the polysaccharide, both in n.m.r. spectroscopy and the temporary protection method.

Acetates↗
PubMed1987

Structure of the capsular polysaccharide of Klebsiella serotype K40.

The capsular polysaccharide from Klebsiella Serotype K40 contains D-galactose, D-mannose, L-rhamnose, and D-glucuronic acid in the ratios of 4:1:1:1. Methylation analysis of the native and carboxyl-reduced polysaccharide provided information about the glycosidic linkages in the repeating unit. Degradation of the permethylated polymer with base established the identity of the sugar unit preceding the glycosyluronic acid residue. The modes of linkages of different sugar residues were further confirmed by Smith degradation and partial hydrolysis of the K40 polysaccharide. The anomeric configurations of the different sugar residues were determined by oxidation of the peracetylated native and carboxyl-reduced polysaccharide with chromium trioxide. Based on all of these results, the heptasaccharide structure 1 was assigned to the repeating unit of the K40 polysaccharide. (Formula: see text)

Carbohydrate Conformation↗
PubMed1988

Structure and anti-complementary activity of pectic polysaccharides isolated from the root of Angelica acutiloba Kitagawa.

Four pectic polysaccharides (AR-2IIa-IId) with anti-complementary activity have been isolated from a hot-water extract of the root of Angelica acutiloba Kitagawa. Each of these polysaccharides contained a large proportion of GalA together with neutral sugars consisting mainly of Rha, Ara, and Gal. Digestion with endo-alpha-(1----4)-polygalacturonase indicated that AR-2IIa-IIc each contained a large proportion of enzyme-sensitive polygalacturonan regions, and that AR-2IId contained a large proportion of enzyme-resistant regions. When AR-2IId was de-esterified, it became sensitive to the enzyme. These polysaccharides also contained small proportions of enzyme-resistant regions (PG-1) which were rich in neutral sugars. Methylation analysis and base-catalysed beta-elimination studies suggested that each PG-1 contained a rhamnogalacturonan moiety in which 2,4-disubstituted Rha was attached to 4-substituted GalA through position 2 of Rha. Carboxyl-reduction and methyl- and de-esterification of these polysaccharides modulated their anti-complementary activities. Digestion with endo-alpha-(1----4)-polygalacturonase decreased the activities of AR-2IIa and -2IIb, but not those of AR-2IIc and -2IId. Although PG-1 fractions from AR-2IIa-IIc were more active than the original polysaccharides, oligogalacturonide fragments obtained by enzymic digestion had weak or negligible activity. AR-2IIa-IIc expressed their anti-complementary activities mainly via the classical pathway, but AR-2IId and each PG-1 expressed their activities via both the classical and alternative pathways.

Complement Activation↗