Individual Polysaccharides Research Articles

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans.

Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), 0.05 M Na2CO3, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na2CO3, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides.

Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae.

Transfer of exponentially growing cells of the yeast Saccharomyces cerevisiae to hyperosmotic growth medium containing 0.7-1 M KCl, 1 M mannitol, and/or 1 M glycerol caused cessation of yeast growth for about 2 h; thereafter, growth resumed at almost the original rate. During this time, formation of fluorescent patches on the inner surface of cell walls stained with Primulin or Calcofluor white was observed. The fluorescent patches also formed in solutions of KCl or when synthesis of the cell wall was blocked with cycloheximide and/or 2-deoxyglucose. The patches gradually disappeared as the cells resumed growth, and the new buds had smooth cell walls. Electron microscopy of freeze-etched replicas of osmotically stressed cells revealed deep plasma membrane invaginations filled from the periplasmic side with an amorphous cell wall material that appeared to correspond to the fluorescent patches on the cell surface. The rate of incorporation of D-[U-14C]glucose from the growth medium into the individual cell wall polysaccharides during osmotic shock followed the growth kinetics. No differences in cell wall composition between osmotically stressed yeast and control cells were found. Hyperosmotic shock caused changes in cytoskeletal elements, as demonstrated by the disappearance of microtubules and actin microfilaments. After 2-3 h in hyperosmotic medium, both microtubules and microfilaments regenerated to their original polarized forms and the actin patches resumed their positions at the apices of growing buds. The response of S. cerevisiae strains with mutations in the osmosensing pathway genes hog1 and pbs2 to hyperosmotic shock was similar to that of the wild-type strain. We conclude that, besides causing a temporary disassembling of the cytoskeleton, hyperosmotic shock induces a change in the organization of the cell wall, apparently resulting from the displacement of periplasmic and cell wall matrix material into invaginations of the plasma membrane created by the plasmolysis.

Development and Validation of an NMR-Based Identity Assay for Bacterial Polysaccharides

A method utilizing NMR spectroscopy has been developed to confirm the identity of bacterial polysaccharides used to formulate a polyvalent pneumococcal polysaccharide vaccine. The method is based on 600 MHz proton NMR spectra of individual serotype-specific polysaccharides. A portion of the anomeric region of each spectrum (5.89 to 4.64 ppm) is compared to spectra generated for designated reference samples for each polysaccharide of interest. The selected region offers a spectral window that is unique to a given polysaccharide and is sensitive to any structural alteration of the repeating units. The similarity of any two spectral profiles is evaluated using a correlation coefficient (ρ), where ρ ≥ 0.95 between a sample and reference profile indicates a positive identification of the sample polysaccharide. This method has been shown to be extremely selective in its ability to discriminate between serotype-specific polysaccharides, some of which differ by no more than a single glycosidic linkage. Furthermore, the method is rapid and does not require extensive sample manipulations or pretreatments. The method was validated as a qualitative identity assay and will be incorporated into routine quality control testing of polysaccharide powders to be used in preparation of the polyvalent pneumococcal vaccine PNEUMOVAX 23. The specificity and reproducibility of the NMR-based identity assay is superior to the currently used colorimetric assays and can be readily adapted for use with other bacterial polysaccharide preparations as well.

Study of cryostructuration of polymer systems. XVIII. Freeze-thaw influence on water-solubilized artificial mixtures of amylopectin and amylose

The effects caused by cryogenic treatment (freezing-frozen storage-thawing) of 0.5-2.0 g/dL solutions of individual starch polysaccharides, namely, amylopectin (solvents: water or 0.35M NaCl aqueous solution) and amylose (solvent: 0.35M NaCl aqueous solution), and also of 0.5 g/dL solutions of their artificial mixtures with various amylopectin/amylose ratios were studied. Freezing of these systems at temperatures from -6 to -24°C for 18 h and subsequent thawing resulted in the formation of cryogelled or cryoprecipitated matter whose morphology and yield depended on the type of polysaccharide, initial polymer concentration, portion of each macromolecular component in the mixed systems, and conditions of cryogenic treatment. Comparison of the process efficiency in the mixed systems during the formation of precipitates (from storage at room temperature) and cryoprecipitates (as a result of cryogenic treatment) revealed the promoting effects of the freezing-thawing influence on polymer-polymer association and showed the presence of some synergism in the mutual interaction of these polysaccharides.

Hyaluronate-alginate combination for the preparation of new biomaterials: investigation of the behaviour in aqueous solutions

With the aim of producing a biomaterial for surgical applications, the alginate-hyaluronate association has been investigated. Crossed techniques were used to assess the existence of polymer interactions in aqueous solutions up to 20 mg/ml. Alginate was obtained from algae and hyaluronate was purified from rooster comb. Viscometry measurements using the capillary technique or the Couette flow, together with circular dichroism investigations, evidenced the moderate significance of interactions between the two polysaccharides in dilute solutions. In addition, the case of more concentrated solutions and containing 20 mg/ml alginate was approached by rheological measurements in the flow mode; the behaviour of the polymer associations appeared as a compromise between those of individual polysaccharides.

Imaging polysaccharides by atomic force microscopy.

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides l-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by kappa-carrageenan and gellan gum.

Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring.

Many common, biologically important polysaccharides contain pyranose rings made of five carbon atoms and one oxygen atom. They occur in a variety of cellular structures, where they are often subjected to considerable tensile stress. The polysaccharides are thought to respond to this stress by elastic deformation, but the underlying molecular rearrangements allowing such a response remain poorly understood. It is typically assumed, however, that the pyranose ring structure is inelastic and locked into a chair-like conformation. Here we describe single-molecule force measurements on individual polysaccharides that identify the pyranose rings as the structural unit controlling the molecule's elasticity. In particular, we find that the enthalpic component of the polymer elasticity of amylose, dextran and pullulan is eliminated once their pyranose rings are cleaved. We interpret these observations as indicating that the elasticity of the three polysaccharides results from a force-induced elongation of the ring structure and a final transition from a chair-like to a boat-like conformation. We expect that the force-induced deformation of pyranose rings reported here plays an important role in accommodating mechanical stresses and modulating ligand binding in biological systems.

Complex serology and immune response of mice to variant high-molecular-weight O polysaccharides isolated from Pseudomonas aeruginosa serogroup O2 strains.

The O antigen of the Pseudomonas aeruginosa lipopolysaccharide is the optimal target for protective antibodies, but the unusual and complex nature of their sugar substituents has made it difficult to define the range of these structures needed in an effective vaccine. Most clinical isolates of P. aeruginosa can be classified into 10 O-antigen serogroups, but slight chemical differences among O polysaccharides within a serogroup give rise to subtype epitopes. These epitopes could impact the reactivity of O-antigen-specific antibodies, as well as the susceptibility of a target strain to protective, opsonic antibodies. To define parameters of serogroup and subtype-epitope immunogenicity, antigenicity, and surface expression on P. aeruginosa cells, we prepared high-molecular-weight O-polysaccharide vaccines from strains of P. aeruginosa serogroup O2, for which eight structurally variant O antigens expressing six defined subtype epitopes (O2a to O2f) have been identified. A complex pattern of immune responses to these antigens was observed following vaccination of mice. The high-molecular-weight O polysaccharides were generally more immunogenic at low doses (1 and 10 microg) than at a high dose (50 microg) and usually elicited antibodies that opsonized the homologous strain for phagocytic killing. Some of the individual polysaccharides elicited cross-opsonic antibodies to a variable number of strains that express all of the defined serogroup O2 subtype epitopes. Combination into one vaccine of two antigens that individually elicited cross-reactive opsonic antibodies to most members of the O2 serogroup inhibited, instead of enhanced, the production of antibodies broadly reactive with most serogroup O2 subtype strains. Thus, immune responses to P. aeruginosa O antigens may be restricted to a limited range of epitopes on structurally complex O antigens, and combining multiple related antigens into a single vaccine formulation may inhibit the production of those antibodies best able to protect against most P. aeruginosa strains within a given O-antigen serogroup.

Germination triggers of zoospore cysts of Aphanomyces euteiches and Phytophthora parasitica

Zoospore cysts of four isolates of Aphanomyces euteiches with different host-specific pathogenicity germinated in response to peptone, pea root extract, CaCl2 or gum arabic but, in tests on one isolate, did not respond to several individual sugars, amino acids or polysaccharides. A. euteiches showed differential responses to substances applied at different times in the encystment process. Gum arabic triggered germination only when added to motile zoospores, which it caused to encyst. CaCl2 triggered germination when added to motile zoospores or during vortex-encystment of zoospores, but seldom immediately ( Zoospore taxis, encystment and germination of A. euteiches and P. parasitica cysts were recorded on pea roots by video microscopy. The speed of germination and pattern of sporeling development on roots were most closely matched by treatment of motile spores with gum arabic in pure culture. The behaviour of A. euteiches conformed to a previous model for Pythium and Phytophthora spp., in which zoospores encyst by recognition of root surface components (simulated by gum arabic), germinate autonomously by a calcium-mediated process (simulated by calcium treatment during vortexing), and the germ-tube produces an assimilative hypha (simulated by peptone or pea root extract).

Immune response to the 23-valent pneumococcal polysaccharide vaccine in lymphoma patients and patients with chronic renal diseases.

Lymphoma patients and patients suffering from chronic renal diseases exhibit an increased risk for pneumococcal infections. Therefore, vaccination with the polyvalent pneumococcal vaccine of such patients is recommended (1). This pneumococcal vaccine consists of purified capsular polysaccharides prepared from 23 types of Streptococcus pneumoniae. A “protective antibody level” is still not clearly defined. For routine estimations, we assume an at least fourfold rise in antibodies to the 23-valent vaccine as a positive immune response when measured in an ELISA using the derivatized whole vaccine as antigen four weeks post immunization as compared with the preimmunization level. Antibodies developed to individual capsular polysaccharides and to the cell wall polysaccharide (discussed as a contaminant of the vaccine) were estimated to compare their relative immunogenicity.

Investigation of Gellan Networks and Gels by Atomic Force Microscopy

Methods previously developed for the atomic force microscopy (AFM) imaging of individual polysaccharides (Kirby, A. R.; Gunning, A. P.; Morris, V. J. Biopolymers 1996, 38, 355−366) have been extended in order to image networks and gels formed by the bacterial polysaccharide gellan gum. Networks were formed by air-drying solutions of K+ gellan onto freshly cleaved mica. AFM images were obtained in the direct current contact mode at constant force under butanol. Network formation can be inhibited by diluting the stock gellan solution or by converting K+ gellan into the tetramethylammonium salt form. Inhibition of network formation led to AFM images of heterogeneous populations of gellan aggregates (gel precursors). Attempts have been made to image the surface of bulk aqueous gellan gels under butanol. The quality of the images obtained improved with increasing gel modulus. For rigid acid-set gellan gels, molecular resolution was achieved, revealing a bifurcated branched fibrous network.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction.

Chemical composition in varieties of rapeseed and turnip rapeseed, including several samples of hull and dehulled seed

AbstractVarieties of spring rapeseed (n = 10), winter rapeseed (n = 12), spring turnip rapeseed (n = 11) and winter turnip rapeseed (n = 3) were analysed for contents of dietary fibre (DF) constituents, crude fat, crude protein and ash. Most of the varieties were of the double‐low type but four single‐low winter rapeseed varieties and seven yellow‐seeded (triple‐low) varieties were also investigated. Principal component analysis (PCA) made it possible to divide the varieties into different groups. One group, including winter rapeseed varieties and the winter turnip rapeseed variety Kova, generally had high crude fat contents (432‐492 g kg−1 whole seed, DM), low crude protein (181‐221 g kg−1) and ash contents (32‐38 g kg−1). Another group, consisting of the yellow‐seeded spring turnip rapeseed varieties, had a lower mean content of DF (150 g kg−1), due to low content of Klason lignin, than the other categories of varieties (mean contents: 176‐183 g kg1‐). Spring rapeseed varieties generally exhibited lower values of neutral polysaccharide residues than the other varieties studied. It was shown—using PCA—that the individual neutral polysaccharide residues primarily varied in unison. In addition, the presence of fucose‐containing xyloglucan, pectin and galacto‐glucomannan in the seeds was indicated. DF content in manually prepared hulls and dehulled seeds was determined for 10 varieties. The content of dietary fibre polysaccharide residues (DFPR) in the dehulled seeds of spring rape was low (range: 59‐71 g kg−1) compared with the other varieties analysed (76–87 g kg−1). The content of Klason lignin in yellow hulls was considerably lower (48–63 g kg−1) than in dark hulls (224–330 g kg−1). Yellow hulls instead had a higher DFPR content (491‐497 g kg−1) than dark hulls (341–401 g kg−1).

Immunogenic and antigenic properties of a heptavalent high-molecular-weight O-polysaccharide vaccine derived from Pseudomonas aeruginosa.

We investigated the chemical and immunologic properties of a heptavalent vaccine composed of high-molecular-weight polymers of the lipopolysaccharide (LPS) O polysaccharides representative of the most common clinical isolates of Pseudomonas aeruginosa. We also evaluated the serum antibody response to nonvaccine strains of P. aeruginosa, including strains expressing structural variants (subtype strains) of the O side chain of the vaccine strains. The polyvalent vaccine, prepared under conditions suitable for human use, contained low levels of contaminants and passed preclinical safety and toxicity tests required for human use. Chemical analyses indicated that individual polysaccharides were composed of both O-side chain and core sugars. Following immunization of C3H/HeN mice and New Zealand White rabbits, antibody titers against vaccine components increased between 32- and 200-fold. Antibodies reactive with LPS isolated from smooth and rough nonvaccine strains were also elicited. Analysis of the opsonic activity against the known LPS subtype variants of the vaccine strains revealed a variable pattern of killing, which ranged from opsonic killing of > or = 69% of bacterial cells representing all subtype variants within a serogroup to opsonization of only a minority of the subtype variant strains. Mouse and rabbit immune sera showed different patterns of opsonic activity against subtype strains, indicating that different epitopes on these antigens are immunodominant in the representatives of these two animal species tested. The polyvalent vaccine was effective at eliciting antibodies to vaccine components in mice and rabbits, but it remains to be determined if the current heptavalent formulation contains sufficient components to provoke human antibodies reactive with a majority of clinical strains of P. aeruginosa.

Neonatal mouse protection against infection with multiple group B streptococcal (GBS) serotypes by maternal immunization with a tetravalent GBS polysaccharide-tetanus toxoid conjugate vaccine

Most cases of neonatal sepsis and meningitis caused by group B streptococci (GBS) are attributable to one of four major capsular serotypes: Ia, Ib, II, or III. Because resistance to infection with GBS has been correlated with the presence of serum antibodies to the type-specific capsular polysaccharides in both experimental animals and human neonates, efforts have been made to elicit protective immunity with GBS capsular polysaccharide vaccines. However, the GBS capsular polysaccharides alone are not highly immunogenic in either animals or human volunteers. Therefore, we and other investigators have attempted to enhance immunogenicity by coupling individual capsular polysaccharides to a carrier protein. Here we report the synthesis and immunogenicity in rabbits of a GBS type Ib polysaccharide-tetanus toxoid vaccine prepared by the direct, covalent attachment of tetanus toxoid to a selected number of sialic acid residues on the type-specific polysaccharide. In addition, the Ib polysaccharide-tetanus toxoid conjugate vaccine was combined with similar tetanus toxoid conjugates of GBS type Ia, II, and III polysaccharides to form a tetravalent GBS conjugate vaccine. Protective efficacy of the GBS tetravalent conjugate vaccine was demonstrated in a mouse maternal immunization-neonatal challenge model of GBS infection. The results support testing in human subjects of a multivalent GBS conjugate vaccine of this design, with the eventual goal of protecting newborns against GBS infection.

Immune responses of systemic and mucosal lymphoid organs to Pnu-Imune vaccine as a function of age and the efficacy of monophosphoryl lipid A as an adjuvant.

A murine model system was established to study immune responses to the Pnu-Imune vaccine, which is made up of 23 different pneumococcal capsular polysaccharides. In this animal model, antibody-forming cell responses to 21 of 23 individual polysaccharides in the vaccine were detected. The Pnu-Imune vaccine elicited good antibody responses from the spleens and mesenteric lymph nodes (MLN) of young mice, whereas a variety of other peripheral lymph nodes were unresponsive. The immunoglobulin M plaque-forming cell (PFC) response in the spleen to the Pnu-Imune vaccine (given intraperitoneally or subcutaneously) decreased dramatically with increasing age. However, the spleen and MLN differed in their susceptibility to an age-associated decline in immune function. While the PFC responses in the spleen declined with age, the PFC response in the mucosa-associated MLN did not decline with age but instead remained constant over the entire age span of 4 to 28 months studied. These studies showed that the spleen, peripheral lymph nodes, and MLN did not demonstrate parallel age-associated defects in antibody responses to pneumococcal polysaccharides when the antigen was administered systematically. Also, the deficient splenic antibody response to Pnu-Imune vaccine in aged mice could be enhanced by injecting a combination of Pnu-Imune vaccine and the nontoxic adjuvant monophosphoryl lipid A. Moreover, an immunoglobulin G response was induced when the immunogen was a mixture of vaccine and adjuvant.

Computer modeling of polysaccharide-polysaccharide interactions: an approach to the kappa-carrageenan-mannan case.

A computer program SAINT has been developed for the investigation of the structure and for the prediction of minimum-energy structure of polysaccharide-polysaccharide complexes. The energy minimization is carried out on internal geometrical parameters--namely bond angles, torsional angles, and five parameters describing the mutual orientations of polysaccharide chains. For this purpose, the nonderivative method of conjugated directions is used. This procedure was applied to computer modeling of an idealized model of the binary gelling kappa-carrageenan and galactomannan system. It is shown that the interaction between two chains influences the structure of the individual polysaccharide molecule and that in the minimum-energy structures of the complex, the conformation of the chains does not correspond to the lowest energy.

Murine monoclonal antibodies against pneumococcal capsular polysaccharide types 4, 8, 22F and 19A/19F.

Monoclonal antibodies (MAbs) were produced against pneumococcal capsular polysaccharides after subcutaneous immunization of BALB/c mice with a 23-valent vaccine (Pneumovax N, Merck, Sharp & Dohme). Selected antibodies were tested in ELISA against individual polysaccharides from 23 different pneumococcal types and in a dot blot assay with heat-killed whole bacteria adhered to nitrocellulose paper. Three MAbs (isotype IgM) were found to be specific for types 4, 8 and 22F, respectively, whereas one (isotype IgA) reacted both with 19A and 19F. Very mild acid hydrolysis of the capsular polysaccharides resulted in loss of reaction with the antibodies.

Structural Polysaccharides in Forages and Their Degradability

AbstractPlant cell walls are organized into complex matrices composed primarily of carbohydrates that represent a potential energy source for ruminants. An overview of the basic structural features of the major groups of polysaccharides that make up forage cell walls, metabolic changes during cell wall development, cell wall component interactions, and some aspects of cell wall degradation are presented. Polysaccharide components that compose the cell wall matrix can be disassociated and fractionated according to similar physio‐chemical properties. Plant structural polysaccharides vary in monosaccharide composition, substitution patterns, and glycosidic linkages between monomers. Chemical fractionation schemes typically separate structural polysaccharides into three major groups. These include pectic polysaccharides rich in galacturonic acid residues, hemicellulosic polysaccharides typically rich in xylose, and the cellulose fraction. Mature walls can be envisioned as a layered matrix with cellulose microfibrils embedded in an amorphous material composed of pectic and hemicellulosic polysaccharides. The amorphous regions of cell walls show the greatest variation during development. Interactions among individual components of the cell wall are key to the formation and structural integrity of the matrix. These include bonding interactions that range from weak hydrogen bonds to covalent cross‐links. During growth of plant cells there is a rearrangement of structural elements within the wall. The matrix undergoes reorganization by disruption of selected intra‐ and intermolecular bonds and de novo synthesis and insertion of new wall polymers. Individual polysaccharides may vary in rates and possibly extent of degradation by rumen microorganisms. These differences are due to the degree of hydrogen bonding of the individual polysaccharides, branching patterns, and associations with other wall constituents.

Antibody response to 14-valent pneumococcal capsular polysaccharide vaccine in pre-school age children

Antibody responses to 14-valent pneumococcal capsular polysaccharide vaccine were measured by Farr-type radioimmunoassay in children younger than 7 years of age. On the basis of immunogenicity in young children individual pneumococcal polysaccharides could be identified as uniformly good, strongly age-dependent or uniformly poor immunogens. Pneumococcal types 6A and 23F, which frequently cause pneumococcal infections in small children, were the poorest immunogens in this age group. The children younger than 2 years of age responded very poorly also to types 19F and 18C whereas older children had good antibody responses to these types. The results support the current view that present pneumococcal polysaccharide vaccines are not beneficial in children younger than 2 years of age and stress the importance of attempts to improve their immunogenicity.