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PubMed1999

A new method of non-cross-linking conjugation of polysaccharides to proteins via thioether bonds for the preparation of saccharide-protein conjugate vaccines.

Bacterial polysaccharides, including capsular polysaccharides, are poor immunogens particularly in young infants. However, conjugation of bacterial polysaccharides to immunogenic carrier proteins generally results in conjugates that induce strong antipolysaccharide T-helper-cell dependent immune responses, also in young infants. The magnitude of the response and the extent of the T-helper-cell dependency is related to the chemical characteristics of the particular conjugate such as presence or absence of polysaccharide-protein cross-linking, presence or absence of spacer arms, character of spacer arms, type of carrier protein, size of conjugated polysaccharide hapten and molar degree of substitution. In the present study a new, general and simple method for the preparation of poly- and oligosaccharide-protein conjugates is presented. This new method is based on spacer-introducing chemistry that allowed for conjugation of a model polysaccharide, dextran, ranging in size from 0.5 to 150 kDa, to tetanus toxoid (TTd). The developed conjugation method involves derivatization of polysaccharide with 2-iminothiolane (2-IT) and activation of carrier protein, such as TTd, with N-hydroxysuccinimide ester of bromoacetic acid. Reaction rates and accordingly the substitution of the conjugates, could be controlled by varying time, pH and concentration of the reactants. Unlike direct reductive amination, the 2-IT based conjugation technology is fast and made it possible to couple fairly large polysaccharides to TTd.

Dextrans↗
PubMed2002

Ultrasound-assisted extraction of water-soluble polysaccharides from the roots of valerian (Valeriana officinalis L.).

The insoluble plant residues, obtained after preparation of medicinal tinctures from the roots of valerian (Valeriana officinalis L.) by classical and ultrasound-assisted extraction with aqueous ethanol in a pilot plant, were subsequently treated with hot water to isolate the accessible polysaccharide cell wall components. At almost equal amounts of the hot-water extractable material, the yields of the recovered polysaccharides were lower in the ultrasonical experiment. This is due to the fact that a part of accessible polysaccharides were already solubilised by the aqueous ethanol and recoverable from the medicinal tincture. Therefore, the net yield of extracted polysaccharides was enhanced in the ultrasonical procedure. This fact as well as the sugar composition and structural features of the isolated polysaccharides suggest that ultrasonication have attacked the integrity of cell walls, released and degraded its most accessible polysaccharides (pectic polysaccharides and starch) and increased also the extractibility of its less accessible components--xylan, mannan and glucan. The water-soluble polysaccharide fractions from both the conventional and ultrasonical experiments exhibit significant immunostimulatory activities in mitogenic and comitogenic thymocyte tests.

Particle Size↗
PubMed2003

Conformational flexibility of the group B meningococcal polysaccharide in solution.

To elucidate the role of secondary structure in the immune response against alpha(2-->8)-linked polysialic acid, the capsular polysaccharide of Group B meningococci, we have investigated its solution dynamics by using specific models of molecular motion and hydrodynamic modeling to interpret experimental NMR data. (13)C-[(1)H] NMR relaxation times and steady-state NOE enhancements were measured for two aqueous solutions of alpha(2-->8)-linked sialic acid polysaccharides. Each contained a unique distribution of polysaccharide chain lengths, with average lengths estimated at 40 or 400 residues. Models for rigid molecule tumbling, including two based on helical conformations proposed for the polysaccharide,(31) could not explain the NMR measurements. In general for these helices, the correlation times for their overall tumbling that best account for the NMR data correspond to polysaccharide chains between 9 and 18 residues in length, far short of the average lengths estimated for either solution. The effects of internal motions incorporated into these helices was modeled with an effective correlation time representing helix tumbling as well as internal motion. This modeling demonstrated that even with extreme amounts of internal motion, "flexible helices" of 25 residues or more still could not produce the NMR measurements. All data are consistent with internal and segmental motions dominating the nuclear magnetic relaxation of the polysaccharide and not molecular tumbling. Statistical distributions of correlation times have been found specifically for the pyranose rings, linkage groups, and methoxy groups that can account for the measured relaxation times and NOE enhancements. The distributions suggest that considerable flexibility attends the polysaccharide in solution, and the ranges of motional frequencies for the linkage groups and pyranose rings are comparable. We conclude that the Group B meningococcal polysaccharide is a random coil chain in solution, and therefore, does not have antigenic epitopes dependent upon a rigid, ordered conformation.

Bacterial Capsules↗
PubMed1998

Mimosa pudica apyrase requires polysaccharide and Ca2+ for the activity.

Mimosa pudica Linn leaves with pulvini contain unique isoforms (I and II) of Apyrase enzyme (EC 3.6.1.5). The activity of isoform I depends on divalent cation Mn2+. This isoform is associated noncovalently with the polysaccharide, containing mainly of galactose and arabinose sugars. The apparent molecular mass of these 2 isoforms are 36 and 34 Kd respectively. The association of the polysaccharide with the isoform I has been found to be Ca2+ dependent which is endogenously present in this isoform. Removal of Ca2+ and polysaccharide from the enzyme (isoform I) leads to an inactivation. The enzyme activity can be restored when both Ca2+ and endogenous polysaccharide fraction were added at an optimal molar ratio of Ca2+:protein of 7:1. The endogenous polysaccharide can be replaced by the standard arabinogalactan. No other sugar or polysaccharide except the arabinogalactan can restore the apyrase activity. Calcium mediates a conformational change in the protein which helps in association of polysaccharide as evidenced from fluorometric and far UV-CD studies to restore the enzymic activity. Neither any interaction of the polysaccharide with the protein is detected in absence of Ca2+ nor the enzyme activity could be recovered under such condition.

Amylases↗
PubMed1972

The sites of synthesis and transport of extracellular polysaccharides in the root tissues of maize.

1. Subcellular fractionation of maize roots resulted in the isolation of the following enriched fractions: cell wall, dictyosome, smooth-membrane and rough-microsomal fractions. In addition, extracellular polysaccharide of the root slime was isolated. 2. Maizeseedling roots were incubated in vivo with d-[U-(14)C]glucose, and the pattern of incorporation of radioactivity into the polysaccharides of each fraction was investigated. 3. The differentiation of maize-root cells with respect to the synthesis of specific extracellular polysaccharide directly relates to the polysaccharide synthesized and transported within the membrane system of the cell. A fucose-containing polysaccharide, characteristic only of root slime, was present only in the membrane system of the root-tip region of the root. Regions of typical secondary wall development within the root were characterized by an increased incorporation of radioactivity into xylose of polysaccharide within the membrane system. 4. The incorporation of radioactivity into glucan polymers in the membrane fractions was very low in all regions of the root. Since in regions of secondary wall development greater than 60% of all radioactive incorporation was into a glucan polymer, it can be inferred that this polymer, most probably cellulose, is not synthesized or transported within the compartments of the membrane system. It is suggested that synthesis of cellulose occurs at the surface of the plasmalemma. 5. Maize-root cells contained 40 times more rough endoplasmic reticulum than dictyosome membrane. The relative specific radioactivities of each fraction indicated that polysaccharide was concentrated in the region of the Golgi apparatus, which showed a 100% increase in specific radioactivity compared with the rough endoplasmic reticulum. The Golgi apparatus can thus be regarded as a localized focal point on the synthetic and transport system of polysaccharide by the intracellular membrane compartments.

Biological Transport, Active↗
PubMed1974

The amounts and rates of export of polysaccharides found within the membrane system of maize root cells.

1. Maize seedling roots were incubated in vivo with d-[U-(14)C]glucose for 2, 5, 10, 15, 30 and 45min. The total incorporation of radioactivity into polysaccharide components in isolated fractions was investigated, and the pattern of incorporation into different polysaccharide components within the rough endoplasmic reticulum, Golgi apparatus and exported material was analysed. 2. The membrane compartments reached a saturation value of radioactivity in polysaccharide components by 30min incubation. Radioactivity in exported polysaccharide continued to increase after that time. The latter was formed and maintained by a steady-state turnover of polysaccharide synthesis and transport from the membrane system. 3. If the only access of the slime polysaccharide to the cell surface is via dictyosome-derived vesicles, the amount of slime components in the Golgi apparatus would have to be displaced every 0.3min in order to maintain the observed rates of increase in slime. This is in contrast with a displacement time of about 2.5min that is necessary for polysaccharide components in the Golgi apparatus to produce the observed increase in cell-wall material. The activity of the membrane system in the production of maize root slime is 8 times as great as that of the membrane system involved in cell-wall synthesis. 4. If the amount of polysaccharide material in the Golgi apparatus is maintained only by inflow of polymeric material from the rough endoplasmic reticulum the total amount of slime components in the rough endoplasmic reticulum would have to be displaced every 7min to maintain a constant amount in the Golgi apparatus. If the endoplasmic reticulum contributed directly to the cell surface in the synthesis of cell-wall material, displacement times necessary to maintain the observed rate of polymer production would be very slow.

Arabinose↗
PubMed2003

Overexpression of UDP-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of K5 polysaccharide.

The Escherichia coli K5 capsular polysaccharide (glycosaminoglycan) chains are composed of the repeated disaccharide structure: -GlcAbeta1,4-GlcNAcalpha1,4-(where GlcA is glucuronic acid and GlcNAc is N-acetyl-D-glucosamine). The GlcA, present in most glycosaminoglycans, is donated from UDP-GlcA, which, in turn, is generated from UDP-glucose by the enzyme UDP-glucose dehydrogenase (UDPGDH). The formation of UDP-GlcA is critical for the biosynthesis of glycosaminoglycans. To investigate the role of UDPGDH in glycosaminoglycan biosynthesis, we used K5 polysaccharide biosynthesis as a model. E. coli was transformed with the complete gene cluster for K5 polysaccharide production. Additional transformation with an extra copy of UDPGDH resulted in an approx. 15-fold increase in the in vitro UDPGDH enzyme activity compared with the strain lacking extra UDPGDH. UDP-GlcA levels were increased 3-fold in overexpressing strains. However, metabolic labelling with [14C]glucose showed, unexpectedly, that overexpression of UDPGDH lead to decreased formation of K5 polysaccharide. No significant difference in the K5 polysaccharide chain length was observed between control and overexpressing strains, indicating that the decrease in K5-polysaccharide production most probably was due to synthesis of fewer chains. Our results suggest that K5-polysaccharide biosynthesis is strictly regulated such that increasing the amount of available UDP-GlcA results in diminished K5-polysaccharide production.

Bacterial Capsules↗
PubMed1991

Biosynthesis of heparin. Use of Escherichia coli K5 capsular polysaccharide as a model substrate in enzymic polymer-modification reactions.

A capsular polysaccharide from Escherichia coli K5 was previously found to have the same structure, [-(4)beta GlcA(1)----(4)alpha GlcNAc(1)-]n, as that of the non-sulphated precursor polysaccharide in heparin biosynthesis [Vann, Schmidt, Jann & Jann (1981) Eur. J. Biochem. 116, 359-364]. The K5 polysaccharide was N-deacetylated (by hydrazinolysis) and N-sulphated, and was then incubated with detergent-solubilized enzymes from a heparin-producing mouse mastocytoma, in the presence of adenosine 3'-phosphate 5'-phospho[35S] sulphate ([35S]PAPS). Structural analysis of the resulting 35S-labelled polysaccharide revealed the formation of all the major disaccharide units found in heparin. The identification of 2-O-[35S]sulphated IdoA (L-iduronic acid) as well as 6-O-[35S]sulphated GlcNSO3 units demonstrated that the modified K5 polysaccharide served as a substrate in the hexuronosyl C-5-epimerase and the major O-sulphotransferase reactions involved in the biosynthesis of heparin. The GlcA units of the native (N-acetylated) E. coli polysaccharide were attacked by the epimerase only when PAPS was present in the incubations, whereas those of the chemically N-sulphated polysaccharide were epimerized also in the absence of PAPS, in accord with the notion that N-sulphate groups are required for epimerization. With increasing concentrations of PAPS, the mono-O-sulphated disaccharide unit-IdoA(2-OSO3)-GlcNSO3- was progressively converted into the di-O-sulphated species -IdoA(2-OSO3)-GlcNSO3(6-OSO3)-. A small proportion of the 35S-labelled polysaccharide was found to bind with high affinity to the proteinase inhibitor antithrombin. This proportion increased with increasing concentration of PAPS up to a level corresponding to approximately 1-2% of the total incorporated 35S. The solubilized enzymes thus catalysed all the reactions required for the generation of functional antithrombin-binding sites.

Animals↗
PubMed1992

Purification of an anti-ulcer polysaccharide from the leaves of Panax ginseng.

Water-soluble and alkaline-soluble crude polysaccharides which were separated from the roots or leaves of Panax ginseng C. A. Meyer, were compared for their anti-ulcer activity. Of these four polysaccharide fractions, the water-soluble crude polysaccharide fraction (GL-2) from the leaves and the alkaline-soluble crude polysaccharide fraction (GRA-2) from the roots prevented HCl/ethanol-induced ulcerogenesis in mice potently. The most potent fraction, GL-2, was further fractionated into four polysaccharide fractions by precipitation with cethyltrimethylammonium bromide, and the weakly acidic polysaccharide fraction, GL-4, showed the most potent inhibition of gastric lesion formation. The activity of GL-4 decreased after treatment with periodate or digestion with endo-polygalacturonase, indicating that the carbohydrate moiety may contribute to the expression of the activity. GL-4 was further purified by anion-exchange chromatography and gel filtration, and the most active purified polysaccharide, GL-4IIb1III was obtained. GL-4IIb1III (average relative molecular mass, 16,000 d) had the nature of a pectic polysaccharide, and was composed mainly of galactose and galacturonic acid with small proportions of rhamnose, arabinose, mannose, glucose, and glucuronic acid. GL-4IIIb1III prevented HCl/ethanol-induced ulcerogenesis in mice dose dependently.

Animals↗
PubMed2002

Intestinal immune system modulating polysaccharides in a Japanese herbal (Kampo) medicine, Juzen-Taiho-To.

A commercially available dried extract (TJ-48) of Japanese herbal (Kampo) prescription, Juzen-Taiho-To has been found to enhance functions of Peyer's patch cells (intestinal immune system modulating activity) in vitro and in vivo. When TJ-48 was fractionated, the dialyzable fraction (F-3) and the polysaccharide fraction (F-5) expressed the in vitro activity. Oral administration of F-5 (150 mg/kg/day) to mice showed the intestinal immune system modulating activity. When the galacturonan moiety of pectic polysaccharides in F-5 was degraded enzymatically by endo-polygalacturonase, the digestion products significantly increased the activity of F-5. Purification the polygalacturonase-digested F-5 indicated that the active substances were composed mainly of the enzyme-resistant or undigestable polysaccharide molecules. Gel filtrations and anion-exchange chromatographies of F-5 gave 12 kinds of polysaccharides, and among them, 7 polysaccharides had significant intestinal immune system modulating activity. Component sugar analysis suggests that some active polysaccharides are grouped into pectic polysaccharides containing arabinogalactan or heteroglycan chains. Single radial gel diffusion analysis using beta-D-glucosyl-Yariv antigen indicated that some of the active polysaccharides comprised arabino-3,6-galactan moiety, and this moiety was suggested to play an important role partly for expression of the activity by single linear regression analysis between degree of the activity and reactivity with beta-D-glyucosyl-Yariv antigen.

Adjuvants, Immunologic↗
PubMed1989

Digestion of fibre polysaccharides of pea (Pisum sativum) hulls, carrot and cabbage by adult cockerels.

Characterization of the carbohydrates of pea (Pisum sativum) hulls, carrot and cabbage using both colorimetric and gas-liquid chromatographic techniques permitted a detailed investigation into the extent of digestion of differing types of fibre. These digestion studies were greatly aided by the development of a rapid bioassay employing starved adult cockerels. Total collection of undigested residues, uncontaminated by food spillage, could be made from trays placed under the cockerels. Chemical analysis showed that pea hulls consisted mainly of fibre with very little available carbohydrate present, whereas more than half of freeze-dried carrot and cabbage consisted of available carbohydrate (sucrose, glucose, fructose, starch) and consequently considerably less fibre was present. The fibre of carrot and cabbage was similarly composed of nearly equal amounts of neutral and acidic polysaccharides, whereas pea-hull fibre had four times as much neutral as acidic polysaccharides. The digestibility of total neutral polysaccharides from all three foodstuffs was extremely low. However, there appeared to be preferential digestion of polysaccharides composed of rhamnose, arabinose and galactose residues, all associated with pectic material, in contrast to the indigestibility of polysaccharides composed of fucose, xylose and glucose. Acidic polysaccharides were digested to a greater extent than neutral ones, and those of carrot and cabbage more so than pea hulls. The polysaccharides which were the most soluble were also the most digestible, but due to the arbitrariness of polysaccharide solubility, quantification of their total digestibility per se was considered not possible.

Animals↗
PubMed1998

Structure and technofunctional properties of protein-polysaccharide complexes: a review.

Food proteins and polysaccharides are the two key structural entities in food materials. Generally, interactions between proteins and polysaccharides in aqueous media can lead to one- or two-phase systems, the latter being generally observed. In some cases of protein-polysaccharide net attraction, mainly mediated through electrostatic interactions, complex coacervation or associative phase separation occurs, giving rise to the formation of protein-polysaccharide complexes. Physicochemical factors such as pH, ionic strength, ratio of protein to polysaccharide, polysaccharide and protein charge, and molecular weight affect the formation and stability of such complexes. Additionally, the temperature and mechanical factors (pressure, shearing rate, and time) have an influence on phase separation and time stability of the system. The protein-polysaccharide complexes exhibit better functional properties than that of the proteins and polysaccharides alone. This improvement could be attributed to the simultaneous presence of the two biopolymers, as well as the structure of the complexes. Consequently, the interesting hydration (solubility, viscosity), structuration (aggregation, gelation) and surface (foaming, emulsifying) properties of these complexes can be used in a number of domains. Among others, these could be macromolecular purification, microencapsulation, food formulation (fat replacers, texturing agents), and synthesis of biomaterials (edible films, artificial grafts).

Biopolymers↗
PubMed1991

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants.

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.

Carbohydrate Sequence↗
PubMedDate not supplied

Chemical structure of and immune response to polysaccharides of Streptococcus pneumoniae.

By use of rabbit antisera, extensive cross-reactions were demonstrated among pneumococcal (Danish) types 6A and 6B, types 9N, 9A, 9L, and 9V, and types 19F and 19A. The structural similarity of the group 6 polysaccharides was associated with extensive immunogenicity in humans. In contrast, immunization of humans with type 19F or 19A may not induce sufficient cross-protection. The type 19F polysaccharide has the structure of a linear polymer of N-acetylmannosamine-glucose-rhamnose phosphate. Danish type 19A (type 57 in the United States system) polysaccharide contains this repeating unit and, in addition, has side chains of N-acetylglucosamine-galactose phosphate and fucose phosphate. Maternal immunization with type 19F or type 6A polysaccharide during pregnancy elicited high antibody formation in the offspring. Young mice, which received an additional dose of polysaccharide at two weeks of age, showed a higher antibody response than did those that did not receive polysaccharide. These studies reveal that the extensive cross-immunogenicity of pneumococcal group polysaccharides has a basis in chemical structure. Maternal immunization with pneumococcal polysaccharides may enhance the immune response of neonates.

Animals↗
PubMedDate not supplied

Studies on vaccine control and immunogenicity of polysaccharides of Streptococcus pneumoniae.

An immunoelectrophoretic method was devised for quantitation of 14 polysaccharide components in the pneumococcal vaccine, and for determination of their stability in the final container. By this method the individual polysaccharide types, 1, 2, 3, 4, 6A, 8, 9N, 12F, 18C, 19F, 23F, and 25 (Danish nomenclature), were found to be present at 80%-123% of the manufacturer's listed concentrations. Pneumococcal polysaccharide types 3, 6A, 9N, and 19F, used as representative types, were heated at 37C for 24 hr and stored at 4 C. The concentrations of these polysaccharides remained constant over a 12-month period, and the molecular sizes of types 3 and 9N were stable during storage. In contrast, the molecular sizes of types 6A and 19F declined gradually during the 12-month storage period. Pneumococcal type 19F polysaccharide was conjugated to various proteins, i.e., bovine serum albumin, human immunoglobulin, and pneumococcal R61 cell wall protein, by the method of reductive amination. Immunization of mice with 19F polysaccharide-protein conjugates resulted in formation of more antibody than was found in the control group. Young mice exposed to pneumococcal type 19F polysaccharide-protein conjugate during gestation and suckling exhibited a greater antibody response than did mice that received no type 19F polysaccharide-protein conjugate while suckling or received the conjugate only when they were only two weeks of old.

Animals↗
PubMed1995

Lectin recognition of host-like saccharide motifs in streptococcal cell wall polysaccharides.

Viridans streptococci that participate in the microbial colonization of teeth have cell wall polysaccharides composed of linear phosphodiester-linked hexa- or heptasaccharide repeating units, each containing a host-like disaccharide motif, either Gal beta 1-->3GalNAc or GalNAc beta 1-->3Gal. Whereas strains with GalNAc beta 1-->3Gal-containing polysaccharides co-aggregated with streptococci that possess GalNAc-sensitive lectins, strains with either host-like motif co-aggregated with Actinomyces spp. The latter interactions reflected the specificity of Actinomyces spp. lectins for common features of Gal beta 1-->3GalNAc and GalNAc beta 1-->3Gal. Thus, alpha-linked glycosides of both disaccharides were much more potent inhibitors of co-aggregation than Gal or GalNAc. Six non-bacterial lectins also reacted with the streptococcal polysaccharides. In general, precipitation of each lectin with each polysaccharide involved binding of Gal or GalNAc within the host-like motifs, but not saccharides outside these regions. The lectins of Ricinus communis, Abrus precatorius, Codium fragile and Agaricus bisporus were most reactive with the Gal beta 1-->3GalNAc-containing polysaccharides, the Wisteria floribunda lectin with the GalNAc beta 1-->3Gal-containing polysaccharides and the Bauhinia purpurea lectin with polysaccharides containing either disaccharide. Thus, lectin recognition of the streptococcal cell wall polysaccharides involved either the common or specific sides of the Gal beta 1-->3GalNAc and GalNAc beta 1-->3Gal motifs present within these molecules.

Actinomyces↗
PubMed1980

Liposomal membranes. VI. Polysaccharide-induced aggregation of multilamellar liposomes of egg lecithin.

Induced aggregation of multilamellar liposomes of egg lecithin with several polysaccharides, such as pullulan, dextran, hydroxyethylstarch, and amylopectin, has been investigated in conjunction with structural characteristics of polysaccharides, incubation temperature, ionic strength, and surface charge of liposomes. Aggregation of liposomes as a function of time was followed by monitoring turbidity of the liposome suspension at 600 nm. The inverse relationship between the minimal concentration and molecular weight of polysaccharides for the induced aggregation of liposomes was observed for all the polysaccharides employed. Polysaccharides having larger excluded volume more easily bind to liposomes, leading to the acceleration of the aggregation of liposomes. Hence, the order of efficiency of polysaccharides for the promotion of liposome aggregation was pullulan greater than dextran greater than hydroxyethylstarch congruent to amylopectin. Increasing the surface charge of liposomes brought about a decrease in aggregation irrespective of the sign. Even when negatively charged liposomes were incubated with the cationic DEAE-dextran, the liposome aggregation was depressed. These results including salt effect and temperature dependency of the induced aggregation of liposomes supported a plausible mechanism for the reaction, the polysaccharide-bridging mechanism, which has been provided for the polysaccharide-promoted agglutination of human erythrocytes.

Chemical Phenomena↗
PubMed1996

Novel O-polysaccharide expression, as a lipid A-core-free form, in a lipopolysaccharide-core-defective mutant of Pseudomonas aeruginosa.

Pseudomonas aeruginosa PML14e is a mutant strain, isolated from strain PML14 (Homma serotype I), that is resistant to all types of R-pyocins. PML14e completely lacked glucose and rhamnose as components of the lipopolysaccharide (LPS) outer core region. Whereas the O-polysaccharide attachment site on the LPS core was considered to be absent, PML14e was agglutinable with anti-serotype-I antibodies. The O-polysaccharide of PML14e was recovered in the supernatant after ultracentrifugation of the aqueous layer from a hot phenol/water extraction. Chromatographic behaviour and chemical analysis indicated that the PML14e O-polysaccharide was not linked to the lipid A. 1H-NMR spectroscopy indicated that the structure of the PML14e O-polysaccharide was the same as that of the O-polysaccharide from PML14. The above evidence indicated that the O-polysaccharide is expressed on the cell surface of the mutant strain PML14e as the lipid A-free form. To examine the nature of the cell surface, the accessibility of monoclonal antibodies (mAbs) against cell surface antigens was tested by enzyme-linked immunosorbent assay. An anti-lipid A mAb and an anti-outer-membrane protein mAb, the epitopes for which are considered to be exposed on rough strains, bound to a greater extent to the PML14e cells than to two other LPS-core-defective rough mutants, PML14b and PML14d. Whereas these mutants appeared to have lesser defects in the LPS core, they expressed less O-polysaccharide than PML14e. The results indicated that the epitopes exposed on rough strains, such as lipid A and outer-membrane proteins, were mainly hindered by covalently linked core oligosaccharide rather than by the O-polysaccharide chain.

Antibodies, Monoclonal↗