A novel carbohydrate-glycosphingolipid interaction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes.

The immunomodulator Betafectin(R) PGG-glucan is a homopolymer of glucose derived from yeast cell walls which has been demonstrated to enhance leukocyte anti-infective activity in vitro and in vivo, without the induction of proinflammatory cytokines. We report here the purification of a PGG-glucan-binding element from human leukocytes and its identification as lactosylceramide, a major glycosphingolipid of neutrophils, which includes the CDw17 epitope. The binding of radiolabeled PGG-glucan to purified lactosylceramide was saturable, specific, and time- and temperature-dependent. Lactosylceramides from human leukocytes were fractionated by high performance liquid chromatography in order to analyze the effect of ceramide structure on binding. A variety of fatty acid chain lengths with varying degrees of unsaturation were found to support binding to radiolabeled PGG-glucan. However, DL-lactosylceramides containing dihydrosphingosine did not bind. Radiolabeled PGG-glucan bound several other neutral glycosphingolipids with a terminal galactose, including galactosylceramide, globotriaosylceramide, and gangliotetraosylceramide. The binding of radiolabeled PGG-glucan to lactosylceramide was not inhibited by glycogen, dextran, mannan, pustulan, laminarin, or a low molecular weight beta-(1-3)-glucan, but was inhibited by high molecular weight beta-(1-3)-glucans and by a monoclonal antibody to lactosylceramide. Although this glycosphingolipid has been shown in numerous reports to bind various microorganisms, this represents the first report of lactosylceramide binding to a macromolecular carbohydrate.

Thermal polymerization of some glucofuranose derivatives.

Methyl alpha,beta-D-glucofuranoside (1) and 1,2-O-isopropylidene-alpha-D-glucofuranose (2) have been polymerized thermally, using 0.1% phosphoric acid as catalyst. The resulting alcohol-insoluble polymers were analyzed by gel-permeation chromatography and methylation analysis. The two starting materials gave polymers that were very similar in molecular-weight distribution and in glycosyl-linkage composition. The proportion of furanosyl residues in the polymer, estimated by the relative proportions of furanosyl and pyranosyl residues that could be determined unequivocally by methylation analysis, was approximately 35-40%; higher than is found when glucopyranosides are polymerized, but indicating that significant furanose-pyranose isomerization had occurred during the polymerization process.

Chemical methods for the analysis of sulphated galactans from red algae.

Methods are reported that facilitate the structural characterization of complex sulphated galactans of the red algae. Two procedures have been developed for the production of alditol acetates from carrageenans and agaroids. Both procedures generate 3,6-anhydrogalactitol acetate from the easily destroyed 3,6-anhydrogalactosyl residues in near quantitative yield. The "double hydrolysis-reduction" method involves preliminary hydrolysis under conditions sufficient to cleave all of the 3,6-anhydrogalactosidic bonds, but mild enough to avoid significant further degradation. The "reductive hydrolysis" method uses the acid-stable 4-methylmorpholine-borane to reduce the 3,6-anhydrogalactose end groups as they are released during acid hydrolysis. An alditol acetate sample can be prepared from a polysaccharide in a single tube, ready for g.l.c. analysis, in less than 2.5 h, i.e. more quickly than by any previous procedure. Problems associated with incomplete methylation of sulphated carrageenans and agaroids by the Hakomori procedure have been overcome by first converting the sulphated polysaccharide into its triethylammonium salt form. The reductive hydrolysis method is effective for the production of partially methylated alditol acetates from the methylated polysaccharides, enabling the rapid determination of the substitution pattern of these polysaccharides. These improved analytical methods have been applied successfully to kappa-, iota-, and lambda-carrageenans, as well as some agars.

Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells.

The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified alpha-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.

Structure and function of plant cell wall polysaccharides.

Studies of the primary structures of polysaccharides of growing plant cell walls have shown that these structures are far more complex than was anticipated just a few years ago. This complexity can best be appreciated by considering xyloglucan, a hemicellulose present in the cell wall of both monocots and dicots, and rhamnogalacturonan II (RG-II) and rhamnogalacturonan I (RG-I), two structurally unrelated pectic polysaccharides. This realization led us to postulate that cell wall polysaccharides have functions beyond determining the size, shape and strength of plants. Some years ago we demonstrated that oligosaccharide fragments of a branched beta-linked glucan of fungal cell walls can elicit the production of phytoalexins (antibiotics) in plants by inducing the formation of the enzymes responsible for synthesis of the phytoalexins. It has now been ascertained and confirmed by synthesis that the elicitor activity resides in a very specific hepta-beta-D-glucoside. The heptaglucoside has been shown to elicit phytoalexins by activating the expression of specific genes, that is, by causing the synthesis of the mRNAs that encode the enzymes that synthesize phytoalexins. In other words, complex carbohydrates can be regulatory molecules. Further experiments established that oligosaccharide fragments of polysaccharides, produced by acid or base hydrolysis or by enzymolysis of primary cell walls of plants, also evoked defence responses in plants. Subsequently, we learned that defined fragments of polysaccharides, released from covalent attachment within plant cell walls, can function as regulators of various physiological processes such as morphogenesis, rate of cell growth and time of flowering and rooting, in addition to activating mechanisms for resisting potential pathogens. Examples of plant oligosaccharides with regulatory properties (called oligosaccharins) will be described.

Osmoregulation in the Avena Coleoptile : CONTROL OF SOLUTE UPTAKE IN PEELED SECTIONS.

Peeled Avena sativa coleoptile sections (i.e. sections from which the epidermis has been removed) have been used to study the control of solute uptake under conditions where the uptake is not limited by the cuticular barrier. In the presence of 2% sucrose, auxin enhances the rate at which the total osmotic solutes increase, but this appears to be a response to the increased growth rate, inasmuch as the auxin effect is eliminated when growth is inhibited osmotically. When sections are incubated in sucrose or in 20 millimolar NaCl, the osmotic concentration increases until a plateau is reached after 8 to 24 hours. Auxin has no effect on the initial rate of increase in osmotic concentration but causes the osmotic concentration to reach a plateau earlier and at a lower osmotic conentration value. This difference in steady-state osmotic concentration is, in part, a response to auxin itself, as it persists when auxin-induced growth is inhibited osmotically. The upper limit for osmotic concentration does not appear to be determined by the turgor pressure, inasmuch as a combination of sucrose and NaCl gave a higher plateau osmotic concentration than did either solute alone. We suggest that the rate of solute uptake is determined by the availability of absorbable solutes and by the surface area exposed to the solutes. Each absorbable solute reaches a maximum internal concentration independent of other absorbable solutes; the steady-state osmotic concentration is simply the sum of these individual internal concentrations.

Osmoregulation in the Avena coleoptile in relation to auxin and growth.

A study has been made of the effects of auxin and growth on the ability of Avena coleoptile sections to osmoregulate, i.e. to take up solutes so as to maintain their osmotic concentration, turgor pressure, and growth rate. The high auxin-induced growth rate of Avena coleoptiles is maintained when cells are provided sucrose, glucose, NaCl, or KCl as a source of absorbable solutes, but not when 2-deoxy-d-glucose or 3-O-methyl-d-glucose is used. In the absence of auxin, cells take up solutes from a 2% sucrose solution and the osmotic concentration increases. The rate of solute uptake is even greater in the presence of auxin or fusicoccin, but the osmotic concentration rises only slightly because of the water taken up during growth. Solute uptake is not stimulated by auxin when growth is inhibited osmotically or by calcium ions. Solute uptake appears to have two components: a basal rate, independent of auxin or growth, and an additional uptake which is proportional to growth. Osmoregulation of sections may be limited by the rate of entry of solutes into the tissue rather than by their rate of uptake into the cells.