Characterization of a Yariv precipitated arabinogalactan-protein from fruits of rye (Secale cereale L.).

An arabinogalactan-protein (AGP) with a molecular mass of 110 kDa was isolated from whole grain of rye (Secale cereale L.) by double precipitation with (beta-D-Glc)3-Yariv-phenylglycoside (3GlcY) and its structure was analyzed. The AGP consists of a hydroxyproline-rich protein backbone of about 7 % and an arabinogalactan moiety of about 93%. By alkaline hydrolysis, hydroxyproline was identified as the main amino acid responsible for the binding between the protein and the carbohydrate subunits via an O-glycosidic linkage. The arabinogalactan moieties are highly branched consisting of 1,3-linked Galp residues, some of them linked in position 6 to 1,6-Galp side chains, terminating in Araf residues. With regard to its structure, the rye AGP is comparable to other cereal AGPs like those from oat or wheat grain.

Immunomodulatory effects of arabinogalactan-proteins from Baptisia and Echinacea.

The influences of different arabinogalactan-proteins (AGPs) on proliferation and IgM-production of mouse lymphocytes as well as nitrite- and IL6-production of mouse macrophages were investigated in vitro. AGPs have been isolated and purified from roots of Baptisia tinctoria and Echinacea pallida and suspension culture of Echinacea purpurea. Comparing the AGPs, there are differences with regard to fine structure as well as to activities. AGPs from roots of B. tinctoria and E. pallida show high activity in all test systems. AGP from cell culture of E. purpurea shows no influence on proliferation of mouse lymphocytes, only weak influence on the IgM-production of mouse lymphocytes and weak stimulation of nitrite- and IL6-production in alveolar mouse macrophage culture.

Binding studies of an arabinogalactan-protein from Echinacea purpurea to leucocytes.

Flow cytometric investigations show binding of an isolated arabinogalactan-protein (AGP) from pressed juice of the aerial parts of Echinacea purpurea to the cell surface of human leucocytes. AGP demonstrates binding to lymphocytes, monocytes and granulocytes of different donors (n=8). Competition assays with two antibodies, directed against CD4 and CD8, revealed no interaction of AGP with these receptors, leading to the conclusion that binding of AGP to leucocytes is mediated via other structures.

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the beta-glucosyl Yariv reagent.

An arabinogalactan-protein (AGP) from pressed juice of Echinacea purpurea herb was isolated from a high molecular weight fraction by precipitation with the beta-glucosyl Yariv reagent, followed by gel-permeation chromatography. It revealed characteristic features of other AGPs: i.e., a high amount of polysaccharide (83%) with a ratio of galactose to arabinose of 1.8:1, some uronic acids (4-5%), and a low protein content (7%) with high levels of serine, alanine and hydroxyproline. The molecular weight was estimated to be 1.2 x 10(6) Da. Linkage and 13C NMR analyses showed that the AGP is composed of a highly branched core polysaccharide of 3-, 6-, and 3,6-linked Galp residues with terminal Araf, GlcAp and terminal units of Araf-(1-->5)-Araf-(1-->. Partial acid hydrolysis resulted in loss of Araf residues at the periphery of the molecule. Complete loss of reactivity toward the beta-glucosyl Yariv antigen was then noticed.

The inhibiting effects of components of stinging nettle roots on experimentally induced prostatic hyperplasia in mice.

Direct implanting of fetal urogenital sinus (UGS) tissue into the ventral prostate gland of adult mice led to a 4-fold weight increase of the manipulated prostatic lobe. The induced growth could be reduced by the polysaccharide fraction (POLY-M) of the 20% methanolic extract of stinging nettle roots by 33.8%.

High molecular weight acidic polysaccharides from Malva sylvestris and Alcea rosea.

Leaves and flowers of Malva sylvestris and Alcea rosea (Malvaceae) were compared by determination of the swelling index as well as the content and viscosity of their mucilages. The investigations showed mucilage from flowers of Alcea to be superior to mucilages from leaves or flowers from Malva. High molecular weight acidic polysaccharides (HMWAPs) were isolated from the mucilages of leaves and flowers of both species. The molecular weight of all HMWAPs was in a range of 1.3 to 1.6 x 10(6) D. HMWAP-content in mucilage from flowers of Alcea was highest compared to content in mucilages from leaves or flowers from Malva. HMWAPs were found to be composed mainly of glucuronic acid, galacturonic acid, rhamnose and galactose. Methylation analysis of the carboxyl-reduced and deuterium labeled sugar derivatives of HMWAPs from flowers of Malva sylvestris ssp. mauritiana and Alcea rosea enabled elucidation of the principal structural features of both polysaccharides.

Pythium aphanidermatum: culture, cell-wall composition, and isolation and structure of antitumour storage and solubilised cell-wall (1----3),(1----6)-beta-D-glucans.

Under optimal conditions for the culture of the fungus Phytium aphanidermatum, no polysaccharides were excreted into the medium. The mycelium contained up to 38% of a slightly branched, storage (1----3),(1----6)-beta-D-glucan with a MW of 20,000. The cell-wall polysaccharides of the mycelium comprised 18% of cellulose and 82% of (1----3),(1----6)-beta-D-glucans. Of the non-cellulosic glucans, approximately 33% could be solubilised by extraction with water at 121 degrees, and they had a MW of 10,000, were highly branched, and contained 6% of (1----6) linkages. Treatment of the cell wall with 0.1 M trifluoroacetic acid released approximately 50% of the non-cellulosic glucans. The acid-soluble cell-wall (1----3),-(1----6)-beta-D-glucans of lower MW (6000) were still highly branched and contained 14% of (1----6) and 8% of (1----4) linkages. The storage glucan and the hot-water-soluble cell-wall glucan exhibited strong activity against the Sarcoma 180 in CD-1 mice, whereas the acid-soluble cell-wall glucans were inactive. The hot-water-soluble cell-wall glucan was also active against the DBA/2-MC.SC-1 fibrosarcoma in DBA/2 mice.

Antitumor activity of cell wall beta-1,3/1,6-glucans from Phytophthora species.

From the cell walls of various Phytophthora species (Oomycetes) different water-soluble beta-1,3/1,6-glucans were isolated and characterized. The glucans with relative molecular masses between 13 and 50 kd exhibited varying degrees of branching in the beta-1,3-linked glucan backbone ranging from 14 to 50%. The dose-dependent antitumor activity of the glucans against allogeneic sarcoma-180 was investigated. It could be demonstrated that beta-1,3/1,6-glucans with relative molecular masses below 50 kd can exhibit a prominent antitumor activity (inhibition rate up to 99%), being comparable to that of the high relative molecular mass (450 kd) beta-1,3/1,6-glucans. Biological activity of the glucans could be shown to be correlated with a low degree of branching. The most potent glucan with a degree of branching of 14% also was active against the syngeneic DBA/2-MC.SC-1 fibrosarcoma (inhibition rate up to 90%) and in combination with a suboptimum dose of diethylstilbestrol against Nobel-Nb prostate carcinoma (inhibition rate up to 90%).

The influence of phytohormones on growth, organ differentiation and fructan production in callus of Symphytum officinale L.

Callus derived from Symphytum officinale L. regenerants was cultured in the presence of various phytohormones. The growth rate of callus was stimulated by all phytohormones at various concentrations. With 1-naphthaleneacetic acid no organ differentiation could be observed. With indole-3-butyric acid at low concentrations only roots were formed, whereas 6-benzylaminopurine, kinetin and zeatin at various concentrations induced either root or shoot formation or the simultaneous regeneration of both. Minor amounts of fructans were formed at high 6-benzylaminopurine-, zeatin- and at all indole-3-acetic acid-concentrations. The concentration of 1-naphthaleneacetic acid had no influence on the fructan content. Highest rates of fructan synthesis occurred at low zeatin-concentrations up to 1.5 mg/l. Only zeatin at all concentrations induced the synthesis of polyfructans, whereas appreciable amounts of oligofructans were formed under the influence of all other phytohormones.

In vitro propagation of Lilium testaceum and structural investigation of the storage ß-1,4-glucomannan.

Bulblet and callus cultures of Lilium testaceum were initiated in vitro from bulbscales. Continous propagation of the bulblet cultures was achieved on a modified Murashige and Skoog agar medium containing 1-naphthalene acetic acid (0.1 mg/l) and kinetin (0.1 mg/l) as phytohormones. The in vitro grown bulbs synthesized large quantities of storage ß-1,4-glucomannans (mannose: glucose = 7∶3; molecular weight = 200 kd) with an identical structure to the glucomannans from the in vivo grown bulbs. Higher 1-naphthalene acetic acid concentrations (1 mg/l) resulted in increased callus formation. Liquid suspension cultures derived from callus exhibited only small amounts of reserve glucomannans.

Calcium ions and polyamines activate the plasma membrane-located 1,3-ß-glucan synthase.

Sucrose-density-gradient centrifugation and partitioning in a polyethylene glycol/dextran two-phase system were used to isolate plasmamembrane vesicles from microsomal preparations of soybean cell suspension cultures. Both methods resulted in the enrichment of the activity of a 1,3-ß-glucan synthase which forms a polymer consisting of more than 99% of 1,3-linked glucose (callose). Digitonin increases the 1,3-ß-glucan synthase activity in the various membrane fractions to a different degree, supporting the suggestion that this enzyme is vectorially arranged in the plasma membrane. The enzyme is greatly activated either by poly-L-ornithine or synergistically by Ca(2+) and spermine, indicating that the same enzyme is affected and exhibits the regulatory properties necessary for callose synthesis.

Xyloglucan (amyloid) formation in the cotyledons of Tropaeolum majus L. seeds.

Mature seeds of Tropaeolum majus L. contain the cell wall polysaccharide xyloglucan (amyloid), protein and lipid as storage substances. The transitory occurrence of starch during the process of seed development could be substantiated.[U-(14)C]-labelled xylose, glucose and glucuronic acid were fed to ripening seeds and the incorporation of radioactivity into xyloglucan, starch and the sugar nucleotide fraction of the cotyledons was determined. The results indicate that exogenous supplied xylose is not incorporated directly into xyloglucan, but is transformed to glucose before incorporation into xyloglucan and starch. Radioactivity from glucuronic acid was predominantly found in the xylose moiety of xyloglucan. Incubation of seeds with [6-(14)C]-labelled glucose resulted in an incorporation of labelled hexoses into amyloid and starch, whereas xylose residues of amyloid remained unlabelled.

Chitosan-elicited callose synthesis in soybean cells as a ca-dependent process.

A new method for the rapid and quantitative fluorometric determination of callose is described. In suspension-cultured cells of Glycine max, synthesis of callose starts within 20 minutes of treatment with chitosan and parallels over hours the accumulation of 1,3-linked glucose in the wall. Poly-l-lysine also elicits callose synthesis. The effect of chitosan is enhanced by Polymyxin B at low concentrations; this antibiotic alone at higher concentrations can also induce callose synthesis. Callose synthesis is immediately stopped when external Ca(2+) is bound by ethylene glycolbis-(2-aminoethyl ether)-N,N'-tetraacetate or cation exchange beads, and partly recovers upon restoration of 15 micromolar Ca(2+).Callose synthesis is observed only when membrane perturbation causing electrolyte leakage from the cells is induced by one of the above treatments. It does not appear to be due to de novo synthesis or proteolytic activation of 1,3-beta-d-glucan synthase. It is concluded that this Ca(2+)-dependent enzyme is directly activated by the influx of Ca(2+) occurring concomitantly with the leakage of cell constituents. This suggestion is also discussed in conjunction with the chitosan-induced synthesis of phytoalexin in the same cells.

Comparison between maize root cells and their respective regenerating protoplasts: wall polysaccharides.

The cell-wall polysaccharides from different parts of maize roots have been analysed. The arabinose, galactose and mannose contents are influenced by cell differentiation, whereas xylose, rhamnose and uronic-acid contents are not. In cap cells, the pectin content is low but rhamnose and fucose are present in larger quantities. The cell-wall polysaccharides from cells of the elongation zone and their respective regenerating protoplasts were also analysed. The walls of the protoplasts contained higher xylose and mannose levels and a much lower level of cellulose than the cells from which they were derived.

Influence of growth conditions on the composition of cell wall polysaccharides from cultured tobacco cells.

The sugar composition of cell wall polysaccharides of two tobacco varieties obtained from mesophyll, regenerating protoplasts and cells grown under various conditions were compared. Regenerating protoplasts developed an unusual cell wall with a low cellulose and a high non-cellulosic glucan content. In the presence of different phytohormones compact and friable calli were obtained with cell walls containing low and high arabinose/xylose ratios. The cell walls of compact calli were comparable to those of genuine mesophyll cells. The sugar constituents of cell walls obtained from cells grown in liquid media were different from those of solid calli. The cell wall composition of suspension cultured cells was hardly affected by various combinations of phytohormones, but was altered by high osmolarity of the medium.

Molecular weight distribution of cellulose in primary cell walls : Investigations with regenerating protoplasts, suspension cultured cells and mesophyll of tobacco.

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized (14)C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension - cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP∼4,000) than in protoplasts or suspension - cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse - chase experiments with either protoplasts or suspension -cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.