Wolfgang Blaschek

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

Characterization of an arabinogalactan-protein isolated from pressed juice of Echinacea purpurea by precipitation with the β-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.

Cell wall regeneration by Nicotiana tabacum protoplast: Chemical and biochemical aspects

Abstract Protoplasts isolated enzymatically from the mesophyll of tobacco leaves ( Nicotiana tabacum L.) were cultured in a defined liquid medium. The composition of the cell wall polysaccharides (CWP) and of the extracellular polysaccharides (ECP) synthesized after 1–4 days of culture was investigated by gas-liquid chromatography (GLC) in comparison with that derived from tobacco mesophyll and tobacco tissue cultured on solid agar or in suspension. The cell wall of regenerating protoplasts was composed mainly of non-cellulosic polysaccharides with glucose predominating (65%) and a content of only 5% cellulose whereas the cell walls of leaves and cultured tissue contained 60% and 45% cellulose, respectively. The cell walls of leaves, callus and suspension cultured cells showed differences in their arabinose/ xylose ratio. Galactose, arabinose and uronic acids were the main constituents in the ECP of regenerating protoplast and of suspension cultured cells indicating the secretion of pectic material into the culture medium. The labelling pattern of polysaccharides synthesized by protoplasts after culture with [U- 14 C]glucose or [U- 14 C]sucrose was in accordance with the chemical composition. No label of the generally used osmoticum mannitol was incorporated into polysaccharides. Radioactivity derived from myo-[ 3 H]inositol was transformed into uronic acids and pentoses, but not incorporated into the different hexoses. Simultaneous feeding of [U- 14 C] glucose and unlabelled myo-inositol reduced incorporation of radioactivity into uronic acids and pentoses. Hence, the myo-inositol pathway is operative in cell wall regenerating tobacco protoplasts.

Inhibition of the intestinal sodium-coupled glucose transporter 1 (SGLT1) by extracts and polyphenols from apple reduces postprandial blood glucose levels in mice and humans.

SCOPE There is a growing interest in food constituents that could reduce intestinal glucose absorption to prevent overshooting plasma glucose and insulin levels in patients with prediabetes and diabetes mellitus type 2. METHODS AND RESULTS We here demonstrate that an extract and individual polyphenols from apple diminish sodium-coupled glucose transporter 1 (SGLT1) mediated glucose uptake in vitro and in vivo. Inhibition of transport of sugars by SGLT1 was shown in Xenopus oocytes and in mice jejunal segments. Strongest inhibition was observed for phlorizin with IC50 values for transport inhibition of 0.46 ± 0.19 and 4.1 ± 0.6 μM in oocytes and intestinal segments, respectively. An oral glucose tolerance test performed in volunteers with prior administration of the apple extract reduced venous blood glucose and plasma insulin levels, similar to findings obtained in C57BL/6N mice. Analysis of human urine samples revealed that the extract increased modestly renal glucose loss that is most likely a result of inhibition of renal glucose reabsorption by phloretin derivatives found in plasma of the volunteers. CONCLUSION Although the apple extract substantially decreased intestinal glucose absorption in all test systems, the finding that there are systemic effects that relate to inhibition of glucose transport processes beyond the intestine addresses safety issues that need further exploitation.

Structural investigations on arabinogalactan-protein from wheat, isolated with Yariv reagent.

For the first time a pure arabinogalactan-protein (AGP) could be isolated from whole grain of wheat ( Triticum aestivum L.) by performing a double precipitation with beta-glucosyl Yariv reagent. The putative bioactive AGP has been characterized with regard to its polysaccharide and protein parts. Analytical investigations by GLC-MS and (13)C NMR revealed a carbohydrate moiety consisting of a 1,3-Galp backbone, linked in position 6 to short 1,6-Galp-chains, terminating in Araf. In the protein part, a high content of hydroxyproline has been found, probably responsible for linkage between protein and polysaccharide moieties. The molecular mass of AGP has been determined by size exclusion chromatography with laser light scattering detection and found to be 125 kDa. Alkaline hydrolysis of the protein resulted in single carbohydrate moieties with a molecular mass of about 20 kDa, indicating that AGP from whole grain of wheat belongs to the wattle blossom type of AGPs.

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.

Characterization and Immunolocalization of Arabinogalactan-Proteins in Roots of Echinacea purpurea

From the high molecular weight fraction of an aqueous extract from roots of Echinacea purpurea L. Moench, arabinogalactan-proteins (AGPs), a class of proteoglycans proposed to be involved in cell differentiation and plant growth, were purified and characterized with regard to amino acid composition and structure of the polysaccharide moiety. The protein content of the AGP was 5.0 % (w/w) with the dominating amino acids Glx, Hyp, Asx, Ser, Thr and Ala. The highly branched polysaccharide moiety shows a linkage composition typical of AGPs with 1,3-, 1,6- and 1,3,6-linked galactopyranosyl residues and arabinofuranosyl residues predominantly as terminal and 1,5-linked residues. Terminal units of glucuronopyranose acid were also detected. Furthermore, a new method for the localization of AGPs in plant tissue has been developed. The synthetic (beta- D-Glc)(3) Yariv phenylgycoside (betaGlcY) is known to specifically bind to AGPs. For immunolocalization, polyclonal betaGlcY-antibodies have been generated and were used to label Yariv-treated thin sections of roots from E. purpurea. After addition of the FITC-conjugated secondary antibody, the sections were analyzed by confocal laser scanning microscopy. AGPs are detected mainly in the central cylinder in the area of the xylem. Cell walls of vessels and tracheids are strongly labelled, especially at the inner area of the wall. Furthermore, there is intense labelling of the pit canals.

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-14C]-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 [6-14C]-labelled glucose resulted in an incorporation of labelled hexoses into amyloid and starch, whereas xylose residues of amyloid remained unlabelled.

In vitro production of specific polysaccharides: isolation and structure of an antitumor active β-glucan from Phytophthora parasitica

Polysaccharides are known to be an important group of biological polymers which are of great interest for industrial use. Besides their physical properties, their chemical structures can be the base for specific applications in medicine. Various polysaccharides are known to exhibit an antitumor activity, which is thought to be host mediated by a stimulation of the hosts immune system. A β-1.3/1.6-glucan P isolated from the cell walls of Phytophthora parasitica Dastur (Mating type A1, 1828) exhibited strong antitumor activity against Sarcoma-180 in mice. Growth of the fungus was markedly stimulated by β-sitosterol and calcium ions. The fungus was shown to be thiamine heterotroph. At optimal growth conditions up to 30 g (dry weight) of fungal mycelium could be harvested within 5 days in 81 of culture medium, which represented ~6 g of cell wall material. A specific water-soluble cell wall glucan P was isolated by extraction with hot water and purification by ion-exchange and Con A chromatography. By methylation analysis it was shown that the β-glucan consisted of 1.3-, 1.3.6- and terminal-glucose residues in a molar ratio of 5:1:1. Sequential Smith degradations combined with methylation analyses showed the presence of mono- as well as di- and trisaccharides as side chain constituents in a ratio of 9:2:3. A structure of the antitumor active β-1.3/1.6-glucan P is proposed.

Natural Products as Lead Compounds for Sodium Glucose Cotransporter (SGLT) Inhibitors

Glucose homeostasis is maintained by antagonistic hormones such as insulin and glucagon as well as by regulation of glucose absorption, gluconeogenesis, biosynthesis and mobilization of glycogen, glucose consumption in all tissues and glomerular filtration, and reabsorption of glucose in the kidneys. Glucose enters or leaves cells mainly with the help of two membrane integrated transporters belonging either to the family of facilitative glucose transporters (GLUTs) or to the family of sodium glucose cotransporters (SGLTs). The intestinal glucose absorption by endothelial cells is managed by SGLT1, the transfer from them to the blood by GLUT2. In the kidney SGLT2 and SGLT1 are responsible for reabsorption of filtered glucose from the primary urine, and GLUT2 and GLUT1 enable the transport of glucose from epithelial cells back into the blood stream.The flavonoid phlorizin was isolated from the bark of apple trees and shown to cause glucosuria. Phlorizin is an inhibitor of SGLT1 and SGLT2. With phlorizin as lead compound, specific inhibitors of SGLT2 were developed in the last decade and some of them have been approved for treatment mainly of type 2 diabetes. Inhibition of SGLT2 eliminates excess glucose via the urine. In recent times, the dual SGLT1/SGLT2 inhibitory activity of phlorizin has served as a model for the development and testing of new drugs exhibiting both activities.Besides phlorizin, also some other flavonoids and especially flavonoid enriched plant extracts have been investigated for their potency to reduce postprandial blood glucose levels which can be helpful in the prevention and supplementary treatment especially of type 2 diabetes.