Thin films of xyloglucans for BSA adsorption

In this work. XG extracted from Tamarindus indica (XGT) and Copaifera langsdorffii (XGC) seeds were deposited onto Si wafers as thin films. The characteristics of XGT and XGC adsorbed layers were compared with a commercial XG sample (TKP, Tamarind kernel powder) by ellipsometry, and atomic force microscopy (AFM). Moreover, the adsorption of oxidized derivative of XGT (To60) onto amino-terminated Si wafers and the immobilization of bovine serum albumin (BSA) onto polysaccharides covered wafers, as a function of pH, were also investigated. The XG samples presented molar ratios Glc:Xyl:Gal of 2.4:2.1:1 (XGC) 2.8: 23: 1 (XGT) and 1.91.91 (TKP). The structure of XGT and XGC was determined by O-methy alditol acetate derivatization and showed similar features, but XGC confirmed the presence of more alpha-D-Xyl branches due to more beta-D-Gal ends. XGT deposited onto Si adsorbed as fibers and small entities uniformly distributed, as evidenced by AFM, while TPK and XGC formed larger aggregates. The thickness of To60 onto amino-terminated surface was similar to that determined for XGT onto Si wafers. A maximum in the adsorbed amount of BSA occurred close to its isoelectric point (5.5). These findings indicate that XGT and To60 are potential materials for the development of biomaterials and biotechnological devices. (C) 2008 Elsevier B.V. All rights reserved.

Structural characterization and biological activity of polysaccharides from infusions used in the popular tradition

The use of plants with medicinal purposes is an ancient practice still very common in developing regions, and is rapidly spreading in industrialized countries. This fact is evidenced by the large number of ethnobotanical studies found in the literature referring that these plants are often used as decoctions and infusions. In most studies the reported biological activities are attributed to the presence of phenolic compounds, due to their antioxidant properties, and to polysaccharides, with its anti-tumoral properties. In “Trás-os-Montes” region, some of the most popular infusions used by the popular medicine are prepared with the dried leaves of Fraxinus angustifolia, the dried shoots of Mentha suaveolens, and the dried inflorescences of Pterospartum tridentatum. However, there are no studies about the polysaccharides present in these infusions. Thus, through the structural characterization of the polysaccharides present in the infusions of F. angustifolia, M. suaveolens, and P. tridentatum, the present PhD thesis intends to evaluate the possible relation between polysaccharides and the immunostimulatory activity that these infusions might present. In a preliminary phase, infusions of F. angustifolia were prepared according to the popular tradition, and it was observed that the obtained water soluble material contained approximately 85% of material non-retained in C18 cartridges, with hydrophilic characteristics, with the remaining 15% comprising retained-material with hydrophobic characteristics. It was also shown that the infusions only contained between 2 and 4% of high molecular weight material (HMWM), which comprised approximately 30% of carbohydrate material. Sugar and methylation analysis of the HMWM suggested the presence of pectic polysaccharides, together with type II arabinogalactans, mannans, and xyloglucans. However, the amount of material obtained is to low for the fractionation, and structural analysis of the polysaccharides present. The 4 h decoction, divided in two periods of 2 h, with water renewal, allowed to increase the HMWM yield, relatively to the infusions traditional infusions. It was also observed that the decoction also allowed to increase the HMWM proportion of carbohydrate material, due to an increase in the proportion of uronic acid present, although the neutral sugar residues seemed to be detected in similar proportions. Therefore, in all the experiments subsequently performed, the HMWM used was obtained through the decoction of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences. x After the fractionation, through ethanol precipitation, and anion exchange chromatography, of the polysaccharides from the HMWM obtained by the decoction of the vegetable material of the distinct studied plants, it was observed the presence of high proportions of pectic polysaccharides, containing type I arabinogalactans, together with minor proportions of type II arabinogalactans, mannans, and xyloglucans. The presence of pectic polysaccharides in the extracts from F. angustifolia was also evidenced through endo-polygalacturonase treatment, and ESI-MS and ESI-MS/MS experiments. The detection of linked pentose and uronic acid residues, also seemed to suggest the presence of xylogalacturonan domains in the pectic polysaccharides from F. angustifolia. The extracts from F. angustifolia dried leaves also contained type II arabinogalactans that exhibited a higher structural diversity than those detected in the M. suaveolens, and P. tridentatum extracts, particularly in the substitution degree of the galactan backbone, and in the extension of the (1→5)-Araf side chains. Moreover, for all the plants studied, it was also observed that the type II arabinogalactans, extracted during the 2nd 2h of the extraction process, exhibited a substitution degree of the galactan backbone higher than those extracted during the 1st 2h. The extracts from P. tridentatum dried inflorescences contained higher proportions of mannans, and also of xyloglucans, both presenting a substitution degree higher than those, which were detected in lower proportion in the extracts of F. angustifolia and M. suaveolens. Through ESI-MS and ESI-MS/MS it was possible to evidence that the mannans present in the extracts of P. tridentatum presented acetyl groups on the O-2 of the mannosyl residues. It was also evidenced that the P. tridentatum mannans were more extensively acetylated than the mannans detected in the coffee infusion, LBG, and other non-conventional mannan sources. Moreover, it was detected the presence of oligosaccharides comprising hexose residues linked to non acetylated pentose residues, suggesting the possible presence of arabinose residues in the mannans from P. tridentatum extracts. The immunostimulatory activity of three fractions isolated from the extracts of F. angustifolia, M. suaveolens, and P. tridentatum, was tested and an increase in the NO production by macrophages, without compromising their cellular viability, was observed. The type I, and type II arabinogalactans detected in the extracts from F. angustifolia, and M. suaveolens seem to have contributed for the observed immunostimulatory activity. For the fraction from P. tridentatum, the mannans acetylation, and the presence of type I, and type II arabinogalactans seemed to contribute for the macrophage immunostimulatory activity observed. The possible presence of storage xyloglucans from the inflorescences seeds, also seems to have contributed for the immunostimulatory activity registered when the macrophages were stimulated with higher extract concentrations. The results obtained allow to conclude that the extracts of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences contained high proportions of pectic polysaccharides, exhibiting type I arabinogalactans, together with other polysaccharides, such as type II arabinogalactans, mannans, and xyloglucans. This polysaccharide mixture seems to have contributed to the immunostimulatory activity of fractions isolated from the extracts of the studied plants. Therefore, as the same type of polysaccharides seem to be present in the decoctions and in the infusions, it seems possible that the polysaccharides might contribute for the therapeutic properties frequently associated by the popular tradition to the infusions of these plants.

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.

Plant Cell Wall Research Related to Evolution and Chemical Defenses

The plant cell wall is composed mainly of polysaccharides some constituted of repeating units of a single sugar, as cellulose or by two or more sugars grouped in repeating oligosaccharide blocks as the galactomannans and xyloglucans. Variations in composition and fine structure of these cell wall polysaccharides have been used as taxonomic markers and in the comprehension of the evolutive process, particularly in the Leguminosae. Partial hydrolysis of these compounds give rise to oligomers, some of which are capable of eliciting the synthesis of defensive substances in plants named phytoalexins. Species which differ in respect to phytoalexin liberation also differ in cell wall composition, particularly in the pectic fraction of the wall. Pectinases (mainly endopolygalacturonases) present in fungi, have been shown to hydrolyze plant cell walls yielding phytoalexin-eliciting oligosaccharides which differ in composition and in eliciting capacity in different species. These differences can be associated with the capacity of a given species to produce phytoalexins. On the other hand, the phytoalexin induction in plants is being used as a method of producing novel bioactive secondary metabolites.

Preparação e caracterização de filmes a base de xiloglucana extraída de sementes de Hymenaea Courbaril (Jatobá)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização funcional e estrutural de uma ?-glucanase GH12 de Aspergillus terreus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The Molecular Characterization of a Predicted Glycosyltransferase from Lycopersicon esculentum

The synthesis of the plant cell wall is very complex, and understanding how this process occurs will lead to many benefits for future research and industries dependent upon cell walls for their products. The recent discovery of the functions of AtMUR3 and AtGT18 in Arabidopsis thaliana as xyloglucan galactosyltransferases has led to the identification of many more putative glycosyltransferases in the Arabidopsis genome. Due to the structural differences between the xyloglucans of Arabidopsis and solanaceous plants, we decided to search for putative arabinosyltransferases in the Solanaceae. Solanaceous xyloglucan is substituted by one to two arabinosyl residues at the second xylose position, and sometimes contains an arabinose at the first xylose position. In contrast, Arabidopsis xyloglucan does not contain arabinose, and is substituted by galactose at the second and third xylose position. Furthermore, the second galactose residue in Arabidopsis xyloglucan is usually fucosylated, a modification not found in solanaceous plants. Searching the database of expressed sequence tags (dbEST), we identified many likely glycosyltransferases in solanaceous plants, including tomato (Lycopersicon esculentum). AtMUR3 and AtGT18 search queries resulted in the identification of three putative glycosyltransferases in L. esculentum, which were tentatively designated LeGT1, Le1GT18, and Le2GT18. Based on phylogenetic considerations, Le2GT18 was thought to be a putative arabinosyltransferase. The gene was transformed into atmur3-3 and atgt18 mutant plants, and the resulting plants will be screened for homozygous plants with the inserted gene. The homozygous T2 plants can then be screened for changes in the composition of their cell walls. Because Le2GT18 is thought to be an arabinosyltransferase, the levels of arabinose may be increased in the xyloglucan fraction of the cell wall. If so, further testing can be performed to reveal the true function of Le2GT18.

Effects of structural variation in xyloglucan polymers on interactions with bacterial cellulose

A cellulose/xyloglucan framework is considered to form the basis for the mechanical properties of primary plant cell walls and hence to have a major influence on the biomechanical properties of growing, fleshy plant tissues. In this study, structural variants of xyloglucan have been investigated as components of composites with bacterial cellulose as a simplified model for the cellulose/xyloglucan framework of primary plant cell walls. Evidence for molecular binding to cellulose with perturbation of cellulose crystallinity was found for all xyloglucan types. High molecular mass samples gave homogeneous centimeter-scale composites with extensive cross-linking of cellulose with xyloglucan. Lower molecular mass xyloglucans gave heterogeneous composites having a range of microscopic structures with little, if any, cross-linking. Xyloglucans with reduced levels of galactose substitution had evidence of self-association, competitive with cellulose binding. At comparable molecular mass, fucose substitution resulted in a modest promotion of microscopic features characteristic of primary cell walls. Taken together, the data are evidence that galactose substitution of the xyloglucan core structure is a major determinant of cellulose composite formation and properties, with additional fucose substitution acting as a secondary modulator. These conclusions are consistent with reported structural and mechanical properties of Arabidopsis mutants lacking specific facose and/or galactose residues.

Fruit cell culture as a model system to study cell wall changes during strawberry fruit ripening

Strawberry (Fragaria x ananassa, Duch.) fruit is characterized by its fast ripening and soft texture at the ripen stage, resulting in a short postharvest shelf life and high economic losses. It is generally believed that the disassembly of cell walls, the dissolution of the middle lamella and the reduction of cell turgor are the main factors determining the softening of fleshy fruits. In strawberry, several studies indicate that the solubilisation and depolymerisation of pectins, as well as the depolymerisation of xyloglucans, are the main processes occurring during ripening. Functional analyses of genes encoding pectinases such as polygalacturonase and pectate lyase also point out to the pectin fraction as a key factor involved in textural changes. All these studies have been performed with whole fruits, a complex organ containing different tissues that differ in their cell wall composition and undergo ripening at different rates. Cell cultures derived from fruits have been proposed as model systems for the study of several processes occurring during fruit ripening, such as the production of anthocyanin and its regulation by plant hormones. The main objective of this research was to obtain and characterize strawberry cell cultures to evaluate their potential use as a model for the study of the cell wall disassembly process associate with fruit ripening. Cell cultures were obtained from cortical tissue of strawberry fruits, cv. Chandler, at the stages of unripe-green, white and mature-red. Additionally, a cell culture line derived from strawberry leaves was obtained. All cultures were maintained in solid medium supplemented with 2.5 mg.l-1 2,4-D and incubated in the dark. Cell walls from the different callus lines were extracted and fractionated to obtain CDTA and sodium carbonate soluble pectin fractions, which represent polyuronides located in the middle lamella or the primary cell wall, respectively. The amounts of homogalacturonan in both fractions were estimated by ELISA using LM19 and LM20 antibodies, specific against demethylated and methyl-esterified homogalacturonan, respectively. In the CDTA fraction, the cell line from ripe fruit showed a significant lower amount of demethylated pectins than the rest of lines. By contrast, the content of methylated pectins was similar in green- and red-fruit lines, and lower than in white-fruit and leaf lines. In the sodium carbonate pectin fraction, the line from red fruit also showed the lowest amount of pectins. These preliminary results indicate that cell cultures obtained from fruits at different developmental stages differ in their cell wall composition and these differences resemble to some extent the changes that occur during strawberry softening. Experiments are in progress to further characterize cell wall extracts with monoclonal antibodies against other cell wall epitopes.