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PubMed2005

Immunostimulating activity of pectic polysaccharide from Bergenia crassifolia (L.) Fritsch.

The pectic polysaccharide named bergenan BC was obtained using extraction of the green leaves of Siberian tea Bergenia crassifolia (L.) Fritsch. by an aqueous ammonium oxalate. The polysaccharide obtained was proved to comprise mainly d-galacturonic acid, galactose, rhamnose, arabinose and glucose residues and appeared to be pectin. Delayed type hypersensitivity (DTH) reaction to aggregated ovalbumin (agOVA) was found to increase in mice that received bergenan solution (2 mg/mL) for 3 weeks. Bergenan BC was observed to enhance the uptake capacity of human neutrophils at a concentration 100 microg/mL and to stimulate the generation of oxygen radicals by mouse peritoneal macrophages in vitro. Bergenan BC was found to increase the spontaneous adhesion of peritoneal leukocytes and failed to influence adhesion stimulated by PMA or adhesion of peritoneal leukocytes incubated in the presence of 5 mm EDTA. Bergenan failed to show cytotoxic action. The viability of peritoneal leukocytes was estimated to be equal to 91% +/- 8% and 90% +/- 7%% in the control and in the pectin solution at a concentration of 1 mg/mL.Thus, bergenan was shown to possess immunostimulating activity in relation to DTH response in vivo and phagocytic activity in vitro.

Adjuvants, Immunologic↗
PubMed1998

Analysis of partially methyl-esterified galacturonic acid oligomers by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Two methods were developed to detect partially methyl-esterified galacturonic acid oligomers, generated by endopolygalacturonase treatment of a 30% methyl-esterified pectin. The enzyme digest was shown, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, to contain sodiated galacturonic acid oligomers with a degree of polymerization of 2-12, containing 0-6 methyl esters. Galacturonic acid (monomer) could not be detected because of matrix ions interference in the low mass region. Using high-performance anion-exchange chromatography, with a sodium acetate gradient at pH 5.0 and postcolumn sodium hydroxide addition to allow pulsed amplified detection, a complex elution profile was obtained with the endopolygalacturonase-treated 30% methyl-esterified pectin. All the components eluted before nonesterified tetragalacturonic acid. The partially methyl-esterified oligogalacturonic acids eluted in a discernible series of oligomers with an identical number of nonesterified carboxylic acid groups; the large, more esterified oligomers eluted before small, less esterified oligomers. The methyl esters may hinder the interaction of the neighboring carboxylic acid groups with the anion-exchange resin, thereby giving the components an apparent lower overall negative charge.

Chromatography, Ion Exchange↗
PubMed1994

Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis.

The Erwinia chrysanthemi kdgR gene encodes a repressor that negatively regulates the expression of genes involved in pectinolysis and in pectinase secretion. The cloned kdgR gene was overexpressed in Escherichia coli by using a phage T7 system. Overproduced repressor was purified to homogeneity by two chromatographic steps. Gel retardation and DNase I protection experiments demonstrated the specific binding of the KdgR protein to the operators of pectinase genes (pelA, pelB, pelC, pelE), to the operator of genes involved in pectin catabolism (kdgT, ogl, kduI-kdgF) and to that of the outT gene involved in pectinase secretion. These interactions involved one (pelA, pelB, kduI-kdgF, outT) or several operator sites (pelC, pelE, ogl, kdgT) that generally overlap the promoter. Despite the presence of potential KdgR binding sites (KdgR-box) in the regulatory regions of four genes involved in pectin catabolism (kdgC, kduD, pem, kdgA) and in a pectinase secretion gene outC, no DNA-repressor complex could be observed by in vitro experiments. By using a missing contact experiment on the coding strand of ogl and pelE regulatory regions, a new KdgR-binding consensus was proposed. This new consensus, constituted by two half motifs (AATGAAAACT)N(NTCGATTTCTA), is well conserved in the operators which interact in vitro with the KdgR repressor. In contrast, this repressor-recognized motif is degenerated in the other operators that cannot interact in vitro with the repressor. These results suggest the existence of different regulation mechanisms mediated by the KdgR protein for the two classes of operators.

Base Sequence↗
PubMed1996

Variations in the structure of neutral sugar chains in the pectic polysaccharides of morphologically different carrot calli and correlations with the size of cell clusters.

Carrot (Daucus carota L.) embryogenic callus (EC) loses its embryogenic competence and becomes non-embryogenic callus (NC) during long-term culture. With the loss of embryogenic competence, the cell clusters become smaller and the extent of intercellular attachments is reduced. Pectic fractions prepared from EC and NC were separated into two subfractions by gel filtration. A difference in sugar composition between EC and NC was found only in the high-molecular-mass (ca. 1300 kDa) subfraction, and the ratio of the amount of arabinose to that of galactose (Ara/Gal) was strongly and positively correlated with the size of cell clusters in several different cultures. From the results of sugar-composition and methylation analyses, and the results of treatment with exo-arabinanase, models of the neutral sugar chains of pectins from Ec and NC are proposed. Both neutral sugar chains are composed of three regions. The basal region is composed of linearly linked arabinan 5-Araf) moieties in both types of callus. The middle galactan region is composed of 6-linked galactose, some of which branches at the 3 and 4 positions, and this region is larger and more frequently branched in NC than in EC. Finally, the terminal arabinan region is composed of 5-linked arabinose, branched at the 3 position, and the size of the terminal arabinan is larger in EC than in NC. The significance of the neutral sugar chains of pectins in the interaction of cell wall components and intercellular attachment is discussed.

Carbohydrate Conformation↗
PubMed1985

Differential effects of dietary fibers on rat intestinal circular muscle cell size.

The relationship between dietary fiber and intestinal circular muscle cell size was investigated in rats by feeding defined diets supplemented with four different sources of fiber. In the first study, a 20% wheat bran supplement was fed to 10 rats for nine weeks. This resulted in larger muscle cell size, with a 22.5% increase in the proximal (P less than 0.02) and 77.9% increase in the distal colon (P less than 0.01) when compared with a control group of 10 rats fed a fiber-free diet. In the second study, which lasted four weeks, a control group of 10 rats was fed a fiber-free diet, while similar sized experimental groups were fed the same basal diet plus either 20% oat bran, 10% pectin, or 10% guar. Muscle cell size was decreased by 20.6% in the proximal jejunum of the oat bran- and pectin-fed groups (P less than 0.05) and by 43% in the proximal colon of the oat bran-fed group, when compared with the controls (P less than 0.05). These results show that the effects of high fiber diets on intestinal muscle cell size depend on the type of fiber consumed.

Animals↗
PubMed1978

Fiber, hypercholesteremia, and atherosclerosis.

Epidemiological data suggest that populations subsisting on high fiber diets are free of a number of the diseases of Western civilization, among them coronary heart disease. Studies in animals and man show that each type of fiber exerts its own specific influence. Thus, in man bran has no effect on serum lipids, but pectin lowers cholesterol levels. In animals fed atherogenic diets, alfalfa and pectin exert some measure of protection, but cellulose does not. To fully understand the mode of action of dietary fiber, it is necessary to ascertain the mechanism(s) of action of each chemical component of that fiber.

Africa↗
PubMed2001

Localization of cell wall polysaccharides in nonarticulated laticifers of Asclepias speciosa Torr.

Asclepias speciosa Torr. has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1-->4) beta-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (1-->5) alpha-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1-->4) beta-D-galactan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to beta-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled by Ricinus communis agglutinin, a lectin that binds to terminal beta-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.

Apocynaceae↗
PubMed2003

Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase.

The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced. The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da. The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp. and Erwinia chrysanthemi. The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure. The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively. The activity of purified PelAHis was inhibited in the presence of excess substrate. Characterization of product formation revealed unsaturated trigalacturonate as the main product. The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin.

Amino Acid Sequence↗
PubMed1997

Growth conditions of Aspergillus sp. ATHUM-3482 for polygalacturonase production.

A wild type of Aspergillus sp. ATHUM-3482 produced extracellular polygalacturonase when grown in liquid medium containing citrus pectin as sole carbon source. A number of factors affecting enzyme activity were investigated. Polygalacturonase activities as high as 4.3 U ml-1 (reducing-group-releasing activity) and 17 U ml-1 (viscosity-diminishing activity) were obtained under optimum growth conditions. With sugar-beet as sole carbon source the respective activities were 6.5 U ml-1 and 40 U ml-1, the highest achieved in this work. Under these conditions no pectin lyase or pectinesterase activity was detected. The above yields of polygalacturonase activity compare favourably with those reported for fungi grown under similar growth conditions.

Aspergillus↗
PubMed2004

Distribution of cell wall components in Sphagnum hyaline cells and in liverwort and hornwort elaters.

Spiral secondary walls are found in hyaline cells of Sphagnum, in the elaters of most liverworts, and in elaters of the hornwort Megaceros. Recent studies on these cells suggest that cytoskeletal and ultrastructural processes involved in cell differentiation and secondary wall formation are similar in bryophytes and vascular plant tracheary elements. To examine differences in wall structure, primary and secondary wall constituents of the hyaline cells of Sphagnum novo-zelandicum and elaters of the liverwort Radula buccinifera and the hornwort Megaceros gracilis were analyzed by immunohistochemical and chemical methods. Anti-arabinogalactan-protein antibodies, JIM8 and JIM13, labeled the central fibrillar secondary wall layer of Megaceros elaters and the walls of Sphagnum leaf cells, but did not label the walls of Radula elaters. The CCRC-M7 antibody, which detects an arabinosylated (1-->6)-linked beta-galactan epitope, exclusively labeled hyaline cells in Sphagnum leaves and the secondary walls of Radula elaters. Anti-pectin antibodies, LM5 and JIM5, labeled the primary wall in Megaceros elaters. LM5 also labeled the central layer of the secondary wall but only during formation. In Radula elaters, JIM5 and another anti-pectin antibody, JIM7, labeled the primary wall. The distribution of arabinogalactan-proteins and pectic polysaccharides restricted to specific wall types and stages of development provides evidence for the developmental and functional regulation of cell wall composition in bryophytes. Monosaccharide-linkage analysis of Sphagnum leaf cell walls suggests they contain polysaccharides similar to those of higher plants. The most abundant linkage was 4-Glc, typical of cellulose, but there was also evidence for xyloglucans, 4-linked mannans, 4-linked xylans and rhamnogalacturonan-type polysaccharides.

Cell Wall↗
PubMed2005

Characterization and in vitro expression patterns of extracellular degradative enzymes from non-pathogenic binucleate Rhizoctonia AG-G.

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source beta-1,3 and beta-1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The beta-(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.

Carboxylic Ester Hydrolases↗
PubMed2006

Characterization of an extracellular endopolygalacturonase from the saprobe Mucor ramosissimus Samutsevitsch and its action as trigger of defensive response in tropical plants.

In recent years, interest in the ability of non-pathogenic microorganisms to induce resistance in plants has grown, particularly with respect to their use as environmentally safe controllers of plant disease. In this study, we investigated the capacity of Mucor ramosissimus Samutsevitsch to release pectinases able to degrade cell walls of Palicourea marcgravii St. Hil., a tropical forest native Rubiaceae on which the spores of this saprobic fungus have been found. The fungus was grown in liquid culture medium containing pectin as the sole carbon source and filtrates were analyzed for pectinase activity. An endopolygalacturonase was partially purified by ion exchange chromatography, gel filtration, and preparative isoelectrofocusing, and characterized. This enzyme was more active upon pectic substrates with a low degree of methyl esterification. The products of hydrolysis of different pectic substrates (including pectin from P. marcgravii) by the action of this endopolygalacturonase elicited to different extents the phytoalexin production in soybean cotyledons. Also, the enzyme itself and the products of its action on the pectic fraction of P. marcgravii elicited the production of defensive compounds in the leaves of the plant. These results suggest that, besides the role in recycling organic matter, saprobes may also play an important role in the induction of defensive mechanisms in wild plants by enhancing their non-specific resistance against pathogens. Furthermore, they set the stage for future studies on the role of saprobic fungi in inducing resistance of host plants to pathogens.

Biological Products↗
PubMed1992

An ultraviolet-spectrophotometric method with 2-cyanoacetamide for the determination of the enzymatic degradation of reducing polysaccharides.

A rapid, facile, and sensitive uv-spectrophotometric assay has been developed for the determination of the enzymatic degradation of polysaccharides that generates reducing sugars. The assay was carried out with 2-cyanoacetamide in a single test tube. The solution was left at pH 9 by the addition of borate buffer within 5 min. Measurement of the reaction mixture at 274 nm allows a simple determination up to 600 mumol/liter of reducing sugars. The coefficient of variation was less than 2% on all measurements. The assay was developed with pectin and polygalacturonic acid from apples and has been compared with the Somogyi-Nelson method. The new assay was then exemplarily used for the determination of the enzymatic hydrolysis products of pectin from cotton.

Calibration↗
PubMed1992

Dietary fibre in the diet of the growing pig: effects on apparent digestibility and resultant implications for its use in reducing the incidence of oesophagogastric parakeratosis.

The effects on apparent digestibility of increasing dietary fibre concentrations in barley-based diets by substituting for barley either bran, oatmeal by-product, guar gum or pectin were studied using 72 pigs. Increasing substitutions of bran up to a maximum of 300 g kg-1 gave progressive decreases in apparent digestibility of dry matter, ether extract and gross energy fractions and in digestible and metabolisable energy contents, progressive increases in apparent digestibility of modified acid detergent fibre but had no consistent effect on either nitrogen or extract free of nitrogen fractions. Increasing substitution of oatmeal by-product up to the same maximum gave significant linear decreases in apparent digestibility of dry matter, extract free of nitrogen and gross energy, significant depressions in apparent digestibility of modified acid detergent fibre and significant increases in ether extract. Guar gum and pectin, both substituted to a maximum of 50 g kg-1 had no significant effects on apparent digestibility. The results are discussed in the context of the concentrations of dietary fibre needed to reduce the incidence of oesophagogastric parakeratosis.

Animal Feed↗
PubMed1989

Long clinostation influence on the localization of free and weakly bound calcium in cell walls of Funaria hygrometrica moss protonema cells.

The pyroantimonate method was used to study the localization of free and weakly bound calcium in cells of moss protonema of Funaria hygrometrica Hedw. cultivated on a clinostat (2rev/min). Electroncytochemical study of control cells cultivated at 1 g revealed that granular precipitate marked chloroplasts, mitochondria, Golgi apparatus, lipid drops, nucleoplasma, nucleolus, nucleus membranes, cell walls and endoplasmic reticulum. In mitochondria the precipitate was revealed in stroma, in chloroplast it was found on thylakoids and envelope membranes. The cultivation of protonema on clinostat led to the intensification in cytochemical reaction product deposit. A considerable intensification of the reaction was noted in endomembranes, vacuoles, periplasmic space and cell walls. At the same time analysis of pectinase localization was made using the electroncytochemical method. A high reaction intensity in walls in comparison to that in control was found out to be a distinctive peculiarity of the cells cultivated on clinostat. It testifies to the fact that increasing of free calcium concentrations under conditions of clinostation is connected with pectinic substances hydrolysis and breaking of methoxy groups of pectins. Data obtained are discussed in relation to problems of possible mechanisms of disturbance in calcium balance of plant cells and the role of cell walls in gomeostasis of cell grown under conditions of simulated weightlessness.

Bryopsida↗
PubMed1984

Hydrocolloid food additives and rat caecal microbial enzyme activities.

Agar, carboxymethylcellulose, carrageenan, guar gum, gum acacia, locust-beam gum or pectin (50 g/kg diet), given to weanling rats for 4 wk, increased the weight of the caecal wall and the caecal contents. Feeding carboxymethylcellulose, guar gum or pectin significantly increased, and feeding carrageenan decreased, the total bacterial population of the caecum. Feeding carboxymethylcellulose significantly increased in vitro activity of bacterial azoreductase, beta-glucosidase, beta-glucuronidase, nitrate reductase, nitroreductase and urease. Guar gum, gum acacia and locust-bean gum each increased at least three of these activities. In contrast, feeding carrageenan greatly decreased all microbial enzyme activities, while agar decreased beta-glucosidase, beta-glucuronidase and nitroreductase activities.

Agar↗
PubMed1996

Evaluation of the safety of sodium pectate as a food ingredient.

New potential uses of pectates in food products have recently stimulated interest in re-evaluating the information available concerning the safety of pectins and pectates as food ingredients. Data relevant to this re-evaluation have been obtained in rats in recent 14-day and 13 wk subchronic feeding studies with sodium pectate. Ames tests and other mutagenicity tests have been conducted with sodium pectate, bleached sodium pectate and mixed sodium/calcium pectate salts. These toxicological studies with pectates have provided further evidence of their safety, and support of the continued GRAS status of pectins and pectate salts.

Animals↗
PubMed1986

Effect of mixtures of dietary fibres on the enzyme activity of the rat caecal microflora.

The enzyme activity of the caecal microflora from weanling rats was determined after feeding 1 of 3 basal diets (purified fibre-free; purified plus cellulose; and stock), with or without additional dietary fibre (pectin, i-carrageenan or carboxymethylcellulose 5% w/w). The wet weight of caecal contents and total bacterial numbers were similar for the purified fibre-free and purified plus cellulose diets, yet were significantly higher in animals fed the stock diet. Pectin supplementation of the basal diets had no effect of caecal bacterial numbers, but significantly increased total nitrate reductase activity per caecum except when added to stock diet. Carrageenan decreased caecal bacterial numbers and most enzyme activities with both purified diets, and to a lesser extent with the stock diet. Carboxymethylcellulose increased bacterial numbers and enzyme activities, particularly beta-glucosidase and nitrate reductase when added to the purified diet but not when added to either the purified diet plus cellulose or the stock diet. The results demonstrate that the effects of dietary fibre components on the rat caecal microflora are dependent upon the initial fibre content of the diet base.

Animals↗