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Tetrad pollen formation in quartet mutants of Arabidopsis thaliana is associated with persistence of pectic polysaccharides of the pollen mother cell wall.

The quartet (qrt) mutants of Arabidopsis thaliana produce tetrad pollen in which microspores fail to separate during pollen development. Because the amount of callose deposition between microspores is correlated with tetrad pollen formation in other species, and because pectin is implicated as playing a role in cell adhesion, these cell-wall components in wild-type and mutant anthers were visualized by immunofluorescence microscopy at different stages of microsporogenesis. In wild-type, callose was detected around the pollen mother cell at the onset of meiosis and around the microspores during the tetrad stage. Microspores were released into the anther locule at the stage where callose was no longer detected. Deposition and degradation of callose during tetrad pollen formation in qrt1 and qrt2 mutants were indistinguishable from those in wild-type. Enzymatic removal of callose from wild-type microspores at the tetrad stage did not release the microspores, suggesting that callose removal is not sufficient to disperse the microspores in wild-type. Pectic components were detected in the primary wall of the pollen mother cell. This wall surrounded the callosic wall around the pollen mother cell and the microspores during the tetrad stage. In wild-type, pectic components of this wall were no longer detectable at the time of microspore release. However, in qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation. This result suggests that failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, and indicates that QRT1 and QRT2 may be required for cell type-specific pectin degradation to separate microspores.

Arabidopsis↗

Cell wall antibodies without immunization: generation and use of de-esterified homogalacturonan block-specific antibodies from a naive phage display library.

Homogalacturonan (HG) is a multi-functional pectic polysaccharide of primary cell walls involved in calcium cross-linking and gel formation, and the regulation of ionic status and porosity of the cell wall matrix, and is a source of oligosaccharins functioning in development and defence. Phase display monoclonal antibodies with specificity for de-esterified stretches ('blocks') of pectic HG have been isolated from a naive phage display library without the need for immunization of animals or conjugation of an oligosaccharide to protein. These antibodies, designated PAM1 and PAM2, bind specifically to de-esterified and un-substituted HG. Assays with a series of pectins de-esterified by the action of plant or fungal pectin methyl esterases indicated that the antibodies were specific to de-esterified blocks resulting from the blockwise action of plant pectin methyl esterases. Analysis of antibody binding to a series of oligogalacturonides indicated that optimal binding required in the region of 30 de-esterified GalA residues. The recognition of such a large epitope by these antibodies allows the HG block architecture of primary cell walls to be identified and localized for the first time. Furthermore, we have demonstrated that monoclonal antibodies with high specificity and avidity to cell wall epitopes can be generated using a 'single pot' phage display approach.

Amino Acid Sequence↗

Detection and tissue distribution of anti-ulcer pectic polysaccharides from Bupleurum falcatum by polyclonal antibody.

Anti-sera against the "ramified" region (PG-1) of an anti-ulcer polysaccharide (bupleuran 2IIc), which was purified from the roots of Bupleurum falcatum L, were obtained by immunization of rabbits, and a polyclonal anti-bupleuran 2IIc/PG-1-antibody of the IgG class was purified by Protein G and "ramified" region (PG-1) immobilized affinity chromatographies. The antigenic specificity of anti-bupleuran 2IIc/PG-1-IgG was examined by a two-site sandwich ELISA which was developed as an improved method for microanalysis of bupleuran 2IIc using a biotinylated antibody. Another pectin from B. falcatum and anti-complementary pectins from Angelica acutilaba and Glycyrrhiza uralensis also showed significant reactivity to anti-bupleuran 2IIc/PG-1-IgG, although these reactivities were lower than that of bupleuran 2IIc. Other polysaccharides tested such as apple pectin, araban, yeast mannan, pullulan, etc., had negligible reactivity. The KDO-containing region and oligogalacturonides, which were obtained by endo-alpha-(1-->4)-polygalacturonase digestion of bupleuran 2IIc, were also not significantly recognized by anti-bupleuran 2IIc/PG-1-IgG. When bupleuran 2IIc was administered to the mice i.v., the polysaccharide disappeared from the circulation within 24 h and was mainly detected in the liver by the two-site sandwich ELISA. However the clearance of bupleuran 2IIc from the circulation was delayed by pretreatment with iota-carrageenan. When the crude polysaccharide fraction (BR-2), containing mainly bupleurans 2IIb and 2IIc from B.falcatum, was administrated orally to the mice, the polysaccharides were detected in the liver and Peyer's patch.

Animals↗

Fermentation of dietary fibre components in the rat intestinal tract.

1. The fermentative breakdown of dietary fibre from various sources in the intestinal tract was studied using rat balance experiments and gas-liquid chromatograhic analysis of dietary fibre monomers in feed and faces. 2. On a basal diet with 690 g maize starch/kg but no added fibre, small but detectable amounts of polymeric glucose, rhamnose, arabinose, xylose, galactose, mannose and uronic acids, i.e. sugars occurring in dietary fibre, were excreted in faeces. 3. Dietary fibre in wheat bran was rather resistant to fermentation; 63% was recovered in the faeces. Guar gum, on the other hand, was almost completely fermented, whereas 19 and 25% of the uronic acids in low and high methoxylated pectin respectively, were excreted in faeces. The various constituents of sugar-beet dietary fibre (approximately equal amounts of arabinose-based hemicellulose, pectin and non-starch glucan (cellulose)) showed quite variable availability for micro-organisms in that 6-12% of the arabinose, 17-25% of the uronic acids, and 52-58% of the cellulose were recovered in the faeces. 4. Faecal nitrogen excretion increased on addition of any one of the dietary fibre preparations studied, resulting in decreased true and apparent protein digestibility values. 5. The faecal dry weight increment was most pronounced when feeding bran and could then almost be accounted for by the remaining fibre and by protein. The less-prominent bulking effect of guar gum and pectins, that were much more extensively fermented, could be only partly explained by dietary fibre and protein.

Animals↗

Effects of structural and non-structural polysaccharides in the diet of the growing pig on gastric emptying rate and rate of passage of digesta to the terminal ileum and through the total gastrointestinal tract.

Six experiments were made in growing pigs to investigate the effects of substituting bran, oatmeal byproduct, pectin and guar gum for barley on gastric emptying rate, pH and dry matter of digesta and on the dry matter content and rate of passage of digesta to the terminal ileum and overall. Twelve pigs with cannulas sited at the pyloric/fundic junction of the stomach were used to measure gastric emptying. Seventy-two pigs were kept in metabolism cages for the total collection of faeces and nine pigs were fitted with simple cannulas 150 mm cranial to the ileo-caecal junction. For the overall transit measurements the barley was ground through a 4.68 mm screen (C) and for the other studies either through this screen or a 1.56 mm screen (F). Gastric emptying rates were significantly faster for F diets than for C diets, but bran and oatmeal by-product substitutions were without significant effect on this variable or on rate of passage to the terminal ileum. Guar gum and pectin significantly speeded up gastric emptying and transit to the terminal ileum in C diets. Guar gum and pectin did not significantly affect overall transit time, but bran accelerated it. The results are discussed in relation to the depressive effects on nutrient utilization which have been recorded for the four polysaccharides: it is postulated that gastric emptying rate and transit time to the terminal ileum are likely to have a minimal effect on overall transit time.

Animal Nutritional Physiological Phenomena↗

Chronic and postprandial responses of plasma insulin, glucose and lipids in volunteers given dietary fibre supplements.

We questioned whether a dietary fibre supplement known to lower fasting plasma cholesterol concentrations can also lower the postprandial plasma cholesterol, glucose and insulin concentrations when it is administered just before a meal. Two studies were conducted in healthy middle-aged volunteers of both sexes in whom the fasting plasma total cholesterol concentrations were above normal. In the first study the dietary fibre treatments (2.2 g) were psyllium and a psyllium-citrus pectin mixture to which the subjects (four males, eight females) had no prior exposure. Controls received no supplement. The meals were high-fat breakfasts and lunches. In the second study the dietary fibre (6 g) was from sugar-beet root and the reference control was alpha-cellulose (2 g); the meal was of glucose. The volunteers (eight males, eight females) had prior exposure to the fibre supplements three times daily for 3 weeks. After adjustments for fasting values and changes in haemodilution, the psyllium and psyllium-citrus pectin mixture in the first experiment had no significant effects on the postprandial measurements of plasma glucose, insulin:glucose ratio, total-, LDL- and HDL-cholesterol, and triacylglycerol. By contrast, the sugar-beet fibre in the second study significantly decreased the area under the glucose response curve by 6.9%, the area under the insulin response curve was lower by 9.6%, although not significantly, and the post-glucose meal HDL-cholesterol concentration was significantly (12%) higher. Additionally, the 3-week treatment with sugar-beet fibre significantly lowered the fasting total- and LDL-cholesterol concentrations, by 8.5% and 9.6% respectively. We conclude that low doses of psyllium and citrus pectin at breakfast and lunch have no effects on the postprandial plasma measurements, but that sugar-beet fibre taken daily for 3 weeks affects both fasting and postprandial plasma metabolites favourably in these individuals with mildly increased risk of ischaemic heart disease. Further, we observed that small changes in haemodilution occur after meals, as indicated by plasma albumin concentration and packed cell volume. Underemphasis of the dietary fibre effects may occur when postprandial haemodilution is not taken into account.

Adult↗

Contribution of acetate to butyrate formation by human faecal bacteria.

Acetate is normally regarded as an endproduct of anaerobic fermentation, but butyrate-producing bacteria found in the human colon can be net utilisers of acetate. The butyrate formed provides a fuel for epithelial cells of the large intestine and influences colonic health. [1-(13)C]Acetate was used to investigate the contribution of exogenous acetate to butyrate formation. Faecalibacterium prausnitzii and Roseburia spp. grown in the presence of 60 mm-acetate and 10 mm-glucose derived 85-90 % butyrate-C from external acetate. This was due to rapid interchange between extracellular acetate and intracellular acetyl-CoA, plus net acetate uptake. In contrast, a Coprococcus-related strain that is a net acetate producer derived only 28 % butyrate-C from external acetate. Different carbohydrate-derived energy sources affected butyrate formation by mixed human faecal bacteria growing in continuous or batch cultures. The ranking order of butyrate production rates was amylopectin > oat xylan > shredded wheat > inulin > pectin (continuous cultures), and inulin > amylopectin > oat xylan > shredded wheat > pectin (batch cultures). The contribution of external acetate to butyrate formation in these experiments ranged from 56 (pectin) to 90 % (xylan) in continuous cultures, and from 72 to 91 % in the batch cultures. This is consistent with a major role for bacteria related to F. prausnitzii and Roseburia spp. in butyrate formation from a range of substrates that are fermented in the large intestine. Variations in the dominant metabolic type of butyrate producer between individuals or with variations in diet are not ruled out, however, and could influence butyrate supply in the large intestine.

Acetates↗

Dietary fat and fiber modulate colonic cell proliferation in an interactive site-specific manner.

A 3 x 3 factorial experiment was conducted to examine how dietary fiber and fat interactively affect cell proliferation in the colon in rats. Groups of 10 male Sprague-Dawley rats were fed one of nine experimental diets for three weeks: three types of fat at 15% by weight (beef tallow, corn oil, and fish oil) x 2 types of fiber (pectin and cellulose) plus a fiber-free diet as a control. Cell proliferation was measured by in vivo incorporation of bromodeoxyuridine into DNA. The main effect of dietary fiber on cell proliferation was found in the proximal colon, where the pectin diet stimulated cell proliferation compared with the cellulose and fiber-free diets (p < 0.05). In comparison, the main effect of dietary fat was in the distal colon, where the beef tallow diet was more promotive with respect to cell proliferation than the fish oil diet and the corn oil diet had an intermediate effect (p < 0.05). In the proximal colon, however, the effect of fiber on cell proliferation was highly dependent on the source of fat in the diet. Pectin exerted a hyperproliferative effect when the source of fat in the diet was corn oil, but not when beef tallow or fish oil was the fat source. These data indicate that dietary fiber and fat modulate cell proliferation of the colon in an interactive site-specific manner.

Analysis of Variance↗

Relationship among colonocyte proliferation, differentiation, and apoptosis as a function of diet and carcinogen.

To determine the relationship among colonocyte proliferation, differentiation, and apoptosis as a function of fiber and carcinogen, we conducted a 2 x 2 factorial design study with two fibers (pectin or cellulose) and two injection protocols (azoxymethane or saline) in male Sprague-Dawley rats. Rats were killed six weeks after the injections, and in vivo cell proliferation was measured by incorporation of bromodeoxyuridine into DNA, differentiation by binding of the lectin Dolichos biflorus agglutinin, and apoptosis by immunoperoxidase detection of digoxigenin-labeled genomic DNA. In the proximal colon, pectin decreased differentiation and apoptosis, resulting in a greater number of cells per crypt column. In the distal colon, pectin increased cell proliferation, resulting in more crypts per millimeter of colon and a greater number of surface cells. Azoxymethane increased cell proliferation and decreased differentiation and apoptosis in the proximal and the distal colon. This resulted in a greater number of surface cells proximally and more crypts per millimeter of colon distally. These results illustrate the importance of considering all three parameters (proliferation, differentiation, and apoptosis) when evaluating neoplastic growth.

Animals↗

Effect of simulated gastrointestinal conditions on drug release from pectin/ethylcellulose as film coating for drug delivery to the colon.

The aim of this work was to investigate the effect of acidic pH representative of gastric fluid on the release of 5-aminosalicylic acid from beads coated with pectin/ethylcellulose as film coating intended for drug delivery to the colon, in media mimicking the lower gastrointestinal (GI) tract and representative of colonic conditions. In this work, the in vitro incubation of the beads in acid medium was found to influence the hydration and the swelling characteristics of pectin after transfer into simulated intestinal fluid and simulated cecal fluid containing pectinolytic enzymes. Moreover, the drug release profiles from the beads in simulated intestinal fluid after incubation for 2 h or 30 min in simulated gastric fluid vs. no acid incubation were found to be very different. The in vitro degradation of pectin in the coat by pectinolytic enzymes in simulated cecal fluid depended on whether the beads were placed in simulated gastric fluid prior to testing in simulated intestinal fluid. The percentage drug release also depended on the ratio of pectin to ethylcellulose in the coat.

Animals↗

Pectin/zein beads for potential colon-specific drug delivery: synthesis and in vitro evaluation.

Novel complex hydrogel beads were prepared from two edible polymers: pectin, a carbohydrate from citrus fruits, and zein, a protein from corn. The pectin/zein complex hydrogels did not swell in physiological environments, but hydrolyzed in the presence of pectinases. An in vitro study showed the capacity of the hydrogels to endure protease attack and residence time variation. The physical and biological properties of the new hydrogels were attributed to molecular entanglement of the two polymers. The pectin networks were stabilized by the bound zein molecules. In turn, the pectin networks shielded the bound zein from protease digestion.

Anti-Inflammatory Agents, Non-Steroidal↗

Isolation and spectroscopic study of pectic substances from kenaf (Hibiscus cannabinus L.).

Pectins were isolated from bark, wood and pith of kenaf (Hibiscus cannabinus L.) variety Cuba108. Imidazole was used as extractant. The isolated pectins were studied by 13C nuclear magnetic resonance (13C-NMR) spectroscopy and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). From the spectroscopic investigation, the isolated pectic substances were found similar to commercial pectins.

Hibiscus↗

Spectroscopic determination of the degree of esterification of pectic substances from kenaf.

Pectins were isolated from bark, wood and pith of four kenaf (Hibiscus cannabinus L.) varieties. The degree of esterification of isolated pectins was studied using Fourier transform infrared spectroscopy. It was determined as being between 57 and 90% and there were no notable differences between kenaf varieties. The higher value of the degree of esterification was presented in wood pectins (86-90%), the interim in pith (75-83%) and the lower in bark (57-64%).

Hibiscus↗

Gastroretentive delivery systems: hollow beads.

The objective of this study was to develop a floatable multiparticulate system with potential for intragastric sustained drug delivery. Cross-linked beads were made by using calcium and low methoxylated pectin (LMP), which is an anionic polysaccharide, and calcium, LMP, and sodium alginate. Beads were dried separately in an air convection type oven at 40 degrees C for 6 hours and in a freeze dryer to evaluate the changes in bead characteristics due to process variability. Riboflavin (B-2), tetracycline (TCN), and Methotrexate (MTX) were used as model drugs for encapsulation. Ionic and nonionic excipients were added to study their effects on the release profiles of the beads. The presence of noncross linking agents in low amounts (less than 2%) did not significantly interfere with release kinetics. For an amphoteric drug like TCN, which has pH dependent solubility, three different pHs (1.5, 5.0, and 8.0) of cross-linking media were used to evaluate the effects of pH on the drug entrapment capacity of the beads. As anticipated, highest entrapment was possible when cross-linking media pH coincided with least drug solubility. Evaluation of the drying process demonstrated that the freeze-dried beads remained buoyant over 12 hours in United States Pharmacopeia (USP) hydrochloride buffer at pH 1.5, whereas the air-dried beads remained submerged throughout the release study. Confocal laser microscopy revealed the presence of air-filled hollow spaces inside the freeze dried beads, which was responsible for the flotation property of the beads. However, the release kinetics from freeze dried beads was independent of hydrodynamic conditions. Calcium-pectinate-alginate beads released their contents at much faster rates than did calcium-pectinate beads (100% in 10 hours vs. 50% in 10 hours). It appears that the nature of cross-linking, drying method, drug solubility, and production approach are all important and provide the opportunity and potential for development of a gastroretentive drug delivery system.

Alginates↗

Effects of manufactured soluble dietary fiber from Quercus mongolica on hepatic HMG-CoA reductase and lipoprotein lipase activities in epididymal adipose tissue of rats fed high cholesterol diets.

This study investigated the effect of a manufactured soluble dietary fiber on lipid metabolism in rats fed high cholesterol diets. Soluble dietary fiber was prepared from wood chips of oak (Quercus mongolica). Male Sprague-Dawley rats weighing 100 +/- 10 g were randomly assigned to either a normal diet or five high cholesterol diets containing 1% cholesterol and different fiber supplements. The high cholesterol groups were subdivided into fiber-free diet (FF), 5% pectin (5P), 10% pectin (10P), 5% manufactured soluble dietary fiber (5QM), and 10% manufactured soluble dietary fiber (10QM) groups. Total serum cholesterol concentrations in all soluble dietary fiber-supplemented groups were lower than in the FF group. The high-density lipoprotein-cholesterol concentration in the FF group was significantly lower, compared with the normal group, but was increased in groups supplemented with soluble dietary fiber. Low-density lipoprotein-cholesterol levels and the atherogenic index had the same tendency as total cholesterol concentration. Compared with the FF group, in the 5P, 5QM, 10P, and 10QM groups hepatic triglyceride concentrations were 12%, 16%, 20%, and 24% lower, respectively, and hepatic cholesterol concentrations were 48%, 52%, 52%, and 58% lower, respectively. Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity in the soluble fiber groups was significantly higher than in the FF groups, but lower than the normal group. When hepatic tissue was observed under a light microscope, the FF group had completely formed lipomas in the hepatic tissue, which led to fat deposits and then a fatty liver. The size and number of lipomas were lower in the soluble dietary fiber-fed groups, as compared with the group not fed dietary fiber. In conclusion, improvements in lipid metabolism were observed as a result of the manufactured soluble dietary fiber from the oak chips, and were similar to that seen for pectin. The preparation method for the soluble dietary fiber from oak chips successfully produced a functional soluble fiber.

Adipose Tissue↗

Plant cell wall fractionation and structural analysis.

The structure of the major plant polysaccharides contributing to dietary fiber, their organization within the plant cell wall, and the problems associated with their analysis are briefly reviewed. Alkaline, acidic, and detergent fractionation methods are described and considered in relation to their specificity. The analysis of pectin, often ignored as a component of dietary fiber, is examined in detail. The solubility of pectins from a number of common plant foods was determined, indicating that a considerable portion of pectin polyuronide is lost to many of the commonly used analytical procedures.

Cell Wall↗

Effect of fiber on breath hydrogen response and symptoms after oral lactose in lactose malabsorbers.

The effect of ingesting cellulose, pectin, and psyllium with orally administered lactose in water or milk was tested in six lactose malabsorbers. Breath hydrogen tests were used to evaluate lactose malabsorption and mouth-to-cecum transit times. Addition of psyllium significantly reduced the breath hydrogen response, and symptoms in each subject; whereas, less diminution of expired hydrogen was seen after cellulose or pectin was added. The effect of each fiber on gastric emptying rates of an equal volume liquid meal was evaluated in three volunteers. Pectin had no effect, while the cellulose and psyllium modestly delayed emptying at approximately 30 min.

Adolescent↗

Fermentability of various fiber sources by human fecal bacteria in vitro.

Certain beneficial effects of fiber in the human diet may be mediated by short-chain fatty acids (SCFAs) produced during anaerobic fermentation in the colon. Two studies, both involving in vitro incubations with human fecal bacteria as inoculum, were conducted to assess fermentation of various fiber sources and to quantitate the SCFAs produced. In experiment 1, substrate fermentability based on total SCFA production ranked as follows: citrus pectin greater than soy fiber greater than sugarbeet fiber greater than pea fiber greater than oat fiber. Fermentation of soy fiber led to higher proportions of propionate and butyrate than did fermentation of other substrates. In experiment 2, fermentation of gum arabic, a mixture of arabic and guar, and apple pectin resulted in greater SCFA production than did fermentation of either oat fiber or corn bran. Fermentation of gums led to more propionate and butyrate production than did that of apple pectin. It may be possible to select fiber sources capable of supporting stipulated amounts of both total and individual SCFA production in the human colon.

Bacteria↗