Cell Wall Composition and Ultrastructural Immunolocalization of Pectin and Arabinogalactan Protein during Olea europaea L. Fruit Abscission.

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as 'Picual' undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in 'Picual' olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

Structures formed by a cell membrane-associated arabinogalactan-protein on graphite or mica alone and with Yariv phenylglycosides.

BACKGROUND: Certain membrane-associated arabinogalactan-proteins (AGPs) with lysine-rich sub-domains participate in plant growth, development and resistance to stress. To complement fluorescence imaging of such molecules when tagged and introduced transgenically to the cell periphery and to extend the groundwork for assessing molecular structure, some behaviours of surface-spread AGPs were visualized at the nanometre scale in a simplified electrostatic environment. METHODS: Enhanced green fluorescent protein (EGFP)-labelled LeAGP1 was isolated from Arabidopsis thaliana leaves using antibody-coated magnetic beads, deposited on graphite or mica, and examined with atomic force microscopy (AFM). KEY RESULTS: When deposited at low concentration on graphite, LeAGP can form independent clusters and rings a few nanometres in diameter, often defining deep pits; the aperture of the rings depends on plating parameters. On mica, intermediate and high concentrations, respectively, yielded lacy meshes and solid sheets that could dynamically evolve arcs, rings, 'pores' and 'co-pores', and pits. Glucosyl Yariv reagent combined with the AGP to make very large and distinctive rings. CONCLUSIONS: Diverse cell-specific nano-patterns of native lysine-rich AGPs are expected at the wall-membrane interface and, while there will not be an identical patterning in different environmental settings, AFM imaging suggests protein tendencies for surficial organization and thus opens new avenues for experimentation. Nanopore formation with Yariv reagents suggests how the reagent might bind with AGP to admit Ca(2+) to cells and hints at ways in which AGP might be structured at some cell surfaces.

Recognition of galactan components of pectin by galectin-3.

It has been reported that modified forms of pectin possess anticancer activity. To account for this bioactivity, it has been proposed that fragments of pectin molecules can act by binding to and inhibiting the various roles of the mammalian protein galectin 3 (Gal3) in cancer progression and metastasis. Despite this clear molecular hypothesis and evidence for the bioactivity of modified pectin, the structural origins of the "bioactive fragments" of pectin molecules are currently ill defined. By using a combination of fluorescence microscopy, flow cytometry, and force spectroscopy, it has been possible to demonstrate, for the first time, specific binding of a pectin galactan to the recombinant form of human Gal3. Present studies suggest that bioactivity resides in the neutral sugar side chains of pectin polysaccharides and that these components could be isolated and modified to optimize bioactivity.

The acacia gum arabinogalactan fraction is a thin oblate ellipsoid: a new model based on small-angle neutron scattering and ab initio calculation.

Acacia gum is a branched complex polysaccharide whose main chain consists of 1,3-linked beta-D-galactopyranosyl units. Acacia gum is defined as a heteropolysaccharide since it contains approximately 2% of a polypeptide. The major molecular fraction (F1) accounting for approximately 88% of the total acacia gum mass is an arabinogalactan peptide with a weight-average molecular weight of 2.86 x 10(5) g/mol. The molecular structure of F1 is actually unknown. From small angle neutron scattering experiments in charge screening conditions, F1 appeared to be a dispersion of two-dimensional structures with a radius of gyration of approximately 6.5 nm and an inner dense branched structure. Inverse Fourier transform of F1 scattering form factor revealed a disk-like morphology with a diameter of approximately 20 nm and a thickness below 2 nm. Ab initio calculations on the pair distance distribution function produced a porous oblate ellipsoid particle with a central intricated "network". Both transmission electron microscopy and atomic force microscopy confirm the thin disk model and structural dimensions. The model proposed is a breakthrough in the field of arabinogalactan-protein-type macromolecules. In particular, concerning the site of biosynthesis of these macromolecules, the structural dimensions found in this study would be in agreement with a phloem-mediated long-distance transport. In addition, the structure of F1 could also explain the low viscosity of acacia gum solutions, and its ability to self-assemble and to interact with proteins.

Mango kernel starch-gum composite films: Physical, mechanical and barrier properties.

Composite films were developed by the casting method using mango kernel starch (MKS) and guar and xanthan gums. The concentration of both gums ranged from 0% to 30% (w/w of starch; db). Mechanical properties, oxygen permeability (OP), water vapor permeability (WVP), solubility in water and color parameters of composite films were evaluated. The crystallinity and homogeneity between the starch and gums were also evaluated by X-ray diffraction (XRD) and scanning electron microscopy (SEM). The scanning electron micrographs showed homogeneous matrix, with no signs of phase separation between the components. XRD analysis demonstrated diminished crystalline peak. Regardless of gum type the tensile strength (TS) of composite films increased with increasing gum concentration while reverse trend was noted for elongation at break (EAB) which found to be decreased with increasing gum concentration. The addition of both guar and xanthan gums increased solubility and WVP of the composite films. However, the OP was found to be lower than that of the control with both gums. Furthermore, addition of both gums led to changes in transparency and opacity of MKS films. Films containing 10% (w/w) xanthan gum showed lower values for solubility, WVP and OP, while film containing 20% guar gum showed good mechanical properties.

Grafting of vinyl acetate-ethylacrylate binary monomer mixture onto guar gum.

Present article reports on guar gum (GG) functionalization through graftcopolymerization of vinylacetate (VAC) and ethylacrylate (EA) from their binary mixtures. The potassium persulfate/ascorbic acid (KPS/AA) redox initiator system has been used for the binary grafting under the previously optimized conditions for VAC grafting at guar gum. The concentration of ascorbic acid (AA), persulfate (KPS), and grafting temperature were varied to optimize the binary grafting. A preliminary investigation revealed that the copolymer has excellent ability to capture Hg(II) from aqueous solution. It was observed that the optimum % grafting sample (CP3) was best at Hg(II) adsorption. CP3 and mercury loaded CP3 (CP3-Hg) have been extensively characterized using Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), and Thermo gravimetric analysis (TGA) and a plausible mechanism for the grafting has been proposed.

Development of microbial trigger based oral formulation of Tinidazole and its Gamma Scintigraphy Evaluation: A promising tool against anaerobic microbes associated GI problems.

Tinidazole is a versatile anti-amoebic and anti-anaerobic drug used in treatment of intestinal infection. The aim of present study was to develop and evaluate a guar gum based novel target release Tinidazole matrix tablet in animal models and healthy human volunteer using Gamma Scintigraphy technique. Anti-anaerobic and anti-protozoal activity of the developed formulation was studied in vitro against Bacteroides fragilis and Dentamoeba fragilis. Tinidazole was successful radiolabelled with (99m)Tc-pertechnetate using stannous chloride as a reducing agent and stable up to 24h in normal saline and serum. Radiolabeled formulation was evaluated in 6 Newzealand white rabbits by gamma Scintigraphy in static manner up to 24h for its retention in gastrointestinal tract (GIT). Similar set of study was conducted in 12 healthy human volunteers for similar objective Scintigraphy images of healthy human volunteer showed retention of optimized formulations in stomach up to 60min, from where it moved to duodenum further and reached ileum in around 5h. However, initiation of drug release was observed from intestine at 7h. Complete dissociation and release of drug was observed at 24h in colon due to anaerobic microbial rich environment. Results drawn from Scintigraphy images indicate that radiolabeled (99m)Tc-Tinidazole tablet transit through upper part of GI without disintegration. Hence the developed matrix tablet may have a role in treatment of intestinal infection caused by anaerobic bacteria.

Mycomembrane and S-layer: two important structures of Corynebacterium glutamicum cell envelope with promising biotechnology applications.

Corynebacteria belong to a distinct Gram-positive group of bacteria including mycobacteria and nocardia, which are characterized by the presence of mycolic acids in their cell wall. These bacteria share the property of having an unusual cell envelope structural organization close to Gram-negative bacteria. In addition to the inner membrane, the cell envelope is constituted of a thick arabinogalactan-peptidoglycan polymer covalently linked to an outer lipid layer, which is mainly composed of mycolic acids and probably organized in an outer membrane like structure. In some species, the cell is covered by a crystalline surface layer composed of a single protein species, which is anchored in the outer membrane like barrier. An increasing number of reports have led to a better understanding of the structure of the cell wall of Corynebacterium glutamicum. These works included the characterization of several cell wall proteins like S-layer protein and porins, genetic and biochemical characterization of mycolic acids biosynthesis, ultrastructural description of the cell envelope, and chemical analysis of its constituents. All these data address new aspects regarding cell wall permeability towards macromolecules and amino acids but also open new opportunities for biotechnology applications.

Synthesis, characterization, and in vitro evaluation of palmitoylated arabinogalactan with potential for liver targeting.

Arabinogalactan (AG), a water soluble polysaccharide with more than 80 mol% galactose units, was hydrophobized by covalent attachment of palmitoyl chains using a base-catalyzed esterification reaction with the objective of effective amalgamation of arabinogalactan in liposomes for targeting asialoglycoprotein receptors (ASGPR) on liver parenchymal cells. Palmitoylated AG (PAG) was characterized by physico-chemical parameters, IR, (1)H NMR, and (13)C NMR and molecular weight determination by gel permeation chromatography. PAG was incorporated in liposomes and the liposomes were characterized by dynamic light scattering, optical microscopy, zeta potential, and transmission electron microscopic (TEM) techniques. The liposomal system was evaluated for acute toxicity in swiss albino mice and was found to be safe. Targeting ability of PAG was confirmed by in vitro binding affinity to Ricinus communis agglutinin (RCA(120)), a lectin specific for galactose. The liposomal system with PAG was evaluated for cytotoxicity on HepG2, MCF7, and A549 cancer cell lines. Cytotoxicity study revealed enhanced activity on ASGPR-expressive HepG2 cells as compared to MCF7.

Preparation and characterization of pH-responsive guar gum microspheres.

Guar gum, being the natural polymer is renewable, nontoxic, biocompatible and biodegradable. Therefore, it is the perfect material to formulate particulates or microspheres for potential applications in pharmaceutical. The formulation of material in nano/microsphere scale offers new rich in application potential. In view of that, novel biodegradable and pH-sensitive hydrogels composed of pH-sensitive methacrylic acid (MAc) and a biodegradable guar gum were synthesized by grafting reactions. Water-in-oil (w/o) emulsion method was used to direct the pH-sensitive material in microspheres shape using bi-functional glutaraldehyde (GA) as crosslinker. The synthesized microspheres were characterized by FTIR and SEM (different magnification). The swelling ratios of hydrogels in buffer solutions showed a pH-dependent profile at physiological pH. In vitro release data was analyzed using Fick's law, which indicated swelling controlled super case II transport of BSA through the synthesized microspheres. Therefore, in conclusion, as ascertained from the results the introduction of -COOH moieties along the guar gum chain drastically increases the end-use performance due to pH-sensitivity.

Modification of hydroxypropyl guar gum with ethanolamine.

A new guar gum derivative containing amino group was synthesized through nucleophilic substitution of p-toluenesulfonate activated hydroxypropyl guar gum with ethanolamine. For the preparation of p-toluenesulfonate esters hydroxypropyl guar gum, the results showed that the reaction rate was optimal at 25 °C and the reaction could reach equilibrium state when it was carried out for 10h at 25 °C. For the nucleophilic substitution of tosyl group with ethanolamine, the reaction was completed after 10h reaction at 50 °C. The structures of products were characterized by NMR and FT-IR spectroscopy. The results showed that the p-toluenesulfonate esters can be effectively substituted by ethanolamine to form the hydroxyethyl amino hydroxypropyl guar gum (EAHPG). The content of nitrogen of EAHPG was determined by acid-base titration and element analysis.

Exploring the ultrastructural localization and biosynthesis of beta(1,4)-galactan in Pinus radiata compression wood.

Softwood species such as pines react to gravitropic stimuli by producing compression wood, which unlike normal wood contains significant amounts of beta(1,4)-galactan. Currently, little is known regarding the biosynthesis or physiological function of this polymer or the regulation of its deposition. The subcellular location of beta(1,4)-galactan in developing tracheids was investigated in Pinus radiata D. Don using anti-beta(1,4)-galactan antibodies to gain insight into its possible physiological role in compression wood. beta(1,4)-Galactan was prominent and evenly distributed throughout the S2 layer of developing tracheid cell walls in P. radiata compression wood. In contrast, beta(1,4)-galactan was not detected in normal wood. Greatly reduced antibody labeling was observed in fully lignified compression wood tracheids, implying that lignification results in masking of the epitope. To begin to understand the biosynthesis of galactan and its regulation, an assay was developed to monitor the enzyme that elongates the beta(1,4)-galactan backbone in pine. A beta(1,4)-galactosyltransferase (GalT) activity capable of extending 2-aminopyridine-labeled galacto-oligosaccharides was found to be associated with microsomes. Digestion of the enzymatic products using a beta(1,4)-specific endogalactanase confirmed the production of beta(1,4)-galactan by this enzyme. This GalT activity was substantially higher in compression wood relative to normal wood. Characterization of the identified pine GalT enzyme activity revealed pH and temperature optima of 7.0 and 20 degrees C, respectively. The beta(1,4)-galactan produced by the pine GalT had a higher degree of polymerization than most pectic galactans found in angiosperms. This observation is consistent with the high degree of polymerization of the naturally occurring beta(1,4)-galactan in pine.