Biomedical subjects

Jean-François Thibault

Publications and source records attributed to Jean-François Thibault.

14 recordsLinked to original sources

PubMed2007

Assessment of in vitro binding of isolated pectic domains to cellulose by adsorption isotherms, electron microscopy, and X-ray diffraction methods.

Isolated pectic domains representative of the pectic backbone and the neutral sugar side chains were tested for their ability to interact with cellulose in comparison to the well-known binding of xyloglucan. Pectic side chains displayed a significant in vitro binding capacity to cellulose, whereas pectic backbone domains exhibited only slight adsorption to cellulose microfibrils. To support the binding results, electron microscopy and X-ray diffraction were applied. Celluloses from bacteria and sugar beet cell walls were used as substrates for the precipitation of isolated pectic domains or xyloglucan by acetone vapor diffusion. Pectic side chains grew attached to the cellulose surfaces, whereas pectic backbone domains were observed separately from cellulose microfibrils. Xyloglucan seeded with cellulose provoked a decrease of microfibrils entanglement, but no clear cross-links between neighboring microfibrils were observed. These results led to the elucidation of the pectic domains responsible for binding with cellulose microfibrils.

Adsorption↗

PubMed2006

Cellulase and protease preparations can extract pectins from various plant byproducts.

The use of protease and cellulase preparations to extract pectins from plant byproducts (chicory, cauliflower) was investigated. Different enzymatic preparations were characterized by their activities toward proteins, cellulose, and pectins. These preparations were then tested regarding pectin extraction, and extraction conditions (nature and concentration of enzyme, incubation time) were optimized. Enzymatic and acidic extractions were compared and also combined in sequential extractions. This study shows that it is possible to extract pectins by using cellulases and proteases. Enzymes can extract pectins with a higher yield ( approximately 35%) than acid (approximately 28%) but enzyme-extracted pectins have a smaller molar mass (300,000 g/mol) than acid-extracted pectins (500,000 g/ mol). Different hypotheses are tested and discussed to explain this mass difference.

Brassica↗

PubMed2005

Sugar beet (Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall.

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after (18)O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (beta-1-->4-linked-dimer), Ara (alpha-1-->5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.

Beta vulgaris↗

PubMed2005

Distribution of pectic epitopes in cell walls of the sugar beet root.

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.

Antibodies, Monoclonal↗

PubMed2005

Mapping sugar beet pectin acetylation pattern.

Homogalacturonan-derived partly methylated and/or acetylated oligogalacturonates were recovered after enzymatic hydrolysis (endo-polygalacturonase+pectin methyl esterase+side-chain degrading enzymes) of sugar beet pectin followed by anion-exchange and size exclusion chromatography. Around 90% of the GalA and 75% of the acetyl groups present in the initial sugar beet pectin were recovered as homogalacturonan-derived oligogalacturonates, the remaining GalA and acetyl belonging to rhamnogalacturonic regions. Around 50% of the acetyl groups present in sugar beet homogalacturonans were recovered as partly methylated and/or acetylated oligogalacturonates of degree of polymerisation 5 whose structures were determined by electrospray ionization ion trap mass spectrometry (ESI-IT-MSn). 2-O-acetyl- and 3-O-acetyl-GalA were detected in roughly similar amounts but 2,3-di-O-acetylation was absent. Methyl-esterified GalA residues occurred mainly upstream 2-O-acetyl GalA. Oligogalacturonates containing GalA residues that are at once methyl- and acetyl-esterified were recovered in very limited amounts. A tentative mapping of the distribution of acetyl and methyl esters within sugar beet homogalacturonans is proposed. Unsubstituted GalA residues are likely to be present in limited amounts (approximately 10% of total GalA residues), due to the fact that methyl and acetyl groups are assumed to be most often not carried by the same residues.

Acetylation↗

PubMed2004

Isolation of diferulic bridges ester-linked to arabinan in sugar beet cell walls.

After degradation of sugar beet cell walls with Driselase and fractionation of the solubilised products by hydrophobic interaction chromatography, a dehydrodiferuloylated oligoarabinan was isolated. Its structure was assigned to two dimers of (1-->5)-linked arabinose units esterified by a central 8-O-4' ferulic dimer. These results provide the first direct evidence that pectic arabinans in sugar beet cell walls may be covalently cross-linked through dehydrodiferulates.

Beta vulgaris↗

PubMed2004

A monoclonal antibody to feruloylated-(1-->4)-beta-D-galactan.

We report the isolation and characterization of a monoclonal antibody, designated LM9, against feruloylated-(1-->4)-beta-D-galactan. This epitope is a structural feature of cell wall pectic polysaccharides of plants belonging to the family Amaranthaceae (including the Chenopodiaceae). Immuno-assays and immunofluorescence microscopy indicated that LM9 binding is specific to samples and cell walls obtained from species belonging to this family. In a series of competitive-inhibition enzyme-linked immunosorbent assays with potential oligosaccharide haptens, the most effective inhibitor was O-[6-O-(trans-feruloyl)-beta-D-galactopyranosyl]-(1-->4)-D-galactopyranose (Gal2F). LM9 is therefore a useful antibody probe for the analysis of phenolic substitution of cell wall pectic polymers and of cell wall structure in the Amaranthaceae including sugar beet (Beta vulgaris L.) and spinach (Spinacia oleracea L.).

Amaranthaceae↗

PubMed2003

A xylogalacturonan epitope is specifically associated with plant cell detachment.

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.

Antibodies, Monoclonal↗

PubMed2003

Preparation of sulfoacetate derivatives of cellulose by direct esterification.

A new and efficient method for the preparation in one step of water-soluble cellulose acetate sulfate derivatives (CAS) is reported. Acetylation and sulfation were carried out simultaneously, using a mixture of acetic anhydride and sulfuric acid in glacial acetic acid. The reaction time and the amount of acetic anhydride were optimized and the method provided water-soluble esters, with a degree of acetylation in the range 1.6 and 2.4 and a degree of sulfation of 0.3. This method has been successfully applied to pure cellulose and to cellulose-enriched materials obtained from agricultural by-products. The product exhibited a high viscosity in aqueous solution suggesting interesting rheological properties.

Acetates↗

PubMed2003

Rheological properties of sulfoacetate derivatives of cellulose.

Water-soluble cellulose acetate sulfate derivatives (CAS) have been prepared through chemical reaction involving sulfuric acid as a catalyst. These CAS have been obtained from cellulosic materials of different origins (pure cellulose, wheat bran, maize bran) and their rheological behavior in salt-free aqueous solution has been estimated in dilute and semi-dilute regime using dynamic viscoelastic and viscosity measurements. Influence of concentration, temperature of solubilization and temperature of measurement has been investigated. Weak gel-like properties were exhibited at elevated concentration (typically above 7-8 g/L). These systems also exhibited thixotropic properties: the structure was partly broken down upon shearing and recovered at rest. They also displayed thermoreversibility with large hysteresis, the melting temperature being approximately 15 degrees C higher than the temperature at which gelation took place. These overall observations clearly indicate that these distinctive properties arise from intermolecular association of the macromolecular chains of the cellulose derivative.

Acetates↗

PubMed2003

Assignment of acetyl groups to O-2 and/or O-3 of pectic oligogalacturonides using negative electrospray ionization ion trap mass spectrometry.

Partially acetylated and methylated oligogalacturonides produced by enzymatic hydrolysis of sugar beet pectin were analysed by negative electrospray ionization ion trap mass spectrometry (ESI-ITMS). The (18)O labelling of the oligomer reducing end allowed the precise assignment of the fragments resulting from glycosidic bond and cross-ring cleavages. The collisional-induced dissociation of the C(i) and Z(j) fragment ions through sequential MS(n) experiments always displayed (0, 2)A-type cross-ring cleavage ions which were related to C(2)H(4)O(2) losses. These (0, 2)A ions appeared to be highly diagnostic ions allowing the precise location of the acetyl groups to the O-2 and/or O-3 of the acetylated galacturonic acid residues.

Acetylation↗

PubMed2003

Cadmium and lead adsorption by a natural polysaccharide in MF membrane reactor: experimental analysis and modelling.

In the present work, Pb(2+) and Cd(2+) adsorption onto a natural polysaccharide has been studied in membrane reactors. The process involves a stirred semi-batch reactor for the adsorption step and a microfiltration (MF) process in order to confine the particles. Due to their lower affinity for the biosorbent, Cd(2+) ions were found to breakthrough the process faster than Pb(2+) cations. The experimental results showed the technical feasibility of the pilot. A mass balance model based on the Langmuir equilibrium isotherm was used to describe the adsorption process. This relation is able to predict experimental data under different operating conditions: the adsorbent and metal concentrations, and the permeate flow rate. Based on these results, it is demonstrated that the biosorbent studied represents an interesting low-cost solution for the treatment of metal ions polluted waters.

Bioreactors↗

PubMed2002

Characterisation of pectin subunits released by an optimised combination of enzymes.

Pectins from sugar beet, lime and apple were degraded by a rhamnogalacturonan hydrolase associated or not with pectin methylesterases and side chain degrading enzymes (galactanase and arabinanase). The composition of the enzymatic mixture was optimised by following the reaction by viscosimetric means. The reaction products were fractionated by ion exchange chromatography. Treatment with all the enzymes released four fractions: (1). 227-247 mg/g of initial pectins and corresponded to neutral sugars from the side chains; (2,3). represented together 184-220 mg/g of pectins and corresponded to rhamnogalacturonan; (4). 533-588 mg/g of pectins and corresponded to homogalacturonan. Lime pectins have the shortest rhamnogalacturonan regions. The molar masses of homogalacturonans were in the range of 16000-43400 g/mol according to the origin of pectins, corresponding to degrees of polymerisation of 85-250. The mode of action of the enzymes used is also discussed.

Beta vulgaris↗

PubMedDate not supplied

Interchain heterogeneity of enzymatically deesterified lime pectins.

Two series of pectins with different levels and patterns of methyl esterification were produced by treatment of a very highly methylated lime pectin with a fungus- or plant-pectin methylesterase. The interchain distribution of free carboxyl groups was investigated by size exclusion and ion exchange chromatography. "Homogeneous" populations with respect to molar mass or charge density were thereby obtained, and their composition, molar mass, and calcium binding properties were investigated. The composition varies from one size exclusion chromatography fraction to another, the highest molar mass fraction being richer in rhamnogalacturonic sequences and exhibiting a slightly higher degree of methylation (DM). Separation of pectins by ion exchange chromatography revealed a narrow charge density distribution for pectins deesterified by fungus-pectin methylesterase, in agreement with a multichain mechanism. Conversely, pectins deesterified by plant-pectin methylesterase exhibited a very large charge density distribution suggesting a processive mechanism. The interchain polydispersity with regard to DM was however shown to have no impact on calcium binding properties of the different fractions. The progressive dimerization through calcium ions with decreasing DM of pectins deesterified by plant-pectin methylesterase seems to be the result of a peculiar intrachain pattern of methyl esterification that can be attributed to a multiple attack mechanism.

Carbohydrates↗