Efficient purification of small unsaturated oligoglucuronides by reversed-phase chromatography.

Ion-exchange chromatography has been applied to purification of unsaturated oligoglucuronans. After an isocratic elution on a strong anion-exchange column, the collected fractions were desalted by low pressure size exclusion chromatography. However, this efficient separation was limited by the time required to desalt. So, we developed a reversed-phase chromatography method using back ionization of oligomers. Two C18 columns were tested with trifluoroacetic acid (TFA 0.7%) as eluent. Different selectivities and column stabilities were observed in this acidic condition. The scale up for semi-preparative applications enabled us to recover pure unsaturated oligoglucuronans without desalting step.

Production of oligoglucuronans by enzymatic depolymerization of nascent glucuronan.

An original bioreactor process for production of oligoglucuronans was developed using the Sinorhizobium meliloti M5N1CS strain that produces glucuronan. This anionic homopolysaccharide was composed of beta-D-(1,4)-glucopyranosyluronic residues variably O-acetylated at C-3 and/or C-2 positions according to culture conditions. It was depolymerized during its biosynthesis by addition of a fungal glucuronan lyase activity in broths. After purification by tangential ultrafiltration and low-pressure liquid chromatography, (1)H NMR and ESI-Q/TOF-MS characterized the poly- and oligoglucuronic acid fractions. This enzymatic bioreactor strategy authorized the production in gram quantity of an unsaturated and no acetylated oligoglucuronan with a degree of polymerization of 3.

Rapid quantification of O-acetyl and O-methyl residues in pectin extracts.

A rapid method for the determination of the degrees of methylation (DM) and acetylation (DA) of pectins was developed. The polymer substitution degree as determined after saponification at 80 degrees C with NaOD during 1H NMR analysis. Under alkaline conditions, the cleavage of O-acetyl and O-methyl linkages allows the detection and the integration of the H-4 signal from galacturonic acid residues in the newly unesterified pectins. So, after a 10-min NMR recording, sodium acetate and sodium methanolate can be easily quantified relative to the clearly identified H-4 signal in galacturonic acid residues. Protons signals from pectin neutral sugars do not interfere with H-4. During the analysis, a limited (<3%) methanol evaporation leading to a weak reduced signal from the methanolate protons was observed. The proposed method allows in few minutes an accurate simultaneous quantification of DM and DA from few mg of pectin extracts, without the need of external standards.

Improved stability of TMS derivatives for the robust quantification of plant polar metabolites by gas chromatography-mass spectrometry.

Plant metabolite profiling is commonly carried out by GC-MS of methoximated trimethylsilyl (TMS) derivatives. This technique is robust and enables a library search for spectra produced by electron ionization. However, recent articles have described problems associated with the low stability of some TMS derivatives. This limits the use of GC-MS for metabolomic studies that need large sets of qualitative and quantitative analyses. The aim of this work is to determine the experimental conditions in which the stability of TMS derivatives could be improved. This would facilitate the analysis of the large-scale experimental designs needed in the metabolomics approach. For good repeatability, the sampling conditions and the storage temperature of samples during analysis were investigated. Multiple injections of one sample from one vial led to high variations while injection of one sample from different vials improved the analysis. However, before injection, some amino acid TMS derivatives were degraded during the storage of vials in the autosampler. Only 10% of the initial quantity of glutamine 3 TMS and glutamate 3 TMS and 66% of α-alanine 2 TMS was detected 48 h after derivatization. When stored at 4 °C until injection, all TMS derivatives remained stable for 12 h; at -20 °C, they remained stable for 72 h. From the integration of all these results, a detailed analytical procedure is thus proposed. It enables a robust quantification of polar metabolites, useful for further plant metabolomics studies using GC-MS.

Exopolysaccharide production by nitrogen-fixing bacteria within nodules of Medicago plants exposed to chronic radiation in the Chernobyl exclusion zone.

Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family (Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family (Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 x 10(6) and 3 x 10(6) Da. Five different compositions of EPS were characterized by physico-chemical analyses and (1)H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs.

Production of arabinoxylan-oligosaccharides from flaxseed (Linum usitatissimum).

Flaxseed mucilage from Linum usitatissimum L. species was constituted by arabinoxylan (about 75%) and pectin (about 25%). A new procedure was developed to obtain only arabinoxylans which implicated treatment of the pectin fraction by enzymatic hydrolysis with pectinase. Then three processes of depolymerization were evaluated on arabinoxylans. First, a thermic hydrolysis in mild acid conditions was performed and an ultrafiltration process was used as purification method. Second, the potential of xylanases from different glycoside hydrolase families for arabinoxylan-oligosaccharides (AXOS) production was tested, and finally a radical depolymerization was conducted. Average molecular weights were determined by high pressure size exclusion chromatography coupled with multiple angle laser light scattering (MALLS), and carbohydrate compositions were determined by high pH anion exchange chromatography pulse amperometric detector (HPAEC-PAD). Both chemical and enzymatic treatments were inefficient to convert arabinoxylans from flaxseed mucilage into AXOS. Only radical depolymerization process was allowed to obtain arabinoxylan-oligosaccharides presenting different molecular weights (11.9 x 10(3) to 1.9 x 10(3) g mol(-1)) with satisfactory yields (75% to 35%).

Oligogalacturonic acid inhibit bone resorption and collagen degradation through its interaction with type I collagen.

In this study, we showed that oligogalacturonic acid (OGA) purified from flax pectin inhibit in vitro osteoclastic bone resorption in a dose-dependent manner. The OGA inhibitory effect was neither linked to an effect on osteoclast apoptosis, nor to an inhibition of cathepsin K activity. By means of an in vitro collagen degradation assay we demonstrated that OGA prevented triple-helical type I collagen cleavage by cathepsin K in a dose and chain length dependent manner. This inhibition was not restricted to cathepsin K, since collagenolytic activity of other lysosomal cysteine proteases, such as cathepsin B and cathepsin L, as well as matrixmetalloproteinases such as MMP-9 were also inhibited. Interestingly, using non-collagen substrates we demonstrated that OGA does not inhibit the proteolytic activity of cathepsin B and L, suggesting that OGA inhibits collagen degradation without affecting the lysosomal cysteine enzyme proteolytic activity. Finally, preliminary study using surface plasmon resonance (SPR) showed that OGA binds to type I collagen but not to albumin, consistent with a specific effect on collagen. These results suggest that the observed inhibition of collagen degradation by OGA may be due to its ability to bind to the collagen molecule. By masking the collagen surface, OGA may render the collagen cleavage site less accessible to enzymes and thus prevent its enzymatic degradation.

Electronics manufacturing infrastructures for education and commercialization.

Many medical and biological applications require custom integrated hardware. Infrastructures for the design of CMOS and MEMS hardware allow the education and commercialization at a low cost and in a short time. This paper is reviewing the most recent developments at CMP for the manufacturing of ICs and MEMS. These custom hardware vehicles allow to target many medical and biological applications. Examples are provided through the paper. Such infrastructures may help the Medical and Biological community the same way they helped the Microelectronics community at the time of the VLSI revolution.

Controlled sulfatation of natural anionic bacterial polysaccharides can yield agents with specific regenerating activity in vivo.

The regenerating activities of chemically modified anionic bacterial polysaccharides by O-sulfonation were investigated using a in vivo model of rat injured muscle regeneration. Glucuronan (GA), a linear homopolysaccharide of -->4)-beta-D-GlcpA-(1--> residues partially acetylated at the C-3 and/or the C-2 position, and glucoglucuronan (GGA), a linear heteropolysaccharide of -->3)-beta-D-GlcpA-(1-->4)-beta-D-Glcp-(1--> residues were sulfated. SO3-DMF sulfatation complex provided polysaccharides with different sulfur contents, however, a depolymerization occurred because we did not use large excess of pyridine to obtain pure modified polysaccharides. A regenerating activity on injured extensor digitorum longus (EDL) muscles on rats was obtained with these two sulfated anionic polymers. The position of sulfate groups on glucoglucuronan (primary or secondary alcohol) seems to have no influence on the biological activity by opposition to the degree of sulfatation both for the glucuronans and the glucoglucuronans. The yield of acetate groups in the glucuronan polymer modulated the specific activity.

Studies of low molecular weight samples of glucuronans with various acetylation degree.

Partially acetylated, high molecular weight glucuronans were produced by a Sinorhizobium meliloti mutant strain. Two native glucuronan samples with various degrees of acetylation were sonicated to obtain lower molecular weight samples and with low viscosity suitable for chemical modification and (13)C NMR experiments. The average degree of substitution (DS) of the polymer was estimated by Fourier transform infrared (FTIR) and NMR. (13)C NMR spectra were obtained and used to suggest a complete assignment of the signals. The nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear multi-bond coherence (HMBC) experiments were used to elucidate connectivities between the various residues and deduce the linkage of these residues within the polysaccharide.