Biomedical subjects

M Monsigny

Publications and source records attributed to M Monsigny.

221 records · Page 13Linked to original sources

PubMedDate not supplied

Novel glycosynthons for glycoconjugate preparation: oligosaccharylpyroglutamylanilide derivatives.

The reducing sugar of an oligosaccharide reacting with the alpha-amino group of an amino acid is converted to an N-oligosaccharylamino acid which can then be stabilized by N-acylation. Oligosaccharides in solution in N,N-dimethylformamide reacted with alpha-glutamyl-p-nitroanilide at 50 degrees C for a few hours, leading to an N-oligosaccharylglutamyl-p-nitroanilide. Then, the gamma-carboxylic group of the glutamyl moiety, activated by adding (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), reacted with the substituted alpha-amino group of the glutamyl residue, leading to an N-oligosaccharylpyroglutamyl-p-nitroanilide within 0.5 h. Such a one-pot two-step reaction was shown to be very efficient in the case of a disaccharide such as lactose, or pentasaccharides such as lacto-N-fucopentaoses, Lewis(a) or Lewis(x). The glycosynthons were characterized by chromatography (HPAEC and HPLC); their molecular mass was determined by electrospray ionization mass spectrometry, and the glycosylamides were shown to have a beta-anomeric configuration on the basis of their proton NMR. The N-oligosaccharylpyroglutamyl-p-nitroanilides are quite stable at room temperature over a large pH range. They are easily converted to N-oligosaccharylpyroglutamyl-p-isothiocyanatoanilides which can be used to prepare glycoconjugates such as cationic glycosylated polylysines suitable for specifically delivering genes or oligonucleotides in a sugar-dependent manner.

Aminocaproic Acid↗

PubMedDate not supplied

Membrane permeabilization and efficient gene transfer by a peptide containing several histidines.

We designed a peptide, H5WYG (GLFHAIAHFIHGGWHGLIHGWYG), that permeabilizes cell membrane at a slightly acidic pH but not at neutral pH. Absorbance, fluorescence, and circular dichroism spectra showed that H5WYG undergoes a dramatic conformational change between pH 7.0 and 6.0 that correlates with the protonation of the histidyl residues. Cell permeabilization studies monitored by flow cytometry on living cells showed that H5WYG permeabilizes the cell membrane with a great efficiency at pH 6.4 but was not active at neutral pH; at pH 6.8, the peptide permeabilized 50% of the cells at 20 degrees C within 10 min. H5WYG increased the expression of genes transferred to cells as glycosylated polylysine-DNA complexes, and the transfection efficiency was not impaired in the presence of serum. Therefore, this peptide containing several histidines that become positively charged when the pH decreased to less than 7.0 is a suitable helper for delivering molecules into the cytosol upon either permeabilization of the plasma membrane induced by lowering the extracellular medium to pH 6.4 or permeabilization of the endosomal membrane induced by acidification of endosomes.

Amino Acid Sequence↗

PubMedDate not supplied

Optimized conditions to couple two water-soluble biomolecules through alkylamine thiolation and thioetherification.

A simple method for introducing, in buffered saline, a reactive sulfhydryl group on water-soluble molecules bearing an alkyl-amino group is described. This method is based on the use of two water-soluble reagents: 2-iminothiolane and 6,6'-dithiodinicotinic acid. The first one is open upon reaction with an amino group, and the generated thiol group is immediately protected by action of the second reagent. The optimal conditions were determined by taking into account the stability and the reactivity of both reagents with regards to pH and temperature. This method was validated through two applications, the substitution of bovine serum albumin with a bromoacetyl peptide and the substitution of an amino link at the 5' end of an oligonucleotide by reaction with either a fluorescent tag, iodoacetamidofluorescein, or a bromoacetyl peptide, upon reduction of the protected disulfide bridge with a third water-soluble reagent, namely tris(2-carboxyethyl)phosphine.

Amino Acid Sequence↗

PubMedDate not supplied

Efficient gene transfer by histidylated polylysine/pDNA complexes.

Plasmid/polylysine complexes, which are used to transfect mammalian cells, increase the uptake of DNA, but plasmid molecules are sequestered into vesicles where they cannot escape to reach the nuclear machinery. However, the transfection efficiency increases when membrane-disrupting reagents such as chloroquine or fusogenic peptides, are used to disrupt endosomal membranes and to favor the delivery of plasmid into the cytosol. We designed a cationic polymer that forms complexes with a plasmid DNA (pDNA) and mediates the transfection of various cell lines in the absence of chloroquine or fusogenic peptides. This polymer is a polylysine (average degree of polymerization of 190) partially substituted with histidyl residues which become cationic upon protonation of the imidazole groups at pH below 6.0. The transfection efficiency was optimal with a polylysine having 38 +/- 5% of the epsilon-amino groups substituted with histidyl residues; it was not significantly impaired in the presence of serum in the culture medium. The transfection was drastically inhibited in the presence of bafilomycin A1, indicating that the protonation of the imidazole groups in the endosome lumen might favor the delivery of pDNA into the cytosol.

Amino Acid Sequence↗

PubMedDate not supplied

Characterization of membrane lectins in human colon carcinoma cells by flow cytofluorometry, drug targeting and affinity chromatography.

Flow cytofluorometry and drug targeting with labelled neoglycoproteins are used as tools to probe for membrane lectins in two human adenocarcinoma cell lines. Both cell lines express activities for galactosides, glucosides and fucosides. Affinity chromatography on gels with immobilized sugar leads to purification of an alpha-galactoside-binding protein at an apparent molecular weight of 64 kDa that also binds to lactose, maltose and fucose and exhibits Ca2+-requirement for binding, a beta-galactoside-binding protein without Ca2+-requirement at an apparent molecular weight of 14 kDa, and an alpha-glucosyl-binding protein without Ca2+-requirement at an apparent molecular weight of 34 kDa from both cell lines. The description of membrane lectins, documented here for the first human tumor cell lines, is an initial step towards a lectin-based improvement of the clinical management of human colon adenocarcinoma.

Adenocarcinoma↗