Biomedical subjects

M Monsigny

Publications and source records attributed to M Monsigny.

At least 73 records · Page 4Linked to original sources

PubMed1995

In vitro toxicity assays for dye ligands used in affinity chromatography.

Some reactive textile dyes have been used for years as biomimetic ligands in protein purification. There has been reluctance, however, to use these dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination. Therefore, toxicological data are necessary to quantify the level of this hazard. This study deals with a series of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy, etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies. Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2 and Reactive Red 120 and their derivatives over the range 10-62.5 micrograms/ml in several assays.

Chromatography, Affinity↗

PubMed1995

Nuclear import of glycoconjugates is distinct from the classical NLS pathway.

The nuclear import of many proteins depends on a short peptide sequence called the nuclear localization signal. However, glycosylated proteins, which lack such a nuclear localization signal, upon their injection into the cytosol by electroporation, enter the nucleus in a sugar-dependent manner. This paper brings new insights on the mechanism of this process, based on a study of neoglycoprotein nuclear uptake by digitonin-permeabilized cells. The nuclear import of neoglycoproteins is energy dependent: it does not occur when cells are maintained at 4 degrees C or when cells are ATP-depleted by treatment with apyrase. The nuclear import of neoglycoproteins occurs through the nuclear pore: it is inhibited by preincubation of cells with wheat germ agglutinin, a lectin which binds the nuclear pore glycoproteins and blocks the translocation step of nuclear localization signal bearing proteins through the nuclear pore. Furthermore, the nuclear import of neoglycoproteins does not use the pathway of nuclear localization signal bearing proteins: nuclear import of nuclear localization signal bearing proteins depends on cytosolic factors and is inhibited by treatment of cells with N-ethylmaleimide, while the nuclear import of neoglycoproteins neither requires added cytosolic factors nor is sensitive to alkylation by N-ethylmaleimide. In addition, upon incubation in the presence of a large excess of nuclear localization signal bearing protein, the nuclear import of neoglycoproteins is not inhibited.

Adenosine Triphosphate↗

PubMed1994

A negative regulatory element of the macrophage-specific human mannose receptor gene represses its expression in nonmyeloid cells.

We have cloned the putative promoter of the human mannose receptor gene using the ligation-mediated polymerase chain reaction. This modified polymerase chain reaction method depends upon the ligation of restricted genomic DNA fragments to a sequence of DNA containing a generic primer site. Approximately 400 bp of genomic DNA sequence immediately upstream from the 5' end of the lectin gene was amplified with this strategy. Primer-extended reverse transcription identified several 5' ends of the mannose receptor mRNA corresponding to differential use of initiation transcription sites. DNA sequence analysis of the 5' flanking sequence of the mannose receptor gene indicated the presence of a TATA box and various putative binding sites for several transcription activators. The insertion of the sequence into a plasmid containing a promoterless luciferase reporter gene reveals a promoter activity with a high cell type specificity, efficient expression upon transfection into macrophage type cells, and lack of efficiency upon transfection into nonmyeloid cells. A series of deletion mutants reveals that this cell-type-specific promoter activity is mediated by a negative regulatory element located at the 5' end of the isolated promoter.

Base Sequence↗

PubMed1994

Characterization and cellular localization by monoclonal antibodies of the 60 kDa mannose specific lectin of human promyelocytic cells, HL60.

Myelomonocytic lineage cells express an M(r) 60,000 mannose specific lectin, MR60 (Pimpaneau et al. (1991), Carbohydr Res 213: 95-108). Under non-reducing conditions, this protein migrates as a 120,000 protein. MR60 does not contain any N-glycan moiety cleavable by the action of N-glycanase. MR60 induces a sugar selective aggregation of beads coated with glycosylated albumin: beads bearing alpha-D-mannosyl residues are aggregated while beads bearing alpha-D-glucosyl residues are not. A monoclonal antibody Lec101B, specific for MR60, recognizes a single M(r) 60,000 protein by Western blotting. This monoclonal antibody does not label the cell surface of cells expressing MR60, but decorates intracellular vesicles upon permeabilization of these cells.

Agglutination Tests↗

PubMed1994

Toxicity studies on native Procion Red HE-3B and released dye from affinity material exposed to degradative chemical conditions.

Leached ligands from chromatographic packing material submitted to drastic regeneration conditions can contaminate pure biological preparations. These contaminants could have adverse effects from a toxicology point of view that are very poorly documented in liquid chromatography for protein separation. Investigations on toxicity level have been made on released material from immobilized Procion Red HE-3B, after formal identification of the nature of the leached chemical material. Toxicity investigations in vitro involved a number of tests on living cells (eucaryotic and procaryotic) covering different aspects. Behaviour of cells in regular cultures, polyploïdia induction, genotoxicity as well as mechanisms of endocytosis have been studied. Results showed no toxic effects within the range of concentration of dye and dye derivatives studied. Genotoxicity studies in particular did not show any toxic effect over a range of concentration much higher than the regular level of dye leakage from the sorbent.

Cell Division↗

PubMed1994

Mannose dependent tightening of the rat ependymal cell barrier. In vivo and in vitro study using neoglycoproteins.

The possible role of carbohydrate binding proteins (lectins) and glycoconjugates in the formation of junctions ensuring tightening between ependymal cells was studied using synthetic glycoconjugates, the neoglycoproteins. These compounds are prepared by substituting bovine serum albumin with sugar residues and additional labelling (or not) with fluorescein or biotin. Injections of these components into the cerebral ventricles of adult rats resulted in a binding pattern which could be related to their carbohydrate composition. Mannose-containing neoglycoproteins were bound to ependymal cell cilia and penetrated rapidly the brain tissue. Such phenomenon was not seen with glucose- or galactose-containing neoglycoprotein molecules. In contrast, mannose-, galactose- and glucose-containing neoglycoproteins bound strongly to some endothelial cells around blood vessels. Fluorescent unglycosylated serum albumin did not bind to any brain structures. In contrast, co-injection of mannose-containing non-fluorescent neoglycoproteins with the other fluorescent compounds (including fluorescent sugar-free BSA) resulted in the penetration of the fluorescent compounds into the brain tissue. This internalization into brain was attributed to disaggregation of junctions between ependymal cells. Cultured ependymal cells behaved likewise. In short term experiments (5 min-1 h), only the mannose-containing neoglycoproteins bound strongly to the ependymal cells, particularly to the cilia. In long term experiments (1-9 days), mannose-containing neoglycoproteins specifically induced the disappearance of junctions between the cultured cells. These results emphasize the importance of mannose-dependent recognition system in the maintenance of junctions between ependymal cells, where a mannose-binding lectin has been previously detected.

Animals↗

PubMed1994

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells.

The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-1 and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin-3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.

3T3 Cells↗

PubMed1994

Purification of an alpha-L-fucoside-binding protein from Rhizobium lupini.

Lectins associated with the bacterial cell surface of Rhizobium lupini strain LL13 were evidenced by erythrocyte agglutination, by aggregation of neoglycoprotein coated beads and by spectrofluorimetry using fluoresceinylated neoglycoproteins. At pH 5.0, a specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucose was observed. The binding of this labelled neoglycoprotein is a saturable phenomenon and is inhibited by the same unlabelled neoglycoprotein. Extracts of R lupini obtained by disrupting a bacterial pellet through a French press were stabilized at pH 5.6 by gel filtration and purified to homogeneity by affinity chromatography on Agarose A4 substituted with alpha-L-fucose. A protein with a M(r) approximately 19,000 was specifically eluted from this affinity column with L-fucose. Isoelectric focusing of this sample yielded a single band with pI near 6.7. This protein specifically aggregated L-Fuc-BSA-coated microspheres. The results obtained in the present study indicate that we have purified from Rhizobium lupini strain LL13, a L-fucose binding protein as a lectin.

Agglutination Tests↗

PubMed1994

Dye-hydrophobic hapten conjugate/anti-dye antibody complex as immunogen: preparation of hydrophobic hapten-specific monoclonal antibodies.

In order to induce the production of antibodies specific for small molecules, it is common to link them to a protein. However, when the small molecule is very hydrophobic it is extremely difficult to prepare such a conjugate. Here, we describe a simple way to obtain an antigenic conjugate under controlled conditions: in a first step a very hydrophobic hapten, cholanic acid, is linked to a dye, basilen blue, in organic solvent; in a second step the cholanic acid-basilen blue conjugate is dissolved in phosphate buffered saline and mixed with rabbit polyclonal anti-basilen blue antibodies previously raised in rabbits against basilen blue-key-hole limpet hemocyanin conjugate. Such a complex, which dissociates very slowly, appears to be a good immunogen in mice. Anti-cholanyl residue monoclonal antibodies were produced and characterized.

Animals↗

PubMed1994

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase.

Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

Animals↗

PubMed1994

Toxicity studies on Reactive Blue-2 leached from affinity material exposed to extreme chemical conditions.

Toxicity effects related to leached ligands from affinity sorbents that can contaminate biological preparations were investigated in the particular case of immobilized Reactive Blue-2. Initially, identification of the real chemical structure of leached dye has been done by HPLC after incubation in extreme conditions. Toxicity investigations in vitro involving several well known tests showed no toxic effects within the studied range of dye concentration. Cell cultures behaved normally when the adhesion phase was successful; polyploidy induction in human cells by the native dye and its derivatives identified as possible leached material was very similar to standard cultures. Genotoxicity studies did not evidence any toxic effect in E. Coli cultures of dyes themselves or of the same dyes after metabolic activation.

Cell Adhesion↗

PubMed1994

[Studies of in vitro and in vivo toxicity of dyes used in affinity chromatography].

Some reactive textile dyes are used for years as biomimetic ligands in protein purification. The reluctance to use these performant systems in large scale for therapeutically applicable proteins is related with the possible dye leakage and consequently with problems of contamination. Therefore, toxicology data are necessary to quantify the level of danger in association with sensitive assays. This study deals with a series of in vitro toxicity studies with eucaryotic cells (growth, polyploidia, ...) as well as with procaryotic cells (E. coli) for genotoxic studies. Both approaches demonstrated a total absence of toxicity for all ranges of concentrations investigated for Reactive Blue 2, Reactive Red 120 and their derivatives. Additional experiments done in vivo by the administration of dye solutions to a series of mice confirmed the non toxic character of these dyes in vitro.

Animals↗

PubMed1993

Sensitive, hydrosoluble, macromolecular fluorogenic substrates for human immunodeficiency virus 1 proteinase.

Hydrosoluble macromolecular fluorogenic substrates specific for the human immunodeficiency virus 1 (HIV-1) proteinase have been prepared. The fluoresceinyl peptide Ftc-epsilon-Ahx-Ser-Phe-Asn-Phe-Pro-Gln-Ile-Thr-(Gly)n, corresponding to the first cleavage site of HIV-1 gag-pol native precursor was linked to a water-soluble neutral (Lys)n derivative. The epsilon-aminohexanoyl residue (epsilon-Ahx) and the glycyl sequence were added in order to improve the stability of the substrate and the accessibility of the cleavage site to the HIV-1 proteinase respectively. This macro-molecular peptidic-substrate conjugate is significantly more water-soluble than the free peptide itself on a substrate molar concentration basis. The assay is based on the quantitative precipitation of the polymeric material by adding propan-2-ol whereas the fluorescent peptide moiety released upon proteolysis remains soluble in the supernatant. The proteinase activity is assessed by measuring the fluorescence of the supernatant. This assay allows the detection of a few fmol of HIV-1 proteinase, even in the presence of cell culture media, plasma or cell lysate and it gives accurate results within a large proteinase concentration range. The hydrosoluble macromolecular substrate is also suitable for determining the HIV-1 proteinase activity using 96-well microplates, allowing us to test accurately and rapidly numerous enzyme samples and/or the potency of new proteinase inhibitors.

Amino Acid Sequence↗

PubMed1993

Specific gene transfer mediated by lactosylated poly-L-lysine into hepatoma cells.

Plasmid DNA/glycosylated polylysine complexes were used to transfer in vitro a luciferase reporter gene into human hepatoma cells by a receptor-mediated endocytosis process. HepG2 cells which express a galactose specific membrane lectin were efficiently and selectively transfected with pSV2Luc/lactosylated polylysine complexes in a sugar dependent manner: i) HepG2 cells which do not express membrane lectin specific for mannose were quite poorly transfected with pSV2Luc/mannosylated polylysine complexes, ii) HeLa cells which do not express membrane lectin specific for galactose were not transfected with pSV2Luc/lactosylated polylysine complexes. The transfection efficiency of HepG2 cells with pSV2Luc/lactosylated polylysine complexes was greatly enhanced either in the presence of chloroquine or in the presence of a fusogenic peptide. A 22-residue peptide derived from the influenza virus hemagglutinin HA2 N-terminal polypeptide that mimics the fusogenic activity of the virus, was selected. In the presence of the fusogenic peptide, the luciferase activity in HepG2 cells was 10 fold larger than that of cells transfected with pSV2Luc/lactosylated polylysine complexes in the presence of chloroquine.

Amino Acid Sequence↗

PubMed1993

Sugar-dependent nuclear import of glycoconjugates from the cytosol.

Large proteins (above 40 kDa) do not migrate from the cytosol to the nucleus except when they contain or are substituted with specific peptides called nuclear localization sequence. Accordingly fluorescein-labeled serum albumin, introduced into the cytosol upon electroporation, does not leave the cytosol. Conversely the same protein substituted with about 25 sugar residues migrate from the cytosol to the nucleus in a manner dependent on the nature of the sugar linked to serum albumin, on the temperature, and on the incubation time. Serum albumin substituted with glucose, fucose, or mannose residues enter the nucleus at 37 degrees C within 30 min while sugar-free serum albumin or serum albumin substituted with galactoside or 6-phosphomannoside residues do not. At 4 degrees C, all of those proteins stay in the cytosol. Thus, glycosylated proteins can enter the nucleus from the cytosol by making use of the sugars borne either by a new type of nuclear import or by making a complex with lectins acting as shuttle between cytosol and nucleus.

Biological Transport↗

PubMed1993

A SV-40 immortalized murine endothelial cell line from peripheral lymph node high endothelium expresses a new alpha-L-fucose binding protein.

Endothelial cells from mouse peripheral lymph nodes were immortalized by cationic liposome-mediated transfection using a plasmid construct containing both the gene coding for the large T antigen of simian virus 40 and a geneticin resistance gene suitable for selection. A cell line (HECa10) was isolated on the basis of its capacity to specifically bind fucoside carrying glycoconjugates; these cells present the main characteristics of endothelial cells: production of angiotensin converting enzyme and of factor VIII-related antigen. Upon stimulation, they express E-selectin which binds oligosaccharides containing the Lewisx determinant (Fuc alpha 3[Gal beta 4 GlcNAc beta 3Gal beta) and the MECA 79 addressin which is characteristic for the peripheral lymph node high endothelium and is a L-selectin ligand. HECa10 cells, as well as peripheral lymph node high endothelial cells in primary culture, express a second fucoside binding protein which differs from E-selectin. Indeed, this new fucoside-binding protein is constitutively expressed on unstimulated cells while E-selectin is not. Furthermore, HECa10 cells mediate selective lymphoid cell adhesion in a selectin/addressin-dependent mechanism, mainly inhibited by MECA 79 antibody and, in a fucose-binding lectin-dependent manner, mainly inhibited by the specific neoglycoprotein.

Animals↗

PubMed1993

Characterization of membrane sugar-specific receptors in cultured high endothelial cells from mouse peripheral lymph nodes.

The culture of specialized high endothelial cells (HEC) from lymphoid organs (peripheral lymph nodes (PLN) and Peyer's patches (PP)) was undertaken in order to study and characterize the cell surface molecules which are involved in lymphocyte recognition and allow homing. Cells were stimulated in vivo by a graft versus host (GVH) type of reaction before isolation and culture. The resulting adherent and growing cells were characterized as endothelial cells because of their typical aspect and their ability to produce angiotensin-converting enzyme and factor VIII-related antigen. They possess tissue-specific endothelial addressins. MECA 79 antigen is present on cells isolated from PLN while MECA 367 antigen is detected on cells from PP. Surface receptors for glycans were studied cytochemically using neoglycoproteins and fluorescence microscopy and quantified by flow cytometry experiments which showed that the specificity of sugar receptors depends upon endothelial cell origin. Indeed, sugar receptors for alpha-L-fucosyl residues were specifically expressed by endothelial cells from PLN. These receptors were inducible upon action of activated lymphocyte-conditioned medium. Further characterization of endothelial cells from peripheral lymph nodes indicates that they indeed mediate adhesion of lymphocytes in vitro. The role of protein-sugar interactions in this process was assessed by inhibition experiments performed with the help of neoglycoproteins. Best inhibitory effects were obtained when endothelial cells had been preincubated with alpha-L-fucosyl-BSA and when lymphoid cells were preincubated with beta-D-galactosyl-BSA. Concomitant inhibition assays indicate the participation of sugar specific receptors--endogenous lectins--on the surface of both endothelial and lymphoid cells to achieve recognition and adhesion.

Animals↗

PubMed1993

Endocytosis of alpha 1-acid glycoprotein variants by human monocytic lineage cells.

Human alpha 1-acid glycoprotein (AGP or orosomucoid) is a major glycoprotein of plasma. AGP can be separated on immobilized concanavalin A into three variants bearing none (AGP A), one (AGP B) or two (AGP C) biantennary glycans. In this paper, we show, using flow cytometry and confocal microscopy, that AGP C which is eluted from concanavalin A with mannose, binds to human monocytes, monocyte-derived macrophages as well as human promonocytic cell lines such as THP1 or U937. Conversely HL60, a promyelocytic cell line, does not express the surface AGP C binding protein. AGP C is internalized and degraded with an efficiency depending on the state of differentiation of these cells. In contrast, AGP A which is not recognized by concanavalin A, does not bind to any of these cells.

Cell Line↗