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Biomedical subjects

M Monsigny

Publications and source records attributed to M Monsigny.

At least 19 recordsLinked to original sources
PubMed1992

Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates.

Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides--substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,--were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides.

Animals↗
PubMed1992

Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex.

A nuclear carbohydrate-binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose-containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP-dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose-conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co-addition of a glucose-recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80-kDa polypeptide both in isolated rat liver nuclear envelopes and pore-complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.

Animals↗
PubMed1992

C32 human melanoma cell endogenous lectins: characterization and implication in vesicle-mediated melanin transfer to keratinocytes.

To optimize skin pigmentation in order to help body prevention against UV radiation, the mechanism of melanin pigment transfer from melanocytes to keratinocytes must be elucidated. Melanin transfer to keratinocytes requires specific recognition between keratinocytes and melanocytes or melanosomes. Cell surface sugar-specific receptor (membrane lectin) expression was studied in human C32 melanoma cells, an amelanotic melanoma, by flow cytometry analysis of neoglycoprotein binding as an approach to the molecular specificity. Sugar receptors on melanocytes are mainly specific for alpha-L-fucose. Their expression is enhanced upon treatment by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol, which can induce melanin synthesis in amelanotic human melanoma cells in a dose-dependent manner. Flow cytometry analyses showed a small-sized population of vesicles distinguishable from large cells by their fluorescence properties upon neoglycoprotein binding. Sorting indicated that the small-sized subpopulation is composed of vesicles produced by melanocytic cells. Upon vesicle formation, a selective concentration of sugar receptors specific for 6-phospho-beta-D-galactosides appears in the resulting melanocytic vesicles. Vesicles are recognized and taken up by cultured keratinocytes and a partial inhibitory effect was obtained upon cell incubation in the presence of neoglycoproteins, indicating a possible participation of sugar receptors in this recognition. The validity for such a model to help in understanding the natural melanin transfer by melanosomes is confirmed by electron microscopy, which demonstrates the presence of melanin inside keratinocytic cells upon incubation with melanocytic vesicles.

Biological Transport↗
PubMed1992

Characterization of two galactosidases extracted from wheat germ with a hydroalcoholic solvent.

Alpha- and beta-D-galactosidases were characterized from a hydroalcoholic extract of wheat germ (Triticum vulgare). Kinetic constants (Vmax and KM) and the optimal pHs for the hydrolysis of p-nitrophenyl galactopyranosides by both enzymes were determined. These enzymes presented a high stability in hydroalcoholic medium and were inhibited by iodoacetamide and sodium p-hydroxy-mercuribenzoate.

Acetates↗
PubMed1992

Antileishmanial drug targeting through glycosylated polymers specifically internalized by macrophage membrane lectins.

Antileishmanial chemotherapy is hampered by the location of the parasite within the phagolysosome of the macrophage, which restricts the bioavailability of many potentially useful antileishmanial drugs. In this study, the possibility of using antileishmanial drugs targeted to the infected macrophages by means of a chemical linkage to a neutral mannose-substituted poly-L-lysine carrier molecule was explored. The study was performed in an in vitro model with Leishmania donovani-infected murine macrophages. The antileishmanial activities of various synthetic constructs were compared with those of the free drugs and the pentavalent antimonial Pentostam, which was used as the positive control. The 50% effective dose of allopurinol riboside linked to the mannosylated poly-L-lysine was below 7.5 x 10(-6) M, while it was up to 3 x 10(-4) M for the free drug, indicating that the drug bound to the polymer was 50 times more active than the free drug. Control experiments with other constructs (e.g., allopurinol riboside linked to the mannose-free polymer) confirmed that the enhancement of activity was indeed achieved by means of the mannose homing device.

Animals↗
PubMed1992

Secretion of macrophage cytotoxic factors induced by new desmuramyl acylpseudopeptide analogs of muramyl dipeptide.

Sixteen desmuramyl analogs of muramyl dipeptide (MDP) were tested for their abilities to stimulate cytotoxic factor secretion by mouse peritoneal macrophages. Among them, the pseudohexapeptide Boc-Gly psi [CH2O]-D-Ala-Ala-D-Glu[Lys(H-Gly)NHEt]-NH2 appeared to be four times more effective than MDP. From this study, the D configuration of the pseudo-alanyl (or lactyl) residue appears to be essential for activity.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1991

Water-soluble macromolecular fluorogenic substrates for assaying proteinases: determination of pancreatic elastase activity.

Water-soluble macromolecular fluorogenic substrates were synthesized in order to develop an easy specific proteinase assay. The validity of this method was tested with porcine pancreatic elastase by using its specific peptidic substrate Ala-Ala-Pro-Ala linked to a hydrosoluble polymer. The octapeptidic sequence FTC-epsilon Aca-Ala-Ala-Pro-Ala-Gly-Gly-Gly was linked to a water-soluble and neutral poly-L-lysine derivative. The aminocaproyl residue and the triglycyl sequence were added in order to improve the stability of the substrate, and the accessibility of the specific sequence Ala-Ala-Pro-Ala to elastase, respectively. The assay is based on the quantitative precipitation of the polymeric substrate in isopropanol while the released soluble fluorescent peptidic moiety is fluorometrically titrated in the supernatant.

Amino Acid Sequence↗
PubMed1991

Characterization and isolation of an intracellular D-mannose-specific receptor from human promyelocytic HL60 cells.

Most mammalian macrophages express D-mannose-specific receptor (membrane lectin, Mr 175,000) allowing endocytosis of their ligands, but cells of the monocytic lineage (HL60, U937, monocyte) lack this receptor. However, after permeabilization, promyelocytic, promonocytic cells and monocytes bound fluoresceinylated D-mannose-terminated neoglycoproteins as evidenced by flow cytometry. Under these conditions, confocal analysis confirmed the intracellular membrane localization of the labeling and the absence of nuclear binding. An intracellular D-mannose-specific receptor was isolated from the human promyelocytic cell line HL60, by affinity chromatography on 4-isothiocyanatophenyl alpha-D-mannopyranoside-substituted Affi-gel as a 60,000-Mr membrane protein requiring divalent cations for the ligand binding. Under the same conditions, mouse macrophages were shown to express a 175,000-Mr D-mannose-specific receptor but not the 60,000-Mr receptor.

Chromatography, Affinity↗
PubMed1991

Changes in the expression of lectins in human T lymphocyte membrane upon mitogenic stimulation.

Surface lectins, specific for given sugar structures, are expressed on human T cells, as shown by flow cytofluorometry using F-neoglycoproteins bearing either beta- and alpha-D-galactosyl, beta-D-galactosyl 6-phosphate, or alpha-L-rhamnosyl groups, but not by F-neoglycoproteins bearing other sugar groups (such as alpha-D-mannosyl groups). After stimulation with Phaseolus vulgaris mitogen, the number of cells that bind beta-D-galactosyl 6-phosphate groups (6-P-beta-D-Galp lec+ cells) increased fourfold during the first five days; these cells are helper (CD4+) T cells. Conversely, cells that bind alpha-L-Rha groups belong to the T suppressor (CD8+) family and their number moderately increased. Upon stimulation by concanavalin A, the number of cells expressing the lectin recognizing alpha-L-Rha groups increased during the first two days and then decreased within the next two days. These results are discussed with regard to the implication of lymphocyte membrane lectins in the suppressor mechanism and in the homing process.

Animals↗
PubMed1991

Hydrosoluble fluorogenic substrates for plasmin.

New hydrosoluble fluorogenic substrates for plasmin gluconoylpeptidyl-3-amido-9-ethylcarbazole were synthesized. The substitution of the N-terminal end of the peptides by a gluconoyl group prevents the substrates from aminopeptidase degradation and highly increases their hydrosolubility. The substitution of the peptide C-terminal end by a 3-amino-9-ethylcarbazole group leads to substrates suitable for direct fluorometric assay of plasmin present in cell supernatants or in cell lysates. On the basis of the kinetic parameters of the substrate hydrolysis by plasmin, it was found that D amino acids in the P2 position decrease systematically the kinetic constants of the substrates. The L configuration of the P2 amino acid appears therefore as essential in optimum substrates for plasmin.

Amino Acid Sequence↗
PubMed1991

Soluble human lymphocyte sugar binding proteins with immunosuppressive activity.

Suppression of the immune response, which involves suppressor factors released from specialized T cells, is inhibited by alpha-L-rhamnose. In this paper, we show the presence of rhamnose-specific receptors on a human CD8+ T cell-rich population and describe a novel method to isolate cells which express a given sugar-binding protein on their surface. We describe the isolation of alpha-L-rhamnose-specific molecules (rhamnose-binding fractions: RBF) from a water-soluble extract from lymphocytes, their purification by affinity chromatography on immobilized neoglycoproteins containing rhamnose residues. RBF kept their ability to bind rhamnose, as shown by the binding of fluorescein-labeled RBF to rhamnosylated BSA-substituted beads. RBF efficiently suppresses DNA synthesis of mitogen-stimulated human lymphocytes as well as B cell immunoglobulin production. Therefore, these rhamnose-binding molecules appear to be antigen-independent suppressor factors.

Carrier Proteins↗
PubMed1991

Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines.

In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors: membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either alpha-L-fucosyl or alpha-L-rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein-coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to alpha-L-rhamnosyl-BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4 degrees C, and the estimation of the endocytosis of F-, neoglycoproteins: F-, alpha-L-Rha-BSA and F-, alpha-L-Fuc-BSA were efficiently internalized. Thereafter, alpha-L-rhamnose-substituted liposomes containing 5-(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL1 alpha, IL1 beta, IL2 and TNF) which are known as biological response modifiers of keratinocytes.

Binding Sites↗
PubMed1991

Peptide derivatives specific for a Plasmodium falciparum proteinase inhibit the human erythrocyte invasion by merozoites.

A specific proteinase of P. falciparum merozoites has been detected by using hydrosoluble fluorogenic peptidic substrates synthesized by classical peptide chemistry; their N-terminal end was acylated by a gluconoyl group that protects them from aminopeptidase degradation and increases their hydrosolubility, and their carboxylic end was substituted by a 3-amino-9-ethylcarbazole group. The sequence Val-Leu-Gly-Lys was found to be the most specific substrate. On this basis, reversible peptidic inhibitors were synthesized by substituting the C-terminal lysyl residue, at the proteolytic site, by different alkylamines and amino alcohols. The activity of these compounds, studied on the P. falciparum proteinase and in in vitro cultures, strongly suggests a specific effect of this peptidic sequence on the reinvasion process. The peptidic inhibitors do not impair the release of merozoites from schizonts, but selectively inhibit the invasion step leading to the formation of rings. Although the natural target of this enzyme is not yet known, these specific peptide inhibitors could lead to a new antimalarial approach.

Amino Acid Sequence↗
PubMed1991

Characterization of an Agrobacterium tumefaciens lectin.

An Agrobacterium tumefaciens suspension induces a strong agglutination of aldehyde-fixed pig erythrocytes at pH 5.0. The agglutination is inhibited by some polysaccharides, such as fucoidin, and also when the pH is raised to 7.0. Lectins (sugar-binding proteins) associated with the bacterial cell wall of A. tumefaciens strain 84.5 were directly evidenced by spectrofluorimetry using fluoresceinylated neoglycoproteins. The specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucoside residues was also optimal at pH 5.0. A lectin was purified by affinity chromatography on agarose substituted with alpha-L-fucopyranoside. Furthermore, the haemagglutination activity of this lectin was inhibited by polysaccharides isolated from poplar leaves.

Agrobacterium tumefaciens↗
PubMed1990

Analysis of nuclear sugar-binding components in undifferentiated and in vitro differentiated human promyelocytic leukemia cells (HL60).

The nuclear sugar-binding components (i.e., lectin-like molecules) were analyzed using isolated and membrane-depleted nuclei after incubation in the presence of fluorescein-labeled neoglycoproteins. This analysis was performed before and during the in vitro differentiation of HL60 cells into monocytes by PMA treatment and into granulocytes by DMSO treatment. The nucleoli of undifferentiated and differentiated HL60 cells were not labeled, unlike the nucleoli of other mammalian cells studied so far. This peculiarity allowed us to quantitatively analyze by flow cytometry the changes in the lectin activity associated with the extranucleolar territories enriched in ribonucleoprotein complexes. The neoglycoprotein binding was found to be significantly lower in differentiated than in undifferentiated cells. The decrease in neoglycoprotein binding was observed within the first 24 h of DMSO or PMA treatment, just before the arrest of DNA synthesis. Taking into account that the granulocytic differentiation required 72 h of chemical treatment, the extra-nucleolar lectins might be involved in modulation of the DNA synthesis rather than in phenotypic differentiation. These data are discussed in an attempt to reconcile the association of lectins with RNP complexes and their possible involvement in modulation of HL60 cell proliferation.

Carrier Proteins↗
PubMed1990

Benzoxazinone kanamycin A conjugate. A new fluorescent probe suitable to detect mycoplasmas in cell culture.

The synthesis of a new benzoxazinone derivative suitable to detect early infection of cultured cells with mycoplasmas is described. p-[beta-(7-dimethylamino 1,4-benzoxazin 2-one 3yl)-vinyl]- phenylpropenoic acid was coupled to kanamycin A, an aminoglycoside leading to a cationic fluorescent probe which fluoresces at 600 nm upon excitation at 490 nm. This fluorescent probe is shown to heavily label the glycocallix of all the mycoplasma strains tested which are found to be associated with contaminated cultured cells and to allow an easy and rapid detection of contamination by fluorescence microscopy and flow cytometry.

Animals↗
PubMed1990

Cell surface lectins of human granulocytes: their expression is modulated by mononuclear cells and granulocyte/macrophage colony-stimulating factor.

This paper presents the characterization of a sugar-specific receptor on the surface of human circulating polymorphonuclear cells. With the help of fluorescent neoglycoproteins and flow cytometry, a receptor was identified as being specific for alpha-L-rhamnosyl residues. The number of receptors was 55,000/cell and their affinity reached 2 x 10(8) l mol-1. This number changed as a function of the biological state of the cells. Indeed, receptor expression was modulated by the presence of other cells. T cells and B cells increased the number of receptors on the granulocyte surface. Expression of the alpha-L-rhamnose-specific lectin was dependent on lymphocyte derived soluble factor(s), which induce(s) growth and differentiation of polymorphonuclear phagocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) specifically produced a significant increase in the number of receptors for alpha-L-rhamnose (2-10-fold/cell). This modulation was independent of protein kinase C activators such as phorbol ester, which produced no effect on alpha-L-rhamnose receptor expression. These findings demonstrate that GM-CSF may stimulate post differentiation functions and properties of mature granulocytes.

Antibodies, Monoclonal↗