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Biomedical subjects

M Monsigny

Publications and source records attributed to M Monsigny.

At least 109 records · Page 6Linked to original sources
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↗
PubMed1990

Psi[CH2O] pseudodipeptide synthesis. An improved approach which allows absolute configuration determination.

An improved way to obtain psi[CH2O] pseudodipeptide units is proposed, involving an intramolecular Williamson's reaction, with displacement of bromine by an alkoxide, instead of the classical intermolecular one. Until now, psi[CH2O] pseudodipeptide synthesis done by this new method, has used a protected form of the amino alcohol hydroxyl group to prepare the acyclic precursor. In the present paper, the use of an active ester of the brominated carboxylic acid avoids this protection step. The pseudodipeptides AcGly psi[CH2O]-D,L-Ala-OH and Ac-Ser(Bzl) psi [CH2O]-D,L-Ala-OH were obtained in high yields, through a delta-lactam intermediate, which furthermore allows the determination of the absolute configuration of compounds using HPLC and appropriate nuclear magnetic resonance (NMR) techniques.

Chemical Phenomena↗
PubMed1990

Distribution of sugar-binding sites within interphase nuclei and mitotic chromosomes of a human cell line.

Using gold labelled neoglycoproteins containing either alpha-D-glucose, N-acetyl-beta-D-glucosamine, alpha-D-mannose, 6-phospho-alpha-D-mannose, and alpha-L-fucose (BSA), we investigated their intranuclear binding sites in the TG human cell line. Although gold-labelled BSA did not give any noticeable labelling, the presence of 1% free BSA in the medium containing the gold labelled neoglycoproteins was revealed to be a key factor of the labelling. During interphase in the presence of free BSA most of the labelling was detected in the nucleoplasm. The border of the condensed chromatin, known to be the site of hnRNA synthesis as well as the interchromatin areas enriched in RNPs were labelled. Condensed chromatin also contained binding-sites. The nucleolus was seen to present low labelling in comparison with the labelling observed over the nucleoplasm. These nucleolar binding sites were located both in the dense fibrillar and granular components. No labelling could be detected over the fibrillar centers which are very conspicuous in this cell line. During mitosis sugar-binding sites were observed over the chromosomes. Data reported here show for the first time that lectin-like proteins and chromatin components are colocalized both during interphase and mitosis. In addition, within the nucleolus the presence of sugar-binding proteins was seen to be restricted to the dense fibrillar and granular components.

Carbohydrate Metabolism↗
PubMed1990

Proteases in malaria-infected red blood cells.

The discrimination between erythrocyte and Plasmodium proteases is now made easier by using synthetic fluorogenic substrates, high-pressure liquid chromatography, reliable methods of cell preparation, as well as radiolabeled extracts from in vitro cultures of P. falciparum. The reinvasion process of an erythrocyte by a merozoite involves specific proteinases, which were recently identified using fluorogenic peptidyl-AEC substrates and by analysis of schizont and merozoite extracts with the gelatin-SDS-PAGE method. The biological targets of both host and parasite proteinases are not yet well characterized because Plasmodium-infected red blood cells contain at least four compartments with different pH values, which could modulate the proteinase activities according to their pH range activity. The processing of the precursor for the major merozoite surface antigens involves cleavage of very specific peptidic bonds by, so far unknown, proteinases. The depletion of the erythrocyte cytoskeleton could depend on a 37 kD proteinase, which cleaves spectrin and the 4.1 component, as shown in P. berghei and P. falciparum species. In contrast to leupeptin, which inhibits the merozoite release from schizont-infected erythrocytes, the structural inhibitor analogous to the Val-Leu-Gly-Lys (or Arg) P. falciparum neutral proteinase substrates appears to block the invasion step of erythrocytes by merozoites and may open new trends in chemotherapeutical strategies.

Animals↗
PubMed1990

Macrophage activation by muramyl dipeptide bound to neoglycoproteins and glycosylated polymers: cytotoxic factor production.

Some biological functions of macrophages can be stimulated by muramyl dipeptide (MDP) in vitro. Such stimulation is more efficient when MDP is bound to macromolecule carriers. The macrophage stimulation by MDP bound to glycosylated serum albumin (BSA) or bound to gluconoylated and glycosylated poly-L-lysine (PLK) is investigated. These two types of MDP conjugates are more efficient than free MDP in rendering mouse peritoneal and rat alveolar macrophages cytostatic against various tumor cells. However, the release of mitogenic factor or cytotoxic factor (CF) by activated macrophages varies according to the nature of the carrier (BSA or PLK) and to the nature and content of sugar residues bound to the macromolecule carrier (mannose or 6-phosphomannose). Macrophages activated by MDP bound to glycosylated BSA release mitogenic factor and CF into the medium; anti-recombinant tumor necrosis factor (rTNF) totally inhibits the cytotoxicity of the supernatant. On the contrary, MDP bound to glycosylated PLK induces no secretion of mitogenic factor and a very small amount of CF in the culture medium. The role of CF in the cytostatic activity of activated macrophages is discussed. The released CF is not involved in the cytostatic activity, but TNF-like molecules, expressed at the membrane level, could be implied because anti-rTNF abrogates 40% of the cytostatic activity of the macrophages.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1989

Purification and identification of a neutral endopeptidase in Plasmodium falciparum schizonts and merozoites.

An endopeptidase specific to the Plasmodium falciparum erythrocytic schizont stage and to free merozoites was detected using the fluorogenic GlcA-Val-Leu-Gly-Lys(or Arg)-AEC substrate. The enzyme was purified by high performance liquid chromatography (HPLC); its optimal activity was around pH 7.5 and its isoelectric point was 4.4. The molecular weight of the enzyme was about 68,000, as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The endopeptidase was strongly inhibited by thiol proteinase inhibitors, leupeptin, and antipain. The possible involvement of this neutral endopeptidase in the reinvasion process is discussed.

Animals↗
PubMed1989

Muramyl dipeptide bound to poly-L-lysine substituted with mannose and gluconoyl residues as macrophage activators.

Poly-L-lysine modified with mannose derivatives, the residual cationic charges of which being neutralized by N-acylation, were synthesized and used as carriers of a macrophage activator (N-acetylmuramyl dipeptide, MDP). The influence of the acylating agent on the targeting efficiency was investigated: a hydrosolubilizing group such as a gluconoyl moiety led to very efficient carrier conjugates, while an acetyl group did not. The effect of sugar and acyl content of the polymers was assessed using these compounds as inhibitors of red blood cell agglutination by Concanavalin A. The binding and specific endocytosis of poly-L-lysine substituted with several mannose derivatives and gluconoyl residues (GlcAx-, Man(y)-PLK) have been determined by a quantitative flow cytometry analysis. MDP bound to these conjugates was much more efficient in vitro than free MDP in macrophage cytostasis assays.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1989

Endocytosis of alpha 1-acid glycoprotein variants and of neoglycoproteins containing mannose derivatives by a mouse hybridoma cell line (2C11-12). Comparison with mouse peritoneal macrophages.

Macrophages from various origins are known to express membrane lectins that mediate the endocytosis of mannose-bearing glycoconjugates. Most macrophage tumor cell-lines lack such receptors. In this paper we show by flow cytometry analysis that a newly generated macrophage hybridoma (2C11-12), which displays several macrophage characteristics, also expresses mannose membrane lectins, resulting in the internalization of fluoresceinylated neoglycoproteins into acidic compartments. Thioglycolate elicited mouse peritoneal macrophages and the 2C11-12 hybridomas were compared by flow cytometry with regard to the binding and endocytosis of alpha 1-acid glycoprotein (AGP) variants separated by affinity chromatography on immobilized concanavalin A. AGP C eluted specifically with methyl alpha-mannopyranoside, which contains two bi-antennary oligosaccharides, was endocytosed as mannosylated serum albumin (Man-BSA). In both types of macrophages, the fluoresceinylated ligands were internalized in acidic compartments as demonstrated by the fluorescence intensity increase upon monensin post-incubation. However the behaviour of the internalized ligands was found to be quite different. AGP C and Man-BSA were rapidly degraded by thioglycolate elicited peritoneal macrophages and excreted in the medium as small peptide fragments; conversely they remained a longer time in the 2C11-12 hybridoma.

Animals↗
PubMed1989

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells.

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.

Animals↗
PubMed1989

Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength.

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.

Animals↗
PubMed1988

p-Benzylthiocarbamoyl-aspartyl-daunorubicin-substituted polytrisacryl. A new drug acid-labile arm-carrier conjugate.

A new type of drug acid-labile arm-carrier is described. A hydrophilic polymer [polytrisacryl, or poly(acryloyl 2-amido-2-hydroxymethyl-1,3-propanediol)], used as a model, is substituted with p-nitrobenzyl groups. The nitrobenzyl groups are reduced to aminobenzyl and transformed into benzylisothiocyanate groups which are allowed to react with aminoacyl daunorubicin. The excess of benzylisothiocyanate is transformed into benzylthiocarbamoyl N-methyl glucamine. A conjugate containing benzylthiocarbamoyl-aspartyl daunorubicin is stable at neutral pH, and releases free daunorubicin when it is exposed to pH 5 or below. This conjugate is about 200-fold less toxic than free daunorubicin, on a molecular basis, when it is added to the medium of Lewis lung carcinoma (3LL) cells in culture.

Acrylates↗
PubMed1988

Kluyveromyces bulgaricus yeast lectins. Isolation of N-acetylglucosamine and galactose-specific lectins: their relation with flocculation.

Kluyveromyces bulgaricus is a yeast which, upon culture in a calcium-enriched glucose-peptone medium, flocculates. Its flocculation can be reversed by the addition of galactose. In this paper, it is shown that two lectins can be isolated either from the concentrated culture broth or from the supernatant of deflocculated cells suspended in galactose solution. The N-acetylglucosamine-specific lectin, at pH 7.4, agglutinates untreated sheep red blood cells, but agglutinates neither untreated rabbit red blood cells nor glutaraldehyde-fixed sheep or rabbit red blood cells. Conversely, at pH 4.5, this lectin agglutinates glutaraldehyde-fixed sheep red blood cells. The galactose-specific lectin, at pH 7.4, agglutinates both untreated and glutaraldehyde-fixed rabbit red blood cells but does not agglutinate untreated or glutaraldehyde-fixed sheep red blood cells. At pH 4.5, this lectin agglutinates both glutaraldehyde-fixed sheep and rabbit red blood cells and induces flocculation of deflocculated K. bulgaricus cells. In all cases, the agglutination and the flocculation induced by one of these two lectins were inhibited by free or conjugated N-acetyl-D-glucosamine or by free or conjugated D-galactose, respectively. No glycosylhydrolase activity could be detected in the purified lectins.

Acetylglucosamine↗
PubMed1988

Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland.

A sugar-binding protein, or endogenous lectin, was localized on fixed and paraffin-embedded thyroid sections by means of fluorescein-labelled neoglycoproteins. Fluorescence microscopy showed the binding of this lectin to be dependent on calcium ions and acidic pH and indicated sugar specificity for GlcNAc moieties only. In human, porcine and murine thyrocytes, specific binding was observed mainly on subcellular organelles. Conversely, in rabbit thyrocytes, specific labelling was seen mostly at the apical cell surface facing the follicular lumen. The possibility that this novel endogenous lectin is involved in the Tg metabolism is considered.

Acetylglucosamine↗
PubMed1988

Colorimetric determination of neutral sugars by a resorcinol sulfuric acid micromethod.

A microscale colorimetric assay for neutral sugars, in which neutral sugars react with resorcinol in the presence of 75% sulfuric acid solution is described. This assay, based on the use of microtiter plates with 96 U-shaped wells is simple and easy to handle; it allows accurate determinations with small samples (20 microliters) containing 1 to 100 nmol of neutral sugars and is quite convenient for detection of glycoconjugates in chromatographic column effluents.

Carbohydrates↗
PubMed1988

Neutral proteases involved in the reinvasion of erythrocytes by Plasmodium merozoites.

Neutral proteases of Plasmodium sp erythrocytic stages were studied by means of a sensitive fluorogenic method and gelatin-SDS-PAGE. The substrates gluconoyl-Val-Leu-Gly-Lys(or Arg)-3-amido-9-ethylcarbazole were selectively hydrolyzed by an endopeptidase from rodent Plasmodium berghei (Pb) and Plasmodium chabaudi (Pc) and from human Plasmodium falciparum (Pf) parasites. These endopeptidases were purified from 100,000-g soluble schizont extract by high pressure liquid chromatography; they have a similar Mr of 68,000 in SDS-PAGE, and an optimal activity at pH 7.4. The Pb 68 and Pf 68 endopeptidases were localized in schizonts and also in merozoites as shown by indirect immunofluorescence on Pb merozoites and by the identification of the Pf 68 endopeptidase activity in free viable merozoites. The Pb 68 and Pf 68 endopeptidases belong to the class of cysteine proteases. Analysis by gelatin-SDS-PAGE of a Pb 68 endopeptidase-enriched fraction showed a reproducible 95,000 proteolytic band. The initial extracts showed a similar 95,000 proteolytic band, and also 2 other 90,000 and 85,000 major bands. During reinvasion experiments, it was possible to recover a 95,000 and a 40,000 protease band from supernates of cultures grown in a semidefined medium without serum. Hydrophilic peptide derivatives related to the substrate of Pf 68 endopeptidase are shown to be potential inhibitors of the Pf reinvasion process in vitro.

Animals↗