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P Midoux

Publications and source records attributed to P Midoux.

At least 37 records · Page 2Linked to original sources
PubMed1995

Membrane permeabilization by alpha-helical peptides: a flow cytometry study.

The permeabilization by alpha-helical peptides of nucleated mammalian cells can be monitored by flow cytometry. Ethidium bromide, a non fluorescent and poorly membrane permeant molecule, becomes strongly fluorescent only upon binding to DNA. On this basis, the permeabilization of the plasma membrane of HL60 promyelocytic cells induced by alpha-helical peptides such as melittin, succinylated melittin and anionic peptides derived from the N-terminus of HA2 subunit of the influenza virus hemagglutinin, was measured. Melittin (GIGAVLKVLTTGLPALISWIKRKRQQ-NH2) caused a rapid (< 5 min) and dose-dependent (ED50 = 0.5 microM) permeabilization of HL60 cells at neutral pH, whereas the succinylated derivative induced cell permeabilization only at pH below 4.5 with an ED50 = 18 microM. The permeabilization by the anionic E5CA peptide (GLFEAIAEFIEGGWEGLIEGCA) containing 5 glutamic residues occurred (ED50 = 11 microM) at pH ranging from 6.5 to 6.0; replacing the tryptophan residue in position 14 by a phenylalanine residue decreased by about 1 unit the pH at which membrane permeabilization was effective. The membrane permeabilization activity of the E5CA peptide was reversibly abolished when the peptide was linked to a protein carrier. These results show that alpha-helical peptide-induced membrane permeabilization can be easily monitored by using flow cytometry in the presence of a non permeant dye. This method allows a rapid screening and an efficient mean of selection of peptides suitable to induce membrane permeabilization.

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

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↗
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↗
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↗
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↗
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

Characterization and biological implications of membrane lectins in tumor, lymphoid and myeloid cells.

Complex carbohydrates and sugar receptors at the surface of eukaryotic cells are involved in recognition phenomena. Membrane lectins have been characterized, using biochemical, biological and cytological methods. Their biological activities have been assessed using labeled glycoproteins or neoglycoproteins. Specific glycoproteins or neoglycoproteins have been used to inhibit their binding capacity in both in vitro and in vivo experiments. In adults, lymphoid and myeloid cells as well as tumor cells grow in a given organ and eventually migrate and home in another organ; these phenomena are known as the homing process or metastasis, respectively. In specific cases, membrane lectins of endothelial cells recognize cell surface glycoconjugates of lymphocytes or tumor cells, while membrane lectins of lymphocytes and of tumor cells recognize glycoconjugates of extracellular matrices or of non-migrating cells. Therefore, membrane lectins are involved in cell-cell recognition phenomena. Membrane lectins are also involved in endocytosis and intracellular traffic of glycoconjugates. This property has been demonstrated not only in hepatocytes, fibroblasts, macrophages and histiocytes but also in tumor cells, monocytes, thyrocytes, etc. Upon endocytosis, membrane lectins are present in endosomes, whose luminal pH rapidly decreases. In cells such as tumor cells or macrophages, endosomes fuse with lysosomes; it is therefore possible to target cytotoxic drugs or activators, by binding them to specific glycoconjugates or neoglycoproteins through a linkage specifically hydrolyzed by lysosomal enzymes. In cells such as monocytes, the delivery of glycoconjugates to lysosomes is not active; in this case, it would be preferable to use an acid-labile linkage. Cell surface membrane lectins are developmentally regulated; they are present at given stages of differentiation and of malignant transformation. Cell surface membrane lectins usually bind glycoconjugates at neutral pH but not in acidic medium: their ligand is released in acidic specialized organelles; the internalized ligand may be then delivered into lysosomes, while the membrane lectin is recycled. Some membrane lectins, however, do bind their ligand in relatively acidic medium as in the case of thyrocytes. The presence of cell surface membrane lectins which recognize specific sugar moieties opens the way to interesting applications: for instance, isolation of cell subpopulations such as human suppressor T cells, targeting of anti-tumor or anti-viral drugs, targeting of immunomodulators or biological response modifiers.

Adjuvants, Immunologic↗
PubMed1987

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry.

The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled alpha-glucosylated serum albumin to 3LL cells at 4 degrees C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37 degrees C in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4 degrees C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4 degrees C, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.

Animals↗
PubMed1986

Analysis of sugar-binding sites in mammalian cell nuclei by quantitative flow microfluorometry.

Quantitative flow microfluorometry of neoglycoprotein (bovine serum albumin coupled to sugar and to fluorescein) binding demonstrated the existence of sugar-binding sites (i.e., lectin-like molecules) in isolated BHK cell nuclei. The very similar labeling intensities obtained with nuclei isolated by cell lysis and with permeabilized karyoplasts obtained by enucleation strengthened the idea that the binding sites are borne by actual nuclear structures and not by cytoplasmic or membrane-derived contaminants. With both nuclei-isolation procedures, neoglycoproteins (containing similar numbers of sugar residues) used as markers can be similarly classified. Fluorescence microscopy further indicated that in both nuclear preparations, the neoglycoprotein binding sites were associated with the nucleoli as well as with nucleoplasmic ribonucleoprotein elements. Nuclei from exponentially growing cells bound much greater amounts of neoglycoprotein than did nuclei from contact-inhibited cells.

Animals↗
PubMed1986

Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules.

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.

Animals↗
PubMed1985

Membrane lectins on human monocytes. Maturation-dependent modulation of 6-phosphomannose and mannose receptors.

Freshly isolated human monocytes, which do not contain cell-surface mannose-specific receptors, bind mannose 6-phosphate and actively endocytose mannose 6-phosphate-bearing neoglycoproteins (6-P-Man-F-BSA). Three days after isolation, human monocytes endocytose very actively 6-P-Man-F-BSA as well as Man-F-BSA, and the endocytosed neoglycoproteins are rapidly degraded. These results were obtained in quantitative flow cytofluorometry by using a panel of fluoresceinylated sugar-substituted serum albumins (neoglycoproteins). Thus, in contrast to mannose receptors which appear only after maturation, mannose 6-phosphate receptors are already present on freshly isolated human monocytes.

Carrier Proteins↗
PubMed1984

Fluorescence quenching of tryptophan by trifluoroacetamide.

Trifluoroacetamide was found to be a good quencher of tryptophan fluorescence, and the quenching was shown to proceed via both a dynamic and a static process. The respective quenching constants were determined by the measurement of the decrease of the fluorescence lifetime in the presence of the quencher. The static and the bimolecular rate quenching constants of N-acetyltryptophanamide are equal to 0.34 1 X mol-1 and 1.9 X 10(9) 1 X mol-1 X s-1, respectively. These values indicate that trifluoroacetamide is an efficient quencher of tryptophan fluorescence. This conclusion is also supported by a complete quenching of bovine serum albumin and wheat germ agglutinin fluorescence. In the case of lysozyme, trifluoroacetamide quenches the fluorescence of tryptophan residues which fluoresce with a maximum at 348 nm but not the buried tryptophan residues which fluoresce with a maximum at 333 nm. Trifluoroacetamide quenching of wheat germ agglutinin emission confirms the homogeneity and the high accessibility of emitting tryptophan residues, in agreement with a previous report (Privat, J.P. and Monsigny, M. (1975) Eur. J. Biochem. 60, 555-567). The tryptophan fluorescence decay of wheat germ agglutinin is biexponential even in the presence of the quencher; the static and bimolecular rate quenching constants are equal to 0.22 1 X mol-1 and 0.92 X 10(9) 1 X mol-1 X s-1, respectively. In the presence of a specific lectin ligand, the methyldi-N,N'-trifluoroacetyl-beta-chitobioside, the quenching of wheat germ agglutinin fluorescence involves a direct contact between tryptophan residues and trifluoroacetamido groups of the ligand and in contrast with the quenching induced by free trifluoroacetamide shows that the tryptophan fluorescence is not fully quenched.

Acetamides↗