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Biomedical subjects

M Monsigny

Publications and source records attributed to M Monsigny.

At least 127 records · Page 7Linked to original sources

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↗

Visualization of exocellular lectins in the entomopathogenic fungus Conidiobolus obscurus.

We demonstrated the presence of lectins binding to glucose and N-acetylglucosamine on the surface of Conidiobolus obscurus spores by using glycosylated serum albumins substituted with fluorescent dyes and colloidal gold. The role of these exocellular lectins was examined in relation to the adhesion of the fungal spores to their host insect and the pathogenicity of the fungus.

Acetylglucosamine↗

[Localized tracheobronchial amyloidosis. A new case studied with x-ray computed tomographic and nuclear magnetic resonance. Review of the literature].

We report a new case of localized primary tracheobronchial amyloidosis revealed by haemoptysis and effort dyspnea. The CT scan image was a homogeneous and circumferential thickening of the bronchial wall measuring 282 on the Hounsfield scale. At magnetic resonance imaging the amyloid substance was a slightly less intense than fat tissue in T1 and T2-weighted sequences. Treatment using Yag laser photoresection stabilized the lesions with one year follow-up. The review of the literature enables us to summarize the clinical, endoscopic, morphological, prognostic and therapeutic features of tracheobronchial amyloidosis, as well as pathogenic hypotheses and relationship with tracheobronchopathia osteoplastica.

Adult↗

Lectin-like components in the macronuclear replication bands of Euplotes eurystomus.

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar-binding components (i.e., lectin-like molecules) are associated with presumed nucleoli and with the macronuclear replication bands. This is the first demonstration that DNA synthesis and sugar-binding components are co-localized in an eukaryotic cell.

Animals↗

The N-acetylglucosamine-specific receptor of the thyroid. Binding characteristics, partial characterization, and potential role.

The binding characteristics of the GlcNAc binding protein present in thyroid membranes (Consiglio, E., Shifrin, S., Yavin, Z., Ambesi-Impiombato, F.S., Rall, J.E., Salvatore, G., and Kohn, L.D. (1981) J. Biol. Chem. 256, 10592-10599) were reinvestigated using neoglycoproteins as probes. Plasma membrane preparations from porcine thyroid specifically bound 125I-GlcNAc35-bovine serum albumin. Binding was dependent on the presence of calcium. Binding of ligand to receptor was minimal at neutral pH and maximal at pH 5.0. Equilibrium binding studies indicated positive cooperativity of binding and a site capacity of about 60 pmol/mg of protein. Competition studies were compatible with a specificity hierarchy of GlcNAc much greater than Gal; no recognition of mannose, fucose, or glucose moieties was noted. The receptor was detergent-solubilized from plasma membrane preparations and on the basis of the defined binding properties, purified by chromatography on a GlcNAc-Sepharose affinity column. The purified GlcNAc thyroid receptor has a subunit molecular size of about 45 kDa and appears to be an oligomer composed of three subunits. The receptor was identified as a component of thyrocytes by in situ cytochemical localization with fluorescent neoglycoproteins. In certain cases it was mainly present on, or near, the apical cell surface. It is suggested that this GlcNAc receptor functions in thyroglobulin metabolism, possibly involved in recycling of internalized thyroglobulin molecules back into the follicular lumen.

Acetylglucosamine↗

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↗

Detection of sugar-binding sites in the fibrillar and the granular components of the nucleolus: an experimental study in cultured mammalian cells.

The intranucleolar distribution of sugar-binding sites (i.e., lectin-like molecules) was analyzed in segregated nucleoli of actinomycin D-treated HeLa cells. The detection of sugar-binding sites was performed by incubation either of permeabilized nuclei in the presence of fluorescein-labeled neoglycoproteins or of ultrathin sections cut through in situ-fixed nuclei in the presence of gold-labeled neoglycoproteins. In the former case, the fluorescent nucleolar components were identified by comparison with the nucleolar components of similarly treated cells observed in electron microscopy. For the first time, this study reveals the presence of sugar-binding sites in both the fibrillar and the granular components of the nucleolus. In view of the data already reported on the biochemical composition of the nucleolus, some of our results led us to conclude that the nucleolar sugar-binding sites are lectin-like proteins. These proteins could be associated with preribosomes since the nucleolus is the site of both synthesis and stockage of ribosomal precursors. Some results from this study, however, show that the possibility of a relationship between some lectins and a structural component cannot be excluded.

Binding Sites↗

Plasmodium berghei and Plasmodium chabaudi: a neutral endopeptidase in parasite extracts and plasma of infected animals.

By using a sensitive fluorometric method with Val-Leu-Gly-Arg-3-amino-9-ethylcarbazole (VLGR-AEC) as a substrate, two endopeptidase activities were identified in two fractions of Sephacryl S-200 gel filtration from soluble P. berghei and P. chabaudi extracts. Controls with normal mouse erythrocytes, with leukocytes, and with reticulocyte enriched blood and different washing procedures during the preparation of soluble P. berghei extracts showed that the MW greater than 200 kDa fraction was a contaminant from erythrocytes and exhibited an optimal pH activity of 8.2. In contrast, the fraction 130 kDa was related to P. berghei and P. chabaudi and exhibited an optimal pH activity of 7.4. The two enzyme activities were compared with eight different substrates. The parasite endopeptidase showed a strong activity with Val-Leu-Gly-Lys-AEC (VLGK-AEC) and Ser-Gly-Lys-AEC (SGK-AEC) as substrates; in contrast, the mouse host endopeptidase poorly cleaved the VLGK-AEC and did not cleave SGK-AEC. Presence of the hydrophobic benzyl group on serine reduced the hydrolizing properties of P. berghei endopeptidase: the reverse was observed with host endopeptidase. The hydrolysis of the N-polyhydroxyalcanoyl-VLGK-AEC substrate by the parasite neutral endopeptidase strongly increased with the schizogonic stage, as shown with synchronized P. chabaudi in mice. By its physiological pH and specificity the release of this enzyme in mouse plasma during the infection could be of interest in a peptidyl-drug strategy.

Animals↗

Amino-sugars inhibit the in vitro cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells.

Twenty-two sugars and related compounds, nine neoglycoproteins, dopamine, four polyamines and oligomers of glucosamine were examined for their effect on the cytoadherence of Plasmodium falciparum-infected erythrocytes to melanoma cells. Inhibition of cytoadherence was high in the presence of the amino-sugars, glucosamine, galactosamine and mannosamine, and dopamine, and significant, although lower, in the presence of the polyamines, spermine, spermidine and putrescine. N-acetylated amino-sugars and the other compounds were not significant inhibitors of cytoadherence.

Amino Sugars↗

Differential changes of nuclear-envelope-associated enzyme activities involved in nucleocytoplasmic mRNA transport in the developing rat brain and liver.

Nucleocytoplasmic transport of rat liver mRNA is thought to be regulated by a nucleoside triphosphatase whose activity in the intact nuclear envelope is stimulated by the 3'poly(A) tail of poly(A)+ mRNA. In contrast to the liver mRNA, the mRNA from rat brain contains a great population of poly(A)- mRNA's that does not appear until after birth. Measurements of the nuclear-envelope-associated enzyme activities involved in mRNA transport, and their dependence on endogenous (isolated cytoplasmic mRNA-transport-stimulating proteins) and exogenous (poly(A), lectins, and neoglycoproteins) factors during prenatal and postnatal rat brain and liver development, revealed marked organ-dependent differences paralleling the appearance of the poly(A)- mRNA unique in the brain.

Animals↗

Interactions between apo-(D-beta-hydroxybutyrate dehydrogenase) and phospholipids studied by intrinsic and extrinsic fluorescence.

Interactions of D-beta-hydroxybutyrate dehydrogenase with phospholipids were investigated by both intrinsic- and extrinsic-fluorescence approaches. The intrinsic fluorescence, mainly caused by tryptophan residues, increased upon re-activation in the presence of phospholipids bearing a positive charge, i.e. phosphatidylcholine, but decreased in the presence of non-re-activating phospholipids with a negative charge. This indicates either that the environment of tryptophan residues is affected by charges rather than by hydrophobic chains of phospholipids, or that the enzyme undergoes different conformational changes depending on the nature of the phospholipids. On the other hand, the graph of the temperature-dependence of the fluorescence intensities of the enzyme embedded in dimyristoylphosphatidylcholine liposomes exhibits a break around 21 degrees C. This indicates either that at least one tryptophan residue is closely in contact with the hydrophobic chains of phospholipids or that there is a change in the environment of tryptophan residues owing to the physical state of the phospholipids. The addition of D-beta-hydroxybutyrate apo-dehydrogenase to phospholipid liposomes containing diphenylhexatriene (a fluorescent probe) increased the diphenylhexatriene fluorescence polarization. Moreover, there was a partial fluorescence energy transfer from tryptophan to diphenylhexatriene. These results strongly favour the possibility that there is a portion of the enzyme polypeptide chain inserted into the phospholipid hydrophobic region. All these results demonstrate that D-beta-hydroxybutyrate apo-dehydrogenase interacts with both polar and hydrophobic parts of phospholipids and leads to small, but essential, conformational changes of the enzyme.

Apoenzymes↗

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↗

Surface Plasmodium sugar-binding components evidenced by fluorescent neoglycoproteins.

A sugar-binding component was shown to be present at the surface of endoerythrocytic Plasmodium berghei and P. chabaudi stages and merozoites by means of a panel of neoglycoproteins using fluorescence microscopy and flow cytofluorometry. The protein nature of this material was ascertained by the loss of sugar-binding capacity upon trypsin treatment. This lectin-like protein primarily bound neoglycoprotein-borne N-acetylglucosamine and secondarily bound neoglycoprotein-borne alpha-D-glucose, beta-D-galactose and alpha-L-fucose. These results suggest the presence of at least one type of lectin-like protein with an extended binding site accomodating several sugar units, and define its localization on the parasite surface.

Animals↗

Identification of two macrophage populations by flow cytometry monitoring of oxidative burst and phagocytic functions.

Two populations of phagocytic cells from trehalose dimycolate-elicited mouse peritoneal cells are demonstrated by flow cytofluorometry, using two fluorescent probes excited at the same wavelength (488 nm). Liposomes containing diethylenetriaminepentaacetate daunorubicin conjugate (maximum emission wavelength: 590 nm) allow the discrimination of phagocytes and non-phagocytic cells. Among the phagocytes, an activated population is revealed by a cell-associated fluorescence of the oxidation product of dichlorofluorescein diacetate (maximum emission wavelength: 520 nm).

Animals↗

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↗

[Tumor angiogenesis].

Tumor angiogenesis is a very important process for growth and proliferation of most solid tumors. It insures the delivery of feeding molecules as well as the elimination of degradation products and allows tumoral cells to escape from the primary tumor site and the further establishment of metastases. Tumor neovascularisation is the result of a cascade of events primarily related to the properties of endothelial cells of capillaries. The main steps are: a) degradation of capillary basal lamina and destruction of the surrounding tissues, b) endothelial cell proliferation and c) endothelial cell migration towards the tumor site. A number of substances either synthetic or of natural origin are known to stimulate one of the above mentioned steps and/or to induce the production of factors which, in turn, stimulate one or several steps of the cascade. Such molecules can also be synthesized by tumoral cells; indeed they have often been evidenced in the fluids surrounding the tumor site. Many factors remain to be identified and their mechanism of action wait to be elucidated. However, it is already clear that several molecules are involved in the various steps of tumor angiogenesis. They display a coordinated sequential action and their synthesis is induced and controlled by the tumor itself. Amongst others, the following molecules play a role in tumor angiogenesis: degradative enzymes, E-prostaglandins, specific oligopeptides, fibronectin and heparin. Furthermore, several metal cations are also involved in tumor angiogenesis.

Animals↗

Involvement of membrane sugar receptors and membrane glycoconjugates in the adhesion of 3LL cell subpopulations to cultured pulmonary cells.

Lewis lung carcinoma cells are able to bind sugar residues, mainly alpha-D-glucosyl and alpha-D-mannosyl derivatives as assessed by fluorescent neoglycoproteins binding assay. We have investigated the binding efficiency and shown that: 3LL tumor cells are heterogeneous with regards to their capability to recognize neoglycoproteins, as shown by fluorescence microscopy and flow cytofluorometry analyses; basically two distinct subpopulations could be evidenced which were called glucose-receptor-rich (or glucose-specific lectin-rich, GLR 3LL) and glucose-receptor-poor (or glucose-specific lectin-poor, GLP 3LL) cells; those two subpopulations could be separated on the basis of their binding properties to neoglycoprotein-substituted microcarriers onto which GLR 3LL cells were able to rapidly adhere (2 h) while GLP 3LL cells were not. Some aspects of the biological behavior of these two selected populations were investigated in order to determine the possible involvement of 3LL cell membrane sugar receptors in cell-cell recognition and adhesion to other cells: namely C57 B1/6 mouse pulmonary cells maintained in primary culture. The two 3LL sublines bind to pulmonary cells but their adhesion kinetics were markedly different. Adhesion inhibition studies showed the adhesion process to be dependent upon the specificity of membrane lectins present on both the tumor cell surface (alpha-D-glucose-specific) and on the pulmonary cells (alpha-L-fucose-specific). Surface sugar-specific receptors on mouse pulmonary cells were shown to bind beta-D-galactose-, alpha-L-fucose and alpha-L-rhamnose substituted serum albumin. A neoglycoprotein bearing alpha-L-rhamnose residues was an efficient binder under the conditions of cell adhesion experiments and a potent cell adhesion inhibitor. A fucose-containing neoglycoprotein was shown to have a high inhibitory activity when used concomitantly to alpha-D-glucose-containing neoglycoproteins. Adhesion inhibition experiments, performed with cells the sugar specific receptors of which have been selectively inactivated, showed that the alpha-L-fucose specific receptors on pulmonary cell surface are partly responsible for the specificity of this cell-cell recognition process.

Animals↗