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P Roussel

Publications and source records attributed to P Roussel.

At least 109 records · Page 6Linked to original sources
PubMed1989

Use of an antiserum against deglycosylated human mucins for cellular localization of their peptide precursors: antigenic similarities between bronchial and intestinal mucins.

Highly glycosylated regions of mucins, or glycopeptides, were obtained by proteolysis of human bronchial mucins. They were deglycosylated by treatment with a trifluoromethane sulfonic acid/anisole mixture and subsequent solvolysis with anhydrous liquid hydrogen fluoride. The resulting peptides were then used to raise an immune serum in rabbit. This immune serum was used to localize the peptide precursors of human respiratory mucins within bronchial cells, using an immunohistochemical method. Two main patterns of labeling were observed in the goblet cells: the entire cytoplasm of some goblet cells was immunoreactive, whereas in other cells the labeling was concentrated around the nucleus. In the respiratory mucous glands, the labeling was localized around or below the nucleus. The serous cells were not stained. Similar labeling was observed in human colon goblet cells. This immune serum seems to be specific for mucin-secreting cells and has a strong affinity for the perinuclear region of these cells.

Amino Acids↗
PubMed1989

Identification of human tracheo-bronchial mucin precursors.

Bronchial mucin peptide chains were obtained by performing a two-step chemical deglycosylation of the highly glycosylated regions (or glycopeptides) which are the most characteristic part of bronchial mucins. The deglycosylated preparation was used to prepare an antiserum directed against mucin peptide epitopes. This antiserum reacted with the area containing rough endoplasmic reticulum of goblet cells and of mucous gland of human bronchial mucosa but not with secretory vesicles. The antiserum was used for immunoprecipitation of radiolabelled mucin precursors in pulse-chase experiments with explants of human bronchial mucosa. SDS-polyacrylamide gel electrophoresis followed by fluorography revealed precursors with a molecular mass in the range of 200 to 400 kDa as early as after 10 min pulse labeling with [3H]threonine. These results suggest that the mucin polydispersity previously visualized by electron microscopy may be explained by the synthesis of several respiratory mucin peptide precursors with different molecular sizes and/or that precursors with different amounts of carbohydrate are rapidly formed.

Bronchi↗
PubMed1988

Isolation and structural characterization of low-molecular-mass monosialyl oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis.

The carbohydrate chains of the respiratory-mucus glycoproteins of a patient suffering from bronchiectasis due to Kartagener's syndrome were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by consecutive straight-phase high-performance liquid chromatography (HPLC) on a silica-based alkylamine column, and reverse-phase HPLC on a silica-based octadecyl column, respectively. The structures of the oligosaccharidealditols were determined by 500-MHz 1H-NMR spectroscopy in combination with sugar composition analysis. The 24 structures established range in size from disaccharides to heptasaccharides. Novel oligosaccharides obtained from the bronchiectasis mucus glycoproteins are: (formula; see text) 23 of the 24 monosialyl oligosaccharides characterized can be conceived of as extensions of neutral oligosaccharides purified from the bronchial mucus of this patient [Klein, A. et al. (1988) Eur. J. Biochem. 171, 631-642; Breg, J. et al. (1988) Eur. J Biochem. 171, 643-654]. The sialic acid residue was found to occur either in alpha (2----3)- or alpha (2----6)-linkage to a galactosyl residue or in alpha (2----6)-linkage to GalNAc-ol.

Amino Acids↗
PubMed1988

Primary structure of neutral oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500-MHz 1H-NMR spectroscopy and quantitative sugar analysis. 1. Structure of 16 oligosaccharides having the Gal beta(1----3)GalNAc-ol core (type 1) or the Gal beta(1----3)[GlcNAc beta(1----6)]GalNac-ol core (type 2).

Carbohydrate chains of respiratory-mucus glycopeptides from a patient (blood group O) suffering from bronchiectasis with a Kartagener's syndrome have been released by alkaline borohydride treatment. Application of high-performance liquid chromatography using subsequently two silica columns, one bonded with aminopropyl groups and the other with octadecyl groups, afforded 39 neutral fractions; 35 oligosaccharide-alditol structures have been characterized by employing 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis. Here, 16 oligosaccharide structures, possessing a core consisting of Gal beta(1----3)GalNAc-ol branching through a GlcNAc residue linked beta(1----6) to the GalNAc residue (core type 2 or core type 1, respectively), are described. Ten oligosaccharide-alditols with these types of cores (2, 3, 10a, 14, 7, 11a, 15a, 16a, 12 and 16c) have been identified previously in human bronchial mucins of patients suffering from cystic fibrosis [Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J.H.G.M., Van Halbeek, H. and Vliegenthart, J.F.G. (1984) Eur. J. Biochem. 143, 227-236]. Of the remaining six compounds, one is a partial structure of oligosaccharides previously described: (Formula: see text). The structures 17a and 20 contain the Y determinant, i.e., Fuc alpha(1----2)Gal beta(1----4)[Fuc alpha(1----3)]GlcNAc beta(1----). High-resolution 1H-NMR spectroscopy is able to distinguish whether the Y determinant is beta(1----3) or beta(1----6) linked in such oligosaccharide-alditols.

Bronchiectasis↗
PubMed1988

Primary structure of neutral oligosaccharides derived from respiratory-mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500-MHz 1H-NMR spectroscopy and quantitative sugar analysis. 2. Structure of 19 oligosaccharides having the GlcNAc beta(1----3)GalNAc-ol core (type 3) or the GlcNAc beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol core (type 4).

A pool of neutral carbohydrate chains was prepared from respiratory mucins of a patient suffering from bronchiectasis. Fractionation by HPLC led to 35 smaller-size oligosaccharide-alditols; the structure of 16 oligosaccharide-alditols with core type 1 or type 2 has been established (Klein, A., Lamblin, G., Lhermitte, M., Roussel, P., Breg, J., Van Halbeek, H. & Vliegenthart, J.F.G., preceding paper in this journal). In this second part, we identified 19 oligosaccharide-alditols possessing core types 3 and 4. Nine of the structures (1, 5, 9, 6, 10b, 13, 19, 15b and 18.1) have been described previously to be present in cystic fibrosis mucins [Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J.G.H. M., Van Halbeek, H. & Vliegenthart, J.F.G. (1984) Eur. J. Biochem. 143, 227-234]. The remaining ten are new structures isolated from bronchial mucins; they are all extensions of the above-mentioned nine oligosaccharides. These compounds are octassaccharide-alditols containing the Y determinant together with the H determinant of either backbone-type 1 or 2, and partial structures thereof: (Formula: see text) and 21b, which is 23c without Fuc alpha(1----3), 18.2, which is 23c without any Fuc in the 6-branch, and 22b, which is 23c without Fuc in the 3-branch.

Bronchiectasis↗
PubMed1988

Pipetting errors in viral titrations: a useful approach.

A simple recursive formula, which yields an estimate for the statistical error resulting from pipetting errors accumulated throughout a dilution procedure, is described. Its influence on the variance of the end-point titre estimate is then inferred.

Biometry↗
PubMed1988

The complexity of mucins.

Mucins represent the main components of gel-like secretions, or mucus, secreted by mucosae or some exocrine glands. These high-molecular-weight glycoproteins are characterized by the large number of carbohydrate chains O-glycosidically linked to the peptide. The determination of mucin molecular weight and conformation has been controversial for several reasons: 1) the methods used to solubilize mucus and to purify mucins are different and 2) the molecules have a strong tendency to aggregate or to bind to other molecules (peptides or lipids). Recently, electron microscopy has shown the filamentous shape of most mucins and their polydisperse character which, in some secretions, might correspond to a polymorphism of the peptide part of these molecules. The recent development of high pressure liquid chromatography and high-resolution proton NMR spectroscopy has allowed major progress in the structural study of mucin carbohydrate chains. These chains may have from 1 to about 20 sugars and bear different antigenic determinants, such as A, B, H, I, i, X, Y or Cad antigens. In some mucins, such as human respiratory mucins, the carbohydrate chain diversity is remarkable, which raises many questions. Mucins are molecules located at the interface between mucosae and the external environment. The carbohydrate chain diversity might allow many interactions between mucins and microorganisms and play a major role in the colonization or the defense of mucosae.

Animals↗
PubMed1988

The binding of anti-pseudomonal antibiotics to macromolecules from cystic fibrosis sputum.

Antibiotics are known to bind to whole cystic fibrosis sputum. However, the composition of sputum varies from one patient to another, making the interpretation of binding studies difficult. This problem has been examined by standardising the macromolecule concentration of sputum from four cystic fibrosis patients and adding tobramycin or ceftazidime directly to the sputum components. Binding to mucin-rich and DNA-rich fractions of sputum was also studied before and after DNase treatment of these fractions. These studies indicated that (i) the degree of tobramycin binding is dependent on the sputum macromolecule concentration, (ii) a significant proportion of tobramycin is bound even at concentrations of 100 mg/l of drug, (iii) tobramycin binds to both the mucin rich fraction and the DNA rich fraction of sputum and (iv) ceftazidime binding to sputum is negligible. Our data indicate that there is a need to standardise sputum in antibiotic binding studies and they provide another rationale for favouring the use of ceftazidime over aminoglycosides in infectious exacerbations of cystic fibrosis caused by Pseudomonas aeruginosa.

Ceftazidime↗
PubMed1987

Evaluation by immunoblotting of the criteria for tubular lesions diagnostic with urinary SDS-PAGE.

Immunoblotting methods have allowed the identification of 14 urinary proteins. From this study, it appears that some low-relative-molecular mass proteins are derived from disrupted high-relative-molecular mass proteins. The only reliable bands for the diagnosis of tubular lesions, on the polyacrylamide gel stained with Coomassie Blue, are retinol-binding-protein and beta 2-microglobulin. alpha 1-microglobulin can overlap with breakdown products of albumin. The visualization of Ig light chains and lysozyme is rather poor after Coomassie Blue stainings and the accurate identification of these bands may be only improved by the use of immunoblotting.

Collodion↗
PubMed1987

Peptides of human bronchial mucus glycoproteins. Size determination by electron microscopy and by biosynthetic experiments.

Secreted human bronchial mucins, directly collected from macroscopically healthy bronchial mucosa, were prepared in the presence of six proteinase inhibitors, and analysed by electron microscopy. These mucins were similar in length distribution to molecules prepared from sputum [Slayter, Lamblin, Le Treut, Galabert, Houdret, Degand & Roussel (1984) Eur. J. Biochem. 142, 209-218], although they were a little longer, their lengths ranging up to about 1,650 nm. This length corresponds to an extended mucin peptide of about 450 kDa. In order to compare these peptide lengths with the molecular size of biosynthetic precursors, an antiserum raised against trifluoromethanesulphonic acid-treated highly glycosylated regions of human bronchial mucins was used to isolate mucin precursors synthesized in explants of human bronchial mucosa during pulse-labelling with [3H]threonine or [3H]glucosamine. A main precursor labelled with [3H]threonine and with an apparent molecular mass of about 400 kDa was detected by fluorography following SDS/polyacrylamide-gel electrophoresis. This band was observed as early as 20 min; it was more intense after a 40 min chase and had disappeared after a chase period of 280 min in unlabelled medium, presumably owing to glycosylation. Much fainter bands at about 200 kDa and between 200 and 400 kDa, also labelled with [3H]threonine, were observed mainly after a 40 min chase and had disappeared after a 280 min chase. None of these bands was labelled with [3H]glucosamine, nor did they disappear after multiple treatments with immobilized lectins. After a 280 min chase, [3H]threonine-labelled material appeared in the stacking gel, which also contained [3H]glucosamine label. The results indicate that the 200-400 kDa species are mucin precursors, whose size is comparable with that obtained by electron microscopy for respiratory mucins collected directly from the macroscopically healthy bronchial mucosa.

Bronchi↗
PubMed1987

Structure of sialyl-oligosaccharides isolated from bronchial mucus glycoproteins of patients (blood group O) suffering from cystic fibrosis.

The carbohydrate chains of the bronchial-mucus glycoproteins of six cystic fibrosis patients with blood group O were released by alkaline borohydride treatment. Low-molecular-mass, monosialyl oligosaccharide-alditols were isolated by anion-exchange chromatography and fractionated by high-performance liquid chromatography. Structural characterization was performed by 500-MHz 1H-NMR spectroscopy in combination with quantitative sugar analysis. The established structures range in size from tetra- up to heptasaccharides. They are all sialyl analogs of neutral oligosaccharides that were characterized previously [Lamblin G., Boersma A., Lhermitte M., Roussel P., Mutsaers J. H. G. M., Van Halbeek H. & Vliegenthart J. F. G. (1984) Eur. J. Biochem. 143, 227-236]. The NeuAc residue was found to occur either in alpha (2----3)-linkage to Gal, or in alpha (2----6)-linkage to GalNAc-ol or Gal.

ABO Blood-Group System↗
PubMed1987

Comparison of hydrolases, peroxidase and protease inhibitors in bronchoalveolar fluid from Macacus cynomolgus and human controls.

1. Serial bronchoalveolar lavages were performed on a subhuman primate (Macacus cynomolgus) in order to give an experimental model for silicosis. 2. We have measured glycosidases, proteases, peroxidase and antiproteases of the BAL fluids from seven normal monkeys. 3. The results obtained were similar to those found in human control BAL fluids. 4. For monkeys, the repetition of the bronchoalveolar procedure does not seem to have an important influence on the values obtained. 5. The present results will now permit sequential follow up studies during the course of experimental silicosis.

Animals↗
PubMed1986

Action of trifluoromethanesulfonic acid on highly glycosylated regions of human bronchial mucins.

Highly glycosylated glycopeptides were prepared from human bronchial mucus. They were heterogeneous and contained an average of 45 residues of glycosylated hydroxyamino acid per 100 amino acid residues. The kinetics of deglycosylation of these glycopeptides by trifluoromethanesulfonic acid-anisole mixtures at 25 degrees was monitored by chemical analysis and by polyacrylamide gel electrophoresis. The peripheral sugars were almost completely cleaved in 45 min with 3:2 and 2:1 CF3SO3H-anisole. A maximum of 75% of the O-linked N-acetylgalactosamine residues were released and mixtures of glycopeptides and peptides were obtained. Increasing the reaction time caused peptide bond cleavage. Rather mild conditions (1.2:1 CF3SO3H-anisole at 25 degrees for 90 min) gave limited deglycosylation of highly glycosylated bronchial glycopeptides, allowing the uncovering of GalNAc-peptide linkages and peptide regions able to induce the formation of specific antibodies in the rabbit.

Amino Acids↗