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Biomedical subjects

P Roussel

Publications and source records attributed to P Roussel.

At least 91 records · Page 5Linked to original sources
PubMed1991

Structure of two sulphated oligosaccharides from respiratory mucins of a patient suffering from cystic fibrosis. A fast-atom-bombardment m.s. and 1H-n.m.r. spectroscopic study.

Human respiratory-mucin glycopeptides were isolated from the sputum of a patient suffering from cystic fibrosis. They were subjected to treatment with alkaline borohydride. Application of ion-exchange chromatography afforded carbohydrate fractions containing sulphate. One of these fractions containing a mixture of sulphated oligosaccharides was subsequently submitted to gel-filtration chromatography and h.p.l.c. Two sulphated oligosaccharide-alditols, A and B, were prepared; their structure was determined by means of 400 MHz 1H-n.m.r. spectroscopy and fast-atom-bombardment m.s. They had a core type 2 and the sulphate was 3-linked to a terminal galactose residue: (Formula; see text)

Carbohydrate Conformation↗
PubMed1991

Multiple apomucin translation products from human respiratory mucosa mRNA.

Poly(A)-rich RNA was purified from a pool of five human tracheobronchial mucosa. After in vitro translation in a reticulocyte lysate and immunoprecipitation of the translated products, using either a polyclonal antiserum or a monoclonal antibody to deglycosylated respiratory mucin peptides, the products were characterized by SDS/PAGE. The respiratory mucin precursors migrated as a very large smear from almost the top of the resolving polyacrylamide gel to an area corresponding to a molecular mass of about 100 kDa. After hybridization with mucin cDNA probe TH 29 described by Crepin et al. [Crepin, M., Porchet, N., Aubert, J. P. & Degand, P. (1990) Biorheology 27, 471-484] respiratory mucin mRNAs also appeared polydisperse. Although degradation or incomplete translation of high-molecular-mass mRNA cannot be entirely ruled out, these results suggest that human respiratory apomucins consist of a family of peptides which share some common epitopes. This possibility is in agreement with (a) the diversity of mucin precursors observed previously with pulse/chase experiments performed with explants of human respiratory mucosa and (b) the polydispersity of secreted respiratory mucins observed by electron microscopy.

Animals↗
PubMed1991

Structures of neutral oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Le(a+b-)) patient suffering from chronic bronchitis.

Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitis patient with blood group O, Le(a+b-). Oligosaccharides were released by alkaline-borohydride treatment and purified by anion-exchange chromatography, size exclusion chromatography, and HPLC on a silica-bonded alkylamine column. Structural studies employed 500-MHz 1H-NMR spectroscopy, fast-atom-bombardment mass spectrometry and methylation analysis. Twenty-four neutral oligosaccharides, ranging in size from disaccharides to hexasaccharides, were fully characterized in this study. None of the structures contained an alpha(1----2)-linked fucose residue, in keeping with the non-secretor status of the patient. Seven of the structures had fucose present in alpha(1----3)-linkage in the X-determinant, while only one oligosaccharide (compound 14b) was seen with fucose alpha(1----4)-linked in the Le(a) determinant. The following eight structures isolated from the mucins of the non-secretor patient had not been found previously in the mucins of secretor individuals: [formula: see text] This study confirms that the blood group status of an individual is reflected in the carbohydrate structures of the secreted mucins. Furthermore, it clearly illustrates the need for detailed carbohydrate structural studies of mucins from different individuals before any attempt can be made to correlate observed differences in oligosaccharide profiles to disease status.

ABO Blood-Group System↗
PubMed1991

Arylneuraminidase activity of Pseudomonas aeruginosa does not degrade natural substrates such as human respiratory mucins.

The culture supernatant from a single Pseudomonas aeruginosa strain has been reported to show neuraminidase activity, leading to the speculation that this bacterium may use this enzyme as a virulence factor to act on host macromolecules. In order to extend this finding, we have examined the activity of concentrated P. aeruginosa culture supernatants and cells on synthetic and natural substrates containing sialic acid, such as human respiratory mucins. Four P. aeruginosa strains showed some activity on the synthetic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid but failed to liberate N-acetylneuraminic acid from six different natural substrates. Attempts to induce enzyme production by use of human respiratory mucins in the culture medium were also unsuccessful. The supernatants also showed N-acetyl-beta-D-glucosaminidase-like activity on a synthetic substrate but did not liberate N-acetylhexosamines from natural substrates. We conclude that the neuraminidase-like activity observed in P. aeruginosa can be defined as an arylneuraminidase and that the possession of a neuraminidase active on natural substrates is not a common attribute of P. aeruginosa strains.

Extracellular Space↗
PubMed1991

Pseudomonas aeruginosa recognizes carbohydrate chains containing type 1 (Gal beta 1-3GlcNAc) or type 2 (Gal beta 1-4GlcNAc) disaccharide units.

The adhesion of Pseudomonas aeruginosa to type 1 (Gal beta 1-3GlcNAc) and type 2 (Gal beta 1-4GlcNAc) disaccharide determinants was studied in a microtiter adhesion assay and a thin-layer chromatography bacterial overlay assay. The oligosaccharides were prepared from human breast milk and human urine and were conjugated to hexadecylaniline to form neoglycolipids that were used in were used in the assays. Both the mucoid and the nonmucoid strains that were studied recognized the disaccharide determinants Sialylation of the oligosaccharides did not suppress binding in the thin-layer chromatography assay, but alpha 2-6-linked sialic acid blocked binding in the microtiter assay. The use of bovine serum albumin instead of gelatin as a blocking agent against nonspecific binding completely suppressed binding in the thin-layer chromatography assay. Isogenic nonpiliated mutants of nonmucoid strains constructed by interrupting the pilin gene retained their adhesive capacity for the disaccharide units, indicating that binding to the disaccharides was mediated by a nonpilus adhesin(s). Furthermore, two monoclonal antibodies that recognize the type 2 disaccharide determinant (Gal beta 1-4GlcNAc) partially inhibited adhesion of a pair of piliated and nonpiliated isogenic strains to mucin. This study suggests that P. aeruginosa utilizes a nonpilus adhesin(s) to bind to disaccharide units commonly found in mucins, in addition to pili and alginate, two previously described adhesins.

Amino Sugars↗
PubMed1991

The carbohydrate diversity of human respiratory mucins: a protection of the underlying mucosa?

Human respiratory mucins consist of a family of glycoproteins with different peptides in which glycosylation, the major post-translational phenomenon, is responsible for about 70 to 80% of the weight of these molecules. This glycosylation generates a remarkable diversity of O-glycosidically linked carbohydrate chains, which are expressed as several hundreds of different chains in a single person. These chains, which can vary from one to about 20 sugars, may be neutral, sialylated, or sulfated. They bear multiple epitopes. Some antigenic determinants such as ABO, Leb antigens in secretor individuals, Lea, or X or Y antigens have been identified. There is increasing evidence that, among other functions, this diversity of chains allows many interactions with microorganisms and may be an important factor in maintaining the sterility of the respiratory tree. In certain pathologic situations such as cystic fibrosis, which is associated with colonization by Pseudomonas aeruginosa, the hypothesis of an alteration of this interaction is open.

Carbohydrate Sequence↗
PubMed1991

Sulfated O-glycoproteins secreted by guinea pig trachea in organ culture.

Organ culture of guinea pig trachea was performed in the presence of [35S]sulfate in order to characterize the sulfated glycoproteins released from the respiratory epithelium and mucosa. The sulfated macromolecules that were synthesized during a 6-h incorporation were separated by CsBr density-gradient centrifugation and gel-filtration chromatography successively. Most of the sulfated secreted macromolecules corresponded to a population of glycoproteins sensitive to reductive beta-elimination but resistant to both chondroitinase ABC and heparinase. These glycoproteins had different buoyant densities (ranging from 1.48 g/ml to 1.16 g/ml) and could be subfractionated according to molecular mass. A major part of the radioactivity was incorporated into high-molecular-mass mucins that were excluded from a Sepharose CL-2B column and did not penetrate into polyacrylamide gel in PAGE. However, a mixture of sulfated O-glycoproteins of much lower molecular mass was also characterized in addition to low amounts of chondroitin sulfate. Epithelial goblet cells are the predominant mucin-containing cells of the respiratory guinea pig trachea. Our results suggest that a wide range of sulfated O-glycoproteins are secreted by the guinea pig tracheal mucosa.

Animals↗
PubMed1991

Chondroitin sulfate in sputum from patients with cystic fibrosis and chronic bronchitis.

In order to ascertain whether or not the presence of glycosaminoglycans in sputa of patients suffering from chronic bronchial disorders was related to tracheobronchial infection, an electrophoretic procedure was set up. The different acidic macromolecular components of sputum, namely nucleic acids, glycosaminoglycans, and bronchial glycopeptides could be identified in proteolyzed sputum using agarose electrophoresis before and after the action of different enzymes: nucleases, chondroitinases, hyaluronidase and heparinase. This procedure was used to analyze 13 sputum samples from patients suffering from cystic fibrosis (CF) and 12 sputum samples from patients suffering from chronic bronchitis. Chondroitin sulfate was identified in 11 infected sputum samples from patients with CF and also in the noninfected sputum from a patient with chronic bronchitis. These data suggest a relationship between the presence of chondroitin sulfate proteoglycans in sputum and severe tracheobronchial infection in CF.

Adolescent↗
PubMed1991

An attempt to evaluate lung aggression in monkey silicosis: hydrolases, peroxidase and antiproteases activities in serial bronchoalveolar lavages.

Exposure to silica can induce fibrosis and/or emphysema. Various factors such as proteases, other hydrolases and oxidants may be involved in the destruction of lung parenchyma. On the other hand, antiproteases play an important role in the protection of lung parenchyma against the action of proteases. We have developed an animal model of silicosis in monkey Macacus cynomolgus and followed these factors by bronchoalveolar lavage (BAL). We have studied glycosidases activities, elastase-like activity, immunoreactive alpha 1-protease inhibitor (alpha 1PI), neutrophil elastase inhibitory capacity (NEIC) and myeloperoxidase. Bronchoalveolar cells in serial BAL were also studied. Six monkeys were exposed to quartz aerosols (100 mg.m-3) for 18 wks. They were followed until they developed X-ray changes, which occurred between 21-64 wks after the end of the dust exposure. Cellular "silicotic nodules" were observed in lung biopsies. A control animal underwent serial BAL. Changes were seen in the differential cell count. The release of superoxide anion by bronchoalveolar cells obtained during the experiment was increased. Separation on a gradient of Percoll showed the presence of young macrophages, which exhibited enhanced release of superoxide anion as compared to the totality of bronchoalveolar cells. The biochemical analysis of BAL fluids obtained during and after the period of dust exposure showed an increase in glycosidases, alpha 1PI and NEIC. Some free elastase-like activity was simultaneously detected in BAL fluids from exposed animals but not from the control. This elastase-like activity was very low compared to NEIC. The increase in enzymatic and antiprotease activities occurred at different points in time for each animal, suggesting large differences in individual responses to dust, but occurred before the chest X-ray abnormalities.

Acetylglucosaminidase↗
PubMed1989

Evidence for the in vivo degradation of human respiratory mucins during Pseudomonas aeruginosa infection.

The comparison of distribution of glycopeptides of sputa from patients suffering from various chronic hypersecretions has already shown an increased acidity with a decreased proportion of neutral glycopeptides in the respiratory secretions of patients suffering from cystic fibrosis, as compared to those of patients with chronic bronchitis. In order to find out whether this decrease is specific to cystic fibrosis mucins or whether it is due to a degradation of mucus by Pseudomonas aeruginosa, which infects most of the sputa from patients with this disease, mucus glycopeptides from patients with different chronic bronchial disorders, infected by Pseudomonas or not, were prepared and fractionated by ion-exchange chromatography. The neutral fraction, which has never been studied in detail, was gel-filtered, and provided two fractions, one containing true mucin glycopeptides and the other containing a mixture of peptides and glycopeptides with a lower molecular mass. In the Pseudomonas-infected samples, the true mucin glycopeptide fraction was greatly diminished as compared to this same fraction in non-Pseudomonas-infected samples; this was not specific to cystic fibrosis secretions. In contrast, the glycopeptide fraction with a lower molecular mass was greatly increased in all the Pseudomonas-infected samples. Polyacrylamide gel electrophoresis of this second fraction showed unique glycopeptide bands between 40-50 kDa in the Pseudomonas-infected samples, regardless of the origin of the samples. These bands were revealed by an antibody directed against whole cystic fibrosis mucin. Infected chronic bronchitis sputa and cystic fibrosis samples without P. aeruginosa did not show these bands. These studies therefore suggest that there are P. aeruginosa-associated changes in mucins which may result from degradation of mucins.

Amino Acids↗
PubMed1989

Identification of plasma proteins contained in bronchoalveolar fluid from Macacus cynomolgus.

1. Serial bronchoalveolar lavages (BAL) were performed on a subhuman primate (Macacus cynomolgus) in order to give an experimental model for silicosis. 2. We have identified and quantified some plasma proteins of monkey BAL fluid. 3. The results were compared to those previously obtained in humans. 4. Two proteins previously detected in human BAL fluid (alpha 1 acid glycoprotein, alpha 1 antichymotrypsin) were not detected in monkey control BAL fluid. 5. Two kinds of transferrins were detected in monkey BAL fluids while only one is described in human. 6. The present results will now permit sequential follow up studies during the course of experimental silicosis.

Animals↗
PubMed1989

Differences in adhesion of Pseudomonas aeruginosa to mucin glycopeptides from sputa of patients with cystic fibrosis and chronic bronchitis.

Pseudomonas aeruginosa is the most prominent colonizer of the respiratory tract of patients with cystic fibrosis, but it is not known why this occurs. P. aeruginosa adheres to mucins from normal individuals, but mucins from cystic fibrosis patients have not been studied. To compare adhesion to mucins from cystic fibrosis with other mucins, we prepared highly glycosylated mucin glycopeptides from cystic fibrosis and chronic bronchitis patients by ion-exchange and gel-filtration chromatography and measured the adhesion of P. aeruginosa 1244 to these glycopeptides. We found (i) that the most mucinlike glycopeptides from P. aeruginosa-infected cystic fibrosis sputa showed less bacterial adhesion than did the corresponding bronchitis samples, (ii) that the most adhesive activity in cystic fibrosis samples came from a fraction that contains O and N glycopeptides and may be in part a degradation product of P. aeruginosa infection, and (iii) that highly glycosylated glycopeptides of the most acidic species (sialylated and sulfated) showed no adhesion at all. A single cystic fibrosis sample not infected by P. aeruginosa showed better binding in the adhesion-positive fractions than did the infected sputa. These studies suggest that cystic fibrosis mucins may be altered after infection is established, resulting in less binding to some fragments. However, since the clinical picture shows heavy mucus colonization, other receptors, such as cellular glycolipids which have been shed into mucus, may be contributing to this colonization.

Bacterial Adhesion↗