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Parietal endometriosis is a rare disease. Its diagnosis and treatment are often difficult. We report 3 cases of parietal endometriosis occurring in cesarean and appendectomy scars. Clinical symptoms are not specific and may lead to erroneous diagnosis. Diagnosis is usually made on the histological exam of the resected lesion. Treatment of choice is complete surgical excision.

High-molecular-mass glycoconjugates are secreted by the continuous cell line MM-39, which has been obtained from cultured human tracheal gland cells transformed by simian virus 40. They were purified on Sepharose(R) CL-4B and then by two steps of density-gradient centrifugation. High-molecular-mass glycoproteins resistant to digestion by hyaluronidase, chondroitin ABC lyase and heparitinase were obtained, in addition to hyaluronic acid and proteoglycans. They were susceptible to beta-elimination. They contained polylactosaminoglycan chains as well as carbohydrate chains with a terminal sialic acid in the NeuAc alpha2-3 sequence. Most of them have a buoyant density of 1.45 g/ml in CsCl-density-gradient centrifugation, except for MUC1. The MM-39 cells were also characterized by a high expression of MUC1 and MUC4 genes, but they did not express MUC2, MUC3, MUC5B and MUC5AC. Therefore the MM-39 cells synthesized mucin-like glycoproteins as well as lysozyme and mucous proteinase inhibitor [Merten, Kammouni, Renaud, Birg, Mattéi and Figarella (1996) Am. J. Respir. Cell. Mol. Biol. 15, 520-528]; they should be considered as having a mixed, both serous and mucous, phenotype.

Sialic acids are a family of 9-carbon carboxylated sugars, where different substitutions of the backbone define over 30 members. Biological roles of these substitutions have been missed until recently because of their low abundance and lability to conventional isolation/purification methods. This new approach characterizes sialic acids using electrospray ionization-mass spectrometry (ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene) derivatives (quinoxalinones). A combination of retention times and spectra characteristics allows definition of the type and position of the various substituents. This approach requires no previous purification, involving a simple derivatization reaction followed by direct injection on the microbore HPLC column. A complete spectrum, including molecular ions and CAD fragments of a sialic acid quinoxalinone, is obtained by injecting 10-20 pmol of the compound. Individual quinoxalinones can be purified by regular RP-HPLC and analyzed by direct-injection ESI-MS or LSIMS. Using this approach, we identified 28 different sialic acids, including the following new species: Neu5Gc9Lt (BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro (horse serum hydrolyzates), KDN5(7)Ac and KDN5(7),9Ac2 (amphibian Pleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydro derivatives of Neu5Gc8Me (glycolipids of the starfish Pisaster brevispinus), and Neu5Ac8S (in addition to Neu5Gc8S, in the glycolipids of the sea urchin Lovenia cordiformis). Results show the usefulness of LC-ESI-MS to study sialic acid diversity, and identification of small amounts of unexpected sialic acids or new members of their family.

Mucin glycopeptides were prepared from the respiratory mucus of a non-secretor, chronic bronchitic 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 high performance anion-exchange chromatography. Structural studies employed 400-MHz 1H-NMR spectroscopy and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Nine monosulfated oligosaccharides ranging in size from tetra- to hexasaccharide, were fully characterized in this study. The sulfate group occurs either on the C-3 of a terminal galactose residue or on the C-6 of a N-acetylglucosamine residue. In keeping with the non-secretor status of the patient, no structure with an (alpha 1-2)-linked fucose residue was found. Five of the structures had fucose present in (alpha 1-3)-linkage in the X determinant, while only one oligosaccharide (compound 7b) was seen with fucose (alpha 1-4)-linked in the Le(a) determinant. Eight structures isolated from the mucins of the non-secretor patient had not been found previously in the respiratory mucins; they are listed below.

Pseudomonas aeruginosa binds to different glycoconjugates in vitro. As six other bacteria, it binds to several glycolipids, mainly asialo GM1 and asialo GM2. Asialo GM1 has been reported to exist at the surface of cystic fibrosis cells. The binding of P. aeruginosa to asialo GM1 involves the pili, especially the C-terminal part of pilin that recognizes the GaINAc(beta 1,4) Gal sequence of asialo GM1.P. aeruginosa may also bind to sialylated membrane-bound glycoproteins. Human salivary and respiratory mucins are also recognized by P. aeruginosa. Mucins represent the main components of mucus. The peptide part (apomucin) of this broad family of secreted glycoproteins is encoded by several mucin genes. The apomucins are covered by a large number of carbohydrate chains that can be remarkably different and represent a mosaic of sites for attachment of microorganisms. The binding of P. aeruginosa to mucins involves outer membrane proteins and mucin carbohydrate chains that are structurally different from the carbohydrate recognized by pillin. Airway and salivary mucins secreted by patients suffering from cystic fibrosis (CF) show alterations in their carbohydrate moiety. The increased sulfation of airway mucins seems to correspond to a primary defect. Other abnormalities such as increased sialylation or fucosylation have also been detected. The binding of P. aeruginosa to airway or salivary mucins is increased in CF. However, the precise link between the carbohydrate alterations and the increased binding of P. aeruginosa to CF mucins remains to be elucidated.

The authors report a case of Campylobacter fetus subsp. fetus gastro-intestinal infection and bacteremia with poly-arthritis, mainly of the hip, in a French patient simultaneously suffering from cirrhosis of the liver. The outcome was eventually favorable, however only after a trial of ineffective pefloxacin-gentamicin therapy. The authors suggest: (i) gentamicin should not be given alone in C. fetus subsp. fetus infections, and (ii) pefloxacin should not be given if antibiotic sensitivities data are not available. The inconclusive reliability of disk diffusion tests for C. fetus subsp. fetus should be recognized.

Human bronchial surface epithelial cells were maintained in secondary culture on a collagen gel substrate in a defined, serum-free medium. These conditions have previously been reported to promote mucous cell differentiation. After 3 wk in culture, approximately 40% of the cells were stained by an antibody directed against human respiratory mucin. Analysis of media from cells cultured in the presence of the radioactive precursors [3H]glucosamine and [35S]sulfate revealed that the cells secreted high molecular weight glycoproteins with properties of typical respiratory mucins. In addition, hyaluronic acid and proteoglycans containing chondroitin sulfate and/or heparan sulfate glycosaminoglycans were identified in cell conditioned media. Finally, Western blot analyses showed that the cells secreted lysozyme and mucous proteinase inhibitor, proteins that are generally considered to be markers for submucosal gland serous cells. These results show that human bronchial cells from the surface epithelium in secondary culture secreted a range of glycoconjugates and proteins that were typical secretory products of both mucous and serous cells.

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 high-performance liquid chromatography on a silica-bonded alkylamine column. Structural studies employed 500-MHz 1H-NMR spectroscopy and fast atom bombardment-mass spectrometry. Twenty-six monosialyl oligosaccharides, ranging in size from di- to octasaccharide, were fully characterized in this study. The sialic acid occurs either alpha(2-->3)- or alpha(2-->6)-linked to a galactosyl residue, or alpha(2-->6)-linked to GalNAc-ol. In keeping with the non-secretor status of the patient, no structures with an alpha(-->2)-linked fucose residue were found. Nine of the structures had fucose present in alpha(-->3) linkage, either in the Le(x) determinant or in the sialyl-Le(x) determinant, or both (structure 11). Only one oligosaccharide (structure 3b) was seen with fucose alpha (1-->4)-linked in the Le(a) determinant. Eight structures isolated from the mucins of the non-secretor patient had not been found previously in the respiratory mucins of secretor individuals; they are listed below. Among these, structure 4 alpha represents a novel type of mucin carbohydrate core structure.

We compared the chemical composition of salivary mucin glycopeptides from cystic fibrosis (CF) and from non-CF subjects and the adhesion of Pseudomonas aeruginosa to these different salivary glycopeptides. Three pools of CF saliva, four pools of non-CF saliva, one individual CF saliva, and one individual non-CF saliva were studied. The soluble fraction of the saliva was treated with pronase, and gel filtration was performed to obtain high and low molecular mass salivary mucin glycopeptides. The yield of total glycopeptides was significantly higher from CF than from non-CF saliva. Furthermore, the chemical composition revealed a significantly higher sialic acid content in CF than in non-CF mucin glycopeptides, and higher sulfate and fucose content in CF than in non-CF high molecular mass glycopeptides. We studied the adhesion of a nonmucoid strain of P. aeruginosa (1244), its nonpiliated isogenic derivative, and a mucoid strain (M35) to salivary mucin glycopeptides from patients with CF and from non-CF subjects. The three strains bound significantly more to the CF salivary glycopeptides than to the corresponding non-CF salivary glycopeptides. The nonpiliated isogenic mutant of P. aeruginosa 1244 also bound to CF salivary glycopeptides, suggesting that the adhesion of P. aeruginosa could involve nonpilus adhesions. Furthermore, neuraminidase treatment of CF glycopeptides decreased the adhesion of P. aeruginosa 1244. Altogether these results suggested that differences in mucins may in part explain the specificity of P. aeruginosa for CF.

The carbohydrate chains of the respiratory-mucus glycoproteins of a patient (blood group O) suffering from bronchiectasis due to Kartagener's syndrome, were released by alkaline borohydride treatment of a pronase digest. The structures of 82 neutral and low-molecular-mass sialylated oligosaccharides have been described previously [van Kuik A., de Waard P., Vliegenthart J. F. G., Klein A., Carnoy C., Lamblin G. Roussel P. (1991) Eur. J. Biochem. 198, 169-182]. In the present work, medium-size sialylated oligosaccharides were obtained after ion-exchange chromatography and were subsequently separated into 36 fractions utilizing gel filtration, HPLC on normal-phase alkylamine-bonded silica and reverse-phase HPLC. From these fractions, the following six sialylated hepta- and octa-saccharide-alditols have been characterized by employing 500-MHz 1H-NMR spectroscopy, in conjunction with fast-atom-bombardment mass spectroscopy and methylation analysis. [formula: see text]

Mucin glycopeptides were prepared from the salivary mucins of 20 healthy donors with blood group O. The carbohydrate chains of the high-molecular-weight mucins were released by alkaline borohydride treatment. Neutral and monosialylated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and HPLC. The structures of the oligosaccharide-alditols were determined by high-resolution 1H-NMR spectroscopy in combination with fast atom bombardment mass spectrometry and methylation analysis. Thirty-seven oligosaccharide-alditols were characterized and illustrate the extreme diversity of the salivary mucins carbohydrate chains. This diversity might represent a mosaic of bacterial adhesion sites and be involved in the early events of the nonimmune defense of the oral cavity. Among these 37 oligosaccharide-alditols, 31 have not been previously described in human saliva and five of these are novel structures: [formula: see text]

Multiple homeobox genes are expressed in haematopoietic cell lineages and their expression is cell-type specific. Thus we hypothesized that certain homeobox genes may play an important role in the process of haematopoiesis. To prove that issue, normal murine bone marrow cells were stimulated with appropriate Colony Stimulating Factors in the presence of mouse homeobox gene (Hox 2.3) sense or antisense oligodeoxynucleotides and the effects on the haematopoietic colony formation were examined. Treatment of the cells to Hox 2.3 antisense oligodeoxynucleotides led to a selective inhibition of myeloid colony formation, both in size and in numbers, but without significant effect on erythroid and megakaryocytic haematopoiesis. Exposure to Hox 2.3 sense oligodeoxynucleotides (no-oligomers), had no such effect. It was further showed that inhibition of myelopoiesis by Hox 2.3 antisense oligodeoxynucleotides was dependent on the differentiation stage of target cells. These findings demonstrated that Hox 2.3 gene plays a critical role in regulating normal murine myelopoiesis.

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.

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.

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.

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.

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.

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effect of purified recombinant human GM-CSF (rhGM-CSF) on induction of TGF-alpha in macrophages. Using Northern blot analysis and immunoassays, we show here that rhGM-CSF markedly stimulates production of TGF-alpha messenger RNA and protein in normal tonsil macrophages. The findings are consistent with macrophages being a normal inducible source of TGF-alpha which may be an important mediator of various activities of GM-CSF both in hematopoietic and non-hematopoietic cells.

The authors report 2 cases of hepatic lymphangioma observed in a 54-year-old woman, and in a 39-year-old man. These tumors were discovered upon ultrasound examination performed for jaundice due to viral hepatitis, and for abdominal right upper quadrant pain respectively. Computed tomography and angiography showed hypervascularized tumors. Diagnosis was established through surgical biopsy specimens. The clinical course was uneventful, during respectively the 9- and 3-year follow-up periods, respectively. From these and the 40 previously reported cases, the authors describe the different features of this unusual tumor.

In order to evaluate the effect of measles on proteins carrying vitamin A, we have examined 58 children with measles (24 have been seen again 2 weeks later) and 52 healthy controls of similar age and nutritional status. During the course of fever we have observed a high urinary excretion of urea and creatinine with proteinuria while the hydroxyproline index is significantly lower than in the controls. Irrespective of the nutritional status the plasma levels of albumin, prealbumin and R.B.P. are consistantly low. Two weeks later, while the albumin level has decreased, the other parameters are aiming towards normal values. The higher levels of prealbumin and R.B.P. suggest a reactivation of the hepatic protein synthesis. The urinary excretion of R.B.P. has not changed significantly during measles. We have observed rather high urinary losses of R.B.P. in the controls. The low levels of plasma prealbumin possibly do not allow the complete binding of the R.B.P.

From bronchial mucins of cystic fibrosis patients with blood group O, the carbohydrate chains were released in the form of oligosaccharide-alditols by alkaline borohydride treatment. Application of high-performance liquid chromatography directly on the pool of neutral oligosaccharides afforded 23 fractions. Twenty oligosaccharide structures were characterized by employing 500-MHz 1H-NMR spectroscopy in conjunction with sugar analysis. Thirteen among these had been revealed before to occur in human bronchial mucins [Van Halbeek, H., Dorland, L., Vliegenthart, J. F. G., Hull, W. E., Lamblin, G., Lhermitte, M., Boersma, A., and Roussel, P. (1982) Eur. J. Biochem. 127, 7-20], when paper-chromatographic fractionation of this pool of neutral oligosaccharides was employed. High-performance liquid chromatography enabled to obtain another seven pentasaccharide and hexasaccharide alditols; the largest-size representatives are: (Formula see text). Thereby, this approach afforded deeper insight into the structural heterogeneity displayed by the carbohydrate chains of bronchial mucins.

The structure of carbohydrate units of bronchial-mucus glycoproteins obtained from cystic fibrosis patients was investigated by 500-MHz 1H-NMR spectroscopy and methylation analysis. To that purpose, the mucin was subjected to alkaline borohydride degradation. Neutral oligosaccharide-alditols, ranging in size from disaccharides to pentasaccharides, were isolated. Eight compounds could be purified to homogeneity; furthermore, three fractions were obtained consisting mainly of two components. For all 14 compounds the primary structure could be elucidated. 500-MHz 1H-NMR spectroscopy was found to be effective in detecting heterogeneity and to be invaluable for the determination of structures in mixtures of oligosaccharide-alditols. The structures can be divided into two groups depending on the core disaccharide. One group contains Gal(beta 1 leads to 3)GalNAc-ol as common structural element, the other GlcNAc(beta 1 leads to 3)GalNAc-ol. Both disaccharides were identified as such; the other compounds can be conceived as extensions thereof. The most complex representatives of the two groups are: (formula; see text) The italicized structural elements, comprising the SSEA-1 determinant and the type-1 blood-group-H determinant, are novel sequences in oligosaccharide chains of mucin-type glycoproteins.

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.

Human bronchial mucus glycoproteins or mucins were isolated from the sputum of two patients by a method avoiding reducing agents and involving water extraction and gel filtration on Sepharose CL-2B in 6 M guanidinium chloride. The chemical analysis indicated approximately 25-40% lipid. The amino acid and carbohydrate analysis differ quantitatively from that of mucins purified after prior reduction of mucus. These fractions also have a higher proportion of aspartic and glutamic acids than that of the mucins from reduced sputum. These mucins are still contaminated by small amounts of peptides but do not seem to contain disulfide-attached cross-linking protein. Human bronchial mucins have a strong tendency to form aggregates except in 6 M guanidinium chloride. Electron microscopy performed with various procedures indicates the presence of both micelles and flexible threads measuring 200-1000 nm. Delipidation removes most of the micellar forms. Thereafter mucins appear mainly as polydisperse flexible extended threads and also as aggregates. These features of bronchial mucins do not fit with the generally accepted idea of mucin subunits linked by disulfide bridges (unless they are linked end to end) and alternatively favour a model where mucin molecules behave like filaments that could easily aggregate according to the solvent system (mucin concentration, absence of dissociating conditions).

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.

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.

Gel=like or fibrillar human bronchial mucus was reduced with mercaptoethanol under dissociating and non-dissociating conditions. Bronchial mucus was solubilized in both conditions; but reduction in phosphate buffer induced more extensive depolymerization of mucus glycoproteins than obtained under reduction in guanidine. Moreover, mucins prepared by reduction in non-dissociating conditions contained less amino acid and more carbohydrate. These data strongly suggest that reduction under non-dissociating conditions does not act only on disulfide bridges but also induces the activation of a mucolytic enzymatic system. They also would explain some of the discrepancies observed in the molecular weight determination and chemical composition of human bronchial mucus glycoproteins purified after reduction of gel-like mucus under different conditions.

The carbohydrate chains of the human, parotid-saliva, proline-rich glycoprotein were released as oligosaccharides by hydrazinolysis and were fractionated by high-pressure-liquid chromatography. Four oligosaccharides were characterized. On the basis of compositional analysis, sequential enzymic degradation, and methylation data, the carbohydrate moiety of the glycoprotein was found to be a complex-type oligosaccharide containing a tri-D-mannosyl-di-N-acetylchitobiose core with two and three lactosamine branches, and an L-fucosyl group linked to O-6 of the asparagine-linked 2-acetamido-2-deoxy-D-glucose residue. In addition, several L-fucosyl groups, linked at O-6 of the penultimate D-galactose residues, are present in variable proportions. The biological significance of these results is discussed.

In order to elucidate the biological role of the sugar residue of the antitumor drug bleomycin, this was deglycosylated by beta-elimination under mild alkaline conditions, and by solvolysis with hydrogen fluoride. The latter procedure proved to be better because it led to the complete deglycosylation without modification of the peptide, thus allowing further biological investigations of this component.

Most of the mucins isolated from the sputum of patients suffering from cystic fibrosis are acidic. Acidic mucins from a child suffering from cystic fibrosis were degraded by alkali treatment. Analysis of the degradation products demonstrated the wide heterogeneity of the carbohydrate chains linked to the mucin polypeptide moiety. However, this heterogeneity of carbohydrate chains is probably not restricted to mucins from patients with cystic fibrosis. Bronchial mucins were prepared either from the sputum of different patients belonging to blood group O or B and suffering from cystic fibrosis, chronic bronchitis and other chronic bronchial diseases, or from bronchial washings performed in macroscopically healthy area of the bronchial tree of subjects belonging to blood group O. The chemical composition of each mucin fraction was established and an average carbohydrate chain length was estimated. Acidic mucins isolated from the sputum of two children suffering from cystic fibrosis were more sulfated than sialylated and their average carbohydrate chain length was relatively large. These characters were not specific for cystic fibrosis since they were also found in acidic mucins of two children suffering from other bronchial diseases. Most of the acidic mucins isolated from the sputum of three adults suffering from chronic bronchitis were more sialylated than sulfated and had a relatively short average carbohydrate chain length. The sputum of these patients also contained a variable proportion of neural or weakly acidic mucins. The mucins isolated from bronchial washings performed in macroscopically healthy areas of the bronchial tree were acidic molecules whose acidic characteristics and average carbohydrate chain length were about the same as for acidic mucins from patients with chronic bronchitis.

Acids mucins were isolated from a mixture of bronchial washings carried out on 5 subjects, in macroscopically healthy areas of the bronchial tree. Neutral mucins were absent.

Glycoproteins were isolated in the contents of two sinus mucoceles by ionic exchange and gel filtration chromatography. These glycoproteins are of the mucin-type and characterized by their richness in carbohydrate, a low amino acid content with a strong proportion of hydroxy amino acids. However, they differ largely by their peptide axis, the length of the carbohydrate chain and their acidity, which is in relation with the presence of sialic acid residue and of sulfate groups. The least acidic mucins are the richest in sialic acid residue and in threonine but have the shortest carbohydrate chains while the most acidic are rich in sulfate, richer in serine and have longer carbohydrate chains. The wall of these two mucoceles has only one type of cell capable of synthetizing the glycoproteins: the epithelium goblet cells revealed by the PAS and the alcian blue at different pH. Glandular formations have never been found in the chorion.

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.

Two lectin fractions (S20W = 6,8 and 4,9 S) were purified from Ricinus communis seeds. The purification was carried out in four steps : ammonium sulfate fractionation, affinity chromatography on Sepharose 4 B, gel filtration on Sephadex G 150 and chromatography on CM celluloes. The purified lectins were glycoproteins whose chemical composition was determined. Amino terminal analysis of the two fractions revealed glycine and serine. Polyacrylamide gel electrophoresis of the higher molecular weight fraction allowed the separation of several components with different affinity for PAS staining.

1. Colony-stimulating factor (CSF-1) was isolated from a large volume of fresh normal human urine by 5 steps of purification and enrichment. 2. The purification factor is 100,000 fold and the purified compound exhibits a 2.16 x 10(7) U/mg of protein sp. act. 3. The isolated CSF-1 is a sialoglycoprotein with 41.5% of carbohydrate. The almost complete removal of this carbohydrate moiety (up to 91%) was achieved by incubation with trifluoromethane sulfonic acid. 4. The deglycosylated CSF-1 (DG-CSF-1) possesses an apparent Mr 38,000 compared to native CSF-1 with an initial Mr 57,000 (Goa et al., 1988). 5. The features of the interaction of radio-iodinated [125I]CSF-1 with single cell suspensions from various human tissues (bone marrow, spleen, blood, peritoneal cavity, alveolar lavage, lymph node and thymus), were studied. 6. The binding activity of peritoneal macrophages was the highest among the cells examined and erythrocytes, thymus and blood granulocytes showed no CSF-1 binding. 7. On incubation with [125I]CSF-1 at 0 degrees C, cellular binding of [125I]CSF-1 reached a stable maximum within 16 hr. This is in contrast to the association behaviour at higher temperature. 8. At 37 degrees C, cellular associated [125I]CSF-1 levels reached, within 90 min, an unstable maximum which was up to 10 times less than that occurring under the same conditions at 0 degree C. From the Scatchard plot analysis, we obtained the affinity constant and the number of receptor(s). 9. The binding site is sensitive to trypsin. 10. The receptor alone, (labelled by cross-linking to [125I]CSF-1 with di-succinylimidyl-suberate), is a polypeptide with an approx. Mr 110,000. 11. Our results showed that the receptor of CSF-1 is a tyrosin-kinase.