Hypofertility with thick cervical mucus: another mild form of cystic fibrosis?
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Adult↗
Biomedical subjects
P Roussel
Publications and source records attributed to P Roussel.
Congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis transmembrane regulator (CFTR): correlation between genotype and phenotype.
To assess better the link between congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis (CF), we compared sweat chloride values, analysis of the CFTR intron 8 poly(T) tract length and analysis of 10 exons in a population of 38 patients with CBAVD. The data indicate that this population can be divided into three groups of patients. In the first group of 15 patients with abnormal sweat chloride (> 60 mmol/l), the frequency of CF mutations is high. In the second group of 18 patients with equivocal sweat chloride (between 40 and 60 mmol/l), the frequency of the 5T variant is high; 6 patients have a delta F508 mutation and a 5T variant and 1 patient is homozygous for the 5T variant; a 5T variant has been detected in 3 other patients, and a delta F508 mutation in another patient. A third group of 5 patients is probably not related to CF: these patients have other congenital abnormalities of the urogenital tract, low chloride values (< 40 mmol/l) and apparently no abnormality of the CF gene.
Adult↗
Adenosine and neuropathic pain.
Recent studies have reported the possibilities of relieving neuropathic pain by administering adenosine or its analogs. In order to determine if there exists a metabolic anomaly of this nucleoside in patients with neuropathic pain, circulating adenosine levels were compared in three patient groups. The first was composed of individuals suffering from neuropathic pain, the second of patients with nervous system lesions in the absence of pain, and the third was composed of patients suffering from pain resulting from excessive nociception. The adenosine blood levels of these patients were compared to those of a control group. Finally, adenosine in the cerebrospinal fluid (CSF) of some patients was also assayed. The results show that there are reduced levels of blood and CSF adenosine in patients with neuropathic pain. This adenosine deficiency could explain the potential therapeutic effects of administering adenosine or its analogs.
Adenosine↗
The rDNA transcription machinery is assembled during mitosis in active NORs and absent in inactive NORs.
In cycling cells, the rDNAs are expressed from telophase to the end of G2 phase. The early resumption of rDNA transcription at telophase raises the question of the fate of the rDNA transcription machinery during mitosis. At the beginning of mitosis, rDNA transcription is arrested, and the rDNAs are clustered in specific chromosomal sites, the nucleolar organizer regions (NOR). In human cells, we demonstrate that the rDNA transcription machinery, as defined in vitro, is colocalized in some NORs and absent from others whatever the mitotic phase: RNA polymerase I and the RNA polymerase I transcription factors, upstream binding factor and promoter selectivity factor (as verified for TATA-binding protein and TATA-binding protein-associated factor for RNA polymerase I [110]), were colocalized in the same NORs. The RNA polymerase I complex was localized using two different antibodies recognizing the two largest subunits or only the third largest subunit, respectively. These two antibodies immunoprecipitated the RNA polymerase I complex in interphase cells as well as in mitotic cells. These results clearly indicated that the RNA polymerase I complex remained assembled during mitosis. In addition, RNA polymerase I and the transcription factors varied in the same proportions in the positive NORs, suggesting stoichiometric association of these components. The fact that the rDNA transcription machinery is not equally distributed among NORs most likely reflects the implication of the different NORs during the subsequent interphase. Indeed, we demonstrate that only positive NORs exhibit transcription activity at telophase and that the level of transcription activity is related to the amount of rDNA transcription machinery present in the NOR. We propose that assembly of rDNA transcription machinery preceding mitosis determines expression of the rDNAs at the beginning of the next cell cycle. Consequently, the association of rDNAs with the rDNA transcription machinery defines the "active" NORs and the level of activity at the transition telophase/interphase.
Cell Nucleolus↗
Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth.
The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis. Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization. In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P. aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth [TSB]). However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding. In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9. Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media. When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB. These results demonstrate that the adhesion of P. aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P. aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.
Bacterial Adhesion↗
Interactions between glycoconjugates from human respiratory airways and Pseudomonas aeruginosa.
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.
Animals↗
Characterization of a sulfotransferase from human airways responsible for the 3-O-sulfation of terminal galactose in N-acetyllactosamine-containing mucin carbohydrate chains.
A galactose 3-O-sulfotransferase activity able to transfer a sulfate group from adenosine 3'-phosphate 5'-phosphosulfate to methyl galactosides or terminal N-acetyllactosamine-containing carbohydrate chains from human respiratory mucins was characterized in microsomal fractions prepared from human respiratory mucosa. The reaction products, methyl alpha- or beta-galactose 3-sulfate and three oligosaccharide alditols containing the sequence HSO3-3Gal beta 1-4GlcNAc beta 1-6GalNAc-itol were identified by high performance anion-exchange chromatography. Using methyl beta-galactoside as a substrate, the optimum activity was obtained with 0.1% Triton X-100, 30 mM NaF, 20 mM Mn2+, and 10 mM AMP in a 30 mM 2-(N-morpholino)ethanesulfonic acid buffer at pH 6.1. The apparent Km for methyl beta-galactoside and for adenosine 3'-phosphate 5'-phosphosulfate were observed at 0.69 x 10(-3) M and at 4 x 10(-6) M respectively. This sulfotransferase is different from that responsible for sulfatide synthesis.
Adenosine Monophosphate↗
Congenital bilateral absence of vas deferens in absence of cystic fibrosis.
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Abnormalities, Multiple↗
Quantitative changes of the two major AgNOR proteins, nucleolin and protein B23, related to stimulation of rDNA transcription.
The relationship between the amount of the two major AgNOR proteins, nucleolin and protein B23, and the rate of ribosomal RNA (rRNA) synthesis was studied in cortisol-treated and regenerating rat hepatocytes after partial hepatectomy. In both experimental models the synthesis of rRNA was greatly stimulated, but only in regenerating hepatocytes was the increased synthesis associated with cells entering the mitotic cycle. Nucleolin and protein B23 were identified on SDS-polyacrylamide gels of nuclear proteins transferred to nitrocellulose and detected (1) by immunoreaction with specific monoclonal antibodies revealed by second antibodies coupled to peroxidase followed by chemiluminescence or (2) by the silver-staining procedure for AgNOR proteins. Nucleolin and protein B23 were then quantified by computerized densitometric analysis of the immunolabeling signals or the silver-stained bands at 105 kDa (nucleolin) and 39 kDa (protein B23). The synthesis of rRNA was measured by evaluating the amount of radioactivity incorporated into pre-rRNA after [3H]orotic acid injection. Densitometric analysis of silver-stained bands and immunolabeling signals showed no change in nucleolin and protein B23 amounts in cortisol-stimulated hepatocytes, whereas a moderate increase was found in regenerating hepatocytes at 12 h after partial hepatectomy. In both cortisol-stimulated and regenerating hepatocytes the synthesis of rRNA was highly increased (2.6-fold and 4.3-fold above the control level, respectively). To ascertain the relationship between quantitative changes in nucleolin and protein B23 and stimulation of rRNA transcriptional activity in regenerating hepatocytes, the quantitative distribution of these proteins was also investigated in the early times of regeneration using silver-stained nitrocellulose-transblotted nuclear proteins. The quantity of protein B23 was unchanged until 12 h after partial hepatectomy, whereas nucleolin appeared to be slightly increased at 9 h (1.15-fold above the control value) after partial hepatectomy. On the other hand, at just 6 h after partial hepatectomy, a significant increase of rRNA synthesis occurred in regenerating rat hepatocytes (1.8-fold above the control value). These data demonstrated that stimulation of rRNA transcriptional activity occurring in rat hepatocytes after cortisol treatment and in the early times after partial hepatectomy was not associated with quantitative changes in the amounts of nucleolin and protein B23.
Animals↗
Interactions between human respiratory mucins and pathogens.
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Adhesins, Bacterial↗
A new monoclonal antibody (3D3) generated with human respiratory mucins and directed against Lewis determinants.
We have prepared a monoclonal antibody (MAb), 3D3, raised against purified human respiratory mucins. This antibody recognized mucins and proteolytically derived glycopeptides. The epitope recognized by the antibody was destroyed by alpha-L-fucosidase, indicating that it was present on the carbohydrate moieties. Structural specificity was determined by adsorption on a variety of synthetic, insolubilized oligosaccharides. Several lines of evidence indicate that the 3D3 MAb reacted strongly with the Lewis (Leb) antigen, but also recognized Le(a) and Le(y) determinants. This antibody might be useful to study mucin secretion.
Adsorption↗
Mucous and serous secretions of human bronchial epithelial cells in secondary culture.
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.
Bronchi↗
Amount variability of total and individual Ag-NOR proteins in cells stimulated to proliferate.
Ribosomal genes are associated with a subset of acidic proteins called Ag-NOR proteins. The amount of nucleolar Ag-NOR proteins varies, depending on nucleolar activity and/or cell proliferation. To understand the linkage between the amount of Ag-NOR proteins, ribosome biogenesis, and cell proliferation, we investigated the variability of Ag-NOR proteins in rRNA-stimulated cells maintained in G1 and in rRNA-stimulated cells entering the mitotic cycle. Rat hepatocytes were stimulated with cortisol for rRNA synthesis (1, 4, and 8 hr) and the cell cycle was induced by hepatectomy in regenerating hepatocytes (3-21 hr). In non-stimulated hepatocytes, nucleolin and protein B23 were the two major Ag-NOR proteins, corresponding to 70% of total Ag-NOR staining. In hepatocytes stimulated for rRNA synthesis in G1, the amount of Ag-NOR proteins was only slightly increased, whereas in cycle-stimulated cells it was increased 3.04-fold. This is the consequence of a differential increase of the major Ag-NOR proteins that appears earlier and is proportionally more important for nucleolin (3.5-fold) than for protein B23 (twofold) and also for the increase of several minor Ag-NOR proteins. We conclude that, in dividing cells, the mean value of the Ag-NOR proteins measured reflects the percentage of cells in the different phases. This could explain why the amount of Ag-NOR proteins can be used as a marker of cell proliferation.
Animals↗
Interaction of fimbriated and nonfimbriated strains of unencapsulated Haemophilus influenzae with human respiratory tract mucus in vitro.
Adherence to mucus may influence bacterial colonization of the respiratory tract. Clinical isolates of nontypable Haemophilus influenzae (NTHi) from the respiratory tract are often fimbriated. We wondered whether fimbriated strains have a different adherence from related nonfimbriated strains. A microtitre plate assay has been developed to study adherence of nontypable H. influenzae to mucus. Wells were coated by incubation either with sol phase of sterile mucoid secretions or with purified preparations of mucins. Two laboratory pairs of fimbriated (F+) and nonfimbriated (F-) nontypable H. influenzae, and six fresh clinical isolates of fimbriated nontypable H. influenzae each with nonfimbriated partners derived by serial passage on agar, were cultured to mid-log phase, washed, and then added to the wells. They were then incubated at 37 degrees C for 30 min before washing to remove unbound bacteria. Adherent bacteria were desorbed by agitation with 0.5% Tween 80 and a viable count performed. The two fimbriated laboratory strains (n = 12 and n = 17), and 5 of the 6 fimbriated clinical isolates were more adherent to sol phase than their respective nonfimbriated partners. Two nonfimbriated clinical isolates were more adherent to plastic than their fimbriated partners. A fimbriated laboratory strain was more adherent than its nonfimbriated partner both to a purified preparation of high molecular mass mucin and to the glycopeptide fraction of the same. We conclude that fimbriated strains of nontypable H. influenzae have increased adherence to sol phase of mucus and purified human respiratory tract mucin. The interactions of fimbriae with mucus are likely to be complex, and may involve both nonspecific and specific interactions.
Bacterial Adhesion↗
Sialylation and sulfation of the carbohydrate chains in respiratory mucins from a patient with cystic fibrosis.
Mucin glycopeptides were prepared from the sputum of a patient suffering from cystic fibrosis. The carbohydrate chains were released by alkaline borohydride treatment. Sialylated and sulfated oligosaccharide-alditols were purified by ion-exchange chromatography, gel filtration, and high performance anion-exchange chromatography. The structures of the oligosaccharide-alditols were determined by high resolution 1H NMR spectroscopy in combination with fast atom bombardment-mass spectrometry. Twenty-four oligosaccharides were characterized and illustrate the diversity of the carbohydrate chains of cystic fibrosis respiratory mucins. Among these 24 oligosaccharide-alditols, 15 are novel structures. Sialylation may occur on the C-6 of the N-acetylgalactosamine involved in the carbohydrate-peptide linkage or on a terminal galactose residue, either on C-3 or on C-6. Sulfation may occur either on the C-3 of a terminal galactose residue or on the C-6 of a N-acetylglucosamine residue. The most complex structures contain sulfated derivatives of the H,X, or Y determinants or a sialylated and sulfated derivative of the X determinant (Structure A).
Carbohydrate Sequence↗
Identification of Ag-NOR proteins, markers of proliferation related to ribosomal gene activity.
During mitosis, ribosomal genes are associated with a set of silver-stained nucleolar proteins designated Ag-nucleolar organizer region (NOR) proteins. The amount of Ag-NOR protein, estimated during interphase, may be used as marker of cell proliferation with a prognostic value for several human cancers. Our objective was to identify the Ag-NOR proteins in human transformed cell lines at specific phases of the cell cycle and in a hamster cell line that serves as model for active ribosomal transcription. During interphase, the major Ag-NOR proteins in both human and hamster cells were nucleolin and protein B23 and also proteins of 42, 40, and 29 kDa, which accounted for a small amount of the silver stain. The pIs of these proteins were between 4.5 and 5.6. During mitosis, the Ag-NOR proteins were either solubilized in the cytoplasm, distributed around the chromosomes, or associated with the ribosomal genes, i.e., in the NORs. The major Ag-NOR proteins associated with the ribosomal genes were the largest RNA polymerase I subunit, the 135-kDa NOR protein, the UBF transcription factor, and a 50-kDa protein. Less than 5% of the total nucleolin remained associated with ribosomal genes during mitosis. Using the purified RNA polymerase I complex from yeast, we demonstrate that the 190-, 43-, and 34.5-kDa subunits are Ag-NOR proteins in this species. This study demonstrates that the major Ag-NOR proteins in nucleoli during interphase are not the same as those associated with the ribosomal genes during mitosis. We conclude that the prognostic test for human cancer cell proliferation is largely based on the amount of the nucleolar proteins, nucleolin, and protein B23, which are not directly involved in ribosomal gene transcription. In contrast, the evaluation of active NORs in karyotypes during mitosis is based on the presence of some proteins of the ribosomal gene transcription machinery.
Antigens, Nuclear↗
Structures of monosialyl oligosaccharides isolated from the respiratory mucins of a non-secretor (O, Lea+b-) patient suffering from chronic bronchitis. Characterization of a novel type of mucin carbohydrate core structure.
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.
Bronchi↗
Pseudomonas aeruginosa outer membrane adhesins for human respiratory mucus glycoproteins.
The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cases of cystic fibrosis. For this purpose, microtiter plate adhesion assays have been developed and have suggested that nonpilus adhesins of P. aeruginosa are the most important ones for binding to human respiratory mucins. In order to characterize these mucin-binding adhesins, outer membrane proteins (OMP) from two adhesive strains, 1244-NP and PAK-NP, and their poorly adhesive rpoN mutants, 1244-N3 and PAK-N1, were prepared by a mild extraction with Zwittergent 3-14. Mucin-binding adhesins were detected after polyacrylamide gel electrophoresis and blotting of the OMP on nitrocellulose replicas, using human bronchial mucins labeled with 125I. The binding properties of these OMP with lactotransferrin, another glycoprotein abundant in respiratory mucus, were also studied. Radiolabeled mucins detected four bands at 48, 46, 28, and 25 kDa with strain PAK-NP. With the nonmucoid strain 1244-NP, five bands were observed at 48, 46, 42, 28, and 25 kDa. The bands at 48 and 25 kDa were also visualized by radiolabeled lactotransferrin. These bands were partially or completely displaced by nonradiolabeled respiratory mucin glycopeptides but not by tetramethylurea, suggesting that they recognized carbohydrate sites. In contrast, the poorly adhesive strains showed weakly binding bands. These results demonstrate that outer membranes from two different nonpiliated P. aeruginosa strains express multiple adhesins with an affinity for human respiratory mucins and/or lactotransferrin.
Adhesins, Bacterial↗