Biomedical subjects
D A Hutton
Publications and source records attributed to D A Hutton.
The MUC2 gene product: a human intestinal mucin.
The MUC2 gene product is the first human secretory mucin protein core to be fully sequenced. Like the other eight human MUC genes identified to date, MUC2 is characterised by tandem and irregular repeat sequences rich in threonine and serine, the potential sites of attachment of the oligosaccharide chains. The MUC2 gene product is more than 5100 amino acids in its commonest allelic form and accounts for one fifth by weight of the mucin glycoprotein molecule (80% oligosaccharide side chains). The MUC2 product is polymerised end to end through disulphide bridges to form large secreted polymeric gel-forming mucins (Mr approximately 10(7)). The primary function of the MUC2 gene product is to provide a protective barrier between the epithelial surfaces and the gut lumen. There is decreased expression of MUC2 in colonic cancer and defective polymerisation of secreted mucin in ulcerative colitis. Elucidation of the MUC2 and other mucin gene sequences has opened the way for a full structural characterisation and an improved understanding of the structure and function of these complex mucus gel secretions.
Heterogeneity in the protein cores of mucins isolated from human middle ear effusions: evidence for expression of different mucin gene products.
High molecular weight mucins were isolated and purified from human middle ear effusions of children with Otitis Media with Effusion (OME) classified into three groups, (1) thick and (2) thin from anatomically normal children and (3) effusions from cleft palate patients. Amino acid analyses of the purified mucins from the three pools were similar but not identical with characteristic contents of serine threonine and proline (32%, 28%, and 38% for pools (1) (2) and (3) respectively). Proteinase resistant glycopeptide fragments corresponding to the tandem repeat domains of cloned mucin genes showed marked differences both between the three mucin pools and with the composition of the tandem repeat sequences of the cloned mucin genes expressed in the airways. Studies on the antigenic identity of middle ear mucins found an epitope likely to be present on MUC5AC, but only accounting for a maximum of 15% by weight and no reactivity was found with antibodies to MUC2 or MUC1. A polyclonal antibody raised to thick effusion mucins reacted strongly with human salivary mucin suggesting the presence of MUC5B epitopes. These studies suggest that more than one mucin gene product is secreted by the human middle ear mucosa and that there may be further mucin genes expressed by the middle ear that have yet to be cloned.
Fragmentation patterns of human paranasal sinus mucins.
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MUC5B expression in middle ear mucosal glands.
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A polydisperse linear random coil model for the quaternary structure of pig colonic mucin.
The distribution of molecular weights for polymeric colonic mucus glycoprotein or "mucin" isolated and solubilised in the presence of protease inhibitors from pig colons is shown to be considerably greater than its "subunit" (thiol reduction product) and papain digested forms using the technique of size-exclusion chromatography coupled to multi-angle laser light scattering, and confirmed by sedimentation equilibrium measurements. The conformation of this mucin is probed by examining the molecular weight-intrinsic viscosity relationship in terms of the Mark-Houwink-Kuhn-Sakurada analysis for its polymeric (or "whole"), reduced and papain-digested forms: an exponent "a" of (1.1 +/- 0.1) is obtained indicating a linear random coil conformation consistent with other mucins. Size-exclusion chromatography coupled to multi-angle laser light scattering is shown to provide a relatively simple complementary technique to sedimentation equilibrium for the molecular weight distribution analysis of polydisperse materials.
Expression of MUC2 and MUC5AC by human nasal mucosa.
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Compositional differences between bilateral middle ear effusions in otitis media with effusion: evidence for a different etiology?
The aim of this study was to clarify the site of primary pathology in otitis media with effusion. Effusions were collected from 64 children with bilateral effusions at the time of myringotomy. The rheological properties and biochemical compositions of effusions were measured for 23 pairs of effusions, and the levels of the inflammatory mediators TNF alpha, IL-1beta, and IL-8 were measured in 41 pairs using specific enzyme-linked immunosorbent assays (ELISAs). Measurements from paired ears were compared using analysis of variance (ANOVA) tests and significant differences were found for reduced specific viscosity, mucin content, protein content, and levels of IL-8. The results demonstrate that the two ears have different immunological processes or rates of processes which might explain the significantly different rheological properties of effusions. This suggests that each ear undergoes pathological changes independently and has implications for using the opposite ear as a control in clinical trials.
Characterization of pig colonic mucins.
Pig colonic mucins isolated from the adherent mucus gel in the presence of proteinase inhibitors were solubilized by homogenization and the component mucins fractionated by CsC1 density-gradient centrifugation. Polymeric and reduced pig colonic mucin were both largely excluded on Sepharose CL-2B, papain-digested colonic mucin was included. The M(r) values of polymeric, reduced and digested mucins were 5.5 x 10(6), 2.1 x 10(6) and 0.6 x 10(6) respectively. This suggests that pig colonic mucin is comprised of 2-3 subunits, each subunit containing 3-4 glycosylated regions. The intrinsic viscosities of polymeric, reduced and digested mucin were 240 ml.g-1, 100 ml.g-1 and 20 ml.g-1 respectively. Polymeric pig colonic mucin comprised 16% protein per mg of glycoprotein and was rich in serine, threonine and proline (43% of total amino acids). There were approx. 150 disulphide bridges and 53 free thiol groups per mucin polymer. A seventh of the protein content was lost on reduction. This protein was particularly rich in proline and the hydrophobic amino acids. Papain-digested pig colonic mucin contained 11% protein per mg of glycoprotein and was rich in serine, threonine, glutamate and aspartate. All types of amino acids with the exception of aspartate were lost on digestion. The amino acid analysis of the proteolytically digested regions of pig colonic mucin are markedly different to the tandem repeat regions of the human mucin genes shown to be expressed in the colon.
The use of wheat germ agglutinin to improve binding of heterogeneous mucin species to nitrocellulose membranes.
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Interaction of polyacrylates with porcine pepsin and the gastric mucus barrier: a mechanism for mucosal protection.
1. The mechanism of interaction of the polyacrylates, carbopols with the mucus barrier in vivo has been investigated in vitro. 2. Carbopol caused a dramatic increase in the viscosity of porcine gastric mucin solutions that was up to 19-fold greater than that of the sum of the individual polymers. 3. The mucin-carbopol interaction was stable after an initial 30 min period for up to 36 h at 25 degrees C or 37 degrees C. It was reduced by increasing the temperature from 20 degrees C to 45 degrees C, was unaffected by pH and ionic strength, but was enhanced by Ca2+. 4. The magnitude of the interaction between mucin and carbopol depended on the polymeric structure of the mucin and the molecular size and level of cross-linking of the carbopol. 5. The interactions were reversible and increased with increasing carbopol and mucin concentration. The dramatic increase in viscosity can be explained in terms of space filling by the mucin molecules leading to predominantly carbopol-carbopol interactions. 6. Carbopol 934P inhibits pepsin hydrolysis and therefore has potential as a mucosal protective agent in vivo.
Preliminary characterization of mucin from effusions of cleft palate patients.
Middle ear effusions from children undergoing myringotomy were classified into three groups-cleft palate, thick (mucoid), and thin (serous). Mucin was purified from each of the three groups using CsCI equilibrium density gradient centrifugation. Analysis of the cleft palate mucin on Sepharose CL-2B showed it was excluded and therefore of large molecular weight. It could be broken down into smaller glycopeptide units by proteolysis and these glycopeptides had, based on elution position, a larger hydrodynamic size than those from the thick mucin. Intrinsic viscosity measurements demonstrated that the intact mucins could be ranked in order of molecular space occupancy; cleft palate > thick > thin. Amino acid analysis showed the cleft palate mucin to have an amino acid composition similar to other mucins, with serine, threonine, and proline constituting 41% by weight of the protein core. Thiol analysis gave evidence of a possible difference in polymerization between the three mucins, in that thin (the smallest mucin) contained the lowest number of thiols. This preliminary analysis of cleft palate mucin suggests a mucin with larger glycopeptide units forming an intact mucin of larger hydrodynamic size than either thick or thin middle ear mucins from anatomically normal children.
Culture of human middle ear mucosal explants; mucin production.
Middle ear mucosal biopsies could be maintained in culture for up to 7 days, the longest time attempted in this study. Mucin biosynthesis and secretion were measured by incorporation of 14C-glucosamine. Three peaks of radioactivity were present when the dialysed medium was chromatographed. Peak I which accounted for about 10% of the total radioactivity had properties characteristic of mucin. The other two peaks were not characteristic of mucins. Labelled macromolecules excluded on Sepharose 2B were also present in the tissue. Autoradiography of the explants showed that the labelled glucosamine was concentrated in the epithelial layer. Morphometry demonstrated that 1-2% of the epithelial cell volume consisted of goblet cells. The proportionate incorporation of radioactivity into macromolecules increased with increasing epithelial cell volume. This system will allow assessment of factors implicated in the pathogenesis of otitis media with effusion and the study of the action of pharmacological agents on biosynthesis and secretion.
Otitis media with effusion: components which contribute to the viscous properties.
Middle ear effusions from children undergoing myringotomy were classified into thick (mucoid) and thin (serous) on the basis of their flow properties. Their composition was analysed and their rheological properties measured. The viscosity of the effusions was measured using a Contraves low shear viscometer and expressed as specific viscosity per mg/ml of non-dialysable solids present. In order to measure the effusion viscosity it was necessary to solubilize the effusion by mild homogenisation in a phosphate buffer pH 6.7 containing a cocktail of proteolytic inhibitors. The viscosity of mucoid effusions was significantly greater than that of the serous effusions. There was a small but measurable amount of proteolytic activity in the effusions, range 0.05-1.79 micrograms/mg of non-dialysable solids. This proteolytic activity was not significantly different between the thick and thin effusions and was therefore unlikely to explain the difference in viscosity. Analysis of the constituents of the effusions showed that glycoprotein and DNA but not protein nor lipid were significantly higher in the mucoid effusions compared to the serous effusions. The viscosity of the effusions correlated with the glycoprotein concentration but not with the protein or lipid concentration. Under certain circumstances the DNA concentration did correlate with the viscosity of the effusion. However, digestion with a proteinase free DNase did not reduce the viscosity of the effusion. These results demonstrate that classifying effusions as thick and thin based on visual inspection and flow properties is valid and that the only constituent present in the effusions that determines viscosity is mucin.
Glycosylated molecules secreted by human middle ear mucosa in culture.
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Mucolysis of the colonic mucus barrier by faecal proteinases: inhibition by interacting polyacrylate.
1. Mucolytic (mucus solubilizing) activity in human faeces has been characterized with both purified human and pig colonic mucin and shown to be mediated by proteolysis. 2. Mucolytic activity was demonstrated by: (i) a drop in mucin viscosity; (ii) a substantial reduction in mucin size, from polymer to degraded subunit, as assessed by Sepharose CL-2B gel filtration; (iii) formation of new N-terminal peptides. 3. Mucolytic activity was also followed in faecal extracts by its proteolytic activity using standard succinyl albumin substrate. Proteolysis extended over the pH range 4.5-11.0. Proteolysis was inhibited at pH 7.5 by soybean trypsin inhibitor and phenylmethanesulphonyl fluoride, suggesting the presence of serine proteinases. 4. The polyacrylate carbomer (934P) inhibited both mucolysis of pig colonic mucin and proteolysis of succinyl albumin. 5. Interaction between the polyacrylate (carbomer 934P) and purified human and pig colonic mucin was demonstrated by a marked synergistic increase in solution viscosity (360% above control). 6. The results demonstrate the presence of a mucolytic activity in the human colonic lumen that has the potential to degrade the mucus barrier, and that polyacrylates inhibit this mucolysis and interact to strengthen the colonic mucus barrier. Polyacrylates may therefore have therapeutic potential in inflammatory bowel disease where luminal proteolytic activity can be raised.
The dependence of size and functional properties of pig gastric mucus on the isolation method used.
The size of isolated gastric mucin is substantially larger if proteolysis is rigorously excluded during the isolation procedure by inclusion of proteinase inhibitors with or without guanidinium chloride. Pig gastric mucin isolated in 0.2 M NaCl (without proteinase inhibitors) so25,w, 33S, Mr approximately 2 x 10(6) has consistent physical and chemical properties, retains the gel-forming properties of the native mucus secretion and is readily susceptible to proteolysis although it has undergone proteolytic "nicking" during isolation. Gastric mucins isolated in proteinase inhibitors with guanidinium chloride are of larger size so25,w, 41-110S but precipitate from solution at gel-forming concentrations. Reduction of all these gastric mucin preparations with either 0.2 M mercaptoethanol or 10 mM dithiothreitol produced subunits of size in the region of Mr approximately 5 x 10(5). Gel filtration studies also showed that reduced mucin subunits prepared from gastric mucins isolated in guanidinium chloride could reassociate to form larger subunits of size approximately Mr approximately 2 x 10(6). The dependence of gastric mucin size on the isolation procedure is discussed.