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D J Thornton

Publications and source records attributed to D J Thornton.

At least 37 records · Page 2Linked to original sources
PubMed1995

Methods for separation and deglycosylation of mucin subunits.

Mucins (density 1.35-1.50 g/ml) prepared from human cervical, gastric, respiratory, and salivary secretions were reduced and alkylated to generate reduced mucin subunits. These subunits were separated into different populations analytically by using agarose gel electrophoresis and preparatively by using anion-exchange chromatography. Both techniques separate the molecules primarily on the basis of their inherent charge. A procedure has been developed for the efficient deglycosylation of small amounts of these reduced mucin subunits immobilized on polyvinylidene difluoride. The combination of these procedures has allowed the isolation and identification of glycoforms of a specific mucin gene product in a respiratory mucous secretion.

Blotting, Western↗
PubMed1995

Analysis of respiratory mucus glycoproteins in asthma: a detailed study from a patient who died in status asthmaticus.

Airway mucus from asthmatics is often unusually solid. The death of a patient in status asthmaticus allowed the collection of 28 g of abnormal airway mucus at autopsy. Its chemical and physical properties were studied to reveal differences from more normal airway mucus. The gel plug taken from the airways could be dispersed in 6 M guanidinium chloride, but it took > 1 wk and 700 ml of extractant to disperse 3 g of exudate completely. In contrast, treatment with 10 mM dithiothreitol, which reduces disulfide bonds, dispersed the gel within seconds. Mucins accounted for 25% of the non-dialyzable material in the gel, while DNA constituted < 1% and proteoglycans could not be detected. The mucins were similar in architecture and general composition to other respiratory mucins and were present at a high concentration (approximately 40 mg/ml). The majority of mucins were of extreme size (mean M(r) 30-40 x 10(6)) and slow to dissolve, but sequential extraction experiments on the gel exudate demonstrated a proportion of mucins (15%), the most readily extracted, which had a higher density, 1.45-1.55 g/ml, a lower M(r) (11.5 x 10(6)) and were markedly more acidic than the bulk of the mucins. Both major and minor mucin populations were extremely heterogeneous in mass distribution. Electron microscopy of the major mucin species demonstrated extensive networks of molecules many microns in length. The major mucin species was distinctly less acidic than mucins previously described from either normal or diseased airways. Amino acid analysis of fractions across the charge distribution suggested the presence of at least two different mucin proteins occurring as distinct glycoforms.

Gels↗
PubMed1994

Identification of two major populations of mucins in respiratory secretions.

Two populations of reduced subunits were present in the mucins purified from pooled normal secretions and asthmatic and chronic bronchitic sputa; their relative level differed between samples. To investigate the nature of this heterogeneity, an asthmatic respiratory mucin preparation from a single individual was reduced and alkylated with 14C-iodoacetamide. This preparation was analyzed by gel filtration, agarose gel electrophoresis, immunoblotting, rate-zonal- and density-gradient centrifugation, and HPLC ion-exchange- and reverse-phase chromatography. Two populations (A and B) of reduced mucin subunits and a high-M(r)protein-rich fraction were identified. Species A has the higher molecular mass, is slowest migrating on agarose electrophoresis, has longer oligosaccharide chains, and expresses the carbohydrate structure sialyl-Le(x). Species B has a lower molecular mass, migrates faster in agarose electrophoresis Species B has a lower molecular mass, migrates faster in agarose electrophoresis, has shorter chains, and does not express sialyl-Le(x). The two subunits have similar but not identical amino acid compositions and 14C-tryptic peptide maps indicating they have different protein cores. The anti-sialyl-Le(x) antibody selectively precipitated subunit A not only from the reduced but also from the nonreduced mucin preparation, demonstrating that subunits A and B are present in different intact mucins.

Amino Acids↗
PubMed1993

Intravascular calcium deposits in a critically ill patient. Light- and electron-microscopic findings.

We describe a case of a 2-year-old girl with an unusual finding of amorphous hematoxyphilic substance in the pulmonary and myocardial vascular lumina. The patient had a prolonged history of intestinal obstruction necessitating extended periods of total parenteral nutrition. The patient terminally had hypercalcemia with levels reaching 4.63 mmol/L. The intravascular substance stains strongly positive for calcium, and weakly positive for fibrin. Electron microscopy shows that the substance has a distinctive configuration suggestive of calcium hydroxyapatite crystals.

Blood Vessels↗
PubMed1991

Mucus glycoproteins from cystic fibrotic sputum. Macromolecular properties and structural 'architecture'.

Mucus glycoproteins (mucins) were isolated from sputum of patients with cystic fibrosis (CF) after separation into sol and gel phases. The mucus gel was solubilized with gentle stirring in 6 M-guanidinium chloride supplemented with proteinase inhibitors, and purification of mucins was subsequently achieved by isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. Density-gradient centrifugation also revealed a heterogeneity of the macromolecules, the pattern of which varied between individuals, and mucins from the gel phase was pooled as 'heavy' and 'light' fractions. Gel chromatography on Sepharose CL-2B showed that the heavy fraction contained a larger proportion of smaller species than the 'light' fraction and that the gel phase mucins were much larger than those from the sol. An apparently homogeneous high-Mr mucin population from one individual contained approx. 70% (w/w) carbohydrate, the major sugars being N-acetylglucosamine (17.8%), N-acetylgalactosamine (6.7%), galactose (20.7%), fucose (13.2%) and sialic acid (11.4%). These mucins had an S020.w of 47 S, and an Mr of 15 x 10(6) -20 x 10(6), and rate-zonal centrifugation revealed a polydisperse size distribution [range (5-30) x 10(6)] with a weight-average Mr of 17 x 10(6). The whole mucins were visualized with electron microscopy as linear and apparently flexible threads, disperse in size. Reduction produced subunits which were included on Sepharose CL-2B, and subsequent trypsin digestion yielded high-Mr glycopeptides which were further retarded. The size distributions and fragmentation patterns of mucin from two other CF patients were the same, as studied by gel chromatography, rate-zonal centrifugation and electron microscopy. We conclude that CF mucins are heterogeneous in both size and buoyant density and that the various populations, though differing in buoyant density, share the same architecture and macromolecular properties and are, in this respect, similar to mucins from normal respiratory secretions [Thornton, Davies, Kraayenbrink, Richardson, Sheehan & Carlstedt (1990) Biochem. J. 265, 179-186] and human cervical mucus [Carlstedt & Sheehan (1989) SEB Symp. XLIII 289-316].

Adolescent↗
PubMed1991

Heterogeneity of mucus glycoproteins from cystic fibrotic sputum. Are there different families of mucins?

High-Mr mucin glycopeptides prepared from sputum of an individual with cystic fibrosis (CF) were studied by ion-exchange h.p.l.c. The glycopeptides were heterogeneous and a number of partially resolved populations were identified. Whole mucins from the gel phase were separated into four fractions by isopycnic density-gradient centrifugation in CsCl, and high-Mr glycopeptides from these fractions were examined by ion-exchange h.p.l.c. The acidic nature of the high-Mr glycopeptides increased with increasing buoyant density of the intact mucins, and a periodate-Schiff (PAS)-rich and an extremely high-iron diamine (HID)-reactive component were present in the lowest and highest density fractions respectively. The various glycopeptide populations were identified in different proportions in mucins from four other individuals with CF. CF sputum thus seems to contain distinct mucin populations containing different oligosaccharide clusters corresponding to these high-Mr glycopeptides.

Centrifugation, Density Gradient↗
PubMed1991

Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy.

Polyclonal antibodies were raised in rabbits towards reduced subunits of human cervical mucus glycoproteins. The reduced subunits almost completely inhibited the antiserum, whereas the intact mucins and the heavily glycosylated fragments obtained after digestion of reduced subunits with trypsin (T-domains) caused only partial inhibition. Periodate oxidation of intact mucins, reduced subunits and T-domains caused no effect on the antibody response, and fragments obtained by more extensive proteolysis of the reduced subunits (P-domains) showed no inhibitory activity. By using electron microscopy, antibodies from T-domain-adsorbed antisera were revealed as bound to cervical mucin reduced subunits, either directly or with colloidal gold-Protein A. Binding sites (100-150 nm apart) were observed at the ends and at internal positions of the reduced subunits. We conclude that the antibodies do not recognize carbohydrate structures but are directed to two kinds of protein epitopes, one shared by whole mucins, reduced subunits and T-domains, and the other specific to the reduced subunit fragment. The latter epitopes are 'cryptic' and are probably shielded within folded protein domains stabilized by disulphide bonds. Human bronchial, cervical, gastric and salivary mucus glycoproteins share some of these cryptic epitopes.

Antibodies↗
PubMed1991

Mucin structure. The structure and heterogeneity of respiratory mucus glycoproteins.

Respiratory mucus glycoproteins purified from both "normal" respiratory secretions and sputa of patients with a variety of hypersecretory conditions are high Mr linear molecules adopting a random coil configuration in solution. Studies on their polydispersity show them to have an Mr in the range 3 to 32 x 10(6) and a distribution of length from 200 nm to beyond 10 microns. These macromolecules are fragmented by reduction of intermolecular disulfide bonds into subunits, with Mr approximately 2 x 10(6) and length from 200 to 600 nm. Reduction not only cleaves the mucin molecule but opens, presumably by breaking intramolecular disulfide bonds, cryptic "naked" protein regions. Trypsin digestion of subunits yields high Mr glycopeptides (Mr, 300 to 500,000), presumably by cleavage of the peptide core within the unfolded "naked" protein domains. Respiratory mucus glycoproteins from infected sputum samples are usually heterogeneous in CsCl density gradients, in contrast to those from "normal" tracheobronchial secretions. The former are characterized by the presence of a number of different mucin species, and the basis for the separation of these mucins appears to be the variable presence of sialic acid and sulfate moieties in the oligosaccharide clusters. This heterogeneity may reflect a difference in cellular origin of the mucins and also may be clinically significant.

Glycoproteins↗
PubMed1990

Mucus glycoproteins from 'normal' human tracheobronchial secretion.

Mucous secretions were collected from tracheas of patients undergoing minor surgery under general anaesthesia with tracheal intubation, and mucus glycoproteins were isolated by using isopycnic density-gradient centrifugation in CsCl/guanidinium chloride. 'Whole' mucins were excluded from a Sepharose CL-2B gel, whereas subunits obtained after reduction were included. Trypsin digestion of subunits afforded high-Mr glycopeptides (T-domains), which were further included in the gel. The latter fragments are heterogeneous and comprise two or three populations, as indicated by gel chromatography and ion-exchange h.p.l.c. Rate-zonal centrifugation showed that the 'whole' mucins are polydisperse in size, with a weight-average Mr of (14-16) x 10(6). The macromolecules were observed by electron microscopy, as linear and apparently flexible thread-like structures. Subunits and T-domains had weight-average contour lengths of 490 nm and 160 nm respectively. It is concluded that mucus glycoproteins are present in secretions from the healthy lower respiratory tract. The 'whole' tracheal mucins are assembled from subunits, which in turn can be fragmented into high-Mr glycopeptides corresponding to the oligosaccharide domains typically found in mucus glycoproteins. The size and macromolecular architecture of the tracheal mucins is thus similar to that observed for mucins from human cervical mucus, chronic bronchitic sputum and pig stomach, providing yet another example of this general design of these macromolecules, i.e. subunits assembled end-to-end into very large linear and flexible macromolecules.

Bronchi↗
PubMed1990

Mucinophilic and chemotactic properties of Pseudomonas aeruginosa in relation to pulmonary colonization in cystic fibrosis.

Representative isolates of nonmucoid Pseudomonas aeruginosa were studied to investigate the hypothesis that mucinophilic and chemotactic properties in this species act as potential factors in the initial stages of pulmonary colonization in patients with cystic fibrosis (CF). Transmission electron microscopy with a surfactant monolayer technique was used in a novel manner to demonstrate the adhesion of all 10 P. aeruginosa strains examined to porcine gastric mucin and tracheobronchial mucin from a patient with CF. Control experiments showed that Escherichia coli K-12 and single representatives of Proteus mirabilis and Klebsiella aerogenes did not bind to these mucins. The Adler capillary technique, used to measure bacterial chemotactic response, showed that purified CF mucin acted as a chemoattractant for most P. aeruginosa strains, with the exception of the nonmotile mutant M2Fla- and the nonchemotactic mutant WR-5. The ability of the major sugar and amino acid components of mucin to act as chemoattractants was investigated. The degree of chemotaxis was strain specific; optimum chemotaxis was observed toward serine, alanine, glycine, proline, and threonine. No strain showed chemotaxis to N-acetylneuraminic acid, but all strains showed a strain-dependent chemotactic response to the sugars L-fucose, D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine. These results provide new information on the mucinophilic and chemotactic properties of nonmucoid P. aeruginosa and support the hypothesis that these properties could play a role in the initial stages of pulmonary colonization in patients with CF.

Amino Acids↗
PubMed1990

The school breakfast program and cognition in adolescents.

We studied cognitive function and metabolic status in a group of healthy adolescents fed the government supplied breakfast (n = 18) versus a control group (n = 16) fed a very low calorie meal. Serum glucose and beta-hydroxybutyrate levels were repeatedly measured at predetermined intervals throughout the testing period. Acute cognitive and mood effect were evaluated in all subjects on a pre-post basis. No significant group differences emerged on a battery of psychological measures that assessed short-term auditory memory, vigilance, impulsivity, and mood. Neither serum glucose or beta-hydroxybutyrate levels correlated significantly with any behavioral measure. Results are discussed in the context of previous studies of nutrition and cognition.

3-Hydroxybutyric Acid↗
PubMed1989

Structural and immunological studies of keratan sulphates from mature bovine articular cartilage.

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.

Animals↗
PubMed1989

Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage.

Two discrete peptido-keratan sulphate fragments were isolated via chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (K(av) = 0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (K(av) = 0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains. The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by 13C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.

Animals↗
PubMed1989

Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes.

A sensitive assay for mucus glycoproteins (mucins) and fragments thereof is presented. The macromolecules are blotted onto nitrocellulose membranes and visualized using a periodate-Schiff (PAS) reaction and the color yield quantitated with an image analysis system used as a reflectance densitometer. At least 50 ng of the macromolecules was detected. "Whole" mucins and subunits were assayed on 0.2-micron pore size nitrocellulose membranes whereas immobilization of the high-molecular-weight mucin glycopeptides (Mr 300-500,000) required pretreatment of membranes with poly-L-lysine. Binding of the glycopeptides to the polylysine-treated membranes was found to decrease with increasing salt concentration suggesting an electrostatic interaction. The data obtained with this method and a solution PAS assay are in good agreement but the former is more sensitive and can be performed on samples dissolved in chaotropic solvents.

Collodion↗