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Biomedical subjects

D J Thornton

Publications and source records attributed to D J Thornton.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a nonmucin glycoprotein (gp-340) from a purified respiratory mucin preparation: evidence for an association involving the MUC5B mucin.

Rate-zonal centrifugation of a reduced and alkylated respiratory mucin preparation identified a protein-rich fraction. This was subjected to trypsin treatment and one of the many liberated peptides was purified and its N-terminal sequence determined. The peptide was identical to a 14 amino acid sequence from the scavenger receptor cysteine-rich domain containing glycoprotein gp-340. A polyclonal antiserum, raised against the peptide, stained the serous cells in the submucosal glands of human tracheal tissue. The glycoprotein was purified from respiratory mucus by density-gradient centrifugation, gel chromatography, and anion exchange chromatography. The molecule exhibited a heterogeneous distribution of buoyant density (1.28-1.46 g/ml) that overlapped with the gel-forming mucins, was included on Sepharose CL-2B and was quite highly anionic. SDS-PAGE indicated a mass greater than 208 kDa and measurements performed across the molecular size distribution indicated an average M(r) of 5 x 10(5) with a range of M(r) from 2 x 10(5) to 1 x 10(6). Gel chromatography of respiratory mucus extracts ("associative" and "dissociative") indicated that this glycoprotein forms complexes that may involve the large gel-forming mucins MUC5AC and MUC5B. Rate zonal centrifugation suggested such complexes are more likely to involve MUC5B rather than MUC5AC mucins.

Amino Acid Sequence↗
PubMed2000

Physical characterization of the MUC5AC mucin: a highly oligomeric glycoprotein whether isolated from cell culture or in vivo from respiratory mucous secretions.

We have isolated the high-M(r) mucins from growth medium of the early stage of an HT-29 cell culture by gel chromatography and isopycnic density gradient centrifugation. The mucins (buoyant density 1.34-1.44 g/ml) were reactive with an anti-peptide antiserum (MAN-5ACI) raised against a sequence from within the MUC5AC mucin. Similar antisera raised against the MUC2 and MUC5B mucins were not reactive. The MUC5AC reduced-mucin subunits exhibited a homogeneous charge distribution on anion-exchange chromatography, but appeared as two bands, one major and one more minor, after agarose gel electrophoresis. The unreduced mucins had an average M(r) in excess of 40 MDa and were visualized in the electron microscope as large, fine filamentous threads (many microns in length) that after reduction were greatly reduced in size (number average length 570 nm). Agarose gel electrophoresis of unreduced MUC5AC mucins identified a major band just entering the gel with evidence of a 'ladder' of faster-migrating minor bands. Partial reduction of the mucins increased the proportion of the faster bands and at least 16 could be discriminated. M(r) measurements showed that these bands differed by single monomer units. The mucins behaved as very stiff extended structures in solution and this characteristic might explain the poor separation of different-sized oligomers in sedimentation-rate experiments. The cell-culture mucin preparation had similar characteristics of charge and buoyant density to MUC5AC mucins from respiratory secretions in vivo. In addition the MUC5AC mucin from respiratory tract secretions exhibited similar behaviour, reduced and unreduced on agarose gel electrophoresis, indicating that the mucin has a similar molecular phenotype in vivo and in vitro.

Cell Line↗
PubMed2000

Identification in vitreous and molecular cloning of opticin, a novel member of the family of leucine-rich repeat proteins of the extracellular matrix.

A prominent 45-kDa component was identified by protein staining following SDS-polyacrylamide gel electrophoresis of a 4 M guanidine hydrochloride extract from bovine vitreous collagen fibrils. Peptide sequences obtained from this component were used as a basis for the cloning (from human retinal cDNA) and sequencing of a novel member of the leucine-rich repeat extracellular matrix protein family that we have named opticin. Opticin mRNA was found by reverse transcription polymerase chain reaction in ligament and skin as well as in retina. An open reading frame containing 332 amino acids was identified, the first 19 amino acids representing a signal peptide. The deduced amino acid sequence of the mature protein encodes a 35-kDa protein with a calculated isoelectric point of 5.4. The central domain of this protein consists of six B-type leucine-rich repeats. This domain is flanked by cysteine clusters including a C-terminal two-cysteine cluster containing an additional leucine-rich repeat. The N-terminal region contains a cluster of potential O-glycosylation sites, and analysis of bovine vitreous opticin demonstrated the presence of sialylated O-linked oligosaccharides substituting the core protein. Opticin shows highest protein sequence identity to epiphycan (42%) and osteoglycin (35%) and belongs to Class III of the leucine-rich repeat extracellular matrix protein family.

Amino Acid Sequence↗
PubMed2000

Characterization of mucins from cultured normal human tracheobronchial epithelial cells.

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.

Bronchi↗
PubMed1999

Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug.

We have previously noted that sequential extraction of an asthmatic mucous exudate with 6 M guanidinium chloride yielded a fraction of the mucins that were most resistant to solubilization and of high Mr [Sheehan, Richardson, Fung, Howard and Thornton (1995) Am. J. Respir. Cell Mol. Biol. 13, 748-756]. Here we show that this mucin fraction is dominated (at least 96% of the total) by the low-charge glycoform of the MUC5B gene product. Seen in the electron microscope the mucins appeared mainly as compact 'island' structures composed of linear threads often emanating from globular 'nodes' rather than the discrete linear threads more typical of mucins that we have previously described. The effect of reducing agents was as expected for other gel-forming mucins, i.e. reduced subunits or monomers of Mr 3x10(6)) were produced within 15 min of treatment. Kinetic experiments on the cleavage of the intact mucins with the proteinase trypsin indicated two clear regimes of fragmentation. An initial rapid cleavage generated mucins ranging from Mr=4x10(6) to 30x10(6) that in the electron microscope appeared as polydisperse threads (500-3000 nm in length), similar to normal and other respiratory mucins that we have previously characterized. A subsequent slower fragmentation over many hours yielded a major fragment of Mr 3x10(6) and length 200-600 nm, very similar in size and Mr to the subunits obtained by reduction. The results suggest that the MUC5B mucin is assembled, first into polydisperse linear threads, which are then linked together via a protein-mediated process. This might involve part of the mucin polypeptide or an as yet unidentified protein(s). The high proteinase susceptibility of the linkage suggests that it might be a point of control for mucin size and thus mucus rheology.

Asthma↗
PubMed1999

Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.

The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin.

Amino Acids↗
PubMed1999

A study of the intracellular and secreted forms of the MUC2 mucin from the PC/AA intestinal cell line.

In this study we present data on the entire population of MUC2 molecules secreted from and within the cell layer of an intestinal cell line. The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin. Oligomerized forms of the mucin were found in both the cell layer and medium; however, precursor forms were confined to the cell layer. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Three different populations of MUC2 were identified: one at low density (>1.3 g/ml) containing the N-glycosylated, non-O-glycosylated polypeptide; a second at intermediate density (1.3-1.35 g/ml) which may represent partially O-glycosylated intermediates; and a third at high density (1.36-1.48 g/ml) containing the mature MUC2 mucins. Rate-zonal centrifugation and agarose electrophoretic analysis of the low-density fraction indicated that the N-glycosylated MUC2 polypeptide was present as putative monomer and dimer/oligomer species. The combination of isopycnic density gradient centrifugation with agarose electrophoresis provides a new and simple approach that allows us to follow the MUC2 gene product from polypeptide through to the mature glycosylated mucin.

Cell Line↗
PubMed1998

Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association.

Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30-120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.

Acetic Anhydrides↗
PubMed1998

Monoclonal antibody recognizing a core epitope on mucin.

Monoclonal antibody TH1 (IgM) was prepared by immunizing mice with deglycosylated (TFMSA-treated) cystic fibrosis mucin. TH1 reacted strongly with TFMSA treated cystic fibrosis mucin but not with the fully glycosylated mucin, indicating reactivity with a core mucin epitope. TH1 showed no reactivity with ovine mucin (98% of glycans as sialyl-Tn) but reacted strongly with desialylated ovine mucin, indicating the epitope for this mab was the Tn-antigen (O-linked GalNAc). However, TH1 showed no reactivity with Tn-positive red blood cells, and the binding of TH1 was not inhibited by GalNAc at 2.5 mg/ml, illustrating the importance of the peptide sequence to which the GalNAc is attached. TH1 stained the majority of cancers of the colon, lung, stomach, ovary, breast, and cervix, and the cellular distribution of this antigen in normal tissue suggested reactivity with immature mucin. This antibody appears to be a useful reagent for the detection of immature mucin.

Amino Acid Sequence↗
PubMed1997

Identification of two glycoforms of the MUC5B mucin in human respiratory mucus. Evidence for a cysteine-rich sequence repeated within the molecule.

It has been demonstrated previously that respiratory secretions contain three oligomeric, gel-forming mucins; one of these was identified as the product of the MUC5AC gene (1). Here we demonstrate that the other two mucins are glycoforms of the MUC5B gene product. This was accomplished by trypsin treatment of the purified reduced mucin subunit populations and N-terminal sequencing of the liberated peptides. The products of trypsin digestion were separated by gel filtration into high molecular weight mucin glycopeptides and low molecular weight tryptic peptides. The latter were fractionated by reverse phase chromatography, and four of the major peptides were sequenced. Three of these peptides were identical to and contiguous within a 51-amino acid sequence deduced from a cDNA clone (JER57) encoding a portion of the MUC5B mucin. The other peptide is also present within this sequence but showed identity in only 9 of its 10 residues. A polyclonal antiserum raised against one of these peptides was reactive with the two putative MUC5B glycoforms. Analysis of the high molecular weight glycopeptides indicated that the MUC5B subunit contained different types and lengths of glycosylated domains; one domain of Mr 7.3 x 10(5), two domains of Mr 5.2 x 10(5), and a third domain of Mr 2.0 x 10(5). The amino acid composition of the larger two glycopeptides was similar in serine, threonine, and proline content but distinct from that of the smallest glycopeptide. Each of these domains in the mucin subunit is separated by a trypsin-sensitive region, and the relative abundance of the major peptides derived by proteolysis of these regions and their occurrence in a contiguous sequence suggest that they contain a common cysteine-rich motif.

Amino Acid Sequence↗
PubMed1997

Increased alkaline phosphatase isoforms in autoimmune diseases.

We found significant increases in ALP and ALP isoform band 10 in the serum of patients with early insulin-dependent diabetes, rheumatoid arthritis, and in those with multiple sclerosis during periods of disease exacerbation as compared with healthy controls. The ALP isoforms were assayed by isoelectric focusing. Our data suggest that the increase in ALP and ALP-10 closely reflects the abnormal activation of T lymphocytes that is common in autoimmune diseases, and that the source of the ALP-10 is activated T lymphocytes. ALP-10 is a sensitive but nonspecific marker of an active autoimmune process and appears to have the ability to detect abnormal T-cell activation. ALP-10 may be a useful test in the screening for autoimmune disorders.

Adolescent↗
PubMed1996

Respiratory mucins: identification of core proteins and glycoforms.

At least eight mucin apoproteins are expressed by the tracheobronchial epithelium, but it is not known which, if any, of these are major constituents of the respiratory secretions responsible for the formation of the mucus gel. To address this we have isolated mucins from normal, asthmatic and chronic bronchitic secretions. The asthmatic mucin reduced subunits were fractionated into four populations (I-IV) by anion-exchange HPLC. Amino acid and monosaccharide compositional analysis, as well as M(r) and size measurements, indicate that two of these populations (I and II) are glycoforms of the same or related apoprotein(s) and that the other populations contain two different apoproteins. A panel of antibodies and antisera recognizing the variable number tandem repeat (VNTR) of specific mucin apoproteins did not, as predicted, react with the glycosylated molecules, but after deglycosylation the majority of these probes (with the exception of those to MUC2, which were negative) reacted at a low level with each of the subunit populations. In contrast, an antiserum against a non-VNTR sequence of MUC5AC identified one of the populations (III) as the MUC5AC mucin. The MUC5AC reduced subunit had an M(r) of 2.2 x 10(6) and an RG (radius of gyration) of 57 nm. The genetic identities of the major mucin (populations I and II) and a minor component (population IV) were not established. The MUC5AC mucin was also identified as a major component in the pooled normal secretions from 20 individuals, whereas in a chronic bronchitic sample it was only a minor constituent. Furthermore, in all these different respiratory secretions the MUC5AC mucin appears as a similar biochemical entity, as assessed by Mono Q chromatography and agarose electrophoresis, suggesting that it may have a well-defined pattern of glycosylation in the respiratory tract.

Amino Acid Sequence↗
PubMed1996

Biosynthesis of the MUC2 mucin: evidence for a slow assembly of fully glycosylated units.

The human colonic cell line PC/AA was grown to near confluency over 24 days and labelled with [14C]proline and [3H]glucose over the last 48 h in culture. The cell layer was extracted with 6 M guanidinium chloride and the mature fully glycosylated mucins were isolated at a density of 1.45 g/ml by using density-gradient centrifugation in CsCl/4 M guanidinium chloride. These mucins were identified as MUC2 with an anti-peptide antibody. The macromolecules were fragmented by reduction into two distinct populations of MUC2 subunits as assessed by agarose electrophoresis. The MUC2 mucin was polydisperse in length, ranging from 500 nm to many microns and its molecular-mass distribution, assessed by rate-zonal centrifugation, ranged from 5 x 10(6) to 40 x 10(6) Da. However, the metabolically labelled MUC2 mucins, though found throughout the whole distribution, were of much smaller average size. Since the entire distribution is not uniformly radiolabelled over 48 h, the formation of the largest species must be preceded by glycosylation and occur slowly, over several days, via the assembly of fully glycosylated units which are likely to be at least dimers [Asker, Baeckstrom, Axelsson, Carlstedt, and Hansson (1995) Biochem. J. 308, 873-880].

Amino Acid Sequence↗
PubMed1996

Identification of glycoproteins on nitrocellulose membranes and gels.

In this article we describe a procedure for the detection of glycoproteins on gels employing the periodic acid-Schiff's reagent. In addition, a number of staining protocols and direct binding ELISA, employing antibodies and lectins, are described for the identification and quantitation of glycoproteins after their immobilization by dot, slot, or Western blotting onto nitrocellulose membranes. We document, in detail, the conditions (i.e., the effect of solvent and detergents) for the immobilization of one specific family of O-linked glycoproteins, namely mucins. However, taking into account our suggestions, these procedures should be applicable to other types of glycoprotein.

Acrylic Resins↗
PubMed1996

Swayback in a blesbok (Damaliscus dorcas phillipsi) and a black wildebeest (Connochaetes gnou).

Ataxia associated with myelopathy and low liver copper concentrations is described in a blesbok (Damaliscus dorcas phillipsi) and black wildebeest (Connochaetes gnou) from the Karoo Nature Reserve, Graaff-Reinet. This syndrome, which occurs in neonatal and delayed forms, has been described in sheep, goats, pigs and domesticated red deer, but reports in other ungulates, including antelope, are very rare and generally unconfirmed by histopathological examination. The 2 animals examined exhibited a typical pattern of Wallerian degeneration in selected tracts of the spinal cord. Many blesbok in the reserve are markedly pale in colour. Fading of the hair is also associated with copper deficiency in ungulates. High lamb mortality occurs as a result of the ataxia and threatens the survival of the blesbok herd in the reserve. Confirmation of low liver copper concentrations in affected animals offers direction towards the solution of the problem.

Animal Nutritional Physiological Phenomena↗