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Biomedical subjects

David J Thornton

Publications and source records attributed to David J Thornton.

9 recordsLinked to original sources
PubMed2006

Endothelial function in HIV-infected persons.

BACKGROUND: Several reports have suggested an increased risk of coronary disease in human immunodeficiency virus (HIV)-infected patients receiving protease inhibitors (PIs). Impaired endothelium-dependent vasodilation is a putative surrogate marker of coronary atherosclerotic disease. METHODS: The present study evaluated the effect of HIV infection and antiretroviral treatment on endothelial vasomotor function, by assessing brachial artery flow-mediated dilation (FMD). A total of 75 HIV-infected patients were compared with 223 control subjects who were presumed to be HIV uninfected. RESULTS: HIV-infected patients had significantly impaired FMD, compared with control subjects (mean +/- SD, 7.3% +/- 4.4% vs. 11.1% +/- 6.3%; P < .0001). When adjustments were made for smoking status, sex, and body mass index, the difference between the 2 groups remained statistically significant (P < .01). In a cross-sectional analysis of the HIV-infected patients, we found significant associations between FMD and current injection drug use, hazardous drinking, HIV load, and alpha-high-density lipoprotein triglyceride levels, but not PI therapy. In a multivariate analysis, only current injection drug use and a lower alpha-high-density lipoprotein triglyceride level were significantly associated with FMD. CONCLUSIONS: HIV-infected patients have significant impairment of endothelial function, and this impairment is worse among those with elevated levels of HIV replication, particularly injection drug users.

Adult↗
PubMed2006

Proteomic analysis of mouse growth plate cartilage.

Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS.

Animals↗
PubMed2004

Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin.

MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately 2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.

Cell Line, Tumor↗
PubMed2004

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis.

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M(r) glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M(r) material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.

Amino Acids↗
PubMed2004

From mucins to mucus: toward a more coherent understanding of this essential barrier.

Mucus is essential for protection of the airways; however, in chronic airway disease mucus hypersecretion is an important factor in morbidity and mortality. The properties of the mucus gel are dictated in large part by the oligomeric mucins and, over the past decade, we have gained a better understanding of the molecular nature of these complex O-linked glycoproteins. We know now that MUC5AC mucins, as well as different glycoforms of the MUC5B mucin, are the predominant gel-forming glycoproteins in airways mucus. Furthermore, the amount, molecular size, and morphology of these glycoproteins can be altered in disease. From more recent data, it has become clear that oligomeric mucins alone do not constitute mucus, and other mucin and nonmucin components must be important contributors to mucus organization and hence airways defense. Therefore, the challenge over the coming decade will be to investigate how the oligomeric mucins are organized to yield "functional" mucus. Such studies will provide a clearer perception of airways mucosal protection and may highlight specific components as potential targets for therapeutic strategies for the treatment of hypersecretory disease.

Animals↗
PubMed2003

Calcium-dependent protein interactions in MUC5B provide reversible cross-links in salivary mucus.

The macromolecular organization within saliva was investigated by tracer diffusion measurements of fluorescent polystyrene microspheres by fluorescence recovery after photobleaching using a confocal microscope (confocal-FRAP). There was a concentration-dependent reduction in microsphere diffusion; this was much greater in the presence of calcium (10 mm) and was reduced by the addition of EGTA (10 mm). These effects on tracer diffusion showed that native saliva contained a macromolecular organization that was sensitive to free calcium concentrations. This was supported by a major increase in the weight average molecular weight of the high molecular weight mucin fraction in saliva (10-62 x 106) and an increase in intrinsic viscosity of saliva (733 to 1203 ml/g) both caused by calcium. Analysis of the change in tracer diffusion in saliva showed a 20-fold increase in the apparent pore size (from 130 nm in 10 mm CaCl2 to 2600 nm in 10 mm EGTA at physiological concentration). The effect was specific for calcium and was unaffected by up to 2 m NaCl. The calcium binding activity was contained in a high buoyant density fraction of saliva excluded from Sepharose CL-2B. Calcium binding to this fraction gave an approximate Kd of 7 x 10-6 m, and the binding was irreversibly destroyed by treatment with 6 m guanidinium chloride and by mild reduction, suggesting it to be to a protein site. This fraction of saliva was shown to contain MUC5B as the single major protein species by positive ion electrospray ionization-tandem mass spectrometry analysis. The results suggested that oligomeric MUC5B in saliva is assembled into much larger linear or branched assemblies through calcium-mediated protein cross-links.

Calcium↗
PubMed2002

Concentrated solutions of salivary MUC5B mucin do not replicate the gel-forming properties of saliva.

We have developed a new approach to study the molecular organization of salivary mucus and salivary mucins using confocal fluorescence recovery after photobleaching (confocal-FRAP). MUC5B mucin, its reduced subunit and T-domains were prepared from saliva and fluorescently labelled. The translational self-diffusion coefficients were determined up to 3.6 mg/ml by confocal-FRAP. The results suggest that, in solutions of purified MUC5B mucin, at concentrations at which the hydrodynamic domains overlap, the intermolecular interactions are predominantly due to dynamic entanglements, and there was no evidence of specific self-association of MUC5B mucin, or of its subunits, or T-domains. The analysis of the salivary mucus gel also showed no specific interactions with the purified MUC5B components, but it was much less permeable than expected from its MUC5B content. The saliva was completely permeable to microspheres of 207 nm diameter, but showed size-dependent effects on the diffusion of larger microspheres (499 nm and 711 nm diameter). From these analyses the salivary mucus was shown to be both permeable and dynamic, and with the characteristics of a semi-dilute transient network at physiological concentration. Comparison of the results from saliva and purified MUC5B mucin solutions showed that the network properties of saliva were equivalent to a solution of purified MUC5B mucin of 10-20 times higher concentration. This showed that saliva has additional structure and organization not present in the purified MUC5B mucin and suggests there are other interactions and/or components within saliva that combine with MUC5B to produce its complete properties.

Chromatography, Gel↗
PubMed2002

Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B.

Respiratory mucus contains a mixture of gel-forming mucins but the functional significance of these different mucin species is unknown. To help gain a better understanding of mucus in airways we therefore need to ascertain the concentration of each of the gel-forming mucins within respiratory secretions. Thus the aim of this study was to determine the amounts of specific gel-forming mucins directly from solubilized secretions of the airways and purified mucin preparations. We investigated the feasibility of using direct-binding ELISA employing mucin-specific antisera but were unable to obtain reliable data owing to interference with the immobilization of the mucins on the assay surface by 6 M urea and high levels of non-mucin proteins. We therefore developed an alternative approach based on quantitative Western blotting after agarose-gel electrophoresis, which was not subject to these problems. Here we demonstrate that this procedure provides reliable and reproducible data and have employed it to determine the amounts of the MUC2, MUC5AC and MUC5B mucins in saline-induced sputa from healthy airways and spontaneous sputa from asthmatic airways. Additionally we have used this procedure to analyse these glycoproteins in mucin preparations purified from cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) mucus. Our findings indicate that MUC5AC and MUC5B are the major oligomeric mucins and that airways mucus contains variable amounts of these glycoproteins. By contrast, the MUC2 mucin comprised, at most, only 2.5% of the weight of the gel-forming mucins, indicating that MUC2 is a minor component in sputum. Finally, we show that the amounts and glycosylated variants of the MUC5AC and MUC5B mucins can be altered significantly in diseased airways with, for instance, an increase in the low-charge form of the MUC5B mucin in CF and COPD mucus.

Asthma↗