The presence of excessive amounts of viscous and tenacious mucus secretions in the tracheobronchial region is the most widely known feature of cystic fibrosis (CF). These mucus secretions cause obstruction of the airways and lead to chronic pulmonary disease which dominates the clinical picture and determines the fate of the majority of CF patients. The objective of this research is to investigate the molecular basis of altered viscoelastic properties of CF tracheobronchial secretions.
The specific aims of this proposal are a) to continue isolation, purification and characterization of mucus glycoproteins (mucins) from tracheobronchial secretions of patients with CF, asthma, bronchitis and from normal healthy individuals; b) to investigate the effects of different sodium chloride concentrations on the molecular aggregation, conformation (shape) and viscoelastic properties of purified CF and non-CF mucins; c) to investigate the biochemical properties of integral polypeptides released upon disulfide bond reduction of purified native CF and non-CF mucins; and d) to isolate and characterize mucin species present in the purified mucins prepared from CF and non-CF secretions. The mucins will be purified using protocols established in this laboratory. The mucin species (from purified CF and non-CF mucins) will be isolated by anion-exchange and immunoaffinity chromatographies. The isolated mucins as well as various mucin species from the CF and non-CF secretions will be characterized for their biochemical, biophysical and immunological properties. Biochemical characterization will include: determination of carbodrate, sulfate, amino acid composition, thiol and dissulfide bond content of the mucin molecules. The molecular size, conformation and aggregation properties will be investigated using state-of-the-art techniques, such as, static light scattering and fluorescence polarization techniques. The viscoelastic studies will be conducted using a magnetic microrheometer. The structure of the protein cores of the mucin will be investigated by immunological and biochemical methods. These studies will aid in the understanding of mechanism(s) responsible for altered viscoelastic properties of CF tracheobronchial secretions. This knowledge will be essential in order to develop a rational approach to the treatment of chronic lung disease in CF which will ultimately require an ability to control the viscosity, rate of secretion and elimination of the secretions.
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