The long term goals of the studies outlined in this proposal are to understand the mechanisms involved in the regulation of mucin biosynthesis and secretion and relationship of these processes to the pathology of hypersecretion associated with chronic obstructive pulmonary diseases, such as, cystic fibrosis (CF). Excess mucus secretions with altered viscoelastic properties clearly contributes to the clinical symptoms. To date, little is known about the mucin macromolecules that constitute the secretion; especially knowledge regarding the primary structures of their core protein(s) is lacking. The objectives of this proposal are to gain insights into organization of the macrostructure of human tracheobronchial mucins (HTMs) and to investigate regulation of their gene expression.
The specific aims are to, a) purify and compare physicochemical properties of mucins HTM-1 and HTM-2 isolated from CF and normal lung secretions, b) prepare and characterize poly- and monoclonal antibodies against deglycosylated mucins, c) prepare synthetic oligonucleotide probes based on select peptide sequences of the mucins, d) construct cDNA library in lambda gt22A from poly(A) +RNA isolated from human tracheal epithelial (HTE) cells, e) screen the library using immunological and synthetic oligonucleotide probes, f) isolate, characterize and sequence cDNA clones encoding for protein cores of the mucins, g) using cDNAs, thus obtained, isolate genomic clones which will also sequenced and g) to investigate the mechanism(s) and effects of select secretagogue(s) on regulation of transcription of the HTM apomucin gene. The mucins will be purified according to protocols established in our laboratory and their physicochemical properties will be determined using established techniques. mRNA will be isolated from fresh HTE cells and cultured mucus secreting HTE cells. These preparations will be used to generate cDNA libraries which will be screened using immunological and synthetic oligonucleotide probes. The cDNA inserts will be sequenced. Clone identity will be confirmed by amino acid sequence analysis of selected peptide fragments. The cDNAs will be utilized to isolate genomic clones which will also be sequenced. Effects of secretagogue(s) on mucin related mRNA synthesis will be detected by Northern blot analysis using mucin cDNA probe(s). This knowledge will be essential in order to develop a rational approach to the treatment of chronic obstructive lung diseases including CF which will ultimately require an ability to control the viscosity and rate of secretion.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL034012-08
Application #
3346510
Study Section
Medical Biochemistry Study Section (MEDB)
Project Start
1984-07-01
Project End
1995-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Oklahoma Health Sciences Center
Department
Type
Schools of Pharmacy
DUNS #
937727907
City
Oklahoma City
State
OK
Country
United States
Zip Code
73117
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Hebbar, Vidya; Damera, Gautam; Sachdev, Goverdhan P (2005) Differential expression of MUC genes in endometrial and cervical tissues and tumors. BMC Cancer 5:124
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Desai, V C; Naziruddin, B; Graves, D C et al. (1991) Production and characterization of monoclonal antibodies to purified deglycosylated cystic fibrosis respiratory mucin: evidence for the presence of four immunologically distinct epitopes. Hybridoma 10:285-96

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