Understanding molecular mechanisms regulating RNA synthesis during growth and differentiation of corneal epithelial cells is important in developing more effective treatment of defects arising from corneal injury or disease. The research in progress represents the first in-depth effort to characterize nuclear enzymes of transcription in mammalian corneal epithelium and to determine mechanisms by which their activity is regulated. Our previous studies have provided evidence for cyclic nucleotide-mediated muscarinic cholinergic regulation of RNA polymerase activity in rabbit corneal epithelial cells.
The specific aims of the proposed research are as follows: (1) To characterize RNA polymerases I, II and III of rabbit corneal epithelium (including electrophoretic and further biochemical/kinetic characterization of chromatographically purified enzymes). (2) To determine regulatory mechanisms by which the cholinergic agonist carbamylcholine enhances activity of RNA polymerases in rabbit corneal epithelium. Hypotheses to be tested concerning mechanisms mediating observed enhancement of RNA polymerase activity by carbamylcholine include the following potential effects of the drug, each of which is tentatively consistent with our kinetic data for one or more RNA polymerases in studies of crude enzymes from isolated, purified nuclei: (a) Effects on posttranslational phosphorylation of RNA polymerases by cyclic nucleotide-dependent (cGMP-dependent) protein kinases (""""""""targeted hypothesis""""""""); (b) Effects on phosphorylation of other chromatin-associated proteins (histones, HMG nonhistone proteins) influencing accessibility of DNA template to RNA polymerases; (c) Effects on RNA polymerase synthesis; (d) Effects on relative proportions of kinetically and electrophoretically distinct RNA polymerase isozymes with different substrate affinities. Experimental approaches include electrophoretic analysis of phosphorylation and synthesis of nuclear proteins in drug-treated and control cells, in conjunction with chromatographic purification and electrophoretic characterization of RNA polymerases I, II and III, histones and HMG proteins. Long-range objectives of this research include (1) elucidating molecular regulatory mechanisms by which cholinergic agents may influence gene expression at the transcriptional level during corneal epithelial cell growth and differentiation and (2) determining the suitability of cholinergic agents, alone or in combination with other drugs, for management of various types of injury and disease of the ocular surface.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
1R01EY008277-01
Application #
3265523
Study Section
Visual Sciences A Study Section (VISA)
Project Start
1989-12-01
Project End
1992-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Georgetown University
Department
Type
Schools of Medicine
DUNS #
049515844
City
Washington
State
DC
Country
United States
Zip Code
20057
Ichijima, H; Jester, J V; Petroll, W M et al. (1994) Laser and tandem scanning confocal microscopic studies of rabbit corneal wound healing. Scanning 16:263-8
Ichijima, H; Cavanagh, H D (1994) Effects of rigid lens extended wear on lactate dehydrogenase activity and isozymes in rabbit tears. Cornea 13:429-34
Imayasu, M; Petroll, W M; Jester, J V et al. (1994) The relation between contact lens oxygen transmissibility and binding of Pseudomonas aeruginosa to the cornea after overnight wear. Ophthalmology 101:371-88
Ichijima, H; Petroll, W M; Jester, J V et al. (1993) In vivo confocal microscopic studies of endothelial wound healing in rabbit cornea. Cornea 12:369-78
Ichijima, H; Petroll, W M; Barry, P A et al. (1993) Actin filament organization during endothelial wound healing in the rabbit cornea: comparison between transcorneal freeze and mechanical scrape injuries. Invest Ophthalmol Vis Sci 34:2803-12
Cavanagh, H D; Petroll, W M; Alizadeh, H et al. (1993) Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease. Ophthalmology 100:1444-54
Cavanagh, H D; Petroll, W M; Jester, J V (1993) The application of confocal microscopy to the study of living systems. Neurosci Biobehav Rev 17:483-98
Ichijima, H; Ohashi, J; Petroll, W M et al. (1993) Morphological and biochemical evaluation for rigid gas permeable contact lens extended wear on rabbit corneal epithelium. CLAO J 19:121-8
Petroll, W M; Cavanagh, H D; Jester, J V (1993) Three-dimensional imaging of corneal cells using in vivo confocal microscopy. J Microsc 170:213-9
Ichijima, H; Petroll, W M; Jester, J V et al. (1992) Effects of increasing Dk with rigid contact lens extended wear on rabbit corneal epithelium using confocal microscopy. Cornea 11:282-7

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