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Title of Thesis

Asifa Ahmed
Institute/University/Department Details
University of Karachi/Department of Biochemistry
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
membrane proteins, corneal epithelium, glycosylation, glycoproteins, protein expression, betoptic, betagan, wound healing

In response to wounding, the corneal epithelium migrates rapidly to cover the defect. Therapeutic means to enhance the migration phase of wound healing after surgery or trauma are limited. An enormous amount of data has accumulated regarding the identification, localization and functions of proteins altered or newly synthesized during the migratory process. However, to date the mechanisms or cell-cell and cell-matrix interactions and the functions of most of the proteins in the migrating of the corneal epithelium have not been elucidated. The present work was undertaken to investigate the differential protein expression occurring during corneal epithelial migration. Organ cultures of rabbit non-migrating (n=126) and migrating (n=224) corneal epithelium were prepared. In addition to investigate the possible role of the proteins expressed during migration the effect of beta-blockers eye drops, Betagan (n=74) and Betoptic (n=74) and their preservative benzalkonium chloride (BAK) (n=74) on epithelial migration with respect to proteins was studied. The migrating corneal epithelium was collected after 48hr of healing in culture.

BAK showed a significant delay in the rate of wound healing (27±0.003 um/hr and 19±0.003 um/hr) (p<0.01) as compared to controls (45±0.092 um/hr) at concentrations of 0.001% and 0.004%, respectivel. At a concentration of 0.0002% of BAK, the rate of wound healing was not significantly different from the normal migrating rate (p>0.05). This concentration of BAK was used to determine the effects of beta-blockers alone (i.e. without preservative). Both Betagan and Betoptic a significant showed delay in the rate of wound healing as compared to their respective control concentrations of BAK (39±0.005 um/hr and 43±0.004 um/hr) (p<0.01). Toxicity at high concentrations shown severe desquamation, detachment and re-opening of wound after long-term use at low concentrations was evident in the histological studies of the beta-blocker treated corneal epithelium. Betagan was found to have a more profound effect. The results of the wound healing and histological studies of beta-blocker and BAK treated samples enabled a reference to suggest a role for the different proteins altered in these samples as compared to the normal migrating pattern.

Using sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis approximately 12 proteins were found to be of importance after silver standing. Of these the most interesting was found to be a 98kd protein with a pl of 5.6 which was found to be present in a 9-fold elevated amount in the migrating as compared to non-migrating corneal epithelium. Following in gel trypsin digestion and analysis of peptide fragments with matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS) this protein was identified as Transglutaminase K (TGase K or 1). The relative quantity of the 98kd protein corresponding to Transglutaminase K was decreased in BAK and Betoptic and was completely absent in Betagan treated samples. This indicated that relevance of transglutaminase in cell-cell interaction.

The glycosylation was increased by approximately 2 times during the migration process as indicated by 14C-glucosamine labeling and fluorography. Glycosylation was observed in the high molecular weight region. Using 3H-mannose and 3H-galactose, it was determined that D-galactose played a significant role in the migration process. D-mannose did not show any significant result, however, D-Galactose was seen to increase in the migrating corneal epithelium as compared to non-migrating corneal epithelium and to facilitate wound healing. Galactose oxidase treatment in combination with DIG labeling also indiated a 98kd galactose-linked glycoprotein on SDS-PAGE.

Apart form the 98kd other proteins were also seen to be elevated in the migrating corneal epithelium. A protein with an apparent molecular weight of 27 kd and pl 8.9 was found to be present in only the normal migrating and BAK and Betoptic treated migrating corneal epithelium. Similar observations were made for a 40kd protein with a pl of 6.8. In addition a 26kd protein with pl of 4.8 was found in only the non-migrating corneal epithelium and Betagan treated migrating corneal epithelium. A 42 kd protein with a pl of 6.3 was also elevated in the migrating corneal epithelium and was present in all three migrating drug-treated samples but was absent in the non-migrating corneal epithelium. A 21kd protein was found exclusively in the normal migrating corneal epithelium.

Two acidic proteins of apparent molecular weight 24kd and 23kd, were seen to be down-regulated in the migrating corneal epithelium. Down-regulation of the 24 kd protein was apparent in all treated migrating corneal epithelium but the 23kd protein was only down-regulated in the Betoptic treated samples.

This study also introduces preliminary work conducted to reveal differential expression of the migrating corneal epithelium at the genetic level. Using differential display-reverse transcriptase polymerase chain reaction, two gene are shown to be expressed in only the migrating corneal epithelium, whereas six were expressed in the non-migrating corneal epithelium only. Further work is reauired for the confirmation of the genes and proteins expressed.

Part of this thesis has been published and presented as:

Ahmad, A., Siddiqui, A.A.,and Ahmed, No. (2000) DDRT-PCR; Use of agarose gels for detection of amplified products. Molecular Vision 6:144-7.

Ahmed, N., Ahmed, A., Siddiqui, A. A. and Shafqat, J. (2001) Differentially expressed proteins during corneal reepithelialization in organ culture. XI International Congres on Gene, Gene Families and Isozymes, Stockholm. (Abs) Ahmed, A., Siddiqui, A. A., and Ahmed, N. (2000) Corneal epithelial migration: Up-regulationof galactose-linked glycoproteins. Proceedings of 18th International Congress of Biochemistry and Molecular Biolgy. 1859 (Abs).

Ahmed, A., Siddiqui, A. A., and Ahmed, N. (1996) Comparison of the protein profile of non-migrating and migrating corneal epithelium in organ culture. Presented at the National Meeting of Chemistry, Quetta.

Ahmed, N., Ahmed, A. and Siddiqui, A.A. (1997) Down-regulation of glycoprotein during wound healing of corneal epithelium. Presented at the 5th International, Symposium of Protein Structure-Function, Karachi.

Download Full Thesis
2561.97 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
197.84 KB
2 1 General Introduction
95.74 KB
  1.1 Historical Background 1-3
  1.2 Experimental Models of Corneal Epithelial Wound Healing 3-4
  1.3 Adhesive Mechanisms in the Corneal Epithelium 4-5
  1.4 Molecular and Cellular Expressions in Corneal Epithelium 5-6
  1.5 Exogenous substances and Corneal Epithelial Wound Healing 6
3 2 General Material and Methods
141.9 KB
  2.1 Experimental Animals 9
  2.2 Preparation of Organ Cultures 9-12
  2.3 Analysis of Corneal Epithelial Wounds 12-3
  2.4 Light Microscopy 13-6
  2.5 Determination of Total Proteins 16-7
  2.6 Determination of Radio Activity 17
  2.7 Harvesting and Extraction of Glycoproteins 17-9
  2.8 Polyacrylamide -Gel Electrophoresis in Sodium Dodecyl Sulfate (SDS-PAGE-10%) 19-1
  2.9 Silver Staining of SDS-Page 21-3
  2.10 Two-Dimensional Polyacrylamide Gel Electrophoresis 23-5
  2.11 Analysis of Protein Bands/Spots 25
4 3 Proteins in the Non-Migrating and Migrating Corneal Epithelium
488.56 KB
  3.1 Introduction 26-28
  3.2 Methods 33
  3.3 Results 37
  3.4 Discussion 60-65
  3.5 Conclusions 66-67
5 4 Glycosylation and Glycoproteins in the Nonmigrating and Migrating Corneal Epithelium
286.29 KB
  4.1 Introduction 68-70
  4.2 Methods 71
  4.3 Results 78
  4.4 Discussion 92-5
  4.5 Conclusions 96
6 5 Differential Gene Expression During Migration of the Corneal Epithelium
228.39 KB
  5.1 Introduction 97-8
  5.2 Methods 99
  5.3 Results 110
  5.4 Discussion 114-16
  5.5 Conclusion 117
7 6 Effect of Beta-Blocker Eye Drops on Corneal Epithelial Migration
972.09 KB
  6.1 Introduction 118-20
  6.2 Methods 121
  6.3 Results 124
  6.4 Discussion 158-62
  6.5 Conclusions 163-64
8 7 General Discussion 165-172
89.97 KB
  7.1 General Discussion 165-170
  7.2 General Conclusions 171-72
9 8 Bibliography 173-06
334.97 KB