In this project, the N-acetyl-ex-D-galactosamine (GalNAc) binding lectin from the green marine alga Codium fragile ssp. tomentosoides (C. fragile) was purified and techniques developed to label the lectin for visualisation by light and electron microscopy and electrophoresis. This represents the first time a histochemical reagent has been developed from a marine alga. The new reagent was initially assessed for transmission electron microscopy using human blood group A1 erythrocytes. A novel method gave a topographical view, and showed the distribution of gold particles on the surface of erythrocytes. The pattern of C. fragile lectin binding to pig normal oral epithelium was studied in the environmental scanning electron microscope to avoid charging artefact, using paraffin wax sections of pig normal epithelium stained with C. fragile lectin-gold conjugate enhanced with silver. X-ray micro analysis demonstrated lectin binding on the plasma membrane surface of epithelial cells at cells to cell contacts suggesting binding to cellular adhesion molecules. Biotinylated lectins binding GalNAc were used to investigate, identify and compare the binding of lectins in pig normal oral epithelium and altered glycoconjugates in cultured malignant cells from human oral tumours, using lectin histochemistry in the light microscope. Lectin from C. fragile was compared with Dolichos biflorus, a lectin from plants, and Helix pomatia (HP A) from snails. Although each lectin binds GalNAc it was shown that their binding pattern to pig normal oral epithelium was different, demonstrating that these lectins could be used to identify altered cellular glycosylation in the normal cellular maturation process. Cultured human oral tumour cells from different grades of malignancy were investigated using this panel of lectins. Binding of GalNAc specific lectins to cultured tumour cells changed in relation to their level of differentiation. This discriminating capability of GalNAc specific lectins offers exciting potential as indicators of tumour prognosis in human oral epithelial tumours. The lectins from C. fragile and HP A gave very similar staining results using histochemistry. Binding of these lectins to cell membrane glycoproteins was investigated using electrophoresis to show that C. fragile lectin binds to more and different cell membrane glycoproteins than lectin from HP A, but did not bind to purified CD44, excluding this adhesion molecule as a candidate for binding these lectins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:271046 |
| Date | January 2002 |
| Creators | Griffin, Raymond Leonard |
| Publisher | University of the West of England, Bristol |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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