Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Frederick M. Enright

Abstract

Responses of cultured mouse fibroblasts, human erythrocytes, bovine erythrocytes, and liposomes with different phosphatidylcholine (PC) and sphingomyelin (SPM) ratios to both natural and synthetic lytic peptides were characterized. Peptide-induced morphological alterations of the plasma membrane of cells were examined by various light microscopic techniques and scanning electron microscopy (SEM). The ability of natural and synthetic peptides to kill cells and cause increased permeability of liposomes was evaluated using Trypan Blue (TB) dye exclusion assay and fluorescent dye leakage assay, respectively. Differential interference contrast microscopy, SEM, and fluorescence studies revealed characteristic structural and lipid changes in the plasma membrane of lytic peptide-treated fibroblasts, and these alterations were accompanied by simultaneous changes in the cell permeability as indicated by the uptake of TB. Formation of membrane vesicles, composed primarily of lipids, was demonstrated in cells treated with a low lethal dose of melittin and eventually resulted in the liberation of membrane lipids. SEM revealed that much of the plasma membrane was lost by 5 minutes following peptide exposure. Confocal microscopy confirmed the translocation of membrane proteins from the cell surface to cytoplasmic areas in peptide-treated cells. A fluorescently labeled peptide was used to demonstrate the reaction of the peptides with the plasma membrane. The measurements of the ability of natural and synthetic peptides to kill fibroblasts allowed classification of these peptides into four groups. The results also suggested that peptide length and substitution of glycine for alanine affected the potency of synthetic peptides. Differential susceptibility of different cell types to destruction by the same peptides was demonstrated in mouse fibroblasts and human and bovine erythrocytes. These peptides were less efficient in lysing erythrocytes than in destroying fibroblasts. This difference in activity was possibly due to the higher content of membrane SPM in the resistant RBCs. Lytic peptides demonstrated less ability to permeate liposomes composed of 70% SPM. Synergistic effects were demonstrated in mammalian cells using weak lytic peptides and exogenous phospholipases. The effect was dependant upon the phospholipid composition of the target cells. Cell susceptibility and synergy between peptides and membrane reactive enzymes are important factors in the selection of therapeutic peptides.

ISBN

9780591204063

Pages

144

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