5x105 L.major promastigotes were cultured in 5 mL modified DMEM supplemented with 10% FCS. At the end of 5 days of culture, the stationary phase promastigotes were harvested and resuspended in Hanks balanced salt solution at a cell density of 4x107/mL. The cell suspension was aspirated into a 1 mL syringe and 50 J.!L was injected into the footpad of mice. The mice were returned to the cage and fed ab-limitum. The onset and progression of cutaneous lesion was monitored at 2 weekly intervals by observing an increase in the thickness of the footpad

L.major infection in mouse footpad

The aggregation of the phospholipid vesicles, induced by ribotoxins, was assayed by titrating 40 nmoles of freshly prepared lipid vesicles with various molar concentrations of restrictocin and its mutants. The samples of I ml volume were prepared in 30 mM Tris-HCl buffer, pH 7.0, containing O.IM NaCl and incubated at 37 °C for I h. The change in absorbance due to an increase in turbidity of the lipid suspension was measured at 400 nm in Lambda Bio 20 spectrophotometer (Perkin Elmer) in the cells of I em optical path length. Appropriate control proteins, not interacting with the membranes and the vesicles without proteins were included in all the experiments. The change in absorbance of the lipid suspension was plotted against the toxin concentration.

Aggregation of Lipid Vesicles

~hCG and HBsAg sequences were accessed from GenBank or NBRF database on a Microvax II computer and sequence analysis performed using the HPLOT and AMPHI programmes. HPLOT is based on the algorithm of Kyte and Doolittle ( 1982 ) and plots the hydrophobic and hydrophilic segments of the protein by scanning the whole length of the sequence in blocks of a few residues. The window size used was 6 amino acids. AMPHI is based on the algorithm of Margalit et al., ( 1985 ) and predicts the amphipathic segments of the proetin which correspond to alpha helices and are therefore, likely candidates for being T cell epitopes.

Computer analysis.