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The interaction of VacA with membranes involves: (i) a low pH activation that induces VacA monomerization in solution, (ii) binding of the monomers to the membrane, (iii) oligomerization and (iv) channel formation. To better understand the structure–activity relationship of VacA, we determined its topology in a lipid membrane by a combination of proteolytic, structural and fluorescence techniques. Residues 40–66, 111–169, 205–266, 548–574 and 723–767 were protected from proteolysis because of their interaction with the membrane. This last peptide was shown to most probably adopt a surface orientation. Both α-helices and β-sheets were found in the structure of the protected peptides.

The CD4 receptor synergizes with the T-cell antigen receptor (TCR) in helper T-cell activation. However CD4 crosslinking in the absence of simultaneous TCR engagement leaves the cells primed to activation dependent apoptosis. To assess the role of the CD4 associated protein tyrosine kinase p56lck in CD4 priming to apoptosis we have constructed Jurkat T-cell lines stably transfected with a constitutively active form of p56lck. These cells were constitutively primed to undergo apoptosis upon TCR crosslinking with specific antibodies. In addition the Jurkat JCaM1 line, which is defective for p56lck expression, was resistant to TCR induced apoptosis. These data indicate that p56lck is required for T-cell apoptosis and that CD4 priming of T-cells for antigen dependent apoptosis is due to inappropriate or partial activation of the p56lck signal transduction pathway.

A mammalian expression vector directing the synthesis of a cytoplasmic single chain Fv version of the Y13—259 anti-Ras antibody was constructed and co-transfected into the human lymphoid cell line Jurkat together with a reporter construct containing the bacterial gene for chloramphenicol acetyl transferase under the transcriptional control of several copies of the binding site for the transcription factor NF-AT. The Ras specific antibody interferes with NF-AT activation upon direct activation of the T-cell antigen receptor, whereas activation by direct protein kinase C stimulation is less sensitive to the anti-Ras antibody. Furthermore, the observed inhibition is dependent on the ratio of antibody to reporter plasmid utilized in the transfection experiments.

Activation of T-cells infected by HIV-1 results in activation of long terminal repeal (LTR)-dependent viral transcription and ultimately the production of infectious virus. Although fulI T-cell activation requires a complex series of intracellular signals, including protein kinase C activation, calcium mobilisation, and less-well defined lymphokine-induced signals, the HIV-1 LTR can be activated by subsets of these signals. We have studied the interaction of these signals in the human lymphoma line, Jurkat, in activation of the HIV-1 LTR. The HIV promoter was induced by IL-1 and phorbol ester activation of PKC but not by a calcium ionophore. The constitutively active form of Ha-ras could replace phorbol ester stimulation of the HIV promoter and of a synthetic promoter containing NFκB binding sites.

The Helicobacter pylori toxin VacA induces large membrane-bound vacuolar compartments of late endosomal/lysosomal origin. Pre-treatment of cells with TPA and butyrate enhances the toxin induced vacuolisation up to 20 times, depending on the cell line, whereas other differentiating factors such as DMSO, EGF, valeric and retinoic acid have no effect. The higher toxin sensitivity induced by TPA does not result from an increased surface binding or endocytosis. The effect of TPA is apparent after several hours from addition and is inhibited by a PKC specific inhibitor. These data suggest that expression of cellular proteins, other than the toxin receptor(s), influences the vacuolating activity of VacA and may contribute to the sensitivity of different cell lines. The present findings define the most sensitive in vitro assay of the activity of VacA.