' Konopa et al. 1980; b Schaller et al., 1996; c Büssing et al. 1999c.

' Konopa et al. 1980; b Schaller et al., 1996; c Büssing et al. 1999c.

album ssp. album lacks 1-PS and U-PS, but Viscum album ssp. abietis contains all six viscotoxins (Schaller et al., 1996).

The viscotoxins exert their cytotoxicity by a rapid permeabilisation of the cell membrane. Within 1-2 h, treatment of lymphocytes with viscotoxins resulted in a loss of cell membrane asymetry with translocation of phosphatidyl serine from the inner to the outer leaflet of the cell membrane, uptake of the DNA-intercalating dye propidium iodide, generation of reactive oxygen intermediates (ROI), swelling of mitochondria with loss of their cristae, and degradation of cytoplasma and chromatin (Büssing et al., 1998c, 1999b, c, f). These rapid changes indicate accidental (necrotic) cell death, with an induction of apoptosis in the "surviving" cells later on (Büssing et al., 1999c, f).

The main important question is how the viscotoxins induce cell death. The capability of thionins to form pores in biological membranes is supported by their 3-dimensional structures, as the amphiphilic thionins may interact with amphiphilic phospholipids (Osario e Castro et al., 1989, 1990; Angerhofer et al., 1990; Teeter et al., 1990; Vernon, 1996). There are several effects observed when cells were treated with Pyrularia thionin: the early responses of an increased order parameter of the phospholipids, an increase in membrane permeability, depolarisation of the cell membrane, and probably opening of Ca2+ channels (Vernon, 1996); long term responses include the formation of membrane blebs, and finally activation of endogenous phospholipase A2 and adenylate cyclase (Evans et al., 1989; Vernon, 1996). It is suggested that the viscotoxins affect membranes, resulting in rapid cell membrane permeabilisation and subsequently an excess generation of ROI that are no longer detoxified by cellular components with redoxidation potential, such as glutathione and membrane cardiolipin. Indeed, neither reduced glutathione nor N-acetyl-L-cysteine (5 and 10 mM) were able to, prevent VT-mediated cytotoxicity in cultured human lymphocytes (Büssing et al., 1999c, f). However, other polycationic and/or amphipathic molecules, such as poly-L-arginine, poly-L-lysine, poly-L-glutamic acid, protamine sulfate, purothionin, and mastoparan I and II, did not sufficiently kill the lymphocytes (unpublished own results), indicating that the polycationic structure alone is not sufficient to affect the cells. The cytotoxicity of viscotoxins was prevented only by cleavage of disulphide bounds; this may affect interaction of viscotoxins with membrane lipids.

Apart from cell death, another relevant property of the viscotoxins is the enhancement of the Escherichia coli-induced phagocytosis and oxidative burst of human granulocytes (Stein et al., 1999a, b). This effect may be due to the polycationic structure of viscotoxins, as they were observed (in contrast to their cytotoxic properties) also with polycationic substances such as protamine sulfate, purothionin, histone, poly-L-arginine, poly-L-lysine, but not with the poly-anionic poly-L-glutamic acid (Stein et al., 1999a).

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