Antifungal activity of the ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris†L.) against the green mould Penicillium digitatum.

The ribosome-inactivating protein BE27 from sugar beet (Beta vulgaris L.) leaves is an apoplastic protein induced by signalling compounds, such as hydrogen peroxide and salicylic acid, which has been reported to be involved in defence against viruses. Here, we report that, at a concentration much lower than that present in the apoplast, BE27 displays antifungal activity against the green mould Penicillium digitatum, a necrotrophic fungus that colonizes wounds and grows in the inter- and intracellular spaces of the tissues of several edible plants. BE27 is able to enter into the cytosol and kill fungal cells, thus arresting the growth of the fungus. The mechanism of action seems to involve ribosomal RNA (rRNA) N-glycosylase activity on the sarcin-ricin loop of the major rRNA which inactivates irreversibly the fungal ribosomes, thus inhibiting protein synthesis. We compared the C-terminus of the BE27 structure with antifungal plant defensins and hypothesize that a structural motif composed of an α-helix and a ß-hairpin, similar to the γ-core motif of defensins, might contribute to the specific interaction with the fungal plasma membranes, allowing the protein to enter into the cell.

In vitro and in vivo effects of an anti-mouse endoglin (CD105)-immunotoxin on the early stages of mouse B16MEL4A5 melanoma tumours.

TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC50 values of 4.2 × 10(-9) M for B16MEL4A5 and 7.7 × 10(-11) M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells.