Towards therapeutic antibodies to membrane oncoproteins by a robust strategy using rats immunized with transfectants expressing target molecules fused to green fluorescent protein.

Cell-surface molecules containing growth factor receptors, adhesion molecules and transporter proteins are often over-expressed in various cancer cells, and could be regarded as suitable targets for therapeutic monoclonal antibodies (mAb). Anti-cancer therapeutic mAb are claimed to bind these cell-surface molecules on viable cancer cells: therefore, it is necessary to produce mAb recognizing epitopes on the extracellular domains of native but not denatured proteins. We have experienced difficulty in obtaining mAb bound to viable cancer cells using synthetic peptides or recombinant proteins produced in bacteria as immunogens, although these immunogens are relatively easy to prepare. In this context, we have concluded that viable cancer cells or cells transfected with cDNA encoding target proteins are suitable immunogens for the production of anti-cancer therapeutic mAb. Furthermore, we selected rats as the immunized animals, because of their excellent capacity to generate diverse antibodies. Because many target candidates are multi-pass (type IV) membrane proteins, such as 7-pass G protein-coupled receptors and 12-pass transporter proteins belonging to the solute carrier family, and their possible immunogenic extracellular regions are very small, production of specific mAb was extremely difficult. In this review, we summarize the successful preparation and characterization of rat mAb immunized against the extracellular domain of type I, type II and type IV membrane oncoproteins fused to green fluorescent protein as an approach using reverse genetics, and also introduce the discovery of cell-death-inducing antibodies as an approach using forward genetics and a strategy to produce reshaped antibodies using mimotope peptides as the immunogen.

Ouabain exerts cytoprotection by diminishing the intracellular K(+) concentration increase caused by distinct stimuli in human leukemic cells.

OBJECTIVES: We tested if modulation of cytosolic K(+) levels by ouabain, an inhibitor of Na(+) /K(+) -ATPase, exerts cytoprotection against distinct stressful stimuli in human leukemic cells. METHODS: The cytosolic K(+) , Na(+) or Ca(2+) levels and the cytotoxicity were evaluated by flow cytometry. KEY FINDINGS: Various cytotoxic chemicals and ultraviolet irradiation induced cell death and increased intracellular concentrations of K(+) , Na(+) or Ca(2+) . Ouabain reduced the cytotoxicity and the elevation of cytosolic levels of K(+) but not those of Na(+) or Ca(2+) . CONCLUSIONS: Our data thus suggest that elevated cytosolic K(+) levels are associated with the cytotoxicity in response to distinct stressful stimuli and that ouabain exerts cytoprotection most probably by regulating intracellular K(+) levels.

Biological activities of synthetic saponins and cardiac glycosides.

Comparisons of the biological activities of diosgenyl (DIO) beta-D-xylopyranosyl- (1 --> 2)-alpha-D-glucopyranoside (2DXalpha) and beta-D-glucopyranosyl- (1 --> 3)-beta-D-glucopyranoside (3GLbeta) or methyl glycyrrhetinate (GAM) 2DXalpha and 3GLbeta with those of the other DIO- or GAM- glycosides confirmed our assumption that both haemolytic and antifungal activities of DIO-glycosides (steroid saponins) generally parallel each other, while almost all haemolytic GAM-glycosides (triterpenoid saponins) have no antifungal activity. By comparisons of digitoxigenyl (DIG) 2DXalpha, beta-D-xylopyranosyl- (1 --> 2)-beta-D-glucopyranoside (2DXbeta) and 3GLbeta with the other DIG-glycosides, the previous supposition on DIG-diglycosides (cardiac diglycosides) has been developed into the assumption that those having a (1 --> 4), (1 --> 3) or (1 --> 2)beta sugar linkage show stronger anti-ATPase and antiviral activities than those with a (1 --> 6) or (1 --> 2)alpha linkage.