Importance of polarity of the α4-α5 loop residue-Asn(166) in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for ion permeation and pore opening.

Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn(166) located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation.

Overcoming multidrug resistance in human lung cancer with novel benzo[a]quinolizin-4-ones.

Aim: To investigate the ability of synthetic benzo[a]quinolizin-4-one derivatives to reverse multidrug resistance (MDR) in lung cancer cells.

Materials And Methods: A cell line with MDR, A549RT-eto, was established by exposure to 1.5 μM etoposide.

Insertion behavior of the Bacillus thuringiensis Cry4Ba insecticidal protein into lipid monolayers.

Toxicity mechanisms of Bacillus thuringiensis Cry insecticidal proteins involve membrane insertion and lytic pore formation in lipid bilayers of the target larval midgut cell membranes. The B. thuringiensis Cry4Ba mosquito-larvicidal protein has been shown to be capable of permeabilizing liposome vesicles and of forming ion channels in planar lipid bilayers.

Specific mutations within the alpha4-alpha5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170.

The widely accepted model for toxicity mechanisms of the Bacillus thuringiensis Cry delta-endotoxins suggests that helices alpha4 and alpha5 form a helix-loop-helix hairpin structure to initiate membrane insertion and pore formation. In this report, alanine substitutions of two polar amino acids (Asn-166 and Tyr-170) and one charged residue (Glu-171) within the alpha4-alpha5 loop of the 130-kDa Cry4B mosquito-larvicidal protein were initially made via polymerase chain reaction-based directed mutagenesis. As with the wild-type toxin, all of the mutant proteins were highly expressed in Escherichia coli as inclusion bodies upon isopropyl-beta-Dthiogalactopyranoside induction.