Two pairs of back-to-back α-helices of Kingella kingae RtxA toxin are crucial for the formation of a membrane pore.

The RtxA cytotoxin, a member of the RTX (Repeats in ToXin) family of pore-forming toxins, is the primary virulence factor of the paediatric facultative pathogen Kingella kingae. Although structure-function studies of RTX toxins have defined their characteristic domains and features, the exact membrane topology of RTX toxins remains unknown. Here, we used labelling of cell-bound RtxA with a membrane-impermeable, lysine-reactive reagent and subsequent detection of the labelled lysine residues by mass spectrometry, which revealed that most of the membrane-bound toxin is localised extracellularly.

The Lysosomal Sequestration of Tyrosine Kinase Inhibitors and Drug Resistance.

The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes.

Estimation of ABCB1 concentration in plasma membrane.

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples.

Apoptosis Induced by the Curcumin Analogue EF-24 Is Neither Mediated by Oxidative Stress-Related Mechanisms nor Affected by Expression of Main Drug Transporters ABCB1 and ABCG2 in Human Leukemia Cells.

The synthetic curcumin analogue, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24), suppresses NF-κB activity and exhibits antiproliferative effects against a variety of cancer cells in vitro. Recently, it was reported that EF-24-induced apoptosis was mediated by a redox-dependent mechanism. Here, we studied the effects of -acetylcysteine (NAC) on EF-24-induced cell death.

Reversal of ABCB1 mediated efflux by imatinib and nilotinib in cells expressing various transporter levels.

Recently, it has been suggested that imatinib (IM) and nilotinib (NIL) could be studied beyond their original application, as inhibitors of the drug efflux pump ABCB1 (P-glycoprotein, MDR1). Since the reversal of ABCB1-mediated resistance has never been successfully demonstrated in the clinic, we addressed the question of whether IM and NIL may actually serve as efficient inhibitors of ABCB1. Here we define an efficient inhibitor as a compound that achieves full (90-100%) reversal of drug efflux at a concentration that does not exhibit significant off-target toxicity in vitro.