Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles

Chloroquine (CQ)-resistant Plasmodium falciparum malaria has been a global health catastrophe, yet much about the CQ resistance (CQR) mechanism remains unclear. Hallmarks of the CQR phenotype include reduced accumulation of protonated CQ as a weak base in the digestive vacuole of the erythrocyte-stage parasite, and chemosensitization of CQ-resistant (but not CQ-sensitive) P. falciparum by agents such as verapamil. Mutations in the P. falciparum CQR transporter (PfCRT) confer CQR; particularly important among these mutations is the charge-loss substitution K→T at position 76. Dictyostelium discoideum transformed with mutant PfCRT expresses key features of CQR including reduced drug accumulation and verapamil chemosensitization.We describe the isolation and characterization of PfCRT-transformed, hematin-free vesicles from D. discoideum cells. These vesicles permit assessments of drug accumulation, pH, and membrane potential that are difficult or impossible with hematin-containing digestive vacuoles from P. falciparum-infected erythrocytes. Mutant PfCRT-transformed D. discoideum vesicles show features of the CQR phenotype, and manipulations of vesicle membrane potential by agents including ionophores produce large changes of CQ accumulation that are dissociated from vesicular pH. PfCRT in its native or mutant form blunts the ability of valinomycin to reduce CQ accumulation in transformed vesicles and decreases the ability of K(+) to reverse membrane potential hyperpolarization caused by valinomycin treatment.Isolated vesicles from mutant-PfCRT-transformed D. discoideum exhibit features of the CQR phenotype, consistent with evidence that the drug resistance mechanism operates at the P. falciparum digestive vacuole membrane in malaria. Membrane potential apart from pH has a major effect on the PfCRT-mediated CQR phenotype of D. discoideum vesicles. These results support a model of PfCRT as an electrochemical potential-driven transporter in the drug/metabolite superfamily that (appropriately mutated) acts as a saturable simple carrier for the facilitated diffusion of protonated CQ

Generation of a new bioluminescent model for visualisation of mammary tumour development in transgenic mice

<p>Abstract</p> <p>Background</p> <p>Numerous transgenic models have been generated to study breast cancer. However, despite many advantages, traditional transgenic models for breast cancer are also burdened with difficulties in early detection and longitudinal observation of transgene-induced tumours, which in most cases are randomly located and occur at various time points. Methods such as palpation followed by mechanical measurement of the tumours are of limited value in transgenic models. There is a crucial need for making these previously generated models suitable for modern methods of tumour visualisation and monitoring, e.g. by bioluminescence-based techniques. This approach was successfully used in the current study.</p> <p>Results</p> <p>A new mouse strain (MMTV-Luc2 mice) expressing Luc2 luciferase primarily in mammary tissue in females, with low-level background expression in internal organs, was generated and bred to homozygosity. After these mice were intercrossed with MMTV-PyVT mice, all double transgenic females developed mammary tumours by the age of 10 weeks, the localisation and progression of which could be effectively monitored using the luminescence-based <b><it>in vivo</it></b> imaging. Luminescence-based readout allowed for early visualisation of the locally overgrown mammary tissue and for longitudinal evaluation of local progression of the tumours. When sampled <b><it>ex vivo</it></b> at the age of 10 weeks, all tumours derived from MMTV-Luc2PyVT females displayed robust bioluminescent signal.</p> <p>Conclusions</p> <p>We have created a novel transgenic strain for visualisation and longitudinal monitoring of mammary tumour development in transgenic mice as an addition and/or a new and more advanced alternative to manual methods. Generation of this mouse strain is vital for making many of the existing mammary tumour transgenic models applicable for <b><it>in vivo</it></b> imaging techniques.</p