SummaryResistance to quinoline antimalarial drugs has emerged in different parts of the world and involves sets of discrete mutational changes in pfcrt and pfmdr1 in the human malaria parasite Plasmodium falciparum. To better understand how the different polymorphic haplotypes of pfmdr1 and pfcrt contribute to drug resistance, we have conducted a linkage analysis in the F1 progeny of a genetic cross where we assess both the susceptibility and the amount of accumulation of chloroquine, amodiaquine, quinine and quinidine. Our data show that the different pfcrt and pfmdr1 haplotypes confer drug-specific responses which, depending on the drug, may affect drug accumulation or susceptibility or both. These findings suggest that PfCRT and PfMDR1 are carriers of antimalarial drugs, but that the interaction with a drug interferes with the carriers' natural transport function such that they are now themselves targets of these drugs. How well a mutant PfCRT and PfMDR1 type copes with its competing transport functions is determined by its specific sets of amino acid substitutions.

The emerging resistance to quinine jeopardizes the efficacy of a drug that has been used in the treatment of malaria for several centuries. To identify factors contributing to differential quinine responses in the human malaria parasite Plasmodium falciparum, we have conducted comparative quantitative trait locus analyses on the susceptibility to quinine and also its stereoisomer quinidine, and on the initial and steady-state intracellular drug accumulation levels in the F1 progeny of a genetic cross. These data, together with genetic screens of field isolates and laboratory strains associated differential quinine and quinidine responses with mutated pfcrt, a segment on chromosome 13, and a novel candidate gene, termed MAL7P1.19 (encoding a HECT ubiquitin ligase). Despite a strong likelihood of association, episomal transfections demonstrated a role for the HECT ubiquitin-protein ligase in quinine and quinidine sensitivity in only a subset of genetic backgrounds, and here the changes in IC50 values were moderate (approximately 2-fold). These data show that quinine responsiveness is a complex genetic trait with multiple alleles playing a role and that more experiments are needed to unravel the role of the contributing factors.

In recent years, the methylotrophic Pichia pastoris has become an increasingly famous host for recombinant protein expressions. As a eukaryote, this yeast offers several advantages, including simplicity of genetic manipulation, stable expression, and low-cost scalable fermentation techniques. Previous study has confirmed the insertion of Dengue Virus Serotype 3 (DENV3) pre-Membrane Envelope (pr-M/E) gene in the recombinant P. pastoris X33 generated in our laboratory. The study has also confirmed the strain's ability to express the protein.In this study, we are reporting the genetic stability of the recombinant strain, confirming the steady expression of the heterologous protein in subsequent generations. The genetic stability test was performed by PCR and DNA sequencing on the recombinant P. pastoris cultured in non-baffled shake flasks. Generation time was estimated based on the yeast growth curve and calculated using a previously published formula. According to the growth curve, the generation time of this correlates recombinant yeast is four hours. It differs from the wild type, which took 4.3 h to complete. PCR of target gene performed at generation 1, 18, 39, 56, 81, and 100 revealed two DNA bands which indicating the presence of full AOX1 gene (2.2 kb) and AOX1 promoter plus pr-M/E gene (2 kb). In addition, sequencing of the PCR products show only minor variation, which might have been genuine or a result of PCR or sequencing errors. However, because the amino acid sequences of generation 100 differed from neither the RefSeq nor the original plasmid, we predict that our recombinant P. pastoris stably contains the gene of interest.

No abstract