At least 53 distinct isoforms of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein are expressed in strains or isolates of P. falciparum malarial parasites from around the globe. These parasites exhibit a range of sensitivities to chloroquine (CQ) and other drugs. Mutant PfCRT is believed to confer cytostatic CQ resistance (CQRCS) by transporting CQ away from its DV target (free heme released upon hemoglobin digestion). One theory is that variable CQ transport catalyzed by these different PfCRT isoforms is responsible for the range of CQ sensitivities now found for P. falciparum. Alternatively, additional mutations in drug-selected parasites, or additional functions of PfCRT, might complement PfCRT-mediated CQ transport in conferring the range of observed resistance phenotypes. To distinguish between these possibilities, we recently optimized a convenient method for measuring PfCRT-mediated CQ transport, involving heterologous expression in Saccharomyces cerevisiae. Here, we use this method to quantify drug transport activity for 45 of 53 of the naturally occurring PfCRT isoforms. Data show that variable levels of CQR likely depend upon either additional PfCRT functions or additional genetic events, including perhaps changes that influence DV membrane potential. The data also suggest that the common K76T PfCRT mutation that is often used to distinguish a P. falciparum CQR phenotype is not, in and of itself, a fully reliable indicator of CQR status.

The function of P. falciparum chloroquine resistance transporter (PfCRT) can be quantified using a S. cerevisiae model system (Baro, N. K., Pooput C and Roepe P.D. Biochemistry. 50, 6701 – 6710). We further optimize this system to distinguish PfCRT isoforms found in P. falciparum strains and isolates from across the globe. We create and express 13 naturally occurring pfcrt alleles associated with a range of chloroquine resistant (CQR) phenotypes. Using galactose induction of PfCRT we quantify PfCRT and chloroquine (CQ) dependent yeast growth inhibition, and [3H]-CQ transport specifically due to a given PfCRT isoform. Surprisingly, we find poor correlation between these parameters vs CQ IC50 observed in strains of malaria harboring the same isoforms. This suggests that increased CQ transport due to PfCRT mutation is necessary, but not sufficient, for the range of CQ IC50 observed in globally distributed CQR P. falciparum isolates.

Southeast Asia is an epicenter of multidrug-resistant Plasmodium falciparum strains. Selective pressures on the subcontinent have recurrently produced several allelic variants of parasite drug resistance genes, including the P. falciparum chloroquine resistance transporter (pfcrt). Despite significant reductions in the deployment of the 4-aminoquinoline drug chloroquine (CQ), which selected for the mutant pfcrt alleles that halted CQ efficacy decades ago, the parasite pfcrt locus is continuously evolving. This is highlighted by the presence of a highly mutated allele, Cam734 pfcrt, which has acquired the singular ability to confer parasite CQ resistance without an associated fitness cost. Here, we used pfcrt-specific zinc-finger nucleases to genetically dissect this allele in the pathogenic setting of asexual blood-stage infection. Comparative analysis of drug resistance and growth profiles of recombinant parasites that express Cam734 or variants thereof, Dd2 (the most common Southeast Asian variant), or wild-type pfcrt, revealed previously unknown roles for PfCRT mutations in modulating parasite susceptibility to multiple antimalarial agents. These results were generated in the GC03 strain, used in multiple earlier pfcrt studies, and might differ in natural isolates harboring this allele. Results presented herein show that Cam734-mediated CQ resistance is dependent on the rare A144F mutation that has not been observed beyond Southeast Asia, and reveal distinct impacts of this and other Cam734-specific mutations on CQ resistance and parasite growth rates. Biochemical assays revealed a broad impact of mutant PfCRT isoforms on parasite metabolism, including nucleoside triphosphate levels, hemoglobin catabolism and disposition of heme, as well as digestive vacuole volume and pH. Results from our study provide new insights into the complex molecular basis and physiological impact of PfCRT-mediated antimalarial drug resistance, and inform ongoing efforts to characterize novel pfcrt alleles that can undermine the efficacy of first-line antimalarial drug regimens.

Chloroquine (CQ) resistance (CQR) in Plasmodium falciparum malaria is widespread and has limited the use of CQ in many regions of the globe. Malaria caused by the related human parasite P. vivax is as widespread as is P. falciparum malaria and has been treated with CQ as extensively as has P. falciparum, suggesting that P. vivax parasites have been selected with CQ as profoundly as have P. falciparum parasites. Indeed, a growing number of clinical reports have presented data suggesting increased P. vivax CQR. Cytostatic (growth inhibitory) CQR for P. falciparum is caused by Plasmodium falciparum chloroquine resistance transporter (PfCRT) mutations, and it has been proposed that mutations in the PvCRT orthologue may simliarly cause P. vivax CQR via increasing CQ transport from the P. vivax digestive vacuole. Here we report the first quantitative analysis of drug transport mediated by all known mutant isoforms of Plasmodium vivax chloroquine resistance transporter (PvCRT) in order to test the protein's potential link to growing P. vivax CQR phenomena. Small, but statistically significant, differences in the transport of CQ and other quinoline antimalarial drugs were found for multiple PvCRT isoforms, relative to wild type PvCRT, suggesting that mutations in PvCRT can contribute to P. vivax CQR and other examples of quinoline antimalarial drug resistance.

Background:We have previously identified potent novel antimalarial compounds with an unknown mode of action. Results: A photo-reactive affinity capture method was used to identify parasite proteins that interact with these antimalarials. Conclusion: ACT-213615 interacts with Plasmodium falciparum multidrug resistance protein 1 (PfMDR1). Significance: This photo-reactive affinity capture method can be generally used to identify drug targets in live cells.

BackgroundRecent work has perfected yeast-based methods for measuring drug transport by the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT).MethodsThe approach relies on inducible heterologous expression of PfCRT in Saccharomyces cerevisiae yeast. In these experiments selecting drug concentrations are not toxic to the yeast, nor is expression of PfCRT alone toxic. Only when PfCRT is expressed in the presence of CQ is the growth of yeast impaired, due to inward transport of chloroquine (CQ) via the transporter.ResultsDuring analysis of all 53 known naturally occurring PfCRT isoforms, two isoforms (PH1 and PH2 PfCRT) were found to be intrinsically toxic to yeast, even in the absence of CQ. Additional analysis of six very recently identified PfCRT isoforms from Malaysia also showed some toxicity. In this paper the nature of this yeast toxicity is examined. Data also show that PH1 and PH2 isoforms of PfCRT transport CQ with an efficiency intermediate to that catalyzed by previously studied CQR conferring isoforms. Mutation of PfCRT at position 160 is found to perturb vacuolar physiology, suggesting a fitness cost to position 160 amino acid substitutions.ConclusionThese data further define the wide range of activities that exist for PfCRT isoforms found in P. falciparum isolates from around the globe.Graphical abstractV5 Western blot quantifying relative PfCRT expression in total (CM) vs vacuolar (VAC) yeast membranes for unmodified HB3 PfCRT vs HB3 and PH1 PMA–PfCRT chimeras.

. (1975). British Journal ofIndustrial Medicine, 32,[228][229][230][231][232][233][234] Chest symptoms in farming communities with special reference to farmer's lung. Surveys were carried out on random samples of the farming population in Devon and Wales in order to estimate the prevalence of respiratory symptoms and of positive precipitin reactions to thermophilic fungi. Bronchitis, as defined, was common among the Welsh hill farmers, and the proportion of positive serological tests was higher in both the areas surveyed in Wales as compared with Devon. All three surveys confirmed a previous finding that the proportion of positive precipitin tests was higher among non-smokers than smokers. Although the numbers were small there was some indication that measurement of peak expiratory flow showed different relationships with age in non-smokers according to the presence or absence of positive precipitin tests. The difficulty of determining prevalence rates for farmer's lung is discussed, but the results suggest a rate not dissimilar to those found in two areas of Scotland which were more than 20 times higher than any figure previously reported in Britain.During the winters 1970/71 and 1971/72, surveys of four representative farming populations were made.

Metastases to the urethra are rare. Treatment options have to be individualised to the extent of the disease and the symptoms of the patient. Immunohistochemical staining can help to a certain extent to point the direction towards the possible primary lesion. Atypical presentations of urethral lesions should be viewed with suspicion. A biopsy of the lesion is the only way of confirming diagnosis.