Results: 4

1.
Figure 2

Figure 2. Identification of vesicles enriched for PfCRT and levels of radiolabeled drug accumulation.. From: Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles.

(A) Semi-quantitative immunoblot (WB) analysis of membrane fractions precipitated at 1000×g (F2), 2000×g (F3), 4000×g (F4), 20,000×g (F5), and 100,000×g (F6). Top, middle, and bottom panels show results from the isolated vesicles of untransformed, SEA-CRT-, and WT-CRT-transformed cells. Fractions F1 and F7 represent samples of whole cells and supernatant, respectively. (B) Electron micrograph of a sample from F5. (C) Labeled drug uptake in F5 vesicles incubated for 10 min with 100 nM [3H]-CQ, [3H]-QN, or [3H]-PPQ in VSB with or without 1 mM ATP. Histograms compare results with fresh (solid bars) and stored frozen F/T vesicles (hashed bars) prepared from untransformed, SEA-CRT-, and WT-CRT-transformed D. discoideum. Error bars indicate standard deviations of measurements from three independent measurements on samples from the F/T vesicle preparation.

Janni Papakrivos, et al. PLoS One. 2012;7(6):e39569.
2.
Figure 3

Figure 3. Effects of buffer conditions on [3H]-CQ accumulation and pH in F/T vesicles from untransformed, SEA-CRT-, and WT-CRT-transformed Dictyostelium discoideum.. From: Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles.

(A) [3H]-CQ uptake by F/T vesicles in VSB vs. vesicles exposed to 80 μM VP in VSB, 2 μM CCCP in VSB, OS, or MCB. Levels in vesicles isolated from whole cells pre-incubated in 100 nM [3H]-CQ and 80 μM VP are shown at the right (VP-C). (B) Radiolabel uptake by F/T vesicles exposed to 100 nM [3H]-CQ in VSB containing various concentrations of NH3 (0.0, 0.1 or 1 mM; left panel) or various concentrations of additional unlabeled CQ (1, 10, 100 or 1000 µM; right panel). (C) pH determinations of isolated FITC-dextran loaded vesicles incubated for 10 min in the presence of various concentrations of CQ or 1 mM ammonia. Error bars indicate standard deviations of measurements from three independent measurements on samples from the F/T vesicle preparation.

Janni Papakrivos, et al. PLoS One. 2012;7(6):e39569.
3.
Figure 1

Figure 1. Measures of [3H]-CQ uptake, ATP levels, and vesicle pH in untransformed, SEA-CRT-, and WT-CRT-transformed Dictyostelium discoideum whole cells.. From: Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles.

(A) Labeled drug uptake by D. discoideum cells in PB containing 100 nM [3H]-CQ and various concentrations of unlabeled CQ and ammonia. Cells were incubated in [3H]-CQ for 10 min before determination of [3H]-CQ uptake. (B) Effects of VP, the protonophore CCCP, and the V-Type ATPase inhibitor CMA on [3H]-CQ uptake. (C) Effects of VP, CCCP, and CMA on cytoplasmic ATP levels. (D–F) Relative effects of VP, CCCP, and CMA on acidic compartment pH traced by 0.5 µM LysoSensorTM Blue DND-167 probe (FC, fluorescence counts; y-axis scales are the same for figures D–F). Slight decreases of fluorescence from cells in PB after 600 s may be due to lysosomal alkalinization from the LysoSensorTM Blue DND-167 probe. Concentrations of 80 μM VP, 2 μM CCCP, and 100 nM CMA were employed in the experiments. Error bars indicate standard deviations from three independent measurements.

Janni Papakrivos, et al. PLoS One. 2012;7(6):e39569.
4.
Figure 4

Figure 4. Influence of ionophores on [3H]-CQ accumulation by whole cells and isolated vesicles from untransformed and PfCRT-transformed Dictyostelium discoideum.. From: Functional Characterization of the Plasmodium falciparum Chloroquine-Resistance Transporter (PfCRT) in Transformed Dictyostelium discoideum Vesicles.

(A) Labeled drug uptake by whole cells (in PB) or vesicles (in VSB) in the presence of 100 nM [3H]-CQ and 2 μM concentrations of monensin (Mon), nigericin (Nig), or valinomycin (Val). Cells were incubated in the buffers for 10 min before determination of [3H]-CQ uptake. Note the similar accumulations of [3H]-CQ by untransformed and SEA-CRT-transformed vesicles in the presence of Val. Error bars indicate standard deviations of measurements from three independent measurements. (B) Representative DiOC5(3) fluorescence traces indicating membrane potential changes in untransformed (black), SEA-CRT-transformed (grey), and WT-CRT-transformed (light grey) F/T vesicles in MCB. Val (2 μM) was added at 200 s, and K+ (25 mM) was added at 300 s. P1, P2, P3, and P4 indicate points where fluorescence intensity measurements were taken from the tracings to determine the magnitude of hyperpolarization by valinomycin (P1, fluorescence level before valinomycin is added; P2, trough fluorescence) and the magnitude of depolarization after addition of 25 mM K+ (P3, peak fluorescence after K+ addition; P4, fluorescence level before K+ is added). FC, fluorescence counts. (C) Average valinomycin response (P1–P2) and recoveries of DiOC5 (3) fluorescence (P3–P4) before and after addition of 25 mM K+ in eight independent experiments. Asterisks (*) indicate significant difference from the average result with untransformed vesicles (P<0.005; n = 8).

Janni Papakrivos, et al. PLoS One. 2012;7(6):e39569.

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