Transmission electron microscopy of parasitized erythrocytes treated with inhibitors.

<p>(A) Sections through an erythrocyte containing a trophozoite-stage parasite exposed to 0.1% DMSO for 6 h, 50 µM INA for 6 h or 250 µM AMO-1618 for 8 h, respectively. (B) Asynchronized parasites were treated with 0.1% DMSO (a–d) or 50 µM INA for 6 h (e–h). Sections from representative stages during intraerythrocytic development: ring- (a and e), early trophozoite- (b and f), mature trophozoite- (c and g) and schizont-stage parasites (d and h) are shown. Nuclei (n), food vacuoles (fv), merozoites (m), nuclei of merozoites (mn) and abnormal gaps between the nuclei and the nuclear envelopes (arrowheads) are indicated. Scale bar is indicated at the bottom of the images.</p

Isoprenoid biosynthetic pathways and known inhibitors in various organisms.

<p>Broken arrows indicate blocks in the biosynthesis due to specific inhibitors. “R” indicates various functional groups specific to individual compounds. CPPS, copalyl-diphosphate synthase (EC 5.5.1.13); KO, <i>ent</i>-kaurene oxidase (EC 1.14.13.78); CAS, cycloartenol synthase (EC 5.4.99.8); LS, lanosterol synthase (EC 5.4.99.7); KS, <i>ent</i>-kaurene synthase (EC 4.2.3.19); PP, pyrophosphate.</p

ED₅₀ of the gibberellin biosynthetic inhibitors.

<p>Growth inhibitory effects of gibberellin biosynthetic inhibitors and enantiomers of INA to <i>P. falciparum</i> and <i>T. gondii</i> are shown. Values are the mean ± standard deviations (SD) from three independent experiments, with each treatment duplicated twice. N.D.; not determined.</p

Effect of INA and AMO-1618 on intraerythrocytic development of <i>P. falciparum</i>.

<p>Tightly synchronized parasite cells that have undergone 5% sorbitol treatment and percoll density gradient centrifugation (window period: 4 h) were treated with 1 µl/ml DMSO, 50 µM INA or 250 µM AMO-1618 at different parasite stages: after 0 h (ring, A), 20 h (immature trophozoite, B), 28 h (mature trophozoite, C) and 36 h (schizont, D). Cultures were examined after 4, 8 and 12 h using Giemsa thin blood smears. Scale bar: 3 µm; all images without a scale bar are displayed at the same scale as the left uppermost image in (A).</p

Fluorescence microscopy of INA-treated parasites.

<p>Infected erythrocytes were stained with (A) acridine orange, (B) Nile Red, (C) rhodamine 123 and (D) LysoTracker™ Red DND-99. INA was introduced at: (A) 100 µM for 9 h, (B) 50 µM for 6 h, and (C and D) 50 µM for 4 h. 100 µg/ml acridine orange was applied to thin blood smears made from intraerythrocytic parasites treated with INA. For other fluorescence dyes, <i>P. falciparum</i> cultures were incubated with probes for 1 h at the following concentrations: Nile Red, 1 µg/ml; rhodamine 123, 10 ng/ml; and LysoTracker® Red DND-99, 75 nM. Cells were not washed prior to fluorescence microscopy to minimize damage due to osmotic changes. Scale bar, 3 µm. An arrow indicates a lipid body in (B).</p

Effects of osmotic pressure to intraerythrocytic parasites treated with INA.

<p>(A) Effects of hyperosmotic stress in INA-treated parasites. <i>P. falciparum</i> cultures treated with 50 µM INA were observed in various dilution ratios of PBS for 8 h. Parasitemia was determined by Giemsa-stained thin blood smears. Student's <i>t</i>-test: *P>0.005. Values are means ± SD of n = 6 in two independent experiments. Data were normalized relative to control cultures (1× PBS) in DMSO- and INA-treated samples, respectively. Representative parasite morphologies are shown for each treatment. Scale bar, 3 µm. (B) Effects of hyposmotic stress, experimentally induced by the addition of water to the culture medium of <i>P. falciparum</i>, after 8 h of 50 µM INA treatment. N = 6 smears each in two independent experiments.</p

GC-MS analysis of isoprenoids in <i>P. falciparum in vitro</i> cultures.

<p>(A) Total ion chromatograms of extracts from unparasitized erythrocytes (Unparasitized), parasites treated with 0.1% DMSO, 250 µM AMO-1618, and 50 µM INA for 6 h. Triangles and circles indicate the peaks of geranylgeraniol mixed as an internal control (retention time = 6.57 min) and cholesterol (11.23 min), respectively. These compounds were identified by direct comparison with authentic samples. (B) Quantification of cholesterol in each treated sample. Cholesterol amount was calculated from the peak areas and normalized relative to the ratio of the internal control geranylgeraniol. Values are means and SD of triplicate measurements of a representative experiment.</p