Gibberellin Biosynthetic Inhibitors Make Human Malaria Parasite Plasmodium falciparum Cells Swell and Rupture to Death

Malaria remains as one of the most devastating infectious disease, and continues to exact an enormous toll in medical cost and days of labor lost especially in the tropics. Effective malaria control and eventual eradication remain a huge challenge, with efficacious antimalarials as important intervention/management tool. Clearly new alternative drugs that are more affordable and with fewer side effects are desirable. After preliminary in vitro assays with plant growth regulators and inhibitors, here, we focus on biosynthetic inhibitors of gibberellin, a plant hormone with many important roles in plant growth, and show their inhibitory effect on the growth of both apicomplexa, Plasmodium falciparum and Toxoplasma gondii. Treatment of P. falciparum cultures with the gibberellin biosynthetic inhibitors resulted in marked morphological changes that can be reversed to a certain degree under hyperosmotic environment. These unique observations suggest that changes in the parasite membrane permeability may explain the pleiotropic effects observed within the intracellular parasites

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Plant Hormone Salicylic Acid Produced by a Malaria Parasite Controls Host Immunity and Cerebral Malaria Outcome

<div><p>The apicomplexan parasite <i>Toxoplasma gondii</i> produces the plant hormone abscisic acid, but it is unclear if phytohormones are produced by the malaria parasite <i>Plasmodium</i> spp., the most important parasite of this phylum. Here, we report detection of salicylic acid, an immune-related phytohormone of land plants, in <i>P</i>. <i>berghei</i> ANKA and <i>T</i>. <i>gondii</i> cell lysates. However, addition of salicylic acid to <i>P</i>. <i>falciparum</i> and <i>T</i>. <i>gondii</i> culture had no effect. We transfected <i>P</i>. <i>falciparum</i> 3D7 with the <i>nahG</i> gene, which encodes a salicylic acid-degrading enzyme isolated from plant-infecting <i>Pseudomonas</i> sp., and established a salicylic acid-deficient mutant. The mutant had a significantly decreased concentration of parasite-synthesized prostaglandin E₂, which potentially modulates host immunity as an adaptive evolution of <i>Plasmodium</i> spp. To investigate the function of salicylic acid and prostaglandin E₂ on host immunity, we established <i>P</i>. <i>berghei</i> ANKA mutants expressing <i>nahG</i>. C57BL/6 mice infected with <i>nahG</i> transfectants developed enhanced cerebral malaria, as assessed by Evans blue leakage and brain histological observation. The <i>nahG</i>-transfectant also significantly increased the mortality rate of mice. Prostaglandin E₂ reduced the brain symptoms by induction of T helper-2 cytokines. As expected, T helper-1 cytokines including interferon-γ and interleukin-2 were significantly elevated by infection with the <i>nahG</i> transfectant. Thus, salicylic acid of <i>Plasmodium</i> spp. may be a new pathogenic factor of this threatening parasite and may modulate immune function via parasite-produced prostaglandin E₂.</p></div

Identification of salicylic acid from <i>Plasmodium berghei</i> ANKA.

<p><i>P</i>. <i>berghei</i> ANKA was purified from infected mice blood, and salicylic acid (SA) was extracted, and analyzed by LC-triple TOF mass spectrometry. (A) Structural formula of SA. (B) LC chromatogram of SA standard (control) and <i>P</i>. <i>berghei</i> ANKA sample. (C) Fragmentation analysis of peaks in (B) (colored in aqua). Collision energy was 20 eV.</p

Parasite SA influences the cerebral malaria outcome.

<p>(A-C) Histological observation of infected mouse cerebellums. (A) Cerebellum infected by <i>nahG</i>-expressing parasites. Note the sequestrated leukocytes in microvessels. The inset image shows a higher magnification of the boxed portion. Phagocytized hemozoin is observed (arrowhead). (B) Brain of a mouse infected with <i>gfp</i>-expressing parasites. Slight microbleeding was observed, but no sequestrated vessels were found. (C) Brain of an uninfected control. Sections were stained by hematoxylin and eosin. (D) Evans blue leakage analysis of the severity of cerebral malaria. Photographs of brains from mice infected with <i>nahG</i>- (left upper) and <i>gfp-</i> (left middle) expressing parasites and uninfected controls (left bottom), and quantification of dye leakage (right). Mice (n = 5) were sacrificed at 6 days post-infection. Solid line, p<0.01; dashed line, p<0.05. C57BL/6 mice at 6 days post-infection were used for all experiments. Bar: 50 μm.</p

Molecular and biological analysis revealed genetic diversity and high virulence strain of Toxoplasma gondii in Japan.

Toxoplasma gondii is classified into 16 haplogroups based on a worldwide genotyping study of the parasite. However, only a few isolates from Japan were included in this analysis. To conduct more precise genotyping of T. gondii, we examined the genotypes of Japanese isolates in this study. DNA sequences of 6 loci were determined in 17 Japanese isolates and compared with those of strains of 16 haplogroups. As a result, Japanese isolates were classified into four groups. We investigated the virulence of some Japanese isolates and found a highly virulent strain in mice, comparable to that of RH strain, although this Japanese isolate was sister to strains of haplogroup 2, which show moderate virulence in mice. We further investigated whether this high virulence isolate had different virulence mechanism and strategy to adapt to Japanese host from other strains by comparing the virulence-related genes, ROP5, 18 and the immunomodulatory gene, ROP16 of the isolate with those of archetypical strains (GT1, ME49 and VEG). This analysis indicated the high virulence of the isolate in mice was partly explained by gene sequences of ROP5 and ROP16. These findings lead to the elucidation of biodiversity of T. gondii and have potential to optimize the diagnostic protocol

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

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

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

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