A Novel Method for Inducing Amastigote-To-Trypomastigote Transformation In Vitro in Trypanosoma cruzi Reveals the Importance of Inositol 1,4,5-Trisphosphate Receptor

Background

Trypanosoma cruzi is a parasitic protist that causes Chagas disease, which is prevalent in Latin America. Because of the unavailability of an effective drug or vaccine, and because about 8 million people are infected with the parasite worldwide, the development of novel drugs demands urgent attention. T. cruzi infects a wide variety of mammalian nucleated cells, with a preference for myocardial cells. Non-dividing trypomastigotes in the bloodstream infect host cells where they are transformed into replication-capable amastigotes. The amastigotes revert to trypomastigotes (trypomastigogenesis) before being shed out of the host cells. Although trypomastigote transformation is an essential process for the parasite, the molecular mechanisms underlying this process have not yet been clarified, mainly because of the lack of an assay system to induce trypomastigogenesis in vitro.

Methodology/Principal Findings

Cultivation of amastigotes in a transformation medium composed of 80% RPMI-1640 and 20% Grace’s Insect Medium mediated their transformation into trypomastigotes. Grace’s Insect Medium alone also induced trypomastigogenesis. Furthermore, trypomastigogenesis was induced more efficiently in the presence of fetal bovine serum. Trypomastigotes derived from in vitro trypomastigogenesis were able to infect mammalian host cells as efficiently as tissue-culture-derived trypomastigotes (TCT) and expressed a marker protein for TCT. Using this assay system, we demonstrated that T. cruzi inositol 1,4,5-trisphosphate receptor (TcIP₃R)—an intracellular Ca²⁺ channel and a key molecule involved in Ca²⁺ signaling in the parasite—is important for the transformation process.

Conclusion/Significance

Our findings provide a new tool to identify the molecular mechanisms of the amastigote-to-trypomastigote transformation, leading to a new strategy for drug development against Chagas disease.     

Real-time single-molecule observations of T7 Exonuclease activity in a microflow channel

T7 Exonuclease (T7 Exo) DNA digestion reactions were studied using direct single-molecule observations in microflow channels. DNA digestion reactions were directly observed by staining template DNA double-stranded regions with SYTOX Orange and staining single-stranded (digested) regions with a fluorescently labeled ssDNA-recognizing peptide (ssBP-488). Sequentially acquired photographs demonstrated that a double-stranded region monotonously shortened as a single-stranded region monotonously increased from the free end during a DNA digestion reaction. Furthermore, DNA digestion reactions were directly observed both under pulse-chase conditions and under continuous buffer flow conditions with T7 Exo. Under pulse-chase conditions, the double-stranded regions of λDNA monotonously shortened by a DNA digestion reaction with a single T7 Exo molecule, with an estimated average DNA digestion rate of 5.7 bases/s and a processivity of 6692 bases. Under continuous buffer flow conditions with T7 Exo, some pauses were observed during a DNA digestion reaction and double-stranded regions shortened linearly except during these pauses. The average DNA digestion rate was estimated to be 5.3 bases/s with a processivity of 5072 bases. Thus, the use of our direct single-molecule observations using a fluorescently labeled ssDNA-recognizing peptide (ssBP-488) was an effective analytic method for investigating DNA metabolic processes.     

Syntheses of water-soluble [60]fullerene derivatives and their enhancing effect on neurite outgrowth in NGF-treated PC12 cells

Water-soluble [60]fullerene (C₆₀) derivatives were synthesized to examine their bioactivities. PC12 cells were used as a model of nerve cells and the bioactivities of synthesized C₆₀ derivatives together with some reported ones were tested. Among the compounds tested, C₆₀/(γ-CyD)₂, C₆₀-bis(γ-CyD) (5) containing C₆₀-mono(γ-CyD) (5′), and C₆₀/PVP were sufficiently soluble in water and showed an enhancing effect on the neurite outgrowth of NGF-treated PC12 cells.     

Mapping of quantitative trait loci controlling tick [Riphicephalus (Boophilus) microplus] resistance on bovine chromosomes 5, 7 and 14

Differences in domestication and selection processes have contributed to considerable phenotypic and genotypic differences between Bos taurus and Bos indicus cattle breeds. Of particular interest in tropical and subtropical production environments are those genetic differences between subspecies that underlie the phenotypic extremes in tolerance and susceptibility to parasite infection. In general, B. taurus cattle are more susceptible to ectoparasites than B. indicus cattle in tropical environments, and much of this difference is under genetic control. To identify genomic regions involved in tick resistance, we developed a B. taurus x B. indicus F(2) experimental population to map quantitative trait loci (QTL) for resistance to the Riphicephalus (Boophilus) microplus tick. About 300 individuals were measured for parasite load in two seasons (rainy and dry) and genotyped for 23 microsatellite markers covering chromosomes 5, 7 and 14. We mapped a suggestive chromosome-wide QTL for tick load in the rainy season (P < 0.05) on chromosome 5. For the dry season, suggestive (P < 0.10) chromosome-wide QTL were mapped on chromosomes 7 and 14. The additive effect of the QTL on chromosome 14 corresponds to 3.18% of the total observed phenotypic variance. Our QTL-mapping study has identified different genomic regions controlling tick resistance; these QTL were dependent upon the season in which the ticks were counted, suggesting that the QTL in question may depend on environmental factors.     

Novel membrane-localizing TEMPO derivatives for measurement of cellular oxidative stress at the cell membrane

Oxidative stress affecting lipid membranes is considered to be closely related to cardiovascular disease and brain ischemia. In this study, we designed and synthesized membrane-localizing TEMPO derivatives and demonstrated that one of these synthesized probes, compound 1, localized and detected oxidative stress in the cell membrane in an endotoxic model of a mouse macrophage-like cell line. Compound 1 is therefore a potentially useful probe for evaluating oxidative stress at the cell membrane.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Design, synthesis, and biological activity of folate receptor-targeted prodrugs of thiolate histone deacetylase inhibitors

Aiming to develop selective anticancer drugs, we designed and synthesized three disulfides bearing a folic acid moiety as candidate folate receptor (FR)-targeted prodrugs of thiolate histone deacetylase inhibitors. Among them, compound 1 displayed growth-inhibitory activity toward folate receptor-positive MCF-7 breast cancer cells. The activity of 1 was significantly reduced by free folic acid, suggesting that cellular uptake of 1 is mediated by FR.     

Treatment with beta2-adrenoceptor agonist in vivo induces human clock gene, Per1, mRNA expression in peripheral blood

This study examined whether in vivo exposure to a β₂-adrenoceptor agonist, tulobuterol, induces human Period1 (hPer1) mRNA expression in cells from peripheral whole blood. In one experiment, oral tulobuterol was administered to five healthy volunteers at 22:00 h, while in another, a transdermally tulobuterol patch was applied to the same five subjects at 20:00 h. In each experiment, serum tulobuterol concentrations were measured at four time points, and total RNA was isolated from peripheral blood cells for determinations of hPer1 mRNA expression by real-time polymerase chain reaction. Both the tulobuterol tablet and the transdermal patch increased hPer1 mRNA expression, suggesting that analyses of human peripheral blood cells could reliably represent peripheral clock gene mRNA expression in vivo.