Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei

TbPARG in Trypanosoma brucei. A) TbPARG localization in untreated (control) and in procyclic cultures exposed to 500 μM H2O2 for 10 min. IFI was carried out as reported in our previous work [33]. TbPARG was identified with our home-made antibody against TcPARG [33]; and PAR was identified with a commercial antibody against PAR (BD). White bar represents 50 μm. B) Western blot analysis of 40 μg protein per lane revealed with a commercial anti-PARG antibody (Antibody Verify) in T. brucei procyclic (PC) and bloodstream (BST) forms. The arrow indicates the band with the expected molecular weight (approximately 60 kDa). The membrane stained with Red Ponceau was used as a loading control. (TIF 4272 kb

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution

Altres ajuts: del Consejo Nacional de Investigaciones Científicas y Técnicas- Argentina (CONICET) i del Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo (IBERCAROT).The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution

Improvement of Aroma in Transgenic Potato As a Consequence of Impairing Tuber Browning

Sensory analysis studies are critical in the development of quality enhanced crops, and may be an important component in the public acceptance of genetically modified foods. It has recently been established that odor preferences are shared between humans and mice, suggesting that odor exploration behavior in mice may be used to predict the effect of odors in humans. We have previously found that mice fed diets supplemented with engineered nonbrowning potatoes (-PPO) consumed more potato than mice fed diets supplemented with wild-type potatoes (WT). This prompted us to explore a possible role of potato odor in mice preference for nonbrowning potatoes. Taking advantage of two well established neuroscience paradigms, the “open field test” and the “nose-poking preference test”, we performed experiments where mice exploration behavior was monitored in preference assays on the basis of olfaction alone. No obvious preference was observed towards -PPO or WT lines when fresh potato samples were tested. However, when oxidized samples were tested, mice consistently investigated -PPO potatoes more times and for longer periods than WT potatoes. Congruently, humans discriminated WT from -PPO samples with a considerably better performance when oxidized samples were tested than when fresh samples were tested in blind olfactory experiments. Notably, even though participants ranked all samples with an intermediate level of pleasantness, there was a general consensus that the -PPO samples had a more intense odor and also evoked the sense-impression of a familiar vegetable more often than the WT samples. Taken together, these findings suggest that our previous observations might be influenced, at least in part, by differential odors that are accentuated among the lines once oxidative deterioration takes place. Additionally, our results suggest that nonbrowning potatoes, in addition to their extended shelf life, maintain their odor quality for longer periods of time than WT potatoes. To our knowledge this is the first report on the use of an animal model applied to the sensory analysis of a transgenic crop

Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi.

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites

Anti-Trypanosoma cruzi Activity of Green Tea (Camellia sinensis) Catechins

The trypanocidal action of green tea catechins against two different developmental stages of Trypanosoma cruzi is reported for the first time. This activity was assayed with the nonproliferative bloodstream trypomastigote and with the intracellular replicative amastigote parasite forms. An ethyl acetate fraction from Camellia sinensis green tea leaves, which contains most of the polyphenolic compounds and the maximal trypanocidal activity, was obtained by fractionation of the aqueous extract with organic solvents. The active compounds present in this extract were further purified by LH-20 column chromatography and were identified by high-performance liquid chromatography analysis with a photo diode array detector and gas chromatography coupled to mass spectroscopy. The following flavan-3-ols derivatives, known as catechins, were identified: catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, gallocatechin gallate, and epigallocatechin gallate. The purified compounds lysed more than 50% of the parasites present in the blood of infected BALB/c mice at concentrations as low as 0.12 to 85 pM. The most active compounds were gallocatechin gallate and epigallocatechin gallate, with minimal bactericidal concentrations that inhibited 50% of isolates tested of 0.12 and 0.53 pM, respectively. The number of amastigotes in infected Vero cells decreased by 50% in the presence of each of these compounds at 100 nM. The effects of the catechins on the recombinant T. cruzi arginine kinase, a key enzyme in the energy metabolism of the parasite, were assayed. The activity of this enzyme was inhibited by about 50% by nanomolar concentrations of catechin gallate or gallocatechin gallate, whereas the other members of the group were less effective. On the basis of these results, we suggest that these compounds could be used to sterilize blood and, eventually, as therapeutic agents for Chagas' disease

Odor exploration behavior in mice.

<p>Percentage of entries (A) and exploration time spent (B) by mice in zones A and B of the open field with food containers filled with freshly cut (0 h) WT or -PPO potato samples. Percentage of entries (C) and exploration time spent (D) by mice in zones A and B of the open field with food containers filled with oxidized (24 h) WT or -PPO potato samples. Stars represent statistically significant differences (<i>P≤0.03</i>) according to the one-sample <i>t</i>-test for difference from 50%. Error bars represent the ±95% confidence interval of eight independent experiments. (E) Hole-board experiment. Mean investigation times (s) ± SEM of six independent measurements are shown for each type of sample. The star represents statistically significant differences (<i>P≤0.05</i>) in investigation time according to the ANOVA test followed by the Newman-Keuls multiple comparison post-hoc test.</p

Mouse open field test experimental validation.

<p>Percentage of entries (A) and exploration time spent (B) by mice in zones A and B of the open field with both food containers empty. (C) Representative 5 min mouse trajectory of a negative experimental control where both food containers were empty. Percentage of entries (D) and exploration time spent (E) by mice in zones A and B of the open field with one container filled with freshly cut WT potato and the other one left empty. (F) Representative 5 min mouse trajectory of a positive experimental control where food container A was empty and food container B was filled with freshly cut WT potato samples. Central darker squares in Figures C and F represent zones A (on the left) and B (on the right). Stars represent statistically significant differences (<i>P≤0.01</i>) according to the one-sample <i>t</i>-test for difference from 50%. Error bars represent the ±95% confidence interval of eight independent experiments.</p