Mismatch repair in Trypanosoma brucei: heterologous expression of MSH2 from Trypanosoma cruzi provides new insights into the response to oxidative damage

Trypanosomes are unicellular eukaryotes that cause disease in humans and other mammals. Trypanosoma cruzi and Trypanosoma brucei are the causative agents, respectively, of Chagas disease in the Americas and sleeping sickness in sub-Saharan Africa. To better comprehend the interaction of these parasites with their hosts, understanding the mechanisms involved in the generation of genetic variability is critical. One such mechanism is mismatch repair (MMR), which has a crucial, evolutionarily conserved role in maintaining the fidelity of DNA replication, as well as acting in other cellular processes, such as DNA recombination. Here we have attempted to complement T. brucei MMR through the expression of MSH2 from T. cruzi. Our results show that T. brucei MSH2-null mutants are more sensitive to hydrogen peroxide (H2O2) than wild type cells, suggesting the involvement of MSH2 in the response to oxidative stress in this parasite. This phenotype is reverted by the expression of either the T. cruzi or the T. brucei MSH2 protein in the MSH2-null mutants. In contrast, MMR complementation, as assessed by resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and microsatellite instability, was not achieved by the heterologous expression of T. cruzi MSH2. This finding, associated to the demonstration that mutation of MLH1, another component of the MMR system, did not affect sensitivity of T. brucei cells to H2O2, suggests an additional role of MSH2 in dealing with oxidative damage in these parasites, which may occur independently of MMR.     

Nest‐site selection by Blue‐black Grassquits in a Neotropical savanna: do choices influence nest success?

ABSTRACT.   Nest-site choice affects individual fitness and possibly reflects natural selection of the capacity of individuals to select appropriate microhabitat features. From 2003 to 2005, we examined nest-site characteristics and nesting success of Blue-black Grassquits ( Volatinia jacarina ) in central Brazil. We compared the characteristics of nest sites and nonused sites, as well as the characteristics of successful and unsuccessful nests. Grassquit nest sites were structurally more complex than nonused sites. Shrub height and the interaction between vegetation height and percentage of ground coverage were the most important predictors of nest placement. Grassquits used only four (20%) of the 20 grass species in the study area, with Paspalum pectinatum used less than expected based on availability and Melinis minutiflora more than expected. The only variable that differed between unsuccessful and successful nests was the distance to nearest conspecific nest; the latter were about twice as far from neighboring nests as unsuccessful nests. The evaluation of microhabitat candidate models indicated that the daily survival probability of nests varied chiefly as a function of the interaction between their external height and inner depth. Greater survival occurred when the external height was minimized in combination with augmentation of internal depth of the nest cup. The link between nest success and the inverse association of external height and internal depth suggests that minimizing the visual cues of nest presence while maintaining a viable incubation chamber can positively affect nest success. Thus, we suggest that nest concealment is the most critical attribute associated with nest site choice for Blue-black Grassquits in the study area. Vegetation cover above the nest seems to be particularly important, perhaps as a strategy to deter visually oriented aerial predators.     

Genes that code for T cell signaling proteins establish transcriptional regulatory networks during thymus ontogeny

Gene expression profiling by cDNA microarrays during murine thymus ontogeny has contributed to dissecting the large-scale molecular genetics of T cell maturation. Gene profiling, although useful for characterizing the thymus developmental phases and identifying the differentially expressed genes, does not permit the determination of possible interactions between genes. In order to reconstruct genetic interactions, on RNA level, within thymocyte differentiation, a pair of microarrays containing a total of 1,576 cDNA sequences derived from the IMAGE MTB library was applied on samples of developing thymuses (14-17 days of gestation). The data were analyzed using the GeneNetwork program. Genes that were previously identified as differentially expressed during thymus ontogeny showed their relationships with several other genes. The present method provided the detection of gene nodes coding for proteins implicated in the calcium signaling pathway, such as Prrg2 and Stxbp3, and in protein transport toward the cell membrane, such as Gosr2. The results demonstrate the feasibility of reconstructing networks based on cDNA microarray gene expression determinations, contributing to a clearer understanding of the complex interactions between genes involved in thymus/thymocyte development.     

Anti-apoptotic treatment reduces transthyretin deposition in a transgenic mouse model of Familial Amyloidotic Polyneuropathy

Tauroursodeoxycholic acid (TUDCA) is a unique natural compound that acts as a potent anti-apoptotic and anti-oxidant agent, reducing cytotoxicity in several neurodegenerative diseases. Since oxidative stress, apoptosis and inflammation are associated with transthyretin (TTR) deposition in Familial Amyloidotic Polyneuropathy (FAP), we investigated the possible TUDCA therapeutical application in this disease. We show by semi-quantitative immunohistochemistry and western blotting that administration of TUDCA to a transgenic mouse model of FAP decreased apoptotic and oxidative biomarkers usually associated with TTR deposition, namely the ER stress markers BiP and eIF2alpha, the Fas death receptor and oxidation products such as 3-nitrotyrosine. Most important, TUDCA treatment significantly reduced TTR toxic aggregates in as much as 75%. Since TUDCA has no effect on TTR aggregation "in vitro", this finding points for the "in vivo" modulation of TTR aggregation by cellular responses, such as by oxidative stress, ER stress and apoptosis and prompts for the use of this safe drug in prophylactic and therapeutic measures in FAP.     

Analysis of coastal lagoon metabolism as a basis for management

This work was carried out in a shallow eutrophic coastal lagoon (St. André lagoon, SW Portugal) which is artificially opened to the sea each year in early spring. Macrophytes, mainly Ruppia cirrhosa, are keystone species in this ecosystem covering up to 60% of its total area with peak biomasses over 500 g DW m^–2. The main objectives were to study ecosystem metabolism, to evaluate the metabolic contribution to the community of the macrophyte stands and their influence in the development of thermal stratification and bottom oxygen depletion.The work combined an experimental and a modelling methodology. The experimental approach included open water, mesocosm and microcosm seasonal experiments. During these experiments several physical, chemical and biological parameters were monitored in the lagoon and in plastic enclosures (mesocosms) for periods of 24 hours. The microcosm experiments followed the light-dark bottle technique. The simultaneous use of these different methodologies allowed the analysis of the contribution of the planktonic and benthic compartments to the ecosystem's oxygen budget.The modelling work was based on the mathematical simulation of heat and gas exchanges in a vertically resolved water column, under different macrophyte densities. Several simulations were carried out, in order to investigate the importance of the macrophytes in the development of water column stratification and anoxia.The simulation results suggest that macrophytes may greatly influence thermocline and oxycline development. This influence is proportional to their biomass and canopy height. It is suggested that controlled macrophyte biomass removal of up to 25% of available biomass in summer, may be useful in preventing bottom anoxia without compromising benthic net primary production.     

Hybrid cluster proteins (HCPs) from Desulfovibrio desulfuricans ATCC 27774 and Desulfovibrio vulgaris (Hildenborough): X-ray structures at 1.25 Å resolution using synchrotron radiation

The structures of the hybrid cluster proteins (HCPs) from the sulfate-reducing bacteria Desulfovibrio desulfuricans (ATCC 27774) and Desulfovibrio vulgaris (Hildenborough) have been elucidated at a resolution of 1.25 A using X-ray synchrotron radiation techniques. In the case of the D. desulfuricans protein, protein isolation, purification, crystallization and X-ray data collection were carried out under strict anaerobic conditions, whereas for the D. vulgaris protein the conditions were aerobic. However, both structures are essentially the same, comprising three domains and two iron-sulfur centres. One of these centres situated near the exterior of the molecules in domain 1 is a cubane [4Fe-4S] cluster, whereas the other, located at the interface of the three domains, contains the unusual four-iron cluster initially found in the D. vulgaris protein. Details of the structures and the associated EPR spectroscopy of the D. desulfuricans protein are reported herein. These structures show that the nature of the hybrid cluster, containing both oxygen and sulfur bridges, is independent of the presence of oxygen in the isolation and crystallization procedure and also does not vary significantly with changes in the oxidation state. The structures and amino acid sequences of the HCP are compared with the recently elucidated structure of the catalytic subunit of a carbon monoxide dehydrogenase from Carboxydothermus hydrogenoformans and related dehydrogenases. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0326-y.     

Increased Oxidative Parameters and Decreased Cytokine Levels in an Animal Model of Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is a highly heterogeneous disorder characterized by impairing levels of hyperactivity, impulsivity and inattention. Oxidative and inflammatory parameters have been recognized among its multiple predisposing pathways, and clinical studies indicate that ADHD patients have increased oxidative stress. In this study, we aimed to evaluate oxidative (DCFH oxidation, glutathione levels, glutathione peroxidase, catalase and superoxide dismutase activities) and inflammatory (TNF-α, IL-1β and IL-10) parameters in the most widely accepted animal model of ADHD, the spontaneously hypertensive rats (SHR). Prefrontal cortex, cortex (remaining regions), striatum and hippocampus of adult male SHR and Wistar Kyoto rats were studied. SHR presented increased reactive oxygen species (ROS) production in the cortex, striatum and hippocampus. In SHR, glutathione peroxidase activity was decreased in the prefrontal cortex and hippocampus. TNF-α levels were reduced in the prefrontal cortex, cortex (remaining regions), hippocampus and striatum of SHR. Besides, IL-1β and IL-10 levels were decreased in the cortex of the ADHD model. Results indicate that SHR presented an oxidative profile that is characterized by an increase in ROS production without an effective antioxidant counterbalance. In addition, this strain showed a decrease in cytokine levels, mainly TNF-α, indicating a basal deficit. These results may present a new approach to the cognitive disturbances seen in the SHR.