Recombinant interleukin-4-treated macrophages, epithelioid cell surrogates, harbor and arrest Mycobacterium avium multiplication in vitro

Our group has previously described that murine peritoneal macrophages treated in vitro for 7 days with recombinant interleukin-4 (rIL-4) acquire morphological and functional characteristics of epithelioid cells (ECs) found in granulomatous lesions. Although EC function has not been clarified so far, it has been suggested that these cells could present antigens and control multiplication of mycobacteria. These aspects have been addressed here using in vitro EC surrogates. Using immunocytochemistry and immunofluorescence methods, we have observed an increased expression of CD11b, CD54, CD86 and CD40 molecules on rIL-4-treated macrophages when compared to untreated ones. Cytokine-treated cells were less phagocytic for latex beads (P<0.03) and more pinocytic for dextran particles than untreated macrophages. T-cell lymphoproliferation assays using ovalbumin (OVA) and Mycobacterium avium as antigens showed that both cultured macrophages were equally efficient as antigen presenting cells (APCs). However, M. avium antigens were better presented in vivo by EC surrogates (P<0.01). Both macrophage cultures were similarly infected by M. avium. However, while the infection level was maintained in the cytokine-treated population, untreated macrophages showed a progressive increase in the number of bacilli/cell with time (P<0.01) and a reduction of about 65% in cell population. After 96 h of M. avium infection, untreated cells secreted higher amounts of tumor necrosis factor-alpha (P<0.005) while rIL-4-treated macrophages showed higher, although not significant, transforming growth factor-beta production. Also, EC surrogates produced less nitric oxide than control macrophages (P<0.05). Hence, EC surrogates restrain M. avium growth and act as APCs in vitro and in vivo.     

The peroxynitrite donor 3-morpholinosydnonimine activates Nrf2 and the UPR leading to a cytoprotective response in endothelial cells

Endothelial dysfunction is associated with the formation of peroxynitrite, described to be toxic. Recent data also suggests that peroxynitrite is able to activate the protective Nrf2 pathway and/or the unfolded protein response (UPR). The aim of our work was to study the response of human endothelial cells to 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, and to highlight the possible protective roles of Nrf2 or the UPR pathway in this response.Immortal and primary human umbilical vein endothelial cells were exposed to SIN-1. SIN-1 incubation led to Nrf2 activation and to the overexpression of Nrf2-regulated genes, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1. We also demonstrated that this defensive response protected cells against cell death induced by serum starvation, by reducing apoptosis (monitored by caspase-3 activity and DNA fragmentation) and favoring autophagosome formation, as evidenced by LC3-II accumulation. Interestingly, we observed an activation of the UPR, with a rapid and significant overexpression of CHOP in serum starved cells stimulated with SIN-1. While siRNA mediated knockdown of CHOP had no effect on DNA fragmentation, the invalidation of Nrf2 or HO-1 by siRNA strongly increased DNA fragmentation, but also reinforced the SIN-1-induced LC3-II accumulation.This study shows that peroxynitrite, at least at sublethal concentrations and within a narrow concentration range, could exert protective effects on endothelial cells by modulating the balance between autophagy and apoptosis, through Nrf2-dependent pathways.     

Emergent growth cone responses to combinations of Slit1 and Netrin 1 in thalamocortical axon topography

How guidance cues are integrated during the formation of complex axonal tracts remains largely unknown. Thalamocortical axons (TCAs), which convey sensory and motor information to the neocortex, have a rostrocaudal topographic organization initially established within the ventral telencephalon [1-3]. Here, we show that this topography is set in a small hub, the corridor, which contains matching rostrocaudal gradients of Slit1 and Netrin 1. Using in vitro and in vivo experiments, we show that Slit1 is a rostral repellent that positions intermediate axons. For rostral axons, although Slit1 is also repulsive and Netrin 1 has no chemotactic activity, the two factors combined generate attraction. These results show that Slit1 has a dual context-dependent role in TCA pathfinding and furthermore reveal that a combination of cues produces an emergent activity that neither of them has alone. Our study thus provides a novel framework to explain how a limited set of guidance cues can generate a vast diversity of axonal responses necessary for proper wiring of the nervous system.     

Mechanisms of Allosteric Activation and Inhibition of the Deoxyribonucleoside Triphosphate Triphosphohydrolase from Enterococcus faecalis

EF1143 from Enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase (dNTPase), converting dNTPs into the deoxyribonucleosides and triphosphate. The dNTPase activity of EF1143 is regulated by canonical dNTPs, which simultaneously act as substrates and activity modulators. Previous crystal structures of apo-EF1143 and the protein bound to both dGTP and dATP suggested allosteric regulation of its enzymatic activity by dGTP binding at four identical allosteric sites. However, whether and how other canonical dNTPs regulate the enzyme activity was not defined. Here, we present the crystal structure of EF1143 in complex with dGTP and dTTP. The new structure reveals that the tetrameric EF1143 contains four additional secondary allosteric sites adjacent to the previously identified dGTP-binding primary regulatory sites. Structural and enzyme kinetic studies indicate that dGTP binding to the first allosteric site, with nanomolar affinity, is a prerequisite for substrate docking and hydrolysis. Then, the presence of a particular dNTP in the second site either enhances or inhibits the dNTPase activity of EF1143. Our results provide the first mechanistic insight into dNTP-mediated regulation of dNTPase activity.     

The origin of the absorption band induced through the interaction between apotransketolase and thiamin diphosphate

It has long been known that formation of a catalytically active holotransketolase from the apoenzyme and coenzyme (thiamin diphosphate) is accompanied by the appearance of a new band, in both the absorption and CD spectra. Binding and subsequent conversion of the substrates bring about changes in this band's intensity. The observation of these changes allows the investigator to monitor the coenzyme-to-apoenzyme binding and the conversion of substrates during the transketolase reaction and thus to kinetically characterize its individual steps. The origin of the thiamin diphosphate induced absorption band has been postulated to be resulted from formation of a charge transfer complex or alternatively from an induced conformational transition of the enzyme. The latter brings aromatic amino acid residues into close proximity and generates the absorption. However, X-ray crystallographic and enzyme point mutation experiments cast doubts on both of these hypotheses. Here we show that the binding of thiamin diphosphate to the apotransketolase leads to the conversion of the 4'-amino tautomeric form of its aminopyrimidine ring into the N(1')H-imino tautomeric form. This imino form emerges as a result of the coenzyme's aminopyrymidine ring incorporation into the hydrophobic pocket of the transketolase active center and is stabilized through the interactions with Glu418 and Phe445 residues. The N(1')H-imino tautomeric form of thiamin diphosphate is thought to be the origin of the holotransketolase absorption band induced through the coenzyme binding.     

Evolutionary conservation of kinetochore protein sequences in plants

The evolutionary conservation of structural/functional kinetochore proteins has been studied on isolated nuclei and pro-/metaphase chromosomes of mono- and dicot plants. The cross-reactivities of antibodies against human CENPC, CENPE and CENPF, and against maize CENPCa with the centromeric regions of mitotic chromosomes of Vicia faba and/or Hordeum vulgare are shown. Putative homologs of the kinetochore protein SKP1 (suppressor of kinetochore protein 1p of yeast) were found in both species and of CBF5p (centromere binding factor 5 of yeast) in barley. Antibodies against synthetic peptides derived from partial sequences encoding these proteins were produced and recognized the centromeric regions on mitotic chromosomes as detected by indirect immunofluorescence.     

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.     

Selenium Levels in the Whole Blood of Children and Teenagers from Two Riparian Communities at the Madeira River Basin in the Western Brazilian Amazon

Selenium (Se) is an essential micronutrient that exerts multiple functions in the organism, and both its deficiency and excess can cause health impairments. Thus, it is important to monitor its levels in the population, especially in vulnerable groups, such as children from the Brazilian Amazon region, where there is a lack of information in this regard. The aim of this research was to study Se levels in the whole blood of children and teenagers (5–16 years old) from two riparian communities at the Madeira River (Cuniã RESEX and Belmont). Se level variations related to the communities’ location, seasonality, diet, and body mass index (BMI) were assessed. Blood samples were collected in both communities for Se determinations, using ICP-MS and hemogram analyses, during May and September of 2011. Food frequency questionnaires were applied to assess consumption rates of specific food items. Non-parametric tests and linear multiple regressions were applied in the data analyses. Median Se levels were significantly higher during May (Cuniã RESEX 149 μg L^?1; Belmont 85 μg L^?1) compared to September (Cuniã RESEX 79 μg L^?1; Belmont 53 μg L^?1). No significant differences were found between the communities regarding BMI measurements and anemia prevalence. However, Se blood levels were significantly higher at the Cuniã RESEX compared to Belmont. In addition, the former showed higher fish and Brazil nut intakes, which may be the main Se sources for this community. These results contribute to a better understanding of Se reference levels for children and teenagers of Western Amazon riparian communities.     

Effect of chronological addition of records to species distribution maps: The case of Tonatia saurophila maresi (Chiroptera,Phyllostomidae) in South America

Ecological niche models have become very popular for analysing the potential distribution of species. Nevertheless, models are strongly influenced by many factors, such as spatial resolution, environmental variables and the quality of distribution records. In this paper, we evaluated how ecological niche models changed with the addition of records accumulated over four decades. Our model species was the stripe‐headed round‐eared bat (Tonatia saurophila). Thus, with data organized in chronological order, we could observe how the models changed in predicting distributions over time in comparison with all known point locations. We tested if partial models could predict the occurrence of new unpublished records for savannah areas in central Brazil, considering that the species is typically associated with forest environments. Our results indicate a high omission rate for models built with point localities from the 1970s and 1980s (58.5% and 50.0% of all known points respectively), and predicted that the species could occur in central Brazil. Although T. saurophila has indeed been recorded recently in central Brazil, it was found in places different from those predicted by the models using these restricted earlier data. Nevertheless, the environmental suitability of such areas is significantly different from sites largely described in earlier records from the Amazonia region, as shown by principal components analysis. We argue that populations of T. saurophila that occupy open habitats in central South America (including Caatinga, Cerrado, Chaco and semi‐deciduous interior forests) deserve further study at the genetic level to determine if bats in these very different habitats are taxonomically distinct from Amazonian populations. Our results also suggest that models based on very limited datasets for species occurrence can lead conservationists or decision makers to wrong conclusions.