Functional characterization of iron transporter from Dictyostelium discoideum as a model of cellular iron homeostasis

Iron plays a central role in a large number of essential cellular functions but it is also potentially toxic being able to generate reactive oxygen species. The social amoeba Dictyostelium discoideum possesses several iron genes with the exception of transferrin, ferritin and TFR (Bozzaro et al., 2013; Peracino et al., 2013) and represents a model for the study of cellular iron homeostasis showing subcellular localization of iron transporters resembling that of macrophages. In particular, D. discoideum expresses the ortholog of Nramp1 transporter in phago-lysosomes and that of Nramp2 in the contractile vacuole. To better understand the function of dd Nramp1, the protein was expressed in Xenopus laevis oocytes by cRNA injection and functionally tested by radiochemical and electrophysiological techniques. To increase the surface localization of dd Nramp1, its N and C termini were replaced with the corresponding regions of the murine DMT1, which shows a high level of expression in the membrane of X. laevis oocytes (Gunshin et al., 1997). Dd Nramp1 is an electrogenic proton-dependent divalent metal ion transporter with a cation selectivity comparable to that of the murine DMT1 (Illing et al., 2012). It transports ferrous but not ferric iron and it is partially inhibited by Na+

New alternative splicing BCR/ABL-OOF shows an oncogenic role by lack of inhibition of BCR GTPase activity and an increased of persistence of Rac activation in chronic myeloid leukemia

In Chronic Myeloid Leukemia 80% of patients present alternative splice variants involving BCR exons 1, 13 or 14 and ABL exon 4, with a consequent impairment in the reading frame of the ABL gene. Therefore BCR/ABL fusion proteins (BCR/ABL-OOF) are characterized by an in-frame BCR portion followed by an amino acids sequence arising from the out of frame (OOF) reading of the ABL gene. The product of this new transcript contains the characteristic BCR domains while lacking the COOH-terminal Rho GTPase GAP domain. The present work aims to characterize the protein functionality in terms of cytoskeleton (re-)modelling, adhesion and activation of canonical oncogenic signalling pathways. Here, we show that BCR/ABL-OOF has a peculiar endosomal localization which affects EGF receptor activation and turnover. Moreover, we demonstrate that BCR/ABL-OOF expression leads to aberrant cellular adhesion due to the activation of Rac GTPase, increase in cellular proliferation, migration and survival. When overexpressed in a BCR/ABL positive cell line, BCR/ABL-OOF induces hyperactivation of Rac signaling axis offering a therapeutic window for Rac-targeted therapy. Our data support a critical role of BCR/ABL-OOF in leukemogenesis and identify a subset of patients that may benefit from Rac-targeted therapies

Feasibility of GPS use to locate wild ungulates in high mountain environment

Fix success and accuracy of GPS (Global Positioning System) telemetry both depend on the number of visible satellites and their geometry. Due to the topography of the mountain environment the efficiency of GPS is compromised, field tests were conducted to analyze how it can affect fix success and rate of 3D locations (≥ 4 visible satellites) as regards slope orientation, using pooled data from GPS collars in solved- and differential-mode. Ten test sites were defined in the periphery of the Vanoise National Park. Fixes were simultaneously recorded at the bottom and top of cliffs facing each of the 4 main orientations or at the bottom of valleys oriented £-W and N-S and at a reference site (open field). Fix success and rate of 3D locations were significantly negatively correlated with an increasing obstruction level. Cliff orientation affected both fix success (significantly higher for cliffs facing the North) and rate of 3D locations (significantly higher for cliffs facing the South).<br><br>[fr]<br />Dans la télémétrie par GPS (Global Positioning System), la fixation d'un point et la précision se montrent conditionnées par le nombre de satellites visibles et la géométrie. Les montagnes provoquant des obstructions topographiques qui réduisent la fiabilité du GPS, nous avons effectué des essais de terrain pour analyser comment l'exposition du versant peut affecter la fixation d'un point ou le taux de localisation 3D (4 satellites visibles ou plus). Pour cela nous avons utilisé un ensemble de données GPS. Nous avons marqué 10 points de référence en périphérie du Parc National de la Vanoise. Puis nous avons enregistré simultanément des points fixes en fond de vallée et en haut des falaises exposées aux quatre directions principales ou au fond des vallées orientées E-W et N-S ainsi que dans un point de référence (champ ouvert). La fixation d'un point ainsi que le taux de localisation en 3D montraient significativement une corrélation négative avec un niveau croissant d'obstruction. L'exposition de la falaise affectait le succès de la localisation (tout spécialement les falaises exposées au nord) et également le taux de localisation en 3D (significativement plus haut pour les falaises exposées au sud). <br />[es]<br />El éxito en la ubicación y la exactitud de la telemetría por medio de GPS (Global Positioning System) depende del número de satélites visibles y de su geometría. Las montañas suponen obstrucciones topográficas, reduciendo así la fiabilidad del GPS. Se realizó un test de campo para analizar su efecto sobre el éxito en la ubicación y la proporción de localizaciones en 3D (≥ 4 satélites visibles), teniendo en cuenta la orientación de la pendiente y usando como fuente datos el GPS de collares emisores. Se seleccionaron diez puntos de muestreo en la periferia del Parque Nacional de La Vanoise. Las localizaciones se tomaron simultáneamente en el fondo y en lo alto de riscos orientados hacia cada uno de los 4 puntos cardinales, en el fondo de valles orientados E-W y N-S y en un lugar de referencia (campo abierto). El éxito en la ubicación y la proporción de localizaciones en 3D fue significativamente negativo para un nivel de obstrucción creciente. La orientación del roquedo afecta tanto al éxito de la ubicación (significativamente mayor para roquedos de orientación norte) como a la proporción de localizaciones en 3D (significativamente mayor para roquedos de orientación sur)

Dictyostelium discoideum as a model host for meningococcal pathogenesis

BACKGROUND: The aim of the present study was to evaluate the possibility of studying meningococcal virulence in a new model organism, Dictyostelium discoideum, a haploid social soil amoeba that is an established host model for several human pathogens, leading to the discovery of novel virulence mechanisms. MATERIAL/METHODS: A number of virulent and hyper-virulent N. meningitidis strains, including isogenic encapsulated, unencapsulated, and lipooligosaccharide (LOS) outer core-defective derivatives, were used to test the ability of D. discoideum to internalize and grow in the presence of bacteria. Intracellular survival of the internalized bacteria was also monitored. RESULTS: Meningococci were internalized and killed by D. discoideum cells. The presence of a capsule did not affect the internalization, but, as in human cells, it increased the resistance of the internalized bacteria. Although both encapsulated and unencapsulated meningococci supported the growth and development of D. discoideum on an agar surface, in liquid medium the encapsulated strains were toxic to the slime mould cells. Toxicity inversely correlated with meningococcal survival in the assay medium that was not favorable to bacterial replication, suggesting that it may be due to some toxic compound released after bacterial autolysis. Intriguingly, unencapsulated isogenic strains efficiently supported Dictyostelium growth in suspension, opening the possibility that the toxicity may be associated with the capsular polysaccharide. CONCLUSIONS: These results suggest that several meningococcal virulence determinants, such as the capsular polysaccharide, may be remarkably effective also in Dictyostelium cells, stimulating the use of this model host to search for novel meningococcal virulence determinants

Functional analysis of iron transporter Nramp2 (SLC11A2) from Dictyostelium discoideum

Iron plays a leading role in many essential cellular functions, but it is also potentially toxic being able to generate reactive oxygen species (ROS) that can damage cell components. The social amoeba Dictyostelium discoideum is a model organism for studying cellular iron homeostasis having a subcellular localization of iron transporters (Nramps) that resembles that of macrophages. The ortholog of Nramp1 (SLC11A1) transporter is expressed in phago-lysosomes and is involved in resistance to bacterial infection mediating the efflux of iron and manganese from the phago-lysosomes to the cytosol; Nramp2 (SLC11A2) ortholog is localized in the contractile vacuole, that may have a role of reservoir of iron in a cell lacking ferritin. Nramp2 was expressed on the membrane of Xenopus laevis oocytes, therefore, the N- and C- termini of the protein were replaced with the corresponding regions of murine transporter DMT1. The cRNA was injected and classical radiochemical techniques and a fluorophore indicator (calcein) quenched by the entrance in the cytoplasm of divalent ions, were used for studying Nramp2 activity. Our experiments showed that Nramp2 iron transport is Na+-dependent and proton independent. It prefers ferrous iron and it can transports, Cd++, Co++, Mn++ and Zn++. Dictyostelium Nramp2, in synergy with Nramp1, appears to contribute to the regulation of the iron homeostasis of the cell allowing the efflux of the ion from the vacuole to the cytosol

Functional, spectroscopic and structural properties of haemoglobin from chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex)

The functional and spectroscopic properties of chamois (Rupicapra rupicapra) and steinbock (Capra hircus ibex) haemoglobin (Hb) have been studied with special reference to the action of allosteric effectors and temperature. Moreover, the amino acid sequences of the N-terminal segments of the alpha- and beta-chains have been determined. The present results indicate that chamois and steinbock Hbs display a low affinity for O2, which appears to be modulated in vivo by Cl- ions rather than 2,3-bisphosphoglycerate. The Bohr effect for O2 binding to chamois and steinbock Hb is higher than for reindeer and bovine Hbs, being similar to that of human Hb. Moreover, the temperature-dependence of oxygenation appears intermediate between that of human and reindeer Hbs. E.p.r. and absorption spectroscopic properties of the ferrous nitrosylated derivative of chamois and steinbock Hbs suggest that both haemoproteins are in a low-affinity conformation even in the absence of InsP6. The reduced effect of polyphosphates on the functional and spectroscopic properties of chamois and steinbock Hb agree with amino acid differences in the N-terminal segment of the beta-chains (i.e. the deletion of Val(NA1) and the replacement of His(NA2), present in human Hb, and Gln(NA2), present in horse Hb, by Met). The molecular mechanism modulating the basic reaction of O2 with chamois and steinbock Hb may be linked to specific physiological needs related to the high-altitude habitats of these two animals