Protein flotation assay to isolate lipid rafts from soft tissue or cells

on-line protocol: Bio-protocol: http://www.bio-protocol.org/wenzhang.aspx?id=854The arrangement in eukaryotic cell membranes of liquid-ordered states surrounded by liquid-disordered phases allows for the existence of organized membrane microdomains called rafts. Rafts play a crusial role in signal-transduction events, in lipid transport and in various internalization processes, and for all these reasons need to be purified for further characterization

Prion Replication in the Hematopoietic Compartment Is Not Required for Neuroinvasion in Scrapie Mouse Model

Fatal neurodegenerative prion diseases are caused by the transmissible PrPSc prion agent whose initial replication after peripheral inoculation takes place in follicular dendritic cells present in germinal centers of lymphoid organs. However, prion replication also occurs in lymphoid cells. To assess the role of the hematopoietic compartment in neuroinvasion and prion replication, we generated chimeric mice, on a uniform congenic C57/BL6J background, by bone marrow replacement with hematopoietic cells expressing different levels of PrP protein. Nine different types of chimeric mice were inoculated intraperitoneally either with the lymphotropic Rocky Mountain Laboratory (RML) strain or the non lymphotropic ME-7 scrapie strain, at different doses. Here, we clearly demonstrate that overexpression of PrP by the hematopoietic system, or the lack of PrP expression by the bone marrow derived cells, does not change the incubation time period of the disease, even when the mice are infected at limiting doses. We conclude that the hematopoietic compartment is more or less permissive to prion replication, both for RML and ME-7, but does not play a role in neuroinvasion

Disrupting the plastidic iron-sulfur cluster biogenesis pathway in Toxoplasma gondii has pleiotropic effects irreversibly impacting parasite viability

International audienceLike many other apicomplexan parasites, Toxoplasma gondii contains a plastid harbouring key metabolic pathways, including the sulfur utilization factor (SUF) pathway that is involved in the biosynthesis of iron-sulfur clusters. These cofactors are key for a variety of proteins involved in important metabolic reactions, potentially including plastidic pathways for the synthesis of isoprenoid and fatty acids. It was shown previously that impairing the NFS2 cysteine desulfurase, involved in the first step of the SUF pathway, leads to an irreversible killing of intracellular parasites. However, the metabolic impact of disrupting the pathway remained unexplored. Here, we generated another mutant of this pathway deficient in the SUFC ATPase, and investigated in details the phenotypic consequences of TgNFS2 and TgSUFC depletion on parasite homeostasis. Our analysis confirms that Toxoplasma SUF mutants are severely and irreversibly impacted in division and membrane homeostasis. Our analysis suggests a defect in apicoplast-generated fatty acids. However, we show that increased scavenging from the host or supplementation with exogenous fatty acids do not fully restore parasite growth, suggesting that this is not the primary cause for the demise of the parasites and that other important cellular functions were affected. For instance, we also show that the SUF pathway is key for generating the isoprenoid-derived precursors necessary for the proper targeting of GPI-anchored proteins and for parasite motility. Thus, we conclude plastid-generated iron-sulfur clusters support the functions of proteins involved in several vital downstream cellular pathways, which implies the SUF machinery may be explored for new potential anti-Toxoplasma targets

PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl.

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP(C), for "cellular prion protein") into an abnormal state (PrP(Sc), for "scrapie prion protein"). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP(C). In contrast to its homologue PrP(C), Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP(Sc)-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP(C) (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the alpha-helical monomer forms soluble beta-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy

Aspergillus conidia activate the complement by the mannan-binding lectin C2 bypass mechanism.

Innate immunity is the major host defense against invasive aspergillosis. To determine whether the collectin mannan-binding lectin (MBL) is involved in the initial protective immunity through complement activation against opportunistic fungal infections caused by Aspergillus, we performed in vitro studies on 29 different strains of Aspergillus conidia from five different species. Incubation of Aspergillus conidia in human normal serum leads to activation of the alternative pathway, whereas neither the classical nor the lectin pathways through C4 and C2 cleavage are activated. Complement response to conidia was investigated using a MBL-deficient serum and reconstitution experiments were conducted with MBL/MASPs complexes. We found that MBL can directly support C3 activation by a C2 bypass mechanism. Finally, a stronger activation of the alternative pathway was observed for the clinical strains isolated from patients with invasive aspergillosis, compared with the environmental strains

PrP^c overexpression by the hematopoietic cells favors prion agent replication in the spleen of reconstituted Prp^0/0 mice.

<p>Spleen of Prp^0/0 mice lethally irradiated reconstituted with femoral bone marrow cells from Tga20 (inverse triangle) or B6 (circle) mice and inoculated with RML (black) or ME-7 (grey) strains were sampled 450 days post-inoculation and subsequently inoculated to Tga20 mice. Days post-inoculation (DPI) are represented. A <i>P</i> value of 0.001 was obtained using the Mann-Whitney t-test when comparing the Tga20→Prp^0/0 and B6→Prp^0/0 mice.</p

No difference in disease incubation period is observed in Tga20 chimeric mice inoculated with limiting doses of the RML strain (10⁻⁷ LogLD50).

<p>Tga20 mice were lethally irradiated, reconstituted with femoral bone marrow cells from B6 (square), Tga20 (triangle) or Prp^0/0 (circle) mice and inoculated with the RML prion scrapie strains. The incubation periods are expressed as days post-inoculation (DPI). At this low dose, only 7/10 mice developed scrapie. A <i>P</i> value of 0.19 was obtained using the Kruskal-Wallis ANOVA test when comparing the three groups of mice.</p

Routes of prion neuroinvasion after peripheral exposure.

<p>(a) Natural prion diseases are often acquired <i>via</i> peripheral exposure such as orally, or through skin lesions. How prion reaches its peripheral targets is not known. (b) Direct invasion of the central nervous system might occur with high doses of prion or exposure to neuroinvasive strains. (c) Whereas after exposure to limiting doses of infectivity or less neuroinvasive strains, replication in FDCs in the germinal centers of local lymphoid tissues might be necessary prior to neuroinvasion via closely associated nerve fibers. FDCs are dependent on the presence of B lymphocytes for maturation signals, such as lymphotoxin. (d) Haematogenous spread of infectivity <i>via</i> circulating bone marrow derived cells would not play a role in direct neuroinvasion.</p

Prion protein (PrPc) immunocytochemistry and expression of the green fluorescent protein reporter gene under control of the bovine PrP gene promoter in the mouse brain.

Expression of the cellular prion protein (PrP(c)) by host cells is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. As a consequence, identification of the cell types expressing PrP(c) is necessary to determine the target cells involved in the cerebral propagation of prion diseases. To identify the cells expressing PrP(c) in the mouse brain, the immunocytochemical localization of PrP(c) was investigated at the cellular and ultrastructural levels in several brain regions. In addition, we analyzed the expression pattern of a green fluorescent protein reporter gene under the control of regulatory sequences of the bovine prion protein gene in the brain of transgenic mice. By using a preembedding immunogold technique, neuronal PrP(c) was observed mainly bound to the cell surface and presynaptic sites. Dictyosomes and recycling organelles in most of the major neuron types also exhibited PrP(c) antigen. In the olfactory bulb, neocortex, putamen, hippocampus, thalamus, and cerebellum, the distribution pattern of both green fluorescent protein and PrP(c) immunoreactivity suggested that the transgenic regulatory sequences of the bovine PrP gene were sufficient to promote expression of the reporter gene in neurons that express immunodetectable endogenous PrP(c). Transgenic mice expressing PrP-GFP may thus provide attractive murine models for analyzing the transcriptional activity of the Prnp gene during prion infections as well as the anatomopathological kinetics of prion diseases