FGF6 mediated expansion of a resident subset of cells with SP phenotype in the C2C12 myogenic line

Fibroblast growth factor 6 (FGF6) is selectively expressed during muscle development and regeneration. We examined its effect on muscle precursor cells (mpc) by forcing stable FGF6 expression in C2C12 cells in vitro. FGF6 produced in genetically engineered mpc was active, inducing strong morphological changes, altering cell adhesion and compromising their ability to differentiate into myotubes. Expression of MyoD and myogenin, but not of Myf5, was abrogated in FGF6 engineered mpc. These effects were reversed by FGF inhibitors. Ectopic expression of MyoD also restored fiber formation indicating that FGF6 interferes with the myogenic differentiation pathway upstream of MyoD. We also report that in the presence of FGF6, the minor (0.5-2%) subpopulation of cells actively excluding Hoechst 33342 in a verapamil-dependent manner (SP phenotype) was increased to 15-20% and the expression of the mdr1a gene (but not mdr1b) was upregulated by 400-fold. Our data establish a previously undescribed link between FGF6--a muscle specific growth factor--and a multidrug resistance gene expressed in stem cells, and suggest a role for FGF6 in the maintenance of a reserve pool of progenitor cells in the skeletal muscle.     

Imprints of glacial refugia in the modern genetic diversity of Pinus sylvestris

Aim To understand the impact of glacial refugia and migration pathways on the modern genetic diversity of Pinus sylvestris. Location The study was carried out throughout Europe. Methods An extended set of data of pollen and macrofossil remains was used to locate the glacial refugia and reconstruct the migrating routes of P. sylvestris throughout Europe. A vegetation model was used to simulate the extent of the potential refugia during the last glacial period. At the same time a genetic survey was carried out on this species. Results The simulated distribution of P. sylvestris during the last glacial period is coherent with the observed fossil data, which showed a patchy distribution of the refugia between c. 40° N and 50° N. Several migrational fronts were detected within the Iberian and the Italian peninsulas, and outside the Hungarian plain and around the Alps. The modern mitochondrial DNA depicted three different haplotypes for P. sylvestris. Two distinct haplotypes were restricted to northern Spain and Italy, and the third haplotype dominated most of the present‐day remaining distribution range of P. sylvestris in Europe. Main conclusions During the last glacial period P. sylvestris was constrained under severe climatic conditions to survive in scattered and restricted refugial areas. Combining palaeoenvironmental data, vegetation modelling and the genetic data, we have shown that the long‐term isolation in the glacial refugia and the migrational process during the Holocene have played a major role in shaping the modern genetic diversity of P. sylvestris in Europe.     

Endothelium-dependent vascular relaxation induced by Globularia alypum extract is mediated by EDHF in perfused rat mesenteric arterial bed

The vasodilatory effect of Globularia alypum L. (GA) extract was evaluated in rat mesenteric arterial bed pre-contracted by continuous infusion of phenylephrine (2-4 ng/mL). Bolus injections of GA elicited dose-response vasodilation, which was abolished after endothelium removal. Addition of a nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (100 μmol/L), alone or in the presence of a cyclooxygenase inhibitor, indomethacin (10 μmol/L), did not significantly affect the vasodilation of the mesenteric arterial bed in response to GA extract. These results suggest that GA-induced vasodilation is endothelium dependent but nitric oxide and prostacyclin independent. In the presence of high K(+) (60 mmol/L), the GA vasodilatory effect was completely abolished, suggesting that the vasodilation effect is mediated by hyperpolarization of the vascular cells. Also, pre-treatment with atropine (a muscarinic receptors antagonist) antagonized the GA-induced vasodilation, suggesting that the vasodilatory effect is mainly mediated by the endothelium-derived hyperpolarizing factor through activation of endothelial muscarinic receptors.     

A model of DENV-3 infection that recapitulates severe disease and highlights the importance of IFN-γ in host resistance to infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ(-/-) mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2(-/-) mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection.     

Caspase-mediated cleavage of HuR in the cytoplasm contributes to pp32/PHAP-I regulation of apoptosis

The RNA-binding protein HuR affects cell fate by regulating the stability and/or the translation of messenger RNAs that encode cell stress response proteins. In this study, we delineate a novel regulatory mechanism by which HuR contributes to stress-induced cell death. Upon lethal stress, HuR translocates into the cytoplasm by a mechanism involving its association with the apoptosome activator pp32/PHAP-I. Depleting the expression of pp32/PHAP-I by RNA interference reduces both HuR cytoplasmic accumulation and the efficiency of caspase activation. In the cytoplasm, HuR undergoes caspase-mediated cleavage at aspartate 226. This cleavage activity is significantly reduced in the absence of pp32/PHAP-I. Substituting aspartate 226 with an alanine creates a noncleavable isoform of HuR that, when overexpressed, maintains its association with pp32/PHAP-I and delays the apoptotic response. Thus, we propose a model in which HuR association with pp32/PHAP-I and its caspase-mediated cleavage constitutes a regulatory step that contributes to an amplified apoptotic response.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

Solvent-controlled regioselective protection of 5'-O-protected thymidine

This paper describes an efficient procedure for selective 3'-O- or 3-N-protection of 5'-O-tert-butyldimethylsilylthymidine, depending on the use of aprotic polar solvents with low or high dielectric constant, respectively. These syntheses were activated by either ultrasound or microwaves. Several alkyl bromides offer a convenient route to prepare 3'-O- or 3-N-protected and functionalized thymidine derivatives.