Fetal muscle contains different CD34+ cell subsets that distinctly differentiate into adipogenic, angiogenic and myogenic lineages

We have previously demonstrated that CD34(+) cells isolated from fetal mouse muscles are an interesting source of myogenic progenitors. In the present work, we pinpoint the tissue location of these CD34(+) cells using cell surface and phenotype markers. In order to identify the myogenic population, we next purified different CD34(+) subsets, determined their expression of relevant lineage-related genes, and analyzed their differentiation capacities in vitro and in vivo. The CD34(+) population comprised a CD31(+)/CD45(-) cell subset exhibiting endothelial characteristics and only capable of forming microvessels in vivo. The CD34(+)/CD31(-)/CD45(-)/Sca1(+) subpopulation, which is restricted to the muscle epimysium, displayed adipogenic differentiation both in vitro and in vivo. CD34(+)/CD31(-)/CD45(-)/Sca1(-) cells, localized in the muscle interstitium, transcribed myogenic genes, but did not display the characteristics of adult satellite cells. These cells were distinct from pericytes and fibroblasts. They were myogenic in vitro, and efficiently contributed to skeletal muscle regeneration in vivo, although their myogenic potential was lower than that of the unfractionated CD34(+) cell population. Our results indicate that angiogenic and adipogenic cells grafted with myogenic cells enhance their contribution to myogenic regeneration, highlighting the fundamental role of the microenvironment on the fate of transplanted cells.     

Analysis of proteasomal proteolysis during the in vitro metacyclogenesis of Trypanosoma cruzi

Proteasomes are large protein complexes, whose main function is to degrade unnecessary or damaged proteins. The inhibition of proteasome activity in Trypanosoma cruzi blocks parasite replication and cellular differentiation. We demonstrate that proteasome-dependent proteolysis occurs during the cellular differentiation of T. cruzi from replicative non-infectious epimastigotes to non-replicative and infectious trypomastigotes (metacyclogenesis). No peaks of ubiquitin-mediated degradation were observed and the profile of ubiquitinated conjugates was similar at all stages of differentiation. However, an analysis of carbonylated proteins showed significant variation in oxidized protein levels at the various stages of differentiation and the proteasome inhibition also increased oxidized protein levels. Our data suggest that different proteasome complexes coexist during metacyclogenesis. The 20S proteasome may be free or linked to regulatory particles (PA700, PA26 and PA200), at specific cell sites and the coordinated action of these complexes would make it possible for proteolysis of ubiquitin-tagged proteins and oxidized proteins, to coexist in the cell.     

The over-expression of TRPC6 channels in HEK-293 cells favours the intracellular accumulation of zinc

TRPC6 are plasma membrane cation channels. By means of live-cell imaging and spectroscopic methods, we found that HEK cells expressing TRPC6 channels (HEK-TRPC6) are enriched in zinc and sulphur and have a reduced copper content when compared to HEK cells and HEK cells expressing TRPC3 channels (HEK-TRPC3). Hence, HEK-TRPC6 cells have larger pools of mobilizable Zn2+ and are more sensitive to an oxidative stress. Synchrotron X-ray fluorescence experiments showed a higher zinc content in the nuclear region indicating that the intracellular distribution of this metal was influenced by the over-expression of TRPC6 channels. Their properties were investigated with the diacylglycerol analogue SAG and the plant extract hyperforin. Electrophysiological recordings and imaging experiments with the fluorescent Zn2+ probe FluoZin-3 demonstrated that TRPC6 channels form Zn2+-conducting channels. In cortical neurons, hyperforin-sensitive channels co-exist with voltage-gated channels, AMPA and NMDA receptors, which are known to transport Zn2+. The ability of these channels to regulate the size of the mobilizable pools of Zn2+ was compared. The data collected indicate that the entry of Zn2+ through TRPC6 channels can up-regulate the size of the DTDP-sensitive pool of Zn2+. By showing that TRPC6 channels constitute a Zn2+ entry pathway, our study suggests that they could play a role in zinc homeostasis.     

Limb bud colonization by somite-derived angioblasts is a crucial step for myoblast emigration

We have combined the use of mouse genetic strains and the mouse-into-chicken chimera system to determine precisely the sequence of forelimb colonization by presomitic mesoderm (PSM)-derived myoblasts and angioblasts, and the possible role of this latter cell type in myoblast guidance. By creating a new Flk1/Pax3 double reporter mouse line, we have established the precise timetable for angioblast and myoblast delamination/migration from the somite to the limb bud. This timetable was conserved when mouse PSM was grafted into a chicken host, which further validates the experimental model. The use of Pax3(GFP/GFP) knockout mice showed that establishment of vascular endothelial and smooth muscle cells (SMCs) is not compromised by the absence of Pax3. Of note, Pax3(GFP/GFP) knockout mouse PSM-derived cells can contribute to aortic, but not to limb, SMCs that are derived from the somatopleure. Finally, using the Flk1(lacZ)(/)(lacZ) knockout mouse, we show that, in the absence of angioblast and vascular network formation, myoblasts are prevented from migrating into the limb. Taken together, our study establishes for the first time the time schedule for endothelial and skeletal muscle cell colonization in the mouse limb bud and establishes the absolute requirement of endothelial cells for myoblast delamination and migration to the limb. It also reveals that cells delaminating from the somites display marked differentiation traits, suggesting that if a common progenitor exists, its lifespan is extremely short and restricted to the somite.     

Analysis of carvedilol enantiomers in human plasma using chiral stationary phase column and liquid chromatography with tandem mass spectrometry

Carvedilol is an antihypertensive drug available as a racemic mixture. (?)‐(S)‐carvedilol is responsible for the nonselective β‐blocker activity but both enantiomers present similar activity on α₁‐adrenergic receptor. To our knowledge, this is the first study of carvedilol enantiomers in human plasma using a chiral stationary phase column and liquid chromatography with tandem mass spectrometry. The method involves plasma extraction with diisopropyl ether using metoprolol as internal standard and direct separation of the carvedilol enantiomers on a Chirobiotic T® (Teicoplanin) column. Protonated ions [M + H]⁺ and their respective ion products were monitored at transitions of 407 > 100 for the carvedilol enantiomers and 268 > 116 for the internal standard. The quantification limit was 0.2 ng ml^?1 for both enantiomers in plasma. The method was applied to study enantioselectivity in the pharmacokinetics of carvedilol administered as a single dose of 25 mg to a hypertensive patient. The results showed a higher plasma concentration of (+)‐(R)‐carvedilol (AUC^0–∞ 205.52 vs. 82.61 (ng h) ml^?1), with an enantiomer ratio of 2.48. Chirality, 2012. © 2012 Wiley Periodicals, Inc.     

Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Is water availability really the main environmental factor controlling the phenology of woody vegetation in the central Sahel?

Rainfall distribution and the soil moisture regime have been recognized to be the key drivers of the phenological rhythms in Sahelian woody plants, although different climate triggers have been assumed to be involved in determining the date of the onset of the phenophase. However, almost no comparisons have been made of the actual relative predictive power of these environmental factors. The aim of our study was to quantify the ability of several factors to predict phenophase occurrence in the dominant woody populations of northern Mali. Canopy leafing, flowering and fruiting were monitored from May 2005 to July 2007. Multiple logistic regressions were used to test the predictive power of cumulative rainfall, soil moisture, air temperature, air humidity and day length, with time lags of up to 2 months. Artificial variables derived from time lags observed in phenophases were included as predictors to account for possible auto-correlation and cross-correlation among phenophases. Surprisingly, a decrease in temperature associated with different time lags was most often found to be the strongest predictor of both leafing and reproductive phenophases. In Sahelian shrubs, morphological and physiological adaptations strongly contribute to the relative independence of their activity from water availability, leaf phenology being a way to adjust the plant water balance to current water availability and atmospheric water content. This study provides insight towards the development of a mechanistic understanding of phenological control in the Sahel, which is becoming increasingly important in the context of expected climate changes.