Overexpression of type 1 angiotensin II receptors impairs excitation-contraction coupling in the mouse heart

Transgenic mice that overexpress human type 1 angiotensin II receptor (AT(1)R) in the heart develop cardiac hypertrophy. Previously, we have shown that in 6-mo AT(1)R mice, which exhibit significant cardiac remodeling, fractional shortening is decreased. However, it is not clear whether altered contractility is attributable to AT(1)R overexpression or is secondary to cardiac hypertrophy/remodeling. Thus the present study characterized the effects of AT(1)R overexpression on ventricular L-type Ca(2+) currents (I(CaL)), cell shortening, and Ca(2+) handling in 50-day and 6-mo-old male AT(1)R mice. Echocardiography showed there was no evidence of cardiac hypertrophy in 50-day AT(1)R mice but that fractional shortening was decreased. Cellular experiments showed that cell shortening, I(CaL), and Ca(v)1.2 mRNA expression were significantly reduced in 50-day and 6-mo-old AT(1)R mice compared with controls. In addition, Ca(2+) transients and caffeine-induced Ca(2+) transients were reduced whereas the time to 90% Ca(2+) transient decay was prolonged in both age groups of AT(1)R mice. Western blot analysis revealed that sarcoplasmic reticulum Ca(2+)-ATPase and Na(+)/Ca(2+) exchanger protein expression was significantly decreased in 50-day and 6-mo AT(1)R mice. Overall, the data show that cardiac contractility and the mechanisms that underlie excitation-contraction coupling are altered in AT(1)R mice. Furthermore, since the alterations in contractility occur before the development of cardiac hypertrophy, it is likely that these changes are attributable to the increased activity of the renin-angiotensin system brought about by AT(1)R overexpression. Thus it is possible that AT(1)R blockade may help maintain cardiac contractility in individuals with heart disease.     

Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (mu scle‐s pecific k inase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. Results. In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the ‐MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid‐ordered phase by methyl‐β‐cyclodextrin abolished this association. We further show that actin and the actin‐nucleation factors, N‐WASP (neuronal Wiscott—Aldrich syndrome protein) and Arp2/3 (actin‐related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N‐WASP activity perturbed agrin‐elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR‐enriched rafts. Conclusions. The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.     

Identification of QTL genes for BMD variation using both linkage and gene-based association approaches

Low bone mineral density (BMD) is a risk factor for osteoporotic fracture with a high heritability. Previous large scale linkage study in Northern Chinese has identified four significant quantitative trait loci (QTL) for BMD variation on chromosome 2q24, 5q21, 7p21 and 13q21. We performed a replication study of these four QTL in 1,459 Southern Chinese from 306 pedigrees. Successful replication was observed on chromosome 5q21 for femoral neck BMD with a LOD score of 1.38 (nominal p value = 0.006). We have previously identified this locus in a genome scan meta-analysis of BMD variation in a white population. Subsequent QTL-wide gene-based association analysis in 800 subjects with extreme BMD identified CAST and ERAP1 as novel BMD candidate genes (empirical p value of 0.032 and 0.014, respectively). The associations were independently replicated in a Northern European population (empirical p value of 0.01 and 0.004 for CAST and ERAP1, respectively). These findings provide further evidence that 5q21 is a BMD QTL, and CAST and ERAP1 may be associated with femoral neck BMD variation.     

Protein resistance of dextran and dextran-poly(ethylene glycol) copolymer films

The protein resistance of dextran and dextran-poly(ethylene glycol) (PEG) copolymer films was examined on an organosilica particle-based assay support. Comb-branched dextran-PEG copolymer films were synthesized in a two step process using the organosilica particle as a solid synthetic support. Particles modified with increasing amounts (0.1-1.2 mg m(-2)) of three molecular weights (10,000, 66,900, 400,000 g mol(-1)) of dextran were found to form relatively poor protein-resistant films compared to dextran-PEG copolymers and previously studied PEG films. The efficacy of the antifouling polymer films was found to be dependent on the grafted amount and its composition, with PEG layers being the most efficient, followed by dextran-PEG copolymers, and dextran alone being the least efficient. Immunoglobulin gamma (IgG) adsorption decreased from ～5 to 0.5 mg m(-2) with increasing amounts of grafted dextran, but bovine serum albumin (BSA) adsorption increased above monolayer coverage (～2 mg m(-2)) indicating ternary adsorption of the smaller protein within the dextran layer.     

Dynamics of haplogroup frequencies and survival rates in a contact zone of two mtDNA lineages of the lizard Lacerta vivipara

The analysis of contact zones between lineages that were previously isolated in allopatry can lead to important insights on evolutionary processes such as selection and adaptation. In this paper we conducted a comparative demographic study of two mitochondrial DNA (mtDNA) lineages of the lizard Lacerta vivipara in the western Pyrénées to provide detail on the dynamics of their contact zone. By surveying haplogroup frequency across the contact area, we revealed the existence of a stable and very narrow contact zone between two parapatric lineages, which we infer to demonstrate a role for selection in the maintenance of this contact zone. We suggest these two lineages evolved in allopatry after retreating to different refugia during the Pleistocene glaciations, and subsequently came into secondary contact after the last glacial maximum. Although haplogroup frequencies were stable over time, we found significant age and environment (temperature) dependent survival differences between mtDNA haplogroups in one contact population sampled yearly from 2002 to 2009. Therefore, temperature‐induced demographic differences between the two mtDNA lineages may be responsible for the stability of this narrow contact zone. This is one of the first demographic studies conducted under natural conditions indicating the possibility of selection on mtDNA.     

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

The expression of heteroligomeric protein complexes for structural studies often requires a special coexpression strategy. The reason is that the solubility and proper folding of each subunit of the complex requires physical association with other subunits of the complex. The genomes of pathogenic mycobacteria encode many small protein complexes, implicated in bacterial fitness and pathogenicity, whose characterization may be further complicated by insolubility upon expression in Escherichia coli, the most common heterologous protein expression host. As protein fusions have been shown to dramatically affect the solubility of the proteins to which they are fused, we evaluated the ability of maltose binding protein fusions to produce mycobacterial Esx protein complexes. A single plasmid expression strategy using an N-terminal maltose binding protein fusion to the CFP-10 homolog proved effective in producing soluble Esx protein complexes, as determined by a small-scale expression and affinity purification screen, and coupled with intracellular proteolytic cleavage of the maltose binding protein moiety produced protein complexes of sufficient purity for structural studies. In comparison, the expression of complexes with hexahistidine affinity tags alone on the CFP-10 subunits failed to express in amounts sufficient for biochemical characterization. Using this strategy, six mycobacterial Esx complexes were expressed, purified to homogeneity, and subjected to crystallization screening and the crystal structures of the Mycobacterium abscessus EsxEF, M. smegmatis EsxGH, and M. tuberculosis EsxOP complexes were determined. Maltose binding protein fusions are thus an effective method for production of Esx complexes and this strategy may be applicable for production of other protein complexes.