The effect of Cyclosporine A on cardiomyocytes differentiation

Cyclosporine A (CsA) is a powerful immunosuppressive drug which significantly improved the success of organ transplantation; however, the major limiting factors for the drug's clinical use are its long and short term adverse effects. The present study was conducted to examine, in a dose-dependent manner, in a model of cardiogenesis, the effect of CsA on cardiomyocytes differentiation.     

Reduced Introgression of Sex Chromosome Markers in the Mexican Howler Monkey (Alouatta palliata × A. pigra) Hybrid Zone

International Journal of Primatology - Interspecific hybridization allows the introgression or movement of alleles from one genome to another. While some genomic regions freely exchange alleles...     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

Abi1 is essential for the formation and activation of a WAVE2 signalling complex

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.     

Reproductive effects and geographical distributions of two Wolbachia strains infecting the Neotropical beetle, Chelymorpha alternans Boh. (Chrysomelidae, Cassidinae)

Wolbachia are maternally inherited endocellular bacteria known to alter insect host reproduction to facilitate their own transmission. Multiple Wolbachia infections are more common in tropical than temperate insects but few studies have investigated their dynamics in field populations. The beetle, Chelymorpha alternans, found throughout the Isthmus of Panama, is infected with two strains of Wolbachia, wCalt1 (99.2% of beetles) and wCalt2 (53%). Populations infected solely by the wCalt1 strain were limited to western Pacific Panama, whereas populations outside this region were either polymorphic for single (wCalt1) and double infections (wCalt1 + wCalt2) or consisted entirely of double infections. The wCalt2 strain was not found as a single infection in the wild. Both strains caused cytoplasmic incompatibility (CI). The wCalt1 strain caused weak CI (approximately 20%) and the double infection induced moderate CI (approximately 70-90%) in crosses with uninfected beetles. The wCalt1 strain rescued about 75% of eggs fertilized by sperm from wCalt2 males. Based on the relationships of beetle mtDNA and infection status, maternal transmission, and repeated population sampling we determined that the double infection invaded C. alternans populations about 100,000 years ago and that the wCalt2 strain appears to be declining in some populations, possibly due to environmental factors. This may be the first study to demonstrate an association between widespread strain loss and environmental factors in the field.     

The role of two isoenzymes of alpha-amylase of Araucaria araucana (Araucariaceae) on the digestion of starch granules during germination

Starch is the principal reserve of Araucaria araucana seeds, and it is hydrolysed during germination mainly by alpha-amylase. There are several alpha-amylase isoenzymes whose patterns change in the embryo and in the megagametophyte from the one observed in quiescent seeds (T(0)) to a different one observed 90 h after imbibition (T(90)). The objective of this research was to study the roles of two purified alpha-amylase isoenzymes by in vitro digestion of starch granules extracted from the tissues at two times of imbibition: one is abundant in quiescent seeds and the other is abundant after 90 h of imbibition. The isoenzymes digested the starch granules of their own stage of germination better, since the isoenzyme T(0) digested starch granules mainly from quiescent seeds, while the isoenzyme T(90) digested starch mainly at 90 h of imbibition. The sizes of the starch granule and the tissue from which these granules originated make a difference to digestion by the isoenzymes. Embryonic isoenzyme T(0) digested large embryonic starch granules better than small and medium-sized granules, and better than those isolated from megagametophytes. Similarly isoenzyme T(90) digested small embryonic starch granules better than medium-sized and large granules, and better than those isolated from megagametophytes. However, a mixture of partially purified megagametophytic isoenzymes T(0) and T(90) digested the megagametophytic granules better than those isolated from embryos. Studies of in vitro sequential digestion of starch granules with these isoenzymes corroborated their specificity. The isoenzyme T(90) digested starch granules previously digested by the isoenzyme T(0). This suggests that in vivo these two isoenzymes may act sequentially in starch granule digestion.     

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.     

Medulloblastoma tumorigenesis diverges from cerebellar granule cell differentiation in patched heterozygous mice

Medulloblastoma is a cerebellar tumor that can arise through aberrant activation of Sonic hedgehog (Shh) signaling, which normally regulates cerebellar granule cell proliferation. Mutations of the Shh receptor PATCHED (PTCH) are associated with medulloblastomas, which have not been found to have loss of PTCH heterozygosity. We address whether patched (Ptc) heterozygosity fundamentally alters granule cell differentiation and contributes to tumorigenesis by increasing proliferation and/or decreasing apoptosis in Ptc+/- mice. Our data show that postnatal Ptc+/- mouse granule cell precursor growth is not globally altered. However, many older Ptc+/- mice display abnormal cerebellar regions containing persistently proliferating granule cell precursors. Since fewer Ptc+/- mice form medulloblastomas, these granule cell rests represent a developmentally disrupted, but uncommitted stage of tumorigenesis. Although Ptc+/- mouse medulloblastomas express neurodevelopmental genes, they diverge from granule cell differentiation in their discordant coexpression of postmitotic markers despite their ongoing growth. Like human medulloblastomas, mouse tumors with reduced levels of the neurotrophin-3 receptor, trkC/Ntrk3, display decreased apoptosis in vivo, illustrating the role of TrkC in regulating tumor cell survival. These results indicate that Ptc heterozygosity contributes to tumorigenesis by predisposing a subset of granule cell precursors to the formation of proliferative rests and subsequent dysregulation of developmental gene expression.