Acetylcholine receptor channels are present in undifferentiated satellite cells but not in embryonic myoblasts in culture

The expression and the physiological properties of acetylcholine receptors (AChRs) of mononucleated myogenic cells, isolated from either embryonic or adult muscle of the mouse, have been investigated using the gigaohm seal patch-clamp technique in combination with immunocytochemistry (with an anti-myosin antibody) and alpha-bungarotoxin binding techniques. Undifferentiated (myosin-negative) embryonic myoblasts, grown either in mass culture or under clonal conditions, were found to be unresponsive to ACh and did not bind alpha-bungarotoxin. On the contrary, undifferentiated satellite cells (from adult muscle) exhibited channels activated by ACh and alpha-bungarotoxin binding sites similar to those observed in differentiated (myosin-positive) embryonic myoblasts and myotubes. Two classes of ACh-activated channels with different opening frequencies were identified. The major class of channels had a conductance of about 42 pS and mean open time of 3.1-8.2 msec. The minor class of channels had smaller conductance (about 17 pS) and similar open time. During differentiation, the conductance of the two channels did not change significantly, while channel lifetime became shorter in myotubes derived from satellite cells but not in myotubes derived from embryonic myoblasts. The relative proportion of small over large channels was significantly larger in embryonic than in adult myogenic cells.     

A muscle cell line from dystrophic mice expressing an altered phenotype in vitro

The isolation and characterization of a myogenic cell line from C57BL/6J/dydy mice is described. This line (DyA4) maintains the morphological, biochemical and electrophysiological characteristics of the primary cultured cells, at least for 20 passages. The cells actively divide as long as they are subcultured in media supplemented with horse serum and embryo extract. If the cells are not subcultured for a few days, they fuse into multinucleated contracting myotubes, which readily synthesize specific muscle products such as acetylcholinesterase and acetylcholine receptor. This dystrophic cell line expresses in vitro the same altered phenotype that is characteristic of dystrophic muscle cells in primary cultures, namely reduced acetylcholine sensitivity and reduced acetylcholine receptor expression. Because they can be grown in large amounts, and represent a pure muscle cell population which express an altered phenotype in an in vitro aneural avascular environment, DyA4 cells provide a very useful model system for investigating the pathogenesis of murine muscular dystrophy.     

Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells

Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.     

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.     

NF023 binding to XIAP‐BIR1: Searching drugs for regulation of the NF‐κB pathway

Inhibitor of Apoptosis Proteins (IAPs) are the target of extensive research in the field of cancer therapy since they regulate apoptosis and cell survival. Smac‐mimetics, the most promising IAP‐targeting compounds specifically recognize the IAP‐BIR3 domain and promote apoptosis, competing with caspases for IAP binding. Furthermore, Smac‐mimetics interfere with the NF‐κB survival pathway, inducing cIAP1 and cIAP2 degradation through an auto‐ubiquitination process. It has been shown that the XIAP‐BIR1 (X‐BIR1) domain is involved in the interaction with TAB1, an upstream adaptor for TAK1 kinase activation, which in turn couples with the NF‐κB survival pathway. Preventing X‐BIR1 dimerization abolishes XIAP‐mediated NF‐κB activation, thus implicating a proximity‐induced mechanism for TAK1 activation. In this context, in a systematic search for a molecule capable of impairing X‐BIR1/TAB1 assembly, we identified the compound NF023. Here we report the crystal structure of the human X‐BIR1 domain in the absence and in the presence of NF023, as a starting concept for the design of novel BIR1‐specific compounds acting synergistically with existing pro‐apoptotic drugs in cancer therapy. Proteins 2015; 83:612–620. © 2015 Wiley Periodicals, Inc.     

Fine mapping,physical mapping and development of diagnostic markers for the <Emphasis Type="Italic">Rrs2</Emphasis> scald resistance gene in barley

The Rrs2 gene confers resistance to the fungal pathogen Rhynchosporium secalis which causes leaf scald, a major barley disease. The Rrs2 gene was fine mapped to an interval of 0.08 cM between markers 693M6_6 and P1D23R on the distal end of barley chromosome 7HS using an Atlas (resistant) × Steffi (susceptible) mapping population of 9,179 F₂-plants. The establishment of a physical map of the Rrs2 locus led to the discovery that Rrs2 is located in an area of suppressed recombination within this mapping population. The analysis of 58 barley genotypes revealed a large linkage block at the Rrs2 locus extending over several hundred kb which is present only in Rrs2 carrying cultivars. Due to the lack of recombination in the mapping population and the presence of a Rrs2-specific linkage block, we assume a local chromosomal rearrangement (alien introgression or inversion) in Rrs2 carrying varieties. The variety analysis led to the discovery of eight SNPs which were diagnostic for the Rrs2 phenotype. Based on these SNPs diagnostic molecular markers (CAPS and pyrosequencing markers) were developed which are highly useful for marker-assisted selection in resistance gene pyramiding programmes for Rhynchosporium secalis resistance in barley.     

Dimeric Smac mimetics/IAP inhibitors as in vivo-active pro-apoptotic agents. Part II: Structural and biological characterization

Novel pro-apoptotic, homodimeric and heterodimeric Smac mimetics/IAPs inhibitors connected through head–head (8), tail–tail (9) or head–tail linkers (10), were biologically and structurally characterized. In vitro characterization (binding to BIR3 and linker-BIR2–BIR3 domains from XIAP and cIAP1, cytotoxicity assays) identified early leads from each dimer family. Computational models and structural studies (crystallography, NMR, gel filtration) partially rationalized the observed properties for each dimer class. Tail–tail dimer 9a was shown to be active in a breast and in an ovary tumor model, highlighting the potential of dimeric Smac mimetics/IAP inhibitors based on the N-AVPI-like 4-substituted 1-aza-2-oxobicyclo[5.3.0]decane scaffold as potential antineoplastic agents.     

Motor representation of actions in children with autism

Background

Children with Autistic Spectrum Disorders (ASD) are frequently hampered by motor impairment, with difficulties ranging from imitation of actions to recognition of motor intentions. Such a widespread inefficiency of the motor system is likely to interfere on the ontogeny of both motor planning and understanding of the goals of actions, thus delivering its ultimate effects on the emergence of social cognition.

Methodology/Principal Findings

We investigate the organization of action representation in 15 high functioning ASD (mean age: 8.11) and in two control samples of typically developing (TD) children: the first one, from a primary school, was matched for chronological age (CA), the second one, from a kindergarten, comprised children of much younger age (CY). We used nine newly designed behavioural motor tasks, aiming at exploring three domains of motor cognition: 1) imitation of actions, 2) production of pantomimes, and 3) comprehension of pantomimes. The findings reveal that ASD children fare significantly worse than the two control samples in each of the inspected components of the motor representation of actions, be it the imitation of gestures, the self-planning of pantomimes, or the (verbal) comprehension of observed pantomimes. In the latter task, owing to its cognitive complexity, ASD children come close to the younger TD children’s level of performance; yet they fare significantly worse with respect to their age-mate controls. Overall, ASD children reveal a profound damage to the mechanisms that control both production and pre-cognitive “comprehension” of the motor representation of actions.

Conclusions/Significance

Our findings suggest that many of the social cognitive impairments manifested by ASD individuals are likely rooted in their incapacity to assemble and directly grasp the intrinsic goal-related organization of motor behaviour. Such impairment of motor cognition might be partly due to an early damage of the Mirror Neuron Mechanism (MNM).