A role for the Wnt3a/ß-catenin signaling pathway in the myogenic program of C2C12 cells.

The Wnt/ß-catenin signaling pathway is a key regulator of embryonic development that was first identified in drosophila. More recent work has suggested a role for this pathway in mammalian, myogenesis but this process remains to be more completely elucidated. The current study was designed to investigate the role of the Wnt3a/ß-catenin signaling module in C2C12 cell differentiation and myotube formation. Activation of the Wnt/ß-catenin pathway in differentiating myoblasts, using lithium chloride supplementation, increased both the number and size of C2C12 myotubes. On the other hand, pharmacological inhibitors of Wnt/ß-catenin signaling (IWR-1, XAV939) resulted in significant decreases in myotube length without affecting their numbers. In separate experiments, a neutralizing antibody to Wnt3a also reduced myotube length without affecting the number of myotubes formed during differentiation. Confocal imaging studies revealed that ß-catenin was primarily localized in the cytoplasmic region of undifferentiated myoblasts and differentiation resulted in its enhanced expression in myotubes. Additionally, LiCl treatment resulted in enhanced expression of ß-catenin in pre-differentiating myoblast cultures. These data suggest that Wnt3a/ß-catenin signaling plays a critical role in C2C12 cell differentiation and provides an appropriate model for elucidating its functions in more detail.

Listeria monocytogenes wall teichoic acid decoration in virulence and cell-to-cell spread.

Wall teichoic acid (WTA) comprises a class of glycopolymers covalently attached to the peptidoglycan of gram positive bacteria. In Listeria monocytogenes, mutations that prevent addition of certain WTA decorating sugars are attenuating. However, the steps required for decoration and the pathogenic process interrupted are not well described. We systematically examined the requirement for WTA galactosylation in a mouse oral-virulent strain by first creating mutations in four genes whose products conferred resistance to a WTA-binding bacteriophage. WTA biochemical and structural studies indicated that galactosylated WTA was directly required for bacteriophage adsorption and that mutant WTA lacked appreciable galactose in all except one mutant - which retained a level ca. 7% of the parent. All mutants were profoundly attenuated in orally infected mice and were impaired in cell-to-cell spread in vitro. Confocal microscopy of cytosolic mutants revealed that all expressed ActA on their cell surface and formed actin tails with a frequency similar to the parent. However, the mutant tails were significantly shorter - suggesting a defect in actin based motility. Roles for the gene products in WTA galactosylation are proposed. Identification and interruption of WTA decoration pathways may provide a general strategy to discover non-antibiotic therapeutics for gram positive infections. © 2016 John Wiley & Sons Ltd.

Increased expression of deltaCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease.

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.

A role for protein kinase C in the supersensitivity of the rat vas deferens following chronic surgical denervation.

Chronic surgical denervation of the rat vas deferens leads to an enhanced contractile response of the tissue to norepinephrine in vitro. Norepinephrine produces a higher rate of protein kinase C translocation to the particulate fraction of denervated tissues as compared with the paired, control vas deferens. Diacylglycerol generation in response to norepinephrine and contractile responses to phorbol diacetate were not altered by chronic denervation of the vas deferens. However, the contractile response to norepinephrine in these tissues was less susceptible to the inhibitory effects of the calcium channel blocker nifedipine. A potential role of protein kinase C in sensitizing the contractile apparatus to mobilized calcium in denervation supersensitivity is discussed.