Galectin-3 and N-acetylglucosamine promote myogenesis and improve skeletal muscle function in the mdx model of Duchenne muscular dystrophy

The muscle membrane, sarcolemma, must be firmly attached to the basal lamina. The failure of proper attachment results in muscle injury, which is the underlying cause of Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene disrupts the firm adhesion. In DMD patients, even moderate contraction causes damage, leading to progressive muscle degeneration. The damaged muscles are repaired through myogenesis. Consequently, myogenesis is highly active in DMD patients, and the repeated activation of myogenesis leads to the exhaustion of the myogenic stem cells. Therefore, approaches to reducing the risk of the exhaustion are to develop a treatment that strengthens the interaction between the sarcolemma and the basal lamina, and increases the efficiency of myogenesis. Galectin-3 is an oligosaccharide-binding protein and known to be involved in cell–cell interactions and cell–matrix interactions. Galectin-3 is expressed in myoblasts and skeletal muscle while its function in muscle remains elusive. In this study, we found evidence that galectin-3 and the monosaccharide N-acetylglucosamine, which increases the ligands (oligosaccharides) of galectin-3, promotes myogenesis in vitro. Moreover, in the mdx mouse model of DMD, treatment with N-acetylglucosamine increased the muscle force production. Our results demonstrate that treatment with N-acetylglucosamine can mitigate the burden of DMD

Phosphodiesterase-III Inhibitor Prevents Hemorrhagic Transformation Induced by Focal Cerebral Ischemia in Mice Treated with tPA

The purpose of the present study was to investigate whether cilostazol, a phosphodiesterase-III inhibitor and antiplatelet drug, would prevent tPA-associated hemorrhagic transformation. Mice subjected to 6-h middle cerebral artery occlusion were treated with delayed tPA alone at 6 h, with combined tPA plus cilostazol at 6 h, or with vehicle at 6 h. We used multiple imaging (electron microscopy, spectroscopy), histological and neurobehavioral measures to assess the effects of the treatment at 18 h and 7 days after the reperfusion. To further investigate the mechanism of cilostazol to beneficial effect, we also performed an in vitro study with tPA and a phosphodiesterase-III inhibitor in human brain microvascular endothelial cells, pericytes, and astrocytes. Combination therapy with tPA plus cilostazol prevented development of hemorrhagic transformation, reduced brain edema, prevented endothelial injury via reduction MMP-9 activity, and prevented the blood-brain barrier opening by inhibiting decreased claudin-5 expression. These changes significantly reduced the morbidity and mortality at 18 h and 7 days after the reperfusion. Also, the administration of both drugs prevented injury to brain human endothelial cells and human brain pericytes. The present study indicates that a phosphodiesterase-III inhibitor prevents the hemorrhagic transformation induced by focal cerebral ischemia in mice treated with tPA

Numerical Analysis of Ca2+ Signaling in Rat Ventricular Myocytes with Realistic Transverse-Axial Tubular Geometry and Inhibited Sarcoplasmic Reticulum

The t-tubules of mammalian ventricular myocytes are invaginations of the cell membrane that occur at each Z-line. These invaginations branch within the cell to form a complex network that allows rapid propagation of the electrical signal, and hence synchronous rise of intracellular calcium (Ca2+). To investigate how the t-tubule microanatomy and the distribution of membrane Ca2+ flux affect cardiac excitation-contraction coupling we developed a 3-D continuum model of Ca2+ signaling, buffering and diffusion in rat ventricular myocytes. The transverse-axial t-tubule geometry was derived from light microscopy structural data. To solve the nonlinear reaction-diffusion system we extended SMOL software tool (http://mccammon.ucsd.edu/smol/). The analysis suggests that the quantitative understanding of the Ca2+ signaling requires more accurate knowledge of the t-tubule ultra-structure and Ca2+ flux distribution along the sarcolemma. The results reveal the important role for mobile and stationary Ca2+ buffers, including the Ca2+ indicator dye. In agreement with experiment, in the presence of fluorescence dye and inhibited sarcoplasmic reticulum, the lack of detectible differences in the depolarization-evoked Ca2+ transients was found when the Ca2+ flux was heterogeneously distributed along the sarcolemma. In the absence of fluorescence dye, strongly non-uniform Ca2+ signals are predicted. Even at modest elevation of Ca2+, reached during Ca2+ influx, large and steep Ca2+ gradients are found in the narrow sub-sarcolemmal space. The model predicts that the branched t-tubule structure and changes in the normal Ca2+ flux density along the cell membrane support initiation and propagation of Ca2+ waves in rat myocytes

Structural insight into the TFIIE–TFIIH interaction: TFIIE and p53 share the binding region on TFIIH

RNA polymerase II and general transcription factors (GTFs) assemble on a promoter to form a transcription preinitiation complex (PIC). Among the GTFs, TFIIE recruits TFIIH to complete the PIC formation and regulates enzymatic activities of TFIIH. However, the mode of binding between TFIIE and TFIIH is poorly understood. Here, we demonstrate the specific binding of the C-terminal acidic domain (AC-D) of the human TFIIEα subunit to the pleckstrin homology domain (PH-D) of the human TFIIH p62 subunit and describe the solution structures of the free and PH-D-bound forms of AC-D. Although the flexible N-terminal acidic tail from AC-D wraps around PH-D, the core domain of AC-D also interacts with PH-D. AC-D employs an entirely novel binding mode, which differs from the amphipathic helix method used by many transcriptional activators. So the binding surface between PH-D and AC-D is much broader than the specific binding surface between PH-D and the p53 acidic fragments. From our in vitro studies, we demonstrate that this interaction could be a switch to replace p53 with TFIIE on TFIIH in transcription

Repair of single-strand DNA interruptions by redundant pathways and its implication in cellular sensitivity to DNA-damaging agents

Single-strand DNA interruptions (SSIs) are produced during the process of base excision repair (BER). Through biochemical studies, two SSI repair subpathways have been identified: a pathway mediated by DNA polymerase β (Pol β) and DNA ligase III (Lig III), and a pathway mediated by DNA polymerase δ/ε (Pol δ/ε) and DNA ligase I (Lig I). In addition, the existence of another pathway, mediated by Pol β and DNA Lig I, has been suggested. Although each pathway may play a unique role in cellular DNA damage response, the functional implications of SSI repair by these three pathways are not clearly understood. To obtain a better understanding of the functional relevance of SSI repair by these pathways, we investigated the involvement of each pathway by monitoring the utilization of DNA ligases in cell-free extracts. Our results suggest that the majority of SSIs produced during the repair of alkylated DNA bases are repaired by the pathway mediated by Pol β and either Lig I or Lig III, although some SSIs are repaired by Pol δ/ε and Lig I. At a cellular level, we found that Lig III over-expression increased the resistance of cells to DNA-damaging agents, while Lig I over-expression had little effect. Thus, repair pathways mediated by Lig III may have a role in the regulation of cellular sensitivity to DNA-damaging agents

Dose-dependent spatiotemporal responses of mammalian cells to an alkylating agent.

Cultured cell populations are composed of heterogeneous cells, and previous single-cell lineage tracking analysis of individual HeLa cells provided empirical evidence for significant heterogeneity of the rate of cell proliferation and induction of cell death. Nevertheless, such cell lines have been used for investigations of cellular responses to various substances, resulting in incomplete characterizations. This problem caused by heterogeneity within cell lines could be overcome by investigating the spatiotemporal responses of individual cells to a substance. However, no approach to investigate the responses by analyzing spatiotemporal data is currently available. Thus, this study aimed to analyze the spatiotemporal responses of individual HeLa cells to cytotoxic, sub-cytotoxic, and non-cytotoxic doses of the well-characterized carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although cytotoxic doses of MNNG are known to induce cell death, the single-cell tracking approach revealed that cell death occurred following at least four different cellular events, suggesting that cell death is induced via multiple processes. We also found that HeLa cells exposed to a sub-cytotoxic dose of MNNG were in a state of equilibrium between cell proliferation and cell death, with cell death again induced through different processes. However, exposure of cells to a non-cytotoxic dose of MNNG promoted growth by reducing the cell doubling time, thus promoting the growth of a sub-population of cells. A single-cell lineage tracking approach could dissect processes leading to cell death in a spatiotemporal manner and the results suggest that spatiotemporal data obtained by tracking individual cells can be used as a new type of bioinformatics data resource that enables the examination of cellular responses to various external substances