The main extracellular matrix binding component of the dystrophin-glycoprotein complex, ␣-dystroglycan (␣-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown ␣-DG to be modified by both O-GalNAc-and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle ␣-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on ␣-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from ␣-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.Defects in protein glycosylation related to human disease were first reported in the 1980s, and since then, about 40 various types of congenital disorders of glycosylation have been reported (1). The term congenital disorders of glycosylation was first used to describe alterations of the N-glycosylation pathway and was later expanded to include the O-glycosylation pathways (1-3). The importance and complexity of O-linked glycosylation have only recently begun to be appreciated (1, 3, 4). In particular, mutations in genes encoding (putative) glycosyltransferases, which catalyze the addition and extension of O-linked mannose-initiated glycans, have garnered increased attention in the last decade given that they are causative for several forms of congenital muscular dystrophy (5, 6).The most common forms of O-glycosylation on secretory proteins are the mucin-like O-GalNAc structures that are initiated by polypeptide N-␣-acetylgalactosaminyltransferases in the endoplasmic reticulum-Golgi intermediate compartment and/or early cis-Golgi (7). Additionally, other O-linked structures are initiated with alternative monosaccharides, such as O-mannose, O-glucose, O-fucose, O-xylose, and O-GlcNAc onSer/Thr residues and the O-galactose modification of hydroxylysine residues in collagen domains (4). The diversity of O-mannosylated proteins in mammals, although quite abundant in some tissues (ϳ30% of O-glycans released from mouse brains (8)), has not been well characterized. The only clearly identified mamma...

Resveratrol, a naturally occurring phytoalexin, is known to induce apoptosis in multiple cancer cell types, but the underlying molecular mechanisms remain unclear. Here, we show that resveratrol induced p53-independent, X-linked inhibitor of apoptosis protein (XIAP)-mediated translocation of Bax to mitochondria where it underwent oligomerization to initiate apoptosis. Resveratrol treatment promoted interaction between Bax and XIAP in the cytosol and on mitochondria, suggesting that XIAP plays a critical role in the activation and translocation of Bax to mitochondria. This process did not involve p53 but required accumulation of Bim and t-Bid on mitochondria. Bax primarily underwent homo-oligomerization on mitochondria and played a major role in release of cytochrome c to the cytosol. Bak, another key protein that regulates the mitochondrial membrane permeabilization, did not interact with p53 but continued to associate with Bcl-xL. Thus, the proapoptotic function of Bak remained suppressed during resveratrol-induced apoptosis. Caspase-9 silencing inhibited resveratrol-induced caspase activation, whereas caspase-8 knockdown did not affect caspase activity, suggesting that resveratrol induces caspase-9-dependent apoptosis. Together, our findings characterize the molecular mechanisms of resveratrol-induced caspase activation and subsequent apoptosis in cancer cells.Anticancer agents induce cell death in cancer and normal cells via mechanisms including apoptosis and autophagy (1-4). Therefore, there is a need for alternative anticancer agents that can promote cancer cell death while avoiding killing of normal, non-cancerous cells. Resveratrol (trans-3,5,4Ј-trihydroxystilbene) is a naturally occurring polyphenolic phytoalexin found at high levels in the skin of grapes and in red wine. It is also present in peanuts and other plant products. Resveratrol has been shown to possess an apoptosis-dependent anticancer activity and minimal toxicity to normal cells (5-11). How resveratrol induces apoptosis or cancer cell death is not clearly known, but available evidence indicates that resveratrol induces p53-dependent signaling, which leads to cell cycle arrest and apoptosis induction (10, 12, 13). Additionally, resveratrol targets mitochondria to induce cytochrome c release and thereby triggers caspase-dependent apoptotic cell death in multiple types of cancer cells (14 -18). How resveratrol induces cytochrome c release and caspase activation to execute apoptosis remains unclear.Caspases are activated by proteolytic processing and are broadly divided into initiator caspases (e.g. procaspase-8 and -9) and executioner caspases (such as procaspase-3 and -7) (19 -22). During apoptosis, the released cytochrome c from mitochondria triggers caspase-9 activation, whereas ligation of death receptors on the plasma membrane activates caspase-8. Active caspase-8 generated upon death receptor ligation requires Bid-mediated cytochrome c release to execute apoptotic cell death in epithelial cancer cells (22)(23)(24)(25). Proapoptotic BH3-o...

The study aimed primarily to evaluate the efficacy of noninvasive ventilation (NIV) and to identify possible predictors for success of NIV therapy in preventing extubation failure in critically ill children with heart disease. The secondary objectives of this study were to assess the efficacy of prophylactic NIV therapy initiated immediately after tracheal extubation and to determine the characteristics, outcomes, and complications associated with NIV therapy in pediatric cardiac patients. A retrospective review examined the medical records of all children between the ages 1 day and 18 years who sustained acute respiratory failure (ARF) that required NIV in the cardiovascular intensive care unit (CVICU) at Lucile Packard Children's Hospital between January 2008 and June 2010. Patients were assigned to a prophylactic group if NIV was started directly after extubation and to a nonprophylactic group if NIV was started after signs and symptoms of ARF developed. Patients were designated as responders if they received NIV and did not require reintubation during their CVICU stay and nonresponders if they failed NIV and reintubation was performed. The data collected included demographic data, preexisting conditions, pre-event characteristics, event characteristics, and outcome data. The outcome data evaluated included success or failure of NIV, duration of NIV, CVICU length of stay (LOS), hospital LOS, and hospital mortality. The two complications of NIV assessed in the study included nasal bridge or forehead skin necrosis and pneumothorax. The 221 eligible events during the study period involved 172 responders (77.8 %) and 49 nonresponders (22.2 %). A total of 201 events experienced by the study cohort received continuous positive airway pressure (CPAP), with 156 responders (78 %), whereas 20 events received bilevel positive airway pressure (BiPAP), with 16 responders (80 %). In the study, 58 events (26.3 %) were assigned to the prophylactic group and 163 events (73.7 %) to the nonprophylactic group. Compared with the nonprophylactic group, the prophylactic group experienced significantly shorter CVICU LOS (median, 49 vs 88 days; p = 0.03) and hospital LOS (median, 60 vs 103 days; p = 0.05). The CVICU LOS and hospital LOS did not differ significantly between the responders (p = 0.56) and nonresponders (p = 0.88). Significant variables identifying a responder included a lower risk-adjusted classification for congenital heart surgery (RACHS-1) score (1-3), a good left ventricular ejection fraction, a normal respiratory rate (RR), normal or appropriate oxygen saturation, prophylactic or therapeutic glucocorticoid therapy within 24 h of NIV initiation, presence of atelectasis, fewer than two organ system dysfunctions, fewer days of intubation before extubation, no clinical or microbiologic evidence of sepsis, and no history of reactive airway disease. As a well-tolerated therapy, NIV can be safely and successfully applied in critically ill children with cardiac disease to prevent extubation failure. The independent predicto...

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