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OBJECTIVE We have developed novel methods for isolating fetal central nervous system (CNS)-derived extracellular vesicles (FCEs) from maternal plasma as a non-invasive platform for testing aspects of fetal neurodevelopment in early pregnancy. We investigate the hypothesis that levels of defined sets of functional proteins in FCEs can be used to detect abnormalities in fetal neuronal and glial proliferation, differentiation, and survival. METHOD Maternal plasma was obtained between 10 and 19 weeks from women with current heavy EtOH exposure and matched controls. FCE levels of synaptophysin, synaptotagmin, synaptopodin, and neurogranin were quantified normalized to the exosome marker CD81. Quantitative RT-PCR was performed with specific primers for miR-9. RESULTS FCE cargo protein levels of synaptophysin, synaptotagmin, synaptopodin, and neurogranin were all significantly reduced in pregnancies exposed to current heavy EtOH use (P

The pathophysiology of human immunodeficiency virus (HIV)-associated cardiomyopathy remains uncertain. We used HIV-1 transgenic (Tg26) mice to explore mechanisms by which HIV-related proteins impacted on myocyte function. Compared to adult ventricular myocytes isolated from nontransgenic (wild type [WT]) littermates, Tg26 myocytes had similar mitochondrial membrane potential (ΔΨ m ) under normoxic conditions but lower Δ Ψ m after hypoxia/reoxygenation (H/R). In addition, Δ Ψ m in Tg26 myocytes failed to recover after Ca 2+ challenge. Functionally, mitochondrial Ca 2+ uptake was severely impaired in Tg26 myocytes. Basal and maximal oxygen consumption rates (OCR) were lower in normoxic Tg26 myocytes, and further reduced after H/R. Complex I subunit and ATP levels were lower in Tg26 hearts. Post-H/R, mitochondrial superoxide (O 2 •- ) levels were higher in Tg26 compared to WT myocytes. Overexpression of B-cell lymphoma 2-associated athanogene 3 (BAG3) reduced O 2 •- levels in hypoxic WT and Tg26 myocytes back to normal. Under normoxic conditions, single myocyte contraction dynamics were similar between WT and Tg26 myocytes. Post-H/R and in the presence of isoproterenol, myocyte contraction amplitudes were lower in Tg26 myocytes. BAG3 overexpression restored Tg26 myocyte contraction amplitudes to those measured in WT myocytes post-H/R. Coimmunoprecipitation experiments demonstrated physical association of BAG3 and the HIV protein Tat. We conclude: (a) Under basal conditions, mitochondrial Ca 2+ uptake, OCR, and ATP levels were lower in Tg26 myocytes; (b) post-H/R, Δ Ψ m was lower, mitochondrial O 2 •- levels were higher, and contraction amplitudes were reduced in Tg26 myocytes; and (c) BAG3 overexpression decreased O 2 •- levels and restored contraction amplitudes to normal in Tg26 myocytes post-H/R in the presence of isoproterenol.

Autophagy is an evolutionarily conserved, selective degradation pathway of cellular components that is important for cell homeostasis under healthy and pathologic conditions. Here we demonstrate that an increase in the level of BAG3 results in stimulation of autophagy in glioblastoma cells. BAG3 is a member of a co-chaperone family of proteins that associate with Hsp70 through a conserved BAG domain positioned near the C-terminus of the protein. Expression of BAG3 is induced by a variety of environmental changes that cause stress to cells. Our results show that BAG3 overexpression induces autophagy in glioma cells. Interestingly, inhibition of the proteasome caused an increase in BAG3 levels and induced autophagy. Further analysis using specific siRNA against BAG3 suggests that autophagic activation due to proteosomal inhibition is mediated by BAG3. Analyses of BAG3 domain mutants suggest that the WW domain of BAG3 is crucial for the induction of autophagy. BAG3 overexpression also increased the interaction between Bcl2 and Beclin-1, instead of disrupting them, suggesting that BAG3 induced autophagy is Beclin-1 independent. These observations reveal a novel role for the WW domain of BAG3 in the regulation of autophagy.

Ethanol induces neuronal cell injury and death by dysregulating several signaling events that are controlled, in part, by activation of MAPK/ERK1/2 and/or inactivation of its corresponding phosphatase, PP1. Recently, we have purified a novel protein of 38 kDa in size, p38SJ, from a callus culture of Hypericum perforatum, which belongs to an emerging DINGG family of proteins with phosphate binding activity. Here, we show that treatment of neuronal cells with p38SJ protects cells against injury induced by exposure to ethanol. Furthermore, pre-treatment of neuronal cells with p38SJ diminishes the level of the pro-apoptotic protein Bax and some events associated with apoptosis such as caspase 3 cleavage. In addition, by inducing stress, alcohol can elevate production of reactive oxygen species (ROS) that leads to a decrease in the activity of superoxide dismutase (SOD). Our results showed that p38SJ restores the activity of SOD in the ethanol treated neuronal cells. These observations provide a novel biological tool for developing new approaches for preventing neuronal cell death induced by ethanol and possibly treatment of neurological disorders associated with alcohol abuse. J. Cell. Physiol. 221: 499–504, 2009. © 2009 Wiley-Liss, Inc.