Characterization of a microsomal subfraction associated with mitochondria of the yeast, Saccharomyces cerevisiae. Involvement in synthesis and import of phospholipids into mitochondria

AbstractIn the yeast, Saccharomyces cerevisiae, similar to higher eukaryotes most phospholipids are synthesized in microsomes. Mitochondria contribute to the cellular biosynthesis of phospholipids insofar as they harbor phosphatidylethanolamine decarboxylase, and enzymes of phosphatidylglycerol and cardiolipin synthesis. In this paper we present evidence that certain enzymes of phospholipid biosynthesis, namely phosphatidylserine and phosphatidylinositol synthase, are enriched in a special microsomal fraction associated with mitochondria, which we named MAM. This fraction was isolated and characterized with respect to marker enzymes, protein and phospholipid composition, and enzymes of phospholipid synthesis. According to these analyses MAMs are a specialized subfraction of the endoplasmic reticulum, which is distinct from other microsomal subfractions. Phosphatidylserine synthesized in MAMs can be readily imported into mitochondria and converted to phosphatidylethanolamine. Reassociation of MAMs with purified mitochondria led to reconstitution of the import of phosphatidylserine into mitochondria. Organelle contact is suggested as a possible mechanism of this process

Are treatment effects of neurofeedback training in children with ADHD related to the successful regulation of brain activity? A review on the learning of regulation of brain activity and a contribution to the discussion on specificity

While issues of efficacy and specificity are crucial for the future of neurofeedback training, there may be alternative designs and control analyses to circumvent the methodological and ethical problems associated with double-blind placebo studies. Surprisingly, most NF studies do not report the most immediate result of their NF training, i.e., whether or not children with ADHD gain control over their brain activity during the training sessions. For the investigation of specificity, however, it seems essential to analyze the learning and adaptation processes that take place in the course of the training and to relate improvements in self-regulated brain activity across training sessions to behavioral, neuropsychological and electrophysiological outcomes. To this aim, a review of studies on neurofeedback training with ADHD patients which include the analysis of learning across training sessions or relate training performance to outcome is presented. Methods on how to evaluate and quantify learning of EEG regulation over time are discussed. "Non-learning" has been reported in a small number of ADHD-studies, but has not been a focus of general methodological discussion so far. For this reason, selected results from the brain-computer interface (BCI) research on the so-called "brain-computer illiteracy", the inability to gain control over one's brain activity, are also included. It is concluded that in the discussion on specificity, more attention should be devoted to the analysis of EEG regulation performance in the course of the training and its impact on clinical outcome. It is necessary to improve the knowledge on characteristic cross-session and within-session learning trajectories in ADHD and to provide the best conditions for learning

Identification of Novel in Vivo Phosphorylation Sites of the Human Proapoptotic Protein BAD: PORE-FORMING ACTIVITY OF BAD IS REGULATED BY PHOSPHORYLATION*

BAD is a proapoptotic member of the Bcl-2 protein family that is regulated by phosphorylation in response to survival factors. Although much attention has been devoted to the identification of phosphorylation sites in murine BAD, little data are available with respect to phosphorylation of human BAD protein. Using mass spectrometry, we identified here besides the established phosphorylation sites at serines 75, 99, and 118 several novel in vivo phosphorylation sites within human BAD (serines 25, 32/34, 97, and 124). Furthermore, we investigated the quantitative contribution of BAD targeting kinases in phosphorylating serine residues 75, 99, and 118. Our results indicate that RAF kinases represent, besides protein kinase A, PAK, and Akt/protein kinase B, in vivo BAD-phosphorylating kinases. RAF-induced phosphorylation of BAD was reduced to control levels using the RAF inhibitor BAY 43-9006. This phosphorylation was not prevented by MEK inhibitors. Consistently, expression of constitutively active RAF suppressed apoptosis induced by BAD and the inhibition of colony formation caused by BAD could be prevented by RAF. In addition, using the surface plasmon resonance technique, we analyzed the direct consequences of BAD phosphorylation by RAF with respect to association with 14-3-3 and Bcl-2/Bcl-XL proteins. Phosphorylation of BAD by active RAF promotes 14-3-3 protein association, in which the phosphoserine 99 represented the major binding site. Finally, we show here that BAD forms channels in planar bilayer membranes in vitro. This pore-forming capacity was dependent on phosphorylation status and interaction with 14-3-3 proteins. Collectively, our findings provide new insights into the regulation of BAD function by phosphorylation