Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain

The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans - stereoconfiguration in the beta-position are described. The water- soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis -isoprene unit in the beta-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc- P-Dol10was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis -isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water- soluble analog may provide an experimental approach to assay the hypothetical "flippase" proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer.      

Minimal model for human ventricular action potentials in tissue

Modeling the dynamics of wave propagation in human ventricular tissue and studying wave stability require models that reproduce realistic characteristics in tissue. We present a minimal ventricular (MV) human model that is designed to reproduce important tissue-level characteristics of epicardial, endocardial and midmyocardial cells, including action potential (AP) amplitudes and morphologies, upstroke velocities, steady-state action potential duration (APD) and conduction velocity (CV) restitution curves, minimum APD, and minimum diastolic interval. The model is then compared with three previously published human ventricular cell models, the Priebe and Beuckelmann (PB), the Ten Tusscher–Noble–Noble–Panfilov (TNNP), and the Iyer–Mazhari–Winslow (IMW). For the first time, the stability of reentrant waves for all four models is analyzed, and quantitative comparisons are made among the models in single cells and in tissue. The PB, TNNP, and IMW models exhibit quantitative differences in APD and CV rate adaptation, as well as completely different reentrant wave dynamics of quasi-breakup, stability, and breakup, respectively. All the models have dominant frequencies comparable to clinical values except for the IMW model, which has a large range of frequencies extending beyond the clinical range for both ventricular tachycardia (VT) and ventricular fibrillation (VF). The TNNP and IMW models possess a large degree of short-term memory and we show for the first time the existence of memory in CV restitution. The MV model also can be fitted to reproduce the dynamics of other models and is computationally more efficient: the times required to simulate the MV, TNNP, PB and IMW models follow the ratio 1:31:50:8084.     

Receptor association and tyrosine phosphorylation of S6 kinases

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.     

Application of an optimized marker amplification protocol in immunohistochemistry — a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.     

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease.