Synergy Between Intercellular Communication and Intracellular Ca2+ Handling in Arrhythmogenesis

Calcium is the primary signalling component of excitation-contraction coupling, the process linking electrical excitability of cardiac muscle cells to coordinated contraction of the heart. Understanding Ca2þ handling processes at the cellular level and the role of intercellular communication in the emergence of multicellular synchronization are key aspects in the study of arrhythmias. To probe these mechanisms, we have simulated cellular interactions on large scale arrays that mimic cardiac tissue, and where individual cells are represented by a mathematical model of intracellular Ca2þ dynamics. Theoretical predictions successfully reproduced experimental findings and provide novel insights on the action of two pharmacological agents (ionomycin and verapamil) that modulate Ca2þ signalling pathways via distinct mechanisms. Computational results have demonstrated how transitions between local synchronisation events and large scale wave formation are affected by these agents. Entrainment phenomena are shown to be linked to both ntracellular Ca2þ and coupling-specific dynamics in a synergistic manner. The intrinsic variability of the cellular matrix is also shown to affect emergent patterns of rhythmicity, providing insights into the origins of arrhythmogenic Ca2þ perturbations in cardiac tissue in situ

Modification of the L1-CAM carboxy-terminus in pancreatic adenocarcinoma cells

The neural cell adhesion molecule L1 has recently been shown to be expressed in pancreatic adenocarcinoma (PDAC) cells. In this report, we demonstrate that L1 is expressed by moderately- to poorly-differentiated PDAC cells in situ, and that L1 expression is a predictor of poor patient survival. In vitro, reduced reactivity of an anti-L1 carboxy-terminus-specific antibody was observed in the more poorly differentiated fast-growing (FG) variant of the COLO357 population, versus its well-differentiated slow-growing (SG) counterpart, even though they express equivalent total L1. The carboxy-terminus of L1 mediates binding to the MAP kinase-regulating protein RanBPM and mutation of T1247/S1248 within this region attenuates the expression of malignancy associated proteins and L1-induced tumorigenicity in mice. Therefore, we reasoned that the differential epitope exposure observed might be indicative of modifications responsible for regulating these events. However, epitope mapping demonstrated that the major determinant of binding was actually N1251; mutation of T1247 and S1248, alone or together, had little effect on C20 binding. Moreover, cluster assays using CD25 ectodomain/L1 cytoplasmic domain chimeras demonstrated the N1251-dependent, RanBPM-independent stimulation of erk phosphorylation in these cells. Reactivity of this antibody also reflects the differential exposure of extracellular epitopes in these COLO357 sublines, consistent with the previous demonstration of L1 ectodomain conformation modulation by intracellular modifications. These data further support a central role for L1 in PDAC, and define a specific role for carboxy-terminal residues including N1251 in the regulation of L1 activity in PDAC cells

Taking down the FLAG! How Insect Cell Expression Challenges an Established Tag-System

In 1988 the preceding journal of Nature Biotechnology, Bio/Technology, reported a work by Hopp and co-workers about a new tag system for the identification and purification of recombinant proteins: the FLAG-tag. Beside the extensively used hexa-his tag system the FLAG-tag has gained broad popularity due to its small size, its high solubility, the presence of an internal Enterokinase cleavage site, and the commercial availability of high-affinity anti-FLAG antibodies. Surprisingly, considering the heavy use of FLAG in numerous laboratories world-wide, we identified in insect cells a post-translational modification (PTM) that abolishes the FLAG-anti-FLAG interaction rendering this tag system ineffectual for secreted proteins. The present publication shows that the tyrosine that is part of the crucial FLAG epitope DYK is highly susceptible to sulfation, a PTM catalysed by the enzyme family of Tyrosylprotein-Sulfo-transferases (TPSTs). We showed that this modification can result in less than 20% of secreted FLAG-tagged protein being accessible for purification questioning the universal applicability of this established tag system

Pre- and syn-eruptive degassing and crystallisation processes of the 2010 and 2006 eruptions of Merapi volcano, Indonesia

The 2010 eruption of Merapi (VEI 4) was the volcano’s largest since 1872. In contrast to the prolonged and effusive dome-forming eruptions typical of Merapi’s recent activity, the 2010 eruption began explosively, before a new dome was rapidly emplaced. This new dome was subsequently destroyed by explosions, generating pyroclastic density currents (PDCs), predominantly consisting of dark coloured, dense blocks of basaltic andesite dome lava. A shift towards open-vent conditions in the later stages of the eruption culminated in multiple explosions and the generation of PDCs with conspicuous grey scoria and white pumice clasts resulting from sub-plinian convective column collapse. This paper presents geochemical data for melt inclusions and their clinopyroxene hosts extracted from dense dome lava, grey scoria and white pumice generated during the peak of the 2010 eruption. These are compared with clinopyroxene-hosted melt inclusions from scoriaceous dome fragments from the prolonged dome-forming 2006 eruption, to elucidate any relationship between pre-eruptive degassing and crystallisation processes and eruptive style. Secondary ion mass spectrometry analysis of volatiles (H2O, CO2) and light lithophile elements (Li, B, Be) is augmented by electron microprobe analysis of major elements and volatiles (Cl, S, F) in melt inclusions and groundmass glass. Geobarometric analysis shows that the clinopyroxene phenocrysts crystallised at depths of up to 20 km, with the greatest calculated depths associated with phenocrysts from the white pumice. Based on their volatile contents, melt inclusions have re-equilibrated during shallower storage and/or ascent, at depths of ~0.6–9.7 km, where the Merapi magma system is interpreted to be highly interconnected and not formed of discrete magma reservoirs. Melt inclusions enriched in Li show uniform “buffered” Cl concentrations, indicating the presence of an exsolved brine phase. Boron-enriched inclusions also support the presence of a brine phase, which helped to stabilise B in the melt. Calculations based on S concentrations in melt inclusions and groundmass glass require a degassing melt volume of 0.36 km3 in order to produce the mass of SO2 emitted during the 2010 eruption. This volume is approximately an order of magnitude higher than the erupted magma (DRE) volume. The transition between the contrasting eruptive styles in 2010 and 2006 is linked to changes in magmatic flux and changes in degassing style, with the explosive activity in 2010 driven by an influx of deep magma, which overwhelmed the shallower magma system and ascended rapidly, accompanied by closed-system degassing