A new diamond biosensor with integrated graphitic microchannels for detecting quantal exocytic events from chromaffin cells

The quantal release of catecholamines from neuroendocrine cells is a key mechanism which has been investigated with a broad range of materials and devices, among which carbon-based materials such as carbon fibers, diamond-like carbon, carbon nanotubes and nanocrystalline diamond. In the present work we demonstrate that a MeV-ion-microbeam lithographic technique can be successfully employed for the fabrication of an all-carbon miniaturized cellular bio-sensor based on graphitic micro-channels embedded in a single-crystal diamond matrix. The device was functionally characterized for the in vitro recording of quantal exocytic events from single chromaffin cells, with high sensitivity and signal-to-noise ratio, opening promising perspectives for the realization of monolithic all-carbon cellular biosensors

Fifty Hertz electromagnetic field exposure stimulates secretion of β-amyloid peptide in cultured human neuroglioma

Recent epidemiological studies raise the possibility that individuals with occupational exposure to low frequency (50-60 Hz) electromagnetic fields (LF-EMF), are at increased risk of Alzheimer's disease (AD). However, the mechanisms through which LF-EMF may affect AD pathology are unknown. We here tested the hypothesis that the exposure to LF-EMF may affect amyloidogenic processes. We examined the effect of exposure to 3.1 mT 50 Hz LF-EMF on Abeta secretion in H4 neuroglioma cells stably overexpressing human mutant amyloid precursor protein. We found that overnight exposure to LF-EMF induces a significant increase of amyloid-beta peptide (Abeta) secretion, including the isoform Abeta 1-42, without affecting cell survival. These findings show for the first time that exposure to LF-EMF stimulates Abeta secretion in vitro, thus alluding to a potential link between LF-EMF exposure and APP processing in the brain

Absolute Protein Amounts and Relative Abundance of Volume-regulated Anion Channel (VRAC) LRRC8 Subunits in Cells and Tissues Revealed by Quantitative Immunoblotting

The volume-regulated anion channel (VRAC) plays an important role in osmotic cell volume regulation. In addition, it is involved in various physiological processes such as insulin secretion, glia-neuron communication and purinergic signaling. VRAC is formed by hetero-hexamers of members of the LRRC8 protein family, which consists of five members, LRRC8A-E. LRRC8A is an essential subunit for physiological functionality of VRAC. Its obligate heteromerization with at least one of its paralogues, LRRC8B-E, determines the biophysical properties of VRAC. Moreover, the subunit composition is of physiological relevance as it largely influences the activation mechanism and especially the substrate selectivity. However, the endogenous tissue-specific subunit composition of VRAC is unknown. We have now developed and applied a quantitative immunoblot study of the five VRAC LRRC8 subunits in various mouse cell lines and tissues, using recombinant protein for signal calibration. We found tissue-specific expression patterns of the subunits, and generally relative low expression of the essential LRRC8A subunit. Immunoprecipitation of LRRC8A also co-precipitates an excess of the other subunits, suggesting that non-LRRC8A subunits present the majority in hetero-hexamers. With this, we can estimate that in the tested cell lines, the number of VRAC channels per cell is in the order of 10,000, which is in agreement with earlier calculations from the comparison of single-channel and whole-cell currents

Antiproton stopping power in hydrogen below 120 keV and the Barkas effect

The simultaneous measurement of the spatial coordinates and times of p¯s annihilating at rest in a H2 target at very low density ρ (ρ/ρ0<10-2, ρ0 being the STP density) gives the possibility of evaluating the behavior of the p¯ stopping power in H2 at low energies (below 120 keV). It is different from that of protons (the Barkas effect). Moreover, it is shown that a rise at low-energy values (≲1 keV) is needed to agree with experimental data

An evolutionary perspective on the kinome of malaria parasites

Malaria parasites belong to an ancient lineage that diverged very early from the main branch of eukaryotes. The approximately 90-member plasmodial kinome includes a majority of eukaryotic protein kinases that clearly cluster within the AGC, CMGC, TKL, CaMK and CK1 groups found in yeast, plants and mammals, testifying to the ancient ancestry of these families. However, several hundred millions years of independent evolution, and the specific pressures brought about by first a photosynthetic and then a parasitic lifestyle, led to the emergence of unique features in the plasmodial kinome. These include taxon-restricted kinase families, and unique peculiarities of individual enzymes even when they have homologues in other eukaryotes. Here, we merge essential aspects of all three malaria-related communications that were presented at the Evolution of Protein Phosphorylation meeting, and propose an integrated discussion of the specific features of the parasite's kinome and phosphoproteome

Collisions of antiprotons with hydrogen molecular ions

Time-dependent close-coupling calculations of the ionization and excitation cross section for antiproton collisions with molecular hydrogen ions are performed in an impact-energy range from 0.5 keV to 10 MeV. The Born-Oppenheimer and Franck-Condon approximations as well as the impact parameter method are applied in order to describe the target molecule and the collision process. It is shown that three perpendicular orientations of the molecular axis with respect to the trajectory are sufficient to accurately reproduce the ionization cross section calculated by [Sakimoto, Phys. Rev. A 71, 062704 (2005)] reducing the numerical effort drastically. The independent-event model is employed to approximate the cross section for double ionization and H+ production in antiproton collisions with H2.Comment: 12 pages, 5 figures, 4 table

Binary Decay of Light Nuclear Systems

A review of the characteristic features found in fully energy-damped, binarydecay yields from light heavy-ion reactions with $20\leq A_{target} + A_{projectile}\leq 80$ is presented. The different aspects of these yields that have been used to support models of compound-nucleus (CN) fission and deep-inelastic dinucleus orbiting are highlighted. Cross section calculations based on the statistical phase space at different stages of the reaction are presented and compared to the experimental results. Although the statistical models are found to reproduce most of the observed experimental behaviors, an additional reaction component corresponding to a heavy-ion resonance or orbiting mechanism is also evident in certain systems. The system dependence of this second component is discussed. The extent to which the binary yields in very light systems $(A_{CN} \leq 32)$ can be viewed as resulting from a fusion-fission mechanism is explored. A number of unresolved questions, such as whether the different observed behaviors reflect characteristically different reaction times, are discussed.Comment: 79 pages REVTeX file, 39 ps Figures included - to be publihed in Physics Report

Involvement of Plasmodium falciparum protein kinase CK2 in the chromatin assembly pathway

<p>Abstract</p> <p>Background</p> <p>Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of <it>Plasmodium falciparum </it>CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2.</p> <p>Results</p> <p>We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α <it>in vitro</it>.</p> <p>Conclusions</p> <p>Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.</p