Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique – the actin co-sedimentation, or pelleting, assay – to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin

The Okun Misery Index in the European Union Countries from 2000 to 2009

The study is composed of four main parts and a summary. The first part, introduction, discusses various measures of the economic system's efficiency that are used in practice. Part two emphasises that the GDP per capita according to purchasing power parity still remains the most popular among those measures. Further, it presents the ranking of the European Union countries taking that measure into account, the research period being 1999-2009. Part three points out that it is also the level of poverty (misery) that determines the economic system's efficiency. That level can be measured by means of various indicators, among others, the so called HPI-2 index calculated by the UN. It will be the Okun misery index, however, computed as the sum of inflation and unemployment rates that will be presented as an alternative being of interest from the macroeconomic point of view. The ranking of the European Union member states according to that measure in the 2000-2004 and 2005-2009 periods will be provided in part four. The article will end in a summary containing synthetic conclusions drawn from earlier observations.Opracowanie składa się z czterech części zasadniczych i podsumowania. W punkcie pierwszym omówiono różnorodne mierniki sprawności systemu gospodarczego wykorzystywane w praktyce. W części drugiej podkreślono, iż nadal najpopularniejszym z nich jest PKB per capita według parytetu siły nabywczej. Zgodnie z tym miernikiem przedstawiono ranking państw Unii Europejskiej w latach 1999-2009. W punkcie trzecim podkreślono, że o sprawności systemu gospodarczego decyduje także poziom ubóstwa. Może być on mierzony różnymi wskaźnikami, m.in. tzw. indeksem HPI-2 obliczanym przez ONZ. Jako ciekawą z makroekonomicznego punktu widzenia alternatywę ukazano jednak miarę wskaźnika ubóstwa Okuna obliczanego poprzez zsumowanie stopy inflacji i stopy bezrobocia. Ranking państw Unii Europejskiej według tej miary w okresach 2000-2004 oraz 2005-2009 zaprezentowano w części czwartej. Całość zamknięto podsumowaniem, w którym zawarto syntetyczne wnioski z przeprowadzonych obserwacji

Extinction of the N=20 neutron-shell closure for 32Mg examined by direct mass measurements

The 'island of inversion' around $^{32}$Mg is one of the most important paradigm for studying the disappearance of the stabilizing 'magic' of a shell closure. We present the first Penning-trap mass measurements of the exotic nuclides $^{29-31}$Na and $^{30-34}$Mg, which allow a precise determination of the empirical shell gap for $^{32}$Mg. The new value of 1.10(3) MeV is the lowest observed shell gap for any nuclide with a canonical magic number.Comment: 6 pages, 4 figures, submitted to Physical Review

Synthesis of a norcantharidin-tethered guanosine: Protein phosphatase-1 inhibitors that change alternative splicing.

Phosphorylation and dephosphorylation of splicing factors play a key role in pre-mRNA splicing events, and cantharidin and norcantharidin analogs inhibit protein phosphatase-1 (PP1) and change alternative pre-mRNA splicing. Targeted inhibitors capable of selectively inhibiting PP-1 could promote exon 7 inclusion in the survival-of-motorneuron-2 gene (SMN2) and shift the proportion of SMN2 protein from a dysfunctional to a functional form. As a prelude to the development of norcantharidin-tethered oligonucleotide inhibitors, the synthesis a norcantharidin-tethered guanosine was developed in which a suitable tether prevented the undesired cyclization of norcantharidin monoamides to imides and possessed a secondary amine terminus suited to the synthesis of oligonucleotides analogs. Application of this methodology led to the synthesis of a diastereomeric mixture of norcantharidin-tethered guanosines, namely bisammonium (1R,2S,3R,4S)- and (1S,2R,3S,4R)-3-((4-(2-(((((2R,3R,4R,5R)-5-(2-amino-6-oxo-1,6- dihydro-9H-purin-9-yl)-2-(hydroxymethyl)-4-methoxytetrahydrofuran-3-yl) oxy) oxidophosphoryl) oxy) ethyl)-phenethyl)(methyl)carbamoyl)-7-oxabicyclo[2.2.1] heptane-2-carboxylate, which showed activity in an assay for SMN2 pre-mRNA splicin

Thermally-induced expansion in the 8 GeV/c π−\pi^- + 197^{197}Au reaction

Fragment kinetic energy spectra for reactions induced by 8.0 GeV/c $\rm{\pi^-}$ beams incident on a $\rm{^{197}}$Au target have been analyzed in order to deduce the possible existence and influence of thermal expansion. The average fragment kinetic energies are observed to increase systematically with fragment charge but are nearly independent of excitation energy. Comparison of the data with statistical multifragmentation models indicates the onset of extra collective thermal expansion near an excitation energy of E*/A $\rm{\approx}$ 5 MeV. However, this effect is weak relative to the radial expansion observed in heavy-ion-induced reactions, consistent with the interpretation that the latter expansion may be driven primarily by dynamical effects such as compression/decompression.Comment: 12 pages including 4 postscript figure

Breakup Density in Spectator Fragmentation

Proton-proton correlations and correlations of protons, deuterons and tritons with alpha particles from spectator decays following 197Au + 197Au collisions at 1000 MeV per nucleon have been measured with two highly efficient detector hodoscopes. The constructed correlation functions, interpreted within the approximation of a simultaneous volume decay, indicate a moderate expansion and low breakup densities, similar to assumptions made in statistical multifragmentation models. PACS numbers: 25.70.Pq, 21.65.+f, 25.70.Mn, 25.75.GzComment: 11 pages, LaTeX with 3 included figures; Also available from http://www-kp3.gsi.de/www/kp3/aladin_publications.htm

Ena/VASP is required for endothelial barrier function in vivo

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are key actin regulators that localize at regions of dynamic actin remodeling, including cellular protrusions and cell–cell and cell–matrix junctions. Several studies have suggested that Ena/VASP proteins are involved in the formation and function of cellular junctions. Here, we establish the importance of Ena/VASP in endothelial junctions in vivo by analysis of Ena/VASP-deficient animals. In the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and late stage embryonic lethality. In endothelial cells, we find that Ena/VASP activity is required for normal F-actin content, actomyosin contractility, and proper response to shear stress. These findings demonstrate that Ena/VASP is critical for actin cytoskeleton remodeling events involved in the maintenance of functional endothelia

The liquid to vapor phase transition in excited nuclei

For many years it has been speculated that excited nuclei would undergo a liquid to vapor phase transition. For even longer, it has been known that clusterization in a vapor carries direct information on the liquid- vapor equilibrium according to Fisher's droplet model. Now the thermal component of the 8 GeV/c pion + 197Au multifragmentation data of the ISiS Collaboration is shown to follow the scaling predicted by Fisher's model, thus providing the strongest evidence yet of the liquid to vapor phase transition.Comment: four pages, four figures, first two in color (corrected typo in Ref. [26], corrected error in Fig. 4

Signals for a Transition from Surface to Bulk Emission in Thermal Multifragmentation

Excitation-energy-gated two-fragment correlation functions have been studied between 2 to 9A MeV of excitation energy for equilibrium-like sources formed in $\pi^-$ and p + $^{197}$Au reactions at beam momenta of 8,9.2 and 10.2 GeV/c. Comparison of the data to an N-body Coulomb-trajectory code shows a decrease of one order of magnitude in the fragment emission time in the excitation energy interval 2-5A MeV, followed by a nearly constant breakup time at higher excitation energy. The observed decrease in emission time is shown to be strongly correlated with the increase of the fragment emission probability, and the onset of thermally-induced radial expansion. This result is interpreted as evidence consistent with a transition from surface-dominated to bulk emission expected for spinodal decomposition.Comment: 11 pages including 3 postscript figures (1 color