Analysis of transient phosphorylation-dependent protein-protein interactions in living mammalian cells using split-TEV

Abstract Background Regulated protein-protein interactions (PPIs) are pivotal molecular switches that are important for the regulation of signaling processes within eukaryotic cells. Cellular signaling is altered in various disease conditions and offers interesting options for pharmacological interventions. Constitutive PPIs are usually mediated by large interaction domains. In contrast, stimulus-regulated PPIs often depend on small post-translational modifications and are thus better suited targets for drug development. However, the detection of modification-dependent PPIs with biochemical methods still remains a labour- and material-intensive task, and many pivotal PPIs that are potentially suited for pharmacological intervention most likely remain to be identified. The availability of methods to easily identify and quantify stimulus-dependent, potentially also transient interaction events, is therefore essential. The assays should be applicable to intact mammalian cells, optimally also to primary cells in culture. Results In this study, we adapted the split-TEV system to quantify phosphorylation-dependent and transient PPIs that occur at the membrane and in the cytosol of living mammalian cells. Split-TEV is based on a PPI-induced functional complementation of two inactive TEV protease fragments fused to interaction partners of choice. Genetically encoded transcription-coupled and proteolysis-only TEV reporter systems were used to convert the TEV activity into an easily quantifiable readout. We measured the phosphorylation-dependent interaction between the pro-apoptotic protein Bad and the adapter proteins 14-3-3ε and ζ in NIH-3T3 fibroblasts and in primary cultured neurons. Using split-TEV assays, we show that Bad specifically interacts with 14-3-3 isoforms when phosphorylated by protein kinase Akt-1/PKB at Ser136. We also measured the phosphorylation-dependent Bad/14-3-3 interactions mediated by endogenous and transient Akt-1 activity. We furthermore applied split-TEV assays to measure the phosphorylation-dependent interactions of Neuregulin-1-stimulated ErbB4 receptors with several adapter proteins. Conclusion Split-TEV assays are well suited to measure phosphorylation-dependent and transient PPIs that occur specifically at the membrane and in the cytosol of heterologous and primary cultured mammalian cells. Given the high sensitivity of the split-TEV system, all assays were performed in multi-plate formats and could be adapted for higher throughput to screen for pharmacologically active substances.</p

P2X1 and P2X5 subunits form the functional P2X receptor in mouse cortical astrocytes

ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority (~65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC50 of ~40 nM). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X1 and P2X5 receptor subunits and some expression of P2X2 subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X1/5 heteromeric receptors studied previously in heterologous expression systems

Isospin in BB Decays and the (B0Bˉ0)/(B+B−)(B^0 \bar B^0)/(B^+ B^-) Production Ratio

Methods are proposed for measuring the ratio $R^{+/0} = \Gamma(\Upsilon(4S) \to B^+ B^-)/$ \Gamma(\Upsilon(4S) \to \bo \ob) without assuming isospin invariance in exclusive hadronic decays such as $B \to J/\psi K$. The validity of isospin invariance in various B decays is discussed. Isospin violations are expected to be especially small in inclusive semileptonic decays, so that $\Gamma(B^+ \to X_c^0 \ell^+ \nu_\ell) = \Gamma(B^0 \to X_c^- \ell^+ \nu_\ell)$ is expected to hold at the sub-percent level. Suggestions are made for utilizing this relation. The accuracy of a ``double-tag'' method for obtaining $R^{+/0}$, such as that used for measuring the D^+ D^-/D^0 \od ratio at the $\psi'' \equiv \psi(3770)$, is also estimated.Comment: 6 pages, no figures, to be published in Phys. Rev. D (Brief Reports). One reference and acknowledgment added; slight text chang

Theoretical X-Ray Absorption Debye-Waller Factors

An approach is presented for theoretical calculations of the Debye-Waller factors in x-ray absorption spectra. These factors are represented in terms of the cumulant expansion up to third order. They account respectively for the net thermal expansion $\sigma^{(1)}(T)$, the mean-square relative displacements $\sigma^2(T)$, and the asymmetry of the pair distribution function $\sigma^{(3)}(T)$. Similarly, we obtain Debye-Waller factors for x-ray and neutron scattering in terms of the mean-square vibrational amplitudes $u^2(T)$. Our method is based on density functional theory calculations of the dynamical matrix, together with an efficient Lanczos algorithm for projected phonon spectra within the quasi-harmonic approximation. Due to anharmonicity in the interatomic forces, the results are highly sensitive to variations in the equilibrium lattice constants, and hence to the choice of exchange-correlation potential. In order to treat this sensitivity, we introduce two prescriptions: one based on the local density approximation, and a second based on a modified generalized gradient approximation. Illustrative results for the leading cumulants are presented for several materials and compared with experiment and with correlated Einstein and Debye models. We also obtain Born-von Karman parameters and corrections due to perpendicular vibrations.Comment: 11 pages, 8 figure

The multispecific thyroid hormone transporter OATP1C1 mediates cell-specific sulforhodamine 101-labeling of hippocampal astrocytes

Sulforhodamine 101 (SR101) is widely used for astrocyte identification, though the labeling mechanism remains unknown and the efficacy of labeling in different brain regions is heterogeneous. By combining region-specific isolation of astrocytes followed by transcriptome analysis, two-photon excitation microscopy, and mouse genetics, we identified the thyroid hormone transporter OATP1C1 as the SR101-uptake transporter in hippocampus and cortex. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00429-013-0645-0) contains supplementary material, which is available to authorized users

TEMPERATURE AND LEVEL DENSITY PARAMETER OF EVAPORATION RESIDUES PRODUCED IN THE REACTION 165Ho + 600 MeV 20Ne

Evaporative and preequilibrium neutrons emitted from evaporation residues in the reaction Ho + 600 MeV neon are exploited to deduce the thermal excitation energy E* and temperature T of the residues. From these quantities the level density parameter is deduced at a temperature of 4.1 MeV

Angular anisotropy of the fusion-fission and quasifission fragments

The anisotropy in the angular distribution of the fusion-fission and quasifission fragments for the $^{16}$O+$^{238}$U, $^{19}$F+$^{208}$Pb and $^{32}$S+$^{208}$Pb reactions is studied by analyzing the angular momentum distributions of the dinuclear system and compound nucleus which are formed after capture and complete fusion, respectively. The orientation angles of axial symmetry axes of colliding nuclei to the beam direction are taken into account for the calculation of the variance of the projection of the total spin onto the fission axis. It is shown that the deviation of the experimental angular anisotropy from the statistical model picture is connected with the contribution of the quasifission fragments which is dominant in the $^{32}$S+$^{208}$Pb reaction. Enhancement of anisotropy at low energies in the $^{16}$O+$^{238}$U reaction is connected with quasifission of the dinuclear system having low temperature and effective moment of inertia.Comment: 17 pages 8 figures. Submitted to Euro. Phys. Jour.

Chromosomal aberration frequency in lymphocytes predicts the risk of cancer: results from a pooled cohort study of 22 358 subjects in 11 countries

Mechanistic evidence linking chromosomal aberration (CA) to early stages of cancer has been recently supported by the results of epidemiological studies that associated CA frequency in peripheral lymphocytes of healthy individuals to future cancer incidence. To overcome the limitations of single studies and to evaluate the strength of this association, a pooled analysis was carried out. The pooled database included 11 national cohorts and a total of 22 358 cancer-free individuals who underwent genetic screening with CA for biomonitoring purposes during 1965–2002 and were followed up for cancer incidence and/or mortality for an average of 10.1 years; 368 cancer deaths and 675 incident cancer cases were observed. Subjects were classified within each laboratory according to tertiles of CA frequency. The relative risk (RR) of cancer was increased for subjects in the medium [RR = 1.31, 95% confidence interval (CI) = 1.07–1.60] and in the high (RR = 1.41; 95% CI = 1.16–1.72) tertiles when compared with the low tertile. This increase was mostly driven by chromosome-type aberrations. The presence of ring chromosomes increased the RR to 2.22 (95% CI = 1.34–3.68). The strongest association was found for stomach cancer [RRmedium = 1.17 (95% CI = 0.37–3.70), RRhigh = 3.13 (95% CI = 1.17–8.39)]. Exposure to carcinogens did not modify the effect of CA levels on overall cancer risk. These results reinforce the evidence of a link between CA frequency and cancer risk and provide novel information on the role of aberration subclass and cancer type

Comparing silvopastoral systems and prospects in six regions of the world

Paper presented at the 12th North American Agroforesty Conference, which was held June 4-9, 2011 in Athens, Georgia.In Ashton, S. F., S.W. Workman, W.G. Hubbard and D.J. Moorhead, eds. Agroforestry: A Profitable Land Use. Proceedings, 12th North American Agroforestry Conference, Athens, GA, June 4-9, 2011.Silvopasture systems combine trees, forage and livestock in a variety of different species and management regimes, depending on the biophysical, economic, and social factors in a region. In some regions, culture and tradition cause producers to primarily focus on management of either the livestock and forage or the trees, while in other regions, the focus is on joint production. We describe and compare silvopastoral systems in six regions within five countries of the world: Misiones and Corrientes provinces, Argentina, La Pampa province, Argentina, the Southeast United States, the North Island of New Zealand, Paraguay and Uruguay. This summarizes the biological and social factors that have led to their adoption by farmers and the current status of these systems in each country.Frederick Cubbage (1), Gustavo Balmelli (2), Adriana Bussoni (3), Elke Noellemeyer (4), A. Pachas (5), Hugo Fassola (5), Luis Colcombet (5), Bel�n Rossner (5), Gregory Frey (6), Hayley Stevenson (1), James Hamilton (7) and William Hubbard (8) ; 1. Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, USA. 2. Instituto Nacional de Investigaci�n Agropecuaria, Tacuaremb�, Uruguay. 3. Universidad de la Rep�blica, Montevideo, Uruguay. 4. Universidad Nacional de La Pampa, Santa Rosa, La Pampa, Argentina. 5. INTA EEA Montecarlo, Montecarlo. Misiones, Argentina. 6. World Bank, Washington, D.C. 7. NC Cooperative Extension Service, Watauga County, Boone, North Carolina, USA. 8. Southern Regional Extension Forester, University of Georgia, Athens, Georgia, USA.Includes bibliographical references