Evolution of quantum criticality in the system CeNi9Ge4

The heavy fermion system CeNi9Ge4 exhibits a paramagnetic ground state with remarkable features such as: a record value of the electronic specific heat coefficient in systems with a paramagnetic ground state, \gamma = C/T \simeq 5.5 J/molK^2 at 80 mK, a temperature-dependent Sommerfeld-Wilson ratio, R=\chi/\gamma, below 1 K and an approximate single ion scaling of the 4f-magnetic specific heat and susceptibility. These features are related to a rather small Kondo energy scale of a few Kelvin in combination with a quasi-quartet crystal field ground state. Tuning the system towards long range magnetic order is accomplished by replacing a few at.% of Ni by Cu or Co. Specific heat, susceptibility and resistivity studies reveal T_N \sim 0.2 K for CeNi8CuGe4 and T_N \sim 1 K for CeNi8CoGe4. To gain insight whether the transition from the paramagnetic NFL state to the magnetically ordered ground state is connected with a heavy fermion quantum critical point we performed specific heat and ac susceptibility studies and utilized the \mu SR technique and quasi-elastic neutron scattering.Comment: 8 pages, 3 figures, will be published in J.Phys.: Conf. Series (Proceedings of the International & Interdisciplinary Workshop on Novel Phenomena in Intergrated Comples Sciences: From Living to Non-living Systems, Japan, held in Kyoto, October 11-14, 2010

Crystal field studies on the heavy fermion compound CeNi8_8CuGe4_4

Substitution of nickel by copper in the heavy fermion system CeNi$_{9-x}$Cu$_x$Ge$_4$ alters the local crystal field environment of the Ce$^{3+}$-ions. This leads to a quantum phase transition near $x\approx0.4$, which is not only driven by the competition between Kondo effect and RKKY interaction, but also by a reduction of an effectively fourfold to a twofold degenerate crystal field ground state. To study the consequences of a changing crystal field in CeNi$_8$CuGe$_4$ on its Kondo properties, inelastic neutron scattering (INS) experiments were performed. Two well-defined crystal field transitions were observed in the energy-loss spectra at 4 K. The crystal field level scheme determined by neutron spectroscopy is compared with results from specific heat measurements.Comment: 4 pages, 3 figures, conference SCES0

Evolution of Quantum Criticality in CeNi_{9-x}Cu_xGe_4

Crystal structure, specific heat, thermal expansion, magnetic susceptibility and electrical resistivity studies of the heavy fermion system CeNi_{9-x}Cu_xGe_4 (0 <= x <= 1) reveal a continuous tuning of the ground state by Ni/Cu substitution from an effectively fourfold degenerate non-magnetic Kondo ground state of CeNi_9Ge_4 (with pronounced non-Fermi-liquid features) towards a magnetically ordered, effectively twofold degenerate ground state in CeNi_8CuGe_4 with T_N = 175 +- 5 mK. Quantum critical behavior, C/T ~ \chi ~ -ln(T), is observed for x about 0.4. Hitherto, CeNi_{9-x}Cu_xGe_4 represents the first system where a substitution-driven quantum phase transition is connected not only with changes of the relative strength of Kondo effect and RKKY interaction, but also with a reduction of the effective crystal field ground state degeneracy.Comment: 15 pages, 9 figure

Sensitive Detection of p65 Homodimers Using Red-Shifted and Fluorescent Protein-Based FRET Couples

BACKGROUND: Fluorescence Resonance Energy Transfer (FRET) between the green fluorescent protein (GFP) variants CFP and YFP is widely used for the detection of protein-protein interactions. Nowadays, several monomeric red-shifted fluorescent proteins are available that potentially improve the efficiency of FRET. METHODOLOGY/PRINCIPAL FINDINGS: To allow side-by-side comparison of several fluorescent protein combinations for detection of FRET, yellow or orange fluorescent proteins were directly fused to red fluorescent proteins. FRET from yellow fluorescent proteins to red fluorescent proteins was detected by both FLIM and donor dequenching upon acceptor photobleaching, showing that mCherry and mStrawberry were more efficient acceptors than mRFP1. Circular permutated yellow fluorescent protein variants revealed that in the tandem constructs the orientation of the transition dipole moment influences the FRET efficiency. In addition, it was demonstrated that the orange fluorescent proteins mKO and mOrange are both suitable as donor for FRET studies. The most favorable orange-red FRET pair was mKO-mCherry, which was used to detect homodimerization of the NF-kappaB subunit p65 in single living cells, with a threefold higher lifetime contrast and a twofold higher FRET efficiency than for CFP-YFP. CONCLUSIONS/SIGNIFICANCE: The observed high FRET efficiency of red-shifted couples is in accordance with increased Förster radii of up to 64 A, being significantly higher than the Förster radius of the commonly used CFP-YFP pair. Thus, red-shifted FRET pairs are preferable for detecting protein-protein interactions by donor-based FRET methods in single living cells

Intracellular protein determination using droplet-based immunoassays

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from 50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells

Imaging Activation of Two Ras Isoforms Simultaneously in a Single Cell

Fluorescence resonance energy transfer (FRET) microscopy approaches have been used to study protein interactions in living cells. Up to now, due to the spectral requirements for FRET detection, this has been limited to the measurement of single protein interactions. Here we present a novel time-resolved fluorescence imaging method for simultaneously monitoring the activation state of two proteins in a single cell. A Ras sensor, consisting of fluorescently labelled Ras and a fluorescently labelled Ras binding domain (RBD) of Raf, which reads out Ras activation by its interaction with RBD as a FRET signal, has been adapted for this purpose. By using yellow (YFP) and cyan (CFP) versions of the green fluorescent protein from Aquorea victoria as donors and a tandem construct of Heteractis crispa Red (tHcRed) as acceptor for both donors, two independent FRET signals can be measured at the same time. Measuring the YFP and CFP donor lifetimes by fluorescence-lifetime imaging microscopy (FLIM) allows us to distinguish the two different FRET signals in a single cell. Using this approach, we show that different Ras isoforms and mutants that localize to the plasma membrane, to the Golgi or to both compartments display distinct activation profiles upon growth-factor stimulation; this indicates that there is a differential regulation in cellular compartments. The method presented here is especially useful when studying spatiotemporal aspects of protein regulation as part of larger cellular signalling networks

Spatio-temporal segregation of Ras signals: one ship, three anchors, many harbors

Dynamic assembly of spatially separated signaling platforms enables a cell to tune cellular outputs in response to different input stimuli. Understanding how a vast diversity in signaling responses can be generated from a limited protein repertoire requires knowledge of how cells maintain the segregation of proteins and thereby orchestrate their local activities. Ras proteins are subject to this type of precise regulation of localization, and thus activity, in space and time. A model emerges where different lipid anchors dynamically shuttle Ras between specific membrane compartments, where differences in the accessibility of signaling environments and in the residence time of Ras therein account for isoform-specific signaling responses

Crystal field studies on the heavy fermion compound CeNi₈CuGe₄

Substitution of nickel by copper in the heavy fermion system CeNi₉₋ₓCuₓGe₄ alters the local crystal field environment of the Ce³⁺-ions. This leads to a quantum phase transition near x ≈ 0.4, which is not only driven by the competition between Kondo effect and RKKY interaction, but also by a reduction of an effectively fourfold to a twofold degenerate crystal field ground state. To study the consequences of a changing crystal field in CeNi₈CuGe₄ on its Kondo properties, inelastic neutron scattering (INS) experiments were performed. Two well-defined crystal field transitions were observed in the energy-loss spectra at 4K. The crystal field level scheme determined by neutron spectroscopy is compared with results from specific heat measurements

An Acylation Cycle Regulates Localization and Activity of Palmitoylated Ras Isoforms

We show that the specific subcellular distribution of H- and Nras guanosine triphosphate-binding proteins is generated by a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors