Expression and characterization of a histidine-rich protein, Hpn: Potential for Ni2+ storage in Helicobacter pylori

Hpn is a small cytoplasmic protein found in Helicobacter pylori, which binds Ni2+ ions with moderate affinity. Consisting of 60 amino acids, the protein is rich in histidine (28 residues, 46.7%), as well as glutamate, glycine and serine residues (in total 31.7%), and contains short repeating motifs. In the present study, we report the detailed biophysical characterization of the multimeric status and Ni2+-binding properties of purified recombinant Hpn under physiologically relevant conditions. The protein exists as an equilibration of multimeric forms in solution, with 20-mers (approx. 136 kDa) being the predominant species. Using equilibrium dialysis, ICP-MS (inductively coupled plasma MS) and UV/visible spectroscopy, Hpn was found to bind five Ni2+ ions per monomer at pH 7.4, with a dissociation constant (Kd) of 7.1 μM. Importantly, Ni2+ binding to Hpn is reversible: metal is released either in the presence of a chelating ligand such as EDTA, or at a slightly acidic pH (pH for half dissociation, pH1/2 ∼6.3). Ni2+ binding induces conformational changes within the protein, increasing β-sheet and reducing α-helical content, from 22% to 37%, and 20% to 10% respectively. Growth curves of Escherichia coli BL21(DE3) both with and without the hpn gene performed under Ni2+ pressure clearly implied a role for Hpn to protect the cells from higher concentrations of external metal ions. Similarly, the accumulation of Ni2+ in these cells expressing Hpn from a plasmid was approx. 4-fold higher than in uninduced controls or control cultures that lacked the plasmid. Similarly, levels of Ni2+ in wild-type H. pylori 26695 cells were higher than those in H. pylori hpn-deletion mutant strains. Hpn may potentially serve multiple roles inside the bacterium: storage of Ni 2+ ions in a 'reservoir'; donation of Ni2+ to other proteins; and detoxification via sequestration of excess Ni2+. © 2006 Biochemical Society.published_or_final_versio

Displacements analysis of self-excited vibrations in turning

The actual research deals with determining by a new protocol the necessary parameters considering a three-dimensional model to simulate in a realistic way the turning process on machine tool. This paper is dedicated to the experimental displacements analysis of the block tool / block workpiece with self-excited vibrations. In connexion with turning process, the self-excited vibrations domain is obtained starting from spectra of two accelerometers. The existence of a displacements plane attached to the tool edge point is revealed. This plane proves to be inclined compared to the machines tool axes. We establish that the tool tip point describes an ellipse. This ellipse is very small and can be considered as a small straight line segment for the stable cutting process (without vibrations). In unstable mode (with vibrations) the ellipse of displacements is really more visible. A difference in phase occurs between the tool tip displacements on the radial direction and on the cutting one. The feed motion direction and the cutting one are almost in phase. The values of the long and small ellipse axes (and their ratio) shows that these sizes are increasing with the feed rate value. The axis that goes through the stiffness center and the tool tip represents the maximum stiffness direction. The maximum (resp. minimum) stiffness axis of the tool is perpendicular to the large (resp. small) ellipse displacements axis. FFT analysis of the accelerometers signals allows to reach several important parameters and establish coherent correlations between tool tip displacements and the static - elastic characteristics of the machine tool components tested

Modulating attentional load affects numerosity estimation: evidence against a pre-attentive subitizing mechanism

Traditionally, the visual enumeration of a small number of items (1 to about 4), referred to as subitizing, has been thought of as a parallel and pre-attentive process and functionally different from the serial attentive enumeration of larger numerosities. We tested this hypothesis by employing a dual task paradigm that systematically manipulated the attentional resources available to an enumeration task. Enumeration accuracy for small numerosities was severely decreased as more attentional resources were taken away from the numerical task, challenging the traditionally held notion of subitizing as a pre-attentive, capacity-independent process. Judgement of larger numerosities was also affected by dual task conditions and attentional load. These results challenge the proposal that small numerosities are enumerated by a mechanism separate from large numerosities and support the idea of a single, attention-demanding enumeration mechanism

In situ micropillar deformation of hydrides in Zircaloy-4

Deformation of hydrided Zircaloy-4 has been examined using in situ loading of hydrided micropillars in the scanning electron microscope and using synchrotron X-ray Laue microbeam diffraction. Results suggest that both the matrix and hydride can co-deform, with storage of deformation defects observed within the hydrides, which were twinned. Hydrides placed at the plane of maximum shear stress showed deformation within the hydride packet, whilst packets in other pillars arrested the propagation of shear bands. X-ray Laue peak broadening, prior to deformation, was associated with the precipitation of hydrides, and during deformation plastic rotation and broadening of both the matrix and hydride peaks were observed. Post-mortem TEM of the deformed pillars has indicated a greater density of dislocations associated with the precipitated hydride packets, while the observed broadening of the hydride electron diffraction spots further suggests that plastic strain gradients were induced in the hydrides by compression

Adenosine-mono-phosphate-activated protein kinase-independent effects of metformin in T cells

The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK

Investigating the inner discs of Herbig Ae/Be stars with CO bandhead and Brγ emission

articleHerbig Ae/Be stars lie in the mass range between low- and high-mass young stars, and therefore offer a unique opportunity to observe any changes in the formation processes that may occur across this boundary. This paper presents medium-resolution Very Large Telescope (VLT)/X-shooter spectra of six Herbig Ae/Be stars, drawn from a sample of 91 targets, and high-resolution VLT/Cryogenic Infrared Echelle Spectrograph (CRIRES) spectra of five Herbig Ae/Be stars, chosen based on the presence of CO first overtone bandhead emission in their spectra. The X-shooter survey reveals a low detection rate of CO first overtone emission (7 per cent), consisting of objects mainly of spectral type B. A positive correlation is found between the strength of the CO v = 2–0 and Brγ emission lines, despite their intrinsic linewidths suggesting a separate kinematic origin. The high-resolution CRIRES spectra are modelled, and are well fitted under the assumption that the emission originates from small scale Keplerian discs, interior to the dust sublimation radius, but outside the corotation radius of the central stars. In addition, our findings are in very good agreement for the one object where spatially resolved near-infrared interferometric studies have also been performed. These results suggest that the Herbig Ae/Be stars in question are in the process of gaining mass via disc accretion, and that modelling of high spectral resolution spectra is able to provide a reliable probe into the process of stellar accretion in young stars of intermediate to high masses.European Union FP7-2011Science and Technology Facilities Counci

Development of interfering RNA agents to inhibit SARS-associated coronavirus infection and replication.

published_or_final_versio

Entropy bounds in terms of the w parameter

In a pair of recent articles [PRL 105 (2010) 041302 - arXiv:1005.1132; JHEP 1103 (2011) 056 - arXiv:1012.2867] two of the current authors have developed an entropy bound for equilibrium uncollapsed matter using only classical general relativity, basic thermodynamics, and the Unruh effect. An odd feature of that bound, S <= A/2, was that the proportionality constant, 1/2, was weaker than that expected from black hole thermodynamics, 1/4. In the current article we strengthen the previous results by obtaining a bound involving the (suitably averaged) w parameter. Simple causality arguments restrict this averaged parameter to be <= 1. When equality holds, the entropy bound saturates at the value expected based on black hole thermodynamics. We also add some clarifying comments regarding the (net) positivity of the chemical potential. Overall, we find that even in the absence of any black hole region, we can nevertheless get arbitrarily close to the Bekenstein entropy.Comment: V1: 14 pages. V2: One reference added. V3: This version accepted for publication in JHE

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

Numerical elimination and moduli space of vacua

We propose a new computational method to understand the vacuum moduli space of (supersymmetric) field theories. By combining numerical algebraic geometry (NAG) and elimination theory, we develop a powerful, efficient, and parallelizable algorithm toextract important information such as the dimension, branch structure, Hilbert series and subsequent operator counting, as well as variation according to coupling constants and mass parameters. We illustrate this method on a host of examples from gauge theory, string theory, and algebraic geometry