Sub MeV Particles Detection and Identification in the MUNU detector ((1)ISN, IN2P3/CNRS-UJF, Grenoble, France, (2)Institut de Physique, Neuch\^atel, Switzerland, (3) INFN, Padova Italy, (4) Physik-Institut, Z\"{u}rich, Switzerland)

We report on the performance of a 1 m$^{3}$ TPC filled with CF$_{4}$ at 3 bar, immersed in liquid scintillator and viewed by photomultipliers. Particle detection, event identification and localization achieved by measuring both the current signal and the scintillation light are presented. Particular features of $\alpha$ particle detection are also discussed. Finally, the ${54}$Mn photopeak, reconstructed from the Compton scattering and recoil angle is shown.Comment: Latex, 19 pages, 20 figure

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

Unwinding of a cholesteric liquid crystal and bidirectional surface anchoring

We examine the influence of bidirectional anchoring on the unwinding of a planar cholesteric liquid crystal induced by the application of a magnetic field. We consider a liquid crystal layer confined between two plates with the helical axis perpendicular to the substrates. We fixed the director twist on one boundary and allow for bidirectional anchoring on the other by introducing a high-order surface potential. By minimizing the total free energy for the system, we investigate the untwisting of the cholesteric helix as the liquid crystal attempts to align with the magnetic field. The transitions between metastable states occur as a series of pitchjumps as the helix expels quarter or half-turn twists, depending on the relative sizes of the strength of the surface potential and the bidirectional anchoring. We show that secondary easy axis directions can play a significant role in the unwinding of the cholesteric in its transition towards a nematic, especially when the surface anchoring strength is large

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

Multiple phosphate positions in the catalytic site of glycogen phosphorylase

The three‐dimensional structure of an R‐state conformer of glycogen phosphorylase containing the coenzyme‐substrate analog pyridoxal‐5′‐diphosphate at the catalytic site (PLPP‐GPb) has been refined by X‐ray crystallography to a resolution of 2.87 Å. The molecule comprises four subunits of phosphorylase related by approximate 222 symmetry. Whereas the quaternary structure of R‐state PLPP‐GPb is similar to that of phosphorylase crystallized in the presence of ammonium sulfate (Barford, D. & Johnson, L.N., 1989, Nature 340, 609–616), the tertiary structures differ in that the two domains of the PLPP‐GPb subunits are rotated apart by 5° relative to the T‐state conformation. Global differences among the four subunits suggest that the major domains of the phosphorylase subunit are connected by a flexible hinge. The two different positions observed for the terminal phosphate of the PLPP are interpreted as distinct phosphate subsites that may be occupied at different points along the reaction pathway. The structural basis for the unique ability of R‐state dimers to form tetramers results from the orientation of subunits with respect to the dyad axis of the dimer. Residues in opposing dimers are in proper registration to form tetramers only in the R‐state

Consideration of the bioavailability of metal/metalloid species in freshwaters: experiences regarding the implementation of biotic ligand model-based approaches in risk assessment frameworks

After the scientific development of Biotic Ligand Models (BLMs) in recent decades these models are now considered suitable for implementation in regulatory risk assessment of metals in freshwater bodies. The approach has been developed over several years and has been described in many peer-reviewed publications. The original complex BLMs have been applied in prospective risk assessment reports for metals and metal compounds and are also recommended as suitable concepts for the evaluation of monitoring data in the context of the European Water Framework Directive. Currently, several user-friendly BLM-based bioavailability software tools are available for assessing the aquatic toxicity of a limited number of metals (mainly copper, nickel, and zinc). These tools need only a basic set of water parameters as input (e.g., pH, hardness, dissolved organic matter and dissolved metal concentration). Such tools seem appropriate to foster the implementation in routine water quality assessments. This work aims to review the existing bioavailability-based regulatory approaches and the application of available BLM-based bioavailability tools for this purpose. Advantages and possible drawbacks of these tools (e.g., feasibility, boundaries of validity) are discussed, and recommendations for further implementation are given

Ric-8A catalyzes guanine nucleotide exchange on G alphai1 bound to the GPR/GoLoco exchange inhibitor AGS3

Microtubule pulling forces that govern mitotic spindle movement of chromosomes are tightly regulated by G-proteins. A host of proteins, including Galpha subunits, Ric-8, AGS3, regulators of G-protein signalings, and scaffolding proteins, coordinate this vital cellular process. Ric-8A, acting as a guanine nucleotide exchange factor, catalyzes the release of GDP from various Galpha.GDP subunits and forms a stable nucleotide-free Ric-8A:Galpha complex. AGS3, a guanine nucleotide dissociation inhibitor (GDI), binds and stabilizes Galpha subunits in their GDP-bound state. Because Ric-8A and AGS3 may recognize and compete for Galpha.GDP in this pathway, we probed the interactions of a truncated AGS3 (AGS3-C; containing only the residues responsible for GDI activity), with Ric-8A:Galpha(il) and that of Ric-8A with the AGS3-C:Galpha(il).GDP complex. Pulldown assays, gel filtration, isothermal titration calorimetry, and rapid mixing stopped-flow fluorescence spectroscopy indicate that Ric-8A catalyzes the rapid release of GDP from AGS3-C:Galpha(i1).GDP. Thus, Ric-8A forms a transient ternary complex with AGS3-C:Galpha(i1).GDP. Subsequent dissociation of AGS3-C and GDP from Galpha(i1) yields a stable nucleotide free Ric-8A.Galpha(i1) complex that, in the presence of GTP, dissociates to yield Ric-8A and Galpha(i1).GTP. AGS3-C does not induce dissociation of the Ric-8A.Galpha(i1) complex, even when present at very high concentrations. The action of Ric-8A on AGS3:Galpha(i1).GDP ensures unidirectional activation of Galpha subunits that cannot be reversed by AGS3

Molecular chaperoning function of Ric-8 is to fold nascent heterotrimeric G protein α subunits

We have shown that resistance to inhibitors of cholinesterase 8 (Ric-8) proteins regulate an early step of heterotrimeric G protein α (Gα) subunit biosynthesis. Here, mammalian and plant cell-free translation systems were used to study Ric-8A action during Gα subunit translation and protein folding. Gα translation rates and overall produced protein amounts were equivalent in mock and Ric-8A-immunodepleted rabbit reticulocyte lysate (RRL). GDP-AlF4(-)-bound Gαi, Gαq, Gα13, and Gαs produced in mock-depleted RRL had characteristic resistance to limited trypsinolysis, showing that these G proteins were folded properly. Gαi, Gαq, and Gα13, but not Gαs produced from Ric-8A-depleted RRL were not protected from trypsinization and therefore not folded correctly. Addition of recombinant Ric-8A to the Ric-8A-depleted RRL enhanced GDP-AlF4(-)-bound Gα subunit trypsin protection. Dramatic results were obtained in wheat germ extract (WGE) that has no endogenous Ric-8 component. WGE-translated Gαq was gel filtered and found to be an aggregate. Ric-8A supplementation of WGE allowed production of Gαq that gel filtered as a ∼100 kDa Ric-8A:Gαq heterodimer. Addition of GTPγS to Ric-8A-supplemented WGE Gαq translation resulted in dissociation of the Ric-8A:Gαq heterodimer and production of functional Gαq-GTPγS monomer. Excess Gβγ supplementation of WGE did not support functional Gαq production. The molecular chaperoning function of Ric-8 is to participate in the folding of nascent G protein α subunits