Structure and properties of earthworm metallothionein-2

Since their discovery in the 1950s, metallothioneins (MTs) have been isolated from an array of organisms, from sea urchins to sheep, and pigs to peas. With the advent of nuclear Overhauser effect nuclear magnetic resonance (NMR) experiments, a powerful route for MT structure determination became available. The contribution of structural data obtained by NMR has enabled identification of hitherto unknown metallothionein motifs, thought to be significant in the ‘tuning’ of MT for specific roles or functions. As the MT superfamily is so broad, there is a wealth of information still to be uncovered for MTs from some of the lesser-studied organisms. Although many in vivo genomics-based investigations have been performed within the invertebrate family, proteomics-based investigations, especially with respect to terrestrial invertebrates, are sparse. This research has investigated the metal-binding behaviour and determined the solution structure of earthworm metallothionein-2 (wMT-2) from Lumbricus rubellus. In addition, a method for purifying both wMTs expressed in adult earthworms is presented; in contrast to previous studies the purification method includes the removal of the Nterminal S●tag. Using modified purification methods, the novel protein wMT-3 was also able to be isolated and characterised. Electrospray-ionisation mass spectrometry (ESIMS) revealed that all three wMTs bind 7 equivalents of divalent metal ions as the major species at pH ≈ 8. An NMR investigation of cleaved Cd-wMT-2 indicated that metalbinding is localised in two discrete clusters. Conversely to vertebrate MTs, but similar to some invertebrate MTs, the stoichiometry of the domains is of an M4Cys11- N-terminal cluster, followed by an M3Cys9- cluster. When challenged with protons, demetallation of cleaved Cd-wMT-2 occurred in a step-wise manner: a 3 metal loss being followed by a 4 metal loss; behaviour which is unique to the cadmium form. A comparison of the NMR TOCSY spectra of cleaved Cd- and Zn-wMT-2 showed significant differences, indicating that wMT-2 has a strong metal-binding preference for cadmium in vitro. In conclusion, the studies performed within this thesis have enabled the calculation of the first 3-dimensional solution structure of an MT from a terrestrial invertebrate, wMT-2. In addition, the novel protein wMT-3 was successfully isolated and purified

Earthworm Lumbricus rubellus MT-2 : metal binding and protein folding of a true Cadmium-MT

Earthworms express, as most animals, metallothioneins (MTs)—small, cysteine-rich proteins that bind d10 metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by 1H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo

Time evolution of the Partridge-Barton Model

The time evolution of the Partridge-Barton model in the presence of the pleiotropic constraint and deleterious somatic mutations is exactly solved for arbitrary fecundity in the context of a matricial formalism. Analytical expressions for the time dependence of the mean survival probabilities are derived. Using the fact that the asymptotic behavior for large time $t$ is controlled by the largest matrix eigenvalue, we obtain the steady state values for the mean survival probabilities and the Malthusian growth exponent. The mean age of the population exhibits a $t^{-1}$ power law decayment. Some Monte Carlo simulations were also performed and they corroborated our theoretical results.Comment: 10 pages, Latex, 1 postscript figure, published in Phys. Rev. E 61, 5664 (2000

Mol Cell Proteomics

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases

Interferometric imaging with the 32 element Murchison Wide-field Array

The Murchison Wide-field Array (MWA) is a low frequency radio telescope, currently under construction, intended to search for the spectral signature of the epoch of re-ionisation (EOR) and to probe the structure of the solar corona. Sited in Western Australia, the full MWA will comprise 8192 dipoles grouped into 512 tiles, and be capable of imaging the sky south of 40 degree declination, from 80 MHz to 300 MHz with an instantaneous field of view that is tens of degrees wide and a resolution of a few arcminutes. A 32-station prototype of the MWA has been recently commissioned and a set of observations taken that exercise the whole acquisition and processing pipeline. We present Stokes I, Q, and U images from two ~4 hour integrations of a field 20 degrees wide centered on Pictoris A. These images demonstrate the capacity and stability of a real-time calibration and imaging technique employing the weighted addition of warped snapshots to counter extreme wide field imaging distortions.Comment: Accepted for publication in PASP. This is the draft before journal typesetting corrections and proofs so does contain formatting and journal style errors, also has with lower quality figures for space requirement

The Murchison Widefield Array

It is shown that the excellent Murchison Radio-astronomy Observatory site allows the Murchison Widefield Array to employ a simple RFI blanking scheme and still calibrate visibilities and form images in the FM radio band. The techniques described are running autonomously in our calibration and imaging software, which is currently being used to process an FM-band survey of the entire southern sky.Comment: Accepted for publication in Proceedings of Science [PoS(RFI2010)016]. 6 pages and 3 figures. Presented at RFI2010, the Third Workshop on RFI Mitigation in Radio Astronomy, 29-31 March 2010, Groningen, The Netherland

Virus Replication as a Phenotypic Version of Polynucleotide Evolution

In this paper we revisit and adapt to viral evolution an approach based on the theory of branching process advanced by Demetrius, Schuster and Sigmund ("Polynucleotide evolution and branching processes", Bull. Math. Biol. 46 (1985) 239-262), in their study of polynucleotide evolution. By taking into account beneficial effects we obtain a non-trivial multivariate generalization of their single-type branching process model. Perturbative techniques allows us to obtain analytical asymptotic expressions for the main global parameters of the model which lead to the following rigorous results: (i) a new criterion for "no sure extinction", (ii) a generalization and proof, for this particular class of models, of the lethal mutagenesis criterion proposed by Bull, Sanju\'an and Wilke ("Theory of lethal mutagenesis for viruses", J. Virology 18 (2007) 2930-2939), (iii) a new proposal for the notion of relaxation time with a quantitative prescription for its evaluation, (iv) the quantitative description of the evolution of the expected values in in four distinct "stages": extinction threshold, lethal mutagenesis, stationary "equilibrium" and transient. Finally, based on these quantitative results we are able to draw some qualitative conclusions.Comment: 23 pages, 1 figure, 2 tables. arXiv admin note: substantial text overlap with arXiv:1110.336

The Murchison Widefield Array: Design Overview

The Murchison Widefield Array (MWA) is a dipole-based aperture array synthesis telescope designed to operate in the 80-300 MHz frequency range. It is capable of a wide range of science investigations, but is initially focused on three key science projects. These are detection and characterization of 3-dimensional brightness temperature fluctuations in the 21cm line of neutral hydrogen during the Epoch of Reionization (EoR) at redshifts from 6 to 10, solar imaging and remote sensing of the inner heliosphere via propagation effects on signals from distant background sources,and high-sensitivity exploration of the variable radio sky. The array design features 8192 dual-polarization broad-band active dipoles, arranged into 512 tiles comprising 16 dipoles each. The tiles are quasi-randomly distributed over an aperture 1.5km in diameter, with a small number of outliers extending to 3km. All tile-tile baselines are correlated in custom FPGA-based hardware, yielding a Nyquist-sampled instantaneous monochromatic uv coverage and unprecedented point spread function (PSF) quality. The correlated data are calibrated in real time using novel position-dependent self-calibration algorithms. The array is located in the Murchison region of outback Western Australia. This region is characterized by extremely low population density and a superbly radio-quiet environment,allowing full exploitation of the instrumental capabilities.Comment: 9 pages, 5 figures, 1 table. Accepted for publication in Proceedings of the IEE

Hadroproduction of the Chi1 and Chi2 States of Charmonium in 800 GeV/c Proton-Silicon Interactions

The cross sections for the hadroproduction of the Chi1 and Chi2 states of charmonium in proton-silicon collisions at sqrt{s}=38.8 GeV have been measured in Fermilab fixed target Experiment 771. The Chi states were observed via their radiative decay to J/psi+gamma, where the photon converted to e+e- in the material of the spectrometer. The measured values for the Chi1 and Chi2 cross sections for x_F>0 are 263+-69(stat)+-32(syst) and 498+-143(stat)+-67(syst) nb per nucleon respectively. The resulting sigma(Chi1}/sigma(Chi2) ratio of 0.53+-0.20(stat)+-0.07(syst), although somewhat larger than most theoretical expectations, can be accomodated by the latest theoretical estimates.Comment: 4 pages, 4 figure