Mechanism of the Intermediary Phase Formation in Ti-20 wt. % Al Mixture during Pressureless Reactive Sintering

This work aims to describe the mechanism of intermediary phases formation in TiAl20 (wt. %) alloy composition during reactive sintering. The reaction between titanium and aluminum powders was studied by in situ diffraction and the results were confirmed by annealing at various temperatures. It was found that the Ti2Al5 phase formed preferentially and its formation was detected at 400 degrees C. So far, this phase has never been found in this alloy composition during reactive sintering processes. Subsequently, the Ti2Al5 phase reacted with the titanium, and the formation of the major phase, Ti3Al, was accompanied by the minor phase, TiAl. Equations of the proposed reactions are presented in this paper and their thermodynamic and kinetic feasibility are supported by Gibbs energies of reaction and reaction enthalpies

Investigating the role of Chk1 in mouse skin homeostasis and tumourigenesis

Chk1 is a key regulator of DNA damage response and genome stability in eukaryotes. To better understand how checkpoint proficiency affects cancer development particularly tumours induced by chemical carcinogens in murine skin, I investigated the effect of conditional genetic ablation of chk1. I found that complete deletion of chk1 immediately prior to carcinogen exposure strongly suppressed papilloma formation, and the few, small lesions that did form always retained Chk1 expression. Remarkably, chk1 deletion was accompanied by spontaneous cell proliferation followed by DNA damage and cell death within the hair follicle. This also affected and led to proliferation and ultimately depletion of label-retaining stem cells (LRCs) within the bulge region of hair follicles, the principal source for carcinogen-induced tumours. At later times, ablated skin became progressively repopulated by Chk1-expressing cells and normal sensitivity to tumour induction was restored if carcinogen treatment was delayed. In marked contrast, papillomas formed normally in chk1 hemizygous skin but showed an increased propensity to progress to carcinomas. I conclude that Chk1 is essential for the survival of incipient cancer cells but that partial loss of function (haploinsufficiency) fosters tumour progression.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Kinetic and thermodynamic description of intermediary phases formation in Ti-Al system during reactive sintering

Reactive sintering is currently considered as a promising production route for titanium aluminides in many research works. However, the published descriptions of the reaction mechanism are contradictory or lacking, especially at the temperatures below the melting point of aluminium. This work aims to fill this gap, providing the description of the reactive sintering process at the temperatures between 400 and 900 degrees C. The phases' formation sequence and reaction kinetics were studied and explained using experimental model (Ti/Al diffusion couple) and real reactively sintered samples of equiatomic Ti-Al compressed powder blend. Moreover, phase formation was thermodynamically assessed. It was revealed that Ti2Al5 phase formed preferentially. This phase has not been reported previously as a starting phase in reactive sintering. According to results obtained by experimental model, its formation is controlled by diffusion at 700 degrees C. This phase reacted with aluminium forming pure TiAl3 phase or with titanium, resulting in TiAl phase. Subsequently, TiAl phase reacted with titanium, leading to the Ti3Al phase, or with already present Ti2Al5 phase yielding TiAl2 intermetallic compound. Titanium-rich Ti3Al phase could form only at the temperature of 600 degrees C or above

Oxidation of Phe454 in the Gating Segment Inactivates Trametes multicolor Pyranose Oxidase during Substrate Turnover.

The flavin-dependent enzyme pyranose oxidase catalyses the oxidation of several pyranose sugars at position C-2. In a second reaction step, oxygen is reduced to hydrogen peroxide. POx is of interest for biocatalytic carbohydrate oxidations, yet it was found that the enzyme is rapidly inactivated under turnover conditions. We studied pyranose oxidase from Trametes multicolor (TmPOx) inactivated either during glucose oxidation or by exogenous hydrogen peroxide using mass spectrometry. MALDI-MS experiments of proteolytic fragments of inactivated TmPOx showed several peptides with a mass increase of 16 or 32 Da indicating oxidation of certain amino acids. Most of these fragments contain at least one methionine residue, which most likely is oxidised by hydrogen peroxide. One peptide fragment that did not contain any amino acid residue that is likely to be oxidised by hydrogen peroxide (DAFSYGAVQQSIDSR) was studied in detail by LC-ESI-MS/MS, which showed a +16 Da mass increase for Phe454. We propose that oxidation of Phe454, which is located at the flexible active-site loop of TmPOx, is the first and main step in the inactivation of TmPOx by hydrogen peroxide. Oxidation of methionine residues might then further contribute to the complete inactivation of the enzyme

Oxidation of methionines.

<p>Methionine-containing peptide fragments detected that show a mass increase of 16 or 32 Da after proteolytic digestion of <i>Tm</i>POx (inactivated during turnover of 100 mM D-glucose, treated with endogenous H₂O₂ or unaffected). Proteolytic digestion was performed with trypsin or Asp-N protease as indicated. The last column indicates whether the particular Met is considerably oxidised during substrate turnover and by H₂O₂ treatment compared to the unaffected sample (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.g002" target="_blank">Fig 2</a>).</p

Mass spectrometric identification of methionine residues oxidized by H₂O₂ during <i>Tm</i>POx inactivation.

<p>MALDI MS spectra were measured for unaffected POx (<b>A</b>), for POx inactivated during D-glucose oxidation (<b>B</b>) or for POx inactivated by endogenous H₂O₂ (<b>C</b>). The selected MALDI spectra in the left panel illustrate that Met497 of the tryptic peptide ITDAYNMPQPTFDFR with a theoretical MH⁺ of 1815.8 was extensively oxidised in <i>Tm</i>POx inactivated either during substrate turnover (<b>B</b>, left panel) or by H₂O₂ treatment (<b>C</b>, left panel). In contrast, some methionine residues were found not to be oxidised during <i>Tm</i>POx inactivation as shown for Met74 of the peptide VAMFDIGEIDSGLK having a MH⁺ of 1494.8 (right panel). The small signals at m/z 1510.8 are related to the oxidized form of the peptide generated due to the presence of air oxygen.</p

Accessibility of methionine residues.

<p>Surface of the <i>T</i>. <i>multicolor</i> POx monomer in the vicinity of (<b>A</b>) Met417, showing the surface-exposed sulphur atom which is oxidised by H₂O₂, and (<b>B</b>) Met380 with its sulphur-containing side chain pointing towards the interior of the polypeptide matrix, in which it is buried and hence is not accessible from the surface.</p

Identification of the exact site of oxidation in the peptide ⁴⁵²DAFSYGAVQQSIDSR⁴⁶⁶ by LC-ESI-MS/MS.

<p>The nearly complete series of C-terminal y-ions in the MS/MS spectrum of the double-charged ion of 830.4 confirms the peptide sequence. The +16 Da mass shift found for the ion y₁₃ further indicates that Phe454 is oxidised during substrate turnover and by H₂O₂ treatment.</p

Active-site geometry of pyranose oxidase from <i>T</i>. <i>multicolor</i>.

<p>In the closed form of the active-site loop of pyranose oxidase from <i>T</i>. <i>multicolor</i> (<i>Tm</i>POx, PDB code 1TT0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>]), which is thought to be relevant for the oxidative half-reaction of POx [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148108#pone.0148108.ref003" target="_blank">3</a>], Phe454 is positioned in the direct vicinity of the isoalloxazine ring and the C4a/N5 locus, at which oxygen is reduced. The figure was generated using PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p

Purification and Characterization of Pyranose Oxidase from the White Rot Fungus Trametes multicolor

We purified an intracellular pyranose oxidase from mycelial extracts of the white rot fungus Trametes multicolor by using ammonium sulfate fractionation, hydrophobic interaction, ion-exchange chromatography, and gel filtration. The native enzyme has a molecular mass of 270 kDa as determined by equilibrium ultracentrifugation and is composed of four identical 68-kDa subunits as determined by matrix-assisted laser desorption ionization mass spectrometry. Each subunit contains one covalently bound flavin adenine dinucleotide as its prosthetic group. The enzyme oxidizes several aldopyranoses specifically at position C-2, and its preferred electron donor substrates are d-glucose, d-xylose, and l-sorbose. During this oxidation reaction electrons are transferred to oxygen, yielding hydrogen peroxide. In addition, the enzyme catalyzes the two-electron reduction of 1,4-benzoquinone, several substituted benzoquinones, and 2,6-dichloroindophenol, as well as the one-electron reduction of the ABTS [2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid)] cation radical. As judged by the catalytic efficiencies (k(cat)/K(m)), some of these quinone electron acceptors are much better substrates for pyranose oxidase than oxygen. The optimum pH of the pyranose oxidase-catalyzed reaction depends strongly on the electron acceptor employed and varies from 4 to 8. It has been proposed that the main metabolic function of pyranose oxidase is as a constituent of the ligninolytic system of white rot fungi that provides peroxidases with H(2)O(2). An additional function could be reduction of quinones, key intermediates that are formed during mineralization of lignin