A kinetic study on inactivation of tilapia myosin Ca-ATPase induced by high hydrostatic pressure

Tilapia myosin (2.5 mg/ml) was treated by hydrostatic pressure (50–300 MPa) for 0–60 min to determine the inactivation kinetics of myosin Ca-ATPase. The process of the pressure-induced inactivation of myosin Ca-ATPase included two steps: the first one was an instantaneous pressure-induced inactivation, and the degrees of lost activities, called instantaneous pressure kill (IPK) values, increased with elevated pressure. The second one, the logarithm of residual activity of myosin ATPase, decreased smoothly during each pressure treatment for 10–60 min. However, D values (the time needed for 90% loss of the activity during a treatment at the same pressure) of Ca-ATPase decreased about 50% with per 50 MPa increase. In this study, 150 MPa was the pressure level that caused apparent myosin denaturation and the typical network structure formation with beyond 50% decrease of myosin Ca-ATPase activity.     

Bioorthogonal Probes for Imaging Sterols in Cells

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C₁₉ alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)‐catalyzed azide–alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two‐color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.     

The development of PTFE/Nafion/TEOS membranes for application in moderate and high temperature proton exchange membrane fuel cells

PTFE/Nafion (PN) and PTFE/Nafion/TEOS (PNS) membranes were fabricated for the application of moderate and high temperature proton exchange membrane fuel cells (PEMFCs), respectively. Membrane electrode assemblies (MEAs) were fabricated by PTFE/Nafion (and PTFE/Nafion/TEOS) membranes with commercially available low and high temperature gas diffusion electrodes (GDEs). The effects of relative humidity, operation temperature, and back pressure on the performance and durability test of the as-prepared MEAs were investigated. Incorporating TEOS into a PNS membrane and adding another layer of carbon onto a GDE would result in low membrane conductivity and low fuel cell performance respectively. However, in this work it is shown that HT-PNS MEAs demonstrate a higher performance than LT-PN MEAs in severe conditions - high temperature (118 °C) and low humidity (25% RH). The TEOS and additional carbon layer function as water retaining agents which are especially important for high temperature and low humidity conditions. The HT-PNS MEA showed good stability in a 50 h fuel cell test at high temperature, moderate relative humidity (50% RH) and back pressure of 14.7 psi.     

Formation and characterization of DLC:Cr:Cu multi-layers coating using cathodic arc evaporation

Chromium and copper-doped diamond-like carbon (DLC:Cr:Cu) films were deposited on SKH 51 tool steel. We have prepared multilayers of DLC:Cr and DLC:Cu by cathodic arc evaporation process using chromium (Cr) and copper (Cu) target arc sources to provide Cr and Cu in the DLC. Acetylene reactive gases were also activated at a pressure of 5 mTorr to 25 mTorr and a temperature fixed at 180 °C to provide the DLC. The resulting DLC:Cr:Cu film contained Cr_xCu_y as well as Cr_xC_y nanoparticles vital for the film mechanical properties. The crystal structure was investigated using X-ray diffraction (XRD) and transmission electron microscopy, while the surface morphology and chemical composition were studied by field emission scanning electron microscopy and X-ray photoelectron spectroscopy. The process parameters were compared by studying the various mechanical properties of the films such as microhardness and residual stress. The result of this process enhanced the DLC:Cr:Cu composite coatings for high toughness and lower friction coefficient (0.08). The profiles of sp³/sp² (XPS) ratios corresponded to the change of microhardness profile by varying the pressure of the hydrocarbon gases (C₂H₂).     

安全评价报告中存在的地质问题的讨论

通过分析煤矿安全评价报告的内容,从资料收集、危险有害因素以及重大危险源的定性和评价结论等方面,进行系统的判断,指出煤矿评价过程中存在的地质方面的问题,为评价报告的改进提出了合理化建议.     

Reactivity of the human thioltransferase (glutaredoxin) C7S, C25S, C78S, C82S mutant and NMR solution structure of its glutathionyl mixed disulfide intermediate reflect catalytic specificity

Human thioltransferase (TTase) is a 12 kDa thiol-disulfide oxidoreductase that appears to play a critical role in maintaining the redox environment of the cell. TTase acts as a potent and specific reducing agent for protein-S-S-glutathione mixed disulfides (protein-SSG) likely formed during oxidative stress or as redox intermediates in signal transduction pathways. Accordingly, the catalytic cycle of thioltransferase itself involves a covalent glutathionyl enzyme disulfide intermediate (TTase-C22-SSG). To understand the molecular basis of TTase specificity for the glutathione moiety, we engineered a quadruple Cys to Ser mutant of human TTase (C7S, C25S, C78S, and C82S) which retains only the active site cysteine residue (C22), and we solved its high-resolution NMR solution structure in the mixed disulfide intermediate with glutathione (QM-TTase-SSG). This mutant which cannot form a C22-S-S-C25 intramolecular disulfide displays the same catalytic efficiency (Vmax/KM) and specificity for glutathionyl mixed disulfide substrates as wild-type TTase, indicating that the Cys-25-SH moiety is not required for catalysis or glutathionyl specificity. The structure of human thioltransferase is characterized by a thioredoxin-like fold which comprises a four-stranded central beta-sheet flanked on each side by alpha-helices. The disulfide-adducted glutathione in the TTase-SSG complex has an extended conformation and is localized in a cleft near the protein surface encompassing the residues from helices-alpha2,alpha3, the active site loop, and the loop connecting helix-alpha3 and strand-beta3. Numerous van der Waals and electrostatic interactions between the protein and the glutathione moiety are identified as contributing to stabilization of the complex and confering the substrate specificity. Comparison of the human thioltransferase with other thiol-disulfide oxidoreductases reveals structural and functional differences.     

Synthetic Aspects and Free-Radical Scavenging Efficiency of Polyhydroxylated C60

The substitution effect of functional groups, attached on the aromatic ring of benzoic acid, to the nucleophilic replacement of fullerenic nitronium intermediates by carboxylic acids was demonstrated. The chemistry was utilized for the synthesis of water-soluble polyhydroxylated fullerenes (fullerenols). High reaction product yields (88-90%) of fullerenols were obtained using functional benzoic acids containing one or two electron-withdrawing substituents as nucleophilic reagents. On the contrary, the use of electron-releasing group substituted benzoic acids resulted in a relatively low product yield (38-62%). A competitive superoxide-radical absorbing reaction between DMPO and fullerenol molecules was carried out in a phosphate buffer using an enzymatic system of xanthine/xanthine oxidase for the generation of superoxide radicals. The overall superoxide-radical scavenging efficiency of fullerenols was found to be higher than that of DMPO molecules. Evidence of preferential attack of superoxide radicals toward fullerenol molecules was obtained by a dose-dependent experiment in the presence of a high concentration of DMPO molecules. Complete elimination of DMPO-OH radicals by fullerenols at a dose level of 8.0 mM within a reaction period of 10 min indicated a higher scavenging rate of superoxide radicals for fullerenols than that for DMPO molecules in a concentration of 80 mM.     

Effect of pretreatment of hydrostatic pressure on physicochemical properties of tilapia muscle protein gels induced by setting

ABSTRACT:  Tilapia meat pastes were subjected to pretreatments of hydrostatic pressure (50 to 300 MPa/4 °C/60 min) followed by setting (50 °C/60 min) with or without subsequent cooking (90 °C/20 min) to investigate the changes of rheological properties, gel-forming ability, whiteness, and protein solubility of gels. The gel by setting only as the control was elastic, rigid, and mainly constituted by covalent bonds. The gel by pretreatments of 50 MPa was similar to the control. A 100-MPa pretreatment induced a viscous and soft gel with mainly noncovalent bonds. The 200-MPa pretreatment produced a gel with strongest breaking force and strain compared with all the treatments in this study; moreover, the gel was mainly constituted by hydrogen bonds. A gel induced by a 300-MPa pretreatment was the most viscous. Via subsequent cooking (90 °C/20 min), all the gels became more rigid and elastic except that induced by a 100-MPa pretreatment.