The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase [CcP(MI)], in which the conserved distal His (His-52) is replaced with Leu. The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength. The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively. This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions. Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation. Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules. The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent. In nitrate-containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pKA = 4.5. Protonation of the group caused a loss of reactivity with peroxide. This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pKA = 5.4.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanistic target of rapamycin (mTOR) is a central mediator of protein synthesis in skeletal muscle. We utilized immunofluorescence approaches to study mTOR cellular distribution and protein-protein co-localisation in human skeletal muscle in the basal state as well as immediately, 1 and 3 h after an acute bout of resistance exercise in a fed (FED; 20 g Protein/40 g carbohydrate/1 g fat) or energy-free control (CON) state. mTOR and the lysosomal protein LAMP2 were highly co-localised in basal samples. Resistance exercise resulted in rapid translocation of mTOR/LAMP2 towards the cell membrane. Concurrently, resistance exercise led to the dissociation of TSC2 from Rheb and increased in the co-localisation of mTOR and Rheb post exercise in both FED and CON. In addition, mTOR co-localised with Eukaryotic translation initiation factor 3 subunit F (eIF3F) at the cell membrane post-exercise in both groups, with the response significantly greater at 1 h of recovery in the FED compared to CON. Collectively our data demonstrate that cellular trafficking of mTOR occurs in human muscle in response to an anabolic stimulus, events that appear to be primarily influenced by muscle contraction. The translocation and association of mTOR with positive regulators (i.e. Rheb and eIF3F) is consistent with an enhanced mRNA translational capacity after resistance exercise.

This study investigated the dissolution-based toxicity mechanism for silver nanoparticles to Escherichia coli K12. The silver nanoparticles, synthesised in the vapour phase, are effective anti-bacterial agents against the Gram-negative bacterium, E. coli K12. The nanoparticles associate with the bacterial cell wall, appearing to interact with the outer and inner membranes, and then dissolve to release Ag(+) into the cell and affect a transcriptional response. The dissolution of these nanoparticles in a modified LB medium was measured by inductively coupled plasma mass spectrometry (ICP-MS) and has been shown to follow a simple first-order dissolution process proportional to the decreasing surface area of the nanoparticles. However, the resulting solution phase concentration of Ag(+), demonstrated by the ICP-MS data, is not sufficient to cause the observed effects, including inhibition of bacterial growth and the differential expression of Cu(+) stress response genes. These data indicate that dissolution at the cell membrane is the primary mechanism of action of silver nanoparticles, and the Ag(+) concentration released into the bulk solution phase has only limited anti-bacterial efficacy.

The pH-dependent adsorption isotherms for the charged chromophore crystal violet, CV(+), have been measured with three different bases by a free-running cavity implementation of evanescent wave cavity ring-down spectroscopy. The ratio of the maximal absorbance measurements at pH 5.10 and 9.05 is consistent with a Q2:Q3 silanol site ratio of 72.8:27.2. The adsorption isotherms have been interpreted in terms a cooperative binding adsorption allowing more than one ionic species to bind to each silanol group. The surface concentration is consistent with a silanol charge density of 1.92 +/- 0.55 nm(-2) and a total neutralized interface layer structure extending 9 nm from the surface. Binding constants and stoichiometric coefficients are derived for CV(+) to both the Q2 and Q3 sites. A variation of the adsorption isotherm with base is observed so that the isotherm at pH 9.05 adjusted with ammonium hydroxide sets up a competitive acid-base equilibrium with the SiOH groups with only 49% of the surface silanol sites dissociated. The implications for functionalized surfaces in chromatography are discussed.

Evanescent wave cavity ring-down spectroscopy (EW-CRDS) has been used to investigate the adsorption of crystal violet (CV + ) to a charged silica-water interface as a function of bulk pH by the direct measurement of the absorbance of the CV + chromophore. Absolute absorbances of order 10 -4 have been routinely detected, showing significant variation in the structure of the silica-water interface. At low ionic strength, the interfacial absorbance of CV + shows a monotonic increase with increasing pH. A simple competitive Langmuir adsorption model, which provides values for the silica surface parameters that are in broad agreement with the existing literature values, has been fit to the data. In addition, interfacial absorbance has been monitored as a function of pH for CV + solutions maintained at high ionic strength with NaCl, KCl, and CaCl 2 . As pH increases, the CV + interfacial absorbance exhibits a pronounced maximum, which occurs at pH 8.7 for Na + and K + and at pH 7.9 for Ca 2+ , followed by a sharp decrease. This trend is attributed to competitive binding between the metal cations and CV + to the silica surface binding site, and it has not been observed in previous measurements using second-harmonic generation. The simple Langmuir model, however, does not accurately describe the high ionic strength behavior.