Mechanisms of protein S-thiolation and dethiolation

Mechanisms of S-thiolation and dethiolation of creatine kinase and glycogen phosphorylase b were studied. S-thiolation of the two proteins was initiated by oxyradicals generated by xanthine oxidase in reaction mixtures containing reduced glutathione (GSH). Both proteins were extensively modified under the conditions in which the oxidation of glutathione was inadequate to cause S-thiolation by thiol-disulfide exchange. Creatine kinase was both S-thiolated and non-reducibly oxidized at low GSH concentration, but with adequate GSH, oxidation was prevented while S-thiolation was still active. S-thiolation of glycogen phosphorylase b was not significantly affected by GSH concentration and non-reducible oxidation was not observed. The results suggested that protein sulfhydryls could react more actively than GSH with superoxide anion. Thus, oxyradicals may react with protein sulfhydryls resulting in S-thiolation by a mechanism that involves the reaction of an activated protein thiol with reduced glutathione. Dethiolation of phosphorylase b was actively catalyzed by a cardiac extract by both an NADPH-dependent and a GSH-dependent process. In contrast, creatine kinase was actively dethiolated only by GSH nonenzymatically. Thus, two proteins were dethiolated by different mechanisms. Cells seem to have very high capacity for dethiolation by these three mechanisms. The NADPH-dependent dethiolase had both a high molecular weight component and a low molecular weight component. These two components were sensitive to inhibition by phenylarsine oxide and the inhibition was reversed by addition of a dithiol. Therefore, the properties of these components were similar to thioredoxin and thioredoxin reductase. Dethiolation of phosphorylase b was also demonstrated by Escherichia coli thioredoxin with either dithiothreitol or cardiac extract and NADPH as a reducing power, while creatine kinase was not dethiolated by this process. In contrast, GSH-dependent dethiolation required a single component of low molecular weight, and it was less sensitive to phenylarsine oxide inhibition;Therefore, protein S-thiolation and dethiolation play an important role in preventing oxidative damage to cellular system during oxidative stress

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Amyotrophic lateral sclerosis (ALS) is a progressively paralytic neurodegenerative disease that can be caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1). Transgenic mice that overexpress mutant SOD1 develop paralysis and accumulate aggregates of mutant protein in the brainstem and spinal cord. Bee venom (BV), which is also known as apitoxin, is extracted from honeybees and is commonly used in oriental medicine for the treatment of chronic rheumatoid arthritis and osteoarthritis. The purpose of the present study was to determine whether BV affects misfolded protein aggregates such as alpha-synuclein, which is a known pathological marker in Parkinson disease, and ubiquitin-proteasomal activity in hSOD1G93A mutant mice. BV was bilaterally administered into a 98-day-old hSOD1G93A animal model. We found that BV-treated hSOD1G93A transgenic mice showed reduced detergent-insoluble polymerization and phosphorylation of α-synuclein. Furthermore, phosphorylated or nitrated α-synuclein was significantly reduced in the spinal cords and brainstems of BV-treated hSOD1G93A mice and reduced proteasomal activity was revealed in the brainstems of BV-treated symptomatic hSOD1G93A. From these findings, we suggest that BV treatment attenuates the dysfunction of the ubiquitin-proteasomal system in a symptomatic hSOD1G93A ALS model and may help to slow motor neuron loss caused by misfolded protein aggregates in ALS models

Effect of Magnolol on the Function of Osteoblastic MC3T3-E1 Cells

Objectives. In the present study, the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to stimulate osteoblast function and inhibit the release of bone-resorbing mediators was investigated in osteoblastic MC3T3-E1 cells. Methods. Osteoblast function was measured by cell growth, alkaline phosphatase activity, collagen synthesis, and mineralization. Glutathione content was also measured in the cells. Bone-resorbing cytokines, receptor activator of nuclear factor-κB ligand (RANKL), TNF-α, and IL-6 were measured with an enzyme immunoassay system. Results. Magnolol caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, and glutathione content in the cells (P < 0.05). Skeletal turnover is orchestrated by a complex network of regulatory factors. Among cytokines, RANKL, TNF-α, and IL-6 were found to be key osteoclastogenetic molecules produced by osteoblasts. Magnolol significantly (P < 0.05) decreased the production of osteoclast differentiation inducing factors such as RANKL, TNF-α, and IL-6 in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. Conclusion. Magnolol might be a candidate as an agent for the prevention of bone disorders such as osteoporosis

Flux Noise in MgB2 Thin Films

We have performed flux noise and AC-susceptibility measurements on two 400 nm thick MgB$_2$ films. Both measurement techniques give information about the vortex dynamics in the sample, and hence the superconducting transition, and can be linked to each other through the fluctuation-dissipation-theorem. The transition widths for the two films are 0.3 and 0.8 K, respectively, and the transitions show a multi step-like behavior in the AC-susceptibility measurements. The same phenomenon is observed in the flux noise measurements through a change in the frequency dependence of the spectral density at each step in the transition. The results are discussed and interpreted in terms of vortices carrying an arbitrary fraction of a flux quantum as well as in terms of different macroscopic regions in the films having slightly different compositions, and hence, different critical temperatures.Comment: 8 pages, 4 figures, conference contribution to "Fluctuations and Noise", Santa Fe, New mexico 1-4 june 200

Growth of superconducting MgB2 thin films via postannealing techniques

We report the effect of annealing on the superconductivity of MgB2 thin films as functions of the postannealing temperature in the range from 700 C to 950 C and of the postannealing time in the range from 30 min to 120 min. On annealing at 900 C for 30 min, we obtained the best-quality MgB2 films with a transition temperature of 39 K and a critical current density of ~ 10^7 A/cm^2. Using the scanning electron microscopy, we also investigated the film growth mechanism. The samples annealed at higher temperatures showed the larger grain sizes, well-aligned crystal structures with preferential orientations along the c-axis, and smooth surface morphologies. However, a longer annealing time prevented the alignment of grains and reduced the superconductivity, indicating a strong interfacial reaction between the substrate and the MgB2 film.Comment: 7 pages, 4 figures include