Search

Rationale Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. Methods Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. Results ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. Conclusions Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.

In vivo effects of N-benzyloxycarbonyl (Cbz)-Leu–Leu-leucinal (MG132) on chymotryptic-like (ChT-L), tryptic-like, and post-glutamyl peptide hydrolytic-like proteasome activities, protein oxidation, lipid peroxidation (LP), glutathione (GSH) level, as well as on the activity of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione-reductase) in the rat liver were studied. The possibility of MG132 provoking the formation of free oxygen radicals was also assayed in primary hepatocytes. The following results were obtained: (1) In vivo, MG132 did not change the spontaneous LP, but increased Fe-induced LP and the amount of oxidized proteins; it decreased the GSH level in liver. From the proteasome activities studied in liver cytosol only ChT-L activity was significantly decreased after MG132 administration. Furthermore, MG132 increased antioxidant enzyme activities of SOD, CAT, and GSH-Px. (2) In vitro, MG132 increased free radical oxygen species in hepatocytes; this effect disappeared in the presence of CAT or mannitol. In conclusion, since nowadays proteasome inhibitors are entering into the swing of laboratory and clinical practice, the present data could provide useful information for MG132 action. Consequently, future in vivo experiments with MG132 could highlight the possibility of its use at different pathological conditions. Copyright © 2008 John Wiley & Sons, Ltd.

Investigations of variations in metabolic efficiency and thermogenesis have a short and turbulent history. In small animals, non-shivering thermogenesis and diet-induced thermogenesis have a great impact on overall body weight, and the question is whether mechanisms to waste energy have evolved also in human energy metabolism. The candidate molecules for this adaptive thermogenesis are the uncoupling proteins. This is a newly discovered family of proteins, consisting of at least five proteins, namely UCP1, UCP2, UCP3, UCP4 and UCP5. Although a role for UCP1 in thermogenesis is unequivocal, the physiological function of the newer uncoupling proteins is as yet unclear. UCP1 is present in brown adipose tissue and has a well-documented role in cold-induced thermogenesis. The targeted disruption of the UCP1-gene rendered animals that were cold sensitive, but not obese. UCP2 mRNA has a ubiquitous distribution in tissue, namely, in skeletal muscle, white and brown adipose tissue, the gastro-intestinal tract, the lung and the spleen. By targeting the UCP2-gene there was no effect on whole body energy metabolism, but instead, a reduced ability to protect against free-radical oxygen species. UCP2 has also been shown to act as a negative regulator for insulin secretion. UCP3 is present in skeletal muscle. Targeted disruption of the UCP3-gene gave no effect on whole body energy metabolism, but showed the mitochondria in muscle to be more coupled. In conclusion, the uncoupling proteins may be important in various specific ways, as protectors of free radical oxygen species and as regulators of ATP-dependent processes.

The development of head and neck cancer may depend not only on exposure to environmental carcinogens but also on a genetically based susceptibility to carcinogen-induced damage. This thesis presents a case-control study that demonstrates the significance of mutagen sensitivity, a measure of an individual's intrinsic DNA repair capacity against free radical damage, as a risk factor for the disease. As part of the case-control analysis, 167 previously untreated patients and 177 age- and sex-matched healthy controls were assessed for various lifestyle factors including tobacco and alcohol habits, occupational exposures, and diet. Mutagen sensitivity expressed by each individual was determined by quantifying bleomycin-induced chromosomal breaks within peripheral blood lymphocytes in vitro. Consistent with our initial observations and those of others, mutagen hypersensitivity was strongly associated with increased risk of head and neck cancer (odds ratio, 4.95; 95% confidence interval, 2.67 to 9.17) after adjusting for age, sex, and race. Low intake of vitamins C and E was also associated with an increased risk of disease and was interactive with mutagen sensitivity in risk estimates. Individuals with both a low intake of various antioxidants and increased chromosomal sensitivity to oxidant-induced DNA damage were at greatest risk. This study supports the concept that the risk of head and neck cancer is determined by a balance of factors that either enhance or protect against free radical oxygen damage, including innate capacities for DNA repair.

Conductive polymeric films have been of great interest recently, although one major drawback to their use is environmental stability. Polypyrroles doped with p-toluenesulfonate, tetrafluoroborate and perchlorate anions were examined under elevated temperatures and environments. The use of d.c. electrical conductivity decay measurements and fitting of kinetic models allows changes in bulk properties to be examined. Surface characterisation performed by X-ray photoelectron spectroscopy (XPS) enables detailed investigation into changes in chemical states of surface atoms. The mechanisms described for the change in electrical and structural properties may be attributed to polymer backbone oxidation through free radical oxygen insertion and electrolyte dopant degradation.