Redox signal transduction, especially the oxidative modification of proein thiols, correlates with many diseases and becomes an expanding research area. However, there was rare method for quick and specific detection of protein thiols and their oxidative modification in living cells. In this article, we review the current chemical strategies for the detection and quantification of protein thiols and related cysteine oxidation. We also look into the future of the development of fluorescent probes for protein thiols and their potential application in the research of reactive cysteine proteomes and early detection of redox-related diseases.
Animals ; Cysteine ; metabolism ; Fluorescent Dyes ; Humans ; Nitrosation ; Oxidation-Reduction ; Proteins ; chemistry ; metabolism ; Reactive Nitrogen Species ; metabolism ; Reactive Oxygen Species ; metabolism ; Sulfenic Acids ; analysis ; Sulfhydryl Compounds ; analysis ; chemistry ; metabolism

AIM AND METHODSBased on the reaction that 2',7'-dichlorodihydrofluorescein (DCFH) can be oxidized by reactive oxygen species (ROS) to yield the highly fluorescent 2',7'-dichlorofluorescin (DCF), ROS production in mitochondria can be observed dynamically as well as quantified directly by spectrofluorometer.

RESULTS AND CONCLUSIONDCF fluorescence showed linear increase in State 4 mitochondria, which suggest ROS produced at constant rate. The slopes of the linear increase in fluorescence with time were computed performing a linear regression that took into account all relevant data points in selected time windows. The slopes were proportional to ROS production in mitochondria. Addition of sodium azide and malonic acid increased and decreased the rate of ROS production respectively during measurement. DCF fluorescence varied linearly with increasing concentration of mitochondria, which showed quantitative relations in definite range. Repeated measures showed low coefficients of variation. This method is reliable and efficient for determining ROS in mitochondria.

Animals ; Fluoresceins ; Fluoroimmunoassay ; methods ; Male ; Mice ; Mice, Inbred Strains ; Mitochondria ; metabolism ; Reactive Oxygen Species ; analysis

Oxidative stress is a common condition suffered by biological systems in aerobic conditions. Human semen also has its own molecular guard against the free radicals created by normal respiratory process or from immune reactions. The equilibrium of the creation and scavenging of free radicals is mandatory in the spermatozoa to fertilize and initiate a full-term pregnancy. The paper is a systematic review of publications that evaluate oxidative stress in semen. The Cochrane Library, Medline (1966-2003), Embase (1988-2003), SciSearch (1981-2003) and the conference papers were searched. When sperm samples from fertile and infertile males were analyzed, some of the mechanisms that determine the oxidative stress level were found to be impaired while others were unaltered. In conclusion, the literature as a whole provides contradictory findings and it is necessary to carry out more work to identify all the enzymatic and non-enzymatic systems involved in oxidative stress in the ejaculate, in order to develop new diagnostic systems and therapeutic strategies for combating detrimental free radical imbalance in the semen.
Antioxidants ; analysis ; metabolism ; Fertility ; Humans ; Infertility, Male ; metabolism ; Male ; Oxidants ; analysis ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis ; metabolism ; Semen ; chemistry ; metabolism ; Spermatozoa ; metabolism ; ultrastructure

The oxidative response of Bacillus sp F26 to different forms of reactive oxygen species (ROS) stress including H2O2, O2- * and OH * were investigated by using diverse generating source of ROS, which were characterized by synthesis of antioxidative enzymes. It was shown that the responses of cells to oxidative stress are largely dependent on species, mode (instantaneous and continual) and intensity of stress. Higher synthesis rate of catalase (CAT) is crucial for Bacillus sp F26 to resist H2O2 stress. The damage of H2O2 to cell was minor if CAT can efficiently decompose H2O2 entering into cell, furthermore, the response can stimulate cell growths and sugar consumption. Conversely, cell growth and synthesis of antioxidative enzymes are greatly inhibited when the intensity of H2O2 stress overwhelms the cell capability of clearing H2O2. Due to the difference in mode and effect on cells between O2- * and H2O2, higher synthesis rates of CAT and superoxide dismutase (SOD) couldn't guarantee cells to eliminate H2O2 and O2- * efficiently. Therefore, the toxicity to cells induced by intracellular O2- * is more severe than H2O2 stress. Unlike response to H2O2 and O2- *, OH stress significantly inhibited cell growth and synthesis of antioxidative enzymes due to the fact OH * is most active ROS. Our results indicated that Bacillus sp F26 will show diverse biological behaviour in response to H2O2, O2- * and OH * of stress due to the discrepancy in chemical property. In order to survive in oxidative stress, cells will timely adjust their metabolism to adapt to new environment including regulating synthesis level of antioxidative enzymes, changing rates of cells growth and substrate consumption.
Adaptation, Physiological ; Bacillus ; enzymology ; metabolism ; physiology ; Catalase ; biosynthesis ; Oxidative Stress ; physiology ; Reactive Oxygen Species ; analysis ; metabolism ; Superoxide Dismutase ; biosynthesis

Catalase ; genetics ; Mycelium ; growth & development ; Polymerase Chain Reaction ; RNA, Messenger ; analysis ; Reactive Oxygen Species ; metabolism ; Sporothrix ; enzymology ; growth & development

OBJECTIVETo evaluate the feasibility of establishing a mouse model of ovarian oxidative stress by intraperitoneal injections of arsenic sodium.

METHODSTwenty adult female Kunming mice were randomized equally into the normal control group and ovarian oxidative stress model group for intraperitoneal injections of 0.5 ml distilled water and 8 mg/kg arsenic sodium solution every other day, respectively. After 8 injections, the mice were sacrificed for histological observation of the ovarian sections and enzyme-linked immunosorbent assay (ELISA) of serum estradiol (E(2)) and pregnenedione (P) levels ande contents of reactive oxygen species (ROS) , malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the ovary homogenate.

RESULTSNumerous atretic follicles were found in the ovaries of mice in the model group with obviously reduced growing follicles. Compared with those in the normal control group, the contents of ROS and MDA increased and SOD and GSH-Px levels in the ovarian homogenate decreased significantly in the model group (P<0.05).

CONCLUSIONA mouse model of ovarian oxidative stress can be established by intraperitoneal injections of arsenic sodium.

Animals ; Arsenites ; Disease Models, Animal ; Female ; Glutathione Peroxidase ; analysis ; Malondialdehyde ; analysis ; Mice ; Mice, Inbred Strains ; Ovary ; metabolism ; physiopathology ; Oxidative Stress ; Reactive Oxygen Species ; analysis ; Superoxide Dismutase ; analysis

BACKGROUND/AIMS: Peroxiredoxin (Prx) belongs to a ubiquitous family of antioxidant enzymes that regulates many cellular processes through intracellular oxidative signal transduction pathways. Silica-induced lung damage involves reactive oxygen species (ROS) that trigger subsequent toxic effects and inflammatory responses in alveolar epithelial cells resulting in fibrosis. Therefore, we investigated the role of Prx in the development of lung oxidant injury caused by silicosis, and determined the implication of ROS in that process. METHODS: Lung epithelial cell lines A549 and WI26 were treated with 1% silica for 0, 24, or 48 hours, following pretreatment of the A549 cells with N-acetyl-L-cysteine and diphenylene iodonium and no pretreatment of the WI26 cells. We transfected an HA-ubiquitin construct into the A549 cell line and then analyzed the cells via Western blotting and co-immunoprecipitation. RESULTS: Silica treatment induced cell death in the A549 lung epithelial cell line and selectively degraded Prx I without impairing protein synthesis in the A549 cells, even when the ROS effect was blocked chemically by N-acetyl-L-cysteine. A co-immunoprecipitation study revealed that Prx I did not undergo ubiquitination. CONCLUSIONS: Silica treatment induces a decrease of Prx I expression in lung epithelial cell lines regardless of the presence of ROS. The silica-induced degradation of Prx does not involve the ubiquitin-proteasomal pathway.
Cell Line ; Epithelial Cells/drug effects/metabolism ; Humans ; Lung/chemistry/*drug effects/metabolism ; Peroxiredoxins/analysis/*physiology ; Protein Isoforms ; Reactive Oxygen Species/metabolism ; Silicon Dioxide/*toxicity ; Ubiquitin/metabolism

The aim of this study was to determine the effects of extremely low frequency electromagnetic field (ELF-EMF) on energy metabolism and oxidative stress in Caenorhabditis elegans (C. elegans). Worms in three adult stages (young adult stage, egg-laying stage and peak egg-laying stage) were investigated under 50 Hz, 3 mT ELF-EMF exposure. ATP levels, ATP synthase activity in vivo, reactive oxygen species (ROS) content, and changes of total antioxidant capacity (TAC) were detected, and worms' oxidative stress responses were also evaluated under ELF-EMF exposure. The results showed that ATP levels were significantly increased under this ELF-EMF exposure, and mitochondrial ATP synthase activity was upregulated simultaneously. In young adult stage, worms' ROS level was significantly elevated, together with upregulated TAC but with a decreased ROS-TAC score indicated by principal component analysis. ROS level and TAC of worms had no significant changes in egg-laying and peak egg-laying stages. Based on these results, we concluded that ELF-EMF can enhance worm energy metabolism and elicit oxidative stress, mainly manifesting as ATP and ROS level elevation together with ATP synthase upregulation and ROS-TAC score decrease in young adult C. elegans.
Adenosine Triphosphate ; metabolism ; Animals ; Caenorhabditis elegans ; radiation effects ; Electromagnetic Radiation ; Energy Metabolism ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

BACKGROUNDTissue inhibitor of matrix metalloproteinase-1 (TIMP-1) is related to the aging of many organs, but few data are available on the change of TIMP-1 in liver aging. The purpose of this study was to investigate the expression and role of TIMP-1, matrix metalloproteinase-2 (MMP-2) and MMP-9 in the process of natural aging in the livers of normal and transgenic mice, and to detect the effects of TIMP-1 on oxidative level and anti-oxidative ability of the livers of transgenic young mice.

METHODSNormal and transgenic mice were divided into 3 groups according to their age: 3-month-old group (n = 5), 12-month-old group (n = 5) and 24-month-old group (n = 5). Histopathological changes of the liver were observed after HE and Masson staining. The messenger RNA (mRNA) levels of TIMP-1, MMP-2 and MMP-9 were determined by semi-quantitative reverse transcriptional polymerase chain reaction; protein expression was measured by Western blot in the livers of normal and transgenic mice of various ages. Changes in levels of superoxide dismutase (SOD), monoamine oxidase (MAO), malondialdehyde (MDA) as well as oxidative and anti-oxidative ability were measured.

RESULTSHistologically, more fatty degeneration and collagen deposition were found in the aging livers of transgenic mice than in those of the normal mice as their age of months increased. The mRNA and protein expressions of TIMP-1 were significantly high in the oldest animals. The histopathological changes, mRNA and protein expressions of TIMP-1 increased significantly in the liver of transgenic mice as compared with normal mice. The expression of MMP-2 and MMP-9 showed a minor change in the process of aging. Liver change and collagen deposition were not observed in young mice, but the activity of SOD decreased (P < 0.05), and the activity of MAO (P < 0.01) and the content of MDA increased in the liver of transgenic mice (P < 0.01).

CONCLUSIONSThe expression of TIMP-1 is significantly high in the liver of transgenic mouse in the process of aging, indicating that the oxidative level increases and the anti-oxidative ability decreases in the liver of transgenic mouse. TIMP-1 plays an important role in the process of liver aging.

Aging ; metabolism ; Animals ; Female ; Liver ; metabolism ; pathology ; Male ; Matrix Metalloproteinase 2 ; analysis ; genetics ; Matrix Metalloproteinase 9 ; analysis ; genetics ; Mice ; Mice, Transgenic ; Monoamine Oxidase ; analysis ; RNA, Messenger ; analysis ; Reactive Oxygen Species ; metabolism ; Superoxide Dismutase ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; analysis ; genetics

To investigate the relationship between uncoupling protein 2 (UCP2) expression and the damage caused by oxygen free radicals in acute liver failure rat models. Thirty-five male Sprague-Dawley rats were randomly divided into two groups: the control group (15 rats) and liver failure group (20 rats). The rats were injected intraperitoneally with thioacetamide (TAA) to induce models of acute liver failure. The levels of endotoxin (ET) were detected by double antibody sandwich enzyme-linked immunosorbent assay. The expression of liver UCP2 mRNA was detected by reverse transcription polymerase chain reaction. The superoxide dismutase (SOD) and malonaldehyde (MDA) were detected by spectrophotometry. The expression of UCP2 protein was observed by immunohistochemistry. The data of the two groups were compared using Mann-Whitney U test or ANOVA. The expression of UCP2 mRNA in liver failure group was higher as compared to the control group (P value is less than 0.01); the level of MDA and endotoxin of liver failure group were higher than that of the control group (P value is less than 0.01). SOD of the liver failure group was lower (P value is less than 0.01). There was a certain correlation between UCP2 mRNA expression and ET, SOD and MDA (r = 0.952, -0.667, 0. 634 respectively, P value is less than 0.05 or 0.01). UCP2 is highly expressed in the livers of liver failure rats. A certain correlation perhaps existed between the expression of UCP2 mRNA and the serous SOD, MDA and ET.
Animals ; Endotoxins ; analysis ; Ion Channels ; metabolism ; Liver ; metabolism ; Liver Failure, Acute ; metabolism ; Male ; Malondialdehyde ; analysis ; Mitochondrial Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; adverse effects ; Superoxide Dismutase ; analysis ; Uncoupling Protein 2