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At least 775 records · Page 43Linked to original sources
PubMed2006

UV radiation down-regulates Dsg-2 via Rac/NADPH oxidase-mediated generation of ROS in human lens epithelial cells.

Ultraviolet radiation-induced cataract has been believed to be associated with degradation of cellular components. We report that, in cultured human lens epithelial cells, UV radiation analogous to H2O2 treatment down-regulates desmosomal protein desmoglein-2. UV radiation induces generation of reactive oxygen species and transiently activates epidermal growth factor receptor, which in turn induces translocation of Rac2 and NADPH oxidase activity. Collectively, our data demonstrate that UV-induced desmoglein-2 down-regulation is mediated reactive oxygen species which are generated through EGFR activation and Rac2/NADPH oxidase activation, suggesting that antioxidants may be applied for protection against UV-induced cataract.

Animals↗
PubMed1990

[Role of reactive oxygen species (Ros) in model immune complex nephritis].

In an attempt to examine the role of reactive oxygen species on bivalent hapten immune complex glomerulonephritis, superoxide dismutase (SOD) concentration in renal tissue was studied by Electron Spin Resonance (ESR) and the protective effect of radical scavengers were evaluated. Injection of immune complex (IC) induced severe glomerulonephritis, characterized by neutrophil and/or monocyte infiltration in glomeruli in an association with proteinuria. SOD concentration in renal tissue decreased when neutrophil and/or monocytes infiltration and proteinuria developed, indicating a possible role of reactive oxygen species on renal injury. SOD, superoxide scavenger, and catalase (CAT), which destroy hydrogen peroxide, apparently reduced proteinuria on 14th day (18.5 +/- 3.17 mg/day, 20.7 +/- 7.35 mg/day, respectively, in comparison with control group, 29.5 +/- 4.21 mg/day), but there was no statistical significance. On the other hand, hydroxy radical scavenger, dimethylthiourea (DMTU) significantly reduced proteinuria (15.3 +/- 4.21 mg/day) and remarkable improvement in renal histology was observed. It is concluded that reactive oxygen species, especially hydroxy radical, play a significant role on renal injury in bivalent hapten immune complex glomerulonephritis.

Animals↗
PubMed1990

[Effects of 1,25 dihydroxyvitamin d3 on the morphology and alkaline phosphatase activity of ROS 17/2,8 osteoblastic cells: influence of time and dose].

Osteosarcoma-derived osteoblastic cells were exposed to 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) either to 0.001-1,000 nmol.l-1 for 4 days or to 10 nmol.-1 for 1-21 days. Between 0.01 and 10 nmol.l-1, a dose-dependent increase in alkaline phosphatase (AP) activity was found, which rose to a maximum level at 10 nmol.l-1 (+55%). At higher doses (100 and 1,000 nmol.l-1), 1,25(OH)2D3 induced a decrease in AP activity of 40%. After 1 day at 10 nmol.l-1 a slight increase in AP activity was noted (+20%) which augmented with the duration of exposure. This stimulatory effect was highest after 8 days (+130%). In contrast, after 15 and 21 days, AP activity decreased by 30%. Under 1,25(OH)2D3, microtubules were observed mainly in the perinuclear region.

Alkaline Phosphatase↗
PubMed1981

RCS rat macrophages exhibit normal ROS phagocytosis.

Peritoneal macrophages from RCS rats exhibit phagocytic capability equal to that found in macrophages from normal strains of rat. The similarity in phagocytic activity was found with rat or bovine rod outer segments as well as with dystrophic retinal debris. Thus the genetic defect in RCS pigment epithelium is not expressed in the macrophages, which are available to clear the debris layer that accumulates in dystrophic RCS retinas.

Animals↗
PubMed2000

Light-mediated activation of diacylglycerol kinase in rat and bovine rod outer segments.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate is regulated by light in retinal rod outer segment (ROS) membranes. We recently reported that the activities of phosphatidylinositol synthetase and phosphatidylinositol 3-kinase are also higher in bleached (light-exposed) ROS (B-ROS). In this study, we investigated the effect of bleaching on diacylglycerol (DAG) kinase (DAG-kinase) activity in bovine and rat ROS membranes prepared from dark-adapted (D-ROS) or bleached (B-ROS) retinas. In bovine ROS, DAG-kinase activity toward endogenous DAG substrate was higher in B-ROS than in D-ROS. Quantification of DAG in both sets of membranes showed that the levels were the same, eliminating the possibility that the greater DAG-kinase activity was due to higher levels of endogenous substrate in B-ROS. DAG-kinase activity was also higher in B-ROS against an exogenous, water-soluable substrate (1, 2-didecanoyl-rac-glycerol), which competed with endogenous DAG substrate and saturated at approximately 2 mM. Immunoblot analysis with an anti-DAG-kinase gamma polyclonal antibody demonstrated that the gamma isoform was present in isolated bovine ROS. Immunocytochemistry of frozen bovine retinal sections confirmed the presence of DAG-kinase gamma immunoreactivity in ROS, as well as other retinal cells. Quantification of the immunoreactive products on western blots showed that more DAG-kinase gamma was present in B-ROS than in D-ROS. In an in vivo experiment, ROS prepared from rats exposed to 30 min of room light had greater DAG-kinase activity than ROS prepared from dark-adapted animals. Taken together, these data suggest that light exposure leads to the translocation of DAG-kinase from the cytosol to ROS membranes and that the greater DAG-kinase activity in B-ROS is due to the presence of more protein associated with ROS membranes.

Animals↗
PubMed1995

Incidence and level of seminal reactive oxygen species in normal men.

OBJECTIVES: Excessive formation of reactive oxygen species (ROS) in human semen has been associated with impaired sperm function and fertility potential. The presence of ROS in semen specimens from normal fertile men emphasizes the importance of defining a normal range of ROS formation. The purpose of this study was to establish a normal range of ROS generation and to investigate the effect of sperm concentration on the ROS level. METHODS: ROS was determined in 15 healthy donors and 20 men with suspected infertility. After the sperm concentration in normal donors was adjusted to 20 x 10(6)/mL, ROS was measured by chemiluminescence using luminol in a Berthold luminometer. A specimen was regarded as positive (abnormal) when the value was at least 10 x 10(4) counts per minute (cpm). ROS was also evaluated at 4 sperm concentrations (60, 30, 15, and 7.5 x 10(6)/mL) from samples obtained from the patients with suspected infertility. In addition, ROS was measured in 7 ROS-positive specimens at a sperm concentration of 15 x 10(6)/mL and 60 x 10(6)/mL. RESULTS: Results showed that ROS formation was negative in all 15 healthy donors (median, 0.9 x 10(4) cpm; interquartile range, 0 to 1.48 x 10(4) cpm). The ROS formation value among all the donors was less than 5.5 x 10(4) cpm. ROS formation was positive in 8 (40%) of the suspected infertile patients. ROS levels were significantly lower at sperm concentrations of 15 x 10(6)/mL or 7.5 x 10(6)/mL compared with 30 x 10(6)/mL or 60 x 10(6)/mL (P = 0.05). The ROS level increased after centrifugation for 10 minutes at 500 g in all 7 specimens at both 15 and 60 x 10(6)/mL. However, the increase in ROS formation at 60 x 10(6)/mL was significantly greater than that at 15 x 10(6)/mL (P < 0.001). CONCLUSIONS: A range of ROS formation of 0 to 5.5 x 10(4) cpm at a sperm concentration of 20 x 10(6)/mL may be considered as normal for healthy donor semen. The positive relationship between ROS formation and sperm concentration at the time of measurement emphasizes the importance of concentration adjustment before analysis when comparing ROS levels between different specimens.

Adult↗
PubMed2005

A study of reactive oxygen species in mainstream of cigarette.

UNLABELLED: Reactive oxygenated species (ROS) not only exist in living organisms, they also exist in our environment. Combustion process and photochemical reactions are the major source of environmental ROS, and combustion process produced ROS has been gradually gaining attention in recent years. The purpose of this study is to determine the concentrations of ROS in the mainstream smoke of cigarettes sold in the marketplace using the DCFH2 fluorescence method and to understand particulate and gaseous concentrations of ROS. This research will also discuss the relationship between ROS and nicotine, found in popular cigarette brands, as well as the effectiveness of cigarette filters to remove ROS. Results indicate that the ROS concentration of mainstream smoke is 18.64-54.81 nmol H2O2/l while the correlation coefficients of nicotine and tar to total ROS are 0.959 and 0.909, respectively. Gaseous ROS concentrations are 14.32-39.03 nmol H2O2/l, and make up 71.21-85.99% of the total. It can be clearly seen therefore, that ROS exist mainly in the gaseous phase. Particulate ROS is dominant at PM2.5 (ROS(TSP)/ROS(PM2.5) is 0.652-0.959). The experimental results involving the tobacco leaves and cigarette ash show that ROS in mainstream smoke comes from the combustion process and not from the tobacco leaves. There is no effective means of eliminating ROS from mainstream smoke, regardless of whether a cigarette filter contains active charcoal. PRACTICAL IMPLICATIONS: This study showed that cigarette combustion will produce high concentration of ROS, and this high concentration of ROS in mainstream cigarette smoke probably is one major factor contributing to a high incidence of lung cancer in smokers. Environmental tobacco smoke (ETS) or second-hand smoke is a major indoor air pollutant that could potentially harm non-smokers. We will try to determine the ROS in ETS in the future.

Filtration↗
PubMed2004

Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells.

BACKGROUND: We previously demonstrated that high glucose up-regulates fibronectin mRNA and protein expression by human peritoneal mesothelial cells (HPMC) through activation of protein kinase C (PKC). PKC is known to induce cellular reactive oxygen species (ROS) and PKC-dependent activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been shown to be responsible, in part, for increased oxidative stress in diabetes. On the other hand, high glucose-induced mitochondrial overproduction of superoxide anion was found to activate PKC. We, therefore, hypothesized that high glucose-induced activation of PKC in HPMC may increase cellular ROS and ROS, in turn, may activate PKC and thus provide signal amplification in high glucose-induced fibronectin up-regulation in HPMC. METHODS: The role of ROS in high glucose- and PKC-induced fibronectin expression was examined by quantification of cellular ROS after stimulation with high glucose and phorbol 12-myristate 13-acetate (PMA), by the effect of hydrogen peroxide (H(2)O(2)) and PMA on fibronectin expression, and finally by inhibition of ROS and PKC. The source of cellular ROS was further examined by inhibition of NADPH oxidase and mitochondrial metabolism. RESULTS: D-glucose increased dichlorofluorescein (DCF)-sensitive cellular ROS in HPMC in a dose-dependent manner. l-glucose did not induce ROS generation and cytochalasin B completely blocked high glucose-induced ROS generation, suggesting that glucose uptake, but not media hyperosmolality, is required in ROS generation in HPMC. PMA increased cellular ROS and fibronectin secretion. A single dose of H(2)O(2) or H(2)O(2) continuously generated by glucose oxidase up-regulated fibronectin expression [corrected]. Antioxidants trolox and catalase inhibited high glucose- and PMA-induced fibronectin mRNA and protein expression. Inhibition of PKC inhibited high glucose-and H(2)O(2)-induced fibronectin secretion. NADPH oxidase inhibitors (diphenyleneiodinium and apocynin) and an inhibitor of mitochondrial electron transport chain subunit I (rotenone) all effectively inhibited high glucose-induced cellular ROS generation and fibronectin secretion. CONCLUSION: The present data demonstrate that high glucose increases cellular ROS in HPMC through activation of PKC, NADPH oxidase, and mitochondrial metabolism and that ROS, thus generated, up-regulate fibronectin expression by HPMC. ROS are not only downstream but also upstream signaling molecules to PKC and provide signal amplification in high glucose-induced fibronectin expression by HPMC. The present data imply that cellular ROS may be potential therapeutic targets in progressive accumulation of extracellular matrix in the peritoneal tissue of long-term peritoneal dialysis patients using high glucose-containing peritoneal dialysis solutions.

Antioxidants↗
PubMed1992

Characterization and application of an in vitro detection system for studying the binding and phagocytosis of rod outer segments by retinal pigment epithelial cells.

Direct and indirect radioactivity and fluorescent assays have been developed to study the interaction of rod outer segments (ROS) with retinal pigment epithelial (RPE) cells. In the direct assays ROS labelled with 125I or fluorescein isothiocyanate (FITC) have been used to measure total phagocytosis, i.e. surface binding and ingestion. In the indirect assays RPE cells were first treated with unlabelled ROS or biotinylated ROS and subsequently probed with [125I]Rho 4D2 antirhodopsin antibody or [125I]streptavidin for radioactivity measurements or with the Rho 4D2 antibody and FITC-goat anti-mouse Ig or FITC-streptavidin for fluorescent counting. In these indirect methods the number of surface bound ROS were distinguished from the number of ingested ROS by comparative labelling of non-permeabilized and permeabilized ROS-treated RPE cells. Using these assays, we have studied the binding and ingestion of bovine ROS with cultured bovine RPE cells. As in the case of newborn cultured rat RPE cells [Hall and Abrams (1987) Exp. Eye Res. 45, 907-22], binding and ingestion of bovine ROS by bovine RPE cells was saturable with respect to ROS concentration and time. At 37 degrees C ROS binding reached a saturating concentration at 1 x 10(7) ROS per well; the number of bovine ROS ingested by bovine RPE cells, however, was less than the number of rat ROS ingested by rat RPE cells. When 1 x 10(7) ROS per well was used, maximal surface binding of bovine ROS to bovine RPE cells was obtained after 2-3 hr, whereas after an initial delay, ingestion rapidly increased to a maximum at 1-2 hr.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells.

Retinal pigment epithelial (RPE) cells selectively phagocytize rod outer segments (ROS) by a process which may be mediated by specific cell surface receptors. We have studied the kinetics of this process using rat RPE cells grown in tissue culture. By cooling RPE cells to 17 degrees C, the binding and ingestion phases of phagocytosis can be separated. Maximum ROS binding with minimum ingestion occurs at 17 degrees C; above 17 degrees C the rate of ingestion increases markedly. Thus it is possible to measure the kinetics of ROS binding to RPE cells at 17 degrees C and of ROS ingestion at 37 degrees C. At 17 degrees C, ROS binding is saturable, both with respect to time and to ROS concentration. ROS ingestion saturates after 4 hr of incubation at 37 degrees C, after which the cells are refractory to further ROS ingestion for 1-2 hr. During this recovery period, rapid digestion of the internalized ROS takes place. Cycloheximide, when present at a concentration (2 x 10(-5) M) which inhibits protein synthesis by 92%, has no effect on ROS phagocytosis or on the recovery of ROS ingestion at 37 degrees C. This suggests that if receptors mediate the ingestion of ROS by RPE cells, they are not degraded after the ROS are internalized. Dystrophic rat (RCS-p+) RPE cells exhibit normal binding, but very limited ingestion of ROS at 37 degrees C. The rate and amount of ROS binding to these cells at 37 degrees C is comparable with that occurring to normal cells at 17 degrees C. These observations support the hypothesis that there are a limited number of receptors which are specific for ROS binding on the surface of normal and dystrophic rat RPE cells.

Animals↗
PubMed2006

Biochemical activity of reactive oxygen species scavengers do not predict retinal ganglion cell survival.

PURPOSE: Retinal ganglion cells (RGCs) die as a result of axonal injury in a variety of optic neuropathies, including glaucoma. Reactive oxygen species (ROS) act as intracellular signaling molecules and initiate apoptosis in nerve growth factor-deprived sympathetic neurons and axotomized RGCs. Determination of the role of specific ROS relies on the use of small molecule or protein scavengers with various degrees of specificity. The pro- or anti-cell-death effect of several ROS generating and scavenging systems in cultured RGCs was correlated with their activity in cell-free assays. METHODS: Neonatal rat retinas were dissociated and incubated with ROS-generating systems for hydroxyl radical, superoxide anion (O2-), and H2O2. Scavengers tested were catalase, polyethylene glycol-superoxide dismutase (PEG-SOD), manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), deferoxamine, and U-74389G. Viability of retrogradely labeled RGCs was determined with calcein-AM 24 hours after plating. O2- and H2O2 scavenging in cell-free assays was measured with dihydroethidium and Amplex Red (Invitrogen, Carlsbad, CA), respectively. RESULTS: Systematic differences were found between ROS scavenging in cell-free assays and the ability of scavengers to protect RGCs in cell culture. Furthermore, many ROS scavengers lost specificity and protected against various ROS, whereas others failed to protect against their unique ROS target. These activities stray from commonly recognized specificities of individual ROS scavengers or generating systems and are important in understanding ROS biology. In addition, antioxidant defense mechanisms used by RGCs and other retinal cells interfere with responses expected from ROS scavengers in well-defined systems. Last, H2O2 induced intramitochondrial O2-, whereas paraquat produced O2- outside of the mitochondria, and these areas of generation can mislead interpretations of ROS scavenger activity and effectiveness. CONCLUSIONS: There is discordance between ROS effects in cultured RGCs and cell-free assays, with several mechanisms accounting for this divergence. To identify the roles of ROS signaling in cell death accurately, several approaches should be used. These include using a panel of ROS scavengers and generators, testing the panel in primary neuronal cultures, and quantifying ROS with cell-free assays.

Animals↗
PubMed1984

Immunotitration analysis of the rod outer segment phosphodiesterase.

Two high-affinity monoclonal antibodies (ROS-1, ROS-2) have been produced to the rod outer segment phosphodiesterase (ROS PDE). These antibodies bind at different antigenic determinants. ROS-2 absorbs a subset of the total PDE activity from a detergent-solubilized retina extract, whereas ROS-1 adsorbs nearly all of the PDE activity. DEAE-cellulose chromatography separates two peaks of activity from a hypotonic extract of rod outer segments. Peak I activity is adsorbed only by ROS-1, whereas peak II activity is adsorbed by both ROS-1 and ROS-2. Both peaks of activity are activated by histone H2B and limited trypsin digestion, and both peaks of activity contain a heat-stable, trypsin-sensitive inhibitor. When analyzed by SDS gel electrophoresis, ROS-1 adsorbed a single peptide from peak I, which comigrated with phosphorylase B, whereas ROS-1 adsorbed two slightly faster migrating peptides from peak II. Histone H2B activated at least 80% of the PDE activity bound to ROS-2 but was less effective in activating the PDE bound to ROS-1. ROS-1 but not ROS-2 was an effective inhibitor of PDE activity, suggesting that ROS-1 may be a specific probe to study the effects of ROS PDE on the light response.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2006

Evaluation of assays for the measurement of bovine neutrophil reactive oxygen species.

During mastitis and other bacterial-mediated diseases of cattle, neutrophils play a critical role in the host innate immune response to infection. Neutrophils are among the earliest leukocytes recruited to the site of infection and contribute to host innate immune defenses through their ability to phagocytose and kill bacteria. The bactericidal activity of neutrophils is mediated, in part, through the generation of reactive oxygen species (ROS). Extracellular release of ROS can induce injury to host tissue as well, and aberrant release of ROS has been implicated in the pathogenesis of certain inflammatory-mediated diseases. Due to their essential role in bacterial clearance and implicated involvement in the pathogenesis of other diseases, there is much interest in the study of neutrophil-generated ROS. Several assays have been developed to measure ROS production, however, many of these have not been evaluated with bovine neutrophils. The objectives of the current study were to evaluate different assays capable of measuring bovine neutrophil ROS, and to compare the results of assays never previously tested with bovine neutrophils to those obtained from more well-established assays frequently used with these cells. Eight different assays were evaluated, including: luminol, isoluminol, and methyl cypridina luciferin analog (MCLA) chemiluminescence assays; Amplex Red, dihydroethidium (DHE), dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA), and dihydrorhodamine 123 fluorescence assays; and the cytochrome c absorbance assay. The assays were evaluated in the context of their abilities to detect ROS produced in response to two agonists commonly used to induce neutrophil activation, phorbol 12-myristate, 13-acetate (PMA) and opsonized zymosan. Diphenyleneiodonium chloride, a NADPH oxidase inhibitor, was used to assess the specificity of the assays to detect ROS. The ability of these assays to discriminate between intra- and extracellular ROS and to specifically detect distinct ROS was evaluated using superoxide dismutase and catalase, which scavenge extracellular superoxide and hydrogen peroxide, respectively. With the exception of the DHE assay, all assays detected bovine neutrophil ROS generation elicited by PMA and zymosan. PMA, but not zymosan, was able to stimulate neutrophil generation of ROS at levels that were detectable with DHE. The MCLA chemiluminescence assay was the only assay that detected ROS produced in response to each of the lowest concentrations of PMA and zymosan tested. To our knowledge, this is the first study to evaluate DHE-, MCLA-, Amplex Red-, and isoluminol-based assays for the measurement of bovine neutrophil ROS, and the most comprehensive comparative study of ROS assays under similar experimental conditions.

Animals↗
PubMed1991

Different subpopulations of developing thymocytes are associated with adherent (macrophage) or nonadherent (dendritic) thymic rosettes.

Thymic rosettes (ROS), structures consisting of thymic lymphoid cells attached to a central stromal cell, were isolated from mouse thymus by collagenase digestion and unit-gravity elutriation. The ROS were then separated into those where the stromal cells were either macrophage-like (M-ROS) or dendritic cell-like (D-ROS), on the basis of the differences in adherence properties or in the level of MAC-1 surface antigen. The ROS were then dissociated and the thymocyte content analyzed by immunofluorescent staining and flow cytometry. M-ROS and D-ROS differed in thymocyte composition, although the major component of both was the CD4+CD8+ cortical thymocyte. D-ROS were enriched in thymocytes expressing high levels of surface T-cell antigen receptor (TcR) and the associated CD3 complex, and these included both CD4+CD8-CD3++ and CD4-CD8+CD3++ mature thymocytes. M-ROS were enriched in CD4-CD8- thymocytes and had a reduced content of thymocytes expressing high TcR-CD3 levels; they nevertheless contained some mature thymocytes, but only of the CD4+CD8-CD3++ category. Several lines of evidence indicated that the mature thymocytes in ROS were cells recently formed in the cortex, and were not from the medullary pool. ROS-associated mature thymocytes expressed lower levels of H-2K than free, mature thymocytes. The CD4+CD8+CD3++ subpopulation, believed to be a developmental intermediate between cortical thymocytes and mature T cells, was present in both ROS populations. Further, late intermediates leading to both mature T-cell categories were evident in D-ROS, but only those leading to CD4+CD8-CD3++ T cells were evident in M-ROS. The results are compatible with a role for ROS in TcR-specificity selection and in the final maturation steps in the thymic cortex.

Animals↗