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At least 919 records · Page 51Linked to original sources

PubMed1999

[In vivo scavenging effect of ethylcysteine on reactive oxygen species in human semen].

PURPOSE: The production of reactive oxygen species (ROS) is a normal physiological event in various organs including the testis. Overproduction of ROS, however, can be detrimental to sperm, being associated with male infertility. In vivo experiments using vitamin E (Vit. E), one of the major membrane protectants against ROS and lipid peroxidation, have shown its significant potential in treating ROS-associated male infertility. There has been no study that the scavenging drugs reduce the level of ROS in human semen. Previously we reported the in vitro scavenging effectiveness of ethylcysteine (EC) against ROS in human semen. The present study was performed in order to determine the effectiveness of the in vivo administration of EC as treatment for ROS-associated male infertility. PATIENTS AND METHODS: Ten cases of male infertility, with the exceptions of azoospermia and pyospermia, were chosen. Patients were divided randomly into two groups. Each group received either 600 mg/day of EC (Group A) or Vit. E 600 mg/day (Group B) for 3 months. Then, after a 1-month wash-out period, the patients were switched to another treatment. Conventional semen analysis, computerized motility assessment, measurement of ROS generation and sperm function assessment by triple stain were performed before and after administration of EC and Vit. E. The levels of EC and Vit. E were also assessed in patients' blood serum and seminal plasma before and after administration of EC and Vit. E. RESULTS: Sperm density and sperm motility did not improve but sperm function had a significant tendency toward improvement after administration of EC and Vit. E. ROS levels significantly decreased only after administration of EC After administration of EC and Vit. E, their levels significantly increased in patients' blood serum but were unchanged in patients' seminal plasma. CONCLUSION: Since our study showed that oral administration of EC produced results similar to those of Vit. E, we conclude that EC is an effective treatment agent for ROS-associated male infertility.

Adult↗

PubMed2000

Lutein and zeaxanthin concentrations in rod outer segment membranes from perifoveal and peripheral human retina.

PURPOSE: In addition to acting as an optical filter, macular (carotenoid) pigment has been hypothesized to function as an antioxidant in the human retina by inhibiting the peroxidation of long-chain polyunsaturated fatty acids. However, at its location of highest density in the inner (prereceptoral) layers of the foveal retina, a specific requirement for antioxidant protection would not be predicted. The purpose of this study was to determine whether lutein and zeaxanthin, the major carotenoids comprising the macular pigment, are present in rod outer segment (ROS) membranes where the concentration of long-chain polyunsaturated fatty acids, and susceptibility to oxidation, is highest. METHODS: Retinas from human donor eyes were dissected to obtain two regions: an annular ring of 1.5- to 4-mm eccentricity representing the area centralis excluding the fovea (perifoveal retina) and the remaining retina outside this region (peripheral retina). ROS and residual (ROS-depleted) retinal membranes were isolated from these regions by differential centrifugation and their purity checked by polyacrylamide gel electrophoresis and fatty acid analysis. Lutein and zeaxanthin were analyzed by high-performance liquid chromatography and their concentrations expressed relative to membrane protein. Preparation of membranes and analysis of carotenoids were performed in parallel on bovine retinas for comparison to a nonprimate species. Carotenoid concentrations were also determined for retinal pigment epithelium harvested from human eyes. RESULTS: ROS membranes prepared from perifoveal and peripheral regions of human retina were found to be of high purity as indicated by the presence of a dense opsin band on protein gels. Fatty acid analysis of human ROS membranes showed a characteristic enrichment of docosahexaenoic acid relative to residual membranes. Membranes prepared from bovine retinas had protein profiles and fatty acid composition similar to those from human retinas. Carotenoid analysis showed that lutein and zeaxanthin were present in ROS and residual human retinal membranes. The combined concentration of lutein plus zeaxanthin was 70% higher in human ROS than in residual membranes. Lutein plus zeaxanthin in human ROS membranes was 2.7 times more concentrated in the perifoveal than the peripheral retinal region. Lutein and zeaxanthin were consistently detected in human retinal pigment epithelium at relatively low concentrations. CONCLUSIONS: The presence of lutein and zeaxanthin in human ROS membranes raises the possibility that they function as antioxidants in this cell compartment. The finding of a higher concentration of these carotenoids in ROS of the perifoveal retina lends support to their proposed protective role in age-related macular degeneration.

Aged↗

PubMed2003

Reactive oxygen species as mediators of cell adhesion.

The intracellular production of reactive oxygen species (ROS) has a fundamental importance in both cell proliferation and apoptosis induction. Moreover, many experimental and epidemiological evidence indicate that ROS contribute to the initiation and promotion of carcinogenesis, and that drugs or treatments aimed to reduce the tissue content of ROS can be chemopreventive and curative against cancer. Recently, important observations on the role of ROS as physiological regulators of intracellular signaling cascades activated by growth factors through their tyrosine-kinase receptors have shed new light on the possible mechanisms that can sustain the promoting activity of ROS. The downstream effect of ROS production is the reversible oxidation of proteins. Redox sensitive proteins include protein tyrosine phosphatases (PTPs) as the active-site cysteine is the target of specific oxidation, and this modification can be reversed by intracellular reducing agents. The reversible oxidation of PTPs family member was demonstrated firstly for PTP1B during EGF signaling and then for LMW-PTP and SHP-2 during PDGF stimulation. The inhibition exerted by ROS on tyrosine-phosphatases helps the propagation of RTK signals mediated by protein tyrosine phosphorylation, generally associated with the proliferative stimulus. Our new data are consistent with a model in which ROS take a role in integrin signaling, and in which synergistic activation of Rac-1 by growth factors and adhesion molecules translates in a critical increase of intracellular oxidants up to a threshold level where inhibition of the tyrosine phosphatase LMW-PTP takes place. In seeking for potential molecular mechanisms for oxidative signaling by integrins, we found that transient oxidation/inactivation of LMW-PTP, a known negative regulator of RTK signaling, occurred during fibroblast adhesion to matrix, with a kinetic which paralleled the generation of ROS. Moreover, overexpression of LMW-PTP in NIH-3T3 fibroblasts delayed cell attachment to the substrate. Finally, constitutively high levels of intracellular ROS, as are observed in cells expressing active Rac, would attenuate anchorage dependence for growth, by substituting for integrin signaling in non adherent cells.

3T3 Cells↗

PubMed1987

Light activation of phospholipase A2 in rod outer segments of bovine retina and its modulation by GTP-binding proteins.

Light stimulates phospholipase A2 activity in rod outer segments (ROS) of bovine retina as measured by the liberation of arachidonate from phosphatidylcholine, in in vitro assays of dark-adapted ROS. A role for GTP-binding proteins (G or N proteins) in the light activation of phospholipase A2 is suggested by the capacity for guanosine 5'-O-(thiotriphosphate) (GTP gamma S) to activate phospholipase A2 in dark-adapted ROS. In contrast, addition of GTP gamma S coincident with light exposure inhibited the light activation of phospholipase A2, suggesting that phospholipase A2 activity in the ROS is under dual regulation by G proteins. Transducin, the major G protein of the ROS, mediates the activation of cGMP phosphodiesterase by light and is a substrate for both cholera and pertussis toxin. Treatment of dark-adapted ROS with either toxin inhibits both basal and light-activated phospholipase A2, mimicking the action of the toxins on the light-induced cGMP phosphodiesterase activity of ROS. There is a loss of light-sensitive phospholipase A2 activity coincident with extraction of transducin from ROS membranes. In addition, the light-sensitive phospholipase A2 activity can be partially restored by the addition of purified transducin to the extracted ROS membranes. Light activation of phospholipase A2 in ROS membranes thus occurs by a transducin-dependent mechanism.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1983

The distribution of actin in cultured normal and dystrophic rat pigment epithelial cells during the phagocytosis of rod outer segments.

In the previous article the authors reported that the ingestion phase of phagocytosis is defective in cultured dystrophic rat pigment epithelial (PE) cells. When these cells are challenged with isolated rod outer segments (ROS), attachment of ROS to the PE cell surfaces occurs to a normal extent. However, only a small number of these bound ROS are subsequently ingested. This raised the possibility that the contractile protein actin might not function normally in the dystrophic rat PE cells, since actin is intimately involved in the ingestion mechanism in other phagocytic cells. Utilizing actin antibodies and the technique of indirect immunofluorescence, we have studied the distribution of actin in cultured normal and dystrophic rat PE cells. Results show that the arrangement of actin fibers in the dystrophic cells appears normal both before and during the attachment of ROS to the cell surfaces. With the additional use of an ROS antiserum to label externally bound ROS, it is also possible to show that actin is involved with the ingestion of ROS by both normal and dystrophic PE cells. Thus, it appears that actin can function normally in dystrophic PE cells, but that the ingestion mechanism becomes activated at only a few sites of ROS attachment. The results of a scanning electron microscope study support this conclusion and also show the presence of a saucer-shaped elaboration of the PE cell plasma membrane beneath attached ROS. These may correspond to the actin feltworks seen with immunofluorescence microscopy at sites of ROS attachment.

Actins↗

PubMed1983

Immunologic and biochemical properties of several retinal proteins bound by antibodies in sera from animals with experimental autoimmune uveitis and uveitis patients.

Sera from guinea pigs and rabbits with and without experimental autoimmune uveitis (EAU) induced by immunization with retina, choroid, optic nerve, retinal rod outer segments (ROS) and purified bovine S-antigen were tested for the ability to immunoprecipitate 125I-labeled, detergent-solubilized bovine retinal proteins. The results demonstrate that three major protein antigens with m.w. of 50,000 (p50), 35,000 (p35) and 27,000 (p27) and several minor activities between 30,000 and 60,000 m.w. are recognized by antibodies from these animals. The p50 component was immunoprecipitated by sera from animals immunized with whole retina homogenate, the high speed supernatant of whole retina homogenate, ROS, and S-antigen, and has been identified as S-antigen in competition experiments. The p35 band appeared when sera were used that were raised against antigen preparations containing membrane-bound retinal protein, i.e., whole retina homogenate, ROS, and washed ROS, and thus appears to be an ROS membrane protein. The p27 band was found when sera raised against ROS, washed ROS, optic nerve and whole retina homogenate were used, suggesting it is a membrane-bound antigen common to ROS and optic nerve. Serum from animals immunized with homologous choroid did not immunoprecipitate a detectable product. S-antigen and p35 were also precipitated by some uveitis patient sera. Because S-antigen is also an ROS protein as is rhodopsin, a putative uveitogenic retinal antigen, ROS appear to be an unusually rich source of autoantigenic proteins. S-antigen was also shown to be synthesized in the retina, and the primary translation product was indistinguishable from purified S-antigen by SDS-PAGE, thus eliminating the possibility that it is derived from or is cross-reactive with the 67,000 m.w. rhodopsin kinase.

Animals↗

PubMed1995

Rod outer segment-associated N-acetylgalactosaminylphosphotransferase.

PURPOSE: To determine the exact location of a cell surface glycosyltransferase (N-acetylgalactosaminylphosphotransferase, (GalNAcPTase) immunochemically identified in mammalian rod outer segments (ROS), to determine whether anti-GalNAcPTase antibody recognizes retinal molecules that possess transferase activity and to characterize ROS transferase enzyme activity and acceptors. The GalNAcPTase is known to be associated with the adhesion molecule N-cadherin in embryonic avian retinas and with E-cadherin in mammalian pancreatic islet cells. METHODS: Purified, fixed ROS were reacted with anti-chick GalNAcPTase antibody followed by secondary antibody conjugated to colloidal gold and were examined by electron microscopy. Fractions of retinal and ROS proteins enriched in the transferase were obtained through batch adsorption on Sepharose, separated by gel electrophoresis, transferred to nitrocellulose, and either reacted with anti-GalNAcPTase antibody or assayed for transferase activity. Interphotoreceptor matrix (IPM) was examined for the presence of immunoreactive GalNAcPTase by gel electrophoresis and immunoblot. The kinetics and endogenous acceptors of the cow ROS transferase were characterized. RESULTS: ROS are specifically labeled by anti-GalNAcPTase antibody at the cell surface. The immunogold label was associated with the cell surface and with flocculent material adherent to the cell surface. In addition, soluble and particulate fractions of the IPM showed GalNAcPTase-like immunoreactivity. The transferase appears as single immunoreactive band at or near 220 kd. Transferase enzyme activity was present at this position on Western transfers of retinal and ROS proteins. In whole ROS, transferase activity was directed toward endogenous acceptors of very high molecular mass. CONCLUSIONS: The GalNAcPTase is localized on ROS in association with the cell surface and with components of the IPM. The molecule recognized by the anti-GalNAcPTase antibody possesses transferase activity toward itself and a few other proteins, but mostly toward very large molecules that may be IPM proteoglycans. It is not yet known whether the enzyme of the adult retina specifically transfers sugar or sugar-phosphate groups to its acceptors. It is proposed that the ROS GalNAcPTase is involved in the modulation of adhesive phenomena between or within photoreceptors or between photoreceptors and the interphotoreceptor matrix.

Animals↗

PubMed1995

[Changes in vitreous structure caused by oxygen free radicals].

Vitreous liquefaction in humans is considered to be part of the normal ocular aging process and is associated with vitreoretinal pathology. Because hyaluronic acid (HA), one of the main components of the vitreous gel structure, is degraded by reactive oxygen species (ROS) including oxygen free radicals, the structural changes in the vitreous may be caused by ROS. To investigate the effect of ROS on the vitreous gel structure, we treated animal vitreous with ROS, which was generated from various sources. Using riboflavin as a photosensitizer, calf vitreous was irradiated with visible light (two 15-W fluorescent lamps) and found to be considerably liquefied. The liquefaction resulted from HA depolymerization induced by ROS. Because of the small amount of riboflavin naturally present in the vitreous, a riboflavin-sensitized photochemical reaction may contribute to age-related vitreous liquefaction. Hematoporphyrin (HP), which is similar in chemical structure to heme in blood, was also used as a photosensitizer. Irradiation with HP destroyed the calf vitreous gel structure and caused liquefaction. A HP-sensitized photochemical reaction may contribute to vitreous liquefaction observed after vitreous hemorrhage. Because metal ions, including Fe2+ and Cu2+, can catalyze to generate ROS, liquefaction occurred when we treated calf vitreous with Fe2+ or Cu2+ at 4 degrees C. Adding ascorbic acid to the vitreous during the reaction increased the rate of liquefaction. Therefore, metal ion catalyzed ROS may also contribute to vitreous liquefaction, such as that found in an injured eye with siderosis. To investigate the relation between inflammatory cell mediated ROS and vitreous liquefaction, an endotoxin-induced uveitis model was created in the rabbit eye. Upon inflammation, the vitreous gel contracted and released a water-like liquid. Because superoxide dismutase can suppress the liquefaction, the destruction of the vitreous gel structure resulted from ROS generated from inflammatory cells. Although many unknown factors contribute to vitreous liquefaction, ROS may be the main cause of vitreous structure alterations. To prevent or delay the progress of vitreous liquefaction in the normal aging process or vitreous pathology, a new therapeutic procedure based on clear scientific studies is needed.

Aging↗

PubMed1997

Reactive oxygen species in human sperm suspensions: production by leukocytes and the generation of NADPH to protect sperm against their effects.

In men from couples consulting for infertility, 72% of washed sperm preparations produced detectable amounts of reactive oxygen species (ROS) compared to only 25% of preparations from a population of fertile donors. High ROS production was associated with oligozoospermia and poor sperm function. The rate of ROS production was closely correlated with the concentration of leukocytes (r = 0.826) and the degrees of stimulation by N-formyl-methionyl-leucyl-phenyl alanine (NFMLP) and by phorbol 12-myristate 13 acetate (PMA) were similar (r = 0.923). The removal of leukocytes with 'Dynabeads' either abolished or substantially decreased ROS production but in a few cases ROS production that could be stimulated by PMA but not NFMLP was observed in leukocyte-free preparations. When sperm preparations which produced ROS were incubated under 95% O2 there was a rapid 40% decrease in the number of sperm that could be stimulated to acrosome react although the acrosome reaction was unaffected by incubation under 95% N2 for up to 6 h. The harmful effect of oxygen was not seen in preparations that produced no ROS and could be prevented by removing leukocytes from the suspension or by adding superoxide dismutase and catalase. We conclude that leukocytes are the predominant source of ROS in human sperm preparations and that the ROS they produce are harmful to sperm. On the other hand these data confirm that highly purified sperm can produce ROS albeit in smaller amounts. We have demonstrated that flux through the pentose phosphate pathway (PPP) in purified sperm preparations increases in response to oxidative stress. This is required to make reduced glutathione available for glutathione peroxidase and we suggest that measurement of PPP flux provides an index of the capacity of glutathione peroxidase to protect sperm against oxidation.

Humans↗

PubMed2001

Oxidative stress in scleroderma: maintenance of scleroderma fibroblast phenotype by the constitutive up-regulation of reactive oxygen species generation through the NADPH oxidase complex pathway.

OBJECTIVE: To explore the role of reactive oxygen species (ROS) in the in vitro activation of skin fibroblasts from patients with systemic sclerosis (SSc). METHODS: Fibroblasts were obtained from involved skin of patients with limited or diffuse SSc. Oxidative activity imaging in living cells was carried out using confocal microscopy. Levels of O2- and H2O2 released from fibroblasts were estimated by the superoxide dismutase (SOD)-inhibitable cytochrome c reduction and homovanilic acid assays, respectively. To verify NADPH oxidase activation, the light membrane of fibroblasts was immunoblotted with an anti-p47phox-specific antibody. Fibroblasts were stimulated with various cytokines and growth factors to determine whether any of these factors modulate ROS generation. Cell proliferation was estimated by 3H-thymidine incorporation. Northern blot analysis was used to study alpha1 and alpha2 type I collagen gene expression. RESULTS: Unstimulated skin fibroblasts from SSc patients released more O2- and H2O2 in vitro through the NADPH oxidase complex pathway than did normal fibroblasts, since incubation of SSc fibroblasts with diphenylene iodonium, a flavoprotein inhibitor, suppressed the generation of ROS. This suppression was not seen with rotenone, a mitochondrial oxidase inhibitor, or allopurinol, a xanthine oxidase inhibitor. Furthermore, the cytosolic component of NADPH oxidase, p47phox, was translocated to the plasma membrane of resting SSc fibroblasts. A transient increase in ROS production was induced in normal but not in SSc fibroblasts by interleukin-1beta (IL-1beta), platelet-derived growth factor type BB (PDGF-BB), transforming growth factor beta1 (TGFbeta1), and H2O2. Treatment of normal and SSc fibroblasts with tumor necrosis factor a (TNFalpha), IL-2, IL-4, IL-6, IL-10, interferon-alpha (IFNalpha), IFNgamma, granulocyte-macrophage colony-stimulating factor (GM-CSP), G-CSF, or connective tissue growth factor (CTGF) had no effect on ROS generation. Constitutive ROS production by SSc fibroblasts was not inhibited when these cells were treated with catalase, SOD, IL-1 receptor antagonist, or antibodies blocking the effect of TGFbeta1, PDGF-BB, and other agonists (IL-4, IL-6, TNFalpha, CTGF). In contrast, treatment of SSc fibroblasts with the membrane-permeant antioxidant N-acetyl-L-cysteine inhibited ROS production, and this was accompanied by decreased proliferation of these cells and down-regulation of alpha1(I) and alpha2(I) collagen messenger RNA. CONCLUSION: The constitutive intracellular production of ROS by SSc fibroblasts derives from the activation of an NADPH oxidase-like system and is essential to fibroblast proliferation and expression of type I collagen genes in SSc cells. Our results also exclude O2-, H2O2, IL-1beta, TGFbeta1, PDGF-BB, IL-4, IL-6, TNFalpha, or CTGF as mediators of a positive, autocrine feedback mechanism of ROS generation.

Acetylcysteine↗

PubMed2006

Reactive oxygen species as signals that modulate plant stress responses and programmed cell death.

Reactive oxygen species (ROS) are known as toxic metabolic products in plants and other aerobic organisms. An elaborate and highly redundant plant ROS network, composed of antioxidant enzymes, antioxidants and ROS-producing enzymes, is responsible for maintaining ROS levels under tight control. This allows ROS to serve as signaling molecules that coordinate an astonishing range of diverse plant processes. The specificity of the biological response to ROS depends on the chemical identity of ROS, intensity of the signal, sites of production, plant developmental stage, previous stresses encountered and interactions with other signaling molecules such as nitric oxide, lipid messengers and plant hormones. Although many components of the ROS signaling network have recently been identified, the challenge remains to understand how ROS-derived signals are integrated to eventually regulate such biological processes as plant growth, development, stress adaptation and programmed cell death.

Apoptosis↗

PubMed1999

Redox regulation of TNF signaling.

TNF is produced during inflammation and induces, among other activities, cell death in sensitive tumour cells. We previously reported an increased generation of ROS in TNF-treated L929 fibrosarcoma cells prior to cell death. These ROS are of mitochondrial origin and participate in the cell death process. Presently, we focus on the identification of parameters that control ROS production and subsequent cytotoxicity. From the cytotoxic properties and susceptibility to scavenging of TNF-induced ROS as compared to pro-oxidant-induced ROS we conclude that TNF-mediated ROS generation and their lethal action are confined to the inner mitochondrial membrane. Oxidative substrates, electron-transport inhibitors, glutathione and thiol-reactive agents but also caspase inhibitors modulate TNF-induced ROS production and imply the existence of a negative regulator of ROS production. Inactivation of this regulator by a TNF-induced reduction of NAD(P)H levels and/or formation of intraprotein disulfides would be responsible for ROS generation.

Animals↗

PubMed1998

Generation of reactive oxygen species in a human keratinocyte cell line: role of calcium.

In the human keratinocyte cell line HaCaT, reactive oxygen species (ROS) were generated in a dose- and time-dependent manner in response to epidermal growth factor (EGF), bradykinin, thapsigargin, and the Ca(2+)-ionophore A23187, agonists that interact with different primary cell targets. ROS formation was assessed by both chemiluminescence- and fluorescence-based methods. The ROS evoked by EGF and bradykinin decayed within 8 and 4 min, respectively, this transient effect resulting probably from down-regulation of the specific agonist receptors or dissipation of the secondary signals. In contrast, the response to thapsigargin and A23187 was sustained for at least 15 min. Extracellular Ca2+ and a rise in intracellular Ca2+ concentration ([Ca2+]i) proved essential for ROS production. Chelation by BAPTA suppressed ROS formation. Direct measurement of [Ca2+]i using fura fluorescence revealed that EGF and bradykinin evoked a modest, transient [Ca2+]i elevation of less than twofold, whereas with thapsigargin and A23187 there was a sustained two- to fourfold elevation. For each agonist, the kinetics of the rise and decay of [Ca2+]i were similar to those of ROS. The enzyme(s) involved in ROS formation were inhibited by diphenyleneiodonium, indicating dependence on FAD. Our results suggest a close link between ROS and changes in [Ca2+]i generated by growth factors and hormones. This is a particularly interesting connection because elevation of ROS and/ or [Ca2+]i has been linked to cell proliferation, differentiation, and apoptosis.

Biphenyl Compounds↗

PubMed2000

The role of RhoA in the germinal vesicle breakdown of mouse oocytes.

We have investigated a new role of RhoA in the germinal vesicle breakdown (GVBD) of mouse oocytes. First, RhoA was identified by immunostaining and ADP-ribosylation in germinal vesicle (GV) stage-oocytes. RhoA was mainly localized in the ooplasmic area, but rarely detected in germinal vesicle. Incubation of oocyte extract with C3 transferase induced a strong ADP-ribosylation at about 25 kDa. Incubation of GV-stage oocytes in culture medium induced the spontaneous maturation to GVBD by about 78 and 87% of total oocytes at 1 and 3 h, respectively. However, microinjection of C3 transferase into GV-stage oocytes significantly inhibited GVBD at 1 (GVBD = 29%) and 3 h (GVBD = 49%). To study the role of reactive oxygen species (ROS) in the oocyte maturation, the level of intra-oocyte ROS was measured using a ROS-specific fluorescent dye H(2)DCFDA during the oocyte maturation. Spontaneous maturation of GV-stage oocytes induced a significant increase of ROS at 3 h by about twofold over the control level and then the increased level was maintained until 6 h. However, microinjection of C3 transferase inhibited the production of intra-oocyte ROS. Incubation with ROS scavengers, N-acetyl-l-cysteine and catalase, blocked the ROS increase. The ROS scavengers also significantly inhibited GVBD, as did C3 transferase. Thus, it was proposed that RhoA was involved in the GVBD, possibly by the production of ROS in mouse oocytes.

ADP Ribose Transferases↗

PubMed2000

Cyanide-induced apoptosis involves oxidative-stress-activated NF-kappaB in cortical neurons.

The central nervous system is one of the main target organs in cyanide toxicity. Primary cultured cortical neurons were used to study the cellular mechanisms underlying cyanide-induced cytotoxicity. After exposure to KCN (100-300 microM) for 24 h, cortical neurons underwent apoptosis as characterized by positive TUNEL staining. Reactive oxygen species (ROS) play an important role in cyanide-induced neuronal apoptosis; immediately after cyanide (100-300 microM) treatment, ROS generation was observed and continued to be elevated for up to 3 h. NMDA receptor activation and subsequent Ca(2+) influx contribute in part to cyanide-induced ROS formation, since the selective NMDA receptor antagonist MK801 and intracellular Ca(2+) chelator BAPTA blocked ROS generation. Interestingly, caspases, recently reported to be involved in neuronal apoptosis, play a role in the late phase of ROS production after cyanide stimulation. Z-VAD, a nonspecific caspase inhibitor, blocked ROS generated 1 h after cyanide treatment, but it had no effect on ROS generated immediately after cyanide treatment. Nuclear factor kappaB (NF-kappaB), a redox-sensitive transcription factor, was activated dose dependently after cyanide treatment. Blockade of ROS generation by MK801, Z-VAD, and various antioxidants also blocked the activation of NF-kappaB. SN50, a synthetic peptide which inhibits the nuclear translocation of NF-kappaB, blocked cyanide-induced apoptotic cell death. These results indicate that NF-kappaB plays an important role in cyanide-induced apoptosis in cortical neurons, and the caspases may contribute in part to the activation of NF-kappaB after cyanide treatment by inducing the late phase of ROS generation.

Animals↗

PubMed2004

Mitochondrial swelling and generation of reactive oxygen species induced by photoirradiation are heterogeneously distributed.

Abundant evidence has been gathered to show that overproduction of reactive oxygen species (ROS) can lead to the opening of the mitochondrial permeability transition pore (MPTP) and result in apoptosis in mammalian cells. The information regarding spatial and temporal regulation of intracellular ROS formation related to the MPTP opening, however, is relatively limited. In this study, we used a fluorescent probe, dihydro-2',7'-dichloroforescin (DCF), to detect intracellular ROS levels in different compartments of the cell in a time-resolved manner. The roles of mitochondrial ROS (mROS) in the MPTP opening and mitochondrial membrane potential drop were investigated by using H(2)DCFDA coloaded with a mitochondrial marker dye MitoTracker Red, and by a mitochondrial membrane potential dye tetramethyl rhodamine ethyl ester. We applied multiphoton laser scanning microscopy to avoid autooxidation and bleaching of DCF so that long-term visualization of intracellular ROS formation could be performed. Moreover, we noted that the resting mROS levels of different mitochondria were not homogeneous. After cells had been exposed to photoirradiation, the intracellular ROS gradually increased but the heterogeneity of mROS was maintained. Later, swelling was observed in mitochondria that contained higher levels of ROS, indicating the opening of the MPTP. In cells in which all the mitochondria swelled, they were translocated to the perinuclear area, which became the site of ROS production. At this stage, mROS reached the highest level concomitantly with a complete loss of mitochondrial membrane potential, indicating full opening of the MPTP. At the end, photoirradiation resulted in apoptotic cell death. In summary, we demonstrated by multiphoton laser scanning microscopy that photoirradiation induces heterogeneous intracellular ROS formation and mitochondrial permeability transition pore opening in single intact cells. These observations imply the existence of a microdomain in the regulation of mROS formation and subsequent opening of the MPTP.

Animals↗

PubMed2004

Mitochondrial reactive oxygen species generation and calcium increase induced by visible light in astrocytes.

Mitochondria contain photosensitive chromophores that can be activated or inhibited by light in the visible range. Rather than utilizing light energy, however, mitochondrial electron transport oxidation-reduction reaction and energy coupling could be stimulated or damaged by visible light. Our previous work demonstrated that reactive oxygen species (ROS) were generated in cultured astrocytes after visible laser irradiation. With confocal fluorescence microscopy, we found that ROS were generated mostly from mitochondria. This mitochondrial ROS (mROS) formation plays a critical role in photoirradiation-induced phototoxicity and apoptosis. In this study, we measured changes of mitochondrial calcium level ([Ca(2+)](m)) in cultured astrocytes (RBA-1 cell line) irradiated with blue light and examined the association between mROS formation and [Ca(2+)](m) level changes. Changes of intracellular ROS and [Ca(2+)](m) were visualized using fluorescent probes 2',7'-dichlorodihydrofluorescein (DCF), and rhod-2. After exposure to visible light irradiation, RBA-1 astrocytes showed a rapid increase in ROS accumulation particularly in the mitochondrial area. Increase in [Ca(2+)](m) was also induced by photoirradiation. The levels of increase in DCF fluorescence intensity varied among different astrocytes. Some of the cells generated much higher levels of ROS than others. For those cells that had high ROS levels, mitochondrial Ca(2+) levels were also high. In cells that had mild ROS levels, mitochondrial Ca(2+) levels were only slightly increased. The rate of increase in DCF fluorescence seemed to be close to the rate of rhod-2 fluorescence increase. There is a positive and close correlation between mitochondrial ROS levels and mitochondrial Ca(2+) levels in astrocytes irradiated by visible light.

Animals↗

PubMed2006

The reactive oxygen species network pathways:an essential prerequisite for perception of pathogen attack and the acquired disease resistance in plants.

Availability of complete Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) genome sequences, together with molecular recourses of functional genomics and proteomics have revolutionized our understanding of reactive oxygen species (ROS) signalling network mediating disease resistance in plants. So far, ROS have been associated with aging, cellular and molecular alteration in animal and plant cells. Recently,concluding evidences suggest that ROS network is essential to induce disease resistance and even to mediate resistance to multiple stresses in plants. ROS are obligatory by-products emerging as a result of normal metabolic reactions. They have the potential to be both beneficial and harmful to cellular metabolism. Their dual effects on metabolic reactions are dosage specific. In this review we focus our attention on cellular ROS level to trigger beneficial effects on plant cells responding to pathogen attack. By exploring the research related contributions coupled with data of targeted gene disruption, and RNA interference approaches, we show here that ROS are ubiquitous molecules of redox-pathways that play a crucial role in plant defence mechanism. The molecular prerequisites of ROS network to activate plant defence system in response to pathogen infections are here underlined. Bioinformatic tools are now available to scientists for high throughput analysis of cellular metabolisms. These tools are used to illustrate crucial ROS-related genes that are involved in the defence mechanism of plants. The review describes also the emerging findings of ROS network pathways to modulate multiple stress resistance in plants.

Animals↗