Protective effect of metallothionein-III on DNA damage in response to reactive oxygen species

Metallothionein (MT)-III is a member of a brain-specific MT family, in contrast to MT-I and MT-II that are found in most tissues and are implicated in metal ion homeostasis and as an antioxidant. To investigate the defensive role of MT-III in terms of hydroxyl radical-induced DNA damage, we used purified human MT-III. DNA damage was detected by single-strand breaks of plasmid DNA and deoxyribose degradation. In this study, we show that MT-III is able to protect against the DNA damage induced by ferric ion-nitrilotriacetic acid and H(2)O(2), and that this protective effect is inhibited by the alkylation of the sulfhydryl groups of MT-III by treatment with EDTA and N-ethylmaleimide. MT-III was also able to efficiently remove the superoxide anion, which was generated from the xanthine/xanthine oxidase system. These results strongly suggest that MT-III is involved in the protection of reactive oxygen species-induced DNA damage, probably via direct interaction with reactive oxygen species, and that MT-III acts as a neuroprotective agent.     

Chloroplast-generated reactive oxygen species are involved in hypersensitive response-like cell death mediated by a mitogen-activated protein kinase cascade

Plant defense against pathogens often includes rapid programmed cell death known as the hypersensitive response (HR). Recent genetic studies have demonstrated the involvement of a specific mitogen-activated protein kinase (MAPK) cascade consisting of three tobacco MAPKs, SIPK, Ntf4 and WIPK, and their common upstream MAPK kinase (MAPKK or MEK), NtMEK2. Potential upstream MAPKK kinases (MAPKKKs or MEKKs) in this cascade include the orthologs of Arabidopsis MEKK1 and tomato MAPKKKalpha. Activation of the SIPK/Ntf4/WIPK pathway induces cell death with phenotypes identical to pathogen-induced HR at macroscopic, microscopic and physiological levels, including loss of membrane potential, electrolyte leakage and rapid dehydration. Loss of membrane potential in NtMEK2(DD) plants is associated with the generation of reactive oxygen species (ROS), which is preceded by disruption of metabolic activities in chloroplasts and mitochondria. We observed rapid shutdown of carbon fixation in chloroplasts after SIPK/Ntf4/WIPK activation, which can lead to the generation of ROS in chloroplasts under illumination. Consistent with a role of chloroplast-generated ROS in MAPK-mediated cell death, plants kept in the dark do not accumulate H(2)O(2) in chloroplasts after MAPK activation, and cell death is significantly delayed. Similar light dependency was observed in HR cell death induced by tobacco mosaic virus, which is known to activate the same MAPK pathway in an N-gene-dependent manner. These results suggest that activation of the SIPK/Ntf4/WIPK cascade by pathogens actively promotes the generation of ROS in chloroplasts, which plays an important role in the signaling for and/or execution of HR cell death in plants.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Protective effects of fluvastatin against reactive oxygen species induced DNA damage and mutagenesis

Oxidative stress may be an important factor in the development of diabetic complications. Advanced glycation end-products have drown attention as potential sources of oxidative stress in diabetes. We investigated the protective effects of fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, on oxidative DNA damage from reactive oxygen species or advanced glycation end-products in vitro, as well as effects of main fluvastatin metabolites and other inhibitors of the same enzyme, pravastatin and simvastatin. Protective effects were assessed in terms of the DNA breakage rate in a single-stranded phage DNA system in vitro. DNA was exposed to either reactive oxygen species or advanced glycation end-products. Fluvastatin and its metabolites showed a strong protective effect comparable to those seen with thiourea and mannitol, though pravastatin and simvastatin did not exert clear protective effects. Furthermore, fluvastatin reduced the mutagenesis by reactive oxygen species or advanced glycation end-products in Salmonella typhimurium test strains. Both pravastatin and simvastatin still lacked protective activity. Fluvastatin and its metabolites protect against oxidative DNA damage and may reduce risk of consequent diabetic complications.     

Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase

Reactive oxygen species (ROS) are implicated in plant innate immunity. NADPH oxidase (RBOH; for Respiratory Burst Oxidase Homolog) plays a central role in the oxidative burst, and EF-hand motifs in the N terminus of this protein suggest possible regulation by Ca(2+). However, regulatory mechanisms are largely unknown. We identified Ser-82 and Ser-97 in the N terminus of potato (Solanum tuberosum) St RBOHB as potential phosphorylation sites. An anti-phosphopeptide antibody (pSer82) indicated that Ser-82 was phosphorylated by pathogen signals in planta. We cloned two potato calcium-dependent protein kinases, St CDPK4 and St CDPK5, and mass spectrometry analyses showed that these CDPKs phosphorylated only Ser-82 and Ser-97 in the N terminus of St RBOHB in a calcium-dependent manner. Ectopic expression of the constitutively active mutant of St CDPK5, St CDPK5VK, provoked ROS production in Nicotiana benthamiana leaves. The CDPK-mediated ROS production was disrupted by knockdown of Nb RBOHB in N. benthamiana. The loss of function was complemented by heterologous expression of wild-type potato St RBOHB but not by a mutant (S82A/S97A). Furthermore, the heterologous expression of St CDPK5VK phosphorylated Ser-82 of St RBOHB in N. benthamiana. These results suggest that St CDPK5 induces the phosphorylation of St RBOHB and regulates the oxidative burst.     

Involvement of mitogen-activated protein kinases and reactive oxygen species in the inotropic action of ouabain on cardiac myocytes. A potential role for mitochondrial KATP channels

Binding of ouabain to Na⁺/K⁺-ATPase activated multiple signal transduction pathways including stimulation of Src, Ras, p42/44 MAPKs and production of reactive oxygen species (ROS) in rat cardiac myocytes. Inhibition of either Src or Ras ablated ouabain-induced increase in both [Ca²⁺]_i and contractility. While PD98059 abolished the effects of ouabain on [Ca²⁺]_i, it only caused a partial inhibition of ouabain-induced increases in contractility. On the other hand, pre-incubation of myocytes with N-acetyl cysteine (NAC) reduced the effects of ouabain on contractility, but not [Ca²⁺]_i. Furthermore, 5-hydroxydecanoate (5-HD) blocked ouabain-induced ROS production and partially inhibited ouabain-induced increases in contractility in cardiac myocytes. Pre-incubation of myocytes with both 5-HD and PD98059 completely blocked ouabain's effect on contractility. Finally, we found that opening of mitochondrial K_ATP channel by diazoxide increased intracellular ROS and significantly raised contractility in cardiac myocytes. These new findings indicate that ouabain regulates cardiac contractility via both [Ca²⁺]_i and ROS. While activation of MAPKs leads to increases in [Ca²⁺]_i, opening of mitochondrial K_ATP channel relays the ouabain signal to increased ROS production in cardiac myocytes.     

Role of exercise-induced reactive oxygen species in the modulation of heat shock protein response

Abstract

The multiple roles that have been associated with heat shock proteins (HSPs), inside and outside cells are remarkable. HSPs have been found to play a fundamental role in multiple stress conditions and to offer protection from subsequent insults. Exercise, because of the physiological stresses associated with it, is one of the main stimuli associated with a robust increase of different HSPs in several tissues. Given the combination of physiological stresses induced by exercise, and the ‘cross-talk’ that occurs between signaling pathways in different tissues, it is likely that exercise induces the HSP expression through a combination of ‘stressors’, among which reactive oxygen species (ROS) could play a major role. Indeed, although an imbalance between ROS production and antioxidant levels results in oxidative stress, causing damage to lipids, proteins, and nucleic acids with a possible activation of the programed cell death pathway, at moderate concentrations ROS play an important role as regulatory mediators in signaling processes. Many of the ROS-mediated responses actually protect the cells against oxidative stress and re-establish redox homeostasis. The aim of this review is to provide a critical update on the role of exercise-induced ROS in the modulation of the HSP's response, focusing on experimental results from animal and human studies where the link between redox homeostasis and HSPs’ expression in different tissues has been addressed.     

Sperm activation: role of reactive oxygen species and kinases

Reactive oxygen species (ROS), such as the superoxide anion (O(2)(-*)), hydrogen peroxide (H(2)O(2)) and nitric oxide (NO*), when generated at low and controlled levels, act as second messengers. ROS regulate sperm capacitation, which is the complex series of changes allowing spermatozoa to bind to the zona pellucida surrounding the oocyte, induce the acrosome reaction (exocytotic event by which proteolytic enzymes are released) and fertilize the oocyte. Capacitating spermatozoa produce controlled amounts of ROS that regulate downstream events: first, the increase in cAMP, protein kinase A (PKA) activation and phosphorylation of PKA substrates (arginine-X-X-serine/threonine motif; 15-30 min); second, the phosphorylation of MEK (extracellular signal regulated kinase [ERK] kinase)-like proteins (30-60 min) and then that of the threonine-glutamate-tyrosine motif (>1 h); finally, the late tyrosine phosphorylation of fibrous sheath proteins (>2 h). Although all these events are ROS-dependent, the regulation by various kinases, protein kinase C, PKA, protein tyrosine kinases, the ERK pathway, etc. is different. ROS also regulate the acquisition of hyperactivated motility and the acrosome reaction by spermatozoa. ROS action is probably mediated via the sulfhydryl/disulfide pair on sperm proteins. Redundancy, cross talk, and multiple systems acting in parallel point to an array of safeguards assuring the timely function of spermatozoa.     

Zinc induces mitogen-activated protein kinase activation mediated by reactive oxygen species in rice roots.

It is well known that zinc (Zn) is one of the micronutrients essential for normal growth and development of plants. However, the molecular mechanisms responsible for the regulation of plant growth by Zn are still not completely understood. The aim of this study was to investigate the signalling transduction pathways activated by Zn. We show that Zn elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot analysis, we suggest that Zn-activated 40- and 42-kDa MBP kinases are mitogen-activated protein kinases (MAPK). Pre-treatment of rice roots with reactive oxygen species (ROS) scavenger, sodium benzoate, was able to effectively prevent Zn-induced MAPK activation. However, phosphoinositide 3-kinase (PI-3K) inhibitor, LY294002, was unable to inhibit Zn-induced MAPK activation. These results suggest that the ROS may function in the Zn-triggered MAPK signalling pathway in rice roots.     

Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation.     

Nonthermal plasma induces head and neck cancer cell death: the potential involvement of mitogen-activated protein kinase-dependent mitochondrial reactive oxygen species

Nonthermal plasma (NTP) is generated by ionization of neutral gas molecules, which results in a mixture of energy particles including electrons and ions. Recent progress in the understanding of NTP has led to its application in the treatment of various diseases, including cancer. However, the molecular mechanisms of NTP-induced cell death are unclear. The purpose of this study was to evaluate the molecular mechanism of NTP in the induction of apoptosis of head and neck cancer (HNC) cells. The effects of NTP on apoptosis were investigated using MTT, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, Annexin V assays, and western blot analysis. The cells were examined for production of reactive oxygen species (ROS) using DCFCA or MitoSOX staining, intracellular signaling, and an animal model. NTP reduced HNC cell viability in a dose-dependent manner and induced apoptosis. NTP resulted in alteration of mitochondrial membrane potential and accumulation of intracellular ROS generated from the mitochondria in HNC cells. Blockade of ROS production by N-acetyl-L-cysteine inhibited NTP-induced apoptosis. NTP led to the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular-regulated kinase. Treatment with JNK and p38 inhibitors alleviated NTP-induced apoptosis via ROS generation. Taken together, these results show that NTP induced apoptosis of HNC cells by a mechanism involving MAPK-dependent mitochondrial ROS. NTP inhibited the growth of pre-established FaDu tumors in a nude mouse xenograft model and resulted in accumulation of intracellular ROS. In conclusion, NTP induced apoptosis in HNC cells through a novel mechanism involving MAPK-mediated mitochondrial ROS. These findings show the therapeutic potential of NTP in HNC.     

Protective role of reactive oxygen species in endotoxin-induced lung inflammation through modulation of IL-10 expression

Reactive oxygen species (ROS) generated by NADPH oxidase are generally known to be proinflammatory, and it seems to be counterintuitive that ROS play a critical role in regulating the resolution of the inflammatory response. However, we observed that deficiency of the p47(phox) component of NADPH oxidase in macrophages was associated with a paradoxical accentuation of inflammation in a whole animal model of noninfectious sepsis induced by endotoxin. We have confirmed this observation by interrogating four separate in vivo models that use complementary methodology including the use of p47(phox-/-) mice, p47(phox-/-) bone marrow chimera mice, adoptive transfer of macrophages from p47(phox-/-) mice, and an isolated perfused lung edema model that all point to a relationship between excessive acute inflammation and p47(phox) deficiency in macrophages. Mechanistic data indicate that ROS deficiency in both cells and mice results in decreased production of IL-10 in response to treatment with LPS, at least in part, through attenuation of the Akt-GSK3-β signal pathway and that it can be reversed by the administration of rIL-10. Our data support the innovative concept that generation of ROS is essential for counterregulation of acute lung inflammation.     

解偶联蛋白2对活性氧的抑制作用

线粒体在能量代谢和自由基代谢中占据十分重要的地位。电子传递过程中形成的活性氧(reactive oxygen species,ROS)履行着众多生理功能,但过多或持续存在的ROS可能与癌症、衰老、糖尿病、动脉硬化、局部缺血或再灌注损伤等的发生有关。解偶联蛋白2(uncoupling protein 2,UCP2)作为线粒体内膜质子转运家族中的一个新成员,通过解偶联作用能降低线粒体内膜电势,使活性氢产生减少。UCP2抑制ROS产生的作用日益受到关注。     

Adapter protein CRKII signaling is involved in the rat pancreatic acini response to reactive oxygen species

Recent studies demonstrate that reactive oxygen species (ROS) are important mediators of acute pancreatitis, whether induced experimentally or in necrotizing pancreatitis in humans; however, the cellular processes involved remain unclear. Adapter protein CrkII, plays a central role for convergence of cellular signals from different stimuli. Cholecystokinin (CCK), which induces pancreatitis, stimulates CrkII tyrosine phosphorylation and CrkII protein complexes, raising the possibility it can be important in the acinar cell responses to ROS. Therefore, our aim was to investigate whether CrkII signaling is involved in the biological response of rat pancreatic acini to H2O2 and the intracellular mediators implicated. Treatment of isolated rat pancreatic acini with H2O2 rapidly stimulates CrkII phosphorylation, measured as electrophoretic mobility shift and by using a phosphospecific antibody (pTyr221). Tyrosine kinase blocker B44 inhibits the higher phosphorylation state, demonstrating that it occurs mainly in tyrosine residues. H2O2-induced CrkII phosphorylation is time- and concentration-dependent, showing maximal effect with 3 mM H2O2 at 5 min. The intracellular pathways induced by H2O2 leading to CrkII tyrosine phosphorylation do not involve PKC, intracellular calcium, PI3-K or the actin cytoskeleton integrity. ROS generation clearly promotes the formation of protein complex CrkII-PYK2. In conclusion, ROS clearly affect the key adapter protein CrkII signaling by two ways: stimulation of CkII phosphorylation and a functional consequence: formation of CrkII-protein complexes. Because of its central role in activating more distal pathways, CrkII might likely play an important role in the ability of ROS to induce pancreatic cellular injury and pancreatitis.     

Suppressive effect of reactive oxygen species on CD40-induced B cell activation

Reactive oxygen species (ROS) produced by the innate immune system work as effectors to destroy pathogens and to control cellular responses. However, their role in the adaptive immune response remains unclear. Here we studied the effect of exogenous ROS on CD40-induced B cell activation. H2O2 treatment inhibited CD40-induced immunoglobulin production of B cells, DNA binding of NF-kappaB, IkappaBalpha degradation and IKK phosphorylation. On the other hand, H2O2 treatment did not induce obvious B cell death after 30 min of stimulation. Although the ligation of anti-CD40 antibody was not disturbed by H2O2, TRAF2 recruitment to CD40 was inhibited. These results suggest that exogenous ROS play a negative role in CD40 signaling during B cell activation.