- Email: [email protected]
ROS is the boss
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A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
3
Department of Pathology, University of Vermont College of Medicine, Burlington, VT, USA 4 Department of Natural Sciences, LaGuardia Community College, City University of New York, Queens, NY, USA 5 Department of Biological Sciences and Geology, Queensborough Community College, City University of New York, Bayside, NY, USA
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Formation of reactive oxygen species by the irradiation of cold atmospheric pressure plasma to water Kazunori Anzai, Tamami Aoki, Satoko Koshimizu, Reina Takaya, Kazunori Tsuchida, Tokuko Takajo
Hyperoxia-induced acute lung injury (HALI) during mechanical ventilation can lead to excessive proinflammatory responses, endothelial and epithelial cell damage, and alveolar edema. We and others have demonstrated that NF-kB activation has a protective effect on cultured lung epithelial cells. The present study investigates the effects of NF-kB activation on ALI using CC10-I- κBαSR transgenic mice that are deficient in the NF-kB activation in airway epithelial cells. These mice showed significantly more severe lung injury than wild type mice after being exposed to 95% oxygen for 3 d, characterized by higher wet/dry weight ratios of lung tissue and increased total protein content in bronchoalveolar lavage fluids (BALF). Severe lung injury was associated with elevated neutrophil infiltration and extracellular HMGB1 accumulation in BALF. In addition, prolonged exposure of cultured human bronchial epithelial (HBE) cells to hyperoxia induced the release of HMGB1. Higher levels of extracellular HMGB1 were observed in HBE cells cultured with BAY 11–7082, an inhibitor of NF- kB activation. These results indicate that the NF-kB signaling pathway in airway epithelial cells plays an important protective role against HMGB1mediated hyperoxic inflammatory lung injury.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.210
Nihon Pharmaceutical University, Ina, Japan
We examined the formation of reactive oxygen species by the irradiation of helium-based cold atmospheric pressure plasma to water using ESR and colorimetric analysis. Cold atmospheric plasma was generated with a generator PN-110TPG (NU global, Japan). The plasma was irradiated to aqueous samples changing the irradiation time and the irradiation distance between water surface and the top of the plasma-generating tip. 1) Formation of reactive oxygen species was confirmed by the decrease of the ESR signal intensity of the solution of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), a stable free radical, by the irradiation. 2) Formation of hydroxyl radical was confirmed by the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap reagent. 3) Formation of hydrogen peroxide was confirmed by the colorimetric method using a peroxidase reaction. In these three methods, the change (decrease or increase) in the intensity was observed as irradiation-time dependent fashion. On the other hand, complex dependency on the irradiation distance was observed. These findings suggest that the reactive oxygen species observed are largely derived from the plasma itself rather than hydrolysis or excitation of water molecules.
E-mail address: [email protected] P-64
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.212
AMPK-dependent and independent modulation of thioredoxin interacting protein
P-66
A.L. Dafre 1, A.E. Schmitz 1, P. Maher 2
ROS is the boss
1
Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil 2 Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA
Thioredoxin interacting protein (Txnip) is known for thioredoxin (Trx) inhibition, but Txnip also present Trx-independent functions, such as insulin resistance and inflammasome activation. Little is known about the specific pathways controlling Txnip expression. The present study aims to investigate mechanisms controlling Txnip and Trx expression in HT22. Several kinaseinhibitors were used to study Txnip modulation. Mouse embryonic fibroblasts cells (MEFs) deficient in AMPK (AMPK-KO) were also used to test the participation of AMPK. Methylglyoxal (0.5 mM) produced a biphasic change in Txnip expression, firstly (o1 h) Txnip decreased in a concentration dependent manner, followed by a peak at 2–3 h, returning to basal levels at 18 h. Data reinforce the idea that this increase (3 h) in Txnip expression is mediated by AMPK, but not the fast decrease (1 h). AMPK-KO MEFs present the fast decrease in Txnip when treated with MGO, suggesting this kinase did not participate. Conversely, the AMPK activator (phenformin) depleted and the inhibitor (compound C) induced Txnip in control MEFs, but not in AMPK-KO MEFs. This clearly shows the role of AMPK in controlling Txnip. Methylglyoxal rapidly depletes Txnip by an AMPK-independent mechanism, while later induction seems to be dependent on AMPK.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.211
Jonas Hahn, Deborah Kienhöfer, Julia Stoof, Janka Zsofia Csepregi, Christiane Reinwald, Chrisitan Maueröder, Vilma Urbonaviciute, Luis E Munoz, Malin Hultqvist, Malgorzata J Podolska, Mona H Biermann, Moritz Leppkes, Martin Herrmann, Thomas Harrer, Attila Mocsai, Rikard Holmdahl, Georg Schett, Markus Hoffmann Department of Medicine 3 UK Erlangen, Department of Medicine 1 UK Erlangen, Department of Physiology Semmelweis University School of Medicine, Section of Medical Inflammation Research Department of Medical Biochemistry and Biophysics Erlangen
The production of reactive oxygen species (ROS) via the oxidative burst has been connected with promotion of inflammation and tissue damage, but in recent years has been implicated in regulation and resolution of inflammation. The aim of this project was to elucidate the impact of the oxidative burst on the development of lupus-like autoimmunity. Lupus was induced by with pristane oil. Ex vivo phagocytosis assays were deployed to assess the uptake of cell debris in ROS deficient mice. The formation of neutrophil extracellular traps (NETs) was analysed by immune fluorescence microscopy. ROS activators were injected into mice to investigate the possible beneficial clinical effects on lupus pathology. Determine the effects neutrophil serine proteases have on the degradation of inflammatory mediators The absence of ROS gives rise to dramatically exacerbated lupus. Aberrant phagocytosis in ROS-deficient animals leading to production of inflammatory
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
mediators, accompanied by diminished pristane-induced but higher spontaneous formation of NETs could be responsible for this phenotype. Treatment with NOX2 agonists ameliorated the clinical course in mice, while application of a ROS scavenger worsened disease outcome.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.213
S65
for the detection of sulfenic acid modified proteins. We found that CLOOH treatment resulted in oxidation of iPLA2γ, as evaluated from the different electrophoretic mobilities of the oxidized and reduced forms. In addition, measurements of recombinant iPLA2γ activity showed that the Vmax values obtained with CLOOH as the substrate were identical to the values obtained with H2O2-activated iPLA2γ and cardiolipin as the substrate. These results indicate that lipid hydroperoxides participate in the regulation of iPLA2γ by reversible oxidation of accessible cysteine residues and identify CLOOHs as both substrates and redox activators of the enzyme.
E-mail address: [email protected] P-67
Diverse strategies in P700 oxidation system for suppressing ROS production were acquired during the evolution of photosynthetic organisms Chikahiro Miyake
Acknowledgements Grant support GA16–04788S and LTAUSA17174.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.215
P-69
Faculty of Agriculture, Kobe University, Japan
We developed the method to produce ROS intrinsically in photosynthetic organisms: repetitive short-pulse (rSP) illumination. rSP illumination fulfills electrons in photosynthetic electron transport (PET) system, where O2 is reduced to superoxide radical and O2 is also excited to singlet O2 at photosystem I (PSI) of PET system. These ROS oxidatively damage PSI in angiosperms. By contrast, mosses/liverworts, ferns, and gymnosperms do not suffer from PSI damages, where a reaction center chlorophyll in PSI, P700, was oxidized to the maximum in the presence of O2, on the other hand P700 was reduced in angiosperm leaves. The reduced P700 shows electron accumulation in PSI to stimulate ROS production. These organisms, other than angiosperms, have flavodiiron (FLV) proteins, which catalyze O2 reduction to H2O using ferredoxin/NADPH. The mutant of liverwort, Marchantia polymorpha, deficient in FLV, could not oxidize P700 and suffered from PSI oxidative damage by rSP-illumination. That is, these photosynthetic organisms rapidly oxidize P700 to suppress ROS production. Recently, we found that diatoms, red algae, brown algae oxidize P700 even in the absence of O2 and did not suffer from any oxidative damages of PSI. These photosynthetic organisms might have a new mechanism to oxidize P700.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.214
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Cardiolipin hydroperoxides are both substrates and redox activators of phospholipase iPLA2γ Jabůrek Martin, Holendová Blanka, Průchová Pavla, Ježek Petr Department of Mitochondrial Physiology, Institute of Physiology CAS, Prague, Czech Republic
The mitochondrial calcium independent phospholipase 2γ (iPLA2γ) catalyzes the hydrolysis of membrane glycerophospholipids with subsequent release of free fatty acids and lysophospholipids. The activity of iPLA2γ is upregulated by H2O in isolated mitochondria and reconstituted system, suggesting activation by redox modification of accessible cysteine residues. Here we tested the hypothesis that iPLA2γ is activated also by lipid hydroperoxides. We treated purified recombinant iPLA2γ with cardiolipin hydroperoxides (CLOOHs) or dithiothreitol, together with approaches
Modification of mitochondrial membrane potential during prolonged cultivation of rat C6 glioma cells in the presence of SWCNT-DNA complexes Lena Golubewa 1, Nikita Vasilieu 1, Tatsiana Kulahava 1, Mikhail Shuba 2, Olesya Paddubskaya 2 1 Department of Biophysics, Physics Faculty, Belarusian State University, Minsk, Belarus 2 Research Institute for Nuclear Problems, Belarusian State University, Minsk, Belarus
Distribution of single-walled carbon nanotubes (SWCNT) in C6 glioma cells was investigated by means of confocal Raman microscopy. The analysis was done using short-length (100–250 nm) SWCNT previously dispersed in DNA aqueous solutions. Cytotoxic concentrations of shortlength SWCNT for rat C6 glioma cells were established. SWCNT-DNA in nontoxic concentrations were found to penetrate and accumulate in cells after 6–8 h of cells cultivation in the presence of the complexes. SWCNTDNA complexes distribution was uneven and maintained in cells during 5 passages. Simultaneous combination of confocal Raman microscopy and fluorescent microscopy using JC-1 or MitoSOX made it possible to establish, that the complexes are located predominantly near actively functioning mitochondria, causing their shape modification and slight mitochondrial membrane potential increase, electron transfer and superoxide production in complexes I and III modification. Prolonged C6 cells cultivation in the presence of SWCNT-DNA complexes led to the decrease in cells proliferative activity during the first 3 passages followed by its further recovery, causing, therefore, activation of cell stress adaptation mechanisms.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.216
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
3
Department of Pathology, University of Vermont College of Medicine, Burlington, VT, USA 4 Department of Natural Sciences, LaGuardia Community College, City University of New York, Queens, NY, USA 5 Department of Biological Sciences and Geology, Queensborough Community College, City University of New York, Bayside, NY, USA
P-65
Formation of reactive oxygen species by the irradiation of cold atmospheric pressure plasma to water Kazunori Anzai, Tamami Aoki, Satoko Koshimizu, Reina Takaya, Kazunori Tsuchida, Tokuko Takajo
Hyperoxia-induced acute lung injury (HALI) during mechanical ventilation can lead to excessive proinflammatory responses, endothelial and epithelial cell damage, and alveolar edema. We and others have demonstrated that NF-kB activation has a protective effect on cultured lung epithelial cells. The present study investigates the effects of NF-kB activation on ALI using CC10-I- κBαSR transgenic mice that are deficient in the NF-kB activation in airway epithelial cells. These mice showed significantly more severe lung injury than wild type mice after being exposed to 95% oxygen for 3 d, characterized by higher wet/dry weight ratios of lung tissue and increased total protein content in bronchoalveolar lavage fluids (BALF). Severe lung injury was associated with elevated neutrophil infiltration and extracellular HMGB1 accumulation in BALF. In addition, prolonged exposure of cultured human bronchial epithelial (HBE) cells to hyperoxia induced the release of HMGB1. Higher levels of extracellular HMGB1 were observed in HBE cells cultured with BAY 11–7082, an inhibitor of NF- kB activation. These results indicate that the NF-kB signaling pathway in airway epithelial cells plays an important protective role against HMGB1mediated hyperoxic inflammatory lung injury.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.210
Nihon Pharmaceutical University, Ina, Japan
We examined the formation of reactive oxygen species by the irradiation of helium-based cold atmospheric pressure plasma to water using ESR and colorimetric analysis. Cold atmospheric plasma was generated with a generator PN-110TPG (NU global, Japan). The plasma was irradiated to aqueous samples changing the irradiation time and the irradiation distance between water surface and the top of the plasma-generating tip. 1) Formation of reactive oxygen species was confirmed by the decrease of the ESR signal intensity of the solution of 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO), a stable free radical, by the irradiation. 2) Formation of hydroxyl radical was confirmed by the ESR spin-trapping technique using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin-trap reagent. 3) Formation of hydrogen peroxide was confirmed by the colorimetric method using a peroxidase reaction. In these three methods, the change (decrease or increase) in the intensity was observed as irradiation-time dependent fashion. On the other hand, complex dependency on the irradiation distance was observed. These findings suggest that the reactive oxygen species observed are largely derived from the plasma itself rather than hydrolysis or excitation of water molecules.
E-mail address: [email protected] P-64
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.212
AMPK-dependent and independent modulation of thioredoxin interacting protein
P-66
A.L. Dafre 1, A.E. Schmitz 1, P. Maher 2
ROS is the boss
1
Department of Biochemistry, Federal University of Santa Catarina, Florianópolis, SC, Brazil 2 Cellular Neurobiology Laboratory, Salk Institute for Biological Studies, La Jolla, CA, USA
Thioredoxin interacting protein (Txnip) is known for thioredoxin (Trx) inhibition, but Txnip also present Trx-independent functions, such as insulin resistance and inflammasome activation. Little is known about the specific pathways controlling Txnip expression. The present study aims to investigate mechanisms controlling Txnip and Trx expression in HT22. Several kinaseinhibitors were used to study Txnip modulation. Mouse embryonic fibroblasts cells (MEFs) deficient in AMPK (AMPK-KO) were also used to test the participation of AMPK. Methylglyoxal (0.5 mM) produced a biphasic change in Txnip expression, firstly (o1 h) Txnip decreased in a concentration dependent manner, followed by a peak at 2–3 h, returning to basal levels at 18 h. Data reinforce the idea that this increase (3 h) in Txnip expression is mediated by AMPK, but not the fast decrease (1 h). AMPK-KO MEFs present the fast decrease in Txnip when treated with MGO, suggesting this kinase did not participate. Conversely, the AMPK activator (phenformin) depleted and the inhibitor (compound C) induced Txnip in control MEFs, but not in AMPK-KO MEFs. This clearly shows the role of AMPK in controlling Txnip. Methylglyoxal rapidly depletes Txnip by an AMPK-independent mechanism, while later induction seems to be dependent on AMPK.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.211
Jonas Hahn, Deborah Kienhöfer, Julia Stoof, Janka Zsofia Csepregi, Christiane Reinwald, Chrisitan Maueröder, Vilma Urbonaviciute, Luis E Munoz, Malin Hultqvist, Malgorzata J Podolska, Mona H Biermann, Moritz Leppkes, Martin Herrmann, Thomas Harrer, Attila Mocsai, Rikard Holmdahl, Georg Schett, Markus Hoffmann Department of Medicine 3 UK Erlangen, Department of Medicine 1 UK Erlangen, Department of Physiology Semmelweis University School of Medicine, Section of Medical Inflammation Research Department of Medical Biochemistry and Biophysics Erlangen
The production of reactive oxygen species (ROS) via the oxidative burst has been connected with promotion of inflammation and tissue damage, but in recent years has been implicated in regulation and resolution of inflammation. The aim of this project was to elucidate the impact of the oxidative burst on the development of lupus-like autoimmunity. Lupus was induced by with pristane oil. Ex vivo phagocytosis assays were deployed to assess the uptake of cell debris in ROS deficient mice. The formation of neutrophil extracellular traps (NETs) was analysed by immune fluorescence microscopy. ROS activators were injected into mice to investigate the possible beneficial clinical effects on lupus pathology. Determine the effects neutrophil serine proteases have on the degradation of inflammatory mediators The absence of ROS gives rise to dramatically exacerbated lupus. Aberrant phagocytosis in ROS-deficient animals leading to production of inflammatory
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
mediators, accompanied by diminished pristane-induced but higher spontaneous formation of NETs could be responsible for this phenotype. Treatment with NOX2 agonists ameliorated the clinical course in mice, while application of a ROS scavenger worsened disease outcome.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.213
S65
for the detection of sulfenic acid modified proteins. We found that CLOOH treatment resulted in oxidation of iPLA2γ, as evaluated from the different electrophoretic mobilities of the oxidized and reduced forms. In addition, measurements of recombinant iPLA2γ activity showed that the Vmax values obtained with CLOOH as the substrate were identical to the values obtained with H2O2-activated iPLA2γ and cardiolipin as the substrate. These results indicate that lipid hydroperoxides participate in the regulation of iPLA2γ by reversible oxidation of accessible cysteine residues and identify CLOOHs as both substrates and redox activators of the enzyme.
E-mail address: [email protected] P-67
Diverse strategies in P700 oxidation system for suppressing ROS production were acquired during the evolution of photosynthetic organisms Chikahiro Miyake
Acknowledgements Grant support GA16–04788S and LTAUSA17174.
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.215
P-69
Faculty of Agriculture, Kobe University, Japan
We developed the method to produce ROS intrinsically in photosynthetic organisms: repetitive short-pulse (rSP) illumination. rSP illumination fulfills electrons in photosynthetic electron transport (PET) system, where O2 is reduced to superoxide radical and O2 is also excited to singlet O2 at photosystem I (PSI) of PET system. These ROS oxidatively damage PSI in angiosperms. By contrast, mosses/liverworts, ferns, and gymnosperms do not suffer from PSI damages, where a reaction center chlorophyll in PSI, P700, was oxidized to the maximum in the presence of O2, on the other hand P700 was reduced in angiosperm leaves. The reduced P700 shows electron accumulation in PSI to stimulate ROS production. These organisms, other than angiosperms, have flavodiiron (FLV) proteins, which catalyze O2 reduction to H2O using ferredoxin/NADPH. The mutant of liverwort, Marchantia polymorpha, deficient in FLV, could not oxidize P700 and suffered from PSI oxidative damage by rSP-illumination. That is, these photosynthetic organisms rapidly oxidize P700 to suppress ROS production. Recently, we found that diatoms, red algae, brown algae oxidize P700 even in the absence of O2 and did not suffer from any oxidative damages of PSI. These photosynthetic organisms might have a new mechanism to oxidize P700.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.214
P-68
Cardiolipin hydroperoxides are both substrates and redox activators of phospholipase iPLA2γ Jabůrek Martin, Holendová Blanka, Průchová Pavla, Ježek Petr Department of Mitochondrial Physiology, Institute of Physiology CAS, Prague, Czech Republic
The mitochondrial calcium independent phospholipase 2γ (iPLA2γ) catalyzes the hydrolysis of membrane glycerophospholipids with subsequent release of free fatty acids and lysophospholipids. The activity of iPLA2γ is upregulated by H2O in isolated mitochondria and reconstituted system, suggesting activation by redox modification of accessible cysteine residues. Here we tested the hypothesis that iPLA2γ is activated also by lipid hydroperoxides. We treated purified recombinant iPLA2γ with cardiolipin hydroperoxides (CLOOHs) or dithiothreitol, together with approaches
Modification of mitochondrial membrane potential during prolonged cultivation of rat C6 glioma cells in the presence of SWCNT-DNA complexes Lena Golubewa 1, Nikita Vasilieu 1, Tatsiana Kulahava 1, Mikhail Shuba 2, Olesya Paddubskaya 2 1 Department of Biophysics, Physics Faculty, Belarusian State University, Minsk, Belarus 2 Research Institute for Nuclear Problems, Belarusian State University, Minsk, Belarus
Distribution of single-walled carbon nanotubes (SWCNT) in C6 glioma cells was investigated by means of confocal Raman microscopy. The analysis was done using short-length (100–250 nm) SWCNT previously dispersed in DNA aqueous solutions. Cytotoxic concentrations of shortlength SWCNT for rat C6 glioma cells were established. SWCNT-DNA in nontoxic concentrations were found to penetrate and accumulate in cells after 6–8 h of cells cultivation in the presence of the complexes. SWCNTDNA complexes distribution was uneven and maintained in cells during 5 passages. Simultaneous combination of confocal Raman microscopy and fluorescent microscopy using JC-1 or MitoSOX made it possible to establish, that the complexes are located predominantly near actively functioning mitochondria, causing their shape modification and slight mitochondrial membrane potential increase, electron transfer and superoxide production in complexes I and III modification. Prolonged C6 cells cultivation in the presence of SWCNT-DNA complexes led to the decrease in cells proliferative activity during the first 3 passages followed by its further recovery, causing, therefore, activation of cell stress adaptation mechanisms.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.216