Browsing by Subject "reactive oxygen species"

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  • Tran, Cuong (Helsingin yliopisto, 2018)
    Salicylic acid (SA) is a well-known phytohormone involved in pathogen defense, development and controlling the cellular redox balance. In response to stresses, Arabidopsis thaliana synthesizes SA in the chloroplasts mainly via the isochorismate (IC) pathway: IC synthase (ICS) uses chorismate to produce IC, which is in turn converted to SA. ICS1 is the rate limiting enzyme in SA biosynthesis. The ICS1 protein localizes in the chloroplasts and contains a chloroplast transit peptide sequence, which is the first 45 amino acids in the N-terminus. Under different light conditions, plants employ different stress defense strategies. In a previous study, wild type (WT) Arabidopsis thaliana ecotype Columbia-0 grown under different light conditions [short day (SD; 8h light/16h dark), long day (LD; 16h light/8h dark) and 12h light/12h dark] was exposed to ozone for 1 hour (350 ppb). In a phosphoproteomic approach to study signaling mechanisms, ICS1 has been found as an in vivo phosphoprotein in ozone-treated plants grown under SD condition by mass spectrometry (MS). To analyze the phosphorylation of ICS1 in vitro, WT and phospho-negative ICS1 proteins were produced recombinantly as GST-fusion proteins in E. coli and used as the substrates for targeted kinase assays. Phospho-negative ICS1 protein was generated by mutating the phospho-sites to alanines by site-directed mutagenesis. ICS1 could be phosphorylated either in the cytosol or in the chloroplasts. Therefore, different cytosolic kinases, which are involved in various stress signaling events, and a chloroplast protein kinase that functions as a dominant regulator of chloroplast processes, were selected as the kinases to be tested. In this study, GST-ICS1 could be successfully phosphorylated in vitro. To a similar extent, GST-phospho-negative ICS1 was phosphorylated, indicating that ICS1 was phosphorylated in vitro at sites that were different from the ones found by MS from plant material. In order to study the identified phospho-sites in vivo, transgenic Arabidopsis carrying WT, phospho-positive, and phospho-negative ICS1 proteins were generated. Transgenic plants were confirmed by genotyping. Western blotting was carried out to evaluate ECFP-WT ICS1 protein expression. However, none of the transgenic plants could be confirmed to carry ECFP-WT ICS1. Additionally, an antibody against AtICS1 produced by the company Agrisera was tested for its specificity against the endogenous ICS1 protein. While the antibody could detect recombinant GST-ICS1 protein, ICS1 could not be detected from a total protein extract.
  • Voothuluru, Priya; Mäkelä, Pirjo; Zhu, Jinming; Yamaguchi, Mineo; Oliver, Melvin J.; Simmonds, John; Sharp, Robert (2020)
    Reactive oxygen species (ROS) can act as signaling molecules involved in the acclimation of plants to various abiotic and biotic stresses. However, it is not clear how the generalized increases in ROS and downstream signaling events that occur in response to stressful conditions are coordinated to modify plant growth and development. Previous studies of maize (Zea mays L.) primary root growth under water deficit stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex, and that the rate of cell production is also decreased. It was observed that apoplastic ROS, particularly hydrogen peroxide (H2O2), increased specifically in the apical region of the growth zone under water stress, resulting at least partly from increased oxalate oxidase activity in this region. To assess the function of the increase in apoplastic H2O2 in root growth regulation, transgenic maize lines constitutively expressing a wheat oxalate oxidase were utilized in combination with kinematic growth analysis to examine effects of increased apoplastic H2O2 on the spatial pattern of cell elongation and on cell production in well-watered and water-stressed roots. Effects of H2O2 removal (via scavenger pretreatment) specifically from the apical region of the growth zone were also assessed. The results show that apoplastic H2O2 positively modulates cell production and root elongation under well-watered conditions, whereas the normal increase in apoplastic H2O2 in water-stressed roots is causally related to down-regulation of cell production and root growth inhibition. The effects on cell production were accompanied by changes in spatial profiles of cell elongation and in the length of the growth zone. However, effects on overall cell elongation, as reflected in final cell lengths, were minor. These results reveal a fundamental role of apoplastic H2O2 in regulating cell production and root elongation in both well-watered and water-stressed conditions.
  • Mirzalieva, Oygul; Jeon, Shinhye; Damri, Kevin; Hartke, Ruth; Drwesh, Layla; Demishtein-Zohary, Keren; Azem, Abdussalam; Dunn, Cory D.; Peixoto, Pablo M. (2019)
    The TIM23 complex is a hub for translocation of preproteins into or across the mitochondrial inner membrane. This dual sorting mechanism is currently being investigated, and in yeast appears to be regulated by a recently discovered subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This result and other findings suggested that Mgr2p controls the lateral release of inner membrane proteins harboring a stop-transfer signal that follows an N-terminal amino acid signal. However, the mechanism of lateral release is unknown. Here, we used patch clamp electrophysiology to investigate the role of Mgr2p on the channel activity of TIM23. Deletion of Mgr2p decreased normal channel frequency and increased occurrence of a residual TIM23 activity. The residual channel lacked gating transitions but remained sensitive to synthetic import signal peptides. Similarly, a G145L mutation in Tim23p displaced Mgr2p from the import complex leading to gating impairment. These results suggest that Mgr2p regulates the gating behavior of the TIM23 channel.
  • Shapiguzov, Alexey; Nikkanen, Lauri; Fitzpatrick, Duncan; Vainonen, Julia P.; Gossens, Richard; Alseekh, Saleh; Aarabi, Fayezeh; Tiwari, Arjun; Blokhina, Olga; Panzarova, Klara; Benedikty, Zuzana; Tyystjärvi, Esa; Fernie, Alisdair R.; Trtilek, Martin; Aro, Eva-Mari; Rintamäki, Eevi; Kangasjarvi, Jaakko (2020)
    The Arabidopsis mutant rcd1 is tolerant to methyl viologen (MV). MV enhances the Mehler reaction, i.e. electron transfer from Photosystem I (PSI) to O-2, generating reactive oxygen species (ROS) in the chloroplast. To study the MV tolerance of rcd1, we first addressed chloroplast thiol redox enzymes potentially implicated in ROS scavenging. NADPH-thioredoxin oxidoreductase type C (NTRC) was more reduced in rcd1. NTRC contributed to the photosynthetic and metabolic phenotypes of rcd1, but did not determine its MV tolerance. We next tested rcd1 for alterations in the Mehler reaction. In rcd1, but not in the wild type, the PSI-to-MV electron transfer was abolished by hypoxic atmosphere. A characteristic feature of rcd1 is constitutive expression of mitochondrial dysfunction stimulon (MDS) genes that affect mitochondrial respiration. Similarly to rcd1, in other MDS-overexpressing plants hypoxia also inhibited the PSI-to-MV electron transfer. One possible explanation is that the MDS gene products may affect the Mehler reaction by altering the availability of O-2. In green tissues, this putative effect is masked by photosynthetic O-2 evolution. However, O-2 evolution was rapidly suppressed in MV-treated plants. Transcriptomic meta-analysis indicated that MDS gene expression is linked to hypoxic response not only under MV, but also in standard growth conditions. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.
  • Elbadri, Khalil (Helsingin yliopisto, 2020)
    An increased attention has been drawn towards porous silicon (PSi) based materials for biomedical applications, due to their promising features demonstrated through several scientific studies. Here, we further investigated the biological responses of PSi nanoparticles (NPs) with different surface chemistries, including immunomodulatory effects, inflammation mitigation and biocompatibility. In this collaborative study, the PSi NPs were investigated both in vitro and in vivo, using different molecular biology and biochemistry techniques, e.g., qPCR, ELISA, cell sorting and cell viability assays. Our results showed the capabilities of these PSi NPs to relieve the inflammatory conditions, whereas significant decrease was recorded of pro-inflammatory cytokines: TNF-α, IL-1β and IL-6. Likewise, these PSi NPs revealed a considerable consumption aptitude of pro-inflammatory reactive oxygen species molecules. Administrating PSi NPs in an acute liver inflammation (ALI) model, showed no conspicuous influence on cellular viability. Thus, the outcome of this study demonstrates the potential biocompatibility of PSi nanomaterials, in addition to their outstanding features as potential candidates for further incorporating in ALI applications.
  • Isakova, Elena P.; Matushkina, Irina N.; Popova, Tatyana N.; Dergacheva, Darya; Gessler, Natalya N.; Klein, Olga; Semenikhina, Anastasya; Deryabina, Yulia; La Porta, Nicola; Saris, Nils-Eric L. (2020)
    In this study, we evaluated the metabolic profile of the aerobic microorganism of Endomyces magnusii with a complete respiration chain and well-developed mitochondria system during long-lasting cultivation. The yeast was grown in batches using glycerol and glucose as the sole carbon source for a week. The profile included the cellular biological and chemical parameters, which determined the redox status of the yeast cells. We studied the activities of the antioxidant systems (catalases and superoxide dismutases), glutathione system enzymes (glutathione peroxidase and reductase), aconitase, as well as the main enzymes maintaining NADPH levels in the cells (glucose-6-phosphate dehydrogenase and NADP(+)-isocitrate dehydrogenase) during aging of Endomyces magnusii on two kinds of substrates. We also investigated the dynamics of change in oxidized and reduced glutathione, conjugated dienes, and reactive oxidative species in the cells at different growth stages, including the deep stationary stages. Our results revealed a similar trend in the changes in the activity of all the enzymes tested, which increased 2-4-fold upon aging. The yeast cytosol had a very high reduced glutathione content, 22 times than that of Saccharomyces cerevisiae, and remained unchanged during growth, whereas there was a 7.5-fold increase in the reduced glutathione-to-oxidized glutathione ratio. The much higher level of reactive oxidative species was observed in the cells in the late and deep stationary phases, especially in the cells using glycerol. Cell aging of the culture grown on glycerol, which promotes active oxidative phosphorylation in the mitochondria, facilitated the functioning of powerful antioxidant systems (catalases, superoxide dismutases, and glutathione system enzymes) induced by reactive oxidative species. Moreover, it stimulated NADPH synthesis, regulating the cytosolic reduced glutathione level, which in turn determines the redox potential of the yeast cell during the early aging process.
  • Huttunen, Kati; Wlodarczyk, Anna J.; Tirkkonen, Jenni; Mikkonen, Santtu; Täubel, Martin; Krop, Esmeralda; Jacobs, Jose; Pekkanen, Juha; Heederik, Dick; Zock, Jan-Paul; Hyvärinen, Anne; Hirvonen, Maija-Riitta; Adams, Rachel; Jones, Tim; Zimmermann, Ralf; BeruBe, Kelly (2019)
    Exposure to moisture-damaged indoor environments is associated with adverse respiratory health effects, but responsible factors remain unidentified. In order to explore possible mechanisms behind these effects, the oxidative capacity and hemolytic activity of settled dust samples (n = 25) collected from moisture-damaged and non-damaged schools in Spain, the Netherlands, and Finland were evaluated and matched against the microbial content of the sample. Oxidative capacity was determined with plasmid scission assay and hemolytic activity by assessing the damage to isolated human red blood cells. The microbial content of the samples was measured with quantitative PCR assays for selected microbial groups and by analyzing the cell wall markers ergosterol, muramic acid, endotoxins, and glucans. The moisture observations in the schools were associated with some of the microbial components in the dust, and microbial determinants grouped together increased the oxidative capacity. Oxidative capacity was also affected by particle concentration and country of origin. Two out of 14 studied dust samples from moisture-damaged schools demonstrated some hemolytic activity. The results indicate that the microbial component connected with moisture damage is associated with increased oxidative stress and that hemolysis should be studied further as one possible mechanism contributing to the adverse health effects of moisture-damaged buildings.
  • Szibor, Marten; Schreckenberg, Rolf; Gizatullina, Zemfira; Dufour, Eric; Wiesnet, Marion; Dhandapani, Praveen K.; Debska-Vielhaber, Grazyna; Heidler, Juliana; Wittig, Ilka; Nyman, Tuula A.; Gärtner, Ulrich; Hall, Andrew R.; Pell, Victoria; Viscomi, Carlo; Krieg, Thomas; Murphy, Michael P.; Braun, Thomas; Gellerich, Frank N.; Schlüter, Klaus-Dieter; Jacobs, Howard T. (2020)
    Abstract Cardiac ischaemia-reperfusion (I/R) injury has been attributed to stress signals arising from an impaired mitochondrial electron transport chain (ETC), which include redox imbalance, metabolic stalling and excessive production of reactive oxygen species (ROS). The alternative oxidase (AOX) is a respiratory enzyme, absent in mammals, that accepts electrons from a reduced quinone pool to reduce oxygen to water, thereby restoring electron flux when impaired and, in the process, blunting ROS production. Hence, AOX represents a natural rescue mechanism from respiratory stress. This study aimed to determine how respiratory restoration through xenotopically expressed AOX affects the re-perfused post-ischaemic mouse heart. As expected, AOX supports ETC function and attenuates the ROS load in post-anoxic heart mitochondria. However, post-ischaemic cardiac remodelling over 3 and 9 weeks was not improved. AOX blunted transcript levels of factors known to be up-regulated upon I/R such as the atrial natriuretic peptide (Anp) whilst expression of pro-fibrotic and pro-apoptotic transcripts were increased. Ex vivo analysis revealed contractile failure at nine but not 3 weeks after ischaemia whilst label-free quantitative proteomics identified an increase in proteins promoting adverse extracellular matrix remodelling. Together, this indicates an essential role for ETC-derived signals during cardiac adaptive remodelling and identified ROS as a possible effector.
  • Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T. (2014)