Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

• Klíčová slova
• HL-60 cells, NADPH oxidase, NOX4, U-937 cells, macrophages, phorbol 12-myristate 13-acetate, protein-centered radicals,
• MeSH
• acetofenony farmakologie   MeSH
• barvení a značení MeSH
• buněčná diferenciace * účinky léků   MeSH
• elektronová paramagnetická rezonance MeSH
• HL-60 buňky MeSH
• hydroxylový radikál MeSH
• lidé MeSH
• monocyty cytologie   účinky léků   metabolismus   MeSH
• NADP metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny metabolismus   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• tvar buňky účinky léků   MeSH
• U937 buňky MeSH
• viabilita buněk účinky léků   MeSH
• volné radikály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetofenony MeSH
• acetovanillone MeSH Prohlížeč
• hydroxylový radikál MeSH
• NADP MeSH
• proteiny MeSH
• tetradekanoylforbolacetát MeSH
• volné radikály MeSH

Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2•-) and the hydroxyl (HO•) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2•- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2•- and HO•, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.

• Klíčová slova
• EPR, mass spectrometry, photosystem II, reactive oxygen species, tocopherol,
• MeSH
• alfa-tokoferol chemie   metabolismus   MeSH
• aminokyseliny chemie   metabolismus   MeSH
• Arabidopsis enzymologie   genetika   účinky záření   MeSH
• fotosyntéza fyziologie   účinky záření   MeSH
• fotosystém II (proteinový komplex) chemie   genetika   metabolismus   MeSH
• hydroxylový radikál chemie   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• intramolekulární transferasy chemie   genetika   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů, beta-řetězec MeSH
• kyslík chemie   metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• oxidace-redukce MeSH
• superoxidy chemie   metabolismus   MeSH
• světlo MeSH
• termodynamika MeSH
• Thermosynechococcus enzymologie   genetika   účinky záření   MeSH
• tylakoidy enzymologie   genetika   účinky záření   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• železo chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• alfa-tokoferol MeSH
• aminokyseliny MeSH
• fotosystém II (proteinový komplex) MeSH
• hydroxylový radikál MeSH
• intramolekulární transferasy MeSH
• kyslík MeSH
• superoxidy MeSH
• tocopherol cyclase MeSH Prohlížeč
• železo MeSH

Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.

• Klíčová slova
• aggregate, fragment, hydroxyl radical, photosystem II, protein, protein radical, reactive oxygen species, singlet oxygen,
• Publikační typ
• časopisecké články MeSH

Mechanical injury or wounding in plants can be attributed to abiotic or/and biotic causes. Subsequent defense responses are either local, i.e. within or in the close vicinity of affected tissue, or systemic, i.e. at distant plant organs. Stress stimuli activate a plethora of early and late reactions, from electric signals induced within seconds upon injury, oxidative burst within minutes, and slightly slower changes in hormone levels or expression of defense-related genes, to later cell wall reinforcement by polysaccharides deposition, or accumulation of proteinase inhibitors and hydrolytic enzymes. In the current study, we focused on the production of reactive oxygen species (ROS) in wounded Arabidopsis leaves. Based on fluorescence imaging, we provide experimental evidence that ROS [superoxide anion radical (O2 •-) and singlet oxygen (1O2)] are produced following wounding. As a consequence, oxidation of biomolecules is induced, predominantly of polyunsaturated fatty acid, which leads to the formation of reactive intermediate products and electronically excited species.

• Klíčová slova
• Arabidopsis, confocal microscopy, fluorescent probes, mechanical injury, wounding,
• Publikační typ
• časopisecké články MeSH

This article contains data related to the research article entitled, "Organic radical imaging in plants: Focus on protein radicals" (Kumar et al., 2018). The data presented herein focus on reactive oxygen species (ROS) and organic radical formed within photosynthetic tissues of Arabidopsis thaliana during high light stress and includes (1) Confocal laser scanning microscopic images using 3'-p-(hydroxyphenyl) fluorescein (HPF) as specific probe for the detection of hydroxyl radical (HO•); (2) Confocal laser scanning microscopic images using Singlet Oxygen Sensor Green (SOSG) as a specific probe for the detection of singlet oxygen (1O2) and; (3) Electron paramagnetic resonance (EPR) spectroscopy using spin traps for the detection of organic radical.

• Klíčová slova
• Hydroxyl radical, Organic radical, Photosystem II, Reactive oxygen species, Singlet oxygen,
• Publikační typ
• časopisecké články MeSH