Reactive oxygen species play a key role in cellular homeostasis and redox signaling at physiological levels, where excessive production affects the function and integrity of macromolecules, specifically proteins. Therefore, it is important to define radical-mediated proteotoxic stress in macrophages and identify target protein to prevent tissue dysfunction. A well employed, THP-1 cell line was utilized as in vitro model to study immune response and herein we employ immuno-spin trapping technique to investigate radical-mediated protein oxidation in macrophages. Hydroxyl radical formation along macrophage differentiation was confirmed by electron paramagnetic resonance along with confocal laser scanning microscopy using hydroxyphenyl fluorescein. Lipid peroxidation product, malondialdehyde, generated under experimental conditions as detected using swallow-tailed perylene derivative fluorescence observed by confocal laser scanning microscopy and high-performance liquid chromatography, respectively. The results obtained from this study warrant further corroboration and study of specific proteins involved in the macrophage activation and their role in inflammations.

• Klíčová slova
• All-trans retinoic acid, Lipid peroxidation, Lipopolysaccharide, Macrophage, Malondialdehyde, Phorbol 12-myristate 13-acetate, Protein oxidation, Reactive oxygen species,
• MeSH
• elektronová paramagnetická rezonance metody   MeSH
• makrofágy * metabolismus   MeSH
• proteiny * chemie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• spin trapping metody   MeSH
• volné radikály analýza   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny * MeSH
• reaktivní formy kyslíku MeSH
• volné radikály MeSH

Singlet oxygen (1O2) is formed by triplet-triplet energy transfer from triplet chlorophyll to O2 via Type II photosensitization reaction in photosystem II (PSII). Formation of triplet chlorophyll is associated with the change in spin state of the excited electron and recombination of triplet radical pair in the PSII antenna complex and reaction center, respectively. Here, we have provided evidence for the formation of 1O2 by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex. Protein hydroperoxide is formed by protein oxidation initiated by highly oxidizing chlorophyll cation radical and hydroxyl radical formed by Type I photosensitization reaction. Under highly oxidizing conditions, protein hydroperoxide is oxidized to protein peroxyl radical which either cyclizes to dioxetane or recombines with another protein peroxyl radical to tetroxide. These highly unstable intermediates decompose to triplet carbonyls which transfer energy to O2 forming 1O2. Data presented in this study show for the first time that 1O2 is formed by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex.

• MeSH
• chlorofyl metabolismus   MeSH
• elektronová paramagnetická rezonance metody   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• kyslík metabolismus   MeSH
• oxidace-redukce MeSH
• peroxid vodíku metabolismus   MeSH
• peroxidy metabolismus   MeSH
• přenos energie fyziologie   MeSH
• singletový kyslík metabolismus   MeSH
• světlo MeSH
• světlosběrné proteinové komplexy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl MeSH
• fotosystém II (proteinový komplex) MeSH
• kyslík MeSH
• perhydroxyl radical MeSH Prohlížeč
• peroxid vodíku MeSH
• peroxidy MeSH
• singletový kyslík MeSH
• světlosběrné proteinové komplexy MeSH

Gamma- and neutron doses in an experimental reactor were measured using alanine/electron spin resonance (ESR) spectrometry. The absorbed dose in alanine was decomposed into contributions caused by gamma and neutron radiation using neutron kerma factors. To overcome a low sensitivity of the alanine/ESR response to thermal neutrons, a novel method has been proposed for the assessment of a thermal neutron flux using the (14)N(n,p) (14)C reaction on nitrogen present in alanine and subsequent measurement of (14)C by liquid scintillation counting (LSC).

• Klíčová slova
• (14)C, Alanine/ESR dosimeter, LSC, Reactor radiation, Simultaneous gamma- and neutron dose assessment,
• MeSH
• alanin chemie   účinky záření   MeSH
• elektronová paramagnetická rezonance metody   statistika a číselné údaje   MeSH
• jaderné elektrárny normy   MeSH
• neutrony * MeSH
• radioizotopy uhlíku analýza   MeSH
• radiometrie metody   statistika a číselné údaje   MeSH
• scintilace - počítání metody   statistika a číselné údaje   MeSH
• vztah dávky záření a odpovědi MeSH
• záření gama * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• radioizotopy uhlíku MeSH

When photosystem II (PSII) is exposed to excess light, singlet oxygen ((1)O(2)) formed by the interaction of molecular oxygen with triplet chlorophyll. Triplet chlorophyll is formed by the charge recombination of triplet radical pair (3)[P680(•+)Pheo(•-)] in the acceptor-side photoinhibition of PSII. Here, we provide evidence on the formation of (1)O(2) in the donor side photoinhibition of PSII. Light-induced (1)O(2) production in Tris-treated PSII membranes was studied by electron paramagnetic resonance (EPR) spin-trapping spectroscopy, as monitored by TEMPONE EPR signal. Light-induced formation of carbon-centered radicals (R(•)) was observed by POBN-R adduct EPR signal. Increased oxidation of organic molecules at high pH enhanced the formation of TEMPONE and POBN-R adduct EPR signals in Tris-treated PSII membranes. Interestingly, the scavenging of R(•) by propyl gallate significantly suppressed (1)O(2). Based on our results, it is concluded that (1)O(2) formation correlates with R(•) formation on the donor side of PSII due to oxidation of organic molecules (lipids and proteins) by long-lived P680(•+)/TyrZ(•). It is proposed here that the Russell mechanism for the recombination of two peroxyl radicals formed by the interaction of R(•) with molecular oxygen is a plausible mechanism for (1)O(2) formation in the donor side photoinhibition of PSII.

• MeSH
• chemické modely MeSH
• elektronová paramagnetická rezonance metody   MeSH
• fotochemie metody   MeSH
• fotosystém II (proteinový komplex) fyziologie   MeSH
• koncentrace vodíkových iontů MeSH
• kyslík chemie   MeSH
• propylgalan chemie   MeSH
• singletový kyslík * MeSH
• spin trapping metody   MeSH
• Spinacia oleracea MeSH
• světlo MeSH
• Synechococcus metabolismus   MeSH
• uhlík chemie   MeSH
• volné radikály MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• kyslík MeSH
• propylgalan MeSH
• singletový kyslík * MeSH
• uhlík MeSH
• volné radikály MeSH

Hydroxyl radical (HO•) production in photosystem II (PSII) was studied by electron paramagnetic resonance (EPR) spin-trapping technique. It is demonstrated here that the exposure of PSII membranes to heat stress (40 °C) results in HO• formation, as monitored by the formation of EMPO-OH adduct EPR signal. The presence of different exogenous halides significantly suppressed the EMPO-OH adduct EPR signal in PSII membranes under heat stress. The addition of exogenous acetate and blocker of chloride channel suppressed the EMPO-OH adduct EPR signal, whereas the blocker of calcium channel did not affect the EMPO-OH adduct EPR signal. Heat-induced hydrogen peroxide (H₂O₂) production was studied by amplex red fluorescent assay. The presence of exogenous halides, acetate and chloride blocker showed the suppression of H₂O₂ production in PSII membranes under heat stress. Based on our results, it is proposed that the formation of HO• under heat stress is linked to uncontrolled accessibility of water to the water-splitting manganese complex caused by the release of chloride ion on the electron donor side of PSII. Uncontrolled water accessibility to the water-splitting manganese complex causes the formation of H₂O₂ due to improper water oxidation, which leads to the formation of HO• via the Fenton reaction under heat stress.

• MeSH
• chloridy metabolismus   MeSH
• elektronová paramagnetická rezonance metody   MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• hydroxylový radikál metabolismus   MeSH
• oxidace-redukce MeSH
• peroxid vodíku farmakologie   MeSH
• spin trapping metody   MeSH
• Spinacia oleracea chemie   MeSH
• vytápění MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloridy MeSH
• fotosystém II (proteinový komplex) MeSH
• hydroxylový radikál MeSH
• peroxid vodíku MeSH

Pressurized hot water extraction (PHWE) was employed to prepare extracts from dried grape skin of two wine grape varieties (St. Laurent and Alibernet) at various temperatures (from 40 up to 120°C) and amounts of sample (0.5, 1.0 and 1.5 g). To assess the antioxidant activity of the extracts, electron paramagnetic resonance (EPR) spectroscopy was applied involving DPPH and ABTS(+) assays. Other extract characteristics including HPLC profile of anthocyanins and total phenolic compound content were obtained as well. PHWE has also been compared with earlier results of extractions of the same grape skin samples with compressed methanol and compressed ethanol under the conditions of pressurized fluid extraction (PFE). From this comparison, PHWE emerges as the more benign and efficient extraction method to recover valuable phenolic antioxidants from grape skins for the prospective use in functional food supplements.

• MeSH
• antioxidancia chemie   MeSH
• chemická frakcionace metody   MeSH
• elektronová paramagnetická rezonance metody   MeSH
• epidermis rostlin chemie   MeSH
• rostlinné extrakty chemie   MeSH
• spektrofotometrie ultrafialová MeSH
• tlak MeSH
• Vitis chemie   MeSH
• voda chemie   MeSH
• vysoká teplota * MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antioxidancia MeSH
• rostlinné extrakty MeSH
• voda MeSH

The reaction of vanadocene dichloride with sodium arsenate gives Cp(2)V(O(2)AsO(2)H). This compound was identified on the basis of the super-hyperfine coupling observed in the solution EPR spectrum. The effect of substitution was studied on the ring-substituted and ansa-bridged compounds.

• MeSH
• arseničnany chemie   MeSH
• cyklopentany chemie   MeSH
• elektronová paramagnetická rezonance metody   MeSH
• komplexní sloučeniny * chemická syntéza   chemie   MeSH
• roztoky MeSH
• sloučeniny vanadu chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arseničnany MeSH
• cyklopentany MeSH
• komplexní sloučeniny * MeSH
• roztoky MeSH
• sloučeniny vanadu MeSH
• sodium arsenate MeSH Prohlížeč
• vanadocene dichloride MeSH Prohlížeč

Electron paramagnetic resonance (EPR) spin trapping spectroscopy is an important method used in free radical research; however, its application in biological systems is hindered by EPR silencing of spin adducts. Previous studies in superoxide-generating chemical systems have shown that spin adducts can be partially stabilized by cyclodextrins. In this work, for the first time, this proposed protective effect of cyclodextrins is investigated in a real biological sample-in isolated thylakoid membranes and photosystem II (PSII) particles with EMPO as a spin trap. It is shown that (i) randomly methylated beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin form inclusion complexes with EMPO-superoxide adducts (EMPO-OOH), (ii) both cyclodextrins increase the intensity of the EMPO-OOH EPR signal in PSII particles up to five times, (iii) higher EMPO-OOH EPR signal intensity is a result of increased stability of EMPO-OOH, and (iv) the extent of the protection of EMPO-OOH adduct provided by cyclodextrins is different in thylakoids and PSII particles. Along with the spin trapping data, the toxicity of cyclodextrins is also discussed with particular focus on photosynthetic preparations. The presented data show that both tested cyclodextrins can be used as valuable tools to improve the sensitivity of spin trapping in biological samples.

• MeSH
• beta-cyklodextriny farmakologie   MeSH
• cyklodextriny farmakologie   MeSH
• elektronová paramagnetická rezonance metody   MeSH
• fotosystém II (proteinový komplex) chemie   izolace a purifikace   metabolismus   MeSH
• hydroxypropyl beta cyklodextrin MeSH
• pyrroly MeSH
• senzitivita a specificita MeSH
• spin trapping MeSH
• Spinacia oleracea MeSH
• superoxidy chemie   metabolismus   MeSH
• techniky in vitro MeSH
• tylakoidy chemie   účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide MeSH Prohlížeč
• beta-cyklodextriny MeSH
• cyklodextriny MeSH
• fotosystém II (proteinový komplex) MeSH
• hydroxypropyl beta cyklodextrin MeSH
• pyrroly MeSH
• superoxidy MeSH

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH.) and singlet oxygen ((1)O2), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 degrees C). Production of OH., evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH. production. These observations reveal that OH. is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH., whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH. is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH. scavengers. This finding indicates that the production of these two species was independent and that the production of (1)O2 is not closely linked to PSII donor side.

• MeSH
• elektronová paramagnetická rezonance metody   MeSH
• fotosystém II (proteinový komplex) chemie   metabolismus   MeSH
• intracelulární membrány metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• spin trapping metody   MeSH
• teplota * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosystém II (proteinový komplex) MeSH
• reaktivní formy kyslíku MeSH

It has been suggested that both free metals and reduced ferredoxin (Fd) participate in the light-induced production of hydroxyl radicals (OH*) in thylakoid membranes of chloroplasts. The most direct evidence for the involvement of Fd in OH* formation under physiological conditions was reported by Jakob and Heber (Plant Cell Physiol., 1996, 37, 629-635), who used the oxidation of dimethylsulfoxide to methane sulfinic acid as an indicator of OH* production. We confirmed their conclusions using a more sensitive and reliable EPR spin-trapping method and extended their work by additional findings. Free metal-dependent and ferredoxin-dependent OH* production was studied simultaneously and strong metal chelator Desferal was used to distinguish between these reaction pathways. The participation of protein-bound iron within photosystem I was confirmed by partial suppression of OH* generation in broken chloroplasts by methyl viologen. The enhancement in the production of OH* in thylakoid membranes by externally added ferredoxin can be considered as a straightforward evidence of the involvement of ferredoxin in OH* formation.

• MeSH
• chloroplasty účinky léků   metabolismus   MeSH
• elektronová paramagnetická rezonance metody   MeSH
• ferredoxiny farmakologie   fyziologie   MeSH
• fotosystém I (proteinový komplex) metabolismus   MeSH
• herbicidy farmakologie   MeSH
• hydroxylový radikál analýza   metabolismus   MeSH
• paraquat farmakologie   MeSH
• spin trapping metody   MeSH
• tylakoidy účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ferredoxiny MeSH
• fotosystém I (proteinový komplex) MeSH
• herbicidy MeSH
• hydroxylový radikál MeSH
• paraquat MeSH