Oxidative stress can lead to various derivatives of the tyrosine residue in peptides and proteins. A typical product is 3-nitro-L-tyrosine residue (Nit), which can affect protein behavior during neurodegenerative processes, such as those associated with Alzheimer's and Parkinson's diseases. Surface enhanced Raman spectroscopy (SERS) is a technique with potential for detecting peptides and their metabolic products at very low concentrations. To explore the applicability to Nit, we use SERS to monitor tyrosine nitration in Met-Enkephalin, rev-Prion protein, and α-synuclein models. Useful nitration indicators were the intensity ratio of two tyrosine marker bands at 825 and 870 cm-1 and a bending vibration of the nitro group. During the SERS measurement, a conversion of nitrotyrosine to azobenzene containing peptides was observed. The interpretation of the spectra has been based on density functional theory (DFT) simulations. The CAM-B3LYP and ωB97XD functionals were found to be most suitable for modeling the measured data. The secondary structure of the α-synuclein models was monitored by electronic and vibrational circular dichroism (ECD and VCD) spectroscopies and modeled by molecular dynamics (MD) simulations. The results suggest that the nitration in these peptides has a limited effect on the secondary structure, but may trigger their aggregation.
- MeSH
- azosloučeniny chemie MeSH
- cirkulární dichroismus MeSH
- peptidy chemická syntéza chemie MeSH
- Ramanova spektroskopie metody MeSH
- sekundární struktura proteinů MeSH
- simulace molekulární dynamiky MeSH
- teorie funkcionálu hustoty MeSH
- tyrosin analogy a deriváty analýza MeSH
- Publikační typ
- časopisecké články MeSH
Nitrotyrosine formation is caused by presence of reactive oxygen and nitrogen species. Nitration is a very selective process leading to specific modification of only a few tyrosines in protein molecule. 2D electrophoresis and western blotting techniques coupled with mass spectrometry are common methods used in analysis of proteome. Here we describe protocol for analysis of peroxynitrite-induced protein nitration in isolated mitochondria. Mitochondrial proteins are separated by 2D electrophoresis and transferred to nitrocellulose membrane. Membranes are then incubated with antibodies against nitrotyrosine. Positive spots are compared with corresponding Coomassie-stained gels, and protein nitration is confirmed with mass spectrometry techniques.
- MeSH
- 2D gelová elektroforéza metody MeSH
- hmotnostní spektrometrie metody MeSH
- imunoblotting metody MeSH
- kyselina peroxydusitá chemie MeSH
- mitochondriální proteiny analýza metabolismus MeSH
- skot MeSH
- srdeční mitochondrie chemie metabolismus MeSH
- tyrosin analogy a deriváty analýza metabolismus MeSH
- zvířata MeSH
- Check Tag
- skot MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Intrinsically disordered proteins (IDPs) are subject to post-translational modifications. This allows the same polypeptide to be involved in different interaction networks with different consequences, ranging from regulatory signalling networks to the formation of membrane-less organelles. We report a robust method for co-expression of modification enzyme and SUMO-tagged IDPs with a subsequent purification procedure that allows for the production of modified IDP. The robustness of our protocol is demonstrated using a challenging system: RNA polymerase II C-terminal domain (CTD); that is, a low-complexity repetitive region with multiple phosphorylation sites. In vitro phosphorylation approaches fail to yield multiple-site phosphorylated CTD, whereas our in vivo protocol allows the rapid production of near homogeneous phosphorylated CTD at a low cost. These samples can be used in functional and structural studies.
- MeSH
- Escherichia coli genetika MeSH
- exprese genu MeSH
- fosforylace MeSH
- lidé MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- posttranslační úpravy proteinů MeSH
- proteinové domény MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- transformace genetická MeSH
- tyrosin analýza genetika MeSH
- vnitřně neuspořádané proteiny chemie genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although reactive oxygen/nitrogen species (ROS/RNS) have a fundamental role in physiological processes, enhanced ROS/RNS production induced by exogenous sources, including drugs and other xenobiotics, may result in serious damage to biomolecules. Oxidative/nitrosative stress is being intensively investigated and might be responsible for a variety of health side effects. The present liquid chromatography-tandem mass spectrometry (LC-MS/MS) method provides reliable and accurate simultaneous measurement of malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) in cultured human hepatoma (HepG2) cells. Sample preparation process involving ultrasonic homogenization, alkaline hydrolysis of protein-bound MDA and 3-NT, deproteination, derivatization of MDA by 2,4-dinitrophenylhydrazine and solid-phase extraction was optimized, ensuring the isolation and purification of desired analytes. Additionally, nonprotein thiols and nonprotein disulfides were measured using HPLC-UV. The established lower limit of quantification (0.025 nmol/mL for MDA; 0.0125 nmol/mL for 3-NT) allowed their LC-MS/MS determination in HepG2 cells exposed to model oxidizing agent, tert-butyl hydroperoxide (t-BOOH). The results show significant changes in MDA and 3-NT concentrations and alterations in thiol redox-state in dependence on the t-BOOH concentration and duration of its incubation in HepG2 cells. Concurrent evaluation of oxidative/nitrosative stress biomarkers in the in vitro model may significantly facilitate assessment of toxicity of newly developed drugs in preclinical trials and thus improve their safety profile.
- MeSH
- buňky Hep G2 MeSH
- chromatografie kapalinová metody MeSH
- lidé MeSH
- limita detekce MeSH
- malondialdehyd analýza MeSH
- nádory jater metabolismus MeSH
- oxidační stres MeSH
- reprodukovatelnost výsledků MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- tyrosin analogy a deriváty analýza MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Markers of oxidative stress and inflammation were analysed in the exhaled breath condensate (EBC) and urine samples of 14 workers (mean age 43 ± 7 years) exposed to iron oxide aerosol for an average of 10 ± 4 years and 14 controls (mean age 39 ± 4 years) by liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry (LC-ESI-MS/MS) after solid-phase extraction. Aerosol exposure in the workplace was measured by particle size spectrometers, a scanning mobility particle sizer (SMPS) and an aerodynamic particle sizer (APS), and by aerosol concentration monitors, P-TRAK and DustTRAK DRX. Total aerosol concentrations in workplace locations varied greatly in both time and space. The median mass concentration was 0.083 mg m(-3) (IQR 0.063-0.133 mg m(-3)) and the median particle concentration was 66 800 particles cm(-3) (IQR 16,900-86,900 particles cm(-3)). In addition, more than 80% of particles were smaller than 100 nm in diameter. Markers of oxidative stress, malondialdehyde (MDA), 4-hydroxy-trans-hexenale (HHE), 4-hydroxy-trans-nonenale (HNE), 8-isoProstaglandin F2α (8-isoprostane) and aldehydes C6-C12, in addition to markers of nucleic acid oxidation, including 8-hydroxy-2-deoxyguanosine (8-OHdG), 8-hydroxyguanosine (8-OHG), 5-hydroxymethyl uracil (5-OHMeU), and of proteins, such as o-tyrosine (o-Tyr), 3-chlorotyrosine (3-ClTyr), and 3-nitrotyrosine (3-NOTyr) were analysed in EBC and urine by LC-ESI-MS/MS. Almost all markers of lipid, nucleic acid and protein oxidation were elevated in the EBC of workers comparing with control subjects. Elevated markers were MDA, HNE, HHE, C6-C10, 8-isoprostane, 8-OHdG, 8-OHG, 5-OHMeU, 3-ClTyr, 3-NOTyr, o-Tyr (all p < 0.001), and C11 (p < 0.05). Only aldehyde C12 and the pH of samples did not differ between groups. Markers in urine were not elevated. These findings suggest the adverse effects of nano iron oxide aerosol exposure and support the utility of oxidative stress biomarkers in EBC. The analysis of urine oxidative stress biomarkers does not support the presence of systemic oxidative stress in iron oxide pigment production workers.
- MeSH
- aldehydy analýza MeSH
- biologické markery analýza MeSH
- dechové testy MeSH
- dinoprost analogy a deriváty analýza MeSH
- dospělí MeSH
- guanosin analogy a deriváty analýza MeSH
- lidé středního věku MeSH
- lidé MeSH
- malondialdehyd analýza MeSH
- nanočástice toxicita MeSH
- oxidační stres účinky léků fyziologie MeSH
- tandemová hmotnostní spektrometrie MeSH
- tyrosin analogy a deriváty analýza MeSH
- železité sloučeniny chemická syntéza MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
An assay of L-tyrosine (Tyr) hydroxylating activity operating in lincomycin biosynthesis is described. The assay development consisted of HPLC procedure development, assessing the effect of reaction mixture components on non-enzymatic Dopa and Tyr oxidation, and sample stability evaluation. The HPLC procedure with isocratic elution and fluorescence detection was developed and validated. The method showed a wide linear range of Dopa determination of 0.125-25 micromol/L with lower limit of quantification (LLOQ) of 0.125 micromol/L, RSD of 7.2% and accuracy of 101.7%. The studied linear range of Tyr was 15.625 mmol/L to 500 mmol/L with LLOQ of 15.625 mmol/L, RSD of 1.1%, and accuracy of 98.1%. Recoveries for Dopa and Tyr were 100.66 +/- 0.89% and 94.76 +/- 0.94%, respectively. The inter- and intra-day accuracies and precisions were all within 10%. Samples of the reaction mixture were stable for at least 24 h at room temperature (RT) and 28 days at -20 degrees C. The method was tested for the enzyme activity monitoring in purified as well as crude preparations and enabled micro preparation of the enzyme product during confirmation of its identity. The influence of pH and ascorbic acid content in reaction mixture was studied with respect to non-enzymatic Tyr oxidation.
- MeSH
- aktivace enzymů MeSH
- biotest MeSH
- časové faktory MeSH
- dopaminové látky analýza chemie MeSH
- financování organizované MeSH
- koncentrace vodíkových iontů MeSH
- levodopa analýza chemie MeSH
- oxidace-redukce MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- stabilita léku MeSH
- teplota MeSH
- tyrosin-3-monooxygenasa analýza metabolismus MeSH
- tyrosin analýza MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
Thyroid hormones are associated with the oxidative and antioxidative status of the organism. Depression of metabolism by hypothyroidism has been reported to decrease oxidant production and thus protect tissues against oxidant damage. The purpose of the present study was to investigate Zn and Cu levels in MMI-induced hypothyroidism and to show whether there is a connection between these trace elements and the oxidant-antioxidant status in experimental hypothyroidism. 3-Nitrotyrosine was measured as a marker of nitro-oxidative stress. In order to examine the antioxidant status of MMI-induced hypothyroidism in rats, GSH and SOD levels were determined as well. Significantly decreased 3-nitrotyrosine, Cu and Zn levels were observed in our experimental model when compared with the controls. On the other hand, GSH and SOD levels remained constant. It may be suggested that Cu and Zn serve as antioxidant molecules and exert their effects in an indirect manner to reduce oxidative stress in experimental hypothyroidism.
3-nitrotyrosin (3NT) je považován za jakýsi „indikátor“ tvorby NO. Byla provedena imunohistochemická analýza vzorků lidské synoviální tkáně, získané od 9 pacientů (3 revmatoidní artritida, 4 osteoartróza (OA), 1 juvenilní idiopatická artritida a 1 Bakerova cysta odebraná od pacienta s pigmentovou vilonodulární synovitidou). Sériové řezy byly barveny monoklonální protilátkou 60-E3 proti 3NT a protilátkami proti CD31, CD68 a CD14, což jsou povrchové markery endoteliálních buněk, makrofágů a monocytů. U většiny vzorků (konkrétně u 7 z 9 vyšetřených vzorků, včetně všech vzorků, získaných od nemocných s osteoartrózou) byla pozorována silná imunoreaktivita vůči 3NT lokalizovaná v endoteliálních buňkách, doprovázená slabým a difuzním barvením tkání v těsném sousedství 3NT-pozitivního endotelu. Silnou pozitivitu 3NT ve stromatu synoviální tkáně jsme pozorovali pouze u dvou silně zánětlivých vzorků, konkrétně u Bakerovy cysty a u jednoho pacienta s revmatoidní artritidou. Závěrem této pilotní studie je tedy konstatování, že v lidské synoviální tkáni dochází k nitraci tyrosinu především v endoteliálních buňkách. 3NT-pozitivní barvení endoteliálních buněk v synoviální tkáni pacienta s OA naznačuje, že k aktivaci endoteliálních buněk dochází i ve zdánlivě nezánětlivé synoviální tkáni pacientů s OA.
3-nitrotyrosine (3NT) is regarded as some kind of a „footprint“ of NO generation. Immunohistochemical analysis was performed on 9 specimens of human synovium (3 rheumatoid arthritis, 4 osteoarthritis (OA), 1 juvenile idiopathic arthritis, and 1 inflamed Baker's cyst derived from a joint of a patient with villonodular synovitis). Serial sections were stained with monoclonal antibody 60-E3 to 3NT, and with antibodies to CD31, CD68, and CD14 reacting to endothelial cells, macrophages and monocytes, respectively. The majority of specimens demonstrated a pattern of strong 3NT staining localized to endothelial cells accompanied by weak immunostaining of tissues closely surrounding the 3NT-positive endothelia (7 of the 9 specimens, including all of the specimens derived from osteoarthritic joints). We observed strong 3NT immunostaining of synovial stroma in only 2 specimens of inflamed synovia, those collected from the Baker's cyst secondary to villonodular synovitis and a knee joint with rheumatoid arthritis. We conclude that in human synovial tissue, tyrosine is primarily nitrated in endothelial cells. 3NT-positive staining of endothelial cells in OA synovia suggests that activation of endothelial cells occurs even in apparently non-inflammatory OA synovial tissue.
- MeSH
- biologické markery analýza MeSH
- finanční podpora výzkumu jako téma MeSH
- imunohistochemie metody MeSH
- lidé MeSH
- osteoartróza metabolismus patofyziologie MeSH
- oxid dusnatý chemická syntéza metabolismus MeSH
- pilotní projekty MeSH
- revmatoidní artritida metabolismus patofyziologie MeSH
- synoviální membrána chemie metabolismus MeSH
- tyrosin analogy a deriváty analýza MeSH
- Check Tag
- lidé MeSH
Liečba zvierat s ischemicko-reperfúznym poškodením mozgu použitím antioxidantov, vychytávačov voľných radikálov, by mohla znížiť mieru závažnosti reperfúzneho poškodenia. V našich experimentoch sme overovali protektívny účinok extraktu z Ginkgo biloba (EGb) na I/R indukovaný oxidačný stres a oxidáciu proteínov. Pozorovali sme signifikantný nárast intenzity fluorescencie tryptofánu po 24 hod. reperfúzii (24 hod. REP) u zvierat s podávaným EGb v porovnaní s 24h REP skupinou bez EGb. Signifikantný pokles intenzity fluorescencie dityrozínu u kontrolných, ischemických a 24 hod. REP skupín s EGb boli pozorované v porovnaní so skupinami bez EGb. Naše výsledky naznačujú, že extrakt z Ginkgo biloba ako predpokladný antioxidant môže zohrávať dôležitú úlohu pri znižovaní ischémiou/repefúziou indukovaného oxidačného stresu a proteínovej oxidácie.
Treatment of the animals with brain ischemic-reperfusion injury using antioxidants, scavengers of free radicals may reduce the severity of reperfusion damage. The present study was designed to check the protective effect of extract of Ginkgo biloba (EGb) on I/R injury induced oxidative stress and protein oxidation. We have observed significant increase of tryptophan fluorescence intensity of 24 h reperfusion (REP) groups with EGb administration in comparison to 24 h REP groups without EGb. Significant decrease of bityrosine fluorescence intensity in control groups, ischemia, 24 h REP with EGb were observed in comparison to groups without EGb. Our results indicate that extract of Ginkgo biloba as a presumed antioxidant could play an important role in reducing the ischemia/reperfusion-induced oxidative stress and protein oxidation.
- MeSH
- antioxidancia farmakologie MeSH
- finanční podpora výzkumu jako téma MeSH
- Ginkgo biloba MeSH
- ischemie mozku metabolismus terapie MeSH
- krysa rodu rattus MeSH
- přední mozek MeSH
- reperfuzní poškození MeSH
- rostlinné extrakty MeSH
- tryptofan analýza MeSH
- tyrosin analýza MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- MeSH
- finanční podpora výzkumu jako téma MeSH
- kosterní svaly metabolismus účinky léků MeSH
- krysa rodu rattus MeSH
- leucin analýza chemie MeSH
- peptidy farmakologie MeSH
- proteiny metabolismus MeSH
- proteosyntéza MeSH
- sepse metabolismus MeSH
- sulfony farmakologie MeSH
- tyrosin analýza MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH