Electron paramagnetic resonance (EPR) spectroscopy represents an established tool to study properties of microenvironments, e.g. to investigate the structure and dynamics of biological and artificial membranes. In this study, the partitioning of the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in ex vivo human abdominal and breast skin, ex vivo porcine abdominal and ear skin as well as normal and inflammatory in vitro skin equivalents was investigated by EPR spectroscopy. Furthermore, the stratum corneum (SC) lipid composition (as determined by high-performance thin-layer chromatography), SC lipid chain order (probed by infrared spectroscopy) and the SC thickness (investigated by histology) were determined in the skin models. X-band EPR measurements have shown that TEMPO partitions in the lipophilic and hydrophilic microenvironment in varying ratios in different ex vivo and in vitro skin models. Ex vivo human abdominal skin exhibited the highest amount of TEMPO in the lipophilic microenvironment. In contrast, the lowest amount of TEMPO in the lipophilic microenvironment was determined in ex vivo human breast skin and the inflammatory in vitro skin equivalents. Individual EPR spectra of epidermis including SC and dermis indicated that the lipophilic microenvironment of TEMPO mainly corresponds to the most lipophilic part of the epidermis, the SC. The amount of TEMPO in the lipophilic microenvironment was independent of the SC lipid composition and the SC lipid chain order but correlated with the SC thickness. In conclusion, EPR spectroscopy could be a novel technique to determine differences in the SC thickness, thus suitably complementing existing methods.
- MeSH
- břicho MeSH
- buněčné mikroprostředí MeSH
- chromatografie na tenké vrstvě MeSH
- cyklické N-oxidy chemie MeSH
- dospělí MeSH
- elektronová paramagnetická rezonance MeSH
- epidermis chemie MeSH
- kůže chemie cytologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipidy chemie MeSH
- mladý dospělý MeSH
- prasata MeSH
- prsy MeSH
- senioři MeSH
- spektrofotometrie infračervená MeSH
- spinové značení MeSH
- tloušťka kožní řasy MeSH
- zevní ucho MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Biomolecule (lipid and protein) oxidation products formed in plant cells exposed to photooxidative stress play a crucial role in the retrograde signaling and oxidative damage. The oxidation of biomolecules initiated by reactive oxygen species is associated with formation of organic (alkyl, peroxyl and alkoxyl) radicals. Currently, there is no selective and sensitive technique available for the detection of organic radicals in plant cells. Here, based on the analogy with animal cells, immuno-spin trapping using spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to image organic radicals in Arabidopsis leaves exposed to high light. Using antibody raised against the DMPO nitrone adduct conjugated with the fluorescein isothiocyanate, organic radicals were imaged by confocal laser scanning microscopy. Organic radicals are formed predominantly in the chloroplasts located at the periphery of the cells and distributed uniformly throughout the grana stack. Characterization of protein radicals by standard immunological techniques using anti-DMPO antibody shows protein bands with apparent molecular weights of 32 and 34 kDa assigned to D1 and D2 proteins and two protein bands below the D1/D2 band with apparent molecular weights of 23 and 18 kDa and four protein bands above the D1/D2 band with apparent molecular weights of 41, 43, 55 and 68 kDa. In summary, imaging of organic radicals by immuno-spin trapping represents selective and sensitive technique for the detection of organic radicals that might help to clarify mechanistic aspects on the role of organic radicals in the retrograde signaling and oxidative damage in plant cell.
- MeSH
- cyklické N-oxidy chemie MeSH
- elektronová paramagnetická rezonance MeSH
- lipidy chemie izolace a purifikace MeSH
- oxidace-redukce MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku chemie MeSH
- peroxidy chemie MeSH
- proteiny chemie MeSH
- reaktivní formy kyslíku chemie MeSH
- spin trapping * MeSH
- spinové značení MeSH
- volné radikály chemie izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Various nitric oxide modulators (NO donors--SNP, GSNO, DEA NONOate and scavengers--PTIO, cPTIO) were tested to highlight the role of NO under Cd excess in various ontogenetic stages of chamomile (Matricaria chamomilla). Surprisingly, compared to Cd alone, SNP and PTIO elevated Cd uptake (confirmed also by PhenGreen staining) but depleted glutathione (partially ascorbic acid) and phytochelatins PC2 and PC3 in both older plants (cultured hydroponically) and seedlings (cultured in deionised water). Despite these anomalous impacts, fluorescence staining of NO and ROS confirmed predictable assumptions and revealed reciprocal changes (decrease in NO but increase in ROS after PTIO addition and the opposite after SNP application). Subsequent tests using alternative modulators and seedlings confirmed changes to NO and ROS after application of GSNO and DEA NONOate as mentioned above for SNP while cPTIO altered only NO level (depletion). On the contrary to SNP and PTIO, GSNO, DEA NONOate and cPTIO did not elevate Cd content and phytochelatins (PC2, PC3) were rather elevated. These data provide evidence that various NO modulators are useful in terms of NO and ROS manipulation but interactions with intact plants affect metal uptake and must therefore be used with caution. In this view, cPTIO and DEA NONOate revealed the less pronounced side impacts and are recommended as suitable NO scavenger/donor in plant physiological studies under Cd excess.
- MeSH
- antioxidancia chemie MeSH
- cyklické N-oxidy chemie MeSH
- donory oxidu dusnatého chemie MeSH
- fluorescenční mikroskopie MeSH
- glutathion chemie MeSH
- heřmánek, heřmánkovec, rmen, rmenec účinky léků MeSH
- imidazoly chemie MeSH
- kadmium chemie MeSH
- konfokální mikroskopie MeSH
- kyselina askorbová chemie MeSH
- nitroprusid chemie MeSH
- oxid dusnatý chemie MeSH
- reaktivní formy kyslíku chemie MeSH
- S-nitrosoglutathion chemie MeSH
- semena rostlinná účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Native hyaluronan (HA) has been oxidized to polyaldehyde polymers with a degree of substitution (DS) of up to 50%. Two different procedures enabling the control of the degree of substitution were followed in this study. Selective oxidation of primary hydroxyl groups of N-acetyl-D-glucosamine of hyaluronan was performed either in an aqueous solution containing AcNH-TEMPO/NaBr/NaOCl or in an aprotic solvent containing Dess-Martin periodinane (DMP). It was found that a change of reaction parameters (reaction time and temperature, type of catalyst, oxidant-to-HA ratio, presence of nitrogen, buffer type, and concentration) had an influence on the degree of substitution and molecular weight. The derivatives were characterized by MS, NMR spectroscopy, and SEC-MALLS. Degradation of hyaluronic acid by the oxidant was observed and confirmed by SEC. The effect of oxidized derivatives of hyaluronan on cells was studied by means of NIH 3T3 fibroblast viability, which indicates that prepared hyaluronan polyaldehydes are biocompatible and suitable for medical applications and tissue engineering. The function of polyaldehyde as precursor for other modification was illustrated in the reaction with lysine.
- MeSH
- acetylglukosamin chemie MeSH
- aldehydy chemická syntéza farmakologie MeSH
- biokompatibilní materiály chemická syntéza farmakologie MeSH
- biopolymery chemie farmakologie MeSH
- bromidy chemie MeSH
- buňky NIH 3T3 MeSH
- chlornan sodný chemie MeSH
- cyklické N-oxidy chemie MeSH
- dusík chemie MeSH
- iminopyranosy chemie MeSH
- katalýza MeSH
- kyselina hyaluronová analogy a deriváty chemická syntéza farmakologie MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulová hmotnost MeSH
- myši MeSH
- oxidace-redukce MeSH
- sloučeniny sodíku chemie MeSH
- teplota MeSH
- viabilita buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
