Jelena Bogdanović Pristov

Extracellular iron diminishes anticancer effects of vitamin C: An in vitro study

In vitro studies have shown that hydrogen peroxide (H2O2) produced by high-concentration ascorbate and cell culture medium iron efficiently kills cancer cells. This provided the rationale for clinical trials of high-dose intravenous ascorbate-based treatment for cancer. A drawback in all the in vitro studies was their failure to take into account the in vivo concentration of iron to supplement cell culture media which are characterized by low iron content. Here we showed, using two prostate cancer cell lines (LNCaP and PC-3) and primary astrocytes, that the anticancer/cytotoxic effects of ascorbate are completely abolished by iron at physiological concentrations in cell culture medium and human plasma. A detailed examination of mechanisms showed that iron at physiological concentrations promotes both production and decomposition of H2O2. The latter is mediated by Fenton reaction and prevents H2O2 accumulation. The hydroxyl radical, which is produced in the Fenton reaction, is buffered by extracellular proteins, and could not affect intracellular targets like H2O2. These findings show that anticancer effects of ascorbate have been significantly overestimated in previous in vitro studies, and that common cell culture media might be unsuitable for redox research.

UV‐irradiation provokes generation of superoxide on cell wall polygalacturonic acid

We examined the redox effects of UV irradiation on cell wall isolates from Pisum sativum leaves, and polygalacturonic and galacturonic acid, in the presence of hydrogen peroxide. For this purpose, electron paramagnetic resonance spectroscopy and two spin-traps (DEPMPO and BMPO), capable of differentiating between various free radicals, were applied. Systems were exposed to UV-B (maximum emission at 312 nm) and UV-A (352 nm) for 10 min (6 J m(-2) s(-1)). Cell wall isolates exposed to UV in the presence of hydrogen peroxide, produced hydroxyl radical, carbon dioxide radical and superoxide. The production of superoxide was observed for cell wall isolates, polygalacturonic acid (in the presence and in the absence of calcium) and galacturonic acid, and it was diminished upon superoxide dismutase supplementation. The production is at least partially based on the reaction of hydroxyl radicals with (poly)galacturonic acid having carbon dioxide radicals as a products. Acting as a strong reducing agent, carbon dioxide radical reacts with molecular oxygen to produce superoxide. The results presented here shed a new light on: (1) the redox-modulating role of cell wall; (2) the production of superoxide in the extracellular compartment; (3) the mechanisms involved in translating UV stress into molecular signaling and (4) some other UV-related phenomena in plants, such as CO(2) emission.

Classification and fingerprinting of different berries based on biochemical profiling and antioxidant capacity

O objetivo deste trabalho foi avaliar a composicao bioquimica de seis especies de pequenas frutas dos generos Fragaria, Rubus, Vaccinium e Ribes. As amostras das frutas foram coletadas em triplicata (com 50 frutas cada) de 18 diferentes especies ou cultivares dos generos mencionados, durante tres anos (2008 a 2010). Os teores individuais de acucares, acidos orgânicos, flavonois e acidos fenolicos foram determinados por cromatografia liquida de alta performance (HPLC), e os fenois totais (FT) e a capacidade antioxidante total (CAT), por meio de espectrofotometria. As analises de componente principal (ACP) e de agrupamento hierarquico (AH) foram utilizadas para avaliar as diferencas no perfil bioquimico das frutas. Os maiores teores de compostos bioativos foram encontrados em Ribes nigrum e em Fragaria vesca, Rubus plicatus e Vaccinium myrtillus. ACP e AH foram capazes de discriminar parcialmente as frutas com base em sua composicao bioquimica. Os acucares totais e individuais, a miricetina, o acido elagico, FT e CAT foram os parâmetros que mais impactaram na avaliacao da composicao bioquimica das pequenas frutas. AH fez a separacao de amora-preta, framboesa e mirtilo como grupos isolados, porem a classificacao do morango, da groselha-preta e da groselha-vermelha, em um grupo especifico, nao ocorreu. Ha grande variabilidade entre e dentro dos diferentes tipos de frutas vermelhas. A identificacao de metabolitos das pequenas frutas mostrou perfis bioquimicos unicos e combinacoes especificas de teores de compostos bioativos.

The interactions of vanadium with Phycomyces blakesleeanus mycelium: enzymatic reduction, transport and metabolic effects

The biological and chemical basis of vanadium action and transport in fungi is relatively poorly understood. In this study we investigated the interactions of vanadium in physiologically-relevant redox states: vanadate (+5) and vanadyl (+4), with mycelium of fungus Phycomyces blakesleeanus using EPR and (31)P NMR spectroscopy and biochemical assays. We determined that P. blakesleeanus reduces V(5+) to V(4+) in the extracellular compartment by the means of cell surface enzyme with ferricyanide reductase activity, which contains molybdenum-molybdopterin as a cofactor. Both, V(5+) and V(4+) bind to cell wall. They enter the cytoplasm via phosphate transporter and cation channels, respectively, and exhibit different metabolic effects. Vanadate provokes increased biomass production, the effects being inverted to toxic at higher V(5+) concentrations. In addition, V(5+) activates the synthesis of sugar phosphates and oligophosphates. On the other hand, V(4+) exhibits toxic effects even at low concentrations. The V(4+) detoxification route involves binding to vacuolar polyphosphates. Altogether our results imply that the mechanism of interaction of vanadium with P. blakesleeanus involves three major steps: extracellular enzymatic V(5+)/V(4+) reduction, V(4+) influx, and vacuolar storage, with an additional step - V(5+) import occurring at higher vanadate concentrations.

Hippocampal antioxidative system in mesial temporal lobe epilepsy

To examine antioxidative system in hippocampi of patients with mesial temporal lobe epilepsy associated with hippocampal sclerosis (mTLE‐HS).

The relationship of physicochemical properties to the antioxidative activity of free amino acids in Fenton system.

Herein we compared antioxidative activities (AA) of 25 free L-amino acids (FAA) against Fenton system-mediated hydroxyl radical (HO(•)) production in aqueous solution, and examined the relation between AA and a set of physicochemical properties. The rank order according to AA was: Trp > norleucine > Phe, Leu > Ile > His >3,4-dihydroxyphenylalanine, Arg > Val > Lys, Tyr, Pro > hydroxyproline > α-aminobutyric acid > Gln, Thr, Ser > Glu, Ala, Gly, Asn, Asp. Sulfur-containing FAA generated different secondary reactive products, which were discriminated by the means of electron paramagnetic resonance spin-trapping spectroscopy. AA showed a general positive correlation with hydrophobicity. However, when taken separately, uncharged FAA exhibited strong positive correlation of AA with hydrophobicity whereas charged FAA showed negative or no significant correlation depending on the scale applied. A general strong negative correlation was found between AA and polarity. Steric parameters and hydration numbers correlated positively with AA of nonpolar side-chain FAA. In addition, a decrease of temperature which promotes hydrophobic hydration resulted in increased AA. This implies that HO(•)-provoked oxidation of FAA is strongly affected by hydrophobic hydration. Our findings are important for the understanding of oxidation processes in natural and waste waters.

The potential physiological implications of polygalacturonic acid-mediated production of superoxide

PGA/OGA/PF represent apoplastic signaling molecules implicated in the control of gene expression and the activity of enzymes involved in defense regulation. However, the underlying mechanisms behind such processes are lacking. Here we unequivocally show using EPR spectroscopy with DEPMPO spin-trap capable of differentiating between •OH and •O2- that PGA and PF can produce •O2- by transforming •OH. The potential physiological implications of this unique property are discussed. We propose that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H2O2 being a downstream secondary messenger.

The reaction of methionine with hydroxyl radical: reactive intermediates and methanethiol production

The mechanisms of reaction of methionine with hydroxyl radical are not fully understood. Here, we unequivocally show using electron paramagnetic resonance spin-trapping spectroscopy and GC–FID and GC–MS, the presence of specific carbon-, nitrogen- and sulfur-centered radicals as intermediates of this reaction, as well as the liberation of methanethiol as a gaseous end product. Taking into account the many roles that methionine has in eco- and biosystems, our results may elucidate redox chemistry of this amino acid and processes that methionine is involved in.

A method for in-gel fluorescent visualization of proteins after native and sodium dodecyl sulfate polyacrylamide gel electrophoresis

We have developed a simple one-step 30-min method for fluorescent visualization of proteins in native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels. The method is based on formation of strong fluorophores via potassium ferricyanide-provoked oxidation of tryptophan (Trp). Following PAGE, gels are soaked in water solution of potassium ferricyanide (100 mM) and NaOH (1 M) and are kept in the dark for 30 min. Gels are then transferred to water and scanned. The sensitivity of the method was slightly lower compared with standard Coomassie Brilliant Blue (CBB) staining. The method can be useful when rapid acquisition of data is of the essence. After preview, gels can be post-stained using the CBB protocol for further analysis. The intensity of fluorescence is dependent on Trp number, so the protocol might find application in the quantification of Trp residues as illustrated here. Importantly, there is room for improvement of the method. Namely, according to excitation-emission matrix analysis of stained protein bands, maximal fluorescence intensity (at 345/460 nm) was 3.5-fold higher compared with the settings that were available on a commercial imager (395/525 nm). As a supplement, we present an upgrade of the previously described method for in-gel detection of non-heme iron-binding proteins that also employs potassium ferricyanide.

Fluorescence-Detected Linear Dichroism of Wood Cell Walls in Juvenile Serbian Spruce: Estimation of Compression Wood Severity.

Fluorescence-detected linear dichroism (FDLD) microscopy provides observation of structural order in a microscopic sample and its expression in numerical terms, enabling both quantitative and qualitative comparison among different samples. We applied FDLD microscopy to compare the distribution and alignment of cellulose fibrils in cell walls of compression wood (CW) and normal wood (NW) on stem cross-sections of juvenile Picea omorika trees. Our data indicate a decrease in cellulose fibril order in CW compared with NW. Radial and tangential walls differ considerably in both NW and CW. In radial walls, cellulose fibril order shows a gradual decrease from NW to severe CW, in line with the increase in CW severity. This indicates that FDLD analysis of cellulose fibril order in radial cell walls is a valuable method for estimation of CW severity.