Characterization of oxidative stress in Leishmaniasis-infected or LPS-stimulated macrophages using electrochemical impedance spectroscopy

The physiological changes caused by external stimuli can be employed as parameters to study pathogen infection in cells and the effect of drugs. Among analytical methods, impedance is potentially useful to give insight into cellular behavior by studying morphological changes, alterations in the physiological state, production of charged or redox species without interfering with in vitro cellular metabolism and labeling. The present work describes the use of electrochemical impedances spectroscopy to simply monitor by modeling impedance plots (Nyquist diagram) in appropriate equivalent circuit, the changes affecting murine macrophage cell line (RAW 264.7) in response to parasite infection by Leishmania amazonensis or to lipopolysaccharide (LPS) treatment. These results demonstrate the ability of electrochemical impedance spectroscopy to discriminate between two opposite cell responses associated to two different stimuli, one caused by the internalization of a parasite, and the other by activation by a bacterium component. Indeed, the study has allowed the characterization, from an electrical point of view, of the extra-cellular NO radical produced endogenously and in great quantities by the inducible form of NO-synthase in the case of LPS-stimulatedmacrophages. This production was not observed in the case of Leishmania-infectedmacrophages for which to survive and multiply, the parasite itself possesses mechanisms which may interfere with NO production. In this latest case, only the intracellular production of ROS was observed. To confirm these interpretations confocal microscopy analysis using the ROS (reactive oxygen species) fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate and electron paramagnetic resonance experiments using Fe(DETC)2 as NO radical spin trap were carried out

Electrochemical impedance spectroscopy to study physiological changes affecting the red blood cell after invasion by malaria parasites

The malaria parasite, Plasmodium falciparum, invades human erythrocytes and induces dramatic changes in the host cell. The idea of this work was to use RBC modified electrode to perform electrochemical impedance spectroscopy (EIS) with the aim of monitoring physiological changes affecting the erythrocyte after invasion by the malaria parasite. Impedance cell-based devices are potentially useful to give insight into cellular behavior and to detect morphological changes. The modelling of impedance plots (Nyquist diagram) in equivalent circuit taking into account the presence of the cellular layer, allowed us pointing out specific events associated with the development of the parasite such as (i) strong changes in the host cell cytoplasm illustrated by changes in the film capacity, (ii) perturbation of the ionic composition of the host cell illustrated by changes in the film resistance, (iii) releasing of reducer (lactic acid or heme) and an enhanced oxygen consumption characterized by changes in the charge transfer resistance and in the Warburg coefficient characteristic of the redox species diffusion. These results show that the RBC-based device may help to analyze strategic events in the malaria parasite development constituting a new tool in antimalarial research

Control of anthocyanin biosynthesis pathway gene expression by eutypine, a toxin from Eutypa lata, in grape cell tissue cultures

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 μmol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose- flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOX was not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed

Electrochemical behavior of indolone-N-oxides: Relationship to structure and antiplasmodial activity

Indolone-N-oxides exert high parasiticidal activity at the nanomolar level in vitro against Plasmodiumfalciparum, the parasite responsible for malaria. The bioreductive character of these molecules was investigated using cyclic voltammetry and EPR spectroelectrochemistry to examine the relationship between electrochemical behavior and antimalarial activity and to understand theirmechanisms of action. For all the compounds (37 compounds) studied, the voltammograms recorded in acetonitrile showed a well-defined and reversible redox couple followed by a second complicated electron transfer. The first reduction (−0.88 VbE1/2b−0.50 V vs. SCE) was attributed to the reduction of the N-oxide function to form a radical nitroxide anion. The second reduction (−1.65 VbE1/2b−1.14 V vs. SCE) was assigned to the reduction of the ketone function. By coupling electrochemistry with EPR spectroscopy, the EPR spectra confirmed the formation of the nitroxide anion radical.Moreover, the experiments demonstrated that a slowprotonation occurs at the carbon of the nitrone function and not at the NO function. A relationship between electrochemical behavior and indolone-N-oxide structure can be established for compounds with R1=―OCH3, R2=H, and electron-withdrawing substituents on the phenyl group at R3. The results help in the design of new molecules with more potent in vivo antimalarial activity

Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species

International audienceBackground : Parasite concentration methods facilitate molecular, biochemical and immunologicalresearch on the erythrocytic stages of Plasmodium. In this paper, an adaptation of magnetic MACS®columns for the purification of human Plasmodium species is presented. This method was useful forthe concentration/purification of either schizonts or gametocytes.Results and conclusions : The magnetic removal of non-parasitized red blood cells (in vivo andin vitro) using magnetic columns (MACS) was evaluated. This easy-to-use technique enrichedschizonts and gametocytes from Plasmodium falciparum in vitro cultures with a very high degree ofpurity. In addition, all haemozoin-containing stages (schizonts and/or gametocytes) from theperipheral blood of infected patients could be concentrated using this method. This method isparticularly useful for the concentration of non-falciparum species, which do not grow in cultureand are otherwise difficult to obtain in large amounts

Ethanol triggers grape gene expression leading to anthocyanin accumulation during berry ripening

Recent studies have shown that low doses of ethanol stimulate the maturation of some fruits. The present work showed that spraying Cabernet Sauvignon grapes, with 5% ethanol at veraison enhances the anthocyanin accumulation. Veraison is the time when the berries turn from green to purple. HPLC analysis showed a marked increase in the total concentrations of the derivatives of delphinidin, cyanidin, petunidin, peonidin and malvidin from the fourth day after the ethanol treatment until harvest. This was not linked to a difference in berry weight in comparison to controls. Two distinct expression patterns were found for anthocyanin biosynthesis genes in the treated and untreated berries. For one group, consisting of chalcone synthase, flavanone-3-hydroxylase, dihydroxyflavonol-4-reductase and leucoanthocyanidin dioxygenase, the expression was inhibited or unchanged by the ethanol treatment, whereas for UDP glucose-flavonoid 3-O-glucosyltransferase (UFGT) there was a marked increase in expression from 1 to 20 days after ethanol treatment. These results suggest that the UFGT gene is a key factor in the observed anthocyanin accumulation following ethanol treatment

Kinetics of Production and Consumption of Organic Acids During Alcoholic Fermentation by Saccharomyces cerevisae

This paper presents a study dealing with the production and consumption kinetics of the main organic acids during alcoholic fermentation carried out by a Saccharomyces cerevisiae strain normally used for winemaking. The experiments were carried out using a synthetic medium in which the initial malic acid concentration and the initial pH value were the parameters studied. The kinetics of malic acid consumption and of some organic acids production was then quantified. The results show that a decrease in pH value favors malic acid consumption, while an increase of the initial malic acid concentration increases the consumed amount. The specific malic acid consumption rate shows that its assimilation was 1nicrobial growth independent, while the other organic acids specific production rates show that their excretion was strongly associated with the microbial growth. The pH evolution during the fermentation was followed and partially explained by the evolution of the global organic acid production

Phenolic composition and prospective anti-infectious properties of Atriplex lindleyi

Objective: To investigate the antiplasmodial, antimicrobial, radical scavenging effects and to identifiy the phenolic constituents of Atriplex lindleyi (A. lindleyi). Methods: A. lindleyi extracts and some isolated compounds were tested in vitro against the chloroquine-resistant strains of lasmodium falciparum. The radical scavenging activity was quantified by using 2,2-diphenyl-2-picrylhydrazyl nitrogen-centered free radical. The IC50 of each extract was compared with references. The in vitro anti-infectious activity of extracts was evaluated against representative Gram-positive and Gram-negative bacterial strains [Staphylococcus aureus CIP 4.83, Enterococcus hirae CIP 5855, Pseudomonas aeruginosa (P. aeruginosa) CIP 82118, Escherichia coli CIP 53126], and fungal species [(Candida albicans (C. albicans) IP 48.72, Aspergillus niger IP 1431.83]. Ethanol extract was investigated for chemical composition through column and high performance liquid chromatography. The isolated compounds were identified by mass spectrometry and nuclear magnetic resonance. Results: Quercetin-7-O-arabinopyranoside-3-O-neohesperidosides (1), quercetin-3-O-arabinopyranosyl(1→6) glucopyranoside (2), quercetin-3-O-glucopyranoside-7-O-rhamnopyranoside (3), quercetin-3-O-glucopyranoside-7-O-arabinoside (4), schaftoside (5), quercetin-7-O-glucopyranoside (6) were isolated for the first time from the ethanol extract of A. lindleyi aerial parts in addition to isorhamnetin-3-O-β-glucopyranoside (7) and quercetin (8). The extracts exhibited moderate antiplasmodial activity with IC50≈ranging from 10–50 µg/mL. Quercetin was the most potent compound with IC50 of 9 µg/mL. P. aeruginosa and C. albicans were the most susceptible organisms. Conclusions: The study implies that A. lindelyi can contribute to the fight against malaria, and be useful as prophylactic against C. albicans and P. aeruginosa

Determination of ethylphenol compounds in wine by headspace solid-phase microextraction in conjunction with gas chromatography and flame ionization detection

Headspace solid phase microextraction (HS-SPME) was investigated as a solvent-free alternative method for the extraction and determination of 4-ethylphenol (EP) and 4-ethylguaiacol (EG) in red wine by capillary gas chromatography with flame ionization detection (FID) and compared to liquid–liquid extraction. For HS-SPME, better results were obtained with saturated sodium chloride samples, at 55°C, using a 85μm polyacrylate fiber. An absorption time of 40min was needed to reach the absorption equilibrium for EG. This 40-min duration corresponds to the beginning of EP equilibrium and was selected for the experiments. In these conditions, the calibration graphs were linear in the range 5–5000μgl−1 and the sensitivity was nearly the same for the two compounds. The detection limits were in the low μgl−1 range. In model wine solutions, result obtained with the liquid–liquid extraction method exhibit a linear calibration between 25 and 10,000μgl−1 with a detection limit of 1μgl−1, but, the relative standard deviations of the EP and EG result in the low concentration range (<50μgl−1) are higher than those obtained by HS-SPME (15% compared to 2% for EP and 12% compared to 5% for EG). Taking into account the numerous volatile compounds in wine, HS-SPME is a rapid and valid alternative technique for use in the determination of ethylphenols at trace levels

Determination of ethylphenol compounds in wine by headspace solid-phase microextraction in conjunction with gas chromatography and flame ionization detection

Headspace solid phase microextraction (HS-SPME) was investigated as a solvent-free alternative method for the extraction and determination of 4-ethylphenol (EP) and 4-ethylguaiacol (EG) in red wine by capillary gas chromatography with flame ionization detection (FID) and compared to liquid–liquid extraction. For HS-SPME, better results were obtained with saturated sodium chloride samples, at 55°C, using a 85μm polyacrylate fiber. An absorption time of 40min was needed to reach the absorption equilibrium for EG. This 40-min duration corresponds to the beginning of EP equilibrium and was selected for the experiments. In these conditions, the calibration graphs were linear in the range 5–5000μgl−1 and the sensitivity was nearly the same for the two compounds. The detection limits were in the low μgl−1 range. In model wine solutions, result obtained with the liquid–liquid extraction method exhibit a linear calibration between 25 and 10,000μgl−1 with a detection limit of 1μgl−1, but, the relative standard deviations of the EP and EG result in the low concentration range (<50μgl−1) are higher than those obtained by HS-SPME (15% compared to 2% for EP and 12% compared to 5% for EG). Taking into account the numerous volatile compounds in wine, HS-SPME is a rapid and valid alternative technique for use in the determination of ethylphenols at trace levels