Bioelectrochemistry (v.54, #2)

When Escherichia coli B was aerobically grown at 43 °C in a medium whose concentration was one-fourth that of the Luria–Bertani (LB) medium supplemented with 1.5 g/l of glutamic acid, drastic cell death was observed after the end of the logarithmic growth phase. However, when the same experiment was conducted under inhomogeneous 5.2–6.1 T magnetic field, cell death was extremely suppressed and the ratio of viable cell number under high magnetic field to that under geomagnetic field reached as much as 100,000. When the magnetic field exposure was restricted to 12 h after the logarithmic growth phase, a similar high degree of suppressive effect on the death was observed. The findings that the amount of sigma S protein encoded by the rpoS gene under the high magnetic field was larger than that under the geomagnetic field, and that the magnetic field effect disappeared when the rpoS gene-deficient strain was cultivated under the high magnetic field, suggest the interaction of magnetic field with a stationary phase specific gene.
Keywords: High magnetic field; Escherichia coli; Suppression of death; Exposure period;

The influence of medium conductivity on electropermeabilization and survival of cells in vitro by Gorazd Pucihar; Tadej Kotnik; Maša Kandušer; Damijan Miklavčič (107-115).
Electropermeabilization and cell death caused by the exposure to high voltage electric pulses depends on the parameters of pulses, as well as the composition of the extracellular medium. We studied the influence of extracellular conductivity on electropermeabilization and survival of cells in vitro. For this purpose, we used a physiological medium with a conductivity of 1.6 S/m and three artificial media with conductivities of 0.14, 0.005, and 0.001 S/m. Measurements of pH, osmolarity, and cell diameter were made to estimate possible side effects of the media on the cells. Our study shows that the percentage of surviving cells increases with the decreasing medium conductivity, while the percentage of electropermeabilized cells remains unaffected. Our results show that cell survival in experiments involving electropermeabilization can be improved by decreasing the medium conductivity. To provide an interpretation of experimental results, we have theoretically estimated the resting transmembrane voltage, the induced transmembrane voltage, the time constant of the voltage inducement, and heating of the cell suspension for each of the media used. These calculations imply that for accurate interpretation of experimental results, both the induced and the resting transmembrane voltage must be considered, taking into account the conductivity and the ionic composition of the extracellular medium.
Keywords: Electropermeabilization; Induced transmembrane voltage; Resting transmembrane voltage; Medium conductivity; Bleomycin; DC3F cells;

Background: A reinvented technique for tumour therapy, electrochemical treatment (EChT), is attracting increasing attention. This study compared results from treatment of liver and mammary tissue focusing on destruction and pH changes in the tissue close to the treatment electrodes. Subsequently, data were compared with a dose-planning model. Methods: Mammary or liver tissue in 50 adult female Sprague Dawley rats was given EChT with a constant, direct current. The electrodes used were Pt/Ir (9:1) with spherical tips. In situ pH measurements were taken with a micro-combination glass electrode. Results: Spherical lesions were produced in both liver and mammary tissue. No significant difference was detected when comparing the size of the lesions in the two kinds of tissue. Similar pH profiles were obtained in tissue surrounding the electrodes, with pH values changing rapidly from unphysiological to neutral status within the space of a few millimetres. The pH at the border of the macroscopic destruction zone, regardless of tissue type or coulomb dosage, correlated well with specific values (4.5–5.5 at the anode and between 9 and 10 at the cathode). Conclusion: The analogous destruction patterns in mammary and liver tissue support the hypothesis that EChT has similar results in at least these two different types of tissue. This implies that the destructive pattern caused by the treatment may be the same also in tumours.
Keywords: Electrochemical treatment; Rat; Mammary; Liver; Dose-planning model;

The electrochemical and spectroelectrochemical behaviors of SOD at cysteine modified gold electrode by Xiaoyun Meng; Xiaqin Wu; Zhesu Wang; Xiaowei Cao; Zongrang Zhang (125-129).
Different promoters were used to enhance the rate of the electron transfer process of superoxide dismutase (SOD). A quasi-reversible cyclic voltammogram (CV) of SOD was observed on the cysteine-modified gold wire electrode. The coupled adsorption/desorption processes and the characteristic Raman spectra of different states of SOD molecules on the electrode surface were studied with electrochemical quartz crystal microbalance (EQCM) and in-situ Raman spectroelectrochemical techniques. The possible promotion mechanism was discussed.
Keywords: Superoxide dismutase(SOD); Cysteine; Cyclic voltammetry; EQCM; Raman spectra;

Elimination voltammetry of adenine and cytosine mixtures by Libuše Trnková; Jiřı́ Friml; Oldřich Dračka (131-136).
The elimination voltammetry with linear scan (EVLS) was used to study adenine and cytosine reduction signals at the mercury electrode. In comparison with the linear scan voltammetry (which provides only one unresolved peak), two elimination functions provide good resolution of individual peaks and significant increase of sensitivity. The first elimination function eliminates the kinetic current (I k) and conserves the diffusion current (I d). The second elimination function eliminates kinetic and charging currents (I k and I c) simultaneously and conserves the diffusion current (I d). Both functions give two well-resolved peaks of adenine and cytosine in a wide concentration range, while the linear sweep voltammetry gives badly resolved peaks due to hydrogen evolution. The best resolution of peaks is observed in acetate buffer at pH 3.8 and the detection limit for both substances is 500 nM. The concentration dependence of EVLS peak heights for one substance at the constant concentration of the other substance is linear. The peak potentials differ in these elimination functions. The difference in EVLS peak potentials gives the possibility to evaluate αn a. Elimination voltammetry with linear scan contributes to the resolution of cathodic signals of purine and pyrimidine bases at very negative potentials near supporting electrolyte discharge.
Keywords: Linear sweep voltammetry; Elimination voltammetry with linear scan; Current transformation; Peak resolution; Reduction of adenine and cytosine;

The electroneutral Cl/HCO3 exchange, present at the apical membrane of rabbit gallbladder epithelium, apparently is converted into a stilbene- and dipyridamole-sensitive, nonrectifying, approximately 5-pS anion channel after the exchange is directly inhibited (inhibitors tested: hydrochlorothiazide (HCTZ), phlorizin, phenylglyoxal and diphenylamine-2-carboxylic acid (DPC)). In intact tissue, in the absence of CO2/HCO3 in the media, the opening of these channels causes an approximately 7-mV depolarization of the apical membrane. This has been shown to be a constant index of the total Cl conductance (G Cl) activated. The effect of exogenous and endogenous CO2/HCO3 on the depolarization has now been investigated in the intact tissue by conventional microelectrodes. The anion exchange has been measured radiochemically. The presence of exogenous or endogenous CO2/HCO3 reduces the depolarization induced by HCTZ, phlorizin and DPC from approximately 7 to 3 mV, but 10−4 mol/l acetazolamide restores the full depolarization. Response time, S 0.5 and Hill number are unchanged in each case. The way of bicarbonate replacement is irrelevant. The depolarization generated by phenylglyoxal, which covalently binds to the transport site of the exchanger and prevents HCO3 binding, is unaffected by CO2/HCO3 presence. HCO3 binding to the transport site is suggested to partially hinder the conversion of the exchanger into the channel.
Keywords: Hydrochlorothiazide; Phlorizin; Phenylglyoxal; Diphenylamine-2-carboxylic acid; Acetazolamide; Anion channel;

Electrochemical reactions of redox cofactors in Rhodobacter sphaeroides reaction center proteins in lipid films by Bernard Munge; Zeus Pendon; Harry A Frank; James F Rusling (145-150).
Cyclic voltammetry of thin films made from the lipid dimyristoylphosphatidyl choline and reaction centers from the purple bacterium Rhodobacter sphaeroides on pyrolytic graphite electrodes in bromide-free pH 8 buffers at 4 °C revealed an oxidation peak at 0.98 V and a reduction peak at −0.17 V vs. NHE. No reverse CV peaks were found, suggesting chemical irreversibility. The reduction peak disappeared for reaction centers depleted of quinones, suggesting that the peak represents reduction of this cofactor. The oxidation peak showed a catalytic current increase in the presence of small amounts of ferrous cytochrome c, and decreased by 85% when illuminated by visible light, suggesting assignment to the primary donor (P) cofactor. While oxidized primary donor P+ is destroyed upon electrochemical formation in the film, reaction of ferrous cyt c with P+ suggests its persistence in the films on the microsecond time scale.
Keywords: Electrochemical reaction; Redox cofactor; Rhodobacter sphaeroides;

Mapping peroxidase in plant tissues by scanning electrochemical microscopy by Huafang Zhou; Hitoshi Shiku; Shigenobu Kasai; Hiroyuki Noda; Tomokazu Matsue; Hiroaki Ohya-Nishiguchi; Hitoshi Kamada (151-156).
Scanning electrochemical microscopy has been firstly used to map the enzymatic activity in natural plant tissues. The peroxidase (POD) was maintained in its original state in the celery (Apium graveolens L.) tissues and electrochemically visualized under its native environment. Ferrocenemethanol (FMA) was selected as a mediator to probe the POD in celery tissues based on the fact that POD catalyzed the oxidation of FMA by H2O2 to increase FMA+ concentration. Two-dimensional reduction current profiles for FMA+ produced images indicating the distribution and activity of the POD at the surface of the celery tissues. These images showed that the POD was widely distributed in the celery tissues, and larger amounts were found in some special regions such as the center of celery stem and around some vascular bundles.
Keywords: Scanning electrochemical microscopy; Peroxidase; Images; Celery tissues;

In an attempt to uncover electric field interactions between PS I and PS II during their functioning, fluorescence induction curves were measured on hydroxylamine-treated thylakoids of Chenopodium album under conditions ensuring low and high levels of photogenerated membrane potentials. In parallel experiments with Peperomia metallica chloroplasts, the photocurrents were measured with patch-clamp electrodes and served as indicator of electrogenic activity of thylakoid membranes in continuous light. Inhibition of linear electron flow at PS II donor side by hydroxylamine (0.1 mM) eliminated a slow rise of chlorophyll fluorescence to a peak level and suppressed photoelectrogenesis. Activation of PS I-dependent electron transport using cofactors of either cyclic (phenazine methosulfate) or noncyclic electron transport (reduced TMPD or DCPIP in combination with methyl viologen) restored photoelectrogenesis in hydroxylamine-treated chloroplasts and led to reappearance of slow components in the fluorescence induction curve. Exposure of thylakoids to valinomycin reduced the peak fluorescence in the presence of KCl but not in the absence of KCl. Combined application of valinomycin and nigericin in the presence of KCl exerted stronger suppression of fluorescence than valinomycin alone but was ineffective in the absence of KCl. In samples treated with hydroxylamine and PS I cofactors (DCPIP/ascorbate and methyl viologen), preillumination with a single-turnover flash or a multiturnover pulse shifted the induction curves of both membrane potential and chlorophyll fluorescence to shorter times, which confirms the supposed influence of PS I-generated electrical field on PS II fluorescence. A model is presented that describes modulating effect of the membrane potential on chlorophyll fluorescence and roughly simulates the fluorescence induction curves measured at low and high membrane potentials.
Keywords: Chloroplast membranes; Electric membrane potential; Chlorophyll fluorescence; Photosystem II; Hydroxylamine;

The penicillamine (Pen) self-assembled monolayer (SAM) modified gold electrode (Pen/Au) is demonstrated to catalyze the electrochemical response of dopamine (DA) by cyclic voltammetry. A pair of well-defined redox waves was obtained and the calculated standard rate constant (k s) is 3.88×10−3 cm/s at the self-assembled electrode. The electrode reaction is a quasi-reversible process. The oxidation peak of DA can be used to determine the concentration of DA. The peak current and the concentration of DA are a linear relationship in the range of 2.0×10−5 M to 8.0×10−4 M. The detection limit is 4.0×10−6 M. By ac impedance spectroscopy the apparent electron transfer rate constant (k app) of Fe(CN)3−/Fe(CN)4− at the Pen/Au electrode was obtained as 2.08×10−5 cm/s. The Pen SAM was characterized with X-ray photoelectron spectroscopy (XPS), grazing angle FT-IR spectroscopy and contact angle goniometer.
Keywords: Penicillamine; Dopamine; Self-assembled electrode; XPS; FT-IR spectroscopy;