Bioelectrochemistry (v.60, #1-2)

Contents (vi).

Quantitative model of small molecules uptake after in vitro cell electropermeabilization by Marko Puc; Tadej Kotnik; Lluis M. Mir; Damijan Miklavčič (1-10).
Electropermeabilization of the cell membrane is a phenomenon caused by exposure of the cell to electric pulses. Permeabilization depends on pulse duration, pulse amplitude, the number of pulses delivered, and also on other experimental conditions. With these parameters properly chosen, the process of permeabilization is reversible and cells return to their normal physiological state. This article describes the development of a model of diffusion-driven transmembrane transport of small molecules caused by electropermeabilization. The process of permeabilization is divided into a short permeabilizing phase that takes place during the pulse, and a longer resealing phase that begins after the end of the pulse. Because both phases of permeabilization are important for uptake of molecules into cells, most of the effort is focused on the optimization of parameters that influence the flow between intracellular and extracellular space. The model describes well the transmembrane transport caused by electropermeabilization, allowing to study the uptake of molecules as a function of elapsed time, voltage and pulse duration. In addition, our results show that the shapes of the curves of cell permeabilization and survival as functions of pulse amplitude can to a large extent be explained by cell size distribution.
Keywords: Transmembrane transport; Electroporation; Electropermeabilization; Resealing kinetics; Pharmacokinetic model;

The interaction of doxorubicin with Fe(III) ions and nicotinamide (NA) has been followed by square-wave voltammetry, cyclic voltammetry and UV–VIS. spectroscopy techniques at aerobic and anaerobic conditions. Fe(III)–doxorubicin complex gives a 1-electron reversible step at −0.494 V and a shoulder at 580 nm. Further, the Fe(III)–doxorubicin complex was found to be more stable at aerobic conditions. In the presence of NA, an intermediate (NA–Fe(III)–DQ) forms at −0.462 V under aerobic conditions. Because of the formation of this intermediate, nicotinamide may reduce the cardiotoxic effect of doxorubicin and cause to its detoxification.
Keywords: Doxorubicin; Nicotinamide; Fe(III)/Fe(II); Electrochemistry; UV–VIS spectroscopy;

Effective gating charge of ion channels induced by toxin syringomycin E in lipid bilayers by Ludmila V. Schagina; Philip A. Gurnev; Jon Y. Takemoto; Valery V. Malev (21-27).
To elucidate the voltage gating of syringomycin E (SRE) ion channels in lipid bilayers, the effective gating charge q was measured under different conditions. It was shown that q and its sign are dependent on membrane surface charge, dipole potential, and the outer potential (Δφ). The q values were positive for charged bilayers and negative for uncharged bilayers bathed in the same 0.1 M NaCl solutions. Effects of dipole modifying agents on the gating properties of SRE channels were measured. In uncharged bilayers, addition of phloretin resulted in an increase of q values. For charged bilayers, the presence of RH-421 or 6-ketocholestanol leads to the reverse in the sign of q from positive to negative. The q values were potential-dependent at higher negative voltages with charged membranes bathed in solutions with high salt concentrations. It is concluded that lipid molecules participating in the SRE channel structure contribute to channel formation work due to Coulomb and dipolar interactions with the electric field applied to a membrane. The potential dependence of q is explained by interactions of charged and uncharged lipids with SRE molecules in the channels.
Keywords: Ion channels; Lipid membranes; Channel voltage gating;

Electrochemical study of heavy metals and metallothionein in yeast Yarrowia lipolytica by Martin Strouhal; René Kizek; Jan Vacek; Libuše Trnková; Miroslav Němec (29-36).
The bioaccumulation of heavy metals (cadmium, nickel, cobalt and zinc) and the effect of these metals on the production of metallothionein and metallothionein-like proteins (MT) in Yarrowia lipolytica was studied by electrochemical methods. The concentrations of heavy metals were determined by differential pulse voltammetry (DPV). A combination of the constant current chronopotentiometric stripping analysis (CPSA) and adsorptive transfer stripping technique (AdTS) was used to determine the content of MT in cells. Both the bioaccumulation of heavy metals and the production of MT in different cell compartments of Y. lipolytica exposed to heavy metals were monitored. The LD50 of each metal was determined from the number of viable cells in yeast cultures: LD50Cd (37.5 μM), LD50Ni (570 μM), LD50Co (700 μM), and LD50Zn (1800 μM). The highest concentrations of heavy metals were found in the cell wall and membrane debris while the lowest concentrations were detected in the cytoplasm. Cadmium and nickel showed the most significant effect on the production of MT. This study provides new insights into the ecophysiology of microorganisms and demonstrates the potential use of these electrochemical methods in biotechnology.
Keywords: Yeast; Yarrowia lipolytica; Differential pulse voltammetry; Adsorptive transfer stripping technique; Constant current chronopotentiometric stripping analysis; Metallothionein; Cadmium, nickel, cobalt and zinc; Bioaccumulation;

The keto-enol tautomerization of p-hydroxyphenylpyruvic acid (pHPP) in aqueous solutions and the complexation reaction between enolic pHPP and boric acid have been studied by electrochemical techniques including linear sweep voltammetry (LSV), pulse voltammetry, and cyclic voltammetry (CV), combining with UV spectrometry. Electrochemical techniques reveal that in aqueous solution, there are two tautomers of pHPP: enolic form and ketonic form; the former exists mainly in freshly prepared pHPP solution, and the latter exists mainly in equilibrium solution. Both enolic and ketonic pHPP are electroactive. The electrochemical oxidation of enolic pHPP gives rise to two anodic waves, Ia and IIa, while the electrochemical oxidation of ketonic pHPP only results in the observation of the second wave IIa. The oxidation process Ia is revealed to be associated with the quasi-reversible, two-electron two-proton oxidation of “CC”group at the side chain of enolic pHPP, and the oxidation process IIa is proposed to result from the irreversible oxidation of phenolic hydroxyl group. It is observed that in aqueous solution, enolic pHPP can quickly complex with boric acid to yield enol-borate complex that can also oxidize at a glassy carbon electrode to yield an anodic wave.
Keywords: Keto-enol tautomerization of p-hydroxyphenylpyruvic acid; Electrochemistry;

The regulatory role of pith cells in the stem in Na+ recirculation in sweet pepper was investigated by evaluating the transport characteristics of the plasma membrane of this cell type and comparison with those of root cells. Ion conductivity and Na+ permeability of the plasma membranes of protoplasts of both cell types were studied with the patch-clamp technique in the whole-cell configuration, before and after addition of NaCl to the bath medium. Protoplasts of both pith and root cells showed outward rectifying currents with a reversal potential (V r) near to the equilibrium potential of K+ (EK). Addition of NaCl to the bath medium caused a stronger shift of the reversal potential, V r, in pith protoplasts than in root protoplasts, indicating that the outward rectified currents are permeable to Na+, especially in the pith cells.After plant exposure to exogenous NaCl via the nutrient solution for 1 week, V r in the root cells was closer to EK than in the control plants and hardly shifted upon addition of Na+. This indicated that the net permeability of the OR channel complement in the plasma membrane to Na+ was lower following exposure to Na+. V r in the pith protoplasts, on the other hand, shifted significantly more than in the control plants, suggesting an increase of the permeability to Na+. Moreover, the Na+ channel blocker amiloride blocked the currents in this cell type. It is concluded that pith cells have appropriate features of outward rectified currents to enable Na+ accumulation or release when NaCl is present in or removed from the nutrient medium. Probably, exogenous NaCl even induced expression and formation of Na+-permeable channels in pith cells.
Keywords: Patch clamp; Conductance; Capsicum annuum L.; Plasma membrane; ORC; Na+;

A water-soluble polyxylylviologen (PXV2+) was characterized with a view to making use of it as a redox electron-transfer (ET) mediator. Cyclic voltammetric and spectropotentiometric studies showed (i) that PXV2+ gives two redox waves centering at −0.40 and −0.83 V (vs. Ag/AgCl (3.3 mol dm−3 KCl)) and (ii) that the lifetime of its monocation radical (PXV) is two orders of magnitude greater than that of the well-utilized dimethyl viologen monocation radical. Subsequently, the reaction of the PXV2+/+· couple with NAD+ was evaluated in the similar manners as above. On the basis of this evaluation and the bioluminescence assay using bacterial NADH/FMN oxidoreductase and luciferase, it was shown (i) that the PXV2+/+· couple functions as a useful electron-transfer mediator and (ii) that PXV reacts with NAD+, leading to generation of the enzymatically active NADH, in the absence of any reductases.
Keywords: Viologen; Electron transfer; Mediator; NAD+; NADH; Bacterial bioluminescence;

Evaluation of redox mediators for amperometric biosensors: Ru-complex modified carbon-paste/enzyme electrodes by Ekaterina V. Ivanova; Viktorya S. Sergeeva; Joshua Oni; Christian Kurzawa; Alexander D. Ryabov; Wolfgang Schuhmann (65-71).
The properties of reagentless amperometric biosensors are mainly governed by the interaction of the used redox enzyme and the redox mediators used to facilitate the electron-transfer reaction. Both the used redox mediators and the redox enzymes differ concerning their hydrophilicity and their properties within the matrix of a carbon-paste electrode. Since there is no general procedure which is applicable for any enzyme in combination with any redox mediator, optimisation is necessary for each possible combination.Three approaches for the development of biosensors were investigated using carbon-paste electrodes enriched with redox mediator as a base in all sensor architectures. A class of redox mediators with the common formula Ru(LL)2(X)2 (where LL are 1,10-phenantroline or 2,2′-bipyridine type ligands, and X is an acido ligand) was investigated. In the first approach, enzymes were integrated into the carbon paste; in the second, the enzymes were adsorbed on the surface of the mediator-containing carbon-paste electrode and held in place by a Nafion film; and in the third approach, enzymes were entrapped in polymer films, which were electrochemically deposited onto the electrode's surface.The properties of the obtained biosensors strongly depend on the sensor architecture and the specific features of the used enzyme. Thus, our investigation using three different sensor architectures can provide valuable information about the possible interaction between a specific enzyme and a redox mediators with specific properties.
Keywords: Ru-complex; Biosensor; Electron transfer; Carbon-paste electrode; Redox mediator;

Effects of ELF and static magnetic fields on calcium oscillations in islets of Langerhans by F. Madec; B. Billaudel; R. Charlet de Sauvage; P. Sartor; B. Veyret (73-80).
Several experimental studies have produced contradictory results on the effects of extremely low frequency (ELF) magnetic fields on cellular processes involving calcium ions. Furthermore, the few positive results have not been independently replicated. In most of these studies, isolated cells were used. Our study used mouse islets of Langerhans, in which very regular oscillations of calcium concentration can be observed at length. These oscillations are sustained by processes that imply energetic and inter-intracellular communication. Various magnetic fields were applied, either sinusoidal at different frequencies (50 Hz or multiples of the natural oscillation frequency) at 0.1 or 1 mT or static at 1 mT. Islets were also exposed to “cyclotron resonance” conditions. There was neither alteration of the fundamental oscillation frequency nor the degree of organisation under all exposure conditions. Using this sensitive model, we could not show new evidence of alterations of calcium processes under exposure to various magnetic fields.
Keywords: Extremely low frequency; Static magnetic fields; Islets of Langerhans;

The action of electromagnetic fields (EMF) on different pathways related to cell physiology, proliferation, toxicity of chemicals, gene expression, etc., are currently being investigated although the results are still not conclusive and even conflicting. In laboratory and animal studies, EMF has been found to produce a great variety of effects such as: increase in ornithine decarboxylase activity in breast, increase in β-galactosidase gene expression and oncogene transcription after exposure to 50/60 Hz. Animal studies have shown that the use of EMF can enhance drug delivery across biological barriers (rat abdominal skin), using benzoic acid as the drug candidate. It has been reported by different authors that pulsed EMF (PEMF) can produce alterations in antineoplastic drugs potency. In the present study, we investigated the effects of PEMF on methotrexate cytotoxicity in MCF-7 breast cancer cells and the effects with simultaneous exposure to FeCl3. The data presented in the current report indicate that PEMF (25 Hz, 1.5 mT) do not induce modulation of the action of methotrexate (with and without iron-III) in MCF-7 cells when they are exposed to PEMF for 2 h/day during 3 days.
Keywords: Pulsed electromagnetic fields (PEMF); Drug resistance; Methotrexate; MCF-7; Human breast cancer cells; Iron ion;

Fluorescence induction curves (F(t)) in low intensity 1s light pulses have been measured in leaf discs in the presence and absence of valinomycin (VMC). Addition of VMC causes: (i) no effect on the initial fluorescence level Fo and the initial (O–J) phase of F(t) in the 0.01–1 ms time range. (ii) An approximately 10% decrease in the maximal fluorescence Fm in the light reached at the P level in the O–J–I–P induction curve. (iii) Nearly twofold increase in the rate and extent of the F(t) rise in the J–I phase in the 1–50 ms time range. (iv) A 60–70% decrease in the rise (I–P phase) in the 50–1000 ms time range with no appreciable effect, if at all, on the rate. System analysis of F(t) in terms of rate constants of electron transfer at donor and acceptor sides have been done using the Three State Trapping Model (TSTM). This reveals that VMC causes: (i) no, or very little effect on rate constants of e-transfer reactions powered by PSII. (ii) A manifold lower rate constant of radical pair recombination (k −1) in the light as compared to that in the control. The low rate constant of radical pair recombination in the reaction center (RC) in the presence of VMC is reflected by a substantial increase in the nonzero trapping efficiency in RCs in which the primary quinone acceptor (QA) is reduced (semi-open centers). This causes an increase in their rate of closure and in the overall trapping efficiency. Data suggest evidence that membrane chaotropic agents like VMC abolish the stimulation of the rate constant of radical pair recombination by light. This light stimulation that becomes apparent as an increase in Fo has been documented before [Biophys. J. 79 (2000) 26]. It has been ascribed to effects of (changes in) local electric fields in the vicinity of the RC. The decrease of the I–P phase is attributed to a decrease in the photoelectric trans-thylakoid potential in the presence of VMC. Such effects have been hypothesized and illustrated [Bioelectrochemistry 57 (2002) 123].
Keywords: Electric field effect; Chlorophyll fluorescence; Photochemical quenching; Trapping mechanism; Photosystem II; Valinomycin;

Modeling of ionic relaxation around a biomembrane disk by László Oroszi; Olaf Hasemann; Elmar Wolff; András Dér (97-106).
A simplified Brownian dynamics model and the corresponding software implementation have been developed for the simulation of electrolyte dynamics on the mesoscopic scale. In addition to direct control simulations, the model system has been verified by a quantitative comparison with the Debye–Hückel theory. As a first application, the model was used to simulate ionic relaxation processes following abrupt intramembrane charge rearrangements in the case of a disk shaped membrane. In addition to its general implications, the obtained properties of the relaxation kinetics confirm the assumptions of the theory of the so-called suspension method, a technique capable of tracing molecular charge motions of membrane proteins in three dimensions.
Keywords: Brownian dynamics; Ionic relaxation; Diffuse double layer; Bacteriorhodopsin;

Electrical energy required to form large conducting pores by John C Neu; Kyle C Smith; Wanda Krassowska (107-114).
This study computes the contribution of the externally induced transmembrane potential to the energy of large, highly conductive pores. This work was undertaken because the pore energy formulas existing in the literature predict qualitatively different behavior of large pores: the original formula proposed by Abidor et al. in 1979 implies that the electrical force expanding the pore increases linearly with pore radius, while later extensions of this formula imply that this force decreases to zero for large pores. Starting from the Maxwell stress tensors, our study derives the formula for the mechanical work required to deform a dielectric body in an ionic solution with steady-state electric current. This formula is related to a boundary value problem (BVP) governing electric potentials and fields in a proximity of a pore. Computer simulations yield estimates of the electrical energy for pores of two different shapes: cylindrical and toroidal. In both cases, the energy increases linearly for pore radii above approximately 20 nm, implying that the electrical force expanding the pore asymptotes to a constant value for large pores. This result is different from either of the two energy formulas mentioned above. Our study traces the source of this disagreement to approximations made by previous studies, which are suitable only for small pores. Therefore, this study provides a better understanding of the energy of large pores, which is needed for designing pulsing protocols for DNA delivery.
Keywords: Electric field; Electroporation; Maxwell stress tensor; Pore energy; Pore expansion rate; DNA delivery;

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate–probe distance by Daisuke Oyamatsu; Yu Hirano; Norihiro Kanaya; Yoshiaki Mase; Matsuhiko Nishizawa; Tomokazu Matsue (115-121).
The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate–probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.
Keywords: SECM; Diaphorase; Shear force; Feedback control;