Electrical cell-to-cell communication using aggregates of model cells.

Cell-to-cell communication via a local current caused by ion transport is elucidated using a model-cell system. To imitate tissues such as smooth muscles and cardiac muscles, liquid-membrane cells mimicking the function of K+ and Na+ channels were made. Connecting these channel-mimicking cells (K+ channel and voltage-gated Na+ channel) in parallel, model cells imitating living cell functions were constructed. Action-potential propagation within the cell aggregate model constructed by multiple model cells was investigated. When an action potential was generated at one cell, the cell behaved as an electric power source. Since a circulating current flowed around the cell, it flowed through neighboring model cells. Influx and efflux currents caused negative and positive shifts of the membrane potential, respectively, on the surface of neighboring model cells. The action potential was generated at the depolarized domain when the membrane potential exceeded the threshold of the voltage-gated Na+ channels. Thus, the action potential spread all over the cell system. When an external electric stimulus was applied to the layered cell-aggregate model system, propagation of the action potential was facilitated as if they were synchronized.

Development Perspective of Bioelectrocatalysis-Based Biosensors.

Bioelectrocatalysis provides the intrinsic catalytic functions of redox enzymes to nonspecific electrode reactions and is the most important and basic concept for electrochemical biosensors. This review starts by describing fundamental characteristics of bioelectrocatalytic reactions in mediated and direct electron transfer types from a theoretical viewpoint and summarizes amperometric biosensors based on multi-enzymatic cascades and for multianalyte detection. The review also introduces prospective aspects of two new concepts of biosensors: mass-transfer-controlled (pseudo)steady-state amperometry at microelectrodes with enhanced enzymatic activity without calibration curves and potentiometric coulometry at enzyme/mediator-immobilized biosensors for absolute determination.

Electrostatic roles in electron transfer from [NiFe] hydrogenase to cytochrome c3 from Desulfovibrio vulgaris Miyazaki F.

Electrostatic interactions between proteins are key factors that govern the association and reaction rate. We spectroscopically determine the second-order reaction rate constant (k) of electron transfer from [NiFe] hydrogenase (H2ase) to cytochrome (cyt) c3 at various ionic strengths (I). The k value decreases with I. To analyze the results, we develop a semi-analytical formula for I dependence of k based on the assumptions that molecules are spherical and the reaction proceeds via a transition state. Fitting of the formula to the experimental data reveals that the interaction occurs in limited regions with opposite charges and with radii much smaller than those estimated from crystal structures. This suggests that local charges in H2ase and cyt c3 play important roles in the reaction. Although the crystallographic data indicate a positive electrostatic potential over almost the entire surface of the proteins, there exists a small region with negative potential on H2ase at which the electron transfer from H2ase to cyt c3 may occur. This local negative potential region is identical to the hypothetical interaction sphere predicted by the analysis. Furthermore, I dependence of k is predicted by the Adaptive Poisson-Boltzmann Solver considering all charges of the amino acids in the proteins and the configuration of H2ase/cyt c3 complex. The calculation reproduces the experimental results except at extremely low I. These results indicate that the stabilization derived from the local electrostatic interaction in the H2ase/cyt c3 complex overcomes the destabilization derived from the electrostatic repulsion of the overall positive charge of both proteins.

Propagation of the change in the membrane potential using a biocell-model.

A new model system of nerve conduction, which has two sites (the potential-sending and the potential-receiving sites) was constructed by the use of some liquid-membrane cells which mimic the function of the K(+) and Na(+) channels. The model system setup was such that the membrane potential of the K(+)-channel cell (resting potential) was different from that of the Na(+)-channel cell (action potential). Initially, the K(+)-channel cell at the potential-sending site was connected to that at the potential-receiving site. After switching from the K(+)-channel cell to the Na(+)-channel cell at the potential-sending site, the membrane potential of the K(+)-channel cell at the potential-receiving site began to vary with the generation of the circulating current. By placing several K(+)-channel cells in parallel at the potential-receiving site, the propagation mechanism of the action potential was interpreted and the influence of the resistor and the capacitor on the propagation was evaluated.

Characteristics of fast mediated bioelectrocatalytic reaction near microelectrodes.

The pseudo-steady-state current due to a mediated enzymatic reaction on a microelectrode is characterized on the basis of theoretical analysis and numerical simulation. The steady-state current is proportional to substrate concentration when the enzymatic reaction is considerably faster than substrate mass transport via nonlinear diffusion. Under such conditions, the reaction plane, where the mass flow of the substrate is converted to that of the mediator, exists near the electrode surface. The steady-state current increases as the diffusion coefficient of the substrate increases. In contrast, the diffusion coefficient and the concentration of the mediator have minor effects on the current. This difference can be explained on the basis of a change in the reaction plane location. When a sufficient amount of enzyme exists in a system, the system can be used as an amperometric biosensor, the response of which is independent of any change in enzyme activity.

Improvement of a direct electron transfer-type fructose/dioxygen biofuel cell with a substrate-modified biocathode.

The fructose/dioxygen biofuel cell, one of the direct electron transfer (DET)-type bioelectrochemical devices, utilizes fructose dehydrogenase (FDH) on the anode and multi-copper oxidase such as bilirubin oxidase (BOD) on the cathode as catalysts. The power density in the literature is limited by the biocathode performance. We show that the DET-type biocathode performance is greatly improved, when bilirubin or some related substances are adsorbed on electrodes before the BOD adsorption. Several data show that the substrate modification induces the appropriate orientation of BOD on the electrode surface for the DET. The substrate-modification method has successfully been applied to air-breathing gas-diffusion-type biocathodes. We have also optimized the conditions of the FDH adsorption on carbon cryogel electrodes. Finally, a one-compartment DET-type biofuel cell without separators has been constructed, and the maximum power density of 2.6 mW cm(-2) was achieved at 0.46 V of cell voltage under quiescent (passive) and air atmospheric conditions.

Direct electron transfer to a metagenome-derived laccase fused to affinity tags near the electroactive copper site.

We demonstrate the efficient direct electron transfer (DET) from an electrode to an engineered laccase isolated from a metagenome. The enzyme has a unique homotrimeric architecture with a two-domain-type laccase subunit. The recombinant laccase-modified mesoporous carbon electrode exhibits an effective catalytic current for oxygen reduction, which depends on the affinity tags attached near the electroactive Cu site of the enzyme. We also investigated the effect of the affinity tags on the orientation of the enzyme on functional thiol-modified Au electrodes. The results suggest that a poly-histidine tag (His-tag) functions as an anchor to control the orientation of the enzyme to enhance the current density of the DET-type bioelectrocatalysis.

Bioelectrocatalytic oxidation of glucose with antibiotic channel-containing liposomes.

We developed an efficient bioelectrocatalytic system for glucose oxidation by introducing hydrophilic glucose-permeable antibiotic channels into liposomes.

Directional propagation of action potential within a single cell and intercellular conduction within a cell aggregate using model cell systems.

The mechanism of directional propagation of action potential throughout a single cell was examined using a liquid-membrane model cell system. In the experiments on the liquid-membrane model cell system, liquid-membrane cells were constructed to mimic the function of K+ and voltage-gated Na+ channels, which play important roles in action potential propagation. These channel-mimicking cells were connected electrically, and a model cell system was composed of four parts within the one cell. When one voltage-gated Na+ channel-mimicking cell was connected to form the action potential and generated the inflow current at the one part, action potential occurred in the surrounding area due to the local circulating current and propagated to the other parts. The action potential propagation throughout the cell by a brief electrical stimulus (10 ms) was easier than that by a long electrical stimulus (2 s). The long electric stimulus thus caused hyperpolarized region within the cell. Moreover, the increase in resistance corresponding to the extracellular fluid weakened the action potential propagation. In the simulation experiments using the software LTspice, the characteristics of K+ and Na+ channel-mimicking cells were reproduced in the electrical circuit also. A model cell aggregate consisting of closely packed three model cells and the extracellular fluid was constructed in the electric circuit. When one cell fired, the electrical signal propagated to the neighboring cells through the intercellular and extracellular fluids. This result suggests that electrical propagation can occur between independent cells in closely packed tissues without chemical transmission or direct propagation across the gap junctions.

Metagenomic analysis of ready biodegradability tests to ascertain the relationship between microbiota and the biodegradability of test chemicals.

Ready biodegradability tests conducted in accordance with the Organisation for Economic Co-operation and Development guidelines (test 301C or test 301F) are performed using activated sludge (AS) prepared by the Chemicals Evaluation and Research Institute (AS-CERI) or that taken from a sewage treatment plant (AS-STP). It had been reported that AS-CERI had lower activity than AS-STP in biodegrading test chemicals, and that biodegradation was accelerated by increasing the volume of the test medium. However, these phenomena have not been clarified from the perspective of the microbiota. In this study, using metagenomic analysis, we first showed that the microbiota of AS-CERI was biased in its distribution of phyla, less diverse, and had greater lot-to-lot variability than that of AS-STP. Second, after cultivation for a long period of time, the microbiota of AS-STP and AS-CERI became more similar to each other in terms of community structure. Third, determining degraders of test substances when each substance was actively biodegraded was found to be an effective approach. Finally, we clarified experimentally that a large volume of test medium increased the number of species that could degrade test substances in the condition where the initial concentrations of each substance and AS-STP were kept constant.

Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts.

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N-linked glycans (N-glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N-glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N-glycans and the effects of N-glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N-glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site.

Inhibition of direct-electron-transfer-type bioelectrocatalysis of bilirubin oxidase by silver ions.

In enzyme-based biosensors, Ag+ eluted from the reference electrode inhibits the enzyme activity. Herein, to suppress the inhibition of bilirubin oxidase (BOD) by Ag+, kinetic analysis was used to examine the effect of Ag+ on the activity of BOD. It was confirmed that the addition of Ag+ decreased the bioelectrocatalytic activity of BOD. Atomic absorption spectroscopy (AAS) suggested that Ag+ was attached to BOD. Moreover, the changes in the visible absorption spectra after Ag+ addition showed that Ag+ was bound to the type I Cu sites in BOD. During oxygen reduction by BOD, the direct-electron-transfer-type bioelectrocatalytic current decreased after Ag+ was added. The decay of the catalytic current was evaluated using kinetic analysis (assuming a pseudo-first-order reaction). Based on the analysis, the inhibition of BOD was suppressed when the Ag+ concentration was below 0.1 µM. Referring to the solubility product of AgCl, Cl- at a concentration of 1 mM suppressed the inhibition of the enzymatic activity by 95%.

Ion transport across bilayer lipid membranes in the presence of tetraphenylborate.

A pair of symmetrical cathodic and anodic peaks is observed in cyclic voltammograms for the ion transport across a bilayer lipid membrane (BLM) between two aqueous phases in the presence of tetraphenylborate (TPhB-). Although TPhB- serves as a carrier of a hydrophilic counter ion (Na+) under the steady-state condition, the reason for the appearance of symmetrical peaks has not been clearly explained until now. From the chronoamperometric analysis, it is turned out that the symmetrical peaks are attributed to the translocation of TPhB- between two adsorbed layers on the surface of the BLM.

Cyanide sensitivity in direct electron transfer-type bioelectrocatalysis by membrane-bound alcohol dehydrogenase from Gluconobacter oxydans.

An overexpression system of membrane-bound alcohol dehydrogenase (ADH) from Gluconobacter oxydans was constructed to examine its bioelectrocatalytic characteristics. The effects of cyanide (CN-) addition on the kinetics of direct electron transfer (DET)-type bioelectrocatalysis by ADH were analyzed. CN- enhanced the bioelectrocatalytic activity, while the catalytic activity in the solution remained unchanged, even in the presence of CN-. Electrochemical methods and electron spin resonance spectroscopy showed the detailed electron transfer pathway in the DET-type bioelectrocatalysis by ADH. Briefly, ADH is suggested to communicate with an electrode via a CN--insensitive and H+-sensitive heme c in DET. These characteristics of ADH with respect to CN- suggest the involvement of ADH in CN--insensitive respiration in G. oxydans.

Effects of N-linked glycans of bilirubin oxidase on direct electron transfer-type bioelectrocatalysis.

Bilirubin oxidase from Myrothecium verrucaria (mBOD) is a promising enzyme for catalyzing the four-electron reduction of dioxygen into water and realizes direct electron transfer (DET)-type bioelectrocatalysis. It has two N-linked glycans (N-glycans), and N472 and N482 are known as binding sites. Both binding sites located on opposite side of the type I (T1) Cu, which is the electrode-active site of BOD. We investigated the effect of N-glycans on DET-type bioelectrocatalysis by performing electrochemical measurements using electrodes with controlled surface charges. Two types of BODs with different N-glycans, mBOD and recombinant BOD overexpressed in Pichia pastoris (pBOD), and their deglycosylated forms (dg-mBOD and dg-pBOD) were used in this study. Kinetic analysis of the steady-state catalytic waves revealed that both size and composition of N-glycans affected the orientation of adsorbed BODs on the electrodes. Interestingly, the most favorable orientation was achieved with pBOD, which has the largest N-glycans. Furthermore, the effect of the orientation control by the N-glycans is cooperative with electrostatic interaction.

Comprehensive understanding of multiple actions of anticancer drug tamoxifen in isolated mitochondria.

Tamoxifen has been widely used in the treatment of estrogen receptor (ER)-positive breast cancer, whereas it also exhibits ER-independent anticancer effects in various cancer cell types. As one of the convincing mechanisms underlying the ER-independent effects, induction of apoptosis through mitochondrial dysfunction has been advocated. However, the mechanism of action of tamoxifen even at the isolated mitochondrial level is not fully understood and remains controversial. Here, we attempted to comprehensively understand tamoxifen's multiple actions in isolated rat liver mitochondria through not only revisiting the actions hitherto reported but also conducting originally designed experiments. Using submitochondrial particles, we found that tamoxifen has potential as an inhibitor of both respiratory complex I and ATP synthase. However, these inhibitory effects were not elicited in intact mitochondria, likely because penetration of tamoxifen across the inner mitochondrial membrane is highly restricted owing to its localized positive charge (-N+H(CH3)2). This restricted penetration may also explain why tamoxifen is unable to function as a protonophore-type uncoupler in mitochondria. Moreover, tamoxifen suppressed opening of the mitochondrial permeability transition pore induced by Ca2+ overload through enhancing phosphate uptake into the matrix. The photoaffinity labeling experiments using a photolabile tamoxifen derivative (pTAM1) indicated that pTAM1 specifically binds to voltage-dependent anion channels (VDACs) 1 and 3, which regulate transport of various substances into mitochondria. The binding of tamoxifen to VDAC1 and/or VDAC3 could be responsible for the enhancement of phosphate uptake. Taking all the results together, we consider the principal impairment of mitochondrial functions caused by tamoxifen.

Multiple electron transfer pathways of tungsten-containing formate dehydrogenase in direct electron transfer-type bioelectrocatalysis.

Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)-a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD+)/NADH redox couples-was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize "direct electron transfer (DET)-type bioelectrocatalysis." However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using structural information, electrostatic interactions between the electrode and the enzyme, and the differences in the substrates. Two electrode-active sites and multiple ET pathways in FoDH1 were discovered.

Pollution Control of Nitrate-selective Membrane by the Inner Solution and On-site Monitoring of Nitrate Concentration in Soil.

A liquid-membrane type nitrate-selective electrode was improved to lower the influence of contaminants by modifying its inner electrode system from Ag | AgCl | Cl- to Ag | Ag+. The NO3--selective electrode displayed a linear response to the concentration of NO3- with a Nernstian slope of -53 ± 1 mV decade-1, in the concentration region between 10-5 and 2 mol dm-3 (M). The NO3- detection limit was about 10-5 M. The electrochemical response of this electrode was stable for more than 30 days. The deterioration in responding characteristics due to the coexistence of Cl- was suppressed by use of the Ag | Ag+ redox couple in the absence of Cl- inside the NO3--selective electrode.

A liposome-based energy conversion system for accelerating the multi-enzyme reactions.

We report the first example of a liposome-based energy conversion system that is useful for entrapping enzymes and NAD coenzyme to accelerate multi-step enzymatic reactions. The liposome generates a much higher catalytic current compared with the non-liposome system, which is in good consistency with numerical simulations.

P53, p21, and p73 gene polymorphisms in gastric carcinoma.

BACKGROUND/AIMS: The aim of this study was to evaluate the association of genetic polymorphisms of p53, p21, and p73 genes with susceptibility to gastric carcinoma (GC), its clinicopathologic features, and prognosis. METHODOLOGY: In a case-control study including 419 controls and 389 patients with sporadic GC, single nucleotide polymorphisms (SNPs) of p53 Arg72Pro, p21 Ser31Arg, and p73 G4C14-to-A4T14 at exon 2 were genotyped. Tumor tissue was immunostained with p53 and examined for mutations in exons 5 to 8 of p53 using PCR -based single strand conformational polymorphism analysis and direct sequencing. RESULTS: The SNPs of p53, p21, and p73 genes were not significantly associated with susceptibility to GC. p53 SNP (Pro/Pro) was significantly associated with increased risks for the following subgroups: invasive infiltration-type carcinoma (odds ratio [OR], 2.09; 95% CI, 1.01 to 4.34; p = 0.048), carcinoma with peritoneal dissemination (OR, 3.42; 95%CI, 1.05 to 11.08; p = 0.04), and carcinoma with distant metastasis (OR, 3.90; 95% CI, 1.14 to 13.38; p = 0.03), but not with p53 immunoreactivity or mutations when compared with wild type. With respect to prognosis, p53 SNP (Pro/Pro) was an independent marker of poor overall survival in GC with TNM IB to IV stages (hazard ratio, 2.31; 95% CI, 1.14 to 4.69; p = 0.02), especially in GC treated by chemotherapy (hazard ratio, 2.17; 95% CI, 1.06 to 4.41; p = 0.03). CONCLUSIONS: This study provides evidence supporting the association of p53 SNP Arg72Pro with GC with invasive phenotype, peritoneal dissemination, distant metastasis, chemoresistance, and poor prognosis.