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.

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.

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.

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.

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.

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.

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.

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.

Automatic Management of Nutrient Solution for Hydroponics-Construction of Multi-ion Stat.

In order to improve plant factories, an appropriate control system on fertilization is urgently required. An automatic management system to control nutrient concentration was constructed using a programmable logic controller (PLC) and ion selective electrodes (ISEs) of nitrate, phosphate, and potassium ion. The concentration of nutrient components in a culture solution was monitored using these ISEs. When the concentration of the nutrient components diminished to the threshold set as an optimum condition (0.1 - 2.0 mM), an appropriate amount of a concentrated solution of each nutrient component was added to the culture solution using solenoid valves connected with the PLC. The present cultivation system was simply constructed without any computers and pumps. Three kinds of automatic control systems simultaneously worked and did not influence each other.

Direct electron transfer-type bioelectrocatalysis of FAD-dependent glucose dehydrogenase using porous gold electrodes and enzymatically implanted platinum nanoclusters.

The direct electron transfer (DET)-type bioelectrocatalysis of flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH) from Aspergillus terreus (AtGDH) was carried out using porous gold (Au) electrodes and enzymatically implanted platinum nanoclusters (PtNCs). The porous Au electrodes were prepared by anodization of planar Au electrodes in a phosphate buffer containing glucose as a reductant. Moreover, PtNCs were generated into AtGDH by an enzymatic reduction of hexachloroplatinate (IV) ion. The modification was confirmed by native polyacrylamide gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses. The AtGDH-adsorbed porous Au electrode showed a DET-type bioelectrocatalytic wave both in the presence and absence of PtNCs; however, the current density with PtNCs (~1 mA cm-2 at 0 V vs. Ag|AgCl|sat. KCl) was considerably higher than that without PtNCs. The kinetic and thermodynamic analysis of the steady-state catalytic wave indicated that inner PtNCs shortened the distance between the catalytic center of AtGDH (=FAD) and the conductive material, and improved the heterogeneous electron transfer kinetics between them.

Fabrication of a Phosphate Ion Selective Electrode Based on Modified Molybdenum Metal.

A phosphate ion-selective electrode using molybdenum metal was constructed. The modified molybdenum electrode responded to HPO42- in the presence of molybdenum dioxide and molybdophosphate (PMo12O403-) on the surface. The electrode exhibited a linear response to HPO42- in the concentration range between 1.0 × 10-5 and 1.0 × 10-1 M (mol dm-3) in the pH range from 8.0 to 9.5 with a detection limit of 1.0 × 10-6 M. The sensor showed near Nernstian characteristics (27.8 ± 0.5 mV dec-1) at pH 9.0. Since the responding potential was attributed to the activity of HPO42-, the potential at a given concentration of phosphate depended on the pH. The electrode indicated a good selectivity with respect to other common anions such as NO3-, SO42-, Cl-, HCO3- and CH3COO-. The modified molybdenum electrode can be continuously used for over a 1 month with good reproducibility. The feasibility of the electrochemical sensor was proved by successful for the detection of phosphate in real samples.

Bioelectrocatalytic performance of d-fructose dehydrogenase.

This review summarizes the bioelectrocatalytic properties of d-fructose dehydrogenase (FDH), while taking into consideration its enzymatic characteristics. FDH is a membrane-bound flavohemo-protein with a molecular mass of 138 kDa, and it catalyzes the oxidation of d-fructose to 5-keto-d-fructose. The characteristic feature of FDH is its strong direct-electron-transfer (DET)-type bioelectrocatalytic activity. The pathway of the DET-type reaction is discussed. An overview of the application of FDH-based bioelectrocatalysis to biosensors and biofuel cells is also presented, and the benefits and problems associated with it are extensively discussed.

The origin of hyperpolarization based on the directional conduction of action potential using a model nerve cell system.

In nerve cells, changes in local membrane potentials are generated and propagated along a nerve axon mainly by the function of K+ and Na+ channels. Generally, concurrent monitoring of multi-points on an axon is performed based on the voltage-clamp method. As the respective membrane potentials have been evaluated by considering the relations between the applied potential, the local current, and conductance, experimental values are not directly evaluated. We directly measured the actual membrane potentials and local currents of the respective cells using a nerve-model system comprising liquid-membrane cells. It was then proven that the action potential spreads along the axon toward the axon terminal due to the function of both the channel-type receptors in the synapse and voltage-gated Na+ channels on the axon, and that hyperpolarization cannot be caused by only the operation of the delayed-K+ and the voltage-gated Na+ channels.

Construction of an Automatic Nutrient Solution Management System for Hydroponics-Adjustment of the K+-Concentration and Volume of Water.

An automatic management system for nutrient solutions was constructed using a programmable logic controller (PLC) and a K+-ion selective electrode (K+-ISE). The concentration of K+ was monitored by the K+-ISE. When the concentration of K+ fell to the threshold limit, an appropriate amount of a concentrated K+ solution was added to the hydroponic solution. The volume was also maintained at a constant level by addition of water. This system can be constructed simply and inexpensively without any computers and pumps.

Nanostructured Porous Electrodes by the Anodization of Gold for an Application as Scaffolds in Direct-electron-transfer-type Bioelectrocatalysis.

In this study, nanostructured porous gold electrodes were prepared by the anodization of gold in the presence of oxalic acid or glucose as a reductant, and applied as scaffolds for direct electron transfer (DET)-type bioelectrocatalysis. Gold cations generated in the anodization seem to be reduced by the reductant to construct a porous gold structure. The DET-type performance of the electrode was examined using two DET-type model enzymes, bilirubin oxidase (BOD) and peroxidase (POD), for the four-electron reduction of dioxygen and the two-electron reduction of peroxide, respectively. BOD and POD on the anodized porous gold electrodes exhibited well-defined sigmoidal steady-state waves corresponding to DET-type bioelectrocatalysis. Scanning electron microscopy images revealed sponge-like pores on the electrodes. The anodized porous gold electrodes demonstrate promise as scaffolds for DET-type bioelectrocatalysis.

Construction of Nitrate-selective Electrodes and Monitoring of Nitrates in Hydroponic Solutions.

A liquid-membrane type nitrate-selective electrode was constructed, in which the responding membrane contained polyvinylchloride, o-nitrophenyloctylether and tetraheptylammonium nitrate. The NO3--selective electrode displayed a linear response to the concentration of NO3- with a Nernstian slope of -53.3 ± 1.0 mV decade-1, in the 10-5 - 10-1 mol dm-3 (M) NO3- concentration range. The NO3- detection limit was about 10-6 M. The electrochemical response of this electrode was stable for more than 30 days. Measurements performed using the NO3--sensor indicated that in the presence of green plants, the concentration of NO3- in a hydroponic solution decreased from 0.20 to 0.05 mM over a three-day period.