Electrochemical Molecular Conversion of α-Keto Acid to Amino Acid at a Low Overpotential Using a Nanoporous Gold Catalyst.

A nanoporous gold (NPG) electrode prepared through a facile anodization technique was employed in the electrochemical reductive amination of biomass-derivable α-keto acids in the presence of a nitrogen source to produce the corresponding amino acids. NPG showed a clear reductive current in the presence of α-keto acid and NH2OH, and the electrolysis experiments confirmed the production of L-amino acid. A reductive voltammetric signal at the NPG electrode appeared at a more positive potential by 0.18-0.79 V, compared with those at the planar-gold electrode without anodization and other previously reported electrode systems, indicating the high activity of the prepared nanostructure for the electrochemical reaction. Maximum Faradaic efficiencies (FEs) of 74-93% in the reductive molecular conversion to amino acids of Ala, Asp, Glu, Gly, and Leu were obtained under the optimized conditions. The FE values were strongly dependent on the applied potential in the electrolysis, suggesting that the hydrogen evolution reaction at the electrode surface was more significant as the applied potential became more negative. The effect of potential at the NPG was lower than that at the planar-gold electrode. These results indicate that nanostructurization decreases the overpotential for the electrochemical reductive amination, resulting in high FE.

Electrochemical Oxidation of Monosaccharides at Nanoporous Gold with Controlled Atomic Surface Orientation and Non-Enzymatic Galactose Sensing.

Non-enzymatic saccharide sensors are of great interest in diagnostics, but their non-selectivity limits their practical diagnostic abilities. In this study, we investigated the electrochemical oxidation of monosaccharides at nanoporous gold (NPG) catalysts with different contributions of surface crystallographic orientations. Fructose elicited no clear electrochemical response, but glucose, galactose, and mannose produced clear oxidative current. The onset potentials for oxidation of these saccharides depended on the surface atomic structure of the NPG. The oxidation potential was approximately 100 mV less positive at the Au(100)-enhanced NPG than at the Au(111)-enhanced NPG. Furthermore, the voltammetric responses significantly differed among the saccharides. Galactose was oxidized at less positive potential and exhibited a higher current response than the other saccharides. This tendency was enhanced in the presence of chloride ions. These features enabled the selective and sensitive detection of galactose at an NPG electrode without enzymes under physiological conditions. A linear range of 10 µM to 1.8 mM was obtained in the calibration plot, which was comparable to those in previously reported enzymatic galactose sensors. Thus, we demonstrated that controlling the crystallographic orientation on the nanostructured electrode surface is useful in developing electrochemical sensors.

Redox State Control of Human Cytoglobin by Direct Electrochemical Method to Investigate Its Function in Molecular Basis.

The direct electron transfer between human cytoglobin (Cygb) and the electrode surface, which would allow manipulating the oxidation states of the heme iron in Cygb, was first observed by immobilizing Cygb on a nanoporous gold (NPG) electrode via a carboxy-terminated alkanethiol. The voltammetric performances of the wild type and mutated Cygb-immobilized NPG electrodes were evaluated in the absence or presence of potential substrates. The obtained results demonstrated that the usefulness of the proposed method in understanding the function of Cygb in molecular basis.

Function Control of Anti-microRNA Oligonucleotides Using Interstrand Cross-Linked Duplexes.

MicroRNA (miRNA)-guided argonaute (Ago) controls gene expression upon binding to the 3' UTR of mRNA. The miRNA function can be competitively inhibited by single-stranded anti-miRNA oligonucleotides (AMOs). In this study, we constructed a novel type of AMO flanked by interstrand cross-linked 2'-O-methylated RNA duplexes (CLs) that confer a stable helical conformation. Compared with other structured AMOs, AMO flanked by CLs at the 5' and 3' termini exhibited much higher inhibitory activity in cells. Anti-miRNA activity, nuclease resistance, and miRNA modification pattern distinctly differed according to the CL-connected positions in AMOs. Moreover, we found that the 3'-side CL improves nuclease resistance, whereas the 5'-side CL contributes to stable binding with miRNA in Ago upon interaction with the 3' part of miRNA. These structure-function relationship analyses of AMOs provide important insights into the function control of Ago-miRNA complexes, which will be useful for basic miRNA research as well as for determining therapeutic applications of AMO.

Electrochemical properties of interstrand cross-linked DNA duplexes labeled with Nile blue.

DNA molecules have attracted considerable attention as functional materials in various fields such as electrochemical sensors with redox-labeled DNA. However, the recently developed interstrand cross-link (ICL) technique for double-stranded DNA can adequately modify the electronic properties inside the duplex. Hence, the electrochemical investigation of ICL-DNA helps us to understand the electron transfer of redox-labeled DNA at an electrode surface, which would develop useful sensors. In this study, the first insight into this matter is presented. We prepared 17-mer DNA duplexes incorporating Nile blue (NB-DNA) at one end as a redox marker and a disulfide tether at the other end for immobilization onto an electrode. The duplexes were covalently cross-linked by bifunctional cross-linkers that utilize either a propyl or naphthalene residue to replace a base pair. Their electrochemical responses at the electrode surface were compared to evaluate the effect of the ICL on the electron-transfer reactions of the redox-labeled DNA duplexes. A direct transfer of electrons between NB and the electrode was observed for a standard DNA, as previously reported, whereas interstrand cross-linked DNA (CL-DNA) strands showed a decrease in the direct electron-transfer pathway. This is expected to result from constraining the elastic bending/flexibility of the duplex caused by the covalent cross-links. Interestingly, the CL-DNA incorporating naphthalene residues exhibited additional voltammetric peaks derived from DNA-mediated electron transfer (through base π stacking), which was not observed in the mismatched CL-DNA. The present results indicate that the ICL significantly affects electron transfer in the redox-labeled DNA at the electrode and can be an important determinant for electrochemical signaling in addition to its role in stabilizing the duplex structure.

Successful management of cancer related pruritus by using mirtazapine: a case report

<b>Introduction</b>: It is difficult to manage that pruritus complicated with jaundice, for invalidity of almost all antihistamines. Recently, effects of paroxetine for pruritus are reported, but the report to invalid cases of paroxetine is rare. We report a case treated effectively with mirtazapine for pruritus of the paroxetine invalid. <b>Case report</b>: A 56-years old woman was diagnosed cancer of head of pancreas and peritoneal dissemination.After stenting by a plastic stent for obstructive jaundice in previous hospital, she came to our hospital. But, her total bilirubin (T-bil) were very high (9.9 mg/d<i>l</i>), and she was suffering from systemic pruritus. The NRS (numerical rating scale) score for pruritus was 9-10. Though she was prescribed an antihistamine in previous hospital, it was invalid. We had changed it to paroxetine, but pruritus were protraction two weeks later. After changing it to mirtazapine, the pruritus became NRS 1 on the next day, and recurrence was not seen subsequently. <b>Conclusion</b>: For pruritus of the paroxetine invalid, mirtazapine is important as one of the choices.

Physiological role and redox properties of a small cytochrome c(5), cytochrome c-552, from alkaliphile, Pseudomonas alcaliphila AL15-21(T).

Cytochrome c-552 from Pseudomonas alcaliphila AL15-21(T), which is a small cytochrome c(5) from Pseudomonas spp., was first purified and characterized in our previous study. Although it has been found that cytochrome c-552 is induced at a high pH under air-limited condition, the physiological role of this cytochrome c has not been clarified yet. Therefore, to understand its physiological role, further characterization of this cytochrome expressed in Escherichia coli was performed. The yield of the recombinant protein reached 2.8 mg/l of culture, which was 76.4-fold larger than that of native cells. Analytical data of the recombinant protein exactly agreed with that of native cytochrome c-552. The recombinant cytochrome c-552 was oxidized by partially purified cb-type cytochrome c oxidase from P. alcaliphila AL15-21(T) at a rate of 9.6 mu mol min(-1) mg oxidase(-1). Unlike reported cytochromes c from other Pseudomonas spp., the E degrees ' values between pHs 5.0 and 10.0 were nearly unchanged. Cytochrome c-552 oxidized very slowly at pHs 8.0 (6.1 x 10(-4) h(-1)), 9.0 (1.4 x 10(-3) h(-1)) and 10.0 (1.6 x 10(-3) h(-1)), whereas it oxidized more rapidly at pH 7.0 (2.5 x 10(-3) h(-1)). On the other hand, horse heart cytochrome c showed higher oxidation rates at pHs 6.0-10.0 than cytochrome c-552. It is considered that the high electron-retaining ability of cytochrome c-552 at high pHs is important for its physiological function in the environmental adaptation of this bacteria for superior growth at high pHs under air-limited conditions.

Selective protein patterning based on the micro-structured organosilane self-assembled monolayer by vacuum ultraviolet light lithography.

We have succeeded to immobilize fluorescent proteins selectively using a micro-structured organosilane self-assembled monolayer as a template. An organosilane layer with amino terminal group was formed on a thermally oxidized Si wafer by liquid-phase method and then was pattern-etched by vacuum ultraviolet light (VUV). The second organosilane layer with thiol terminal group was deposited on the etched area by chemical vapor surface modification method (CVSM). These micro-structured organosilane layer containing two reactive terminal groups were chemically modified using bi-functional linkers. Two kinds of fluorescent protein, Enhanced Cyan Fluorescent Protein (ECFP) and R-phycoerythrin were selectively immobilized on the chemically modified surface.

Electrochemically driven drug metabolism by membranes containing human cytochrome P450.

Rapid analyses of drug metabolism reactions by human cytochrome P450s (CYPs) that metabolize 95% of all current drugs are very important in drug development processes and effective therapies. Since CYPs need electrons to metabolize drugs, electrons supplied from electrodes to activate CYP molecules are expected to be very useful to develop rapid assay methods for CYP reactions. Although several studies on the direct electrochemistry of isolated and purified human CYPs have been reported, the use of microsomes (membranes) containing CYP is more suitable, because they are frequently used in drug research due to their easy preparation and low cost. Herein, we demonstrate electrons supplied from an electrode to microsomes containing CYP and CYP-reductase (CPR) on an electrode coated with hydrophobic thin films and observe electrochemically driven drug metabolisms by voltammetry for the first time. We tested the immobilization of microsomes on gold electrodes coated with several thiolates and found that microsomes immobilized on thin hydrophobic surfaces of aromatic compounds showed well-defined redox peaks. Furthermore, electrochemically driven drug metabolism reactions of CYP3A4 were clearly observed by voltammetry, and these reactions were inhibited by a CYP3A4 inhibitor, ketoconazole. We also found that metabolism reactions were facilitated in the presence of CPR.

A novel membrane-anchored cytochrome c-550 of alkaliphilic Bacillus clarkii K24-1U: expression, molecular features and properties of redox potential.

A membrane-anchored cytochrome c-550, which is highly expressed in obligately alkaliphilic Bacillus clarkii K24-1U, was purified and characterized. The protein contained a conspicuous sequence of Gly(22)-Asn(34), in comparison with the other Bacillus small cytochromes c. Analytical data indicated that the original and lipase-treated intermediate forms of cytochrome c-550 bind to fatty acids of C(15), C(16) and C(17) chain lengths and C(15) chain length, respectively, and it was considered that these fatty acids are bound to glycerol-Cys(18). Since there was a possibility that the presence of a diacylglycerol anchor contributed to the formation of dimeric states of this protein (20 and 17 kDa in SDS-PAGE), a C18M (Cys(18) --> Met)-cytochrome c-550 was constructed. The molecular mass of the C18M-cytochrome c-550 was determined as 15 and 10 kDa in SDS-PAGE and 23 kDa in blue native PAGE. The C18M-cytochrome c-550 bound with or without Triton X-100 formed a tetramer as the original cytochrome c-550 bound with Triton X-100, as determined by gel filtration. The midpoint redox potential of cytochrome c-550 as determined by redox titration was +83 mV, while that determined by cyclic voltammetric measurement was +7 mV. The above results indicate that cytochrome c-550 is a novel cytochrome c.

Comparison of enzymatic recycling electrodes for measuring aminophenol: development of a highly sensitive natriuretic peptide assay system.

Several redox enzymes were examined for enzymatic/electrochemical-recycling systems in order to measure p-aminophenol (PAP) with high sensitivity. Glucose oxidase (GOD) and diaphorase (DI) worked well as catalysts for recycling electrode systems: these enzymes effectively reduced p-iminoquinone (PIQ), the electrochemically-oxidized form of PAP, and caused an enhancement in the electrochemical signals (anodic currents in the voltammogram and amperogram) by approximately 100 fold. The lower detection limits for PAP were estimated to be 50 nM with the GOD system and 2 nM with the DI system. We combined the enzymatic-recycling electrode using DI with an enzyme immunoassay system to measure atrial natriuretic peptide (ANP), an important marker peptide hormone involved in heart diseases. ANPs from serum samples at ppt-levels were determined appropriately using the present assay system.

Direct electrochemistry of engineered cytochrome b562 molecules with a ligand binding pocket.

The rapid and reversible electron transfer reaction of cytochrome b562 was observed at an In2O3 electrode. The estimated heterogeneous electron transfer rate constant (k0') was k0' > or = 5.0 x 10(-3) cm s(-1) at pH 6.5. When the methionine-7 (Met-7) residue, which coordinates to the heme iron as an axial ligand, of the wild-type cytochrome b562 was replaced by an Ala or Gly residue, a water molecule bound to the heme iron and the electron transfer rate constants decreased to 1.3 x 10(-3) and 1.8 x 10(-3) cm s(-1), respectively. This decrease in the electron transfer rate would be due to the larger reorganization energy for the structural change at the redox site. The midpoint potential of cytochrome b562 was shifted negatively by approximately 135 mV by replacing Met-7 with Ala or Gly. Similar dissociation kinetics of cyanide for the mutated molecules as compared to native myoglobin was obtained.

Notable deuterium effect on the electron transfer rate of myoglobin.

The electron transfer reaction of wild-type myoglobin at an electrode was significantly facilitated in a D2O buffer as compared with that in an H2O buffer, with k(0)'(H2O)/k(0)'(D2O)= 0.13, while a minimal deuterium kinetic isotope effect on the myoglobin with modification at distal histidine (His-64) was observed.

Functional evaluation of heme vinyl groups in myoglobin with symmetric protoheme isomers.

We replaced protoheme-IX in native myoglobin with the symmetric protohemes-III and -XIII, in order to investigate the role of heme vinyl-globin contacts on Mb function. The UV-visible spectra and the resonance Raman spectra in the high-frequency region (containing oxidation, spin, and coordination state marker lines) of the two reconstituted Mbs were very similar. However, the signal intensity of the Soret band in the CD spectra and the resonance Raman lines for vinyl bending modes in the low-frequency region notably differed, thereby reflecting altered heme peripheral contacts. The redox potentials, formal heterogeneous electron-transfer rates, and thermal denaturation temperatures of the two reconstituted Mbs were also indistinguishable. In addition, the oxygen binding properties of the ferrous deoxy Mbs were comparable. These results demonstrate that altered heme vinyl-globin interactions only slightly affect the physical properties of Mb. It is therefore likely that the orientation of protoheme-IX about the alpha,gamma-axis in the heme pocket is not necessarily a crucial factor for oxygen binding to native Mb.