Identification of amino acid residues participating in the energy coupling and proton transport of Streptomyces coelicolor A3(2) H+-pyrophosphatase

AbstractThe H+-translocating inorganic pyrophosphatase is a proton pump that hydrolyzes inorganic pyrophosphate. It consists of a single polypeptide with 14–17 transmembrane domains (TMs). We focused on the third quarter region of Streptomyces coelicolor A3(2) H+-pyrophosphatase, which contains a long conserved cytoplasmic loop. We assayed 1520 mutants for pyrophosphate hydrolysis and proton translocation, and selected 34 single-residue substitution mutants with low substrate hydrolysis and proton-pump activities. We also generated 39 site-directed mutant enzymes and assayed their activity. The mutation of 5 residues in TM10 resulted in low energy-coupling efficiencies, and mutation of conserved residues Thr409, Val411, and Gly414 showed neither hydrolysis nor pumping activity. The mutation of six, five, and four residues in TM11, 12, and 13, respectively, gave a negative effect. Phe388, Thr389, and Val396 in cytoplasmic loop i were essential for efficient H+ translocation. Ala436 and Pro560 in the periplasmic loops were critical for coupling efficiency. These low-efficiency mutants showed dysfunction of the energy-conversion and/or proton-translocation activity. The energy efficiency was increased markedly by the mutation of two and six residues in TM9 and 12, respectively. These results suggest that TM10 is involved in enzyme function, and that TM12 regulate the energy-conversion efficiency. H+-pyrophosphatase might involve dynamic linkage between the hydrophilic loops and TMs through the central half region of the enzyme

Method for Assessing X-ray-Induced Hydroxyl Radical Scavenging Activity of Biological Compounds/Materials

A method for correctly assessing hydroxyl radical scavenging activity of antioxidative chemicals and/or biological compounds/materials was proposed. This method can simultaneously assess two factors, i.e. hydroxyl radical scavenging and 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide (hydroxyl radical adduct of 5,5-dimethyl-1-pyrroline-N-oxide) reducing ability, as antioxidative properties. In this paper, some biologically common hydrophilic molecules, cell culture media, and rat plasma were tested. X-ray induced hydroxyl radical can be detected using the electron paramagnetic resonance spin trapping technique. Using X-ray irradiation of the reaction mixture as the hydroxyl radical source, the true hydroxyl radical scavenging ability of the subjected antioxidant can be assessed. In addition, the method simultaneously measures the reduction of 5,5-dimethyl-2-hydroxy-1-pyrrolidine-N-oxide, to estimate the reducing ability of the antioxidant. Biological materials, such as sugars and proteins, could abolish hydroxyl radical at the biological concentration. Ascorbic acid showed reducing ability at the biological concentration. The simultaneous assessment of hydroxyl radical scavenging and reducing ability of antioxidants can be an informative index for antioxidants

Compact tree plus algorithms for application-level multicast communications in multihome networks

Application-level multicast (ALM) communications replicate packets on host level to deliver them from a single source to multiple clients, so that it can efficiently realize a variety of network applications using moving pictures such as video conferences, distance learning, and video-on-demands. In this paper, we propose the CT+ (compact tree plus) algorithm for finding a better ALM routing tree in terms of delay minimization between hosts. CT+ consists of a tree construction stage from the existing CT algorithm, and a newly added iterative tree improvement stage. Then, we define the extended ALM routing problem and its heuristic algorithm ExCT+, to optimize the effectiveness of the multihome network in ALM communications by selecting multihomed hosts and connections in the ALM routing tree simultaneously. For their evaluations, we construct a network simulation model named MINET (multiple-ISP network simulator), where the topology is composed of multiple ISP backbone networks with IX connections, and the network traffic is generated by following the M/M/1 queuing process. The simulation results using MINET verify the effectiveness of our algorithms.</p

Optical-drop wavelength assignment problem for wavelength reuse in WDM ring metropolitan area networks

This paper presents a formulation of the optical-drop wavelength assignment problem (ODWAP) and its heuristic algorithm for WDM ring networks. The wavelength-division multiplexing (WDM) technology has been popular in communication societies for providing very large communication bands by multiple lightpaths with different wavelengths on a single optical fiber. Particularly, a double-ring optical network architecture based on the packet-over-WDM technology such as the HORNET architecture has been studied as a next generation platform for metropolitan area networks (MANs). Each node in this architecture is equipped with a wavelength-fixed optical-drop and a tunable transmitter so that a lightpath can be established between any pair of nodes without wavelength conversions. In this paper, we formulate ODWAP for efficient wavelength reuse under heterogeneous traffic in this network. Then, we propose a simple heuristic algorithm for ODWAP. Through extensive simulations, we demonstrate the effectiveness of our approach in reducing waiting times for packet transmissions when a small number of wavelengths are available to retain the network cost for MANs

Changes in biotin levels during production of natto, Japanese fermented soybean

The change of biotin level during production of natto (Japanese fermented soybean) was investigated in this study.  The total biotin level was measured by an agar plate bioassay using Lactobacillus plantarum ATCC 8014.  The total biotin level decreased during water soaking, but increased after the fermentation of soybeans using Bacillus subtilis var. natto (B. natto) and reached a maximum level.  The increase of total biotin was not affected by Asp, Arg, and Ile which promoted the growth of L. plantarum in high concentrations.  The peak level of biotin in the fermented soybeans was significantly higher than that of dry soybeans.  The fermented soybeans at the biotin peak level were adequate for food.  In addition, we detected 9 and 4 biotinylated polypeptides in the soybeans and B. natto used in this study, respectively.  We speculated that the increase of biotin level may depend on the increase of the 4 biotinylated polypeptides and free biotin in B. natto

Estimation of the local concentration of the markedly dense hydroxyl radical generation induced by X-rays in water

The density of X-ray-induced markedly dense hydroxyl radicals (•OH) in water was estimated based on EPR spin-trapping measurement using DMPO as the spin-trapping agent. A lower (0.13 mM‒2.3 M) concentration series of DMPO water solutions and higher (1.7‒4.8 M) concentration series of DMPO water solutions plus neat DMPO liquid (8.8 M as DMPO) were irradiated with 32 Gy of X-rays. Then, the yield of DMPO-OH in DMPO water solutions and the total spin-adduct of DMPO in neat DMPO were quantified. Estimating the EPR signal peak area by the line fitting method is only available for well-separated single EPR lines. For the higher concentration DMPO series, the EPR signal peak area was estimated by double integration. The baseline correction of the integral spectrum is necessary for accurate estimation of the double integration of EPR spectrum. In addition, using the suitable standard sample corresponding to the EPR sensitivity, which was variable by the electric permittivity according to DMPO concentration, was quite important for quantification of DMPO-OH especially in the higher DMPO concentration beyond 2 mM. The local density of •OH generation in water by X-ray irradiation was estimated from the inflection point on the plot of the DMPO-OH yield versus DMPO density. The density of X-ray-induced markedly dense •OH was estimated as 1184 μm-1, which was converted to 0.84 nm as the intermolecular distance and 2.8 M as the local concentration

Trial of Brain Redox Imaging and Estimation of Radiation-Induced Redox Change in Mouse Brain

The in vivo T1-weighted contrasting abilities and signal decay behaviors of several nitroxyl contrast agents, which have been used as redox responsive contrast agents in several magnetic resonance-based imaging modalities, in mouse brain were compared. In addition, daily variations of redox behavior in mouse brain after irradiation of X-ray or carbon-ion beams (C-beam) were tried to estimate based on the in vivo reduction rate of amphiphilic nitroxyl contrast agents.Injection solutions of five types of five-membered-ring nitroxyl contrast agents, i.e. 3-carboxy-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CxP), 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (CmP), 3-methoxy-carbonyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl (MCP), acetoxymethyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-carboxylate (CxP-AM), and 4-(N-methylpiperidine)-2,2,5,5-tetramethylpyrroline-N-oxyl (23c), and a six-membered-ring nitroxyl contrast agent, i.e. 4-hydroxyl-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), were prepared. The nitroxyl contrast agent was i.v. injected to a mouse through tail vein. Then, the distributions and pharmacokinetics of nitroxyl contrast agents were compared based on the time course of T1-weighted MRI. The MRI experiments using CMP or TEMPOL were repeated for mice irradiated by X-ray or C-beam to their head on several deferent timings, i.e. 1, 2, 4, 8 day(s) after irradiation. C-beam was irradiated at Heavy-Ion Medical Accelerator in Chiba (HIMAC, National Institute of Radiological Sciences/ National Institutes for Quantum and Radiological Science and Technology).The blood-brain-barrier (BBB)-impermeable CxP could not be distributed in the brain. The slightly lipophilic CmP showed slight distribution only in the ventricle, but not in the medulla and cortex. The amphiphilic MCP and TEMPOL had good initial uniform distribution in the brain and showed typical 2-phase signal decay profiles. A brain-seeking nitroxyl probe, CxP-AM, showed an accumulating phase, and then its accumulation was maintained in the medulla and ventricle regions, but not in the cortex. The lipophilic 23c was well distributed in the cortex and medulla, but slightly in the ventricle, and showed relatively rapid linear signal decay.Decay rates of MCP in mouse brain after irradiation of 8 Gy X-ray, 8 Gy C-beam or 16 Gy C-beams did not show marked clear changes, however relatively little decreasing were observed at day 1 and day 2 after irradiation. Decay rates of TEMPOL was increased 1 after irradiation then gradually recovered to the control level. MCP and TEMPOL showed opposite responses but the timing of redox change may be 1 or 2 days after irradiation.Nitroxyl contrast agents equipped with a suitable lipophilic substitution group could be BBB-permeable functional contrast agents. MR redox imaging, which can estimate not only the redox characteristics but also the detailed distribution of the contrast agents, is a good candidate for a theranostic tool. Irradiation of ionized radiation to head could cause alternation of redox status in the brain. Detail of redox mechanisms were still in progress.第7回国際放射線神経生物学会大

Analysis of the Antioxidative Function of the Radioprotective Japanese Traditional (Kampo) Medicine, Hangeshashinto, in an Aqueous Phase

Oral mucositis (OM) is a common and painful complication of radiotherapy for head and neck cancer. Hangeshashinto (HST), a Japanese traditional medicine, is known to alleviate radiotherapy and/or chemotherapy-induced OM; however, the detailed mechanism has not yet been clarified. The aim of the present study is to clarify the details of the antioxidative functions of HST against reactive oxygen species (ROS) produced by radiation. The hydroxyl radical (•OH) scavenging ability and reduction ability was simultaneously measured using a modified electron paramagnetic resonance (EPR) spin trapping method. The superoxide (O2•−) scavenging ability was estimated by an EPR redox probing method. Water suspension of powdered HST and its seven constitutive crude drugs were tested. In addition, some of the main water soluble ingredients of the crude drugs were also tested. HST was found to scavenge both •OH and O2•−. Furthermore, HST was observed to reduce relatively stable nitroxyl radicals. Glycyrrhizae Radix (kanzo), Ginseng Radix (ninjin), Zizyphi Fructus (taiso), and glycyrrhizin (an ingredient of kanzo) were all found to be relatively good •OH scavengers. Scutellariae Radix (ogon) and Coptidis Rhizoma (oren) demonstrated reducing ability. In addition, acteoside and berberine chloride, which are water soluble ingredients of ogon and oren, respectively, also demonstrated reducing ability. Oren exhibited oxidative ability at higher concentrations, which may have a function to maintain catalytic redox action. The antioxidative function of HST probably worked in a balance of scavenging ROS, reducing stable free radicals and some minor oxidative effects

EPR based Estimation of Radiation-Induced Reactive Oxygen Species

Generation of reactive oxygen species (ROS) is considered as essential trigger of biological effects of ionizing radiations, and may be deeply linked with the radiation quality.Amounts of total oxidation reactions (i.e. oxidative free radical species, •OH and HO2•), H2O2 generations, Oxygen consumptions, and •OH generations induced by X-ray, 20 keV/μm carbon beam, and 80 keV/μm carbon beam were estimated using EPR based techniques.Total oxidation reactions were estimated as 3, 1.3, and 0.66 μmol/L/Gy, amount of H2O2 generations were 0.2, 0.57, and 0.35 μmol/L/Gy, oxygen consumptions were 0.4, 0.39, and 0.15 μmol/L/Gy for X-ray, 20 keV/μm carbon beam, and 80 keV/μm carbon beam, respectively. The ratio of H2O2 generation per oxygen consumption were increased with LET, and were 0.5, 1.46, 2.33 for X-ray, 20 keV/μm carbon beam, and 80 keV/μm carbon beam, respectively. The •OH generations expected to be localized on the track/range of the radiation beam/ray, and both sparse (≈ 3.3 mM) and very dense (> 1.7 M) •OH generations were suggested. Percentage of sparse •OH generation decreased with LET becoming higher.The SFRBM\u27s 23rd Annual Meeting, a joint meeting with the Society for Free Radical Research International (SFRBM/SFRRI 2016

課題解決型授業における福井市の伝統野菜「河内赤かぶら」を用いた取り組み

Kawachi akabura is a traditional vegetable of Fukui City. It is characterized by its beautiful red fruit and large, handsome leaves. In fact, it is eaten with various arrangements such as tsukemono（Japanese pickles）and pound cake, but the leaves are discarded in the field at the time of harvest. Therefore, students who had taken the special education subjects of the Department of Human Life Science, “Nutrition Experiment” and “Graduation Research and Graduation Production”, took the lead in examining the utilization of Kawachi Akakabura leaf. As a result, it became clear that it could be used not only for dishes such as “takikomi gohan” and “gyoza,” but also for confectioneries such as “pudding,” “macaron” and “baumkuchen.” From this, it became clear that Kawachi akabura leaves, which are currently disposed of in large quantities, are easy to arrange