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Some carotenoids are found in the Euglena gracilis, including β-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated β-carotene to β-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of β-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be β-carotene monohydroxylase, but not β-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga.

There are still considerable gaps in knowledge regarding the biological effects of chronic ionising radiation exposure in amphibians. To fill these gaps, Tohoku hynobiid salamanders, Hynobius lichenatus (Amphibia, Caudata), were chronically irradiated with 137Cs γ-rays from embryonic to adult stages over 1954 days, and the effects on their growth and sexual maturation were examined under laboratory conditions. Irradiation at a dose rate of 33 μGy h−1 had some stimulatory effects on growth (body weight increase) of H. lichenatus, while growth was temporarily or permanently suppressed at 150 or 510 μGy h−1, respectively. On day 1802, secondary sexual characteristics (a tubercle at the anterior angle of the cloacal vent for males and ovisac development for females) were observed in 91% of the salamanders irradiated at 33 μGy h−1, and in a similar percentage of non-irradiated controls. At 150 and 510 μGy h−1, secondary sexual characteristics were not observed in any individuals. These results suggest that the derived consideration reference level (DCRL) of the International Commission on Radiological Protection (ICRP) for Reference Frog, i.e. 40–400 μGy h−1, is applicable for the protection of H. lichenatus, and that growth and sexual maturation of this salamander may not have been adversely affected even in the most severely contaminated area in Fukushima, where the highest dose rate to salamanders was estimated to be 50 μGy h−1. However, observations in the contaminated area are required to confirm this conclusion, considering the possible confounding factors which may make this salamander more sensitive to radiation in the natural environment than under laboratory conditions.

Summary Sensory processing is essential for motor control. Climbing fibers from the inferior olive transmit sensory signals to Purkinje cells, but how the signals are represented in the cerebellar cortex remains elusive. To examine the olivocerebellar organization of the mouse brain, we perform quantitative Ca2+ imaging to measure complex spikes (CSs) evoked by climbing fiber inputs over the entire dorsal surface of the cerebellum simultaneously. The surface is divided into approximately 200 segments, each composed of ∼100 Purkinje cells that fire CSs synchronously. Our in vivo imaging reveals that, although stimulation of four limb muscles individually elicits similar global CS responses across nearly all segments, the timing and location of a stimulus are derived by Bayesian inference from coordinated activation and inactivation of multiple segments on a single trial basis. We propose that the cerebellum performs segment-based, distributed-population coding that represents the conditional probability of sensory events.

We report the fabrication of novel nanofibers using naturally occurring antimicrobial honey incorporated in poly(1,4-cyclohexane dimethylene isosorbide trephthalate) (PICT) for the potential wound dressing applications. We fabricated PICT/honey using three blend ratios 90:10, 85:15 and 80:20 respectively. Morphology of PICT nanofibers and PICT/honey nanofibers was observed under Scanning Electron Microscope and it showed bead-free nanofibers. Fourier Transform Infrared Spectroscope was used to confirm the presence of honey in PICT electrospun nanofibers. Tensile strength of PICT/honey nanofibers was slightly reduced with variation in effect of elongation. Water contact angle measurements were done with the static contact angle by a contact angle meter, which showed that hydrophobicity was decreased by adding the honey. The XPS spectra showed that honey was present in the PICT/honey nanofibers. The release behavior of honey was investigated by UV-visible Spectrophotometer. The release was complete in 15min and the maximum release of honey was 72mg/L in 10min. Therefore, PICT/honey nanofibers having 15% concentration of honey are suitable for good elastic behavior and tensile strength as compared to other concentrations of honey.

Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B12 deficiency, although the underlying disease mechanisms associated with vitamin B12 deficiency are poorly understood. Vitamin B12 deficiency was found to significantly increase cellular H2O2 and NO content in Caenorhabditis elegans and significantly decrease low molecular antioxidant [reduced glutathione (GSH) and L-ascorbic acid] levels and antioxidant enzyme (superoxide dismutase and catalase) activities, indicating that vitamin B12 deficiency induces severe oxidative stress leading to oxidative damage of various cellular components in worms. An NaCl chemotaxis associative learning assay indicated that vitamin B12 deficiency did not affect learning ability but impaired memory retention ability, which decreased to approximately 58% of the control value. When worms were treated with 1 mmol/L GSH, L-ascorbic acid, or vitamin E for three generations during vitamin B12 deficiency, cellular malondialdehyde content as an index of oxidative stress decreased to the control level, but the impairment of memory retention ability was not completely reversed (up to approximately 50%). These results suggest that memory retention impairment formed during vitamin B12 deficiency is partially attributable to oxidative stress., Graphical abstract fx1

We report the effects of lattice dynamics on thermodynamic stability of binary $R_{1-x}$Fe$_x$ $(0, Comment: 12 pages, 6 figures

High-pressure components are generally designed with safety factors based on the tensile strength (TS) of the material; accordingly, materials with higher TS permit designed components with thinner walls, which reduce the weight and cost of the parts. However, many high-strength metals are severely degraded by hydrogen. To this point, efforts to develop a high-strength metal with a TS far beyond 1000 MPa and excellent resistance to hydrogen embrittlement (HE) have failed. This study introduces a high-strength metal with an excellent HE resistance, composed of a precipitation-hardened copper-beryllium alloy with the TS of 1400 MPa. Slow strain rate tensile (SSRT) tests of both smooth and notched specimens were performed in 115-MPa hydrogen gas at room temperature (RT). The alloy had a relative reduction in area RRA ≈ 1 and a relative notch tensile strength RNTS ≈ 1, without degradation in either characteristic.

Euglena gracilis exhibits photomovements in response to various light stimuli, such as phototactic and photophobic responses. Our recent study revealed that carotenoids in the eyespot apparatus are required for triggering phototaxis in this alga. However, the role of chloroplasts in eyespot formation is not understood. Here, we isolated carotenoid-less (cl) strains of E. gracilis from cells silenced gene expression of phytoene synthase (EgcrtB). Unlike WT, the culture colors of cl1, cl3, and the non-photosynthetic mutant SM-ZK were orange, while that of cl4 was white. Electron microscope observations showed that SM-ZK, cl1, and cl3 had no developed chloroplast and formed a normal eyespot apparatus, similar to that of WT, but this was not the case for cl4. Carotenoids detected in WT were diadinoxanthin, neoxanthin, and β-carotene. However, the most abundant species of SM-ZK, cl1, and cl3 was zeaxanthin, and there was no diadinoxanthin or neoxanthin. Photomovement analysis showed that SM-ZK, cl1, and cl3 exhibited negative phototactic and photophobic responses, similar to those of WT, whereas cl4 lacked negative phototaxis. Taken together, the formation of the eyespot apparatus required for phototaxis is independent of chloroplast development in E. gracilis, suggesting that this property is different from other photosynthetic flagellates.

It is widely accepted that the cerebellum acquires and maintain internal models for motor control. An internal model simulates mapping between a set of causes and effects. There are two candidates of cerebellar internal models, forward models and inverse models. A forward model transforms a motor command into a prediction of the sensory consequences of a movement. In contrast, an inverse model inverts the information flow of the forward model. Despite the clearly different formulations of the two internal models, it is still controversial whether the cerebro-cerebellum, the phylogenetically newer part of the cerebellum, provides inverse models or forward models for voluntary limb movements or other higher brain functions. In this article, we review physiological and morphological evidence that suggests the existence in the cerebro-cerebellum of a forward model for limb movement. We will also discuss how the characteristic input–output organization of the cerebro-cerebellum may contribute to forward models for non-motor higher brain functions.

The radiological risks to the Tohoku hynobiid salamanders (class Amphibia), Hynobius lichenatus due to the Fukushima Dai-ichi Nuclear Power Plant accident were assessed in Fukushima Prefecture, including evacuation areas. Aquatic egg clutches (n = 1 for each sampling date and site; n = 4 in total), overwintering larvae (n = 1-5 for each sampling date and site; n = 17 in total), and terrestrial juveniles or adults (n = 1 or 3 for each sampling date and site; n = 12 in total) of H. lichenatus were collected from the end of April 2011 to April 2013. Environmental media such as litter (n = 1-5 for each sampling date and site; n = 30 in total), soil (n = 1-8 for each sampling date and site; n = 31 in total), water (n = 1 for each sampling date and site; n = 17 in total), and sediment (n = 1 for each sampling date and site; n = 17 in total) were also collected. Activity concentrations of (134)Cs + (137)Cs were 1.9-2800, 0.13-320, and 0.51-220 kBq (dry kg) (-1) in the litter, soil, and sediment samples, respectively, and were 0.31-220 and0.29-40 kBq (wet kg)(-1) in the adult and larval salamanders, respectively. External and internal absorbed dose rates to H. lichenatus were calculated from these activity concentration data, using the ERICA Assessment Tool methodology. External dose rates were also measured in situ with glass dosimeters. There was agreement within a factor of 2 between the calculated and measured external dose rates. In the most severely contaminated habitat of this salamander, a northern part of Abukuma Mountains, the highest total dose rates were estimated to be 50 and 15 μGy h(-1) for the adults and overwintering larvae, respectively. Growth and survival of H. lichenatus was not affected at a dose rate of up to 490 μGy h(-1) in the previous laboratory chronic gamma-irradiation experiment, and thus growth and survival of this salamander would not be affected, even in the most severely contaminated habitat in Fukushima Prefecture. However, further studies of the adult salamanders may be required in order to examine whether the most severe radioactive contamination has any effects on sensitive endpoints, since the estimated highest dose rate to the adults exceeded some of the guidance dose rates proposed by various organisations and programmes for the protection of amphibians, which range from 4 to 400 μGy h(-1). Conversely, at one site in Nakadori, a moderately contaminated region in Fukushima Prefecture, the dose rate to the adult salamanders in spring of 2012 was estimated to be 0.2 μGy h(-1). Estimated dose rates to the overwintering larvae in spring of 2012 were 1 and 0.2 μGy h(-1) at one site in Nakadori, and in Aizu, a less contaminated region in Fukushima Prefecture, respectively. These results suggest that there is a low risk that H. lichenatus will be affected by radioactive contamination in these districts, though further studies on dose rate estimation are required for definitive risk characterisation.

Background: Visceral fat accumulation is associated with insulin resistance and various metabolic complications. We aimed to determine the effect of ipragliflozin (sodium-dependent glucose transporter-2 inhibitor) versus metformin with a dipeptidyl peptidase-4 (DPP-4) inhibitor (sitagliptin) in reducing visceral fat in Asian patients with insufficiently controlled type 2 diabetes. Methods: A prospective, multicenter, blinded-endpoint, randomized controlled study was conducted to evaluate the efficacy of treatment with either ipragliflozin or metformin combined with sitagliptin on visceral fat reduction and glycemic control among Japanese patients with type 2 diabetes, HbA1c 7-10%, and BMI ≥22 kg/m2. Patients who met the eligibility criteria were randomly assigned (1:1) to receive ipragliflozin 50 mg (n=51) or metformin 1000-1500 mg daily (n=52). The primary outcome was rate change in visceral fat area measured by computed tomography at 24 weeks of therapy. Two radiologists, blinded to patients' clinical information and randomized treatment assignment, analyzed the images. Secondary outcomes included effects on blood glucose, fasting insulin level, and lipids. The full analysis set was used for analysis. Findings: Mean percent reduction in visceral fat area was significantly greater in the ipragliflozin group than in the metformin group (-12·06% vs. -3·65%, group difference [95% CI] -8·40%; [-16·4 to -3·38], P=0·040). The ipragliflozin group also showed significant lowering of fasting insulin (-20·73% vs. 0·85%, group difference [95% CI] -21·58%, [2·80 to 34·20], P=0·018), while HbA1c decreased in the metformin group compared with ipragliflozin group. Interpretation: Ipragliflozin significantly reduced the visceral fat area compared with metformin, as the secondary agent used in combination with DPP-4 inhibitor. Since the fasting insulin level decreased, the reduction in visceral fat associated with ipragliflozin administration might have contributed to the improvement in insulin resistance. Trial Registration Number: Trial registration: UMIN 000015170. Funding Statement: To conduct this study, an agreement was signed between Chiba University and Astellas Pharma Inc. (Tokyo, Japan). Astellas Pharma Inc. funded this work. Declaration of Interests: KY received research grants from Astellas Pharma Inc. and MSD K.K. (Tokyo, Japan), and received a lecture fee from Astellas Pharma Inc. and Sumitomo Dainippon Pharma (Tokyo, Japan). No conflicts of interest are declared for other authors. Ethics Approval Statement: The study protocol was approved by the Institutional Review Boards (IRBs), Ethics Review Committees, or Ethics Committees of the following institutions: Chiba University Hospital (ID number: G26009), Asahi General Hospital (ID number: 2014091602), National Hospital Organization Chiba Medical Center, Seirei Sakura Citizen Hospital, Chiba Rosai Hospital (ID number: 26-21), Toho University Sakura Medical Center (ID number: 2014-077), Tokyo Women’s Medical University Yachiyo Medical Center (ID number: 150303), Chiba Aoba Municipal Hospital, Kimitsu Chuo Hospital, Funabashi Central Hospital (ID number: H27-1), and Chiba Kaihin Municipal Hospital. In other facilities, approval was provided at Chiba University Hospital (which had the centralized IRB).

We have developed a structure searching technique, which is referred to as “potential energy surface trekking”. In this method, the system is forced to climb up a potential energy surface to its ridges by the inversion of restoring forces and then to climb down toward other local minima of the surface by the release from the inversion across the ridges. One quickly obtains multiple stable structures existing around a starting point of the simulation by increasing the number of trekking paths. We have combined the technique with first-principles calculations based on the density functional theory and applied it to carbon at terapascal pressures. As a result, distorted simple hexagonal, distorted simple cubic, β -tin, and bc8-like structures have been obtained in addition to earlier-predicted bc8, r8, and simple cubic structures. While each structure has its own mechanically stable pressure region within 0.7–3.2 TPa, Gibbs free energy comparisons show that the earlier-predicted sequence of the phase transitions, cubic diamond → bc8 → simple cubic, eventually emerges up to at least 6000 K.

Anthocyanins are important for preventing photoinhibition and photodamage. By comprehensive reverse genetic analysis of chloroplast-produced H2O2-responsive genes, we isolated here an anthocyanin-deficient mutant under photooxidative stress, which lacked ferulate 5-hydroxylase 1 (FAH1) involved in the phenylpropanoid pathway. Interestingly, the expression of anthocyanin biosynthesis-associated genes was also inhibited in this mutant. These findings suggest that FAH1 is essential for expression of anthocyanin biosynthesis-associated genes and anthocyanin accumulation under photooxidative stress in Arabidopsis. Furthermore, we found that estrogen-inducible silencing of thylakoid membrane-bound ascorbate peroxidase, which is a major H2O2-scavenging enzyme in chloroplasts, enhances the expression of FAH1 and anthocyanin biosynthesis-associated genes and accumulation of anthocyanin without any application of stress. Thus, it is likely that chloroplastic H2O2 activates FAH1 expression to induce anthocyanin accumulation for protecting cells from photooxidative stress.

We examined the effects of atorvastatin on urinary podocyte excretion. Thirteen patients with type 2 diabetes receiving 2.5 mg of rosuvastatin were recruited and the medication was switched to 10 mg of atorvastatin for a 24-week period. With the switch to atorvastatin, the urinary excretion of podocytes was significantly reduced.

A spinel-type compound CuCr2S4 has the Curie temperature T c ≃ 380 K. A family (Cu1−xAgx)(Cr0.50Sn0.50)2S4 indicates a double non-magnetic substitution on A- and B-sites in the spinel, with a fixed composition of 0.50 on B-site. An exotic hump anomaly of the magnetization arises over 30–130 K, while a conventional spin-glass behavior is observed below T g =17 K. Irreversible behavior between zero-field-cooled (ZFC) and field-cooled (FC) magnetizations has been detected in these two different temperature regions T 17 K and 30 K T 130 K . Experimental fine specification for causing this hump will be presented, with an emphasis on the sample preparation. The hump emerges from the restricted composition 0.45 ≤ x ≤ 0.58 with annealing procedure, on the contrary, the quenched specimen extinguishes this hump. Magnitude of the hump varies intensely with applied magnetic field. A simple model to explain this novel hump will be discussed on the basis of the ionic radius of the substituted elements.

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.

To clarify genetically the involvement of two Arabidopsis NADPH oxidases (AtrbohD and AtrbohF) in the jasmonic acid (JA) signaling pathway, we characterized single knockout mutants lacking either Atrboh. The accumulation of reactive oxygen species (ROS) and expression of the genes regulated by MYC2, a transcription factor involved in the JA-evoked response, were significantly suppressed by treatment with methyl JA (MeJA) in both mutants. Further experiments using knockout mutants lacking CORONATINE-INSENSITIVE1 (COI1), a master regulator of the JA-evoked response, and MYC2 indicated a possibility that the production of ROS via Atrbohs depends on the function of COI1, but not MYC2.

We aimed at elucidating the roles of transforming growth factor (TGF)-β and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-β/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-β on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-β/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-β significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-β on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-β inhibited adipogenesis independent from the Wnt and β-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-β/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance.

A dual non-magnetic substitution system on A- and B-sites in the spinel structure has been studied. The mother compound is a ferromagnet CuCr 2 S 4 with the Curie temperature T c ≃ 380 K . A system of Cu 1 ‐ x Ag x CrSnS 4 , which is the same notation as ( Cu 1 ‐ x Ag x ) ( Cr 0.50 Sn 0.50 ) 2 S 4 , has been prepared over the entire range of 0.00 ≤ x ≤ 1.00 although the Cr-Sn sublattice is unchanged in the fixed composition of 0.50 on B-sites. All these compounds exhibit the spin-glass phase with the freezing temperature T g approximately at 16 K in 100 Oe. Since only Cr ions have the magnetic moment on the B-sites, the substitution of Ag for Cu on the A-sites does not influence strongly the spin-glass freezing behavior over the whole composition range. Nevertheless, the magnetization of Cu 1 ‐ x Ag x CrSnS 4 with x = 0.50 and 0.55 cause a broad upturn hump over 30–130 K where the spin-glass phase is broken. Strong magnetic field dependence of this hump anomaly has been observed with an irreversibility between zero-field-cooled (ZFC) and field-cooled (FC) magnetizations even though above T g . The hump is suppressed in higher fields and collapsed down at approximately 1.0 kOe with a tiny trace quantity of the anomaly where the difference between the ZFC and FC processes disappears. The specimen with x = 0.45 shows a small hump anomaly in low field 20 Oe which corresponds to a precursor of the huge anomaly for x = 0.50 . The hump anomaly could be attributed to a formation of the cluster-glass. The spin-clusters are embedded in the matrix of spin-glass elements in high degree of disorder without long-range order. All the spins eventually are frozen below T g . The strange magnetic freezing originates from the delicate dual substitutions. The mechanism of the anomaly is far from a complete picture and remains enigmatic.

We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.

At the National Institute of Radiological Sciences (NIRS), we constructed a microbeam system in 2003, the single particle irradiation to cell, SPICE. From the beginning of 2005, we redesigned it to improve the stability of the optical alignment of the system, and obtained an reduction of the beam size proportional to the vertical dimension of the object slits. As a result, SPICE is now capable of producing a beam size of approximately 10 lm diameter, and the particle numbers controllable to an intensity as low as single particles per second, and therefore a single particle irradiation facility has been successfully implemented. Moreover, these conditions can be easily reproduced with a employing a routine procedure. We describe in detail the modifications of the beam line and results, demonstrating the improvements. In addition, results from our first biological experiments are shown.

We have investigated the effect of Fe on the induction of ascorbate peroxidase (APX) in Fe-deficient Euglena cells that completely lacked APX activity. Addition of 0.13 mM Fe to Fe-deficient cells caused a two-phase induction of the APX activity: a sharp rapid increase in the first 3 h, followed by a steady gradual increase from 3 through 24 h. Cycloheximide had no effect on the initial increase of activity, but completely inhibited the subsequent increase. Immunoblot analyses using a monoclonal antibody (EAP1) raised against Euglena APX indicated that the former increase is due to activation of APX apo-protein and the latter one is due to de novo synthesis of the enzyme protein. However, Northern analysis revealed that the levels of APX transcripts remained constant, suggesting that the induction of Euglena APX by Fe is regulated at the post-transcriptional stage. The second increase of APX activity was also suppressed by the addition of succinyl acetone, an inhibitor of heme synthesis, suggesting that heme synthesis is essential for the synthesis of APX protein.

In March 1999, a HVEE Tandetron was installed in the Electrostatic Accelerator Building of National Institute of Radiological Sciences (NIRS) for particle induced X-ray emission (PIXE) analysis. The specifications of the Tandetron accelerator system operating at NIRS are as follows: the accelerating voltage is 0.4–1.7 MV, and the maximum beam current is 500 nA at 3.4 MeV. The accelerator facility incorporates three beam lines for conventional, in-air and microbeam PIXE analysis. The scanning microbeam PIXE analysis line is based around an Oxford Microbeams OM2000 nuclear microscope end stage. This system provides the ability of multi-elemental mapping over sample areas up to 2 × 2 mm area with spatial resolutions routinely at 1 × 1 μm. The scheduled operation of this facility started in April 2000 and is controlled by the Division of Technical Service and Development. The result of beam resolution tests carried out in 2001 are as follows: for scanning transmission ion microscopy, the estimated beam size is 100 × 200 nm, measured using a 2.6 MeV proton beam scanned over a 12.7 μm repeat distance copper grid. For PIXE operation at 50 pA beam current the estimated best spot size is 0.4 × 0.6 μm. The microbeam facility is being used for research into the elemental distribution of small biological samples such as biological cells and tissue.

X-ray photoelectron spectroscopy (XPS), Auger electron spectroscopy (AES), and scanning electron microscopy (SEM) measurements of polycrystalline AlN surfaces exposed to XeF 2 vapor at 1.8×10 −4 Torr at sample temperatures ranging from T s =300 to 920 K are reported. No change in the chemical composition and morphology of the AlN surface exposed at T s =300 K was observed from a virgin AlN surface. The AlN surface exposed at T s =700 K is partially fluorinated, forming an AlF 3 layer, whereas those exposed at T s =750 and 800 K are found to be completely covered with an AlF 3 layer although no change was observed in morphology. Above T s =850 K, however, partially fluorinated AlF x ( x =1, 2) species and exposed AlN surface area were observed but no AlF 3 -passivated layer was detected, probably because the AlF 3 formed is desorbed as soon as it forms. Therefore, it is concluded that, above T s =850 K, the fast corrosion reaction of AlN by XeF 2 proceeds and thus the AlN surface is strongly etched.

When Euglena cells are grown heterotrophically in the dark, the chloroplast development stops at the proplastid stage. Upon exposure to light, the cells rapidly develop chloroplasts and the photosynthesis resumes. Here, we have investigated the regulation of cytosolic ascorbate peroxidase (APX) in Euglena gracilis during light adaptation of dark-grown cells. Both the activity and protein levels of APX increased by nearly fourfold in about 24 h of illumination. Northern hybridization with a partial cDNA of Euglena APX as the probe revealed a constant level of APX transcripts during the light adaptation. Similarly, cycloheximide almost completely inhibited APX induction whereas transcription inhibitors did not have a significant effect, suggesting that the light induction of APX is post-transcriptionally regulated. APX induction was abolished when the development of chloroplast was suppressed by norflurazon, which inhibits the carotenoid synthesis. However, treatment of the dark-grown cells with H 2 O 2 or methyl viologen did not induce APX.

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.

We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.

The spinach stromal, thylakoid-bound, and cytosolic ascorbate peroxidase isozymes (EC 1.11.1.11) were overexpressed in Escherichia coli, and their enzymatic properties were compared with the respective native isozymes. The purification of the recombinant stromal and cytosolic ascorbate peroxidases using the conventional column chromatography yielded 0.73 and 2.2 mg of protein/liter of bacteria culture with enzyme activities of 800 and 486 micromol min-1 mg protein-1, respectively. In every respect, the recombinant stromal, thylakoid-bound, and cytosolic ascorbate peroxidase isozymes exhibited identical enzymatic properties with each native isozyme. Specifically, the recombinant stromal and thylakoid-bound ascorbate peroxidase isozymes showed high utilization of ascorbate as an electron donor and had a very short lifetime in ascorbate-depleted medium. Polyclonal antibodies raised against both purified recombinant stromal and cytosolic ascorbate peroxidase isozymes were prepared. Both antibodies showed a cross-reaction with the recombinant and native ascorbate peroxidase isozymes.

In komatsuna ( Brassica rapa ) leaves, the cytosolic ascorbate peroxidase was the major enzyme, but the chloroplastic stromal ascorbate peroxidase accounted for approximately 20% of the total activity, judging from the separate assay of ascorbate peroxidase isozymes reported by Amako et al. [11]. The cytosolic ascorbate peroxidase was purified to electrophoretic homogeneity from komatsuna leaves. The purified enzyme was a monomer with a molecular mass of 28 kDa using gel filtration and SDS-PAGE. The enzyme activity was maximal at pH 6.5 and 38°C, and stable between pH 6.5 and pH 7.5 to 35°C. The inhibition of the enzyme by cyanide and azide showed that it is a hemoprotein. This enzyme was not a glycoprotein. When the enzyme was diluted with the ascorbate-depleted medium, the half inactivation time was approximately 80 min. The komatsuna cytosolic ascorbate peroxidase cross-reacted by western blotting with the monoclonal antibody raised against Euglena cytosolic ascorbate peroxidase.

Microbial production of a 6-hydroxy-3-pyridylmethyl compound from 3-pyridylmethyl compound was investigated. The hydroxylation of 3-(methylaminomethyl)pyridine to 5-(methylaminomethyl)-2(1 H )-pyridinone, tautomer of 2-hydroxy-5(methylaminomethyl)pyridine, by resting cells of Arthrobacter ureafaciens JCM3873 was found to proceed regio- and chemo-selectively with an almost quantitative yield. The addition of molybdate ion and nicotine as an inducer to the culture medium was required for the preparation of cells containing high hydroxylation activity. The optimal temperature and pH for the hydroxylation by using resting cells were 35°C and around 7, respectively. This hydroxylation enzyme does undergo inhibition by the substrate. The inhibitory effect could be eliminated by stepwise feeding of the substrate. Under adequate conditions, 23 mg/ml of 5-(methylaminomethyl)-2(1 H )-pyridinone was produced with a molar yield of nearly 100% from 3-(methylaminomethyl)pyridine.

Ascorbate peroxidase (EC 1.11.1.11) has been purified to electrophoretic homogeneity from Euglena gracilis Z. The enzyme showed a molecular mass of 58 kDa on SDS-PAGE and gel filtration, indicating that Euglena ascorbate peroxidase exists as a monomeric form. The substrate specificity for an electron donor and the stabilty of the purified enzyme were similar to those of cytosolic isozymes from higher plants. One of the characteristic properties was that Euglena ascorbate peroxidase reduces organic hydroperoxides as well as hydrogen peroxide. The N-terminal amino-acid sequence showed no significant similarity to any other ascorbate peroxidase from higher plants. However, the sequence of the peptides from the purified enzyme exhibited a high degree of homology to sequences of cytosolic and chloroplastic ascorbate peroxidases. Monoclonal antibodies against the purified Euglena ascorbate peroxidase were prepared. Two monoclonal antibodies (EAP1 and EAP2) showed high homology to cytosolic ascorbate peroxidases of higher plants, as judged by Western blot analysis. The EAP1 was also specific for chloroplastic ascorbate peroxidase from spinach. These findings indicate that Euglena ascorbate peroxidase exists in highly homologous regions with the ascorbate peroxidases of higher plants.

Euglena gracilis shows an absolute requirement for iron for growth. Iron depletion causes Euglena cells to arrest cell division and the provision of iron allows the iron-deficient cells to return to the normal state. Iron-deficient cells exhaustively take up iron within 1 h of the addition of iron. Incorporated iron exists in a bound form, but not in a free form. The ascorbate peroxidase activity is not found in iron-deficient cells. Lipid peroxides (thiobarbituric acid-reactive substances, TBARS) in iron-deficient cells is much higher than those in iron-sufficient cells. The experimental results reported here suggest that iron is involved in the expression of ascorbate peroxidase activity and Euglena ascorbate peroxidase may possess a second important function in the form of a lipid peroxide-scavenging system, in addition to its action of destroying hydrogen peroxide.

Hydrogen peroxide (H2O2) generation in intact mitochondria and chloroplasts was investigated in Euglena gracilis. In mitochondria, the addition of respiratory substrate caused the formation of H2O2. Lactate, the most effective substrate, yielded H2O2 at the rate of 9.5 nmol min−1 mg protein−1. Chloroplasts showed a rate of H2O2 generation of 5 μmol mg chlorophyll−1 hr−1 under illumination. The results reported here demonstrate that H2O2 is generated in mitochondria and chloroplasts and immediately diffuses from each organelle into the cytosol.

isual detection of motion is one of cardinal property of human brain. In his study, we investigated how spatial extent of motion stimulus affects uman brain response using 306 ch whole head magnetoencephalograph MEG). We recorded MEG response to motion onset of sinusoidal gratng with different size (2.2× 2.9◦ to 44.8× 57.4◦) and speed (2.9◦/s to 3.5◦/s). The first component of MEG response (peaked at 114–216ms) as observed around occipito-temporal area. The latency and amplitude as affected by the motion speed as previous study for the second and hird stimulus sizes. The latency was also affected by the stimulus size nd was the shortest at the second largest stimulus size. In contrast, the mplitude was not affected by the size. The results show that the motion ize affects human brain response in a manner different from motion peed, suggesting that the neural process of motion detection interacts ith neural information about spatial extent of motion image.