A new application for the quantification of apoplastic redox radicals of plant roots using pre-fluorescent probe.

New application of fluorescence probe to detect apoplastic redox radicals from plant roots were sought. This probe can detect radicals selectively. Calibration curve for radicals was obtained using nitrogen monoxide as radical standard produced by NOC7. Apoplastic radicals released constitutively were quantified and the release rate was 60 µmol L-1 h-1. Oxidative burst triggered by chitin was distinguished from constitutive radical release.

Ongoing rubella outbreak among adults in Tokyo, Japan, June 2012 to April 2013.

INTRODUCTION: A large rubella outbreak has been occurring in Tokyo, Japan since June 2012. Rubella vaccination, introduced in Japan in 1976, has targeted different age groups, resulting in a large proportion of the current population being unvaccinated. METHODS: Rubella cases reported in Tokyo from 2 January 2012 to 21 April 2013 were analysed. A clinical case had generalized maculopapular rash, fever and lymphadenopathy; a laboratory-confirmed case was a clinical case with a positive serology or polymerase chain reaction test for rubella. A descriptive analysis of cases by age, sex, vaccination history and other epidemiological information was conducted. RESULTS: A total of 2382 cases were reported from all areas of Tokyo. Three-quarters were male (n = 1823; 76.5%); the highest number of cases occurred among males aged 35-39 years and females aged 20-24 years. About a third of males (27%) and females (32%) reported never receiving rubella vaccination, with 68% and 56%, respectively, having an unknown vaccination status. DISCUSSION: This outbreak reflects the changing, yet incomplete, immunization policies for rubella in Japan that may increase the risk of congenital rubella syndrome (CRS). To suppress the outbreak of rubella and prevent CRS cases, we recommend vaccination for the entire susceptible population.

Luminescence amplification by enzymatic Eu2+ oxidation to Eu3+ for time-resolved peroxidase activity measurement.

A novel peroxidase activity assay was developed for horseradish peroxidase (HRP) and myeloperoxidase (MPO), in which substrate Eu(2+) was catalytically oxidized to Eu(3+), and the Eu(3+) luminescence was enhanced by the addition of sensitizer 4,4'-bis(1″,1″,1″,2″,2″,3″,3″-hepatafluoro-4″,6″-hexanedione-6″-yl)chlorosulfo-o-terphenyl (BHHCT) for time-resolved measurement of the BHHCT-Eu(3+) complex. Since BHHCT-Eu(3+) has a long lifetime (more than 500 µs), typical of Eu(3+) oxidation state, and the emission wavelength (615 nm) is totally different from those of Eu(2+) complexes, time-resolved luminescence measurement of the Eu(3+) complex enabled suppressed background and high signal/background ratio. The present method was successfully applied to monitor the oxidative stress level, which is closely associated with peroxidase activity level, in rat heart muscle homogenates. Notable parallel temporal change was observed for peroxidase activity and 4-hydroxynonenal (HNE) concentration after lipopolysaccharide (LPS) injection for induction of oxidative stress in rats. Such a relation does not contradict the oxidative stress mechanism that HNE is produced via lipid peroxidation, which is caused by the (•)OH radical generated by peroxidase activity.

Determination of monosaccharides derivatized with 2-aminobenzoic Acid by capillary electrophoresis.

Reducing monosaccharides were derivatized with 2-aminobenzoic acid (2-AA) through reductive amination using sodium cyanoborohydride as a reductant, and the derivatives were separated by capillary zone electrophoresis with UV detection using 50 mM sodium phosphate (pH 5.5) or 150 mM sodium borate-50 mM sodium phosphate (pH 7.0) running buffer. The derivatives of monosaccharides, which are major components of various carbohydrate materials, were completely separated within 25 min.

Osmotic stress responses and plant growth controlled by potassium transporters in Arabidopsis.

Osmotic adjustment plays a fundamental role in water stress responses and growth in plants; however, the molecular mechanisms governing this process are not fully understood. Here, we demonstrated that the KUP potassium transporter family plays important roles in this process, under the control of abscisic acid (ABA) and auxin. We generated Arabidopsis thaliana multiple mutants for K(+) uptake transporter 6 (KUP6), KUP8, KUP2/SHORT HYPOCOTYL3, and an ABA-responsive potassium efflux channel, guard cell outward rectifying K(+) channel (GORK). The triple mutants, kup268 and kup68 gork, exhibited enhanced cell expansion, suggesting that these KUPs negatively regulate turgor-dependent growth. Potassium uptake experiments using (86)radioactive rubidium ion ((86)Rb(+)) in the mutants indicated that these KUPs might be involved in potassium efflux in Arabidopsis roots. The mutants showed increased auxin responses and decreased sensitivity to an auxin inhibitor (1-N-naphthylphthalamic acid) and ABA in lateral root growth. During water deficit stress, kup68 gork impaired ABA-mediated stomatal closing, and kup268 and kup68 gork decreased survival of drought stress. The protein kinase SNF1-related protein kinases 2E (SRK2E), a key component of ABA signaling, interacted with and phosphorylated KUP6, suggesting that KUP functions are regulated directly via an ABA signaling complex. We propose that the KUP6 subfamily transporters act as key factors in osmotic adjustment by balancing potassium homeostasis in cell growth and drought stress responses.

OsTZF1, a CCCH-tandem zinc finger protein, confers delayed senescence and stress tolerance in rice by regulating stress-related genes.

OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of ß-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with ß-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3' untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.

Efficacy of continuous follow-up for preventing the involuntary readmission of psychiatric patients in Japan: a retrospective cohort study.

BACKGROUND: Providing aftercare to psychiatric inpatients is important for preventing frequent readmissions; however, the lack of social resources is a problem in Japan. The prefectural Tama-Fuchu Public Health Centre has attempted to establish a new continuous follow-up system for all discharged psychiatric patients in order to reduce the frequency of readmissions. AIMS: This study aims to evaluate the efficacy of this system. METHODS: The subjects of the present study were 200 psychiatric inpatients from the Tokyo catchment area. The continuous follow-up system was applied to 130 subjects for one year in addition to conventional standard care (the intervention group). Seventy subjects received conventional care alone (the comparison group). The incident rate ratios (IRR) of total and involuntary readmission to hospital were compared by survival analysis. RESULTS: During the observation period, there were 41 readmissions and 29 involuntary readmissions in 49,731 person-days. The patients subjected to continuous follow-up showed a trend towards a lower overall risk of readmission (IRR = 0.56, 95% CI: 0.29-1.10, p = .057) and a significantly reduced risk of involuntary admission (IRR = 0.48, 95% CI: 0.22-0.96, p = .047). CONCLUSION: This study provides empirical evidence that providing continuous follow-up examinations as aftercare for discharged psychiatric patients significantly reduces the incidence of involuntary readmission.

Salt stress-induced changes in the transcriptome, compatible solutes, and membrane lipids in the facultatively phototrophic bacterium Rhodobacter sphaeroides.

Responses to NaCl stress were investigated in phototrophically grown Alphaproteobacterium Rhodobacter sphaeroides by transcriptome profiling, mutational analysis, and measurements of compatible solutes and membrane phospholipids. After exposure to salt stress, genes encoding two putative glycine betaine uptake systems, proVWX and betS, were highly upregulated. Mutational analysis revealed that BetS, not ProVWX, was the primary transporter of this compatible solute. Upon the addition of salt, exogenous glycine betaine was taken up rapidly, and maximal intracellular levels were reached within minutes. In contrast, synthesis of another important compatible solute in R. sphaeroides, trehalose, increased slowly following salt stress, reaching maximal levels only after several hours. This accumulation pattern was consistent with the more gradual increase in salt-induced transcription of the trehalose biosynthesis operon otsBA. Several genes encoding putative transcription factors were highly induced by salt stress. Multiple copies of one of these factors, crpO (RSP1275), whose product is a member of the cyclic AMP receptor protein/fumarate and nitrate reduction regulator (CRP/FNR) family, improved NaCl tolerance. When crpO was provided in multicopy, expression of genes for synthesis or transport of compatible solutes was unaltered, but the membrane phospholipid composition became biased toward that found in salt-stressed cells. Collectively, this study characterized transcriptional responses to salt stress, correlated changes in transcription with compatible solute accumulation rates, identified the main glycine betaine transporter and trehalose synthase, characterized salt-induced changes in phospholipid composition, and uncovered a transcription factor associated with changes in phospholipids. These findings set the stage for deciphering the salt stress-responsive regulatory network in R. sphaeroides.

Synechocystis ferredoxin-NADP(+) oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin.

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP(+) oxidoreductase (FNR( S )). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR( S ) may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR( S ) in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR( S ) is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR( S ) was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.

Aluminum stress increases carbon-centered radicals in soybean roots.

The formation of radical species was examined in roots of soybean seedlings exposed to aluminum (Al). Electron spin resonance (ESR) spectra of root homogenates with the spin-trapping reagent 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) indicated the presence of carbon-centered radicals in plants not exposed to Al. Plants exposed to 50 microM Al showed a similar spectrum, with increased signal intensity. These radicals were likely produced through a H-atom abstraction reaction by hydroxyl (*OH) radicals, the synthesis of which was initiated by the formation of superoxide (O2*-) anions. The increased production of the carbon-centered radicals may be responsible for the lipid peroxidation in Al-treated roots.

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction.

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.

Cell culture and motility study on a polymer surface with a nanometer-scaled stripe structure.

We developed a large cell culture surface with a nanostripe structure by paving polydimethylsiloxane (PDMS) replicas of a glass mold. The stripe structure has a height of 180 nm and top width of 500 nm with 400-nm intervals between stripes. Human stomach cancer SH-10-TC cells cultured on the surface changed their morphology to elongated shapes parallel to the nanostripes. In addition, cell motility parallel to the stripes was greatly enhanced. These findings strongly suggest that the nanostripe structure affected the cell physiology.

Revision of analytical conditions for determining ligand molecules specific to soft metal ions using dequenching of copper(I)-bathocuproine disulfonate as a detection system.

Reoptimization of analytical conditions was performed for a high-performance liquid chromatographic (HPLC) detection system for Cu(I) chelators based on the dequenching of Cu(I)-bathocuproine disulfonate complexes that occurs in the presence of Cu(I) chelators. The revision corrects for emission and excitation wavelengths that were in fact second-order light of the actual optimal wavelengths and for the composition of the postcolumn solution. These revisions resulted in an order of magnitude decrease in detection limits of phytochelatins, a class of cysteine-rich, heavy metal-binding peptides. The revised technique is capable of phytochelatin quantitation at femtomole quantities.

Specific Interactions between the ferredoxin and terminal oxygenase components of a class IIB Rieske nonheme iron oxygenase, carbazole 1,9a-dioxygenase.

Carbazole 1,9a-dioxygenase (CARDO) consists of terminal oxygenase (Oxy), ferredoxin (Fd), and ferredoxin reductase (Red) components and is a member of the Rieske nonheme iron oxygenases. Rieske nonheme iron oxygenases are divided into five subclasses (IA, IB, IIA, IIB, and III) based on the number of constituents and the nature of their redox centers. Each component of a class IIB CARDO from Nocardioides aromaticivorans IC177 was purified, and the interchangeability of the electron transfer reactions with each component from the class III CARDOs was investigated. Despite the fact that the Fds of both classes are Rieske-type, strict specificities between the Oxy and Fd components were observed. On the other hand, the Fd and Red components were interchangeable, even though the Red components differ in cofactor composition; the class IIB Red contains flavin-adenine-dinucleotide (FAD)- and NADH-binding domains, whereas the class III Red has a chloroplast-type [2Fe-2S] cluster in addition to the FAD- and NADH-binding domains. The crystal structures of the class IIB Oxy and Fd components were compared to the previously reported Fd:Oxy complex structure of class III CARDO. This comparison suggested residues in common between class IIB and class III CARDOs that are important for interactions between Fd and Oxy. In the class IIB CARDOs, these included His75 and Glu71 in Fd and Lys20 and Glu357 in Oxy for electrostatic interactions, and Phe74 and Pro90 in Fd and Trp21, Leu359, and Val367 in Oxy for hydrophobic interactions. The residues that formed the interacting surface but were not conserved between classes were thought to be necessary to form the appropriate geometry and to determine electron transfer specificity between Fd and Oxy.

Influence of borate complexation on the electrophoretic behavior of 2-AA derivatized saccharides in capillary electrophoresis.

A complete separation with baseline resolution of the 2-AA derivatized saccharides, including mono-, di-, and oligosaccharides, was achieved using 50 mM sodium phosphate-150 mM borate solution, pH 7.0 as running buffer by capillary electrophoresis. It was thought to be a result of the inclusion of 150 mM borate in the running electrolyte solution. The formation of borate complexes was observed by means of (11)B and (13)C NMR spectroscopy and the electrophoretic mobilities of the various derivatives were calculated. It was found that steric factors play an important role in the stability of the formed borate complexes, which depends strongly on the configuration of the three vicinal hydroxyl groups at C-2, C-3, and C-4. 2-AA-Glc mainly forms stable 1,2-diester complexes with borate and 2-AA-Mal can form stable 1,2-monoesters. In turn, for 2-AA-Rib the formation of complexes is difficult to take place. The results implied that the configurational difference between the hydroxyl groups could cause the difference in formation of borate complexes leading to significant difference among saccharide molecules in their migration time on CE analysis.

Construction of a dimethyl sulfoxide sensor based on dimethyl sulfoxide reductase immobilized on a au film electrode.

A novel dimethyl sulfoxide (DMSO) sensor using DMSO reductase and film electrodes was constructed. The Au and Ag electrodes were fabricated on slide glass by vacuum deposition and the application of a photolithographic technique. The micro-chamber (4 x 50 x 1 mm, volume 200 microl) was fabricated on a poly(dimethylsiloxane) (PDMS) polymer. The Pt electrode was implanted in a PDMS polymer. DMSO reductase was immobilized on a Au film electrode with bovine serum albumin (BSA)-glutaraldehyde. This sensor could determine DMSO in an unpurged aqueous solution with glucose oxidase (GOD) and catalase (CAT) for oxygen removal. The DMSO sensor showed a linear response within 1 mM DMSO with a correlation coefficient of 0.999. The detection limit was 200 microM (3sigma), and the sensitivity was 23.8 mA M(-1) cm(-2). The relative standard deviations at each concentration were within 3.6%.

Microbioassay system for an anti-cancer agent test using animal cells on a microfluidic gradient mixer.

We developed a novel microbioassay system equipped with a gradient mixer of two solutions, and we applied the microfluidic system to an anti-cancer agent test using living animal cells on a microchip. A microchannel for the gradient mixing of two solutions and eight other microchannels for cell assay were fabricated on a poly(dimethylsiloxane) substrate using a soft-lithography method. The functions necessary for this bioassay, i.e., cell culturing, chemical stimulation, cell staining, and fluorescence determination, were integrated into the microfluidic chip. Eight gradient concentrations of the fluorescein solution, ranging from 1 to 98 microg/ml, were archived at 0.1 microl/min on a microchip. A stomach cancer cell line was cultured, and a cell viability assay was conducted using 5-Fluorouracil as an anti-cancer agent on the microchip. Cell viability changed according to the estimated concentration of the agent solution. With the microbioassay system, an anti-cancer agent test was conducted using living cells simultaneously in eight individual channels with the gradient concentration of the agent on a microchip.

Role of trehalose synthesis pathways in salt tolerance mechanism of Rhodobacter sphaeroides f. sp. denitrificans IL106.

The photosynthetic bacterium Rhodobacter sphaeroides (R. sphaeroides) f. sp. denitrificans IL106 accumulates trehalose as the major organic osmoprotectant in response to a salt stress. An analysis of the R. sphaeroides 2.4.1 genome sequence revealed the presence of five different genes encoding enzymes belonging to three putative trehalose biosynthesis pathways (OtsA-OtsB, TreY-TreZ, and TreS). The function of the different pathways of trehalose was studied by characterizing strains defective in individual trehalose biosynthetic routes. A phenotypic comparison revealed that trehalose synthesis in R. sphaeroides f. sp. denitrificans IL106 is mediated mainly by the OtsA-OtsB pathway and, to some extent, by the TreY-TreZ pathway. Strains with the simultaneous inactivation of these two pathways were completely unable to synthesize trehalose. On the other hand, treS mutants showed an increase in the trehalose level. These results suggest that treS plays a role in trehalose degradation. In addition, treS was found to be important in reducing trehalose after osmotic stress was removed. In this report, we show that the strains that accumulate the most trehalose adapt to salt stress earlier. This is the first report of an organism using multiple pathways to synthesize trehalose solely for use as a compatible solute against salt stress.

Microbioassay system for antiallergic drug screening using suspension cells retaining in a poly(dimethylsiloxane) microfluidic device.

This article describes an antiallergic drug-screening system by the detection of histamine released from mast cells (suspension cells) on a multilayer microchip. In this study, the elastmeric material, poly(dimethylsiloxane) (PDMS), was employed to fabricate microchannels and microchambers. The microchip consists of two sections: a histamine-releasing one, which has a cell chamber, and a histamine-derivatizing one. Both were laminated to one microchip. Rat peritoneal mast cells were retained in the cell chamber (1.2 microL) with a filtering system using a cellulose nitrate membrane. This filtering system could easily retain suspension cells without cell damage. Mast cells were viable for a sufficient time to conduct the assay on the cell chamber. The cells were stimulated with a chemical release compound 48/80 (C48/80), and then histamine flowed into the lower layer, where it was derivatized to the fluorescent molecules with o-phthalaldehyde and its fluorescence was detected on the microchip. This flow system could detect the time course of the histamine release, and this microchip system required only 20 min for the assay. By this integrated system, 51 pmol of histamine released from 500 cells was detected, and the number of cells required for the assay was reduced to 1% compared with conventional bulk systems. By comparing the released histamine levels with and without drugs, their effect could be evaluated. The inhibition ratio of C48/80 induced-histamine release using an antiallergic drug, disodium cromoglicate (DSCG), was related to the concentration of DSCG. This flow system was applicable for antiallergy drug screening by rapid measurement of the inhibition of histamine release from a very small amount of mast cells.