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Conductive carbons can boost anaerobic microbial metabolism by assisting extracellular electron transfer (EET), and their chemistry affects microbial metabolism. Graphene oxide (GO), a chemically oxidized sheet of graphite, has been used in various bioelectrochemical systems, although its mechanism is rarely understood. This study revealed specific metabolic responses to reduced GO (rGO) in an electrogenic strain R4 of Citrifermentans bremense, recently renamed from "Geobacter bremensis," in comparison to that with graphite felt (GF). Specifically, the change in growth from planktonic cells to biofilm with an enlarged outer membrane. The mRNA profile supported the fact that rGO upregulated the 14 genes related to the exopolysaccharides (EPS) secretion and biofilm formation, which is more than that in GF (4 genes). While GF upregulated the 35 genes involved in cell motility, which is more than that in rGO (8 genes). The heme protein profile suggested that both carbons induced similar EET pathways involving OmcA/MtrC and OmcS; however, GO specifically induced PilQ. These findings show that the chemistry of conductive carbon differentiates metabolism, especially affecting cellular morphology or living form, rather than electron transfer metabolism.

There are many reports of falsified medicines that may cause harm to patients. A rapid and simple method of identifying falsified medicines that could be used in the field is required. Although Raman scattering spectroscopy has become popular as a non-destructive analysis, few validation experiments on falsified medicines that are actually distributed on the market have been conducted. In this study, we validated a discriminant analysis using an ultra-compact, portable, and low-cost Raman scattering spectrometer combined with multivariate analysis. The medicines were three types of erectile dysfunction therapeutic tablet and one type of antifungal tablet: tadalafil (Cialis), vardenafil hydrochloride (Levitra), sildenafil citrate (Viagra), and fluconazole (Diflucan), which is sometimes advertised as female Viagra. For each medicine, the authentic standard product and products obtained by personal import via the internet (genuine or falsified) were used. Discriminant analyses were performed on the Raman spectra combined with soft independent modeling of class analogy (SIMCA) and partial least squares discriminant analysis (PLS-DA). It was possible to identify all falsified samples by SIMCA using the standard product model for all four products. Using the PLS-DA using the PLS models of the four standard products, falsified Levitra and Diflucan samples were classified correctly, although some falsified Cialis and all Viagra samples also belonged to the standard class. In this study, SIMCA might be more suitable than PLS-DA for identifying falsified medicines. A spectroscopic module that combines the low-cost Raman scattering spectroscopy with SIMCA might contribute to the rapid identification of falsified medicines in the field.

Various substrates have been used to stimulate habitat microbes in chloroethene-contaminated groundwater, however, the specific efficiency and minimum growth of microbes have rarely been studied. This study investigated the effects of seven substrates on trichloroethene (TCE) dechlorination by augmentation of groundwater with Dehalococcoides mccartyi NIT01 and its contribution to the microbial community. Three out of eight test groups completed dechlorination of 1 mM TCE-to-ethene in varying durations; groundwater supplemented with formate (FOR) required 78 days, whereas the microcosms with lactate (LAC) and citrate (CIT) required approximately twice as long (143 days). The calculated efficiency of how much produced H

Certain Desulfovibrio sp. (anaerobic sulfate-reducing bacteria) are indigenous to swine cecum and colon, which are also common habitats for parasitic amoebae such as Entamoeba polecki and Entamoeba suis. In this study, we evaluated the growth-promoting effects of D. desulfuricans co-cultured with Escherichia coli (DH5α) and its products [e.g., hydrogen sulfide (H2S) and certain iron-sulfide (FeS) compounds] using Robinson's medium, on the 4 amoeba isolates from swine-Entamoeba polecki subtype (ST)-1, E. polecki ST-3, Entamoeba suis, and Endolimax sp., and, consequently, a continuous culture system for these amoebae was established. However, this novel culture system was required to regulate the excess H2S dissolved in the medium by increasing air space as amoeba isolates thrive only in large air spaces (30-40%). The effects of air space and H2S and FeS compounds on the growth of E. polecki ST-1 (TDP-5) were determined. E. polecki ST-1 (TDP-5) thrived well in culture bottles with an air space of 30-40% (aerobic) (H2S: ~250-400 μmoles/L), but did not grow at all in an air space < 5% (microaerobic) ( H2S:~800 μmoles/L) and in anaerobic vessels (H2S: 20-30 μmoles/L). In both H2S-depleted and FeS compound-depleted conditions, the amoebae sp. could not thrive either. It was hypothesized that an appropriate concentration of H2S and FeS compounds might function as important physiologically active components of electron carriers such as FeS and ferredoxin.

A novel slow-growing, facultatively anaerobic, filamentous bacterium, strain MO-CFX2T, was isolated from a methanogenic microbial community in a continuous-flow bioreactor that was established from subseafloor sediment collected off the Shimokita Peninsula of Japan. Cells were multicellular filamentous, non-motile and Gram-stain-negative. The filaments were generally more than 20 µm (up to approximately 200 µm) long and 0.5–0.6 µm wide. Cells possessed pili-like structures on the cell surface and a multilayer structure in the cytoplasm. Growth of the strain was observed at 20–37 °C (optimum, 30 °C), pH 5.5–8.0 (pH 6.5–7.0), and 0–30 g l−1 NaCl (5 g l−1 NaCl). Under optimum growth conditions, doubling time and maximum cell density were estimated to be approximately 19 days and ~105 cells ml−1, respectively. Strain MO-CFX2T grew chemoorganotrophically on a limited range of organic substrates in anaerobic conditions. The major cellular fatty acids were saturated C16 : 0 (47.9 %) and C18 : 0 (36.9 %), and unsaturated C18 : 1ω9c (6.0 %) and C16 : 1ω7 (5.1 %). The G+C content of genomic DNA was 63.2 mol%. 16S rRNA gene-based phylogenetic analysis showed that strain MO-CFX2T shares a notably low sequence identity with its closest relatives, which were Thermanaerothrix daxensis GNS-1T and Thermomarinilinea lacunifontana SW7T (both 85.8 % sequence identity). Based on these phenotypic and genomic properties, we propose the name Aggregatilinea lenta gen. nov., sp. nov. for strain MO-CFX2T (=KCTC 15625T, =JCM 32065T). In addition, we also propose the associated family and order as Aggregatilineaceae fam. nov. and Aggregatilineales ord. nov., respectively.

Hydrodynamics has received considerable attention for application in improving microbial fuel cell (MFC) performance. In this study, a method is proposed to calculate the effect of fluid flow on MFC current production from sewage wastewater. First, the effect of flow velocity in an up-flow channel was evaluated, where an air-core MFC was polarized with external resistance (Rext). When tested at a flow velocity ranging from 0 to 20 cm s−1, the MFC with the higher flow velocity produced more current. In sewage wastewater with a chemical oxygen demand (COD) of 76 mg L−1, the MFC polarized with 3 Ω of Rext, and a flow velocity of 20 cm s−1 had 5.4 times more current than the MFC operating in a no-flow environment. This magnitude decreased with higher Rext and COD values. The Michaelis–Menten equation, modified herein by integrating COD and flow velocity, demonstrated the production of current by MFC operating under different conditions of flow. Calculation of current by MFC in a virtual fluid suggested that the flow surrounding the MFC varied with the configuration and affected the current production.

Chloroethenes (CEs) are widespread groundwater toxicants that are reductively dechlorinated to nontoxic ethene (ETH) by members ofDehalococcoides. This study established aDehalococcoides-dominated enrichment culture (designated “YN3”) that dechlorinates tetrachloroethene (PCE) to ETH with high dechlorination activity, that is, complete dechlorination of 800 μM PCE to ETH within 14 days in the presence ofDehalococcoidesspecies at5.7±1.9×107 copies of 16S rRNA gene/mL. The metagenome of YN3 harbored 18rdhAgenes (designatedYN3rdhA1–18) encoding the catalytic subunit of reductive dehalogenase (RdhA), four of which were suggested to be involved in PCE-to-ETH dechlorination based on significant increases in their transcription in response to CE addition. The predicted proteins for two of these four genes, YN3RdhA8 and YN3RdhA16,showed 94% and 97% of amino acid similarity with PceA and VcrA, which are well known to dechlorinate PCE to trichloroethene (TCE) and TCE to ETH, respectively. The other twordhAs,YN3rdhA6andYN3rdhA12,which were never proved asrdhAfor CEs, showed particularly high transcription upon addition of vinyl chloride (VC), with75±38and16±8.6mRNA copies per gene, respectively, suggesting their possible functions as novel VC-reductive dehalogenases. Moreover, metagenome data indicated the presence of three coexisting bacterial species, including novel species of the genusBacteroides, which might promote CE dechlorination byDehalococcoides.

A Gram-negative, anaerobic, non-motile, non-spore-forming bacterial strain, designated YN3PY1T, was isolated from a chloroethene-dechlorinating consortium originally enriched from river sediment. The strain enhanced the dechlorination of cis-dichloroethene to ethene by Dehalococcoides, especially at the early stages of cultivation. Strain YN3PY1T was the first isolate of the genus Bacteroides, obtained from animal-independent environments, and its 16S rRNA gene had the highest sequence similarity (97.1%) with Bacteroides luti JCM 19020T in the ‘Coprosuis’ clade of the genus Bacteroides. Strain YN3PY1T formed a phylogenetic cluster with other phylotypes detected from sediments and paddy soil, and the cluster was affiliated with a linage of so-called free-living Bacteroides detected from animal-independent environments, suggesting specific adaptations to sediment-like environments. The strain showed typical phenotypes of Bacteroides, i.e., polysaccharolytic anaerobe having anteiso-C15:0 as the most abundant fatty acid and MK-11 as one of the major respiratory quinones. Additionally, the strain uniquely transforms glucose to lactate and malate, has MK-12 as another major respiratory quinone, and grows at comparatively low temperatures, i.e. 10–40°C, with an optimum at 28°C. Based on the presented data, strain YN3PY1T (= KCTC 15656T = NBRC 113168T) can be proposed as a novel species of the genus Bacteroides and named as Bacteroides sedimenti sp. nov.

Anaerobic mineralization of 2,4,6-tribromophenol (2,4,6-TBP) was achieved by a synthetic anaerobe community comprising a highly enriched culture of Dehalobacter sp. phylotype FTH1 acting as a reductive debrominator; Clostridium sp. strain Ma13 acting as a hydrogen supplier via glucose fermentation; and a novel 4-chlorophenol-degrading anaerobe, Desulfatiglans parachlorophenolica strain DS. 2,4,6-TBP was debrominated to phenol by the combined action of Ma13 and FTH1, then mineralized into CO2 by sequential introduction of DS, confirmed using [ring-(14)C(U)] phenol. The optimum concentrations of glucose, SO4(2-), and inoculum densities were 0.5 or 2.5mM, 1.0 or 2.5mM, and the densities equivalent to 10(4)copiesmL(-1) of the 16S rRNA genes, respectively. This resulted in the complete mineralization of 23μM 2,4,6-TBP within 35days (0.58μmolL(-1)d(-1)). Thus, using a synthetic microbial community of isolates or highly enriched cultures would be an efficient, optimizable, low-cost strategy for anaerobic bioremediation of halogenated aromatics.

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant on the market. It has been detected in various environmental samples, and a growing body of evidence has demonstrated its toxic effects on living organisms. In this study, we report the enrichment and phylogenetic identification of bacteria that debrominate TBBPA to bisphenol A in the presence of humin. Incubation experiments indicated that humin was required for this debromination activity. Of the five compounds examined for inclusion in the TBBPA-debrominating culture, formate was the optimal electron donor. A 16S rRNA gene library showed that the culture was dominated by three known dehalogenator genera: Dehalobacter, Geobacter, and Sulfurospirillum. Further investigation indicated that Dehalobacter was responsible for the debromination of TBBPA. PCR-denaturing gradient gel electrophoresis analysis showed that Dehalobacter grew in the culture by utilizing TBBPA. Moreover, the copy number of the Dehalobacter 16S rRNA genes increased by about two orders of magnitude in the cultures without the addition of TBBPA, whereas it increased by approximately four orders of magnitude when TBBPA was present. The incubation experiments showed that Dehalobacter was reliant on humin for its debromination activity, indicating a new type of metabolism in Dehalobacter that is linked to humin respiration.

Biodegradable plastics (BPs) were evaluated for their applicability as sustainable and solid H(2) donors for microbial reductive dechlorination of 4,5,6,7-tetrachlorophthalide (fthalide). After a screening test of several BPs, the starch-based plastic (SP) that produced the highest levels of H(2) was selected for its use as the sole H(2) donor in this reaction. Fthalide dechlorination was successfully accomplished by combining an H(2)-producing SP culture and a KFL culture containing Dehalobacter species, supplemented with 0.13% and 0.5% SP, respectively. The efficiency of H(2) use in dechlorination was evaluated in a combined culture containing the KFL culture and strain Clostridium sp. Ma13, a new isolate that produces H(2) from SP. Results obtained with this culture indicated increased H(2)-fraction for fthalide dechlorination much more in this culture than in compared with a KFL culture supplemented with 20mM lactate, which are 0.75 H(2)·glucose(-1) and 0.015 H(2)·lactate(-1) in mol ratio, respectively.

Graphene oxide (GO) has been shown to be reduced by several microorganisms. Recent studies of the growth of Geobacter species in the presence of GO and electricity production by recovery of electrons on the reduced form of GO (rGO) have indicated substantial benefits of GO and GO-respiring bacteria (GORB) in microbial electrochemical systems. In this study, we enriched GORB from a coastal sample to investigate the distribution and phylogenetic variety of GORB in seawater environments. X-ray photoelectron spectroscopy (XPS) and four-terminal probing revealed that the enriched microbial community (designated as CS culture) reduced GO and self-aggregated into a conductive hydrogel complex with rGO (the CS-rGO complex). In the process of GO reduction, certain bacterial populations grew in a manner that was dependent on GO respiration coupled with acetate oxidization. High-throughput sequencing of 16S rRNA as a biomarker revealed the predominance of Desulfomonas species at 92% of the total bacterial population in the CS culture. The CS-rGO complex produced electricity with acetate oxidization, exhibiting less than 1 Ω/cm3 of charge transfer resistance. Thus, these results suggested that Desulfomonas species could grow on rGO and produce electricity via the reduced form of GO.

Aerobic aromatic-hydrocarbon-degrading bacteria from a semi-anaerobic microbial microcosm that exhibited apparent complete dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were isolated through enrichment and plating culture procedures with dibenzofuran as the model substrate. By 16S rRNA gene sequence comparisons, these dibenzofuran-degrading isolates were identified as being members of the phyla Actinobacteria, Firmicutes, and Proteobacteria, among which those of the genera Paenibacillus and Rhizobium were most abundant. All of the isolates utilized naphthalene as the sole carbon and energy source and degraded dibenzofuran metabolically or co-metabolically; however, they hardly attacked monochlorinated dibenzofuran and dibenzo-p-dioxin. By PCR cloning and sequencing, genes predicted to encode aromatic-ring-hydroxylating dioxygenase (AhDO) were detected in all test isolates. Real-time quantitative PCR assays with specific primer sets detected approximately 10⁵ copies of the AhDO large subunit genes g⁻¹ wet wt in the microcosm from which the isolates were obtained. This order of the copy number corresponded to approximately 1% of the 16S rRNA gene copies from "Dehalococcoides" and its relatives present as potent dechlorinators. These results suggest that aerobic AhDO-containing bacteria co-exist and play a role in the oxidative degradation of less chlorinated and completely dechlorinated products in the PCDD/F-dechlorinating process, thereby achieving the apparent complete dechlorination of PCDD/Fs.

Endogenous tocochromanols in extracted lipids from rice brans of the five cultivars were determined by high-performance liquid chromatography, and were investigated in relation to the fatty acid (FA) distribution of triacylglycerols (TAG) and phospholipids (PL). The dominant tocols were α-tocopherol and γ-tocotrienol, followed by α-tocotrienol and with much smaller amounts of γ-tocopherol and δ-tocotrienol. The lipids of these rice brans comprised mainly TAG (80.6-86.0wt.%), free FA (4.2-9.0wt.%), and phospholipids (5.5-6.7wt.%), whilst other components were also detected in minor proportions (0.2-2.1wt.%). The PL components included phosphatidyl choline (31.8-46.8wt.%), phosphatidyl ethanolamine (25.0-38.9wt.%) and phosphatidyl inositol (20.2-23.2wt.%). Comparison of these different cultivars showed, with a few exceptions, no significant differences (P>0.05) in FA distribution. FA distribution of TAG among the five cultivars was evident in the rice brans: unsaturated FA were predominantly concentrated at the sn-2 position and saturated FA primarily occupying the sn-1 or sn-3 position. These results suggest that the tocopherol content, lipid component, and FA distribution in rice brans are not dependent on the cultivation areas during the growing season.

Fatty acid (FA) distributions and molecular species of triacylglycerols (TAG) isolated from total lipids extracted from adzuki beans ( Vigna angularis ) were analysed by a combination of AgNO 3 -TLC and GC, and were investigated in relation to the content of endogenous antioxidants determined by HPLC. δ-Tocopherol was present in the highest concentration (30.5 mg/kg beans), and γ-tocopherol in small amounts (12.8 mg/kg beans). The major lipid components were phospholipids (74.3%), TAG (13.5%), hydrocarbons (4.6%) and steryl esters (4.0%), whilst other components were also present in minor proportions (0.5–1.3%). Seventeen different molecular species were detected. The major TAG components were SMD (5.0%), S 2 T (19.2%), SD 2 (13.7%), SMT (9.3%), MD 2 (4.5%), SDT (7.0%), D 3 (8.8%) and ST 2 (15.8%) (where S, M, D, and T denote a saturated FA, a monoene, a diene, and triene, respectively). These results would be useful to both consumers and producers for manufacture of traditional adzuki foods in Japan.

Fatty acid distribution of triacylglycerols (TAG) and phospholipids (PL) obtained from the four cultivars of broad beans ( Vicia faba ) was investigated. Total lipids extracted from the beans were separated by thin-layer chromatography into eight fractions. The major lipid components were TAG (47.7–50.1%) and PL (47.5–50.5%), while hydrocarbons, steryl esters, free fatty acids, diacylglycerols (1,3-DAG and 1,2-DAG) and monoacylglycerols were present in minor proportions (1.8–2.4%). The major PL components were phosphatidylcholine (56.4–58.4%), phosphatidylethanolamine (20.3–21.7%) and phosphatidylinositol (16.6–18.6%). Phosphatidylinositol was unique in that it had the highest saturated fatty acid content among the three PL. No significant differences ( P > 0.05) in fatty acid distribution existed when the different cultivars were compared. The fatty acid distributions in the TAG were evident among the four cultivars: unsaturated fatty acids were predominantly concentrated in the sn −2 position, and saturated fatty acids primarily occupied the sn −1 or sn −3 position in the oils. These results could be useful to both consumers and producers for manufacture of traditional foods in Japan and elsewhere.

The dechlorination of polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins was examined in an enrichment culture (KFL culture) that contained two phylotypes of Dehalobacter, FTH1 and FTH2. The KFL culture dechlorinated 2,3,4,5-tetrachlorobiphenyl, 2,3,4-trichorobiphenyl (2,3,4-TriCB), and 1,2,3-trichlorodibenzo-p-dioxin (1,2,3-TriCDD). Quantitative real-time PCR targeting FTH1 and FTH2 revealed significant increases with the addition of PCBs and 1,2,3-TriCDD, suggesting halorespiring growth of the Dehalobacter species in the KFL culture. This study demonstrated the reductive dechlorination of PCBs and 1,2,3-TriCDD by Dehalobacter species in a sediment-free culture and a novel dechlorination pathway, the conversion of 2,3,4-TriCB to 4-monochlorobiphenyl via 3,4-dichlorobiphenyl.

The anaerobic degradation of biphenyl was investigated in four uncontaminated Japanese paddy soils and one river sediment sample contaminated with benzene and chlorinated aliphatics. Two of the paddy soils and the sediment were capable of degrading biphenyl anaerobically without any additional medium or electron acceptors. The half-lives of biphenyl biodegradation in the three samples were 212 d in the Kuridashi soil, 327 d in the Kamajima soil, and 429 d in the river sediment. The Kuridashi soil metabolized 1+/-0.3% of [U-14C]-biphenyl into CO2 and 5+/-2% into water-soluble metabolites after 45 d of incubation. Submerged conditions, which result in lower nitrate and iron oxide contents, and neutral pH, appeared to be the common properties among the samples that influenced their degradation capacities. The addition of 10mM sulfate and 20mM Fe(III) as electron acceptors did not enhance the biphenyl degradation rate, whereas 10mM nitrate completely inhibited biphenyl degradation. The addition of different electron donors (lactate, acetate, or pyruvate) slightly slowed the degradation. Molybdate (an inhibitor of sulfate-reducing bacteria) had an inhibitory effect on biphenyl biodegradation, but bromoethanesulfonic acid (an inhibitor of methanogens) did not. Most biphenyl degradation was observed when only water was added, with no other electron acceptors or donors. These results suggest that sulfate-reducing bacteria and fermentative microbial populations play important roles in anaerobic biphenyl biodegradation in paddy soil.

Polychlorinated biphenyls (PCBs) in Kanechlor-300 and -400 mixtures dissipated significantly compared with a sterilized control under anaerobic conditions in three Japanese paddy soils with no history of PCB contamination, demonstrating the anaerobic microbial degradation of PCBs. The PCB-degrading activity was maintained successfully in a static flooded soil medium for more than 3 years by serial transfer at intervals of 56 days (13 transfers). Ortho-, meta-, and para-substituted PCBs, 15.2 ± 9.9 mol% in total, were significantly degraded after 56 days of incubation. Analysis of menaquinones-6 and -7 and cloning of 16S rRNA gene fragments from a polymerase chain reaction denaturing gradient gel electrophoresis (DGGE) profile indicated the predominance of Firmicutes in the consortium. A PCR-based identification of the gene fragments showed the frequent presence of Desulfitobacterium sp., but not Dehalobacter sp. or Dehalococcoides sp., in the consortium. It is proposed that Japanese paddy soils with no history of PCB contamination contain an anaerobic microbial consortium consisting predominantly of Firmicutes that have the potential for anaerobic degradation of PCB.

Terpenoids form a large and structurally diverse family of natural products and are ingredients of various herbal medicines. We have investigated possible interactions between herbal medicines and conventional medicines, and recently reported that monoterpenoids contained in Zanthoxyli Fructus can be potent inhibitors of P-glycoprotein (P-gp). In the present study, the influence of 70 kinds of terpenoids present in natural products on P-gp-mediated efflux transport was investigated. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used to screen the terpenoids. Large increases in the intracellular accumulation of [(3)H]digoxin were observed in the presence of (R)-(+)-citronellal, (S)-(-)-beta-citronellol, alpha-terpinene, terpinolene, (-)-beta-pinene, abietic acid, ophiobolin A, cucurbitacin I, and glycyrrhetic acid. A study of the concentration-dependency revealed that the IC(50) of ophiobolin A, glycyrrhetic acid, (R)-(+)-citronellal, abietic acid, and cucurbitacin I was smaller than that of verapamil. The transcellular transport of [(3)H]digoxin across Caco-2 cell monolayers was then examined in the presence of (R)-(+)-citronellal, abietic acid, and glycyrrhetic acid. Significant increases in the apical-to-basolateral transport and decreases in the basolateral-to-apical transport and efflux ratio were demonstrated. These findings suggest that some natural products containing these terpenoids may inhibit P-gp-mediated transport and interact with P-gp substrates in the intestinal absorption process.

The manipulation of mammalian metaphase II (mII) oocytes has illuminated the mechanisms of fertilization and early embryogenesis and is central to nuclear transfer. Although RNA interference (RNAi) would greatly facilitate this type of manipulation, its application to mature, developmentally competent mII oocytes has not been evaluated. We report efficient RNAi by the injection of short interfering RNAs (siRNAs) into mII oocytes. The levels of the target mRNA and corresponding protein were rapidly and efficiently reduced. The siRNAs were effective when injected in the subnanomolar to nanomolar range and induced concurrently RNAi of multiple targets, revealing the kinetic parameters of RNAi in mII oocytes. Coinjection of sperm with siRNA functionally abolished the transcripts in the resultant blastocysts and in cloned embryos into which siRNA was coinjected during somatic cell nuclear transfer. The RNAi method was used to dissect the early mitotic roles of meiotic regulators, which suggests that CDC20 is essential for the first mitotic division, while EMI1 and EMI2 are not essential for this process. Our results show that siRNA injection of oocytes confers temporal control of RNAi in the analysis and manipulation of key processes in mammalian meiosis and early embryogenesis.

A redox dye-staining method using 5-cyano 2,3-ditolyl tetrazolium chloride (CTC) was modified for differential detection of metabolically active gram-positive and gram-negative bacteria. Exponentially growing cells of representative species of the phyla Actinobacteria, Firmicutes and Proteobacteria were stained with CTC in the presence of a substrate mixture, Meldola’s Blue and KCN, and post-treated with acetone. Epifluorescence microscopy and spectrophotometric experiments showed that the acetone treatment resulted in the almost complete extraction of CTC formazan crystals from the gram-negative species, whereas it had little or no effect on CTC-stained gram-positive bacteria. Metabolically active gram-positive and gram-negative bacteria in municipal-sewage activated sludge measured by the modified CTC-staining method with microscopic detection composed 24 and 29% of the total count on average, respectively, both of which corresponded to approximately 74% of the viable gram-positive and gram-negative counts detected with a ViaGram Red + Bacterial Gram Stain and Viability kit. Flow cytometric analyses of the CTC-stained and acetone-extracted activated sludge samples gave similar counts to those measured by epifluorescence microscopy. These results indicate that the modified CTC stain procedure with epifluorescence microscopy or flow cytometry can be used as a simple and rapid method for the estimation of metabolically active populations of gram-positive bacteria and gram-negative bacteria in the environment.

We examined the role of SigC (Sll0184), a sigma factor of RNA polymerase (RNAP), in a unicellular cyanobacterium, Synechocystis sp. strain PCC 6803. On the inactivation of sigC, which is an Escherichia coli rpoD homolog, cells were viable but had a low survival rate in the stationary phase of growth under normal physiological conditions, indicating that SigC is a group 2 type sigma factor. In analyses of transcript and protein levels using the sigC knockout strain, it was found that expression of glnB, a nitrogen key regulatory gene, is controlled by SigC in the stationary phase. Primer extension revealed that the glnB nitrogen promoter (P2) was specifically recognized by SigC in the stationary phase under conditions of nitrogen starvation. In vitro studies with purified enzymes indicated effective transcription, on supercoiled DNA templates, from P2 by SigC-RNAP with NtcA which is an activator for nitrogen gene transcription. DNase I footprinting also indicated binding and related sites of NtcA and/or RNAP with SigC on the nitrogen promoter. The unique promoter architecture and the mechanism of transcription by RNAP with SigC are also discussed.

A fluorochrome-staining method using 5-cyano-2,3-ditoryl tetrazolium chloride (CTC) was improved to enhance the sensitivity of detection of metabolically active bacteria in activated sludge. The highest efficiency of CTC staining was obtained with 6 mM CTC in MOPS buffer at pH 6.5. CTC staining efficiency was also increased by adding a substrate mixture (0.05% peptone, 0.05% yeast extract and 1 mM glucose), Meldola's Blue as an electron transfer mediator and KCN. The bacterial counts detected by the CTC staining method thus improved accounted for 41-76% of the total counts in activated sludge with the average value being 54%. This value was approximately two-fold higher than those obtained by the traditional CTC staining assay which has been performed in phosphate buffer or phosphate-buffered saline without any addition of external substrate or electron transfer mediator. There was a high positive correlation between CTC-stained direct counts and the plate counts of aerobic chemoorganotrophic bacteria or viable cell counts measured using a LIVE/DEAD BacLight Bacterial Viability kit. The viable population in the activated sludges as measured by BacLight staining and plate counting accounted for 73±7.6% and 8.7±4.7% of the total population, respectively. These data suggested that most of the "viable but nonculturable" bacteria (i.e., BacLight-positive but unculturable population) potentially existing in the sludge system were so metabolically active as to be detectable by the improved CTC staining method.

Aerobic chemoorganotrophic bacteria in soils polluted with different levels of polychlorinated dioxins (6.8 to 4,600 pg-TEQ g-1 dry weight) were isolated by the quantitative agar-plating method and tested for their ability to degrade dibenzofuran (DF) using DF-overlaid agar and DF-containing liquid media. For comparison, bacteria isolated from river sediments were also tested. Out of the 5,069 strains thus isolated, 23 strains were found to be able to degrade DF, and the majority produced soluble yellow metabolites during the degradation. A higher isolation frequency for DF degraders was obtained with samples containing higher concentrations of polychlorinated dioxins. Most of the DF-degrading isolates were identified as members of the class Actinobacteria, particularly of the genera Nocardioides and Rhodococcus. These results suggest that particular actinobacterial species constitute the major populations of culturable DF-degrading bacteria in dioxin-polluted environments.

The direct total count of bacteria and the concentration of isoprenoid quinones were measured in 82 samples from different aquatic and soil environments. The geometric mean of the total count (TC)/total quinine (TQ) ratios obtained with different environments ranged from 1.1 to 2.2×103 cells fmol-1. When the concentration of the total respiratory quinone (TRQ), i.e., ubiquinones and menaquinones, were taken into account, the geometric mean of the TC/TRQ ratios fell into a range of 2.2 to 4.1×103 cells fmol-1. A high positive correlation was noted between TC and TQ (r2=0.9864) or TRQ (r2=0.9990). Based on the relationship between TC and TRQ, 1 nmol of the total quinone was estimated to be equivalent to 2.5×109 cells of bacteria on average. These results indicate that the concentration of the respiratory quinones can be used as a good measure of bacterial counts and biomass in the environment.

Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

The complexation of Zn(II) with L-histidyl-L-histidylglycylglycine (HisHisGlyGly) and L-histidylglycyl-L-histidylglycine (HisGlyHisGly) was studied by proton nuclear magnetic resonance spectroscopy over the pH range from 3 to 11 at room temperature. In weakly acidic solutions below pH 6, both HisHisGlyGly and HisGlyHisGly coordinated to Zn(II) through amino and imidazole nitrogens at the N-terminal histidyl residue. The imidazole nitrogens of the histidyl residues in the second and third positions which are protonated at this pH could not coordinate to the Zn(II) ion. As the pH was raised, however, the imidazolium protons were released and associated with the metal ion. When the peptide bond between His-His in HisHisGlyGly was deprotonated over pH 7, the imidazole nitrogen of the histidyl residue in the second position was likely to coordinate to the metal ion in place of the imidazole nitrogen at the N-terminal histidly residue. A kinetically stable complex in which the Zn(II) coordinated to three nitrogens from the termianl amino, deprotonated peptide-bond, and imidazole of the histidyl residue in the second position was newly produced. The fourth coordination site of Zn(II) was probably occupied with the imidazole nitrogen of the histidyl residue at the N-terminal of another HisHisGlyGly complex.On the other hand, since HisGlyHisGly did not undergo deprotonation of the peptide bond between His-Gly at the N-terminal, the coordination mode with Zn(II) did not change over pH 6.

Endogenous tocopherols in extracted lipids from Jack beans (Canavalia gladiata DC.) were determined by high-performance liquid chromatography (HPLC), and were investigated in relation to the fatty acids (FA) distribution of triacylgycerols (TAG) and phospholipids (PL). The dominant tocopherols were (δ)-tocopherol (78.9-96.5mg%) and (γ)-tocopherol (42.1-56.1mg%) with much smaller amounts of (α)-tocopherol (1.1-1.3mg%). The lipids of Jack beans comprised mainly TAG (34.6-38.6 wt.%) and PL (54.8-57.4 wt.%), and other components were also detected in minor proportions (0.3-3.8 wt.%). The PL components included phosphatidyl choline (46.2-48.7 wt.%), phosphatidyl inositol (23.4-29.6 wt.%) and phosphatidyl ethanolamine (18.5-21.2 wt.%). Comparison of these different beans showed, with a few exceptions, no significant differences (P>0.05) in FA distribution. The FA distribution of TAG among the five beans was evident in the Jack beans: unsaturated FA (93.3-95.3 wt.%) were predominantly concentrated at the sn-2 position and saturated FA (33.6-34.4 wt.%) primarily occupying the sn-1 position or sn-3 position. The results obtained from this work would provide useful information to both producers and consumers for manufacturing functional foods or beverages in Japan and elsewhere.

The distribution of pink-pigmented facultative methylotrophs (PPFMs) on the leaves of various vegetables was studied. All kinds of vegetable leaves tested gave pink-pigmented colonies on agar plates containing methanol as sole carbon source. The numbers of PPFMs on the leaves, colony-forming units (CFU)/g of fresh leaves, differed among the plants, although they were planted and grown at the same farm. Commercial green perilla, Perilla frutescens viridis (Makino) Makino, gave the highest counts of PPFMs (2.0-4.1×10(7) CFU/g) of all the commercial vegetable leaves tested, amounting to 15% of total microbes on the leaves. The PPFMs isolated from seeds of two varieties of perilla, the red and green varieties, exhibited high sequence similarity as to the 16S rRNA gene to two different Methylobacterium species, M. fujisawaense DSM5686(T) and M. radiotolerans JCM2831(T) respectively, suggesting that there is specific interaction between perilla and the PPFMs.

The central tegmental tract (CTT) is mainly the extrapyramidal tract connecting between the red nucleus and the inferior olivary nucleus. There are only a few case reports describing CTT abnormalities on magnetic resonance imaging (MRI) in children. Our purpose was to evaluate the frequency of CTT lesions and their characteristics on MRI, and to correlate the MR imaging findings with clinical features. We reviewed retrospectively the MR images of 392 children (215 boys and 177 girls) ranging in age from 1 to 6 years. To evaluate symmetrical CTT hyperintense lesions, we defined a CTT lesion as an area of bilateral symmetrical hyperintensity in the tegmentum pontis on both T2-weighted images and diffusion-weighted images in more than two slices. We measured the ADC (apparent diffusion coefficient) values of symmetrical CTT hyperintensity, and compared them with those of children without CTT abnormality. CTT lesions were detected in 20 (5.1%) of the 392 children. The mean ADC value for these 20 children was significantly lower than that of the normal CTT (p

Complex formation of gramicidin (GA) and desformylgramicidin (des-GA) with sterols was investigated by measuring the intrinsic Trp fluorescence. In organic solvents, the Trp fluorescence of momeric GA was quenched upon binding either cholesterol or ergosterol, but that of monomeric des-GA was not quenched by adding cholesterol. Both dimeric GA and des-GA bound highly to ergosterol, but not to cholesterol, determined by quenching of Trp fluorescence. Furthermore, GA- and des-GA-loaded lysophosphatidylcholine micelles were incubated with phosphatidylcholine vesicles containing cholesterol or ergosterol. The results showed that both monomeric and dimeric peptides hardly bound to cholesterol incorporated into phospholipid vesicles, but markedly bound to ergosterol incorporated into the bilayer membranes. Interestingly, des-GA bound more specifically to the two sterols than GA. In addition, fluorescence resonance energy transfer analysis showed that des-GA bound more specifically to the two sterol than GA.

医薬保健研究域薬学系, Background: Weight-loss medicines, including crude drugs and herbal supplements disguised as diet-aid products, are readily obtainable and distributed widely, especially in Southeast Asia. Even if such products are unapproved or prescription-only medicines, consumers can purchase them through an agency or directly on the Internet. We evaluated the quality and safety of herbal products purchased on the Internet to reveal their influence on public health. Methods: Diet-aid products containing Bukuryo (Poria sclerotium), Bakumondo (Ophiopogonis tuber), or Daio (rhubarb rhizome) were purchased through websites that did not provide physical addresses or which advertised misleading medicines (e.g., unapproved Cialis 100 mg tablets, Viagra 100 mg tablets) on websites. We carefully noted details in the descriptions on package inserts or accompanying product characteristics and analyzed the ingredients using qualitative and quantitative methods, namely high-performance liquid chromatography equipped with a photodiode array detector. We requested the respective manufacturers to authenticate their products through a structured questionnaire. Results: We purchased 15 items from 15 Internet sites and imported all 15 items to Japan. One item stated to contain rhubarb rhizome was identified as a prescription medicine; the others were dietary supplements and not medicines. Even though we did not analyze the constituents of all crude drugs, we found some active ingredients in the items. Sibutramine was detected in items confirmed to be supplements, including those containing Poria sclerotium and Ophiopogonis tuber. Each capsule contained ≈ 12 mg of sibutramine, which is the daily dose for anti-obesity medicines. Sibutramine is not approved for use in Japan and its sale has been suspended in Europe and the USA owing to serious adverse effects on the circulatory system. Conclusions: Our findings indicate that dietary supplements containing injurious ingredients are distributed to Japanese consumers and potentially to a broader international audience, and that purchasing them through unreliable websites bears potential health risks. To avoid potential adverse events, there should be adequate alerts about the risks of taking products without appropriate indications. © 2015 Yoshida et al.

While there is an abundance of literature describing the association of chromosome aberrations with epilepsy, only a few refer to the detailed features of epilepsy. It is important to investigate the associations between specific chromosome abnormalities and features of epilepsy to identify genes involved in epilepsy and treat them more effectively. We investigated the correlation between specific chromosome aberrations and epilepsy by sending questionnaires to the members of Kyoto Multi-institutional Study Group of Pediatric Neurology. Seventy-six patients were collected from 10 institutions. Chromosome abnormalities included: Down syndrome (n = 19); Angelman syndrome (n = 8); Prader-Willi syndrome (n = 4); 4p- syndrome (n = 3); 1q- syndrome (n = 2); 5p- syndrome (n = 2); Miller-Dieker syndrome (n = 2); 18q- syndrome; (n = 2); Klinefelter syndrome; (n = 2); and 32 other individual chromosomal aberrations. Overall, the severity of mental retardation correlated with the severity of epilepsy. We could abstract characteristic features of epilepsy in some syndromes. In Angelman and Prader-Willi syndromes, febrile seizures occurred frequently, the onset of epilepsy was in early childhood and seizure phenotype was multiple. Paroxysmal discharge of the occipital region and diffuse high voltage slow wave on electroencephalography were characteristic in Angelman syndrome. In Down syndrome, West syndrome and focal epilepsy were common and the prognosis of epilepsy in West syndrome with Down syndrome was good. In 4p- syndrome, febrile seizures were often seen, and unilateral or generalized clonic or tonic-clonic status epilepticus were characteristic. For the other chromosomal aberrations investigated here, the patient numbers were too small to abstract common features of epilepsy.

Human mononuclear leukocytes (MNL), probably OKT4-positive T cells, produced an eosinophil chemotactic factor (ECF) when they were cocultured with irradiated BALL-1, a B cell lymphoma line. Treatment of MNL, with anti-IL-2 antibody failed to suppress BALL-1-induced ECF production. Periodate-lysine-paraformaldehyde-fixed but not acetone- and ethanol-fixed BCLL induced evident ECF production. These results suggested that some cell surface molecules play a role in the induction of ECF production. Isoelectric point of BALL-1-induced ECF was around pH7, whereas that of IL-2-induced ECF was around pH 5. The molecular weight of BALL-1-induced ECF was between 10 and 30 kD. Although a combination of MoAb against IL-3, IL-5, and GM, CSF suppressed the activity of IL-2-induced ECF, it failed to suppress that of BALL-1-induced ECF. Furthermore, BALL-1-induced ECF suppressed fMLP-induced respiratory bursts of eosinophils, while IL-2-induced ECF failed. We propose that at least one reason for eosinophil infiltrate into the stroma of tumors is that the tumor cells stimulate T cells to produce BALL-1-induced ECF, and the eosinophils attracted by the ECF exhibit different functions from those by other ECF.

The complexation of Zn(II) with glycyl-L-histidylglycine and its deuterated derivatives, glycyl-d2-L-histidylglycine and glycyl-L-histidylglycine-d2, was studied by proton nuclear magnetic resonance spectroscopy over the pD range, from 3.4 to 11.0, at 25 degrees C. Addition of Zn(II) to the peptide made the resonances of both imidazole C2-H and C4-H and both methylene-protons of glycyl residues at the amino-terminal and carboxylate end split to three lines in the pD range above 7.0. From the behavior of the C2-H and C4-H chemical shifts, formation of at least two species, in which the imidazole and amino nitrogens coordinated to the metal ion forming a chelate ring, was suggested. Those two species, the ratio of which varied depending on the total concentrations of complexes, seemed to be interconvertible; one referred to as B is a monomer and the other, referred to as C, may be a dimeric or polymeric complex.

医薬保健研究域薬学系, Objective: To explore the circulation of anti-obesity medicines via the internet and their quality. Design: Cross-sectional study. Setting: Internet pharmacies and pharmaceutical suppliers accessible from Japan. Participants: Anti-obesity medicines were purchased using relevant keywords on Japanese Google search engine. Blogs and advertisement-only sites were excluded. Primary and secondary outcome measures: The authenticity of the samples was investigated in collaboration with the manufacturers of the samples and medicine regulatory authorities. Quality of the samples was assessed by pharmacopoeial analyses using high-performance liquid chromatography. Results: 82 samples were purchased from 36 internet sites. Approximately half of the sites did not mention a physical address, and 45% of the samples did not contain a package insert. A variety of custom declarations were made for the shipments of the samples: personal health items, supplement, medicines, general merchandise, tea and others. Among 82 samples, 52 samples were analysed to check their pharmacopoeial quality. Authenticity responses were received from only five of 20 manufacturing companies. According to the pharmacopoeial analyses and authenticity investigation, three of the samples were identified as counterfeits and did not contain any active ingredients. Two of these samples were confirmed as counterfeits by the manufacturer of the authentic products. The manufacturer of the other sample did not respond to our request for an authenticity check even after several communication attempts. These counterfeit cases have been reported at the rapid alert system of Western Pacific Region of the WHO. Conclusions: Many counterfeit and unapproved anti-obesity medicines may be easily bypassing regulatory checks during shipping and are widely circulated through the internet. Regulatory authorities should take measures to prevent these medicines from entering countries to safeguard their citizens.

Chemotactic responses of eosinophils from patients with allergic rhinitis to 5 STO-2-derived eosinophil chemotactic factors (ECF), IL-3, IL-5 and GM-CSF were examined. The patients were divided into two groups: patients with perennial allergic rhinitis sensitive to Dermatophagoides farinae and those with seasonal allergic rhinitis sensitive to Japanese cedar pollen. There was no essential difference between chemotactic response of eosinophils from the former to STO-2-derived ECF and that from healthy individuals. However, eosinophils from patients with seasonal allergic rhinitis failed to respond to one of the STO-2-derived ECFs, ECF-P19. Before and after nasal antigen provocation, no change occurred in the chemotactic profiles. Furthermore, we found that eosinophils from both types of patients with allergic rhinitis responded not only to IL-3 and GM-CSF but also to IL-5 unlike those of healthy individuals.

An indirect immunofluorescence test using a flow cytometer (FACS420) was useful for detection of antineutrophil antibodies (anti-NA2 antibodies) in a case of monozygous twins with alloimmune neonatal neutropenia. When high-dose intravenous gammaglobulin (300 mg/kg for five consecutive days) was given to one twin, the increase in neutrophils was not obvious. Although neutropenia persisted for about three months, no infections were observed in either of the twins.

Anomalous glycosylation shift values of the signals for C-1 (the anomeric carbon atom) and C-2 (the glycosyloxylated carbon atom) were sometimes observed for 2- O -β- d -glycopyranosyl (or -β- d -xylopyranosyl)-β- d -glucopyranosides, whereas no remarkable displacements of the other sugar-carbon signals were observed in these cases. This can be explained in terms of change of the orientation of the glycosyl linkages, owing to strong, steric interaction between the 2- O -glycosyl group and the 1- O -aglycon (or -sugar) group. Various 2- O -glycosylated α- and β- l -arabinopyranosides were synthesized. In the case of α- l -arabinopyranosides, the processes of 2- O -β- d -glucosylation, -β- d -xylosylation, and -α- l -arabinosylation resulted in unexpected, upfield shifts of the C-3, -4, and -5 signals, together with displacement of the C-1 and -2 resonances. Furthermore, significant alteration of the values of 3 J H1,H2 and 1 J C1,H1 was also observed for the 2- O -glycosylated α- l -arabinopyranoside moiety, indicating an increase in the contribution of the 1 C 4 conformation of the glycosylated α- l -arabinopyranoside in these cases. On the other hand, no remarkable variation in the signals of C-3, -4, and -5, or in the 3 J H1,H2 and 1 J C1,H1 values, was found for 2- O -α- l -rhamnosylation, except for 4-epihederagenin-3-yl 2- O -α- l -rhamnopyranosyl-α- l -arabinopyranoside. In the present study, such unusual 2- O -glycosylation shifts were not encountered for the 2- O -glycosylation of β- l -arabinopyranosides.

A proton nuclear magnetic resonance (1H-NMR) study of the interaction of Zn (II) ion with histidine-containing peptide, L-histidylglycylglycine (his-gly-gly), was carried out. The 1 : 2 complex of Zn (II)-(his-gly-gly) takes different coordination structures depending on the pH. In the region of pH 4.5-6.5, the complex is a tautomer in which the Zn (II) ion is exchanged dynamically among amide carbonyl oxygen, imidazole nitrogen, and amino nitrogens above pH 6.5.