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We analyzed the bacterial flora and chemical properties of the Japanese traditional anchovy product called salted Etari, which is distributed in Nagasaki Prefecture in the western part of Japan. The pH and NaCl concentrations ranged from 6.4 to 6.6 and from 9.6% to 13.9%, respectively. The lactic acid and total free amino acid concentrations were determined to be between 122 and 344 mg/100 g of sample and between 2850 and 4783 mg/100 g of sample. The bacterial cell counts were determined to be 103–108 and 104–108 cells/g under aerobic and anaerobic conditions using a plate medium containing 10% NaCl, respectively. The isolates were classified into six groups on the basis of the results of PCR-restriction fragment length polymorphism (RFLP) analysis targeting the 16S rRNA gene. According to the results of 16S rRNA gene sequencing, the predominant PCR–RFLP group was identified as the halophilic lactic acid bacterium Tetragenococcus halophilus, and the remaining groups were identified as bacteria belonging to the genera Chromohalobacter, Halomonas, and Staphylococcus. The results of culture-independent analysis, namely, terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the 16S rRNA gene, indicated that T. halophilus is the bacterium commonly found in salted Etari.

Bacteria can migrate towards more suitable environments by rotating flagella that are under the control of sensory signal transduction networks. The bacterial flagellum is composed of the long helical filament functioning as a propeller, the flexible hook as a universal joint and the basal body as a rotary motor powered by ion motive force across the cell membrane. The flagellar motor consists of a rotor and multiple stator units, each of which couples the ion flow through its ion channel with force generation. The flagellar building blocks and motor proteins are highly conserved among bacterial species, but structural and functional diversity of flagella has also been revealed. It has been reported that the structure and function of the flagellar motor of a Gram-positive bacterium, Bacillus subtilis, differ from those of Escherichia coli and Salmonella. The flagellar motor of the B. subtilis BR151MA strain possesses two distinct types of stator complexes, H+-type MotAB and Na+-type MotPS, around the rotor. These two types of stator units dynamically assemble to and disassemble from the rotor in response to environmental changes such as viscosity and external Na+ concentrations. In this mini-review article, we describe our recent understanding of the structure and dynamics of the B. subtilis flagellar motor.

A novel actinobacterium producing biosurfactant, designated OTB305T, was isolated from marine sediment sampled at Otsuchi Bay, Iwate Prefecture, Japan and its taxonomic position was examined using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences exhibited that strain OTB305T was closely related to Streptomyces bohaiensis JCM 19630T (98.8 %) and Streptomyces lonarensis DSM 42084T (98.8 %). The chemotaxonomic characteristics of strain OTB305T corresponded to those of the genus Streptomyces as follows: the diamino acid of the cell-wall peptidoglycan was ll-diaminopimelic acid; whole-cell hydrolysates contained glucose and lacked characteristic major sugars; the predominant isoprenoid quinones were MK-9(H8) and MK-9(H6); the polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unidentified phospholipid; the major cellular fatty acids were iso-C16 : 0, C16 : 0 and C16 : 1 ω7c; and the genomic DNA G+C content was 72.83 mol%. However, genomic relatedness analysis based on the average nucleotide identity and some phenotypic characteristics revealed that strain OTB305T was distinguished from closely related Streptomyces species. Therefore, strain OTB305T represents a novel species of the genus Streptomyces , for which the name Streptomyces otsuchiensis sp. nov. is proposed. The type strain is OTB305T (=NBRC 113255T=TBRC 9682T).

Seriniquinone was originally isolated as a melanoma-selective anti-cancer agent from a culture broth of marine bacteria. Pharmacological studies on its selectivity and unique target are ongoing. A new dihydronaphthothiophene (1) was synthesized by the biological transformation of seriniquinone using marine-derived actinomycete Streptomyces albogriseolus OM27-12, and its derivatives (2-4) were chemically synthesized. Compounds 1-4 exhibited selective cytotoxic activity against melanoma and improved solubility.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Tellurium (Te) has been increasingly used as a semiconductor material in copious amounts, with a concomitant increase in its discharge from industrial effluents and mining wastewater into the environment. However, soluble Te, such as tellurate (VI) and tellurite (IV), is toxic to organisms. Thus, highly efficient technologies need to be developed for a double-benefit detoxification and recovery of soluble Te from industrial and mining wastewater. Since industrial wastewater contains high concentrations of salt, salt-tolerant microorganisms that metabolize rare metals such as Te have been the subject of focus for the effective detoxification and recovery of Te. In the present study, a total of 52 salt-tolerant tellurate-reducing microorganisms were isolated from marine environmental samples. Of these, 18 strains achieved greater than, or equal to, 50% removal of water-soluble Te from a medium containing 0.4 mM tellurate after 72 h incubation. The 18 isolated strains belonged to 13 species of the following 9 genera: Sulfitobacter, Ruegeria, Hoeflea, Alteromonas, Marinobacter, Pseudoalteromonas, Shewanella, Idiomarina, and Vibrio. No microorganism has been reported to reduce tellurate and tellurite from six of the aforementioned genera, namely, Sulfitobacter, Ruegeria, Alteromonas, Marinobacter, Idiomarina, and Vibrio. Especially, one of the isolates Sulfitobacter sp. strain TK39B, removed 82% (w/w) of soluble Te with a 4% NaCl tolerance. These results showed that salt-tolerant tellurate-reducing bacteria that can be used in the detoxification and recovery of Te are widely present in the marine environment.

Bacillus subtilis possesses two distinct types of stator protein complexes for the flagellar motor: H+-type MotAB and Na+-type MotPS. The MotPS complex is used when both the external Na+ concentration and viscosity are high. Because deletion of the motPS genes does not affect swimming motility of cells, a physiological role of MotPS-dependent motility remains unclear. Here, we report that the MotPS stator complex is required for efficient biofilm maturation. Depletion of the MotPS complex did not cause a significant delay in the initiation of biofilm formation but reduced the number of viable cells in the biofilm. The MotAB stator units were efficiently replaced by the MotPS complexes with an increase in the viscosity of the environments. Therefore, we propose that MotPS-dependent motility of motile cells in the biofilm structure is required to efficiently keep the bacterial society in the biofilm healthy.

The bacterial flagellum is a motility organelle consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC, for flagellar filament formation and autonomously switches their expression at a frequency of 10&minus, 3&ndash, 10&minus, 4 per cell per generation. We report here differences in their structures and motility functions under high-viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than one expressing FliC under high viscosity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure was nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions.

The bacterial flagellum is a motility organelle, consisting of a long helical filament as a propeller and a rotary motor that drives rapid filament rotation to produce thrust. Salmonella enterica serovar Typhimurium has two genes of flagellin, fljB and fliC, for flagellar filament formation and autonomously switches their expression at a frequency of 10-3–10-4 per cell per generation. We report here differences in their structures and motility functions under high viscosity conditions. A Salmonella strain expressing FljB showed a higher motility than the one expressing FliC under high viscousity. To examine the reasons for this motility difference, we carried out structural analyses of the FljB filament by electron cryomicroscopy and found that the structure is nearly identical to that of the FliC filament except for the position and orientation of the outermost domain D3 of flagellin. The density of domain D3 was much lower in FljB than FliC, suggesting that domain D3 of FljB is more flexible and mobile than that of FliC. These differences suggest that domain D3 plays an important role not only in changing antigenicity of the filament but also in optimizing motility function of the filament as a propeller under different conditions.

Malachite green (MG) has been focused on as a biotreatment target and its biological properties have also been an issue in food fish aquaculture. An MG-removing bacterium was isolated from aquaculture fish pond sediment samples in Thailand. The isolate, strain T-5-2, is a Gram-negative, aerobic rod-shaped bacterium, and has been identified as a member of the Pseudomonas putida group. Proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis of a broth culture medium containing MG showed that the concentration of MG decreased markedly and that other molecules, including leucomalachite green (LMG), were generated. Moreover, liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis showed that the MG concentration in the broth culture medium continuously decreased. This analysis also demonstrated that the concentration of LMG initially increased and then gradually decreased. Furthermore, gas chromatography–mass spectrometry (GC–MS) analysis showed 4-(dimethylamino)benzophenone (4DABP) as a degradation component of MG, which was confirmed by 1H-NMR and LC–MS/MS analysis. These findings suggest that this bacterial strain can remove MG in broth culture and degrade it to certain metabolites, including LMG and 4DABP. This study is the first detailed evaluation by the combination of LC–MS/MS, GC–MS, and 1H-NMR analyses of an MG-removing bacterium isolated from Thai aquaculture fish ponds.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anthocyanin pigments accumulated in a cell line derived from storage-root explants of sweet potato (Ipomoea batatas L.) cv 'Ayamurasaki'. Somatic pro-embryos were induced on the explants cultured on Murashige and Skoog medium supplemented with 1 mg/l 2,4-D. The pro-embryo structures produced callus when transferred to MS medium with 0.5 mg/l 2,4-D. A cell line was isolated from this callus which accumulated anthocyanin pigment. The color value of the pigment extracted after 27 days of culture in MS medium with 2 mg/l 2,4-D was 8.2, which was very close to that of a pigment extracted from roots, which was 8.9. Most of the pigments from the cell extract were hydrophilic and appeared on the ODS-column HPLC with a lower retention time than the main anthocyanins of the root tissues. The majority of the pigments were identical with the root anthocyanins. Cell line-specific anthocyanins were detected.

Histamine-producing bacteria were isolated from various types of canned salted anchovies with cooking oil, which were purchased from retail groceries over a study period of eight years. Thirty-two histamine-producing bacterial isolates were collected from 13 samples, which were equivalent to 43 % of the total canned anchovies manufactured in four different countries. All of these histamine-producing bacterial isolates were Gram-positive, catalase-negative, tetrad-forming cocci, and were identified as the lactic acid bacterium Tetragenococcus muriaticus by 16S rRNA gene sequence analysis. These findings indicated that histamine-producing T. muriaticus cells are widely distributed in the canned anchovies that were purchased from retail groceries. In addition, to evaluate the suppression of histamine production using a model of the storage condition of canned anchovies, the histamine-producing ability of our isolates under various culture conditions was examined. Low-pH and low-temperature conditions were demonstrated to be useful for inhibiting the histamine production of these isolates.

The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force, however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli &Delta, motAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis &Delta, motAB&Delta, motPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation.

Bacteria use a type III protein export apparatus for construction of the flagellum, which consists of the basal body, the hook, and the filament. FlhA forms a homo-nonamer through its C-terminal cytoplasmic domains (FlhAC) and ensures the strict order of flagellar assembly. FlhAC goes through dynamic domain motions during protein export, but it remains unknown how it occurs. Here, we report that the FlhA(G368C) mutation biases FlhAC toward a closed form, thereby reducing the binding affinity of FlhAC for flagellar export chaperones in complex with their cognate filament-type substrates. The G368C mutations also restrict the conformational flexibility of a linker region of FlhA (FlhAL), suppressing FlhAC ring formation. We propose that interactions of FlhAL with its neighboring subunit converts FlhAC in the ring from a closed conformation to an open one, allowing the chaperon/substrate complexes to bind to the FlhAC ring to form the filament at the hook tip.

In our screening program on marine-derived actinomycetes, Nonomuraea sp. AKA32 isolated from deep-sea water collected from a depth of 800 m in Sagami Bay, Japan was found to produce compounds cytotoxic to cancer cells. Activity-guided purification led to the isolation of a new aromatic polyketide, akazamicin (1), along with two known compounds, actinofuranone C (2) and N-formylanthranilic acid (3). Structures of these compounds were elucidated through the interpretation of NMR and MS spectroscopic data. Compounds 1, 2, and 3 displayed cytotoxicity against murine B16 melanoma cell line with the IC50 value of 1.7, 1.2, and 25 μM, respectively.

Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14−CD1c+ conventional DCs (cDCs) and CD14−CD141+ cDCs, in addition to CD14+ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123+ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c+ myeloid cells, CD141+ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3+ cells within splenic CD4+ T cells were significantly increased in the presence of GM-CSF. Foxp3+ T cells could be subdivided into two subpopulations, CD45RA−Foxp3hi and CD45RA−Foxp3lo T cells. Whereas CD45RA−Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA−Foxp3lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3+ T cells. The correlation analysis demonstrated that the development of these Foxp3+ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.

Cooperative remodeling of the FlhA ring terminates hook assembly and initiates filament assembly at the hook tip., The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.

The screening of malachite green (MG)-degrading microorganisms was carried out using various sources, namely, fish farms and a traditional fermented fishery product in Myanmar and Thailand. The enrichment culture method was performed using MG-containing broth media, and colonies that showed the decolorization of MG on plate media were isolated as MG-degrading candidates. From the results of the sequencing of the D1/D2 domain of the 26S rRNA gene, strain M3, a representative strain of MG-degrading candidates was identified as a halotolerant yeast, Debaryomyces nepalensis. Liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis indicated that during the incubation of this strain, the MG concentration gradually decreased and eventually reached undetectable levels. Conversely, the concentration of leucomalachite green (LMG) increased, and the final amount of LMG in the broth culture was estimated to be approximately 40 % of the initial MG amount. In addition, results of proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis also showed that MG and the tautomer of MG or other aromatic decomposition products of MG were not detected as a major component at the end of incubation. These results suggest that strain M3 removed MG and changed approximately 40 % to LMG and 60 % to some metabolites other than LMG.

Diketopiperazines, inhibitors of sterol O -acyltransferase, produced by a marine-derived Nocardiopsis sp. KM2-16

This study is a detailed description of the microflora of a traditional fishery product in Myanmar which is fermented with boiled rice. We approached this analysis from two viewpoints; namely, the culture-dependent and culture-independent methods. In Southeast Asia, there are various types of traditional fermented fishery products. In this study, we isolated and characterized lactic acid bacteria (LAB) from small freshwater fish (tinfoil barb) fermented with boiled rice, a typical Myanmar fermented product, to contribute to the understanding of its fermentation process. Eight fermented fishery products were purchased from different markets in Yangon. Forty-three of the 46 isolates were identified as LAB, and they were classified into two groups: 40 homofermentative and three heterofermentative isolates, on the basis of their phenotypic characteristics. From the results of PCR-restriction fragment length polymorphism (RFLP) analysis and 16S rRNA gene sequencing, our isolates were identified as Lactobacillus plantarum-group, Lactobacillus farciminis, Lactobacillus futsaii, Lactobacillus reuteri, Weissella paramesenteroides, and Pediococcus pentosaceus. In addition, L. plantarum and L. farciminis were identified as γ-aminobutyric acid (GABA)-producing LAB. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also carried out using DNA samples extracted from these fermented products. In comparison with culture-dependent methods, the results of T-RFLP analysis did not seem to have major contradictions.

Delphinidin 3-sambubioside (Dp3-Sam), a Hibiscus anthocyanin, was isolated from the dried calices of Hibiscus sabdariffa L, which has been used for folk beverages and herbal medicine although the molecular mechanisms are poorly defined. Based on the properties of Dp3-Sam and the information of inflammatory processes, we investigated the anti-inflammatory activity and molecular mechanisms in both cell and animal models in the present study. In the cell model, Dp3-Sam and Delphinidin (Dp) reduced the levels of inflammatory mediators including iNOS, NO, IL-6, MCP-1, and TNF-α induced by LPS. Cellular signaling analysis revealed that Dp3-Sam and Dp downregulated NF-κB pathway and MEK1/2-ERK1/2 signaling. In animal model, Dp3-Sam and Dp reduced the production of IL-6, MCP-1 and TNF-α and attenuated mouse paw edema induced by LPS. Our in vitro and in vivo data demonstrated that Hibiscus Dp3-Sam possessed potential anti-inflammatory properties.

The bacterial flagellar motor consists of a rotor and a dozen stator units and regulates the number of active stator units around the rotor in response to changes in the environment. The MotPS complex is a Na+-type stator unit in the Bacillus subtilis flagellar motor and binds to the peptidoglycan layer through the peptidoglycan-binding (PGB) domain of MotS to act as the stator. The MotPS complex is activated in response to an increase in the Na+ concentration in the environment, but the mechanism of this activation has remained unknown. We report that activation occurs by a Na+-induced folding and dimer formation of the PGB domain of MotS, as revealed in real-time imaging by high-speed atomic force microscopy. The MotPS complex showed two distinct ellipsoid domains connected by a flexible linker. A smaller domain, corresponding to the PGB domain, became structured and unstructured in the presence and absence of 150 mM NaCl, respectively. When the amino-terminal portion of the PGB domain adopted a partially stretched conformation in the presence of NaCl, the center-to-center distance between these two domains increased by up to 5 nm, allowing the PGB domain to reach and bind to the peptidoglycan layer. We propose that assembly of the MotPS complex into a motor proceeds by means of Na+-induced structural transitions of its PGB domain.

A novel Gram-stain-positive actinobacterium, designated HT7-17T, was isolated from a sediment sample collected from the estuary of the Tama River, Japan, and its taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain HT7-17T was closely related to members of the genus Lysinimicrobium , with a similarity range of 97.1–98.2 %. The peptidoglycan type of strain HT7-17T was A4α, the predominant menaquinone was demethylmenaquinone DMK-9(H4) and the major fatty acids were anteiso-C15 : 0, C16 : 0 and iso-C16 : 0. The DNA G+C content was 69.9 mol%. These chemotaxonomic features corresponded to those of the genus Lysinimicrobium . Meanwhile, the differences in some phenotypic characteristics, along with the result of DNA–DNA hybridization, indicated that strain HT7-17T should be distinguished from the recognized species of the genus Lysinimicrobium . Therefore, strain HT7-17T represents a novel species of the genus Lysinimicrobium , for which the name Lysinimicrobium sediminis sp. nov. is proposed. The type strain is HT7-17T (=NBRC 112286T=TBRC 7037T).

The flagellar motor of Bacillus subtilis possesses two distinct H+-type MotAB and Na+-type MotPS stators. In contrast to the MotAB motor, the MotPS motor functions efficiently at elevated viscosity in the presence of 200 mM NaCl. Here, we analyzed the torque-speed relationship of the Bacillus MotAB and MotPS motors over a wide range of external loads. The stall torque of the MotAB and MotPS motors at high load was about 2,200 pN nm and 220 pN nm, respectively. The number of active stators in the MotAB and MotPS motors was estimated to be about ten and one, respectively. However, the number of functional stators in the MotPS motor was increased up to ten with an increase in the concentration of a polysaccharide, Ficoll 400, as well as in the load. The maximum speeds of the MotAB and MotPS motors at low load were about 200 Hz and 50 Hz, respectively, indicating that the rate of the torque-generation cycle of the MotPS motor is 4-fold slower than that of the MotAB motor. Domain exchange experiments showed that the C-terminal periplasmic domain of MotS directly controls the assembly and disassembly dynamics of the MotPS stator in a load- and polysaccharide-dependent manner.

Synechocystis sp. PCC 6803, a unicellular cyanobacterial species, changes intracellular nitrogen and carbon metabolism through the transcription factor NtcA in response to nitrogen deficiency. In this study, the change in metabolites in carbon storages and the related metabolites after nitrogen depletion was analysed using ntcA-overexpressing strain. Levels of polyhydroxybutyrate (PHB), a biodegradable polyester, increased in the ntcA-overexpressing strain after nitrogen depletion. Capillary electrophoresis-mass spectrometry (CE-MS) analysis revealed that the levels of fumarate and malate increased and phosphoenolpyruvate, isocitrate, 2-oxoglutarate, succinate, phosphorylated monosaccharides and dihydroxyacetone phosphate decreased by ntcA overexpression. Respiratory activity decreased by ntcA overexpression, possibly resulted from the alteration of carbon metabolism and perturbation of redox balance. These results have revealed that NtcA is involved in the re-distribution of intracellular carbon sources to PHB and organic acids in the tricarboxylic acid cycle in this cyanobacterium.

Flavones and anthocyanins were isolated from the leaves and flowers of 14 Ajuga taxa (Lamiaceae), which are all native or naturalized in Japan. Of 13 flavones obtained from the leaves, 11 were characterized as apigenin, luteolin, 6-hydroxyluteolin and acacetin glycosides. Ten flavones were isolated from the flowers. Ten anthocyanins were isolated from the flowers. Six of these anthocyanins were identified as acylated delphinidin glycosides and four were shown to be acylated cyanidin glycosides. Japanese Ajuga taxa were chemotaxonomically discussed by their distribution patterns, especially foliar flavonoids.

The aim of this study was to clarify the transport behaviour and mechanism of caffeic acid analogue bearing a sugar-moiety, 6-O-caffeoylsophorose (CS), in Caco-2 cells. The absorption of CS was investigated by its transport across Caco-2 cell monolayers using a high-performance liquid chromatography-time-of-flight-mass spectrometry (LC-TOF-MS). The permeation of CS was concentration-dependent and reached the plateau at >6 mM. The apparent permeability (P(app)) of CS in the apical-to-basolateral direction was 5.4×10(-7) cm/s, while in the reversed direction the P(app) value was significantly reduced (1.9×10(-7) cm/s). CS transport was competitively inhibited by phloretin, an inhibitor of monocarboxylic acid transporter (MCT). Benzoic acid, an MCT substrate, also reduced CS transport. A less significant change of CS transport was observed across Caco-2 cell monolayers pretreated with quercetin, a suppressor of tight-junction. These findings strongly indicate that CS, a caffeic acid analogue bearing sophorose moiety, can be transported across Caco-2 cell monolayers via the MCT pathway.

The aim of the present study was to develop a strain-specific polymerase chain reaction (PCR) primer set for the detection of Bifidobacterium bifidum OLB6378 (OLB6378) that can serve as suitable probiotics for infants. The random amplified polymorphic DNA (RAPD)-PCR technique was used to obtain OLB6378-specific PCR products. One OLB6378-specific RAPD-PCR product was obtained after testing 97 RAPD primers, and was sequenced. Thirteen PCR primer sets were designed from the sequence. One PCR primer set was found to amplify one PCR product when genomic DNA of OLB6378 was used as template. The primer set did not amplify any PCR product when the other genomic DNA was used as template. The primer set was tested with 47 strains of B. bifidum and 20 strains of the other Bifidobacterium species. As a result, we developed an OLB6378-specific primer set, one that should be useful not only for the detection of OLB6378 but also for the quantification of OLB6378.

A non-destructive method for analyzing crystalline tulobuterol (TBR; a bronchodilator [β(2)-blocker]) in transdermal drug delivery system tapes with a crystal reservoir system was developed. A near infrared spectroscopy (NIRS) and a near infrared spectroscopic imaging (NIRI) were used to investigate the distribution of TBR crystals in transdermal tapes. The characteristic peak derived from a first overtone of secondary amine which appears based on crystal growth was used for the detection of crystals. NIR images were composed by the integrated values of that peak at each pixel. The time-course analysis by NIRS showed that the intensity of the peaks gradually increased, and the intensity reached a plateau between day 30 and day 42 after preparation of the model tapes. The authors observed the growth and distribution of TBR crystals in small areas in several types of matrices by NIRI time-course measurement. The authors also found that a macroscopic map can provide a rough distribution map of crystalline TBR in a whole matrix. In the case in which a tape distributed from the innovator was examined, the characteristic peak was also detected through a liner or a supporting board, by transmittance-reflectance NIR measurement.

Rotation of bacterial flagellar motor is driven by the interaction between the stator and rotor and the driving energy is supplied by ion influx through the stator channel. The stator is composed of the MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA and MotB are four- and single-transmembrane proteins, respectively. To generate torque, the MotA/MotB stator unit changes its conformation in response to the ion influx and interacts with the rotor protein FliG. Here, we overproduced and purified MotA of the hyperthermophilic bacterium Aquifex aeolicus. A chemical crosslinking experiment revealed that MotA formed a multimeric complex, most likely a tetramer. The three-dimensional structure of the purified MotA, reconstructed by electron microscopy single particle imaging, consisted of a slightly elongated globular domain and a pair of arch-like domains with spiky projections, likely to correspond to the transmembrane and cytoplasmic domains, respectively. We show that MotA molecules can form a stable tetrameric complex without MotB and for the first time, demonstrate the cytoplasmic structure of the stator.

Two new acylated anthocyanins were isolated from beninabana, Brassica campestris var. chinensis, in addition to two known anthocyanins. The structures were established by spectral analyses.

Aim: The aim of this study was to clarify the relationship between pathological effects and the prognosis of patients with stage III non-small cell lung cancer (NSCLC) treated with induction chemoradiation. Methods: Patients who were untreated and potentially resectable with stage III NSCLC were enrolled. They received carboplatin and docetaxel with concurrent radiotherapy (5 × 2 Gy/week with a total dose of 40 Gy) followed by surgery. We assessed the relationship between the pathological effect (Ef) (Ef 1: slight pathological response, Ef 2: moderate pathological response, Ef 3: complete pathological response) and prognosis. Results: In all, 30 patients with stage III NSCLC (24 men and 6 women, mean age 60.7 years, 17 with adenocarcinomas and 13 with squamous cell carcinomas, 21 with clinical stage IIIA and nine with stage IIIB) participated in the trial and underwent induction chemoradiation. A total of 27 patients (90%) with complete response, partial response and stable disease had surgical resection. The pathological effect was Ef 1 and Ef 2 in 10 patients each, and Ef 3 in seven patients. Median survival was 10.9 months in patients with Ef 1 and 49.6 months in patients with Ef 2. Six out of seven Ef 3 patients are alive at the time of writing with a mean survival of 77.1 months (14–104 months). There was a significant difference in overall survival based on pathological effect rating (P = 0.0036). Conclusion: The Ef rating was well correlated with prognosis after induction chemoradiation.

A new depsidone, named 7-chlorofolipastatin, and five known structurally related depsidones were isolated from the culture broth of the marine-derived fungus Aspergillus ungui NKM-007 by solvent extraction and HPLC using an octadecylsilyl column. The structure of 7-chlorofolipastatin was elucidated by various spectroscopic data including 1D and 2D NMR spectroscopy. 7-Chlorofolipastatin inhibited sterol O-acyltransferase (SOAT) 1 and 2 isozymes in cell-based and enzyme assays using SOAT1- and SOAT2-expressing Chinese hamster ovary (CHO) cells.

Putative esterase genes were isolated from environmental DNA by using pre-amplified inverse PCR. The sequence analysis of the isolated genes showed 32–80% amino acid sequence identities to known esterases/lipases in public databases. The isolated genes were subsequently expressed in recombinant Escherichia coli . Insoluble proteins were noted in the expression of the majority of the isolated genes. The findings suggest that it is difficult to isolate these genes by using activity-based screening with construction of metagenome library. For the enzymes characterized, we examined substrate specificity, optimal temperature, optimal pH, and thermal stability. The substrate specificity of all the enzymes was high for p -nitrophenyl acetate, but almost undetectable for p -nitrophenyl decanoate. The results indicate that the obtained enzymes are defined as esterases. The enzymes were active in a broad range of temperature. The optimum activity was observed at 25–70 °C and at pH 8.0–9.0. Some enzymes have moderate thermostability and would be useful for industrial enzymes. This study illustrates that pre-amplified inverse PCR, which is one of the sequence-based approach, is potentially applicable to the isolation of diverse genes from environmental DNA.

Potatoes come in many various colors of tuber peels and/or fleshes. Anthocyanin pigment is responsible for red and purple colors of colored potato varieties. We report anthocyanin determination of a red potato cultivar Kintoki-imo and other colored cultivars grown in Hokkaido, Japan, using DAD-HPLC and ESI-TOF/MS.13C-NMR and 1H-NMR analyses showed that the major Kintoki-imo anthocyanin were pelargonidin 3-(6-O-(4-O-(E)-p-coumaroyl-α-L-rhamnopyranosyl)-β-D-glucopyranoside)-5-β-D-glucopyranoside (pelanin), and pelargonidin 3-(6-O-(4-O-(E)-feruloyl-α-L-rhamnopyranosyl)-β-D-glucopyranoside)-5-β-D-glucopyranoside. The anthocyanin content per 100 g flesh tuber was 2-816 mg. The anthocyanin composition patterns of potato cultivars were classified into five types: type A contained a lot of pelanin and Pg 3-Fr.Rut-5-Glc; type B was similar to type A, except that it included purple pigments such as peonanin and Pn 3-Rut-5-Glc; type C was also close to type A, except that the peonanin content was high; type D had pelanin as the main pigment, but no peonanin; and type E had petanin as the main pigment, but no pelanin.

This paper presents a successful synthesis of transparent organic–inorganic nanocomposites from poly(arylene ether ketone) (PAEK)-based matrix polymer and BaTiO3 nanoparticles. The dispersibility of nanoparticles was significantly improved by both the introduction of phosphonic acid moiety into the polymer chain and the organic modification of the nanoparticle surface. The structure of phosphonic-acid-modified PAEK polymer was studied by gel permeation chromatography, 31P nuclear magnetic resonance spectroscopy and X-ray fluorescence spectroscopy. Thermal properties of nanocomposites were studied by thermogravimetry and differential scanning calorimetry. Optical clarity of nanocomposites was evaluated by a hazemeter and regular transmittance spectroscopy. Dispersion of nanoparticles was also demonstrated by transmission electron microscopic observation. Fourier transform infrared analysis was carried out to investigate the interaction between the polymer matrix and nanoparticles. Refractive indices of the obtained transparent nanocomposites, which contain up to 44.1 wt% of nanoparticles, were measured. A high refractive index of 1.72 at 589 nm was achieved. Transparent organic–inorganic nanocomposites were successfully synthesized from poly(arylene ether ketone)-based matrix polymer and BaTiO3 nanoparticles. The dispersibility of nanoparticles was significantly improved by both the introduction of phosphonic acid moiety into the polymer chain and the organic modification of the nanoparticle surface. Refractive index of the obtained transparent nanocomposite, which contains 44 wt% of BaTiO3, increased to 1.72 @ 589 nm.

The new acylated polyphenols were isolated from a red-colored vinegar produced via fermentation with purple-fleshed sweet potato storage roots, and identified mainly by MS and NMR. The three acylated sophoroses were determined as 6-O-(E)-caffeoyl-(2-O-(6-O-acyl)-beta-d-glucopyranosyl)-d-glucopyranoses, where acyl was (E)-caffeoyl, p-hydroxybenzoyl, and (E)-feruloyl, respectively. The four acylated anthocyanins were also determined as cyanidin 3-O-(6-O-(E)-caffeoyl-(2-O-(6-O-(E)-feruloyl)-beta-d-glucopyranosyl)-beta-d-glucopyranoside), in addition to peonidin 3-O-(6-O-(E)-caffeoyl-(2-O-(6-O-acyl)-beta-d-glucopyranosyl)-beta-d-glucopyranosides), where acyl was (E)-caffeoyl, p-hydroxybenzoyl, and (E)-feruloyl, respectively. The diacylated sophoroses showed higher antioxidant capacity than that of monoacylated analogue 6-caffeoylsophorose, so the multiacylation established to enhance their antioxidant capacity. Similarly, 5-deglucosylated anthocyanins also gave somewhat stronger antioxidation than corresponding sweet potato anthocyanins. In rat intestinal alpha-glucosidase inhibition study, the diacylated sophoroses preferably inhibited maltase rather than sucrase with an IC(50) value of

Transparent organic–inorganic nanocomposites were successfully synthesized from sulfonic acid-modified poly(bisphenol A carbonate) (SPC) and TiO2 or ZrO2 nanoparticles. The dispersibility of nanoparticles was significantly improved by both the surface treatment of nanoparticles with phosphoric acid 2-ethylhexyl esters (PAEH) and the introduction of a sulfonic acid moiety into the PC chain. It was found that in some cases, crystallization of the matrix caused a reduction in transparency. Efficient dispersion of nanoparticles and the absence of crystallization resulted in highly transparent nanocomposites with up to 42 wt% TiO2 and 50 wt% ZrO2 nanoparticles. The refractive indices of the nanocomposites based on SPC increased with the increasing amount of nanoparticles. Theoretical equation based on Maxwell–Garnett effective medium theory provided reasonably close estimation of the refractive indices to the experimentally observed values. The prepared nanocomposites had lower thermal stability than the host matrix polymers.

11品種の有色サツマイモ（白色 : 3，黄色 : 2，橙色 : 2，紫色 : 3）の凍結乾燥品から高速溶媒抽出装置を用いて抽出液を調製し，DPPHラジカル消去能とORACを測定した．サツマイモのDPPHラジカル消去活性は肉色が白，黄，橙では同程度であったが，紫サツマイモは他の肉色のサツマイモと比べて約10倍高い活性を示した．ORAC値もDPPHラジカル消去活性と同様な傾向であり，ETとHATを反応原理とする2つの評価系で紫サツマイモが高い抗酸化活性を示した．20品種・系統の紫サツマイモにおいて，主要アントシアニンの含量とORAC値との間に正の相関（R＝0.833）が認められ，アントシアニンがORAC法での主要な抗酸化成分であると判断された．また，紫サツマイモのDPPHラジカル消去活性がORAC値と高い相関（R＝0.894）を示したことから，DPPHラジカルを用いた評価法がORAC値を見積もるための方法として利用できると考えられた．

紫甘しょやその加工食品に含まれる真のAN含量を決定するため，HPLCによる一斉定量法の確立を検討した．酸性化溶媒にギ酸を選択し，種々のHPLC分析条件を検討した結果，品種「アヤムラサキ」塊根中に含まれる主要な8種（YGM-1a, -1b, -2, -3, -4b, -5a, -5b, -6）のANピークが良好に分離する条件を設定できた．本条件下では1回の分析に要する時間は40分であり，これまでに比べて分析時間が短縮された．基準色素YGM-6のHPLC分析では，10-6～10-3Mの濃度範囲でピーク面積との間に相関係数が1.000と良好な直線性が得られ，検出限界は11ng以下であった．また，基準であるYGM-6と各YGMとのピーク面積を比較し，定量係数を設定することで，一斉定量を簡便化できた．本法をPSとその乾燥粉末ならびに加工飲料に適用した結果，「アヤムラサキ」青果物では主要ANの合計は369.1mg/100gであった．また，YGM-4bが最も多く含まれており，ペオニジン系ANの合計が全体の81％を占めていた．「アヤムラサキ」乾燥粉末，清涼飲料水，および醸造酢の主要AN含量はそれぞれ196.9mg/100g, 118.7mg/100g, および15.3mg/100gであり，その組成比はいずれも原料PS青果物と類似していた．