A simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated for the quantification of bulleyaconitine A (BLA) in human serum. Samples were extracted from human serum by protein precipitation. Domperidone was used as internal standard (I.S.). Chromatography separation was performed on a Waters Sunfire C18 column (2.1×50 mm, 5 μm) with a gradient mobile phase. Detection was performed in positive mode using multiple reaction monitoring (MRM) of the transition m⁄z 644.2>584.3 for BLA and m⁄z 426.3>175.3 for I.S. The proposed method was validated in a linear range of 0.0587–11.7 ng/ml. Intra- and inter-day precision was better than 9.88% and 7.76%. The recovery for BLA was 97.90%, and for I.S. 89.40%. This method provides a simple, rapid, specific, and sensitive tool for the quantitativedetermination of BLA in human serum, and can be used to monitor serum levels in patients treated with BLA.

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TIME-EA4 is an ATPase that measures time intervals as a diapause-duration clock found in diapause eggs of the silkworm, Bombyx mori. In the current studies, we report the molecular heterogeneity of TIME-EA4 protein regarding not only amino acid L62V, but also the numbers and linkage patterns of the sugar chain attached to the Asn²² residue. These sugar chain structures were determined in a pico-molar amount of the protein by combining the methods of chemical modification (Smith degradation) and nano-HPLC-electrospray ionization-quadrupole-time of fight-mass spectrometry (ESI-Q-TOF-MS) and -MS/MS. The Japanese and bi-voltine Thai silkworm strains were compared to show the heterogeneity represented by four kinds of molecular species. Judicious choice of the combination methods led us to find the first example of a linkage-position difference in the glycosidic bonds even in sugar moieties of the same molecular weight; thus, the Man(1-6)Man(1-4)GlcNAc(1-4)GlcNAc structure in the C108 pure strain and Man(1-3)Man(1-4)GlcNAc(1-4)GlcNAc structure in the Kinshu-Showa hybrid. A total of five kinds of molecular heterogeneity was determined, including the amino acids in TIME-EA4 protein. This paper describes the details for determining the sugar chain linkage in TIME-EA4 from the diapause eggs of various silkworm strains.

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The syntheses of all stereoisomers of tetrasubstituted tetrahydrofuran lignan were accomplished, and the antimicrobial activity was examined. The 9,9′-diol compound bearing (7R,7′R,8R,8′R) and (7R,7′S,8R,8′R) stereochemistry showed the strongest antibacterial activity against Listeria denitrificans and Bacillus subtilis, respectively. It was also found that (−)-virgatusin bearing (7S,7′R,8S,8′S) stereochemistry had strongest antifungal activity.

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Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produce a novel sex pheromone composed of three methyl-branched ketones (I–III) in a ratio of 2:1:1. In order to confirm the structure of III (6,14-dimethyl-2-octadecanone), a mixture of its four stereoisomers was synthesized via chain elongation by two Wittig reactions, starting from 1,7-hexanediol. GC-MS data of the synthetic III were satisfactorily coincident with those of the natural component. In addition to the racemic mixtures of I (6-methyl-2-octadecanone) and II (14-methyl-2-octadecanone), previously synthesized, the activity of III was evaluated in the Iriomote Islands, and effective male attraction was observed for the 2:1:1 mixture of I–III. This result indicates that the females do not produce only one stereoisomer for each component or that the response of the males is not disturbed by the other stereoisomers of natural isomers produced by the females. The field test also revealed that the two-component lure of I and II captured as many males as the mixture of I–III, while lures baited with two components in other combinations and with only one component scarcely exhibited any male attraction ability.

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A new compound was isolated from the water-soluble fraction of a methyl alcohol extract obtained from the larva of the dobsonfly (Protohermes grandis Thunberg). The novel compound had a 12-membered ring and was confirmed to be a cyclic dipeptide of D-ornithine by a chiral analysis, using high-performance liquid chromatography, 2D-nuclear magnetic resonance, and field desorption mass spectrometry.

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An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibodies have great potential, and in particular, efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, FcεRIα, using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with FcεRIα-expressed KU812F cells. Subsequently a combined phage antibody library 6×10³ in the size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant FcεRIα and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against FcεRIα. These results suggest that one can obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.

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In this study, a previously cloned β-glucosidase gene, umbgl3B, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. The recombinant enzyme was stable over a wide range of pH values (5.0–9.0) and below 30 °C. It displayed optimum enzymatic activity at pH 6.5 at 40 °C, under condition similar to that in the rabbit cecum, suggesting an active role of the native enzyme in vivo. The recombinant β-glucosidase Umbgl3B showed high activity to aryl β-D-glucosides and low activity to cellooligosaccharides, with a polymerization degree of less than 5. The enzyme had no activity toward long cellooligosaccharides or polysaccharides. The aspartic acid residue, D772, of the wild-type Umbgl3B was predicted as a nucleophile. Mutant D772A was constructed. It showed less than 1/10,000 activity of the wild-type enzyme, but had the same properties, suggesting that residue D772 plays a key role in the enzyme’s activity.

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Peptide vaccine was found to be an effective and powerful approach to a variety of pathogens. To explore multi-epitope based peptide vaccines against infectious bronchitis virus (IBV), the immunogenic peptides were fused to the 3′ terminal of glutathione S transferase gene (GST) and expressed in Escherichia coli. ELISA and Western blot analysis showed that the purified fusion proteins had excellent immune activity with chicken anti-IBV serum. During the vaccination course, the candidate peptide vaccines induced strong humoral and cellular response, and provided up to 80.0% immune protection, while all non-immunized chickens in the negative control group manifested obvious typical symptoms and died after virus challenge. Our finding provides a new way to develop multi-epitope based peptide vaccine against IBV.

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The presence of amylosucrase in 12 Alteromonas and Pseudoalteromonas strains was examined. Two Alteromonas species (Alteromonas addita KCTC 12195 and Alteromonas macleodii KCTC 2957) possessed genes that had high sequence homology to known amylosucrases. Genomic clones containing the ASase analogs were obtained from A. addita and A. macleodii, and the deduced amino acid sequences of the corresponding genes (aaas and amas, respectively) revealed that they were highly similar to the ASases of Neisseria polysaccharea, Deinococcus radiodurans, and Deinococcus geothermalis. Functional expression of amas in Escherichia coli was successful, and typical ASase activity was detected in purified recombinant AMAS, whereas the purified recombinant AAAS was nonfunctional. Although maximum total activity of AMAS was observed at 45 °C, the ratio of transglycosylation to total activity increased as the temperature decreased from 55 to 25 °C. These results imply that transglycosylation occurs preferentially at lower temperatures while hydrolysis is predominant at higher temperatures.

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Synthetic D- and L-amino acid type cationic 9-mer peptides (all sequences were synthesized as D- or L-amino acids) derived from the active sites of insect defensins were tested for their ability to modify the growth of blood-stream form African trypanosomes in vitro. One of them, the D-type peptide A (RLYLRIGRR-NH₂), irreversibly suppressed proliferation of the Trypanosoma brucei brucei GUTat3.1 parasite. The presence of negatively charged phosphatidylserine on the surface of the parasites was demonstrated, suggesting electrostatic interaction between the peptide and the phospholipids. Furthermore, this peptide was found to alter trypanosome membrane-potentials significantly, an effect apparently due to the removal of the parasite’s plasma membrane. The potential toxic effects of D-peptide A on mammalian cells was assessed using human brain microvascular endothelial cells. Only minor effects were found when the endothelial cells were exposed for 16 h to peptide concentrations of less than 200 μM. These findings suggest that insect defensin-based peptides represent a potentially new class of membrane-disrupting trypanocidal drugs.

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Solute carrier family member 2 (SLC10A2) reabsorbs bile acids at the distal terminus of the ileum in an Na⁺-dependent manner. Alignment of deduced amino acid sequences of SLC10 family members and homologous genes in various species revealed a highly conserved region that corresponds to Gly¹⁰⁴–Pro¹⁴² of SLC10A2. To elucidate the functional importance of this region, uncharged polar residues and Pro in the distal one-third of this region in mouse Slc10a2 (mSlc10a2) were submitted to mutational analysis, and taurocholic acid uptake and cell surface localization were evaluated. In addition to mutations that abolished almost all of the transport activity with and without cellular localization failure (P142V and T130A respectively), a mutation that perhaps affected affinity for taurocholic acid was identified (T134A). These results suggest that the highly conserved region contains residues involved in the substrate interaction, function, and cellular localization of mSlc10a2.

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Recombinant antibodies (rAbs) are a new diagnostic test for immulogical detection. To date, there are no reports about anti-pyrethrins rAbs. Here we describe the generation of monomeric and dimeric single chain variable fragments (scFvs) with affinity for six esters of pyrethrins using a subtractive phage display technology. First, scFv libraries with long-linker (Ger₄Ser)₃ and short-linker (Ger₄Ser) were established to contain 1.04×10⁷ or 6.07×10⁶ transformants. After four rounds of panning, phage ELISA demonstrated that three clones (E2, F2, and H7) showed higher affinity from the long-linker library, and clones (h6, a5) exhibited better antibody activity to pyrethrin I and II from the short-linker library. The scFv candidates were sequenced to identify the specific antibody response against pyrethrins. Isolated scFvs constitute valuable tools for real-time detection of pyrethrins. In addition, the subtractive phage display provides a simple approach for isolation of scFvs.

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To investigate the roles of protein kinase C (PKC) isoforms in Echinoderms, we cloned starfish cDNAs for novel, atypical, and conventional PKCs. They showed highest homology with PKCδ, ι, and α isoforms respectively. It was predicted from the whole genome sequence and by RT-PCR that sea urchin has only one isoform of each PKC subgroups. It is thus likely that these isoforms are the prototypes or ancestors of the PKC subgroups. The phylogenetic tree suggests that atypical PKC was first formed by evolution from the common prototype of AGC protein kinase family, and novel and conventional PKCs next. RT-PCR analysis indicated that novel and atypical PKC mRNAs are expressed ubiquitously in all tissues of adult starfish, whereas conventional PKC mRNA is expressed mainly in the ovary and oocytes, and only slightly in the tube foot and stomach. Upon heterologous expression, only atypical PKC was expressed in the functional form in insect cells.

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Fission yeast requires nutritional starvation to switch the mitotic cell cycle to sexual differentiation, but sam mutants, of which we had isolated nine alleles, mate without the starvation condition. These mutants are useful for understanding the mechanism underlying the way cells sense nutritional starvation and change the cell cycle. To identify the sam allele, we first sought phenotypes other than the original sam phenotype. We found that all nine sam mutants were sensitive to 1 M KCl, that sam2, sam3, sam4 and sam9 were sensitive to 0.1 M CaCl₂, and that only the sam4 mutant was sensitive to 150 J/m² UV. This peculiar phenotype of sam4 suggested to us that sam4 might be an allele of rad24, which encodes a 14-3-3 protein. In fact, the Rad24 protein disappeared in sam4 and the rad24 mRNA was not transcribed in sam4. In addition, the mutation that changed Gln to a stop codon was found in the rad24 locus of sam4. Hence we concluded that sam4 is an allele of rad24. We also found that over-expression of rad24 or rad25 (a paralog of rad24) has a suppressive effect on sam1, and that sam1 was not an allele of rad24 nor rad25. Thus 14-3-3 proteins are deeply involved in the switching of the mitotic cell cycle to the sexual differentiation of fission yeast.

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Cytochrome bo is a terminal quinol oxidase in the aerobic respiratory chain of Escherichia coli. Subunit I binds all four redox centers, and electrons are transferred from quinols to high-spin heme o and Cu_B through a bound uniquinone-8 and low-spin heme b. To explore the role of conserved charged amino acid residues, we examined the one-electron transfer processes in subunit I mutants. We found that all the mutants examined increased the electron transfer rate from the bound quinone to heme b more than 40-fold. Tyr288 and Lys362 are key residues in the K-channel for charge compensation of the heme o-Cu_B binuclear center with protons. The Tyr288Phe and Lys362Gln mutants showed 100-fold decreases in heme b-to-heme o electron transfer, accompanied by large increases in the redox potential of heme o. Our results indicate that electromagnetic coupling of hemes is important for facilitated heme-heme electron transfer in cytochrome bo.

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Pig edema disease is a bacterial disease caused by Shiga toxin 2e-producing Escherichia coli belonging mainly to serotypes O138, O139, and O141. The B subunit of Shiga toxin 2e (Stx2eB) is a candidate protein for use in a vaccine against edema disease. We produced this protein in transgenic lettuce (Lactuca sativa), an edible plant that can be cultivated in a factory setting. In a transient expression system, we found that NtADH 5′-untranslated region (5′-UTR) functions as a translational enhancer in lettuce cells, and that Stx2eB accumulates most efficiently in the endoplasmic reticulum (ER) of lettuce cells. Stx2eB was produced in stable transgenic lettuce plants expressing a modified Stx2eB gene fused with the NtADH 5′-UTR and sequence encoding ER localization signals.

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ETR101, a human homolog of rat pip92, is a cellular immediate early gene induced by extracellular stimuli such as serum growth factors. ETR101 encodes a short-lived, proline-rich protein (ETR101) exhibiting no significant sequence similarity to any other known protein, and little is known about its function. We investigated the functioning of ETR101 as a transcriptional activator for the gene ISYNA1, which encodes human inositol 1-phosphate synthase. We constructed a yeast strain in which the chromosomal region of the PSS1 promoter was replaced with the human ISYNA1 promoter. Using this yeast strain, we screened human cDNAs, which activated the ISYNA1 promoter, and thus expressed the PSS1 as a reporter gene. We obtained two types of cDNA, E2F1, known as a gene encoding Rb-binding protein, and ETR101. The E2F1 gene product (E2F1) is known to bind to and activate the ISYNA1 promoter. In a manner similar to E2F1, ETR101 binds to and activates the ISYNA1 promoter.

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We created a protein library consisting of 647 Arabidopsis transcription factors (TFs) using a wheat cell-free system. The quality of proteins in the library was checked by binding assay of bZIP family proteins. Screening of TFs binding to 5′-regulatory regions of FLC and LFY was conducted using the library, and MYB67 and GBF1 were found to be binding factors.

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Upon searching plant extracts for inhibitors of the Ca²⁺ signaling pathway using the zds1Δ-yeast proliferation based assay, a crude rhizome extract of Boesenbergia pandurata was found to be strongly positive, and from this extract pinostrobin, alpinetin, and pinocembrin chalcone were isolated as active components. Further biochemical experiments confirmed that pinostrobin possesses inhibitory activity on the Ca²⁺ signals involved in the control of G2/M phase cell cycle progression in Saccharomyces cerevisiae.

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The chemically modified thanatins with the methyl group (CH₃), ethyl group (C₂H₅), and normal-octyl group (C₈H₁₇) at the side-chain of cysteine residues were synthesized. The octyl group modified form exhibited 8-fold higher antimicrobial activity against Micrococcus luteus than wild type thanatin. It was found that there was an equilateral correlation between antimicrobial activity and side-chain hydrophobicity at the cysteine residues in thanatin.

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Beta-amyloid (Aβ) has been suggested to induce neurotoxicity in Alzheimer’s disease. We evaluated the neuroprotective effects of delphinidin, an anthocyanidin commonly present in pigmented fruits and vegetables, against Aβ-induced toxicity. Aβ (25–35) significantly decreased the viability of PC12 cells, and this was accompanied by an increase in intracellular calcium levels and tau phosphorylation. However, treatment with delphinidin rescued PC12 cells from Aβ by attenuating the elevation of intracellular calcium levels and tau phosphorylation. Taken together, these results suggest that delphinidin protects PC12 cells against Aβ-induced toxicity by attenuating intracellular calcium influx and tau hyperphosphorylation.

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Here we report the phenotypic characteristics of a novel hypernodulation mutant, Ljrdh1 (root-determined hypernodulation 1) of Lotus japonicus. At 12 weeks after rhizobial inoculation, there were no differences between the growth of Ljrdh1 and, wild-type. However, Ljrdh1 showed 2 to 3 times higher nitrogen-fixing activity, and seed and pod yields, were approximately 50% higher than the wild-type. This is the first report of a legume hypernodulation mutant showing normal growth and a high-yielding characteristic under optimal cultivation conditions.

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Symmetric chloroplast division requires a prokaryote-derived division regulator protein MinD, whose subchloroplastic localization remains to be completely established. We investigated the localization and functionality of AtMinD1 (Arabidopsis thaliana MinD) fused with a dual hemagglutinin epitope (dHA) or a yellow fluorescent protein (YFP). AtMinD1-dHA, which successfully complemented the arc11/atminD1 mutant phenotype, was predominantly located at the envelope membrane and the mid-chloroplast constriction site. Meanwhile, AtMinD1-YFP was non-functional and showed suborganellar localization partly similar to that of AtMinD1-dHA. This prompts us to reevaluate earlier transgenic and transient expression studies using fluorescent protein-tagged AtMinD1.

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The protective effect of selenoarginine against oxidation resistance was investigated in D-galactose (D-gal)-induced aging mice. The mice were divided into four groups (n=15): a normal group, a model group, a lowdose selenoarginine group (8.35 μg of Se/kg b.w./d), and a high dose selenoarginine group (16.78 μg of Se/kg b.w./d). The aging model was induced by s.c. injection D-galactose dissolved in 0.9% normal saline of a dose of 150 mg/kg/d for 6 weeks. The mice in the normal group received s.c. injection of sterile normal saline at the same dose and frequency. The results showed that oxidative stress in the liver, kidney, and brain tissues and the serum of the mice was induced by D-galactose, but selenoarginine had an obviously protective effect against D-galactose-induced aging mice. Lowdose selenoarginine performed better than high dose selenoarginine. The protective effect of selenoarginine on D-galactose-induced aging mice can be attributed to elevation of the activity of antioxidase and enhanced antioxidant defenses.

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Toll-like receptors (TLRs) are primary sensors that detect a wide variety of microbial components involving induction of innate immune responses. After recognition of microbial components, TLRs trigger the activation of myeloid differential factor 88 (MyD88) and Toll-interleukin-1 (IL-1) receptor domain-containing adapter inducing interferon-β (TRIF)-dependent downstream signaling pathways. 6-Shoagol, an active ingredient of ginger, inhibits the MyD88-dependent signaling pathway by inhibiting inhibitor-κB kinase activity. Inhibitor-κB kinase is a key kinase in nuclear factor κB (NF-κB) activation. However, it is not known whether 6-shogaol inhibits the TRIF-dependent signaling pathway. Our goal was to identify the molecular target of 6-shogaol in the TRIF-dependent pathway of TLRs. 6-Shogaol inhibited the activation of interferon-regulatory factor 3 (IRF3) induced by lipopolysaccharide (LPS) and by polyriboinosinic polyribocytidylic acid (poly[I:C]), overexpression of TRIF, TANK-binding kinase1 (TBK1), and IRF3. Furthermore, 6-shogaol inhibited TBK1 activity in vitro. Together, these results suggest that 6-shogaol inhibits the TRIF-dependent signaling pathway of TLRs by targeting TBK1, and, they imply that 6-shogaol can modulate TLR-derived immune/inflammatory target gene expression induced by microbial infection.

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We examined the effects of lactic acid fermented soymilk, in which part of the soymilk was replaced with okara (soy yogurt), on plasma and hepatic lipid profiles in rats fed a cholesterol-free diet. Additionally, we investigated the effects of soy yogurt on hepatic gene expression in rats using DNA microarray analysis. Male Sprague-Dawley rats aged 5 weeks (n=5/group) were fed a control diet (AIN-93) or a test diet in which 20% of the diet was replaced by soy yogurt for 7 weeks. Soy yogurt consumption did not affect body weight or adipose tissue weight as compared with control diet. In the soy yogurt group, the liver weight and hepatic triglyceride content were significantly lower than the control group, and the level of plasma cholesterol was also lower. Furthermore, DNA microarray analysis indicated that soy yogurt ingestion down-regulated the expression of the SREBP-1 gene and enzymes related to lipogenesis in the rat liver, while expression of β-oxidation-related genes was up-regulated. These results suggest that soy yogurt is beneficial in preventing hepatic lipid accumulation in rats.

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A novel simple method using liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) and LC/UV was established for the quantification of saponins in an extract from green tea leaves. The amount of saponins in a fraction with high in vitro antihypercholesterolemic activity, Fr2-3, was determined to be 72%. An in vivo experiment showed that the addition of 0.5% Fr2-3 to a high-cholesterol diet suppressed the increase in serum cholesterol levels in rats. Fr2-3 induced a decrease in the liver cholesterol and triglyceride levels and an increase in the fecal excretion of cholesterol. These results indicate tea-leaf saponins to be the active components in Fr2-3 and that these saponins exhibited antihypercholesterolemic activity by inhibiting cholesterol absorption in the intestines.

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Lactic acid bacteria (LAB) are popularly used as probiotics, and some strains of LAB have anti-allergic functions in vivo. Although in vitro studies show that LAB modulate the T helper type (Th) 1/Th2 balance and inhibit IgE secretion by inducing IL-12, it is not known how LAB regulates allergies in vivo. In this study, we evaluated in vivo IL-12 production after oral administration of Lactobacillus paracasei KW3110, a strain reported to improve allergies, to mice. Orally administered KW3110 interacted with CD11b positive cells and induced IL-12 mRNA expression at Peyer’s patch. In addition, blood IL-12 levels increased transiently 10 h after administration of KW3110. Based on these results, we found that oral administration of KW3110 induces IL-12 in vivo. Our findings should contribute to understanding of the in vivo function of LAB.

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A three-dimensional (3-D) bio-imaging technique was developed for visualizing and quantifying the 3-D distribution of yeast in frozen bread dough samples in accordance with the progress of the mixing process of the samples, applying cell-surface engineering to the surfaces of the yeast cells. The fluorescent yeast was recognized as bright spots at the wavelength of 520 nm. Frozen dough samples were sliced at intervals of 1 μm by an micro-slicer image processing system (MSIPS) equipped with a fluorescence microscope for acquiring cross-sectional images of the samples. A set of successive two-dimensional images was reconstructed to analyze the 3-D distribution of the yeast. The average shortest distance between centroids of enhanced green fluorescent protein (EGFP) yeasts was 10.7 μm at the pick-up stage, 9.7 μm at the clean-up stage, 9.0 μm at the final stage, and 10.2 μm at the over-mixing stage. The results indicated that the distribution of the yeast cells was the most uniform in the dough of white bread at the final stage, while the heterogeneous distribution at the over-mixing stage was possibly due to the destruction of the gluten network structure within the samples.

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Cell-surface engineering (Ueda et al., 2000) has been applied to develop a novel technique to visualize yeast in bread dough. Enhanced green fluorescent protein (EGFP) was bonded to the surface of yeast cells, and 0.5% EGFP yeasts were mixed into the dough samples at four different mixing stages. The samples were placed on a cryostat at −30 °C and sliced at 10 μm. The sliced samples were observed at an excitation wavelength of 480 nm and a fluorescent wavelength of 520 nm. The results indicated that the combination of the EGFP-displayed yeasts, rapid freezing, and cryo-sectioning made it possible to visualize 2-D distribution of yeast in bread dough to the extent that the EGFP yeasts could be clearly distinguished from the auto-fluorescent background of bread dough.

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Vibrio cholera is a major foodborne pathogen in Thailand. It is present in raw and lightly cooked foods, and it causes cholera. Natural products inhibiting it can be used to improve the safety of foods. In this study, elephant garlic oil was studied for its major diallyl sulfide content and its antimicrobial activity against V. cholerae. The oil had a very low concentration of diallyl monosulfides (1.62%) in comparison with the other diallyl sulfides (25.09% for diallyl disulfide, 16.04% for diallyl trisulfide, and 10.58% for diallyl tetrasulfide). In an in vitro study, the oil was found to have a bacteriocidal effect on all tested strains of V. cholerae, with varied minimal inhibitory concentrations (MICs) ranging from 3.13 to 25 μg/ml. It was also found that elephant garlic oil retarded the growth of the bacteria or reduced the bacterial cell load in the food model, depending on its concentration.

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Dioxins enter the body mainly through the diet, bind to the aryl hydrocarbon receptor (AhR), and cause various toxicological effects. In this study, we found that oral administration of kaempferol or ginkgo biloba extract (EGb) containing 24% flavonol at 100 mg/kg body weight suppressed AhR transformation induced by 3-methylcholanthrene at 10 mg/kg body weight in the liver of mice. The suppressive effect of kaempferol was enhanced by verapamil, an inhibitor of P-glycoprotein (P-gp), in ex vivo experiments using a hepatic cytosolic fraction and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Enhancement of the suppressive effect by verapamil was also observed in mouse hepatoma Hepa-1c1c7 cells, accompanied by an increase in the uptake of kaempferol into the cells. In conclusion, inhibition of P-gp enhanced the suppressive effect of kaempferol on AhR transformation through an increase in the intracellular kaempferol concentration.

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Screening of potential probiotic LAB for aquaculture from adult common carp intestine was performed seasonally. Lactococcus lactis h2 and Lactococcus raffinolactis h47, which show cholic acid resistance and strong antibacterial activity against fish pathogens, were selected from predominant LAB in summer and winter respectively. Enterococcus pseudoavium h50, with the strongest antimicrobial activity among the strains isolated through 1 year, was also selected. Streptococcus iniae I1, with strong antimicrobial activity, was selected from predominant LAB in young common carp intestine. Direct screening of LAB with cholic acid resistance was also carried out seasonally. The antibacterial activity of the isolates was tested, and Lactobacillus fuchuensis K11 was selected from the summer isolates. In addition, five candidate strains were selected from the winter samples. The candidates’ levels of cholic acid resistance and antibacterial activity were better than or at the least matched those of their corresponding type strains. All the candidates grew over a wide range of temperatures.

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An endophytic bacterial strain, Jaas ed1, was isolated from the interior of eggplant (Solanum melongena L.) stems in Jiangsu Province, China. According to the morphological and physiological characteristics and phylogenetic analysis of the 16s rDNA sequence, it was identified as Bacillius subtilis. Strain Jaas ed1 and its cell-free filtrate had strong antifungal activity against Verticillium dahliae. The strain was an internal colonizer within the eggplant without any harmful side effects to the plant. In greenhouse experiments, the strain cell suspension effectively controlled Verticillium wilt of eggplant, and its control efficiency was far more significant than that of the cell-free filtrate after inoculation of V. dahliae.

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We examined the production of an antimicrobial component, 3-hydroxypropionaldehyde (3-HPA), in laboratory-scale silage inoculated with Lactobacillus coryniformis strain 394, which ferments glycerol to 3-HPA. A modified colorimetric method that used an NaOH-treated blank and determined the absorption spectrum of the samples was employed to detect a 3-HPA-like component (HLC) that was assumed to be 3-HPA. Inoculation with Lb. coryniformis 394 plus glycerol in ensiling produced HLC at 10–460 ppm and contributed to inhibition of butyric fermentation and retardation of aerobic spoilage. HLC was considered to be 3-HPA from its absorption spectrum. These results suggest that the production of 3-HPA by Lb. coryniformis 394 is useful in ensiling and that the modified colorimetric method is effective to detect 3-HPA in silage.

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Four marine yeasts isolated from the Pacific Ocean off Japan (Siki No. 4, Siki No. 15, Hach No. 6, and Inub No. 11), which showed high γ-aminobutyric acid (GABA) producing abilities, were identified and classified by physiological and biochemical characteristics and gene sequence analyses. Analysis of biochemical data suggested that while Siki No. 15 was identical to Candida, the remaining three isolates belonged to the genus Pichia. However, these data were insufficient to resolve their identity at the species level. Subsequently, analysis of the 5.8S rRNA genes and the two internal transcribed spacer regions (ITS) sequences revealed that Siki No. 15 belongs to Pichia guilliermondii, while the remaining three isolates corresponded to Pichia anomala. Since Siki No. 4 showed slightly different biochemical properties than the other two isolates, which were otherwise identical, we sought to investigate the sequences of the intergenic spacer region 1 (IGS1). We observed few nucleotide changes, suggesting that the Hach No. 6 and Inub No. 11 isolates belong to different but new strains for which we propose the names P. anomola MR-1 and MR-2 respectively.

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Aspergillus nidulans has many histidine-to-aspartate (His-Asp) phosphorelay components, including 15 histidine kinases (HKs), four response regulators (RRs), and a histidine-containing phosphotransfer intermediate (HPt). Of these, NikA (HK) is highly conserved in many filamentous fungi. It has been found that NikA is responsible for the responses of filamentous fungi to fungicides such as iprodione and fludioxonil. Two RRs, SskA and SrrA, are also involved in the fungicide response, providing a typical example of the His-Asp phosphorelay system, in which NikA functions as a sensor upstream of SskA and SrrA in response to fungicides. To gain further insight into the physiological roles of the NikA-SskA/SrrA phosphorelay system, we constructed a pair of ΔnikAΔsskA and ΔnikAΔsrrA double mutants. Here we provide evidence regarding the crucial involvement of the NikA-SskA/SrrA phosphorelay system in both osmotic adaptation and asexual development, including conidia formation. Based on these results, a general insight into the A. nidulans His-Asp phosphorelay network is also discussed.

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An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog’s specificity. It showed maximum activity at pH 7.0 and 40 °C. Its K_m and V_max values toward HPPO were 20.1 mM and 3.4 μmol min⁻¹ mg protein⁻¹ respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.

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Desulfotignum balticum utilizes benzoate coupled to sulfate reduction. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis was conducted to detect proteins that increased more after growth on benzoate than on butyrate. A comparison of proteins on 2D gels showed that at least six proteins were expressed. The N-terminal sequences of three proteins exhibited significant identities with the α and β subunits of electron transfer flavoprotein (ETF) from anaerobic aromatic-degraders. By sequence analysis of the fosmid clone insert (37,590 bp) containing the genes encoding the ETF subunits, we identified three genes, whose deduced amino acid sequences showed 58%, 74%, and 62% identity with those of Gmet_2267 (Fe-S oxidoreductase), Gmet_2266 (ETF β subunit), and Gmet_2265 (ETF α subunit) respectively, which exist within the 300-kb genomic island of aromatic-degradation genes from Geobacter metallireducens GS-15. The genes encoding ETF subunits found in this study were upregulated in benzoate utilization.

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We established a procedure for analyzing gene transcripts in a microbial community of unknown genomic background. Analysis of a crude oil-degrading marine microbial community detected the expression of genes related to the biodegradation of fatty acids and the biosynthesis of glycolipids probably involved in the emulsification of crude oil.

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The possibility of using two kinds of sorghum as raw materials in consolidated bioprocessing bioethanol production using Flammulina velutipes was investigated. Enzymatic saccharification of sweet sorghum was not as high as in brown mid-rib (bmr) mutated sorghum, but the amount of ethanol production was higher. Ethanol production from bmr mutated sorghum significantly increased when saccharification enzymes were added to the culture.

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In this study, we isolated Pseudomonas sp. strain HB01 from a soil sample contaminated with brominated pollutants, and found that it degraded γ-hexabromocyclododecane (γ-HBCD). The strain degraded 81% of 1 mM γ-HBCD within 5 d of culture. Furthermore, it demonstrated biodegradation of structurally related (bromo) alkanes. To the best of our knowledge, this is the first report that outlines the isolation of a bacterial strain that degrades γ-HBCD.

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