Albizzia saman, a leguminous plant, is known to open its leaves in the daytime and sleep at night with the leaves folded. β-D-Glucopyranosyl 12-hydroxyjasmonate (1) was isolated as an endogenous chemical factor controlling this leafmovement. We developed a concise synthesis of optically pure (−)-1 in 9 steps from (+)-2 with a total yield of 58%. Similarly, such analogs of 1 as epi-LCF (13), enantiomer (14), and galactoside (19) were synthesized for a structure activity relationship (SAR) study. The results of this SAR study strongly suggest that the mechanism for the leaf-closing activity of 1 would be different from that of methyl jasmonate, and also suggest the involvement of a different kind of target protein which recognizes the trans-isomer of a jasmonate derivative.

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The antioxidant activity of butane-type lignans was evaluated. Secoisolariciresinol (SECO) and dihydroguaiaretic acid (DGA) showed higher radical scavenging activity than that of 7,7′-dioxodihydroguaiaretic acid (ODGA). SECO and DGA inhibited the oxidation of unsaturated fatty acid. Both enantiomers of DGA were also lipoxygenase inhibitors, but neither enantiomer of SECO inhibited the lipoxygenase activity.

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Analogs of cortistatins, a series of anti-angiogenic compounds isolated from the Indonesian marine sponge Cortisium simplex, were synthesized from estrone by using the Suzuki-Miyaura coupling reaction as the key step. The estrone-isoquinoline hybridized compound showed selective inhibitory activity against the proliferation and VEGF-induced migration of HUVEC.

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A new N-acylated serinol, inconspicamide (1), was isolated from the marine sponge, Stelletta inconspicua, together with a glyceryl ether (2). Their structures were determined on the basis of spectroscopic data and the modified Mosher analysis. They exhibited moderate cytotoxic activity against HeLa human cervical cancer cells.

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Kohamaic acid A is a potent DNA polymerase inhibitor isolated from the Okinawan marine sponge Ircinia sp. A series of structurally simplified analogs of kohamaic acid A were synthesized with the aim of evaluating structure-activity relationships.

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Starch branching enzyme (SBE) catalyzes the cleavage of α-1.4-linkages and the subsequent transfer of α-1.4 glucan to form an α-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the α-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other α-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 Å on a synchrotron X-ray source.

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Fatty acid (FA) compositions in leaves were investigated for two families of F₁ hybrids of Larix gmelinii var. japonica × L. kaempferi (F₁) and their parent clones. Twenty-one FAs, from C₁₂ to C₃₂, were found in the leaves of both adult trees and seedlings. The levels of 18:1/(18:2 + 18:3) increased in the order L. kaempferi, F₁, and L. gmelinii var. japonica, with significant differences between L. gmelinii var. japonica and F₁ in adult trees, but these differences were not found in the seedlings. Moreover, in the adult trees, the 18:1/(18:2 + 18:3) levels in the neutral phospholipid fraction and the ΣC₁₈/ΣC₁₆, especially in the glycolipid fraction, showed significant differences among the three species. These characteristics are discussed from the viewpoint of lipid synthesis in the endoplasmic reticulum and chloroplasts, and of the activities and substrate specificities in sequential FA desaturation. Linear discriminant analysis suggested that the FA compositions are useable as an index in the identification of hybrid seedlings.

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Natural rubber is synthesized as rubber particles in the latex, the fluid cytoplasm of laticifers, of Hevea brasiliensis. Although it has been found that natural rubber is biosynthesized through the mevalonate pathway, the involvement of an alternative 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is uncertain. We obtained all series of the MEP pathway candidate genes by analyzing expressed sequence tag (EST) information and degenerate PCR in H. brasiliensis. Complementation experiments with Escherichia coli mutants were performed to confirm the functions of the MEP pathway gene products of H. brasiliensis together with those of Arabidopsis thaliana, and it was found that 1-deoxy-D-xylulose-5-phosphate reductoisomerase, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase, and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase of H. brasiliensis were functionally active in the E. coli mutants. Gene expression analysis revealed that the expression level of the HbDXS2 gene in latex was relatively high as compared to those of other MEP pathway genes. However, a feeding experiment with [1-¹³C] 1-deoxy-D-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is not involved in rubber biosynthesis, but is involved in carotenoids biosynthesis in H. brasiliensis.

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During protein import into chloroplasts, one of the Hsp70 proteins in pea (Hsp70-IAP), previously reported to localize in the intermembrane space of chloroplasts, was found to interact with the translocating precursor protein but the gene for Hsp70-IAP has not been identified yet. In an attempt to identify the Arabidopsis homolog of Hsp70-IAP, we employed an in vitro protein import assay to determine the localization of three Arabidopsis Hsp70 homologs (AtHsp70-6 through 8), predicted for chloroplast targeting. AtHsp70-6 and AtHsp70-7 were imported into chloroplasts and processed into similar-sized mature forms. In addition, a smaller-sized processed form of AtHsp70-6 was observed. All the processed forms of both AtHsp70 proteins were localized in the stroma. Organelle-free processing assays revealed that the larger processed forms of both AtHsp70-6 and AtHsp70-7 were cleaved by stromal processing peptidase, whereas the smaller processed form of AtHsp70-6 was produced by an unspecified peptidase.

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In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man_8-9GlcNAc₁, became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-β-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of α-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.

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Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.

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Genes encoding α-methylserine hydroxymethyltransferase from Aminobacter sp. AJ110403 and Ensifer sp. AJ110404 were cloned and expressed in Escherichia coli. The purified enzymes were homodimers with a 46-kDa subunit and contained 1 mol/mol-subunit of pyridoxal 5′-phosphate. The V_max of these enzymes catalyzing the conversion of α-methyl-L-serine to D-alanine via tetrahydrofolate was 22.1 U/mg (AJ110403) and 15.4 U/mg (AJ110404).

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Maltosides of butanol, octanol, and lauryl alcohol were found for the first time to serve as substrates for cyclomaltodextrin glucanotransferase (CGTase), and glycosyl residue was transfered from dextrin to the substrate affording novel maltosides with 3–4 glucose units.

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In Escherichia coli, CitB, a cognate response regulator of CitA, specifically bound to the promoter regions for mdh, citA, citC, and exuT. Transcription of these genes was induced by citrate under anaerobic conditions in a CitAB-dependent manner. Taking this together, we conclude that CitAB is the master regulatory system that activates the set of genes involved in citrate fermentation in E. coli.

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The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.

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In Arabidopsis thaliana, a set of type-A authentic response regulator (ARR) genes, consisting of 10 homologous members, is induced primarily in response to the phytohormone cytokinin. Among these, we found that the expression of ARR9 is uniquely regulated through the circadian clock in a cytokinin-independent manner. This finding appears to be compatible to the current idea that some ARR genes (namely, ARR3, ARR4, ARR8, and ARR9) are implicated in an additional level of regulation of the circadian clock. Hence, the result of this study provided us with a new insight into the complex molecular mechanisms underlying both the cytokinin signaling and circadian rhythm.

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A new binary vector, pZT4B, containing the UDP-N-acetylglucosamine: dolichol phosphate N-acetylglucosamine-1-P transferase (GPT) gene as a selection marker, was constructed. The green fluorescent protein (GFP) gene was inserted into pZT4B, and the resulting plasmid was used in the transformation of Arabidopsis. All of six independent transformants obtained after selection with 0.3 mg/l tunicamycin contained the transgene and showed GFP fluorescence.

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In the phaseolotoxin biosynthetic gene cluster of Pseudomonas syringae pv. phaseolicola 1448A, the PSPPH_4299 gene encodes a novel L-amino acid ligase. The PSPPH_4299 protein synthesized various hetero-dipeptides containing basic amino acids in an ATP-dependent manner, and also synthesized alanyl-homoarginine, part of the phaseolotoxin scaffold.

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4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from 4-hydroxyphenylpyruvate and O₂. In plants, HPPD has been identified as a molecular target for herbicides. We report the isolation and characterization of a cDNA encoding a HPPD from cultured Coptis japonica cells. Recombinant CjHPPD showed significantly higher half-maximum inhibitory concentration (IC₅₀) values for the HPPD-inhibiting herbicide destosyl pyrazolate than other plant HPPDs.

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Elastic fibers in the dermis play an important role in skin elasticity. The desmosine crosslinking structure constructed of lysyl oxidase (LOX) in elastic fibers contributes to elasticity, while elastic fibers are primarily degraded by one of the matrix metalloproteinases (MMPs), MMP-12. We investigated the gender differences and diurnal variation of these enzymes. Gender-based differences in LOX mRNA expression were detected, and were significantly lower in females. In contrast, higher MMP-12 mRNA expression was observed in the light period, suggesting that elastic fibers might be degraded in the light rather than the dark period.

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We have previously demonstrated that collagen from jellyfish simulated immunoglobulin and cytokine production by human-human hybridoma line HB4C5 cells and by human peripheral blood lymphocytes (hPBL). The mode of action of the collagen as an immunostimulatory factor was investigated. The expression levels of immunoglobulin mRNAs in HB4C5 cells, and those of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and transforming growth factor (TGF)-β in hPBL were up-regulated by jellyfish collagen. In addition, this collagen activated IgM production by transcription-suppressed HB4C5 cells that had been treated with actinomycin D. This collagen also enhanced IgM production by translation-suppressed HB4C5 cells that had been treated with sodium fluoride, but was ineffective in accelerating IgM production by HB4C5 cells treated with cycloheximide. Moreover, the intracellular IgM level in HB4C5 cells treated with the post-translation inhibitor, monensin, was increased by this collagen. These results suggest that collagen from jellyfish stimulated not only the transcription activity, but also the translation activity for enhanced immunoglobulin and cytokine production.

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Ginseng root is known to induce anti-diabetic activity, but the key components involved are unknown. We investigated which major ginsenosides in ginseng enhanced glucose homeostasis by in vitro studies. Rb1 and Rg1 reduced the triglyceride accumulation in 3T3-L1 adipocytes by activating PKA with increased intracellular cAMP. However, the insulin-stimulated glucose uptake was enhanced by Rb1 and Rg1 via activation of phosphatidylinositol-3 kinase. Rb1 and Rg1 promoted glucose-stimulated insulin secretion and cell viability in Min6 cells through PKA which augmented IRS2 expression to enhance insulin/IGF-1 signaling. These results suggest that Rb1 and Rg1 improved glucose homeostasis through the activation of a PKA like glucagon-like peptide-1 receptor agonist.

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Many studies have suggested that the 11S/7S ratio in soybeans affects the coagulation reaction at the first step. In this study, the 11S/7S ratio in soybeans showed significantly negative correlation with MgCl₂ concentrations for the maximum breaking stress of tofu for six Japanese varieties. To determine the effect of the 11S/7S ratio, soymilk was fractionated by centrifugation after the addition of MgCl₂, and the distribution of lipids and proteins was studied. The amount of precipitate increased as the MgCl₂ concentration or the 11S/7S ratio increased. More triglyceride was incorporated into the precipitate as the MgCl₂ concentration or the 11S/7S ratio increased. The stain intensity of bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) indicated that the ratio of oleosin, a membrane protein of the oil body, increased in the precipitate as the MgCl₂ concentration or the 11S/7S ratio increased, while the ratios of glycinin and β-conglycinin were less variable. These results indicate that the 11S/7S ratio and coagulant concentration may have an effect on the amount of coagulum and the concentration of oil globules in the coagulum at the beginning of coagulation.

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We investigated how gustatory and olfactory information contributes to the preference for dried bonito broth in mice. In the two-bottle preference test, intact mice consumed dried bonito broth in preference to water or an amino acid-nucleotide (AN) solution containing the same concentration of amino acids and nucleotides as that in dried bonito broth. It was observed that mice with transected bilateral chorda tympani (CT) nerves, those with transected bilateral glossopharyngeal (GL) nerves, and those that were intranasally administered with zinc sulfate preferred dried bonito broth to water. Zinc sulfate was used to produce a temporary loss of olfaction. In the two-bottle preference test with dried bonito broth and an AN solution, the preference for the former was reduced in mice with transected bilateral GL nerves and in those with an olfactory blockade, but not in mice with transected bilateral CT nerves. These results suggest that dried bonito broth was preferred over the AN solution, and that simultaneous inputs from olfaction and the GL nerve contributed to this preference.

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Lactic acid bacteria (LAB) might switch the Th2 biased immune response in allergic patients towards a balanced Th1/Th2 immune profile, leading to amelioration of allergy. To select strains of LAB that could be of potential application for foods in controlling allergy, 35 bacterial strains were screened in vitro using murine splenocytes and peritoneal exudate cells (PECs). Streptococcus thermophilus AHU1838 (FERM AP-21009), and Lactobacillus paracasei subsp. casei AHU1839 (FERM AP-21010) enhanced the secretion of Th1 cytokines such as interferon-γ (IFN-γ) and interleukin-12 (IL-12). The two strains of LAB also up-regulated the expression of CD40, and CD86 in dendritic cells (DCs), and activated cytotoxic T lymphocytes (CTL). These two strains could therefore be used in producing fermented food products that can enhance the Th1 immune profile which is important in ameliorating allergy.

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The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 °C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 °C, but only 1 d at 37 °C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 °C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 °C, but only 1.2 d and 1.9 d for the respective activities at 37 °C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.

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We investigated the effect of dietary Korean proso-millet protein concentrate (PMP) on glycemic responses, plasma lipid levels, and the plasma level and gene expression of adiponectin in obese type 2 diabetic mice under normal and high-fat feeding conditions. The findings were that the feeding of PMP clearly elevated plasma high-density lipoprotein cholesterol (HDL cholesterol) and adiponectin levels and brought about effective reduction in the levels of glucose and insulin in mice under high-fat diet conditions as compared with a control diet. Gene expression study revealed that the diet up-regulated expression of adiponectin and down-regulated tumor necrosis factor-α (TNF-α). Considering the central role of adiponectin and HDL cholesterol in improving and ameliorating type 2 diabetes, obesity, and cardiovascular disease, our findings imply that PMP may have potential for therapeutic intervention in type 2 diabetes.

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A new real-time PCR method using capturing oligo-immobilized PCR tubes is described. This method was used to detect specific genes for soybean and genetically modified (GM) soybean in food matrices. In a standard reaction using soybean genomic DNA and a capturing oligo for the lectin gene (Le1) immobilized on the tube, we examined the effects of such hybridization conditions as the location, length, and amount of the capturing oligo, and the incubation time and temperature. Under optimized conditions, the copy number of Le1 was determined in a concentration-dependent manner from soybean genomic DNA and soybean lysate (DNA 10–1000 ng, r=0.99; lysate 1–100%, r=0.99). The copy number of a Roundup Ready soybean (RRS) gene was also successfully detected in a concentration-dependent manner (1–100%, r=0.99) from GM soybean lysate, using PCR tubes with an immobilized capturing oligo for the transgene. Our data indicate that this is a rapid and simple method to determine specific genes for soybean and GM soybean in food matrices.

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Organosulfur compounds have been established to possess anticancer effects. To provide a better understanding of the biological function of dimethyl sulfides, dimethyl monosulfide (Me₂S), dimethyl disulfide (Me₂S₂), dimethyl trisulfide (Me₂S₃) and dimethyl tetrasulfide (Me₂S₄) were used as experimental materials to investigate their effects on apoptosis induction in human leukemia Jurkat cells and HL-60 cells. Treatment with 20 μM dimethyl sulfides for 24 h decreased the viability of both cells. The cell viability-reducing effect of these sulfides was in the following order: Me₂S₄ ≈ Me₂S₃ > Me₂S₂ ≈ Me₂S for Jurkat cells and Me₂S₄ > Me₂S₃ > Me₂S₂ ≈ Me₂S for HL-60 cells. Me₂S₃ and Me₂S₄ significantly induced DNA fragmentation and caspase-3 activation. The addition of GSH or NAC completely suppressed the sulfide-induced apoptosis. Our results indicate that dimethyl sulfides with a larger number of sulfur atoms more strongly induced apoptosis in both human leukemia cells via ROS production and caspase-3 activation.

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Chives, a member of the Alliaceae family, have been used in food and medicine in Thailand for a long time. Diallyl sulfides (diallyl monosulfide, dially disulfide, diallyl trisulfide, and diallyl tetrasulfide) are believed to be responsible for the antimicrobial activity of plants in this family. In this study, chive oil was examined for its diallyl sulfide content and its antimicrobial activity against some strains of food-borne pathogenic bacteria. Chive oil had a very low concentration of diallyl monosulfide in comparison with the other diallyl sulfides. They inhibited all pathogenic bacteria used in this study with a different degree of inhibition. Chive oil was also shown to be able to inhibit Escherichia coli O157:H7 in a food model. This study is the first report describing not only the diallyl disulfide content of chive oil, but also its antimicrobial activity against food-borne pathogens in both a test tube and food model.

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We investigated the taste synergy between L-theanine and the flavour enhancer, inosine 5′-monophosphate (IMP), by using a human sensory evaluation. When L-theanine was added to IMP, only the umami taste was enhanced. We then investigated this synergistic effect of L-theanine in mice by gustatory nerve recording. We confirmed the synergism between L-theanine and IMP for the umami taste.

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Myrica rubra leaf ethanol extract was added to culture medium of Madino-Darby canine kidney (MDCK) cells inoculated with influenza virus, and the inhibition of influenza virus replication was measured. Myrica rubra leaf ethanol extract showed anti-influenza virus activity irrespective of the hemagglutinin antigen type in the influenza virus type A (H1N1), its subtype (H3N2), and type B.

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(−)-Epigallocatechin-3-gallate (EGCG)-induced apoptosis was along both the extracellular signal-regulated protein kinase (ERK) and c-jun N-terminal kinase (JNK) pathways in Jurkat cells. Co-treatment with EGCG potentiated the cytotoxicity induced by benzyl isothiocyanate (BITC) and H₂O₂, both being inhibited by ERK and JNK inhibitors. These results suggest the significant role of mitogen-activated protein kinase (MAPK) signaling in the apoptosis induction regulated by EGCG alone and in combination.

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Circadian clocks, especially peripheral clocks, can be strongly entrained by daily feedings, but few papers have reported the effects of food components on circadian rhythm. The effects of resveratrol, a natural polyphenol, on circadian clocks of Rat-1 cells were analyzed. A dose of 100 μM resveratrol, which did not show cytotoxicity, regulated the expression of clock genes Per1, Per2, and Bmal1.

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Tocotrienol (T3) is an unsaturated vitamin E having health benefits (e.g., anti-angiogenesis). We measured T3 in commercial eggs, and developed T3-fortified eggs by adding rice bran scum oil (RBO, containing 1.3% T3) to the feed. Commercial eggs contained about 0.11 mg of T3/egg, while the T3 content was improved to 0.62 mg/egg after RBO supplementation to the feed of hens for 7 d.

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The coding sequence of a novel cellulolytic factor, swollenin, was isolated from the cellulolytic fungus Trichoderma pseudokoningii S38. The full-length swo2 gene encodes a protein of 494 amino acids with a calculated molecular mass of 51,393 Da, which includes a putative 22-amino-acid signal peptide. Sequence analysis revealed significant identity between isolated swollenin and that from Trichoderma reesei. The swollenin gene was further expressed and purified in T. reesei QM9414. The expressed swollenin protein was consequently purified by two-step ion exchange chromatography. The purified swollenin had subtle hydrolytic activities on xylan and yeast cell wall glucan, while no apparent activities on carboxymethy cellulose, cotton fiber, filter paper, or cellulose powder CF11 were observed. These results indicate that although swollenin maintains unidentified glycohydrolytic activities, it is inactive against β-1,4-glycosidic bonds in cellulose. Its exact role in lignocellulose hydrolysis calls for further analysis.

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To understand microbial communities in petroleum crude oils, we precipitated DNA using high concentrations of 2,2,4-trimethylpentane (isooctane) and purified. Samples of DNA from five crude oils, (Middle East, 3; China, 1; and Japan, 1) were characterized based upon their 16S rRNA gene sequences after PCR amplification and the construction of clone libraries. We detected 48 eubacterial species, one cyanobacterium, and one archaeon in total. The microbial constituents were diverse in the DNA samples. Most of the bacteria affiliated with the sequences of the three oils from the Middle East comprised similar mesophilic species. Acinetobacter, Propionibacterium, Sphingobium and a Bacillales were common. In contrast, the bacterial communities in Japanese and Chinese samples were unique. Thermophilic Petrotoga-like bacteria (11%) and several anaerobic-thermophilic Clostridia- and Synergistetes-like bacteria (20%) were detected in the Chinese sample. Different thermophiles (12%) and Clostridia (2%) were detected in the Japanese sample.

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High osmolarity and glucose deprivation cause rapid shutdowns of both actin polarization and translation initiation in yeast. Like these stresses, administration of local anesthetics and of antipsychotic phenothiazines caused similar responses. All these drugs have amphiphilic structures and formed emulsions and permeabilized the cell membrane, indicating that they have the same features as a surfactant. Consistently with this, surfactants induced responses similar to those of local anesthetics and phenothiazines. Benzethonium chloride, a cationic surfactant, showed a more potent shutdown activity than phenothiazines, whereas SDS, an anionic surfactant, transiently depolarized actin without inhibiting translation initiation, suggesting that a cationic charge in the amphiphile is important to the shutdown of both reactions. The clinical drugs and the cationic surfactants at low concentrations caused shutdown without membrane permeabilization, suggesting that these compounds and stresses activate shutdown, via perturbation rather than disruption of the cell membrane.

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We have reported increased glutamate production by a mutant of Corynebacterium glutamicum ATCC14067 (strain F172-8) with reduced H⁺-ATPase activity under biotin-limiting culture conditions (Aoki et al. Biosci. Biotechnol. Biochem., 69, 1466–1472 (2005)). In the present study, we examined valine production by an H⁺-ATPase-defective mutant of C. glutamicum. Using the double-crossover chromosome replacement technique, we constructed a newly defined H⁺-ATPase-defective mutant from ATCC13032. After transforming the new strain (A-1) with a C-terminal truncation of acetohydroxyacid synthase gene (ilvBN), valine production increased from 21.7 mM for the wild-type strain to 46.7 mM for the A-1 in shaking flask cultures with 555 mM glucose. Increased production of the valine intermediate acetoin was also observed in A-1, and was reduced by inserting acetohydroxyacid isomeroreductase gene (ilvC) into the ilvBN plasmid. After transformation with this new construct, valine production increased from 38.3 mM for the wild-type strain to 95.7 mM for A-1 strain. To the best of our knowledge, this is the first report indicating that an H⁺-ATPase-defective mutant of C. glutamicum is capable of valine production. Our combined results with glutamate and valine suggest that the H⁺-ATPase defect is also effective in the fermentative production of other practical compounds.

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The linear chromosome of Streptomyces griseus IFO13350 contains not only atypical telomere sequences but also probable pseudogenes for two typical telomeric proteins. Two identical operons (SGR98t-SGR97t near one telomere and SGR7041t-SGR7042t near the other telomere) in the terminal inverted repeat sequence were predicted to encode a novel pair of telomeric proteins. SGR97t, a 185-amino-acid protein showing only 18% amino acid sequence identity to typical terminal proteins of Streptomyces, was found to be attached to the chromosomal ends, as determined by immunological analysis. On the other hand, electrophoretic mobility shift assays showed that SGR98t, an 837-amino-acid protein having a DnaB-like helicase C-terminal domain, was capable of binding specifically to the single-stranded terminal DNA corresponding to the 3′ overhang of the replication intermediate. These results indicate that SGR97t (and SGR7042t) and SGR98t (and SGR7041t) were the functional telomeric proteins in the replication of the linear chromosome of S. griseus IFO13350.

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The unfolded protein response (UPR) is a regulatory system to maintain the homeostasis of ER functions. Here we report a comparison of express levels of UPR relevant genes in Aspergillus oryzae between solid-state and submerged cultivation. The results were that up-regulation of the UPR mechanism in solid-state culture was higher than in submerged culture (heat-shock or non-stress conditions). This might have been a result of changing culture conditions.

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To produce (S)-α-methylbenzylamine (MBA) from acetophenone, recombinant Escherichia coli co-expressing ω-transaminase and acetolactate synthase was used as a whole-cell biocatalyst. The solvent-bridge reaction system increased the yield of the whole-cell reaction by 2.5-fold, and the inhibitory (S)-α-MBA produced in the ω-transaminase reaction solution (pH 8.0) moved into the extraction solution (pH 3.0) via an organic solvent.

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Penicillium strains (n=394) preserved at NBRC (the NITE Biological Resource Center) were compared as to groupings (11 species-clusters) based on phylogeny and the production of bioactive compounds. The strains in two clusters, of which P. chrysogenum and P. citrinum are representative, showed higher rates of positive strains with multi-biological activities.

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Using a DNA microarray, we found that expression of the genes related to lactate metabolism was upregulated in a lactate-producing recombinant Saccharomyces cerevisiae strain. Disruption of the CYB2 gene encoding L-lactate dehydrogenase improved the L-lactate production by S. cerevisiae under low pH condition.

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