A number of 10-membered lactones have been isolated as secondary metabolites, and many of them have interesting and potent activities. Because of synthetic and biological interests, many synthetic studies of these compounds have been made. This review summarizes synthetic approaches to three natural 10-membered lactones, mueggelone, microcarpalide, and Sch 642305. Mueggelone is an inhibitor of fish development, microcarpalide is a microfilament disrupting agent, and Sch 642305 is an inhibitor of bacterial DNA primase.

抄録全体を表示

Conjugated dienyl compounds make one of the main groups of lepidopteran sex pheromones, and GC has been frequently used to determine the configurations of the double bonds. However, the separation of two geometric isomers of a terminal-conjugated diene, such as 7,9-decadien-1-ol secreted by a nettle moth Parasa lepida lepida (Limacodidae), is assumed to be difficult. In order to clarify the chromatographic separation of the terminal dienes, 7,9-decadienyl and 9,11-dodecadienyl compounds (alcohols, acetates, and aldehydes) were analyzed by GC and HPLC. On a capillary GC column, the (E)-isomers flowed out slightly faster than the corresponding (Z)-isomers, but their peaks almost overlapped. On the other hand, HPLC equipped with an ODS column completely separated the two geometric isomers examined and the (Z)-isomers eluted from the column faster than the (E)-isomers without dependence on a functional group. In addition to undergoing direct HPLC analysis without derivatization, the dienyl alcohols were converted into 3,5-dinitrobenzoates and analyzed by LC-ESI-MS operated under the same reversed-phase condition. The two separated geometric isomers were sensitively monitored by negative ions at m⁄z 211, M, M+1, M+17, and M+31, which were characteristically derived from the benzoates. Based on these results, a pheromone extract of P. l. lepida was examined, and it was confirmed that the female moths exclusively produced the (Z)-isomer of the 7,9-diene. Furthermore, a GC-EAD analysis and a field evaluation with both geometrical isomers indicated that the mating communication of P. l. lepida is predominantly mediated with the (Z)-isomer.

抄録全体を表示

A new reagent, N-[2-(diphenylphosphino)ethyl]-4-(1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-8-yl)benzamide (DPPEA-BODIPY), was designed and synthesized for analysis of hydroperoxides. DPPEA-BODIPY fluoresces at low levels in the visible range (λ_ex⁄λ_em = 502 nm/515 nm) and reacts with hydroperoxides to produce DPPEA-BODIPY oxide, which fluoresces at high levels. The fluorescence intensity of the reaction mixture was observed to be linearly related to the methyl linoleate hydroperoxide (MeLOOH) concentration.

抄録全体を表示

To clarify the cause of the difference in blue and red color development of hydrangea sepals, Hydrangea macrophylla, we analyzed the organic and inorganic components in the colored cells. To obtain colored protoplasts, each blue and red sepal tissue was treated with a combination of cellulase and pectinase, and then from the suspension of the olored and colorless protoplast mixture colored cells of the same hue were collected with a micro-pipette. The content of organic components (delphinidin 3-glucoside, chlorogenic acid, neochlorogenic acid and 5-O-p-coumaroylquinic acid) and Al³⁺ in each colored cell was quantified respectively by semimicro-HPLC and graphite furnace atomic absorption spectroscopy (GFAAS). In the blue cells 13 eq. of 5-O-acylquinic acids and 1.2 eq. of Al³⁺ to anthocyanin were contained. Contrary to this result, in the red cells, only 3.6 eq. of 5-O-acylquinic acids and 0.03 eq. of Al³⁺ were detected. A reproduction experiment of each blue and red sepal color by mixing those components concluded that, for blue coloration, both 5-O-acylquinic acids and Al³⁺ were essential.

抄録全体を表示

The biosynthesis of lasiodiplodin (1) and its (5S)-5-hydroxylated derivative (2) were investigated by the administration of ¹³C-labeled acetates to Lasiodiplodia theobromae. The labeling patterns of biosynthetically ¹³C-labeled 1 and 2 were determined by ¹³C-NMR and INADEQUATE spectra, demonstrating the octaketide origins of 1 and 2. Taking into account the biosynthetic study of resorcylic acid lactones, the involvement of highly reduced acyl intermediates in the biosynthesis of lasiodiplodins was presumed; thus, we synthesized ²H-labeled hypothetical acyl intermediates of 1, 9-hydroxydecanoic acid (4) and its N-acetylcysteamine thioester (SNAC, 5). When L. theobromae was incubated with 5 mM of a ²H-labeled intermediate, the ²H-label from the intermediate was incorporated at the expected position of 1. These incorporation studies revealed that 1 was produced via a pathway which closely resembles that of resorcylic acid lactone biosynthesis.

抄録全体を表示

We have purified and characterized an oligoxylosyl transfer enzyme (OxtA) from Bacillus sp. strain KT12. In the present study, a N-terminally His-tagged recombinant form of the enzyme, OxtA(H)^E, was overproduced in Escherichia coli and applied to the reaction with xylan and hydroquinone to produce 4-hydroxyphenyl β-D-oligoxylosides, β-(Xyl)_n-HQ (n=1–4), by one step reaction. The obtained β-(Xyl)_n-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC₅₀ values of β-Xyl-HQ, β-(Xyl)₂-HQ, β-(Xyl)₃-HQ, and β-(Xyl)₄-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. β-(Xyl)₄-HQ showed 35-fold more potent inhibitory activity than β-arbutin (4-hydroxyphenyl β-D-glucopyranoside), of which the IC₅₀ value was measured to be 6.3 mM. Kinetic analysis revealed that β-(Xyl)₂-HQ, β-(Xyl)₃-HQ, and β-(Xyl)₄-HQ competitively inhibited the enzyme, and the corresponding K_i values were calculated to be 0.20, 0.29, and 0.057 mM respectively.

抄録全体を表示

Phospholipase D (PLD) is a biocatalyst in the synthesis of bioactive compounds and a key enzyme in a variety of biological signal transductions. A combination of unnatural phosphatidyl acceptor, N,N,N-triethyl-N-2-hydroxyethylammonium bromide 6, as a substrate for PLD, and tandem electrospray ionization mass spectrometry (ESI MS) was found to provide information as to whether a given phospholipid serves as a substrate for the PLD-catalyzed reaction. Thus 2-(13′-hydroperoxy-octadecadienoyl)-1-palmitoylglycerophosphocholine 1, and its degradation products 2-(13′-oxo-octadecadienoyl)-1-palmitoylglycerophosphocholine 9 and 2-(13′-hydroxy-octadecadienoyl)-1-palmitoylglycerophosphocholine 11, in a mixture were found to be a substrate of the PLD-catalyzed transphosphatidylation. The sensitivity of this method was exemplified by the observation that PLD activity in cabbage leaves was detected using a small amount of crude crushed leaves with little pretreatment. This simple method can be used in screening for PLD activity and searching for inhibitors of the enzyme from various natural sources.

抄録全体を表示

The genes encoding ω-laurolactam hydrolases from Cupriavidus sp. T7, Acidovorax sp. T31, Cupriavidus sp. U124, and Sphingomonas sp. U238 were cloned and sequenced. Nucleotide and amino acid sequence analysis of the four genes indicated that the primary structures of these ω-laurolactam hydrolases are significantly similar to the 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12). These genes were expressed in Escherichia coli, and the ω-laurolactam hydrolysing activity of the recombinant enzymes was compared with that of 6-aminohexanoate-cyclic-dimer hydrolase from Arthrobacter sp. KI72. The enzyme from Acidovorax sp. T31 was most successfully expressed in E. coli. Cell-free extract of the recombinant strain was used for the synthesis of 12-aminolauric acid from ω-laurolactam by “enzymatic transcrystallization,” because crystalline ω-laurolactam added into the enzyme solution was converted to crystalline 12-aminolauric acid (≧97.3% yield). Under the optimum conditions, 208 g/l of 12-aminolauric acid was produced in 17 h. The resulting pure product was identical to authentic 12-aminolauric acid.

抄録全体を表示

Transglutaminase (TGase) from Streptomyces mobaraensis is a Ca²⁺ independent enzyme that cross-links proteins to high molecular weight aggregates. A dispase autolysis inducing protein (DAIP) was identified as an intrinsic TGase substrate exhibiting accessible glutamine and lysine residues. DAIP modification during culture by TGase resulted in deamidation of reactive glutamines, formation of glutamic/lysine residue pairs, and failure of cross-linking. The reactivity of modified DAIP can be restored to some extent by N-lauroylamido-3-N′,N′-dimethylpropyl amine, thus exposing concertedly buried glutamines and lysines. The novel TGase substrate differs considerably from the well known Streptomyces subtilisin inhibitors in higher molecular mass (37 kDa), lower pI (7.1–7.2), moderate thermo-stability, and the mode of erasing dispase activity. Our experiments suggested that DAIP induces autolysis without removal of essential metals, such as Ca²⁺ and Zn²⁺. Among other endoproteases, only thermolysin was similarly affected, but at considerably higher DAIP concentrations, due to simultaneous degradation of DAIP.

抄録全体を表示

In previous reports we have reported that theobroxide induces characteristic accumulation of allene oxide cyclase (AOC; EC 5.3.99.6) protein and jasmonic acid (JA) in Pharbitis nil. In the present study, PnAOC, an AOC gene from Pharbitis nil was cloned. Immunofluorescence assays indicated that the AOC protein is located in the chloroplast of vascular bundles in Pharbitis nil leaves. The PnAOC cDNA sequence lacking the chloroplast signal peptide was successfully expressed in Escherichia coli, and a gas chromatography-mass spectrum assay suggested the relative AOC activity of the recombinant PnAOC protein in comparison with Arabidopsis AOC2. Interestingly, a biphasic expression of PnAOC was induced by theobroxide, which is consistent with the accumulation patterns of AOC protein and JA. All these results indicate that AOC is the primary target of theobroxide regulation and suggest that feedback regulation of PnAOC by JA occurs upon theobroxide treatment in Pharbitis nil.

抄録全体を表示

The expression levels of three chitinase genes in Arabidopsis thaliana, AtChiA (class III), AtChiB (class I), and AtChiV (class IV), were examined under various stress conditions by semi-quantitative RT-PCR. Under normal growth conditions, the AtChiB and AtChiV genes were expressed in most organs of Arabidopsis plants at all growth stages, whereas the AtChiA gene was not expressed at all. The class III AtChiA gene was expressed exclusively when the plants were exposed to environmental stresses, especially to salt and wound stresses. Treatment of Arabidopsis plants with allosamidin, which inhibits class III chitinases, did not affect the growth rate. Surprisingly, however, the plants treated with allosamidin were more tolerant of abiotic stresses (cold, freezing, heat, and strong light) than the control plants. It also appeared that allosamidin enhances AtChiA and AtChiB expression under heat and strong light stresses. Allosamidin is likely to enhance abiotic stress tolerance, probably through crosstalk between the two signaling pathways for biotic and abiotic stress responses.

抄録全体を表示

One of the limiting factors for the application of Trichoderma reesei to degrade cellulosic biomass is its low β-glucosidase activity, required to convert cellobiose to glucose. The egl3 and the xyn3 promoters were used to express β-glucosidase 1 gene bgl1 through homologous recombination to improve the cellulose degradation ability of T. reesei. The recombinant strains expressing β-glucosidase 1 (BGLI) under the control of either the egl3 or the xyn3 promoter had 4.0 and 7.5 fold higher β-glucosidase activity than the native strain, which compares well to the finding that in wild-type T. reesei PC-3-7, the levels of egl3 and xyn3 mRNA expression were 6.0 and 12 fold higher respectively than that of bgl1. Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis of proteins secreted by the recombinant strains demonstrated that BGLI was overproduced. The increase in the transcription of bgl1 and the concomitant elevated level of BGLI in these recombinant strains were sufficient to degrade the cellobiose and cellotriose formed during the degradation of pretreated cedar powder.

抄録全体を表示

In addition to well-known curcuminoids, three colored metabolites were isolated from cultured cell clumps that had been induced from buds on turmeric rhizomes. The isolated compounds were identified as dihydro derivatives of curcuminoids, dihydrocurcumin (dihydroCurc), dihydrodesmethoxycurcumin-a (dihydroDMC-a), and dihydrobisdesmethoxycurcumin (dihydroBDMC). The cell clumps did not contain dihydroDMC-b, an isomer of dihydroDMC-a. A comparison of the distribution profiles of curcuminoids and dihydrocurcuminoids in the cell clumps with those in the rhizomes, leaves, and roots revealed the following differences: Unlike rhizomes, the cell clumps, leaves, and roots contained dihydrocurcuminoids as the major colored constituents. Whereas dimethoxy compounds, curcumin and dihydrocurcumin, respectively, were most abundant in the rhizomes and leaves, one of the monomethoxy derivatives, dihydroDMC-a, was found most abundantly in the cell clumps and roots. While both dihydroDMC-a and b were detected in the rhizomes, dihydroDMC-b was not detectable in the cell clumps, leaves, or roots. The occurrence of only one of the two possible isomers of dihydroDMC suggests biosynthetic formation of dihydrocurcuminoids in turmeric.

抄録全体を表示

In prokaryotes, menaquinone is used for respiration. In Escherichia coli, menaquinone is biosynthesized from chorismate by seven enzymes. However, very recently, we identified an alternative pathway (the futalosine pathway), which operates in some bacteria, including Streptomyces coelicolor, Helicobacter pylori, Campylobacter jejuni, and Thermus thermophilus. We describe the steps of this pathway, which branches at chorismate in a manner similar to the known pathway, but then follows a different route. This new pathway includes futalosine, an unusual nucleoside derivative consisting of inosine and o-substituted benzoate moieties, as a biosynthetic intermediate. In this study, a recombinant futalosine hydrolase (TTHA0556) of T. thermophilus, which participates in the second step of the pathway and catalyzes the reaction releasing hypoxanthine from futalosine, was prepared and used in functional analyses. Recombinant TTHA0556 formed a homotetramer and reacted only with futalosine; other structurally related nucleotides and nucleosides were not accepted. Recombinant TTHA0556 required no cofactors, and the optimum pH and temperature were 4.5 and 80 °C. The Km value was calculated to be 154.0±5.3 μM and the kcat value was 1.02/s. Recombinant TTHA0556 was slightly inhibited by hypoxanthine, with a Ki value of 1.1 mM.

抄録全体を表示

The SLC39A family of zinc transporters can be divided into four subfamilies (I, II, LIV-1, and gufA) in vertebrates, but studies of their functions have been restricted exclusively to members of subfamilies II and LIV-1. In this study, we characterized SLC39A9 (ZIP9), the only member of subfamily I in vertebrates. Confocal microscopy demonstrated that transiently expressed, HA-tagged human ZIP9 (hZIP9-HA) was localized to the trans-Golgi network regardless of zinc status. Disruption of the ZIP9 gene in DT40 cells did not change the growth rate, sensitivity to high zinc and manganese concentrations during long-term culture, or cellular zinc status after short-term incubation with zinc. The alkaline phosphatase activity of ZIP9^−⁄− cells did not change in cells cultured in medium containing normal zinc levels. In contrast, the activity of this enzyme decreased in wild-type cells cultured in zinc deficient medium but less so in ZIP9^−⁄− cells under these conditions. Stable over-expression of hZIP9-HA moderately decreased alkaline phophatase activity. These results suggest that ZIP9 functions to regulate zinc homeostasis in the secretory pathway without significantly altering cytosolic zinc homeostasis.

抄録全体を表示

The P1 and P1′ residue specificities of physarolisin I were investigated using combinatorial peptide substrates. The results indicated that certain hydrophobic residues and acidic residues are preferred at the P1 position and some hydrophobic residues at the P1′ position. This P1 specificity, different from other serine-carboxyl peptidases, appears to be explained partially by the nature of the S1 subsite residues.

抄録全体を表示

A chitinase gene from Bacillus cereus was cloned and expressed in Escherichia coli. The purified recombinant chitinase had much higher (128 fold) specificity to pNP-β-(GlcNAc)₃ than to pNP-β-(GlcNAc), suggesting endochitinase. Thirty-three amino acids in the N-terminal were recognized and cut off during expression, which consequently made the M_r not correspond to that predicted.

抄録全体を表示

HutB, the periplasmic hemin binding protein of Photobacterium damselae subsp. piscicida, was produced as a recombinant protein. UV-Vis spectrophotometrical analysis showed absorption spectral changes in hemin upon mixing it with the recombinant protein, indicating complex formation. Spectrophotometric titration of HutB with hemin showed saturation at a heme/HutB ratio of 1:1 and a binding affinity (K_d) of 10 μM.

抄録全体を表示

Inhibition of branched-chain α-ketoacid dehydrogenase kinase (BDK) by thiamine pyrophosphate (TPP) was analyzed at two potassium ion (K⁺) concentrations. IC₅₀ values of 4.6 and 8.0 μM and inhibition constant values of 3.2 and 16.4 μM were obtained in the presence of 20 and 100 mM K⁺, respectively. These results suggest that BDK is less sensitive to TPP inhibition under physiological TPP and K⁺ concentrations.

抄録全体を表示

To analyze aberrant spindle formation by microtubule-targeting drugs, live cell imaging was performed using multi-fluorescent human MDA-MB-435 cells in which several spindle components were visualized. Time-lapse images revealed that nocodazole and vinblastine induced additional perinuclear asters at the onset of mitosis. These results imply that these drugs stimulate the microtubule-organizing activity, despite their microtubule-destabilizing properties.

抄録全体を表示

A homologous transformation system was developed using the endogenous ATP-sulfurylase gene, AasC, as a selectable marker in Aspergillus aculeatus. Spontaneous mutation was proved to be beneficial in isolating AasC-deficient mutants. Molecular analysis of sC⁺ transformants revealed that the frequency of single copy integration at ATP-sulfurylase locus was more than 40%.

抄録全体を表示

A proteinaceous α-amylase inhibitor (CLAI) was purified from Cicer arietinum seeds. It had a molecular mass of 25.947 kDa and inhibited α-amylases from plants and mammals. Analysis of the amino acid sequence of a polypeptide from CLAI showed that it was different from other known α-amylase inhibitors, but had high identity to legumins from Cicer arietinum (100%) and Vicia faba var. minor (90%).

抄録全体を表示

CEL-III is a hemolytic lectin isolated from Cucumaria echinata. Although recombinant CEL-III (rCEL-III) expressed in Escherichia coli showed very weak hemolytic activity compared with native protein, it was considerably enhanced by refolding in the presence of Ca²⁺. This suggests that Ca²⁺ supported correct folding of the carbohydrate-binding domains of rCEL-III, leading to effective binding to the cell surface and subsequent self-oligomerization.

抄録全体を表示

We cloned and expressed two genes encoding azoreductase homologes, AzrB and AzrC, from Bacillus sp. B29. Purified recombinant AzrB and AzrC were homodimers with 23 kDa identical subunits, and were flavoproteins. NADH was preferred as electron donors for both azoreductases. The azoreductases showed optimal activities at 70 °C (AzrB) and 55 °C (AzrC), and retained activities up to 55 °C (AzrB) and 50 °C (AzrC) after incubation for 1 h. Other enzymatic properties, including the substrate specificities of both azoreductases, were also investigated.

抄録全体を表示

Human prion protein has four distinct Cu-binding motifs that catalyze the generation of superoxide coupled to oxidation of phenols and amines. Here, the thermostability of the superoxide-generating prion-derived peptides was tested. Among the peptides tested, two maintained high catalytic activity even after heating and repeated freezing/thawing cycles. The biological roles for these thermostable catalysts are discussed.

抄録全体を表示

The enzymatic, selective hydrogenation of cinnamaldehyde to cinnamyl alcohol is reported here. Yeast alcohol dehydrogenase was used in a substrate-coupled process with cofactor recycling. Both 100% selectivity and aldehyde conversion were achieved within 3 h. The reaction took place under very mild conditions, in the absence of toxic organic solvent. The overall process proved inexpensive and deserves further optimization studies in order to evaluate industrial applications.

抄録全体を表示

A cysteine residue was systematically introduced into three homologous cytochromes c from Hydrogenobacter thermophilus, Hydrogenophilus thermoluteolus, and Pseudomonas aeruginosa at a conserved position. The H. thermoluteolus variant showed the most decreased thermal stability as compared with the wild type, which might have been due in part to crosslinked polymer formation. The effects of cysteine introduction differed even at the conserved position in these homologous proteins.

抄録全体を表示

Yeast two-hybrid screening of mouse cDNA libraries was performed to identify proteins interacting with selenocysteine lyase (SCL), which decomposes L-selenocysteine. Several proteins related to spermatogenesis, protein synthesis, and cell viability/apoptosis were identified as potential interactors. Major urinary proteins 1 and 2 interacted with SCL and inhibited its activity. Coimmunoprecipitation revealed interactions between SCL and each of two selenophosphate synthetase isozymes.

抄録全体を表示

Phosphinothricin (PPT) is the active ingredient in bialaphos, which specifically inhibits glutamine synthetase in land plants. We isolated a novel PPT-resistant gene from a soil bacterium, Nocardia sp., and characterized it. The encoded protein, consisting of 177 amino acids, showed significant similarity to bacterial N-acetyltransferases, and we originally designated the gene MAT (methionine sulfone N-acetyltransferase). The recombinant MAT protein exhibited functions as a methionine sulfone and PPT N-acetyltransferase in vitro. The PPT N-acetyltransferase activity reached the maximum at pH 8–8.5, indicating that the protein might optimally function in chloroplasts. We therefore constructed a MAT gene, encoding the enzyme with a chloroplast-localizing signal in its amino-terminus. Plant transformation with the construct resulted in the generation of PPT-resistant rice and Arabidopsis. Furthermore, the transformed Arabidopsis was selectable in a synthetic medium containing PPT. The MAT gene thus facilitated establishment of herbicide-resistant plants, and as a new selectable gene marker.

抄録全体を表示

Cellular phototoxicity induced by UVA irradiation and its potential application to therapy has been reported. In the present study, the induction of apoptosis induced by β-carotene and dimethyl tetrasulfide (Me₂S₄) assisted by UVA irradiation in HL-60 cells was assessed. β-carotene assisted by UVA significantly decreased the cell viability and induced DNA fragmentation in HL-60 cells. Me₂S₄ combined with β-carotene and assisted by UVA significantly inhibited the cell viability, and enhanced the caspase-3 activity which was completely inhibited by N-acety-L-cysteine. β-carotene was significantly degraded by UVA, but this was not accelerated by Me₂S₄ in a cell culture system. The photodegradation products of β-carotene prepared by UVA irradiation regardless of the addition of Me₂S₄ showed lower cytotoxicity than β-carotene itself in HL-60 cells. These results suggest that the ROS- and caspase-3-dependent apoptosis induced by β-carotene and Me₂S₄ assisted by UVA was due to a synergistic action rather than to the sole effect of the photodegradation products of β-carotene in HL-60 cells.

抄録全体を表示

The main aim of this study was to elucidate whether thermosensitive transient receptor potential channels (thermoTRPs) play a role in controlling autonomic thermoregulation. We investigated whether the activation of certain thermoTRPs, TRPV1, TRPV3, TRPM8, and TRPA1, would induce autonomic thermoregulation by administering chemical agonists derived from spices and aroma chemicals of these channels to anesthetized mice. We discovered the following: Capsaicin, a TRPV1 agonist, enhanced thermogenesis and heat diffusion; thymol and ethyl vanillin, TRPV3 agonists, did not have any effect on thermogenesis or heat diffusion; menthol and 1,8-cineole, TRPM8 agonists, enhanced thermogenesis; and allyl isothiocyanate and cinnamaldehyde, TRPA1 agonists, enhanced thermogenesis and inhibited heat diffusion. These results suggest that these thermoTRP agonists derived from spices and aroma chemicals modulate autonomic thermoregulation, except for TRPV3 agonists. Our findings suggest the possibility that each thermoTRP is a key sensor inducing reasonable autonomic thermoregulation according to its own activated temperature range.

抄録全体を表示

The effects of a pumpkin paste concentrate and its components on oral glucose tolerance and serum lipid levels were determined in non-obese type 2 diabetic Goto-Kakizaki (GK) rats. In the oral glucose tolerance test, the pumpkin paste concentrate-fed group maintained a lower glucose level than the control group between 15 and 60 min. The compounds considered to be effective in improving glucose tolerance and contained in the methanol extract of the pumpkin in relatively abundant amounts were isolated and identified as trigonelline (TRG) and nicotinic acid (NA).
Feeding a diet containing TRG and NA respectively improved and tended to improve glucose tolerance. The insulin level increased after 15 min in the TRG-fed GK rats and then gradually decreased over the next 120 min. In contrast, a gradual increase was seen in the insulin level over 120 min in the control GK rats not fed with TRG, suggesting that TRG could improve the insulin resistance. The serum and liver triglyceride (TG) levels in the TRG- and NA-fed GK rats were lower than those in the control GK rats. Lower activity of liver fatty acid synthase (FAS), and higher activity of liver carnitine palmitoyl transferase (CPT) and glucokinase (GLK) in the TRG- and NA-fed GK rats than in the control GK rats were observed. This suggests that the regulation of these enzyme activities by TRG and NA was closely related to the suppression of both TG accumulation and the progression of diabetes.

抄録全体を表示

Ginseng (Panax ginseng C.A. Meyer) has a wide range of therapeutic uses including cancer treatment. Human promyelocytic leukemia cells differentiate into monocytes or granulocytes when treated with 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] or all-trans retinoic acid (ATRA). Treatment of HL-60 cells with zero to 100 μg/ml of a methanol extract of ginseng for 72 h induced a small increase in cell differentiation. Surprisingly, a synergistic induction of differentiation was observed when HL-60 cells were treated with ATRA or 1,25-(OH)₂D₃ and the extract. The inhibitors of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), but not of phosphoinositide 3-kinase (PI3-K), inhibited the HL-60 differentiation induced by the extract in combination with ATRA or 1,25-(OH)₂D₃, signifying that PKC and ERK were involved in the cell differentiation enhancement by the extract. These results suggest that the ability of a methanol extract of ginseng to enhance the differentiation potential of ATRA or 1,25-(OH)₂D₃ may improve the ultimate outcome of acute promyelocytic leukemia therapy.

抄録全体を表示

In order to establish the marker constituents of the natural antioxidant food-additive Eucalyptus leaf extract, the UV-absorbing constituents of two eucalyptus leaf extracts registered as food additives (eucalyptus A and B) were investigated. Several major peaks on the reversed-phase HPLC chromatogram of eucalyptus A were characterized as gallic acid, ellagic acid, 3-O-β-D-glucuronides of quercetin and kaempferol, and a hydrolyzable tannin dimer, oenothein B, by direct comparison with authentic specimens isolated from Eucalyptus globulus leaves. A new gallotannin was found in the E. globulus leaf extract, and its structure was found to be 1,2,3,6-tetra-O-galloyl-β-D-galactose. Two major peaks on the HPLC chromatogram of eucalyptus B were identified as gallic acid and ellagic acid, indicative of degradation products from hydrolyzable tannins in the leaves. Considering the evaluation of antioxidant activity by radical scavenging ability, a standardization of eucalyptus leaf extract, including the antioxidative polyphenol, oenothein B, is proposed.

抄録全体を表示

The effects of a water extract prepared from the leaves of Salacia reticulata on the absorption of sugars in normal and type 1 diabetic mice were investigated. The simultaneous oral administration of the extract at a dose of 1.0 mg/mouse with maltose or sucrose inhibited the postprandial elevation of the plasma glucose and insulin levels and intestinal α-glucosidase activities in mice. In addition, the supply of a 0.01% solution of the extract as drinking water prevented the elevation of the plasma glucose level and intestinal α-glucosidase activities in type 1 diabetic mice. This treatment also prevented the elevation of the plasma, pancreatic, and kidney lipid peroxide levels, lowering of the plasma insulin level, and elevation of the kidney aldose reductase activities in diabetic mice. These results suggest that the water extract of the leaves of S. reticulata could be a beneficial food material for the prevention of diabetes and obesity because of its multiple effects.

抄録全体を表示

Various native Korean plants were screened to find an effective acetylcholinesterase (AChE) inhibitor for the treatment of Alzheimer’s disease (AD). Among these plants, the ethanol extract of Poncirus trifoliate was selected for isolating the AChE inhibitor because it exhibited the highest inhibitory activity (47.31%). To separate the active compound from Poncirus trifoliate, solvent partition, open column chromatography, thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) were utilized. The putative chemical structure of the AChE inhibitor was identified as methoxsalen by successive analysis with electron ionization mass spectrometry (EI-MS) and ¹³C/¹H-nuclear magnetic resonance (NMR). To confirm the attenuating effect of the Poncirus trifoliate extract against trimethyltin (TMT)-induced neurotoxicity, in vivo behavior tests were carried out. Our findings suggest that the Poncirus trifoliate extract significantly reversed TMT-induced learning and memory impairment. These results demonstrate that the Poncirus trifoliate extract could possess a wide range of beneficial activities for neurodegenerative disorders, notably AD.

抄録全体を表示

We investigated the effects of an extract of Radix Rosa laevigata (R. R. laevigata) on Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS)-induced acute liver injury. The plasma alanine aminotransferase (ALT) activity was significantly elevated by an intravenous injection of heat-killed P. acnes at a dose of 0.4 mg/mouse and then with LPS at 0.1 μg/mouse after 5 d. However, the elevated ALT activity was significantly reduced by the administered of R. R. laevigata (125 and 500 mg/kg/d) for 7 d before the LPS injection. In addition, the extract treatment reduced the number of liver mononuclear cells (MNCs), and malondialdehyde (MDA) and nitric oxide (NO) contents, but improved the liver oxygen radical absorbance capacity (ORAC) and mitochondrial membrane potential. Moreover, the chemical profile of R. R. laevigata was performed by high-performance liquid chromatography (HPLC) and the main peaks were identified as a series of polyphenol compounds which had been confirmed as the significantly active components by their anti-oxidative and NO inhibitory effects. These results suggest that the extract of R. R. laevigata offered good efficacy for preventing liver injury.

抄録全体を表示

Changes in metabolites in fermented soymilk prepared with selected Bifidobacterium and Streptococci strains were analyzed using a ¹H-NMR-based metabolomic technique. Principal components analysis (PCA) allowed the clear separation of 50% methanol extracts from fermented soymilk with different fermentation times by combining principal components PC1 and PC3, which accounted for 55.1% of the total variance. Loading plot analysis was performed to select major compounds contributing to the separation, and the relative levels of selected metabolites were determined. In addition, the free-radical scavenging activities of each sample were investigated, and the underlying mechanisms were elucidated by determining the total phenolics and total flavonoids contents of each sample. The present study suggests the usefulness of combining ¹H-NMR with PCA in discriminating fermented soymilk samples with different fermentation times, and elucidates of the factors affecting free-radical scavenging activities of fermented soymilk.

抄録全体を表示

The suppression of cytochrome P450 1A1 (CYP1A1) expression was examined in mouse hepatoma Hepa-1c1c7 cells treated with serum prepared from (−)-epigallocatechin-3-gallate- and green tea extract-administered rats. Catechins were found in the rat plasma after the administration. In Hepa-1c1c7 cells, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 expression was suppressed by treatment with the rat serum. It is concluded that catechins can possibly modulate CYP1A1 expression.

抄録全体を表示

Unilamellar liposomes of 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine (PLPC) were exposed to ultraviolet A (UVA) light at 37 °C. The UVA-induced PLPC peroxidation was inhibited by free radical scavengers, while singlet oxygen quenchers showed slight inhibition. When pre-existing hydroperoxides were reduced, UVA irradiation could not initiate the lipid peroxidation. The UVA-induced reaction of PLPC hydroperoxides with α-tocopherol produced addition products. The results indicate that UVA irradiation might dissociate hydroperoxides to alkoxyl and peroxyl radicals which stimulate lipid peroxidation.

抄録全体を表示

5-Aminolevulinic acid (ALA) and the biomass of photosynthetic bacteria, Rhodopseudomonas palustris KG31, have very high potential for development and exploitation as bioherbicide and biofertilizer respectively. In this work, the effects of two precursors and an inhibitor of aminolevulinic dehydratase (ALAD) added to the VFA culture medium on the production of ALA and biomass were investigated. The experimental runs were carried out according to a Box-Behnken design. The precursors were added to the medium at the beginning of cultivation, while the inhibitor was added after 24 h. Statistical analysis indicated that levulinic acid (LA) has a positive effect on ALA production while glycine has a negative effect on biomass production. In order to enhance both ALA and biomass products, the most suitable medium was VFA medium supplemented with 3.0 mM glycine and 10 mM LA, giving ALA and biomass of 182.91 μM and 3.1 gDCW/l within 54 h.

抄録全体を表示

The endoglucanase Cel5A from an anaerobic celluloltyic bacterium, Clostridium josui, contains family 17 CBM (CjCBM17) and family 28 CBM (CjCBM28) in tandem. Individual CjCBM17 and CjCBM28 and tandem CjCBM17/28 were constructed to determine the binding characteristics of CjCBM17/28 and to compare the binding affinity of the three CBMs. CjCBM17/28 bound to non-crystalline cellulose, soluble cellulose derivatives, and oat-spelt xylan, but not to birchwood xylan or starch. The thermodynamic parameters for the binding of the CBMs with cellooligosaccharides were determined by isothermal titration calorimetry. The binding of CjCBM28 to cellotetraose and cellopentaose was enthalpically driven (large negative ΔH value), while that of CjCBM17 was entropically driven (positive ΔS value). These two CBMs had different mechanisms for binding to cellooligosaccharides. They showed similar binding constants (K_a) for cellopentaose, but in the case of insoluble polysaccharides, the K_a of CjCBM17/28 was approximately 3–7 times higher than that of individual CBMs.

抄録全体を表示

The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa. Amino acid residues known to be involved in copper-binding domains were conserved in the deduced amino acid residues of LeTyr. Transcriptional and translational expression of Letyr in the gills of the fruit-body increased during preservation after harvest. This correlation between Letyr expression and fruit-body preservation suggests that tyrosinase gene expression contributes to gill browning.

抄録全体を表示

Soft carbohydrates, defined as readily-recoverable carbohydrates via mere extraction from the biomass or brief enzymatic saccharification, were found in significant amounts in rice straw as forms of free glucose, free fructose, sucrose, starch, and β-1,3-1,4-glucan. In this study, we investigated their amounts in rice straw (defined as culm and leaf sheath), and developed an easy method for glucose and fructose recovery from them with heat-pretreatment and subsequent 4-h enzymatic saccharification with an enzyme cocktail of cellulase and amyloglucosidase. The recovery of glucose and fructose exhibited good correlation with the amounts of soft carbohydrates. The maximum yields of glucose and fructose in the rice straw per dry weight at the heading stage and the mature stage were 43.5% in cv. Habataki and 34.1% in cv. Leafstar. Thus, rice straw with soft carbohydrates can be regarded as a novel feedstock for economically feasible production of readily-fermentable glucose and fructose for bioethanol.

抄録全体を表示

β-1,3-Glucanase (BglF) from Nocardiopsis sp. F96 is composed of only a catalytic domain. To improve the enzymatic properties of BglF, we attempted to construct chimeric enzymes consisting of BglF and some carbohydrate-binding modules, such as the C-terminal additional domain (CAD) and the N-terminal additional domain (NAD) of β-1,3-glucanase H from Bacillus circulans IAM1165 and the chitin-binding domain (ChBD) of chitinase from alkaliphilic Bacillus sp. J813. CAD-fused BglF (BglF-CAD), NAD-fused BglF (NAD-BglF), both NAD- and CAD-fused BglF (NAD-BglF-CAD) and ChBD-fused BglF (BglF-ChBD) were constructed and characterized. The addition of CAD caused increases in binding abilities and hydrolytic activities toward insoluble β-1,3-glucans. As well as BglF-CAD, the binding ability and hydrolytic activity of BglF-ChBD toward pachyman were also increased. The hydrolytic activity of BglF-CAD at pH 9–10 was higher than that of BglF. The relative activities of BglF-CAD and BglF-ChBD at around 50–70 °C were higher than that of BglF.

抄録全体を表示

Flocculating yeasts are highly useful in fermentation processes because these cells can be separated easily from the fermentation mash. However, native yeasts are usually non-flocculating, including Kluyveromyces marxianus, which exhibits a potent high-temperature ethanol fermentation ability. We describe here the construction of flocculent K. marxianus strains via the introduction of the FLO1, FLO5, FLO9, and FLO10 genes from Saccharomyces cerevisiae. The S. cerevisiae FLO genes were overexpressed by upstream insertion of the constitutive TDH3 promoter, resulting in flocculent S. cerevisiae strains. These TDH3p-FLO sequences were then amplified by PCR and introduced directly into a K. marxianus strain. These K. marxianus strains showed a flocculation phenotype, indicating that the introduced S. cerevisiae TDH3 promoter and all FLO genes were functional in this strain. Moreover, a flocculating K. marxianus strain showed the same ethanol production profile as that of its wild-type parent. The K. marxianus flocculating strains we generated should be useful in the future development of cost-effective fed-batch and continuous fermentation systems at high temperatures.

抄録全体を表示

An industrial strain of Bacillus subtilis (natto) was used to produce poly-gamma-DL-glutamate (γPGA), a polymer of DL-glutamate linked by a γ-peptide bond. In spite of efforts to improve γPGA production by modifying the medium, little attention has been paid to the expression of the γPGA synthetase gene. In this study, we investigated the expression of the γPGA synthetic gene and the γPGA product under various conditions with the LacZ-fusion of the synthetic gene (pgsB-lacZ). The 5′ upstream regulatory region of the pgsB gene was also investigated by constructing deletion mutations of lacZ-fusion. The pgsB-lacZ was clearly expressed in the early stationary phase and was abolished by degU gene disruption. The results showed that pgsB-lacZ expression was repressed in rich media, and that γPGA production was limited by the substrate supply rather than by the amount of synthetase. Adding D-glutamate to the medium reduced γPGA production and synthetic gene expression. The transcription start point was determined by primer extension, and it was found that up to −721 bp (translation start point = +1) of the 5′ untranslated region (UTR) was required for optimal pgsB-lacZ fusion gene expression.

抄録全体を表示

Chitin is one of the major cell wall components of ascomycete filamentous fungi, and chitin synthesis plays important roles in the morphogenesis of hyphae. In the Aspergillus nidulans genome, there are two genes, csmA and csmB, that encode a myosin motor-like domain (MMD) at their N-termini and a chitin synthase domain (CSD) at their C-termini. In our previous studies, we found that the MMD of CsmA was required for its functionality, and that CsmA and CsmB had certain overlapping functions essential for polarized filamentous growth. In this study, we constructed a strain that produced CsmB lacking the MMD (CSΔMB). This strain exhibited defects similar to those of the csmB deletion mutant. FLAG-tagged CSΔMB (CSΔMB-FLAG) did not properly localize to the hyphae. CsmB was co-immunoprecipitated with actin in vivo, whereas CSΔMB was not. These results suggest that the MMD of CsmB is crucial for its proper localization via interaction with actin.

抄録全体を表示

We demonstrated the prebiotic effect of lacto-N-biose I (Galβ1-3GlcNAc) on bifidobacteria in vitro. Lacto-N-biose I, a building unit of the type-I milk oligosaccharides, enhanced the growth of many bifidobacteria, especially Bifidobacterium bifidum, B. breve, and B. longum, which are predominant in the intestines of breast-fed infants. It might be a substantial, natural prebiotic in human colostrums.

抄録全体を表示

In the last few decades, enzymatic production of 3,4-dihydroxyphenyl-L-alanine (L-dopa) using tyrosine phenol-lyase (Tpl) has been industrialized. This method has an intrinsic problem of tyrosine contamination because Tpl is synthesized under tyrosine-induced conditions. Herein, we constructed a hyper-L-dopa-producing strain by exploiting a mutant TyrR, an activator of tpl. The highest productivity was obtained for the strain grown under non-induced conditions. It was 30-fold higher than that obtained for tyrosine-induced wild-type cells.

抄録全体を表示