New nematicides named fumiquinones A (1) and B (2), together with spinulosin (3), LL-S490β (4), and pseurotin A (5), were isolated from Aspergillus fumigatus and their structures were established by spectroscopic methods including 2D-NMR. Compound 1 showed effective nematicidal activities against Bursaphelenchus xylophilus and Pratylenchus penetrans without inhibiting plant growth except for lettuce seedlings. Compound 2 showed effective nematicidal activity against B. xylophilus, but had no inhibitory activity against P. penetrans. Compounds 3–5 showed effective nematicidal activities against B. xylophilus without any plant growth inhibition. Compounds 1–5 had no nematicidal activity against Caenorhabditis elegans. This is the first report of the nematicidal activities of compounds 3–5.

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The effect of oxidation degree at the benzylic position of 2,3-dibenzyl-4-butanolide and 3,4-dibenzyltetrahydrofuran lignans on the antimicrobiological activity was examined. The highest oxidation degree at the benzylic position of 2,3-dibenzyl-4-butanolide gave the greatest activity, and 3,4-dibenzoyltetrahydrofuran showed the highest antifungal activity. The relationship between stereochemistry and activity was also examined. Both enantiomers of cis-matairesinol were synthesized for the first time, one of the cis-matairesinols showing antibacterial activity.

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Two new taxanes were isolated from the needles of Taxus canadensis. Their structures were established as 10β,13α-diacetoxytaxa-4(20),11-diene-5α,9α,14β-triol (1) and 4α,13α-diacetoxy-2α,20-epoxy-11(15→1)abeotaxa-11,15-diene-5α,7β,9α,10β-tetraol (2) on the basis of a spectroscopic analysis.

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A novel compound (2) and a known one (1) were isolated from the mushroom, Sparassis crispa. Both compounds inhibited melanin synthesis and MRSA growth.

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Bacillus mojavensis strain RO-H-1 produces a posttranslationally modified hexapeptide, the ComX_RO-H-1 pheromone, that stimulates natural genetic competence controlled by quorum sensing. LC/ESI-MS analysis of partially purification of the ComX_RO-H-1 pheromone suggested a precise modification in its tryptophan residue. The corresponding ComX_RO-H-1 pheromone prepared by solid-phase synthesis was identical to the natural pheromone, and showed significant biological activity. These results indicated that the posttranslational modification of the ComX_RO-H-1 pheromone was geranylation on the tryptophan residue, resulting in the formation of a tricyclic structure. The ComX_RO-H-1 pheromone was immediately dehydrated by acid because of its extreme acid lability.

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β-N-Acetylglucosaminidase is a major glycosidase involved in several physiological processes, such as fertilization, metamorphosis, glycoconjugate degradation, and glycoprotein biosynthesis in insects. A search using the Bombyx mori cDNA database revealed the existence of two putative β-N-acetylglucosaminidase genes. Their full-length cDNAs were cloned by rapid amplification of cDNA ends and polymerase chain reaction using specific primers, and named BmGlcNAcase1 and BmGlcNAcase2. A BLAST search revealed that BmGlcNAcase1 and BmGlcNAcase2 are homologous to a β-subunit homolog encoded by Drosophila melanogaster HEXO2 and the Spodoptera frugiperda β-N-acetylglucosaminidase gene respectively. The recombinant proteins of BmGlcNAcase1 and BmGlcNAcase2 without putative transmembrane domains were expressed in the yeast Pichia pastoris. Both enzymes showed broad substrate specificity, and cleaved terminal N-acetylglucosamine residues from the α-3 and α-6 branches of a biantennary N-glycan substrate, and also hydrolyzed chitotriose to chitobiose.

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The relationships between the structural and energetic domains of lentil seedling amine oxidase (LSAO) were investigated using modifiers that target the active site and the carbohydrate moiety of the enzyme. An irreversible inhibitor, aminoguanidine, specifically modified the active site of the lentil enzyme, whereas sodium metaperiodate cleaves carbohydrate moieties covalently bound to the native enzyme. Differential scanning calorimetry (DSC) measurements were made on the modified LSAOs. Deconvolution of the reversible thermal DSC profiles of the modified enzyme gave three subpeaks (energetic domains), each of which was assigned to one of the three structural domains of the native protein. Our results led us to conclude that deglycosylation of LSAO has no effect on thermal stability, whereas binding of the inhibitor imparts more stability to the enzyme.

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In skeletal muscle, AMP-activated protein kinase (AMPK) is a metabolic master switch regulating glucose and lipid metabolism. Recently, AMPK has been implicated in the control of protein synthesis in skeletal muscle, but the effect of AMPK activation on myofibrillar protein degradation has yet to be elucidated. The present study was designed to examine the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of myofibrillar protein degradation and the expression of atrophy-related genes (atrogin-1/MAFbx, MuRF1, proteasome C2 subunit, calpains, cathepsin B, and caspase-3) in C2C12 myotubes. AICAR stimulated myofibrillar protein degradation (as measured by N^τ-methylhistidine release), while also increasing the levels of atrogin-1/MAFbx and MuRF1 mRNA, but the expression of other atrophy-related genes was not enhanced by AICAR treatment in C2C12 myotubes. AICAR also stimulated the level of FOXO transcription factors mRNA and protein in C2C12 myotubes. These results indicate that activation of AMPK stimulates myofibrillar protein degradation through the expression of atrogin-1/MAFbx and MuRF1 by increasing FOXO transcription factors in skeletal muscles.

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To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH₃OGPP) as allylic substrate homologs.
The wild-type FPP synthase reaction of HOGPP (and CH₃OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation.
On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.

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Catechol 2,3-dioxygenase (C23O), a key enzyme in the meta-cleavage pathway of catechol metabolism, was purified from cell extract of recombinant Escherichia coli JM109 harboring the C23O gene (atdB) cloned from an aniline-degrading bacterium Acinetobacter sp. YAA. SDS–polyacrylamide gel electrophoresis and gel filtration chromatography analysis suggested that the enzyme (AtdB) has a molecular mass of 35 kDa as a monomer and forms a tetrameric structure. It showed relative meta-cleavage activities for the following catechols tested: catechol (100%), 3-methylcatechol (19%), 4-methylcatechol (57%), 4-chlorocatechol (46%), and 2,3-dihydroxybiphenyl (5%). To elevate the activity, a DNA self-shuffling experiment was carried out using the atdB gene. One mutant enzyme, named AtdBE286K, was obtained. It had one amino acid substitution, E286K, and showed 2.4-fold higher C23O activity than the wild-type enzyme at 100 μM. Kinetic analysis of these enzymes revealed that the wild-type enzyme suffered from substrate inhibition at >2 μM, while the mutant enzyme loosened substrate inhibition.

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Agrobacterium tumefaciens KAT23 isolated from peach root causes crown gall disease in a number of grain legume plants, including the common bean (Phaseolus vulgaris) and soybean (Glycine max). KAT23 caused tumor formation in each of these plants more effectively than strain C58. Biotype determination suggested that this strain is biotype II. KAT23 was able to utilize nopaline as a carbon source. Partial sequence analysis indicated that KAT23 harbors a nopaline-type Ti plasmid, designated pTiKAT23, which was highly homologous with other nopaline-type Ti plasmids (pTiC58 and pTiSAKURA).
KAT23 transferred not only the T-DNA of the Ti plasmid but also introduced T-DNA of the binary vector efficiently. The common bean inoculated with KAT23 (pIGFP121-Hm) showed crown galls, and some plants showed β-glucuronidase (GUS) and sGFP (S65T) gene expression. This virulent ability of KAT23 indicates its potential application to legumes, especially to soybean transformation.

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A new functional Corynebacterium glutamicum insertion sequence (IS) element, IS13655, was isolated using a suicide vector. The IS element was 1,293 bp in size and contained 26-bp imperfect inverted repeats (IRs) and 3-bp target site duplication as direct repeats (DRs). IS13655 harbored two ORFs with high similarity to the transposase of IS1206, an IS3 family element. IS13655 revealed relatively high transposition efficiency, with low target site selectivity along the Corynebacterium glutamicum R genome, making it a potentially useful genetic engineering tool.

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The tfdB gene encoding chlorophenol hydroxylase and its homolog were found in 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading strain RD5-C2, which belongs to Bradyrhizobium sp. of α-Proteobacteria. The nucleotide and deduced amino acid sequence identities of the two genes, designated tfdBa and tfdBb, were 60% and 57% respectively. Their nucleotide sequences most closely matched those of previously reported tfdB, which consisted of those from 2,4-D-degrading β- and γ-Proteobacteria and Sphingomonas sp. in α-Proteobacteria, with 61–67% identity. The TfdBa expressed in Escherichia coli showed the highest activity for 2,4-dichlorophenol but a narrower range of activity for the other chlorophenols than previously reported TfdBs. In the case of TfdBb, however, no observable activity for any chlorophenols or phenol was detected, although production of a protein with an appropriate molecular size was observed. Based on codon usage patterns and the GC content of the genes, it probable that the tfdBa genes in the 2,4-D-degrading Bradyrhizobium sp. were obtained through horizontal gene transfer.

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cDNAs encoding three α-glucosidases (HBGases I, II, and III) from European honeybees, Apis mellifera, were cloned and sequenced, two of which were expressed in Pichia pastoris. The cDNAs for HBGases I, II, and III were 1,986, 1,910, and 1,915 bp in length, and included ORFs of 1,767, 1,743, and 1,704 bp encoding polypeptides comprised of 588, 580, and 567 amino acid residues, respectively. The deduced proteins of HBGases I, II, and III contained 18, 14, and 8 putative N-linked glycosylation sites, respectively, but at least 2 sites in HBGase II were unmodified by N-linked oligosaccharide. In spite of remarkable differences in the substrate specificities of the three HBGases, high homologies (38–44% identity) were found in the deduced amino acid sequences. In addition, three genomic DNAs, of 13,325, 2,759, and 27,643 bp, encoding HBGases I, II, and III, respectively, were isolated from honeybees, and the sequences were analyzed. The gene of HBGase I was found to be composed of 8 exons and 7 introns. The gene of HBGase II was not divided by intron. The gene of HBGase III was confirmed to be made up of 9 exons and 8 introns, and to be located in the region upstream the gene of HBGase I.

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The shell of the Japanese pearl oyster, Pinctada fucata, consists of two layers, the prismatic layer on the outside and the nacreous layer on the inside, both of which comprise calcium carbonate and organic matrices. Previous studies indicate that the nacreous organic matrix of the central layer of the framework surrounding the aragonite tablet is β-chitin, but it remains unknown whether organic matrices in the prismatic layer contain chitin or not. In the present study, we identified chitin in the prismatic layer of the Japanese pearl oyster, Pinctada fucata, with a combination of Calcofluor White staining with IR and NMR spectral analyses. Furthermore, we cloned a cDNA encoding chitin synthase (PfCHS1) that produces chitin, contributing to the formation of the framework for calcification in the shell.

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Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.

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The intracellular behavior of human FCHO1 protein was investigated by live-cell imaging microscopy. The fluorescence intensity of green fluorescent protein (GFP)-FCHO1 fluctuated periodically in a perinuclear region approximately every 100 s, reminding us of the periodic fluctuations of clathrin reported in our recent work. The periodicity of FCHO1 was temporally correlated with that of clathrin, suggesting that FCHO1 is involved in clathrin-coated vesicle formation.

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We established an improved purification procedure for Streptomyces caespitosus neutral protease (ScNP) from culture supernatants of S. caespitosus. The procedure comprises sequential ammonium sulfate fractionation and column chromatography procedures with anion exchange chromatography, followed by hydrophobic-interaction chromatography and gel filtration. Purified ScNP revealed a single band with a molecular mass of 14 kDa by SDS–PAGE under reduced conditions and did not contain any detectable pigment, which has not been completely removed by other methods. We also purified another protease with a molecular mass of 40 kDa from the culture supernatants. The pure preparation of ScNP obtained by this procedure is suitable for spectrophotometric measurement of its catalytic activity.

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Nobiletin contributes to pharmacological activities such as anti-cancer and anti-inflammatory effects, but little is known about its effect on melanogenesis. In this study, we found that nobiletin increased melanin content and tyrosinase activity in murine B16/F10 melanoma cells. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK) pathway with U0216 resulted in inhibition of nobiletin-induced melanin synthesis and tyrosinase expression.

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To obtain cDNAs encoding oxidosqualene cyclase (OSC), we cloned two cDNAs, KcCAS and RsCAS, from roots of Kandelia candel (L.) Druce and leaves of Rhizophora stylosa Griff. by homology based PCR method respectively. The deduced amino acid sequences of both OSCs showed 82% homology to cycloartenol synthases from Lotus japonicus (OSC5) and Ricinus cummunis (RcCAS), suggesting that these are cycloartenol synthases of K. candel and R. stylosa. The genes obtained were expressed in a lanosterol synthase deficient Saccharomyces cerevisiae (ERG7) strain, GIL77. GC–MS analysis identified the accumulated reaction product in the yeast transformant to be cycloartenol, indicating that both KcCAS and RsCAS encode cycloartenol synthase.

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After injection with 0.1 mmol diquat/kg body weight, survival time was markedly shorter in Fischer-344 rats fed a purified diet than in rats fed a regular diet, and much more severe hepatotoxicity and nephrotoxicity were observed in the former than in the latter. The longer the feeding period on the purified diet, the shorter the survival time after diquat administration. These results indicate that the purified diet lacked components present in the regular diet that had protective effects against diquat toxicity. These two diets had nearly the same composition and content of vitamins and minerals. We tested the ingredients of the regular diet to determine which ones reduce diquat toxicity. We found that wheat bran had a protective effect, but that rice bran and bean-curd refuse (okara) did not.

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The intestinal absorption characteristics of anthraquinones emodin and chrysophanol were observed by measuring the intracellular accumulation across Caco-2 cells by the reverse-phase high performance liquid chromatography. The intracellular accumulation of chrysophanol was much greater than that of emodin, the maximum absorption of emodin and chrysophanol being 414.02±15.28 and 105.56±11.57 nmol/l·mg·protein, respectively. The absorption of each anthraquinone was significantly lower at 4 °C than that of 37 °C. The effects of the transport inhibitors, verapamil, cyclosporine and phloridzin, on the intracellular accumulation were also examined. Verapamil and cyclosporine increased the absorption of emodin and chrysophanol, while phloridzin inhibited their absorption, all in a dose-dependent manner. These results suggest that the absorption characteristics of emodin and chrysophanol were closely related to their special structure with the hydroxy groups. It is also likely that a specific transport system mediated the intracellular accumulation of emodin and chrysophanol across the Caco-2 cells.

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A PCR method to detect porcine DNA was developed for verifying the allergen labeling of foods and for identifying hidden pork ingredients in processed foods. The primer pair, F2/R1, was designed to detect the gene encoding porcine cytochrome b for the specific detection of pork with high sensitivity. The amplified DNA fragment (130 bp) was specifically detected from porcine DNA, while no amplification occurred with other species such as cattle, chicken, sheep, and horse. When the developed PCR method was used for investigating commercial food products, porcine DNA was clearly detected in those containing pork in the list of ingredients. In addition, 100 ppb of pork in heated gyoza (pork and vegetable dumpling) could be detected by this method. This method is rapid, specific and sensitive, making it applicable for detecting trace amounts of pork in processed foods.

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Furfural is an important intermediate compound of the Maillard reaction of pentose or ascorbic acid. We examined the browning of furfural and lysine by heating and found a yellow compound, called furpipate, (E)-3-(2-furylmethylidene)-3H, 4H, 5H, 6H-pyridine-2-carboxylic acid. Furpipate is a novel pipecolic acid derivative and shows absorption maxima at 375 nm and 310 nm under acidic and alkaline conditions, respectively. This compound was the major colored compound of the heated solution containing lysine and furfural.

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The DPPH radical-scavenging abilities of the naturally occurring phenolic acid, 2,3,4-trihydroxybenzoic acid, and its methyl ester were evaluated. Both compounds in acetonitrile scavenged as many as four radicals compared to three or fewer radical consumption in acetone or ethanol. Only the ester showed relatively high ability in methanol. Oxidation with o-chloranil in acetonitrile resulted in methyl 2,3,4-trihydroxybenzoate giving a novel benzocoumarin-type dimer, its chemical structure being confirmed by spectroscopic evidence. The formation of this dimer might partly account for the higher radical-scavenging efficiency of the ester in acetonitrile or methanol.

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The formation of HEMF[2(or 5)-ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-furanone], the aroma component specific to miso and soy sauce, was promoted by cultivating the halo-tolerant yeast, Zygosaccharomyces rouxii, in a medium including the amino-carbonyl reaction products based on ribose and glycine. The glucose concentration in the medium influenced the HEMF formation by Z. rouxii.

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Soy isoflavone aglycones (IFAs) have a wide range of biological actions. We investigated in this study the effect of IFAs on myeloid cells. The cell surface expression of both CD80 and CD86 was up-regulated by treating myeloid cells with IFAs in vitro and in vivo. The findings suggest that IFAs could modulate the myeloid cell function.

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A sensitive qualitative detection method for walnut (Juglans regia) using polymerase chain reaction (PCR) was developed. For detection of walnuts with high specificity, the primer pair WAL-F/WAL-R was designed based on walnut matK genes. Trace amounts of walnuts in commercial food products can be qualitatively detected using this method.

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Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM α-ketobutyrate, and 100 mM L-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mM Tris–HCl buffer (pH 7.5) containing 2 mM MnCl₂·4H₂O at 28 °C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplex AKU 626. Mn²⁺ and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.

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We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.

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The Aspergillus nidulans high-osmolarity glycerol response (AnHOG) pathway is involved in osmoadaptation. We found that fludioxonil, a fungicide, causes improper activation of HogA mitogen-activated protein kinase (MAPK) in A. nidulans. Here we present novel reporter systems for monitoring activation of the AnHOG pathway. The promoter region of gfdB (glycerol-3-phosphate dehydrogenase), whose expression depends on the presence of HogA, was fused to a β-glucuronidase uidA gene (GUS) to construct the reporter, which was introduced into A. nidulans wild type and hogAΔ. Increased GUS activity was detected in the wild type only when it was treated with high osmolarity or fludioxonil, while reporter activity was scarcely stimulated in the hogAΔ mutant. These results indicate that the reporter activity is controlled via HogA activation. Furthermore, we present possible applications of the reporter systems in screening new antifungal compounds.

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Production of extracellular β-1,4-xylanase, α-L-arabinofuranosidase, feruloyl esterase, and acetyl xylan esterase from Aspergillus kawachii was higher in a culture supplemented with ferulic acid than in a counterpart. Culture supernatant grown on oat spelt xylan supplemented with ferulic acid exhibited an increase in ferulic acid-releasing activity from insoluble arabinoxylan relative as compared to that from the ferulic acid-free culture.

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Solid-state culture encourages high-level enzyme secretion by Aspergillus oryzae. Using the real-time quantitative reverse transcriptase-polymerase chain reaction, we confirmed that expression of the glucoamylase-encoding gene in A. oryzae cultured in solid-state culture depends on the water content of the culture.

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Microorganisms growing in natural habitats are constantly confronted with a wide variety of external stresses. Here we provide several lines of experimental evidence for the thesis that the filamentous fungus Aspergillus nidulans has a homolog of the AP-1-like bZip transcription factor, which is known to play general roles in oxidative responses in many types of yeast.

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