The outer membrane, an essential organelle of Gram-negative bacteria, is composed of four major components: lipopolysaccharide, phospholipids, β-barrel proteins, and lipoproteins. The mechanisms underlying the transport of these components to outer membranes are currently under extensive examination. Among them, the sorting of lipoproteins to the outer membrane of Escherichia coli has been clarified in detail. The Lol system, composed of five proteins, catalyzes outer membrane sorting of lipoproteins. Various Lpt proteins have recently been identified as factors involved in the transport of lipopolysaccharide to the outer membrane, although the mechanism remains largely unknown. Proteins with α-helical membrane spanning segments are found in the inner membrane, whereas amphipathic β-barrel proteins span the outer membrane. These β-barrel proteins are inserted into the outer membranes through a central core protein BamA (YaeT) with the help of four outer membrane lipoproteins. In contrast, little is known about how phospholipids are transported to the outer membrane.

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An increment of alanine aminotransferase (ALT) or aspartate aminotransferase (AST) in human serum indicates an abnormal symptom of the liver. Hence, an electrochemical biosensor array that uses micro electro mechanical systems technology is required for rapid and integrated measurement of ALT/AST. Here we describe a biosensor array consisting of two glutamate sensors. It turned out that porous silicon layers formed on each working electrode were useful to increase the effective surface area. This biosensor array was constructed with platinum electrodes and a polydimethylsiloxane (PDMS) microchannel. Electrodes in sampling wells minimized a cross-interference effect and permitted multiple sampling by immobilization with glutamate oxidase using a silanization technique. The device sensitivities derived from semi-logarithmic plots were 0.145 μA/(U/l) for ALT and 0.463 μA/(U/l) for AST over a range of 1.3 U/l to 250 U/l. Hene, this ALT/AST biosensor array can be applied in diagnostic and home use.

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Glycans play important roles in various biological phenomena, but the lack of a systematic procedure for producing complex structures of glycans severely restricts their application in the medical and industrial fields. In this paper, we propose a basic strategy for the preparation of substantial amounts (>100 mg) of N-linked oligosaccharides, where the structure of each glycan is mapped with its elution position in liquid chromatography as well as the empirical yield. In model experiments using hen egg white and yolk as starting materials, the former provided a series of agalactosylated complex-type and hybrid-type N-linked oligosaccharides containing bisecting N-acetylglucosamine (GlcNAc) in addition to two high-mannose type glycans. In contrast, egg yolk gave predominantly α2-6sialylated biantennary glycans together with a high-mannose type one, reflecting the difference in the origins of the tissues. Due to the total identity of the glycans obtained to human ones, the present strategy should provide a practical scheme for the production of human-type N-linked oligosaccharides.

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Glycosaminoglycans (GAGs) are important polysaccharides with various biological functions, but their structures in solution are affected in a complex manner by their component residues. We successfully measured the vacuum-ultraviolet circular dichroism (VUVCD) spectra of six GAGs (chondroitin, chondroitin sulfates, hyaluronic acid, and heparin) and their component sugars (N-acetylaminosugars and uronic acid) from 240 to 160 nm by a synchrotron-radiation VUVCD spectrophotometer in aqueous solutions at 25 °C. These comprehensive VUVCD spectra revealed the characteristic contribution of the constituent functional groups (sulfate, carboxyl, hydroxyl, hydroxymethyl, acetamido, etc.) and intersaccharide linkages to the higher energy transition of oxygen lone-pair electrons, giving basic information for understanding the detailed structures of GAGs in solution and for their theoretical assignment.

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Three positional isomers of 6¹,6ⁿ-di-O-(α-L-fucopyranosyl)-β-cyclodextrin [6¹,6ⁿ-di-O-(α-L-Fuc)-βCD, n=2–4] were chemically synthesized by using the corresponding authentic compounds, 6¹,6ⁿ-di-O-(tert-butyldimethylsilyl)-βCD (n=2–4) as fucosyl acceptors and 2,3,4-tri-O-benzyl-L-fucopyranosyl trichloroacetimidate as a fucosyl donor. Their structures were analyzed by HPLC, MS and NMR spectroscopy. The hemolytic activities of the α-L-Fuc-βCDs were lower than that of βCD, while the water solubility of these branched βCDs was much higher than that of βCD. The molecular interaction between these compounds and the fucose-binding lectin, Aleuria aurantia lectin (AAL), was investigated by using an optical biosensor based on the surface plasmon resonance (SPR) technique. The order of binding affinity, as a function of the fucose-binding position, was 6¹,6⁴- > 6¹,6³- > 6¹,6²-di-O-(α-L-Fuc)-βCD > 6-O-(α-L-Fuc)-βCD.

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The first diastereoselective synthesis of (1S,6R)-1-hydroxy-2,7(14),10-bisabolatrien-4-one, an antifeedant against Acusta despesta and Locusta migratoria, was produced from Cryptomeria japonica (commonly known as Japanese cedar), starting from (R)-(−)-carvone via (R)-(−)-cryptomerione. The enantiomer was transformed into (1S,3R,6R)-1-hydroxy-7(14),10-bisaboladien-4-one, a novel antifeedant against L. migratoria from the same tree, by 1,4-selective reduction of the enone moiety.

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The culture broth of Daedalea dickinsii was found to predominantly contain the tyrosinase inhibitor, (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene, daedalin A (1). Ongoing research into bioactive metabolites resulted in the identification of two new 2H-chromenes, 6-hydroxy-5,7-dimethoxy-2,2-dimethyl-2H-chromene (3) and 6-hydroxy-2-hydroxymethyl-5-methoxy-2-methyl-2H-chromene (4), together with 6-hydroxy-2,2-dimethyl-2H-chromene (2). Comparative studies of isolated compounds 1–4 and related compounds (±)-1 and 1a–1c showed 1 to have the strongest tyrosinase inhibitory activity. These results suggest that the hydroxyl groups at positions 6 and 9 of 1 were important for the potent activity. A Lineweaver-Burk plot for a kinetic analysis indicates that 1 competed with L-tyrosine for tyrosinase. Compound 1 also suppressed melanogenesis in a human skin model (up to 49% at 2.8 μmol/tissue application) without affecting the cell viability. Compounds 1, 1b and 1c also showed 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity comparable to that of α-tocopherol.

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Magnolia species are widely cultivated in Japan as garden plants, and have been found to contain various compounds, including alkaloids, terpenoids, lignans, and neolignans. The constituents of the mature fruits of M. denudata were investigated, and two new phenolic derivatives, named denudalide and denudaquinol, were isolated and characterized, together with a known neolignan compound (denudatin A). Denudalide and denudaquinol showed cytotoxicity against the SFME and r/mHM-SFME-1 cell lines.

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As a part of our research project on the elucidation of the chain-breaking antioxidation mechanism for natural phenols in food components, the antioxidation products from a sinapic acid methyl ester in lipid oxidation were identified. Sinapic acid is a potent antioxidative phenolic acid widely distributed in edible plants. The structural determination of the four isolated products revealed them each to have a tricyclic structure consisting of ethyl linoleate, methyl sinapate and molecular oxygen, and these were isomeric compounds for the substituents on the tricyclic moiety.

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Taxusecone, 2α,7β,9α,10β-tetraacetoxy-5α,12-dihydroxy-11,12-secotax-4(20)-ene-11,13-dione (1), a novel taxane with an unprecedented skeleton, was isolated from the needles of Taxus cuspidata.

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In vitro assays using recombinant enzymes enabled three cDNAs encoding ent-copalyl diphosphate synthases to be identified in wheat (Triticum aestivum): TaCPS1, TaCPS2, and TaCPS3. The phylogenetic tree and expression analyses suggest that TaCPS3 is responsible for gibberellin biosynthesis, while TaCPS1 and TaCPS2 are possible functional homologs of diterpene cyclase genes OsCPS2 and OsCPS4 involved in phytoalexin biosynthesis in rice.

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One new curcuminoid, 3′-demethoxycyclocurcumin (1), was isolated from Curcuma xanthorrhiza as an antibabesial compound, together with p-hydroxybenzaldehyde (2) and cleomiscosin A (3) from Brucea javanica and (+)-epiloliolide (4) from Excoecaria cochinchinensis. The antibabesial activities were examined in vitro, and compounds 1–4, and diminazene aceturate were studied with IC₅₀ values of 16.6, 7.6, 15.6, 10.0, and 0.6 μg/ml, respectively.

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An ethyl-labeled phosphatidylcholine hydroperoxide (PC-OOH/Et 2) was synthesized as a molecular probe for naturally occurring PC-OOH 1. Applying the precursor ion scan mode in tandem ESI mass spectrometry at m⁄z 198, a signal of the PC-OOH/Et 2 alone could be selectively detected even in the presence of a large excess of a complex mixture of phospholipids in the blood. Furthermore, molecular species that formed from PC-OOH/Et 2 by its degradation in the blood were also observed in the same spectrum. Since the molecular probe-and-mass spectrometry-assisted analytical method presented herein requires no separation process by HPLC or TLC and is speedy, requiring less than 1 h, it may be useful in lipid analysis.

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The first total synthesis of 4-methylthio-3-butenyl glucosinolate (MTBG), a natural bioactive compound and a precursor of radish phototropism-regulating substances, was achieved from commercially available 1,4-butanediol. The glucosinolate framework was prepared by coupling of an oximyl chloride derivative and tetraacetyl thioglucose. A methylthio group was introduced to the framework by a Wittig reaction between triphenylphosphonium thiomethylmethylide and an aldehyde intermediate of glucosinolate. The synthetic route should facilitate preparation of various derivatives needed for probe synthesis based on MTBG.

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Cell death can proceed via at least two distinct pathways, apoptosis and oncosis. Apoptosis is an energy-dependent process characterized morphologically by cell shrinkage, whereas oncosis is defined as a prelethal pathway leading to cell death associated with cellular swelling, organelle swelling, and increased membrane permeability. In this study, we found that overexpression of chromatin modifying protein 6 (CHMP6) induced cell death by a series of experiments, including morphological observation, intracellular ATP determination, caspase-3 activity, and flow cytometry. Typical morphological characteristics consistent with oncosis were observed by transmission electron microscopy. Simultaneously, we obtained some results that indicated apoptosis, but the anti-apoptotic gene Bcl-xL and caspase family inhibitor Z-VAD-FMK had little effect on CHMP6-induced cell death. These results suggest that CHMP6 overexpression can cause cell death, predominantly via oncosis and to a certain extent via apoptosis, and that CHMP6 might be a novel regulator involved in both oncosis and apoptosis.

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Nocardioides sp. DF412 degrades dibenzofuran (DF) to salicylate through the sequential actions of DF dioxygenase (dfdA), extradiol dioxygenase (dfdB), and hydrolase (dfdC). The involvement of a TetR-type regulator gene dfdS in the dfdB and dfdS gene expression was investigated. A reporter assay using a luciferase gene indicated repression of dfdB and dfdS promoter activities in the presence of the dfdS gene product, DfdS. Gel shift analysis indicated specific binding of DfdS to the dfdB promoter region. Both the presence of a DF-degradation intermediate, 2,2′,3-trihydroxybiphenyl (2,2′,3-THBP) or its analog, 2,3-dihydroxybiphenyl (2,3-DHBP), and mutation in the inverted repeat (IR) in the dfdB promoter, canceled repression by DfdS in vivo and the binding of DfdS to the dfdB promoter fragment in vitro. These results suggest derepression of DfdS in the presence of 2,2′,3-THBP and 2,3-DHBP and the involvement of the IR in the repression by DfdS.

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Quinohemoprotein amine dehydrogenase (QH-AmDH) from Paracoccus denitrificans is an αβγ heterotrimeric enzyme catalyzing oxidative deamination of amines with large substitutes, such as butylamine and benzylamine. The smallest γ subunit has cross-linking cysteine tryptophylquinone (CTQ) as a catalytic center. A hemoprotein similar to QH-AmDH in molecular mass, subunit structure, and UV-vis spectral property, but having no enzymatic activity, was isolated. The enzymatically silent form (sQH-AmDH) was activated slowly by the substrates of QH-AmDH, and quickly by reductants, dithionite and dithiothreitol. Electrolysis of sQH-AmDH yielded the active form at potentials more negative than −0.17 V (vs. SHE). The activated protein reacted with a carbonyl reagent, 4-nitrophenylhydrazine, giving a typical spectrum of 4-nitrophenylhydrazone, while sQH-AmDH did not give such a spectrum. The γ subunit of sQH-AmDH showed a sharp peak at 390 nm in UV-vis spectrum clearly distinguishable from that of QH-AmDH. Electrospray ionization mass spectrometric analysis showed that the molecular mass of the γ subunit of sQH-AmDH was larger than that of QH-AmDH by 15.6. The data suggest that the CTQ-like moiety of sQH-AmDH is an oxime. This hypothesis was confirmed by subsequent hydroxylamine treatment of QH-AmDH. QH-AmDH was treated with hydroxylamine yielding an oxime derivative. The UV-vis spectral properties of the γ subunit of hydroxylamine-treated QH-AmDH were identical to those of sQH-AmDH. The hydroxylamine-treated QH-AmDH was also reactivated by butylamine, as in the case of sQH-AmDH.

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Cyclosporine A (CsA) is a effective and widely used immunosuppressive agent. Nevertheless, its intense nephrotoxicity restricts clinical application. In this study, wistar rats were randomly divided into a control group, a low-dose group and a high-dose group. The rats in the low-dose group and those in the high-dose group were given CsA at a daily dose of 5 mg per kg and 100 mg per kg by gavage for 7 d respectively, while the control group was given distilled water of equal volume. The extent of renal impairment was evaluated by renal function assay, and urease and pathological observation. The results showed that the rats in the low-dose group did not suffer remarkable renal impairment, while the rats in the high-dose group did. Two-dimensional gel electrophoresis (2DE) was utilized to resolve the plasma protein profile. Six spots showing stable and significantly different expression were identified by MALDI-TOF-MS and ESI-TOF-MS/MS respectively, the two induced proteins were identified respectively as α-1-acid glycoprotein (AAG) and clusterin (CLUS), and the one inhibited was haptoglobin (Hp).

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In the silkworm, Bombyx mori, antimicrobial peptide (AMP) genes are upregulated in the larval fat body by injection of bacteria and peptidoglycans (PGNs). The DAP-type PGN from Escherichia coli and Bacillus subtilis exhibited stronger elicitor activity for expression of AMP genes in B. mori than Lys-type PGN from Staphylococcus aureus, suggesting that differences in bacterial influence on the induction levels of these genes depend on the differences in types of PGN. BmRelish1 mRNA was more abundant than BmRel mRNAs in the larval fat bodies. Moreover, the ability of the BmRelish1 active form to enhance the promoter activity of AMP genes was higher than that of BmRels. The difference was related to the binding affinity of Rel family proteins to κB sites. Our results suggest that different amounts and different transcriptional activities of Rel family proteins result in differential activation of AMP genes by PGN type and bacterium species.

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To elucidate the ichthyotoxic mechanism of the harmful dinoflagellate Cochlodinium polykrikoides, a bioassay using damselfish was conducted. After exposure to a live-cell suspension of C. polykrikoides, all the fish were died within 90 min. In the presence of catalase and superoxide dismutase (SOD), no significant reduction in the toxicity of C. polykrikoides on the fish was observed. Furthermore, no significant levels of reactive oxygen species (ROS) were detected in five strains of C. polykrikoides isolated in Japan. Our results support the idea that certain toxic substances, rather than ROS, are mainly responsible for the fish-killing activity of C. polykrikoides.

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Human ABCG subfamily proteins ABCG1, ABCG2, ABCG4, ABCG5, and ABCG8 are half-type ATP-binding cassette (ABC) proteins that transport sterols or xenobiotics. ABCG1, ABCG2, and ABCG4 function as homodimers on the plasma membrane. In contrast, ABCG5 and ABCG8 function as heterodimers on the plasma membrane, and the homodimer of either ABCG5 or ABCG8 is retained in the endoplasmic reticulum (ER). To examine the molecular mechanisms of the regulated trafficking of ABCG5 and ABCG8, the subcellular localizations of chimeric proteins, fused with ABCG1 or ABCG2, were analyzed. Homodimers of chimeric proteins, in which the N-terminal cytosolic domain of ABCG1 or ABCG2 was fused to the C-terminal transmembrane domain of ABCG5 or ABCG8 localized to the plasma membrane, whereas chimeric proteins in which the N-terminal cytosolic domain of ABCG5 or ABCG8 was fused to the C-terminal transmembrane domain of ABCG1 or ABCG2 localized to the ER. Mutations in ER-retrieval motif-like sequences in ABCG5 or ABCG8 did not affect their subcellular localization. This suggests that the N-terminal cytosolic domains of ABCG5 and ABCG8 are involved in ER retention of their homodimers, and that novel ER-retention or -retrieval motifs exist within these domains.

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A complementary DNA encoding a TIA-1-type RNA-binding protein (SfTRN-1) was isolated from cultured cells of the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), to characterize its function. The deduced 388-amino acid sequence of SfTRN-1, which possessed three RNA recognition motifs (RRMs) followed by a C-terminal auxiliary domain, showed significant homology with mammalian TIA-1/TIAR and silkworm BmTRN-1, factors important in the metabolism of transcripts. It was found that inhibition of SfTRN-1 gene expression by a transfected oligonucleotide encoding the antisense sequence led to a marked increase in the production of a reporter protein and the amount of reporter transcript in the cultured cells. In addition, overexpression of the recombinant full-length SfTRN-1 open reading frame in the cultured cells led to a decrease in reporter protein production, but the truncated RRM1-3 domain lacking the C-terminal auxiliary domain lost its activity. Analysis using a GFP-fused recombinant protein revealed that, unlike mammalian TIA-1/TIAR, SfTRN-1, most likely shuttling between the nucleus and cytoplasm, had the characteristic of being largely distributed in the cytoplasm, where it perhaps acts to reduce the amount of transcripts, and that RRM1 and RRM3 were related to its nuclear accumulation, but RRM2 to its nuclear export. Furthermore, the posterior half of the auxiliary domain was also found to be related to its nuclear export. This study indicates that respective RRM subdomains of SfTRN-1 play distinct roles important to its subcellular distribution, and it identified unique systems for the distribution and functional regulation of the TIA-1 family in insect cells, ones which are clearly different from those in mammalian cells.

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Ischemic strokes are often accompanied by serious brain injury and poor prognosis, but the molecular mechanisms of primary and secondary injury have not been fully understood. The aim of the present study was to investigate the protein profile in the rat pituitary, adrenal gland, and splenic lymphocyte using proteomics techniques, and to elucidate potential changes in the immune neuroendocrine system following cerebral ischemia injury in rats. Out of the 41 differentially expressed protein spots identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-MS-TOF), 13 proteins were closely related to the immune and/or the neuroendocrine system, and the other proteins might have different functions through other mechanisms in middle cerebral artery occlusion (MCAO) rats. The results showed that (i) the immune neuroendocrine system was obviously changed, and the changes might be important pathological mechanisms in brain injury after cerebral ischemia, and (ii) ischemic brain damage is co-regulated by several mechanisms. The results might lay the foundations for further research on pathological mechanisms in cerebral ischemia.

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The full-length cDNA encoding a coronatine insensitive-1 (COI1) protein, designated HbCOI1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCOI1 contained a 2,187 bp open reading frame encoding 597 amino acids. The deduced HbCOI1 protein, which showed high identity to COI1 protein of other plant species, was predicted to possess F-box and LRRs domains. The promoter region of HbCOI1 was isolated by the PCR-based DNA walking method. TATA box and other core configurations were found in the promoter. Several sequences similar to the eukaryotic cis regulatory element were found in the 5′-untranslated region (UTR) proximal 5′ flanking sequence of HbCOI1. Southern blot analysis indicated that the HbCOI1 is present as a single copy in Hevea brasiliensis. Transcription pattern analysis revealed that HbCOI1 had high transcription in laticifer, low in barks and leaf. Transcription of HbCOI1 in latex was induced by jasmonate and tapping.

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Four enantiomeric 9-mer peptides, D-peptides A (RLYLRIGRR-NH₂), B (RLRLRIGRR-NH₂), C (ALYLAIRRR-NH₂), and D (RLLLRIGRR-NH₂), were designed and synthesized on the basis of a beetle defensin antimicrobial peptide. These D-9-mer peptides have been reported to exhibit multiple functions, including antimicrobial and antiprotozoan activity, without cytotoxicity on normal fibroblasts and leukocyte cells. In this study, we found that the D-9-mer peptides inhibited telomerase activity (IC₁₀₀ = 40 μM). A new peptide, D-peptide C2 (ALYLAIRRRRRRRR-NH₂), designed from D-peptide C to translocate into the cytoplasm by a penetrating sequence (octa-arginine), showed extremely strong telomerase inhibitory activity (IC₁₀₀ = 0.1 μM). D-Peptide C2 exhibited a great increase in cytotoxicity against various cancer cell lines (IC₅₀ = 3.4–26.4 μM). However, the immediate death of the cells suggested that the high cytotoxicity was not an effect of telomerase inhibitory activity. Mitochondrial swelling assay and microscopical observations of mitochondria indicated that the major target of the D-peptide C2 was the mitochondrial membrane.

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Glucagon-like peptide-1 (GLP-1) has considerable potential as a possible therapeutic agent for type-2 diabetes. Unfortunately, this glucoincretin is short lived due to degradation by dipeptidyl-peptidase IV and rapid clearance by renal filtration. In this study, we attempted to extend GLP-1 action through the attachment of a lysine residue at the N-terminal of GLP-1 (named KGLP-1), and to make a fusion protein with human serum albumin (HSA) in Pichia pastoris. The protein, designated KGLP-1/HSA, was purified by an immunomagnetic separation technique. High performance liquid chromatography (HPLC) showed that the purified protein had an overall purity of 92.0%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the expected molecular mass of 70,297.8 Da. Additionally, the N-terminal sequence of KGLP-1/HSA was confirmed by N-terminal sequencing. The stability and biological activity of KGLP-1/HSA were then evaluated in vitro and in vivo. The findings indicated that fusion KGLP-1/HSA preserved the action of native GLP-1, and the active duration was greatly prolonged.

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A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgaris and designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRC increased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitro pull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella.

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A lectin, PPL, was purified from the mushroom Pleurocybella porrigens. The results of SDS–PAGE, gel filtration, and MALDI-TOF-mass of PPL indicated that its molecular mass was 56 kDa, and it was composed of four 14 kDa subunits with no disulfide bonds. In hemagglutination inhibition assay, PPL exhibited the strongest binding specificity toward GalNAc among the mono- and oligo-saccharides tested. Among the glycoproteins, asialo-bovine submaxillary mucin (asialo-BSM) showed the strongest inhibitory effect. In surface plasmon resonance analysis, asialo-BSM, porcine stomach mucin (PSM), and BSM exhibited potent binding affinity. The complete amino acid sequence was determined by amino acid sequencing of intact and of enzyme-digested PPL. The cDNA of PPL was cloned from RNA extracted from the mushroom. The open reading frame of the cDNA of the protein consisted of 411 bp, encoding 137 amino acids. This is the first report of isolation of a lectin of the genus Pleurocybella.

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Coptotermes formosanus is one of the most destructive termites in the southern part of Japan as well as in the United States. Hemicellulose is a noncellulosic polysaccharide found in plant cell walls, and xylan is the major constituent of hemicellulose. Since hemicellulose prevents access of cellulolytic enzymes to cellulose, enzymatic hydrolysis of hemicellulose is beneficial for cellulose digestion. We purified three functional xylanases to homogeneity from C. formosanus for the first time. Elution profiles from the whole termite extract suggest that these three xylanases play major roles in xylan digestion in the gut of the termites. The corresponding cDNAs were successfully cloned based on the N-terminal amino acid sequences, encoding GHF11 xylanases. Reverse transcription-PCR using manipulated protozoan cells in the hindgut revealed that the corresponding genes were expressed in the symbiotic flagellate Holomastigotoides mirabile. These results suggest that the GHF11 xylanases that are produced by the symbiotic flagellates play a primary role in xylan degradation in C. formosanus.

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Culture medium used in antibody purification contains growth factors, but is usually discarded after antibody purification. We examined to determine whether the growth factors can be (i) recovered and (ii) used in subsequent cell culture. Using this medium enhanced the survival of hybridoma cells and improved antibody production. Culture medium previously used in antibody purification offers an attractive resource for mammalian cell culture.

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Replacement of glycine by serine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius and Bacillus sphaericus respectively strikingly decreased enzyme activity toward aromatic amino acids and resulted in an elevation of relative activity toward aliphatic amino acids. The mutant from B. badius preferentially dehydrogenated branched-chain amino acids, while that from B. sphaericus acted on amino acids with straight-chain amino acids.

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In cyanobacteria, nutrient deficiency-induced phycobilisome degradation is controlled by the NblA gene. Red algae also have an NblA-related gene, Ycf18, in their chloroplast genomes. To elucidate the role of Ycf18, the expression pattern of Ycf18 in a red alga, Porphyra yezoensis, was investigated. Ycf18 expression was low in nitrate medium, but was greatly promoted in ammonium medium. Nitrogen starvation caused bleaching, but did not affect the expression of Ycf18. The responses of Ycf18 to nitrogen-starvation and the supply of ammonium were distinct from those of NblA, suggesting that Ycf18 has a role other than the regulation of phycobilisome degradation.

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Coronin cDNA was cloned from the plasmodia of Physarum polycephalum. The amino acid sequence deduced from the cDNA was comprised of 449 residues and showed 60% identity to that of Dictyostelium discoideum coronin. Southern blot analysis suggested that the coronin gene present in the P. polycephalum genome might be a single copy. Coronin was expressed in diploid plasmodia, while it was not detected in haploid amoebae or spores.

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A Saccharomyces cerevisiae strain deficient in phosphatidylethanolamine methyltransferase that exhibits choline auxotrophy grew in the presence of dioctanoyl phosphatidylcholine (diC8PC). We isolated and characterized mutants defective in growth in the diC8PC-containing medium. Mutations in LEM3/ROS3 impaired growth in that medium, indicating that Lem3p is involved in the utilization of extracellular phosphatidylcholine (PC) with short acyl chains.

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A novel purification system for inactive precursor proteins without conventional time-consuming purification steps was established. We purified an inactive precursor protein, procarboxypeptidase Y (proCPY), which was displayed on the cell surface of yeast, due to difficulty of purifying it by a system in which it is produced in cells.

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Prenylated flavonoids are natural products that exhibit diverse biological effects and often represent the active components of various medicinal plants. This study demonstrated the production of prenylated naringenin by biotransformation using transgenic yeast expressing naringenin 8-dimethylallyltransferase, a membrane-bound enzyme, without feeding of prenyl donors. This method provides the possibility of generating prenylated flavonoids that occur rarely in nature.

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We describe our findings on HLH54F, a basic helix-loop-helix transcription factor gene that was highly expressed in the prothoracic gland, an organ producing the insect steroid ecdysone. HLH54F was uncovered by the use of an expressed sequence tag database of the silkworm Bombyx mori. It was also highly expressed in the prothoracic gland of the fruit fly Drosophila melanogaster.

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Branched-chain α-ketoacid dehydrogenase complex (BCKDC) is a rate-limiting enzyme in the branched-chain amino acid catabolic pathway. We have developed a method of BCKDC purification from rat liver using hydrophobic interaction column chromatography (Shimomura et al., Arch. Biochem. Biophys., 283, 293–299 (1990)). Here we report a modification of the method designed to obtain the purified enzyme with high reproducibility.

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SK₆₆-his, a novel glycine-rich peptide derived from the CG13551 gene of Drosophila, was directly expressed in Escherichia coli with the help of the glucose effect of the lac repressor and efficiently purified in high yield (10.063 mg/l). It showed significant activity against Gram-positive bacteria. We determined a MIC value of 9 μg ml⁻¹ with Bacillus thuringiensis.

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We have found that fermented barley extract (FBE), prepared from barley shochu residue, alleviates allergic rhinitis in OVA-sensitized mice. In this study, we examined to determine whether FBE suppresses the development of atopic dermatitis (AD)-like skin lesions in NC/Nga mice. The development of AD-like skin lesions in a 5% FBE containing diet group was significantly inhibited, and scratching behavior, one of aggravating factors, was also suppressed. Neither serum immunoglobulin E (IgE) levels nor interleukin (IL)-4 production by spleen cells in the 5% FBE diet group was found to be significantly reduced. On the other hand, interferon-gamma (IFN-γ) and IL-17 production by spleen cells in the 5% FBE diet group was significantly reduced. Hence it was assumed that FBE alleviates AD-like skin lesions in NC/Nga mice, probably by modulating the cytokine production involved in chronic inflammation, such as IFN-γ or IL-17.

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Fermented soybean paste (doenjang, FSP) is a traditionally fermented Korean food produced by fermentation with various microorganisms that is known to exhibit various beneficial bioactivities. To investigate the changes in nonvolatile metabolites of FSP during fermentation, samples produced with six fermentation times were analyzed using an ¹H nuclear magnetic resonance spectroscopy (NMR)-based metabolomics technique. This revealed clear separation of 50% methanol extracts of the FSP samples with different fermentation times in the principal component plots by combining PC1 and PC2, which cumulatively accounted for 94.2% of the variance. Major compounds contributing to the separation of 50% methanol extracts of FSP with various fermentation times were isoleucine/leucine, lactate, alanine, acetic acid, glutamine, choline, tyrosine, and phenylalanine. In addition, the ¹H NMR spectra of chloroform extracts were separated mainly by a combination of PC1 and PC3, which accounted for 72.6% of the variance. The present study suggests the usefulness of a ¹H NMR-based metabolomics approach to discriminate FSP samples subjected to different fermentation times, and this is the first report regarding metabolomic profiling of FSP.

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The bioavailability of polyphenols in human and rodents has been discussed regarding their biological activity. We found different metabolite profiles of quercetin in rat plasma between two administration procedures. A single intragastric administration (50 mg/kg) resulted in the appearance of a variety of metabolites in the plasma, whereas only a major fraction was detected by free access (1% quercetin). The methylated/non-methylated metabolites ratio was much higher in the free access group. Mass spectrometric analyses showed that the fraction from free access contained highly conjugated quercetin metabolites such as sulfo-glucuronides of quercetin and methylquercetin. The metabolite profile of human plasma after an intake of onion was similar to that with intragastric administration in rats. In vitro oxidation of human low-density lipoprotein showed that methylation of the catechol moiety of quercetin significantly attenuated the antioxidative activity. These results might provide information about the bioavailability of quercetin when conducting animal experiments.

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In vitro, the inhibition by ε-polylysine depends on how the substrate is presented to the lipase. We therefore examined whether ε-polylysine can interact with the lipid emulsion and prevent lipase activity in digestive organs. To confirm lipase inhibition by ε-polylysine, a ¹⁴C-trioleoylglycerol emulsion with or without ε-polylysine was orally administered to rats, and the radioactive lipid distribution determined at regular intervals. The radioactive plasma lipid was decreased, and radioactive fecal lipid was increased by the administration of ε-polylysine. The peak of radioactive lipids in the intestine was delayed by the administration of ε-polylysine.
We used 20-week-old rats as a model for the middle-aged and elderly to test the effect of ε-polylysine on the body weight increase. ε-Polylysine significantly prevented any elevation in body weight and weight of the liver and epididymal adipose tissues. These data show that ε-polylysine inhibited the lipase activity in the digestive organ and had an anti-obesity function in the middle-aged rats.

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Salicornia herbacea (SH) is a halophyte that grows in the salt marshes along the coastline of South Korea, and is known to have antioxidative activity. In this study, the antioxidative and skin-whitening effects of SH aqueous extract were investigated in human dermal fibroblasts (HDFs) and B16 melanoma cells. The water extract of SH had potent antioxidative capacity and protected HDFs from tert-butyl hydroperoxide (tbOOH)-induced oxidative stress in a dose-dependent manner. In a cell cycle analysis, pretreatment with SH reversed the apoptotic cell death induced by tbOOH in HDFs. Additionally, the incubation of SH in mushroom tyrosinase inhibited the oxidation of l-dopa to o-dopaquinone, which implies that SH is a potent tyrosinase inhibitor. An SH treatment to B16 melanoma cells decreased the synthesis of melanin and inhibited tyrosinase activity. These results collectively indicate that SH had antioxidative and whitening effects on skin and would be a good candidate for skin rejuvenating agent.

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We have reported that orally administrated acetate contributed to suppression of lipogenesis in the liver and to reduction of lipid accumulation in the adipose tissue of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The aim of this study was to investigate the effect of acetate on skeletal muscle and adipose tissues. Treatment with acetate showed a higher rate of oxygen consumption and a smaller size of lipid droplets in white adipose and brown adipose tissues. An analysis by Northern blotting revealed that the transcripts of myoglobin and Glut4 genes in the abdominal muscle of the OLETF rats were increased by acetate treatment, while the transcripts of lipolytic genes increased in the white adipose and brown adipose tissues. It is possible that acetate has effects on lipid metabolism in the skeletal muscles and the adipose tissues, and has functions that work against obesity and obesity-linked type 2 diabetes.

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We investigated effects of the non-methylene-interrupted polyunsaturated fatty acid, sciadonic acid (all-cis-5,11,14-eicosatrienoic acid), on the lipid metabolism in rats, to identify the mechanism for the plasma and hepatic triacylglycerol-lowering effects of Japanese torreya (Torreya nucifera) seed oil. Sciadonic acid was isolated from torreya seed oil by the combination of urea-adduct with lipase-esterification. Sprague-Dawley (SD) male rats were fed with experimental diets containing 5% and 10% sciadonic acid based on corn oil for 2 weeks. The serum and liver triacylglycerol levels were lower in the rats fed with sciadonic acid. Considerable amounts of sciadonic acid were detected in the triacylglycerol and phospholipid in both the serum and liver of the rats fed with sciadonic acid. These observations demonstrate that sciadonic acid could modify the lipid metabolism in rats.

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Lactosucrose (LS, 4^G-β-D-galactosylsucrose) is a non-digestible oligosaccharide, and the consumption of LS selectively increases the proportion of intestinal bifidobacteria. We examined in this study the hypolipidemic potential of LS. An oral triolein tolerance test on rats indicated that LS reduced the elevation of serum triglyceride (TG) and free fatty acids (FFA). Furthermore, LS inhibited the enzymatic digestion of triolein by pancreatic lipase in vitro. NMR spectroscopy showed that LS formed an intermolecular complex with triolein. The long-term consumption of a diet containing 5% LS for 8 weeks significantly decreased the weight of abdominal adipose tissue when compared with that of the control group. Thus, LS may reduce adipose tissue accumulation by inhibiting intestinal lipid absorption via a direct interaction with TG.

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The mechanism for the hypocholesterolemic action of a cattle heart protein hydrolysate (HPH) is clarified. The micellar solubility of cholesterol in vitro was significantly lower in the presence of HPH than in the presence of casein. The suppression of cholesterol uptake by Caco-2 cells was significantly higher in the cholesterol micelles containing HPH than in the cholesterol micelles containing casein. The serum cholesterol concentrations and atherogenic index were significantly lower in the rats fed with HPH than in those fed with casein. The cholesterol absorption measured by [³H]-cholesterol was significantly lower by HPH feeding than by casein feeding in rats in vivo accompanying the changes in fecal steroid excretion. Thus, the hypocholesterolemic action of HPH involved the inhibition of jejunal cholesterol absorption. The cattle heart protein hydrolysate ultra-filtrate (HPHU, MW < ca. 1,000 Da peptide fraction) derived from HPH imparted stronger hypocholesterolemic activity than HPH in rats.

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Vietnamese coriander (Persicaria odorata Lour.) belongs to a group known as cilantro mimics with the ‘cilantro’ flavor, in which C10 and C12 aldehydes and alcohols have been found as the potent odor compounds. Their composition isolated by different extraction methods varied. The enzyme activity was assayed, and the reductase acting on some aliphatic aldehydes with NADH/NADPH as a coenzyme was found in a crude enzymatic system of fresh leaves. The maximum activity was observed at pH 8.0 in Na-phosphate and at pH 8.5 to 9.0 in a glycine-NaOH buffer, using heptanal as a substrate. The activated reductase that caused the alcohol generation to increase after a time was inhibited by p-hydroxymercuribenzoate. Our results, which are the first to be reported on Vietnamese coriander leaves, reveal the presence of aliphatic aldehyde dehydrogenase, which is responsible for acid formation, and elucidate the strong activity of the aliphatic aldehyde reductase.

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The acceptor specificity and transfer potential of a β-D-glycosidase (G I), which had been purified from the China white jade snail, were further investigated by using various sugars as acceptors. G I had broad monosaccharide acceptor specificity for its transglycosylation activity. More specifically, it efficiently catalyzed the transfer of the β-D-fucosyl, β-D-glucosyl or β-D-galactosyl moiety from the corresponding p-nitrophenyl-β-D-glycopyranosides to various monosaccharides. The transfucosylation efficiency of G I was studied by using p-nitrophenyl-β-D-fucopyranoside (pNPFuc) as the donor and glucose and xylose as the acceptors. The yields under conditions of non-initial velocity were 88% for glucose and 93% for xylose. The transfer product with glucose as the acceptor was isolated and identified as β-fucosyl-1,6-glucose by an NMR analysis. The data from these analyses indicate that G I had broad acceptor specificity and high efficiency for transglycosylation. These uncommon properties of G I could make it a valuable biocatalyst for the synthesis of various disaccharides.

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