Filamentous fungi usually inhabit normoxic environments by utilizing oxygen as a substrate for respiration and for the biosynthesis of some essential cellular components. This review examines the metabolic mechanisms used by filamentous fungi under oxygen-limited (hypoxic) conditions. Denitrification is one mechanism through which Fusarium oxysporum and other fungi reduce nitrate or nitrite to nitrous oxide, generating nitric oxide as a reaction intermediate. The involvement of cytochrome P450nor as a nitric oxide reductase is a unique feature of fungal denitrification, as opposed to cytochrome bc-type nitric oxide reductase, which is unique to the bacterial mechanism. Ammonia fermentation is the mechanism through which nitrate is reduced to ammonium, and it allows fungal growth under hypoxic conditions. Studies of the model filamentous fungus Aspergillus nidulans have revealed that niaD and niiA encoding NAD(P)H-dependent nitrate and nitrite reductases are essential for producing ammonia. Since niaD and niiA have been identified as genes for nitrate utilization by the fungus, ammonia fermentation and nitrate utilization mechanisms probably share a nitrate-reducing mechanism. I also discuss recent progress in studies of the hypoxic response of A. nidulans.

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The formulation of new biotherapeutics without human serum albumin (HSA) could decrease the potential risk of blood-transmitted diseases and those caused by infectious viruses and other pathogens. In the present study, arginine was examined as a potential alternative to HAS, and bovine lactoferrin (bLf) was used as a representative model glycoprotein since bLf has potential immunomodulatory and antiviral activity. The optimal formulation for the mixture was determined to be 10 mM arginine, 15% (w/v) trehalose, and 0.02% (v/v) Tween 80, using a statistical analysis program, Minitab. Analyses were performed using reverse-phase high-performance liquid chromatography (HPLC) and SDS–PAGE. The blf HSA-free formulations lost only 12–20% of blf compared with 46% for control (without additives) after 28 d of storage. Based on long-term stability studies, the HSA-free formulation developed in this study had a stronger effect on the stability of bLf (1.4-fold) than HSA formulation under various storage conditions over 6 months.

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Archaea have unique membrane lipids typified by ether linkages of the glycerol-to-isoprenoid chains with sn-2,3 stereochemistry that runs against the naturally occurring sn-1,2 stereochemistry of the glycerophospholipids of Bacteria and Eukarya. Membrane lipids were extracted and analyzed from the hyperthermophilic archaeon, Thermococcus kodakaraensis, cultivated at various temperatures. At all growth temperatures examined, both the diphytanylglycerol diether (archaeol, C₂₀) and diphytanyldiglycerol tetraether (caldarchaeol, C₄₀) were identified as saturated forms, and no other lipids could be identified. The ratio of caldarchaeol to archaeol increased with increasing growth temperature, particularly at 93 °C. A larger amount of archaeol was detected from cells in the logarithmic phase than from those in the stationary phase at all temperatures examined. These results indicate that T. kodakaraensis modulated the membrane lipid composition depending on both the growth phase and the growth temperature, and suggest that the membrane fluidity to environmental change was maintained by altering the length of the hydrocarbon chains, and not by side-chain saturation such as double-bond hydrogenation nor by such a modification as cyclopentane ring formation.

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PISP1, a heteropolysaccharide isolated from fruiting bodies of Phellinus igniarius by hot aqueous extraction and purified by DEAE-Sepharose anion-exchange and gel filtration chromatography, is composed of fucose, galactose, mannose, and 3-O-Me-galactose in a ratio of 1:2:1:2. Methylation, monosaccharide analysis, and NMR studies (¹H NMR, ¹³C NMR, COSY-45°, TOCSY, ROESY, HSQC, and HMBC) revealed that PISP1 had a backbone consisting of 1,6-disubstituted-3-O-Me-α-D-galactopyranosyl residue, 1,3,6-trisubstituted-α-D-manopyranosyl residue, 1,4-disubstituted-α-D-galactopyranosyl residue, and 1,2-disubstituted-α-D-galactopyranosyl residue, and had a 1-substituted-α-L-fucopyranosyl terminal attached to O-3 of a manopyranosyl residue. Preliminary bioactivity tests conducted in vitro revealed that PISP1 stimulated the proliferation of mouse spleen lymphocytes.

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The antimicrobial activity of all stereoisomers of morinols A and B was tested. All stereoisomers of morinols A and B showed antifungal activity against Alternaria alternata, especially (−)-morinol B which showed the strongest activity. The natural component, (+)-morinol A, and unnatural stereoisomer, (7S,7′S,8R,8′R)-morinol B, showed antibacterial activity against the gram-positive bacteria, Bacillus subtilis and Listeria denitrificans.

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Elucidation of enzymatic polyether formation is a long-standing controversial issue in organic chemistry. To address this intriguing issue, identifying the actual substrate for epoxidation and sequential cyclization is essential. We selected the representative polyether ionophore, lasalocid, which has been proposed to undergo no modification at the late stage of biosynthesis. Cloning and a sequence analysis revealed seven polyketide synthase (PKS) genes, epoxidase and epoxide hydrolase genes for sequential ether formation, and several putative genes for supplying ethylmalonyl-CoA. Based on bioinformatic data, we propose the lasalocid biosynthetic pathway which involves characteristic aromatic ring formation and sequential cyclic ether formation. The finding of a thioesterase domain at the C-terminal of the seventh PKS indicates that intriguing oxidative cascade cyclization would occur after cleavage of the polyketide intermediate from PKS. Based on this observation, we have recently reported the enzymatic transformation of a bisepoxide intermediate to lasalocid with the recombinant epoxide hydrolase, Lsd19.

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Optically active sydonic acid (1) was isolated for the first time from a culture broth of Glonium sp. The absolute stereochemistry was established to be (S) by comparing the circular dichroism (CD) spectrum with that of (+)-curcutetraol after conversion into (+)-sydonol (2).

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Novel water-soluble conjugates of 1,2,4,5-tetraoxane bis(quaternary ammonium salts) were synthesized in a relatively stable crystalline form via four steps starting from methyltrioxorhenium-catalyzed endo-peroxidation of ethyl 4-oxocyclohexanecarboxylate with hydrogen peroxide in hexafluoro-2-propanol. The assay for the in vitro toxicity of water-soluble tetraoxanes 5a–5d to malaria parasites indicate that they were inactive against the Plasmodium falciparum FCR-3 strain.

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A new sesquiterpenoid (1) was isolated from the mushroom, Sparassis crispa, along with a known sesquiterpenoid (2). The structure of 1 was determined as (3R^*, 3aS^*, 4S^*, 8aR^*)-3-(1′-hydroxy-1′-methylethyl)-5,8a-dimethyldecahydroazulen-4-ol by the combination of NMR and ESI-MS analyses.

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Two new N-hydroxypyrroles, surugapyrroles A and B, were isolated as DPPH radical-scavengers from a culture broth of Streptomyces sp. USF-6280 strain. The structures of the two compounds were elucidated by spectroscopic evidences and derivatization. Their DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity was as high as that of butylhydroxytoluene (BHT): surugapyrrole A, ED₅₀ 50.3 μM; surugapyrrole B, ED₅₀ 75.8 μM.

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Enzymatic syntheses of pyridoxine glycosides were carried out in di-isopropyl ether organic medium using β-glucosidase isolated from sweet almond. Optimum conditions determined for the reaction with D-glucose were 40% (w/w D-glucose) β-glucosidase at 0.18 mM (1.8 ml) of pH 5 acetate buffer over a 72 h incubation period. Of 11 carbohydrates employed, β-glucosidase gave 7-O-(α-D-glucopyranosyl)pyridoxine 5a, 7-O-(β-D-glucopyranosyl)pyridoxine 5b, 6-O-(α-D-glucopyranosyl)pyridoxine 5c, 7-O-(α-D-galactopyranosyl)pyridoxine 6a, 7-O-(β-D-galactopyranosyl)pyridoxine 6b, 6-O-(α-D-galactopyranosyl)pyridoxine 6c, 7-O-(α-D-mannopyranosyl)pyridoxine 7a, 7-O-(β-D-mannopyranosyl)pyridoxine 7b, and 6-O-(α-D-mannopyranosyl)pyridoxine 7c in yields ranging from 23 to 40%.

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A major collagenase was purified about 96-fold from a crude enzyme sample of Streptomyces parvulus by chromatography on Q-Sepharose, Sephacryl S-200, and butyl-Toyopearl. The purified enzyme showed a relative molecular mass of approximately 52,000 on SDS–PAGE and a pH optimum at about 9.0, and was strongly inhibited by metal-chelating agents. It also cleaved 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg specifically at the Leu-Gly bond, with a K_m value of 0.60 mM at pH 9.0 at 37 °C. Based on the amino acid sequences of the N-terminal region and internal tryptic peptides, the corresponding gene was cloned. The DNA sequence of the cloned gene indicated that the enzyme is produced as an 864-residue precursor protein with a 408-residue prepro sequence followed by a 456-residue mature enzyme moiety. The enzyme is most homologous with the collagenase from S. coelicolor, the identity being 73%, and it is thought to be a member of the Vibrio collagenase subfamily.

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In order to gain insight into the gene expression profiles associated with anterior regeneration of the earthworm, Perionyx excavatus, we analyzed 1,159 expressed sequence tags (ESTs) derived from cDNA library early anterior regenerated tissue. Among the 1,159 ESTs analyzed, 622 (53.7%) ESTs showed significant similarity to known genes and represented 338 genes, of which 233 ESTs were singletons and 105 ESTs manifested as two or more ESTs. While 663 ESTs (57.2%) were sequenced only once, 308 ESTs (26.6%) appeared 2 to 5 times, and 188 ESTs (16.2%) were sequenced more than 5 times. A total of 803 genes were categorized into 15 groups according to their biological functions. Among 1,159 ESTs sequenced, we found several gene encoding signaling molecules, such as Notch and Distal-less. The ESTs used in this study should provide a resource for future research in earthworm regeneration.

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Disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae and Aspergillus nidulans led to remarkable morphological defects, and these phenotypes were suppressed under hyperosmotic conditions. In this study, we investigated to determine whether non-KexB proteases might complement the in vivo function of KexB in the two Aspergillus kexB disruptants. Neither overexpression of opsA or opsB encoding A. oryzae aspartyl proteases homologous to yeast yapsins (YPS1/2) suppressed the kexB mutation, although yapsins are multicopy suppressors for the yeast kex2 mutation. A. nidulans and A. oryzae kexB disruptants grown under hyperosmotic conditions processed a recombinant fusion protein carrying a synthetic dibasic processing site (Lys-Arg) although the disruptants grown under normal growth conditions did not cleave the site. These results suggest that the two Aspergilli have other potential processing proteases that are induced and/or activated under hyperosmotic conditions and consequently complement, at least in part, the in vivo function of KexB.

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Typical early pathogen-associated molecular pattern (PAMP) responses include the generation of reactive oxygen species (ROS) and MAP kinase (MAPK) activation, but little is known about the molecular mechanisms that link receptor activation to intracellular signal transduction. In this study, we found that in agb1-2 (AGB1 null mutation) mutants, ROS production triggered by flg22 or elf18 was significantly reduced and that elf18-stimulated PAMP-triggered immunity (PTI) against Agrobacterium tumefaciens was impaired. Thus AGB1 appears to integrate PAMP perception into downstream ROS production, and also to transmit the EF-Tu signal to the defense response, leading to reduced transformation by A. tumefaciens.

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Plant seed cells amass storage proteins that are synthesized on the endoplasmic reticulumn (ER) and then transported to protein storage vacuoles (PSVs). Many dicotyledonous seeds contain 11S globulin (11S) as a major storage protein. We investigated the accumulation behaviors of pea and pumpkin 11S during seed maturation and compared them with soybean 11S biogenesis (Mori et al., 2004). The accumulation of pea 11S in seeds was very similar to that of soybean 11S at all the development stages we examined, whereas pumpkin 11S condensed in the ER. The determinant of accumulation behavior might be the surface hydrophobicity of 11S. Further, we examined the accumulation of 11Ss in tobacco BY-2 cells to analyze behavior in the same environment. 11Ss expressed in BY2 cells were all observed in precursor form (pro11S). Pro11S with high surface hydrophobicity might be transported to vacuoles in a multivesicular body-mediated pathway when the expression level remains low.

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We searched the genome database of the basidiomycete Coprinopsis cinerea (Coprinus cinereus) and found five genes encoding the glycoside hydrolase family 6 (GH6) enzyme, CcCel6A, CcCel6B, CcCel6C, CcCel6D, and CcCel6E, designated in order of increasing locus number (CC1G_01107.1, CC1G_04166.1, CC1G_08276.1, CC1G_08277.1, and CC1G_10605.1). The amino acid sequence of CcCel6A suggests a two-domain structure consisting of an N-terminal family 1 carbohydrate-binding module (CBM1) and a GH6 catalytic domain, while the other genes lack CBM1. The transcripts of CcCel6A were observed at the active growth stage in cellulose culture, whereas they were absent from glucose culture. Cellobiose strongly induced transcription of CcCel6A. On the other hand, transcripts of CcCel6B, -D, and -E were detected in both glucose and cellulose cultures, and transcription of them was induced weakly by cellobiose. The transcript level of CcCel6C was not influenced by glucose or cellobiose.

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We isolated two major zwitterionic glycosphingolipids (ZGLs) from the phytopathogenic filamentous fungus Trichoderma viride. Structural analyses showed that the ZGLs (designated Tv-ZGL2 and Tv-ZGL3) were the same as the glycosphingolipids ZGL2 and ZGL4 from Acremonium sp., which are described in our previous paper. ZGLs have the following structure: Man(α1-6)GlcN(α1-2)Ins-P-Cer (Tv-ZGL2) and phosphocholine (PC) → 6Man(α1-6)GlcN(α1-2)Ins-P-Cer (Tv-ZGL3). To determine whether these ZGLs have functional roles in plant-fungus interaction, we tested to determine whether they would induce defense responses in cultured rice cells. We found that T. viride’s ZGLs elicited expression of the PAL and PBZ1 genes, both of which are associated with pathogen resistance. Tv-ZGL2 induced cell death at a moderate rate. Tv-ZGL3, which contains a PC moiety, induced a high level of cell death in rice cells.

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Nicotinic acid and nicotinamide belong to the water-soluble vitamins, and they have many physiological and pharmacological functions in various organisms. In this study, we investigated the differentiation-inducing ability of nicotinic acid-related compounds in chronic myelogenous leukemia K562 cell line. Proliferation of K562 leukemia cells was inhibited by several nicotinic acid-related compounds. Hemoglobin content was increased by nicotinic acid and by isonicotinic acid. Isonicotinic acid increased γ-globin mRNA expression as much as sodium butyrate did. The nuclei of nicotinic acid and of isonicotinic acid-treated cells decreased in size and the chromatin became more condensed. It was verified that nicotinic acid and isonicotinic acid induced erythroid differentiation in K562 cells. Expression of glycophorin A was increased by sodium butyrate. In contrast, it was decreased by nicotinic acid and by isonicotinic acid, suggesting that these compounds differentiate K562 to erythrocytes through different pathways than sodium butyrate does. Our data perhaps provide useful information as to the mechanisms of cell differentiation.

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Chitin is an insoluble component in the shells of several molluscan species. It is thought to play important roles, in biomineralization and shell structure. To date, however, reports are scarce and sometimes contradictory, and suffer from methodological problems. Only in a single cephalopod species has the chitin been identified as β-chitin. We present data on chitin occurrence in 22 species of shell-bearing Mollusca (Conchifera) and Polyplacophora, including the first evidence for scaphopods, based on pyrolysis gas chromatography, mass spectrometry (GC-MS), and infrared spectroscopy (IR). Pyrolysis GC-MS detected chitin in every tested member of the Conchifera. IR spectroscopy before and after chitinase treatment revealed at least three distinct patterns of peak changes. The contents of the insoluble shell organics included not only chitin and proteins, but also insoluble polysaccharides, e.g., glucan. We conclude that chitin was present in the last common ancestor of the Conchifera and that its abundance in the shell matrix depends on the differentiation of the shell.

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The β-1,4-mannanase 5C gene (man5C) of Vibrio sp. strain MA-138 was cloned and expressed in Escherichia coli. The man5C gene consisted of 2,010 bp nucleotides encoding a protein of 669 amino acids with a predicted molecular weight of 76,309. β-1,4-Mannanase (Man5C) is a modular enzyme composed of a catalytic module belonging to glycoside hydrolase family 5, a linker region, and a putative carbohydrate-binding module (CBM) belonging to family 27. Recombinant Man5C exhibited maximal activity at 50 °C at pH 7.0, and it had a K_m of 0.6 mg ml⁻¹ and a V_max of 556.2 μmol min⁻¹ μmol⁻¹ for glucomannan. Binding studies revealed that the C-terminal putative CBM27 had the ability to bind soluble β-mannans and contributed to increasing the rate of depolymerization by binding to the polymeric substrate. Man5C of Vibrio sp. MA-138 is the first non-extremophile enzyme to be identified as a β-mannanase possessing CBM27.

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We designed and constructed six major toxin-antitoxin disruptants (ΔchpBIK, ΔdinJ-yafQ, ΔhipBA, ΔmazEF, ΔrelBE, and ΔyefM-yoeB) of Escherichia coli K-12 W3110. On prolonged cultivation of these disruptants with minimal M9 medium, the ΔhipBA cells exhibited a significantly longer life span than that of the other disruptants and of wild-type cells, as analyzed with a LIVE/DEAD BacLight kit (Invitrogen, Carlsbad, CA) in combination with flow cytometry analysis. The gene expression level of hipA in the wild-type cells was highest at the stationary phase of 40 h. The ΔhipBA cells showed higher macromolecular synthesis activity than the wild-type cells at the stationary phase. Stationary phase cells of ΔhipBA and the wild-type strain showed a significantly extended life span under anaerobic conditions. Furthermore, the ΔhipBA cells showed higher resistance to H₂O₂ than the wild type. These results suggest that HipBA induces cell death with oxidative stress during prolonged cultivation. This is the first report that an E. coli toxin-antitoxin (TA) system affects frequency of survival during the long-term stationary phase.

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In this study, we purified and characterized an α-mannosidase to homogeneity from mature red tomato fruits. Purified α-mannosidase (α-Man LE-1) gave two separate bands, of molecular masses of 70 kDa (L-subunit) and 47 kDa (S-subunit), on SDS–PAGE under non-reducing and reducing conditions. On the other hand, the molecular weight was estimated to be 230 kDa by gel filtration, indicating that α-Man LE-1 functions in a tetrameric structure in plant cells. The N-terminal sequence of the L-subunit and the S-subunit were determined to be L-Y-M-V-Y-M-T-K-Q-G- and X-X-L-E-Q/K-S-F-S-Y-Y respectively. When pyridylaminated N-glycans were used as substrates, α-Man LE-1 showed optimum activity at about pH 6 and at 40 °C, and the activity was completely inhibited by both swainsonine and 1-deoxy-mannojirimycin. α-Man LE-1 hydrolyzed the α-mannosidic linkages from both high-mannose type and plant complex type N-glycan, but preferred a truncated plant complex type structure to high-mannose type N-glycans bearing α1-2 mannosyl residues.

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KIBRA is a WW domain-containing protein that can bind to protein kinase Cζ (PKCζ). The SNP of the ninth intron of the KIBRA gene is associated with human episodic memory performance. Protein kinase Mζ (PKMζ), a brain-specific variant of PKCζ, plays important roles in memory formation. Here we examined the interaction of KIBRA and PKMζ in the adult mouse brain. Immunoprecipitation using newly-raised anti-KIBRA antibody revealed the interaction between KIBRA and PKMζ in the brain. KIBRA was co-localized with PKMζ in a single cultured neuron. Distribution analysis by immunohistochemistry and in situ hybridization indicated that KIBRA was highly localized with PKMζ in the hippocampal CA1, CA3, and dentate gyrus. These results suggest that KIBRA functions in memory performance via interaction with PKMζ.

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The aim of this study was to use gene therapy via the Sleeping Beauty (SB) system to increase telomerase promoter activity to target hepatocellular carcinoma (HCC). In previous studies, we identified selective and increased expression of luciferase and suicide genes controlled by the hTERT (human telomerase reverse transcriptase) promoter and the SV40 enhancer in telomerase-positive cancer cell lines. Because telomerase is activated in about 80% of HCCs, it is likely that increasing the activity of the telomerase promoter with a suicide gene will effectively eradicate HCCs. We found that the telomerase promoter mediated SB system can efficiently insert transgene into HCC genomes. Also, telomerase promoter activity was increased using a SB vector expressing suicide gene HSV-TK (herpes simplex virus thymidine kinase) controlled by the hTERT promoter and a SV40 enhancer for the induction of telomerase-positive cancer-specific cell death. HCC cell lines transfected with pT.hTp.HSV-tk.Con with active helper plasmid and ganciclovir (GCV) significantly inhibited cancer cell growth. These results indicate that Sleeping Beauty transposon mediated suicide gene expression can be used in HCC-targeted cancer gene therapy.

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In plants, salicylic acid induces both systemic acquired resistance (SAR) and flowering. We found that flower induction in the Lemna species was promoted by reactive oxygen species-generating conditions and SAR inducers. The present paper suggests that SAR in plants and flowering in Lemna share common inducers.

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We determined for the first time the crystal structure of diatom cytochrome c₆ from Phaeodactylum tricornutum at 1.5 Å resolution. The overall structure of the protein was classified as a class I c-type cytochrome. The physicochemical properties of the protein were examined by denaturation with guanidine hydrochloride and urea, and compared with those of other algal cytochrome c₆.

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Two genes for alternative σ factors, σ^E2 and σ^E3, classified in the extracytoplasmic function σ family for RNA polymerases, were identified in the deep-sea piezophilic bacterium Shewanella violacea DSS12. Amino acid alignments revealed that the domains for transcriptional functions were comparatively conserved compared with Escherichia coli σ^E in both proteins. Core-binding analysis suggested that both proteins function as σ factors.

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In this study, we analyzed the structures of complex type N-glycans linked to glycoproteins in mature red tomato to determine the relative ratio of high-mannose type structure and complex type structure. Structural analysis of pyridylaminated N-glycans revealed that the truncated plant complex type structure accounted for about 70% of total conjugated N-glycans, while the high-mannose type structure accounted for about 22%.

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Human macrophage dectin-1, a type II transmembrane β-glucan receptor, was expressed as a fusion protein with an N-terminal hexahistidine tag and glutathione S-transferase in an Escherichia coli cell-free translation system, and assayed for binding specificity. Recombinant dectin-1 specifically bound to some β-glucans, but not to other carbohydrates. The β-glucan binding of recombinant dectin-1 was inhibited by laminarin, a soluble β-glucan, and by laminarioligosaccharides, but not by other carbohydrates. These results suggest that recombinant human dectin-1 can be used as a useful probe in identifying ligands in humans and tonic foods due to its strict binding specificity.

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The effects of the disinfectants NaClO and calcinated calcium on the food-borne pathogens Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella spp. attached to shredded cabbage leaves were examined. After these bacteria were attached to shredded leaves for 1 h, the leaves were treated with NaClO and/or calcinated calcium. About 2.6-log and 3.5-log reductions of E. coli O157 were achieved by treatment with NaClO (100 ppm, pH 6.0, 10 min) and calcinated calcium (0.1%, 20 min), respectively. The combination of 100 ppm NaClO and 0.1% calcinated calcium resulted in a 3- to 4-log reduction in the pathogen populations without apparent deteriorative effects. The bacterial numbers in the treated cabbage did not increase during storage at 4 °C. However, sensory evaluation including appearance and flavor indicated that the quality of the treated cabbage declined during storage. In conclusion, the combination of NaClO and calcinated calcium was useful in treatment before eating.

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To identify commercial Japanese monovarietal green tea and imported green tea samples, leading Japanese cultivars were fingerprinted by using six simple sequence repeat markers analyzed by a capillary sequencer. Two well-authenticated imported Chinese monovarietal green tea samples were also fingerprinted by the same markers, one of which, was Fuyun, was a clonally propagated cultivar, and the other, Jiukengzhong, was seed-propagated. At least three markers used in this study identified 16 leading Japanese cultivars and Fuyun. Although Jiukengzhong was a mixed population with diverse genotypes, some individuals had a unique allele in one simple sequence repeat marker that was not detected in the 16 leading Japanese cultivars, an additional 39 cultivars, and Fuyun. This allele was effective as a detection marker for Jiukengzhong. These results support the use of simple sequence repeat markers for the identification of Japanese monovarietal green tea and also of imported green tea made from foreign cultivars.

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As we have reported, flaxseed lignan, (+)-secoisolariciresinol (SECO), (−)-SECO, and meso-SECO were stereoselectively synthesized and their biological functions were evaluated. In the present study, we focused on the effects of SECOs on the regulation of 3T3-L1 adipocytes, and identified the structure-activity relationships. Optically active SECO and meso-SECO were tested for their effects on lipid metabolism in 3T3-L1 adipocytes. (−)-SECO accelerated adiponectin production of 3T3-L1 adipocytes. On the other hand, (+)- and meso-SECO suppressed the production of adiponectin. In addition, triglyceride (TG) accumulation in 3T3-L1 adipocytes was significantly suppressed by all three SECOs tested here, as was 17β-estradiol, when the SECOs were added to the medium during induction of 3T3-L1 preadipocytes to adipocytes. Especially, (−)-SECO strongly reduced TG accumulation. It is well-known that SECO has estrogen-like activity. Hence the estrogen-like activity of each SECO compound was assessed. Only (−)-SECO had estrogen-like activity.

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Tri a Bd 27K is the predominant allergen in wheat. In the present study, this allergen was purified to homogeneity from wheat flour. The N-terminal amino acid sequences of the purified allergen and the peptides obtained by its digestion, with trypsin were determined, and the allergen was shown to be a glycoprotein with an Asn-linked sugar moiety containing fucose residues.
A cDNA encoding the allergen was obtained by polymerase chain reaction (PCR). The cDNA codes for a protein of 203 amino acid residues, with a molecular mass of 22,803 Da, that has two tentative sites glycosylated at Asn residues. Homology analysis suggested that the allergen might belong to a family of γ-interferon-inducible thiol reductases. The cDNA was expressed as a fusion protein with glutathione S-transferase in Escherichia coli. However, unlike the allergen purified from wheat, recombinant Tri a Bd 27K was not immunoblotted with IgE antibodies in the serum of a wheat-sensitive patient.

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Changes in the thiamin contents in three types of porcine muscle and porcine liver during growth were investigated. The muscular thiamin content was lower at the newborn stage than at fetal stage, and increased after the weaning period. The liver thiamin content, however, remained unchanged from the fetal stage to 5 months old. The changes in thiamin contents were different between Landrace and Meishan pigs.

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We evaluated the effects of soluble fibers on β-carotene and lutein micellization during simulated digestion in vitro, and on carotenoid uptake from mixed micelles by Caco-2 cells. Medium- and high-viscosity alginates and pectins inhibited carotenoid micellization and cellular uptake relative to the fiber-free control. Alginates, carboxy-methylcelluloses, and methylcelluloses inhibited β-carotene uptake mainly by increasing medium viscosity, but pectins might inhibit carotenoid uptake by additional mechanisms.

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Peanut skin (PS) is characterized by almost exclusively consisting of polyphenols and fiber. We fractionated PS into a water-soluble fraction (WSF) and water-insoluble fraction (WIF), and further fractionated WSF into a soluble dietary fiber fraction (DF) and dietary fiber-free, water-soluble fraction (DFF-WSF). Male Sprague-Dawley rats were fed on high-cholesterol diets supplemented with PS and its fractions. PS, WSF, and DFF-WSF decreased the serum lipid and cholesterol levels and increased those in feces. This effect was probably due to the polyphenols that inhibited intestinal cholesterol absorption.

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A number of 2′,3′,4′-trihydroxy-2-phenylacetophenone derivatives were synthesized and examined for growth inhibition of several kinds of bacteria. 2′,3′,4′-Trihydroxy-2-phenylacetophenone itself exhibited no antibacterial activity, but some of its derivatives showed various antibacterial activities depending on functional groups introduced on the 2-phenyl ring. Eighteen out of 24 compounds synthesized in this study appeared to possess antibacterial activities against at least two Gram-positive strains of Bacillus subtilis and Staphylococcus aureus, 2-(biphenyl-4-yl)-2′,3′,4′-trihydroxyacetophenone being the most active with LC₅₀ of 5.8 μM and 5.6 μM respectively. However, none of the synthesized compounds exhibited inhibitory effects on Gram-negative strains, such as Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella enterica, suggesting that anti-Gram-positive specificity of the antibacterial compounds.

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In order to develop practical recombinant DNA techniques in the industrially important yeast Candida utilis, at least six plasmids harboring autonomously replicating sequences (ARSs) were isolated from a C. utilis genomic library. Two ARSs were subjected to detailed analysis. Sequences of 1.9 and 1.8 kb were found to be necessary to exert ARS activity in a plasmid as assessed by transformation efficiency and mitotic stability. Both fragments were found to be rich in AT content (69.5% and 70.8% respectively), and to contain an 11-bp ARS consensus sequences (10 and 13 motifs with one base difference respectively). Using the ARS-containing plasmid as a promoter-cloning vector, several DNA fragments having promoter activities were cloned and characterized. Co-transformation of C. utilis with an integrating DNA fragment and a replicating plasmid yielded plasmid-free transformants harboring the fragment integrated into the C. utilis genome.

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To develop a strategy for improved production of clavulanic acid (CA), we investigated the effect of using oils on cell growth and CA production during the fermentation of Streptomyces clavuligerus NRRL 3585. In this analysis, triolein, whose fatty acid is oleic acid only, was the best oil source for CA production, but free fatty acids generated from the hydrolysis of oils in a culture broth negatively impacted CA production and cell growth. Hence, we screened for mutants that were resistant to high concentrations of oleic acid. From this screen we identified a mutant S. clavuligerus, OL13, that had a minimum inhibitory concentration (MIC) to oleic acid of 2.1 g/l, much higher than that of S. clavuligerus NRRL 3585, at 0.4 g/l. Not only was cell growth improved, but maximum CA production, at 1,950 mg/l, was approximately 2.0-fold higher than that of the parent strain.

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Extracellular enzymes produced by six strains of thraustochytrids, Thraustochytrium, Schizochytrium, and Aurantiochytrium, were investigated. These strains produced 5 to 8 kinds of the extracellular enzymes, depending on the species. Only the genus Thraustochytrium produced amylase. When insoluble cellulose was used as substrate, cellulase was not detected in the six strains of thraustochytrids. This study indicates that marine eukaryotes, thraustochytrids, produced a wide variety of extracellular enzymes.

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Isopentenyl diphosphate isomerase (idi) and farnesyl diphosphate synthase (ispA) genes were overexpressed in Escherichia coli. The resulting transformant showed 6.8-fold higher production of farnesol (389 μg/l). In a similar manner, overexpression of idi and mutated ispA led to high production of geranylgeraniol (128 μg/l).

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Aspergillus oryzae AS 3.951 is widely used in Chinese soy sauce manufacture, but little is known about the profiles of the extracellular proteins from the culture of soybean koji. In this study, we carried out MALDI-TOF/TOF MS analysis of extracellular proteins during koji culture. Besides well-known proteins (TAA and Oryzin), a variety of aminopeptidase and proteases were identical at the proteome level. This suggests that A. oryzae AS 3.951 has a powerful capacity to digest soybean protein.

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PG-F2 and PG-HMW from Panax ginseng are pectin-type polysaccharides and PG-HMW might be an arabinogalactan. They demonstrated strong anti-adhesive activities against oral and skin pathogens to host cell lines in a dose-dependent manner from 0.1 to 2.0 mg/ml. While enzymatic hydrolysis caused complete loss of anti-adhesive activities, partial hydrolysis produced oligosaccharides with anti-adhesive properties. PG-F2 and PG-HMW might have a selective anti-adhesive effect against certain pathogenic bacteria without adverse effects on commensal bacteria.

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We have developed WeGAS, a Web based microbial Genome Annotation System, which provides features that include gene prediction, homology search, promoter/motif analysis, genome browsing, gene ontology analysis based on the COGs and GO, and metabolic pathway analysis with web-based interfaces. Most raw data and intermediate data from genome projects can be managed with the WeGAS database system, and analysis results, including information on each gene and final genome maps, are provided by its visualization modules. Especially, a pie-view browser displaying circular maps of contigs and a COG-GO combination browser are very helpful for an overview of projects. Major public microbial genome databases can be imported, searched, and browsed through the WeGAS modules. WeGAS is freely accessible via web site http://ns.smallsoft.co.kr:8051.

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Glutamate decarboxylase (GAD) from the archaeon Pyrococcus horikoshii was successfully expressed and purified, with the aim of developing a hyperthermostable GAD for industrial applications. Its biochemical properties were different from those reported for other GADs. The enzyme had broad substrate specificity, and its optimum pH and temperature were pH 8.0 and >97 °C.

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Membrane-bound glucono-δ-lactonase (MGL) was purified to homogeneity from the membrane fraction of Gluconobacter oxydans IFO 3244. After solubilization with 1 M CaCl₂, MGL was purified in the presence of Ca²⁺ and detergent. A single band corresponding to 60 kDa appeared in SDS–PAGE. The molecular weight of MGL was judged to be 120k. Differently from cytoplasmic lactonases, MGL showed optimum pH in an acidic range of 5–5.5. It was highly sensitive to metal-chelating agents such as EDTA, and the lost MGL activity was restored to the original level by the addition of divalent cations such as Ca²⁺ or Mg²⁺. The purified MGL was strictly dependent on Ca²⁺ and underwent rapid denaturing precipitation on Ca²⁺ depletion even in the presence of detergent. This communication can be the first one dealing with the solubilization, purification and properties of MGL.

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