In this review, I describe the yeast glycans and their biosynthetic pathways, especially in the budding yeast Saccharomyces cerevisiae. The biosynthetic pathway of N-glycan in the endoplasmic reticulum is similar to that of mammalian cells, while the pathway in the Golgi is different from that of mammalian cells, but the biosynthetic pathway of O-glycan, mainly composed of O-oligomannoses, appears to be specific to yeast cells. Yeast systems are useful not only to understand the basic mechanisms of glycan synthesis but also to produce therapeutic proteins with human-type glycans. Protein modification by glycosylphosphatidylinositol is one of the major post-translational modifications in which oligosaccharides are involved. The biosynthetic pathway and the physiological function of glycosylphosphatidylinositol in S. cerevisiae are described in relation to lipid microdomains (also called “lipid rafts”), focusing on the latest findings related to lipid remodeling of GPI-anchored proteins.

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Food allergy is one of the important health problems, and countermeasures are socially required. We have been undertaking studies on wheat allergens and their epitopes, and have developed a method for producing hypoallergenic wheat flour by enzymatic modification. The hypoallergenic products are now provided to patients. More noteworthy, by taking hypoallergenic cupcakes over a long period, more than half of patients are hyposensitized and become able to eat normal wheat products. This suggests that the hypoallergenic wheat flour can act as anti-allergenic via allergen-specific immunotolerance.
This series of studies was followed by expansive research on food allergy: analysis of epitopes of bovine serum albumin (the major beef allergen), isolation and identification of inhibitory peptides for allergen absorption at the intestine, evaluation of hesperetin as an inhibitor of degranulation of mast cells, and the development of PCR detection methods for verifying allergen labeling and for identifying hidden allergic ingredients in processed foods.

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Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers’ redox potential.

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Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide, the ComX pheromone, that stimulates natural genetic competence controlled by quorum sensing. The ComX_RO-C-2 pheromone from strain RO-C-2 must be modified with a farnesyl group on the Trp residue, but the precise structure is not known. Here we report the precise nature of posttranslational farnesylation of ComX_RO-C-2 pheromone on the Trp residue, resulting in the formation of a tricyclic structure. The ComX₁₆₈ pheromone, produced by the standard laboratory strain used in the study of B. subtilis, is also posttranslationally farnesylated according to phylogenetic resemblance.

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Lycopene is known to decrease cardiovascular risks. The objective of this study was to investigate the molecular mechanisms of tomato paste containing approximately 0.1% lycopene in regulating lipid metabolism and oxidation. Hamsters fed 3% or 9% tomato paste containing 0.2% cholesterol were subjected to total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), and triglyceride (TG) measurements. Our results showed reduced rates of serum TC and LDL levels due to 9% tomato paste were 14.3% and 11.3% respectively. Concentrations of 3% and 9% of tomato paste after 8 weeks of feeding significantly increased serum HDL levels, by 19.4% and 28.8% respectively. After ingestion of tomato paste for 8 weeks, the plasma malondialdehyde (MDA) levels significantly decreased, by 80.18% and 89.33% respectively, as compared to the cholesterol group. MDA and diene conjugation assays indicated the potent antioxidant activity of the tomato paste. The increased activities of superoxide dismutases (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), further supported the antioxidant effects of the tomato paste. Two dimension-gel electrophoresis (2-DE) analysis revealed that carbonic anhydrase III (CAIII) and adenylate kinase 2 (AK2) may be two important regulators involved in the anti-lipid and antioxidant effects of tomato paste, opening new insight into the nutritional value of tomato in public health promotion.

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To understand the response of soil bacteria to the surrounding environment, it is necessary to examine the gene expression profiles of the bacteria in the soil. For this purpose, we developed a new method of extracting RNA from soil reproducibly. Using this new method, we extracted RNA from a field soil, which was sterilized and inoculated with Rhodococcus sp. strain RHA1, a biphenyl degrader isolated from γ-hexachlorocyclohexane-contaminated soil. Data from agarose gel electrophoresis indicated that the extracted RNA was purified properly. This new method can be applied easily in the preparation of large amounts of RNA. Real-time reverse transcription-polymerase chain reaction (RT-PCR) experiments performed by the TaqMan method suggested that the bphAa gene in this strain, which is involved in the degradation of biphenyl, was induced in the biphenyl amended soil.

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In this study, the channel catfish (CC) matrix metalloproteinase-9 (MMP-9) gene was cloned, sequenced, and characterized at both the cDNA and the genomic DNA levels. The complete sequence of the CC MMP-9 cDNA consisted of 2,551 nucleotides, including one open reading frame and 5′- and 3′-end untranslated regions. The open reading frame potentially encoded a 686-amino-acid peptide with a calculated molecular mass (without glycosylation) of approximately 77.4 kDa, which included a signal peptide and potentially heavy O-glycosylation sites. CC MMP-9 did not have the tripeptide Arg-Gly-Asp motif. The degree of conservation of the CC MMP-9 amino acid sequence to human and mouse counterparts was 55%, while to those of other fish species was 67–74%. The full-length CC MMP-9 genomic DNA comprised 5,663 nucleotides, much shorter than human or mouse counterparts. The exon-intron structure followed the splice acceptor/donor consensus rule, and the sequence contained 13 exons. The MMP-9 transcript was constitutively expressed in restrictive CC tissues. This result should provide fundamental information for further exploration of the role of MMP-9 in fish pathophysiology.

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In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher k_cat and 15 fold-lower K_m than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-β-D-N-N′-N″-triacetylchitotrioside, while BJL200-ChiC1 was 10–15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of β-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)₂ (main product), GlcNAc, (GlcNAc)₃, (GlcNAc)₄, and (GlcNAc)₅, indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.

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When 10 strains of lactic acid bacteria were incubated with 5′-hydroxyaverantin (HAVN), a precursor of aflatoxins, seven of them converted HAVN to averufin; the same reaction is found in aflatoxin biosynthesis of aflatoxigenic fungi. These bacteria had a dehydrogenase that catalyzed the reaction from HAVN to 5′-oxoaverantin (OAVN), which was so unstable that it was easily converted to averufin. The enzyme was purified from Lactobacillus brevis IFO 12005. The molecular mass of the enzyme was 100 kDa on gel filtration chromatography and 33 kDa on SDS polyacrylamide gel electrophoresis (SDS–PAGE). The gene encoding the enzyme was cloned and sequenced. The deduced protein consisted of 249 amino acids, and its estimated molecular mass was 25,873, in agreement with that by time of flight mass spectrometry (TOF MS) analysis. Although the deduced amino acid sequence showed about 50% identity to those reported for alcohol dehydrogenases from L. brevis or L. kefir, the commercially available alcohol dehydrogenase from L. kefir did not convert HAVN to OAVN. Aspergillus parasiticus HAVN dehydrogenase showed about 25% identity in amino acid sequence with the dehydrogenase and also with these two alcohol dehydrogenases.

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The crystal structure of the Alba protein (PhoAlba) from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, was determined at a resolution of 2.8 Å. PhoAlba structurally belongs to the α⁄β proteins and is similar not only to archaeal homologues but also to RNA-binding proteins, including the C-terminal half of initiation factor 3 (IF3-C) from Bacillus stearothermophilus, an Esherichia coli protein implicated in cell division (Yhhp), and an Arabidopsis protein of unknown function. We found by gel shift assay that PhoAlba interacts with both ribonuclease P (RNase P) RNA (PhopRNA) and precursor-tRNA^Tyr (pre-tRNA^Tyr) in P. horikoshii. However, the addition of PhoAlba to reconstituted particles composed of PhopRNA and four or five protein subunits had little influence on either the pre-tRNA processing activity or the optimum temperature for the processing activity. These results suggest that PhoAlba contributes little to the catalytic activity of P. horikoshii RNase P.

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Recently, we identified a novel human platelet-derived growth factor (PDGF) receptor α mRNA (type II) that differs from the known human PDGF receptor α mRNA (type I) only in its 5′-untranslated region and is regulated by E2F-1. In this study, we determined the nucleotide sequence of the mouse type II PDGF receptor α mRNA and found that the expression of this transcript was regulated by E2F-1 through an E2F-1-responsive sequence located +711/+718 downstream of the transcription start site of the type I transcript. Furthermore, we observed embryo-predominant expression of the type II transcript.

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We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.

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The effects of cellulose crystallinity, hemicellulose, and lignin on the enzymatic hydrolysis of Miscanthus sinensis to monosaccharides were investigated. A air-dried biomass was ground by ball-milling, and the powder was separated into four fractions by passage through a series of sieves with mesh sizes 250–355 μm, 150–250 μm, 63–150 μm, and <63 μm. Each fraction was hydrolyzed with commercially available cellulase and β-glucosidase. The yield of monosaccharides increased as the crystallinity of the substrate decreased. The addition of xylanase increased the yield of both pentoses and glucose. Delignification by the sodium chlorite method improved the initial rate of hydrolysis by cellulolytic enzymes significantly, resulting in a higher yield of monosaccharides as compared with that for untreated samples. When delignified M. sinensis was hydrolyzed with cellulase, β-glucosidase, and xylanase, hemicellulose was hydrolyzed completely into monosaccharides, and the conversion rate of glucan to glucose was 90.6%.

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In this study, we characterized the DnaK chaperone system from Tetragenococcus halophilus, a halophilic lactic acid bacterium. An in vivo complementation test showed that under heat stress conditions, T. halophilus DnaK did not rescue the growth of the Escherichia coli dnaK deletion mutant, whereas T. halophilus DnaJ and GrpE complemented the corresponding mutations of E. coli. Purified T. halophilus DnaK showed intrinsic weak ATPase activity and holding chaperone activity in vitro, but T. halophilus DnaK did not cooperate with the purified DnaJ and GrpE from either T. halophilus or E. coli in ATP hydrolysis or luciferase-refolding reactions under the conditions tested. E. coli DnaK, however, cross-reacted with those from both bacteria. This difference in the cooperation with DnaJ and GrpE appears to result in an inability of T. halophilus DnaK to replace the in vivo function of the DnaK chaperone of E. coli.

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To understand better the role of subsites E and F in lysozyme-catalyzed reactions, mutant enzymes, in which Arg114, located on the right side of subsites E and F in hen egg-white lysozyme (HEL), was replaced with Lys, His, or Ala, were prepared. Replacement of Arg114 with His or Ala decreased hydrolytic activity toward an artificial substrate, glycol chitin, while replacement with Lys had little effect. Kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)₅, revealed that the replacement for the Arg residue reduced the binding free energies of E-F sites and the rate constant of transglycosylation. The rate constant of transglycosylation for R114A was about half of that for the wild-type enzyme. ¹H-NMR analysis of R114H and R114A indicated that the structural changes induced by the mutations were not restricted to the region surrounding Arg114, but rather extended to the aromatic side chains of Phe34 and Trp123, of which the signals are connected with each other through nuclear Overhauser effect (NOE) in the wild-type. We speculate that such a conformational change causes differences in substrate and acceptor binding at subsites E and F, lowering the efficiency of glycosyl transfer reaction of lysozyme.

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A carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. was purified and characterized. The enzyme efficiently oxidized β-(1→4) linked sugars, such as lactose, xylobiose, and cellooligosaccharides. The enzyme also oxidized maltooligosaccharides, D-glucose, D-xylose, D-galactose, L-arabinose, and 6-deoxy-D-glucose. It specifically oxidized the β-anomer of lactose. Molecular oxygen and 2,6-dichlorophenol indophenol were reduced by the enzyme as electron acceptors. The Paraconiothyrium enzyme was identified as a carbohydrate:acceptor oxidoreductase according to its specificity for electron donors and acceptors, and its molecular properties, as well as the N-terminal amino acid sequence. Further comparison of the amino acid sequences of lactose oxidizing enzymes indicated that carbohydrate:acceptor oxidoreductases belong to the same group as glucooligosaccharide oxidase, while they differ from cellobiose dehydrogenases and cellobiose:quinone oxidoreductases.

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In order to investigate the substrate binding feature of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26 with respect to farnesyl diphosphate and a reaction intermediate, (Z,E,E)-geranylgeranyl diphosphate, we examined the reactivity of artificial substrate analogs, 3-desmethyl farnesyl diphosphate and 3-desmethyl Z-geranylgeranyl diphosphate, which lack the methyl group at the 3-position of farnesyl diphosphate and Z-geranylgeranyl diphosphate, respectively. Undecaprenyl diphosphate synthase did not accept either of the 3-desmethyl analogs as the allylic substrate, indicating that the methyl group at the 3-position of the allylic substrate is important in the undecaprenyl diphosphate synthase reaction. These analogs showed different inhibition patterns in the cis-prenyl chain elongation reaction with respect to the reactions of farnesyl diphosphate and Z-geranylgeranyl diphosphate as allylic substrate. These results suggest that the binding site for the natural substrate farnesyl diphosphate and those for the intermediate allylic diphosphate, which contains the cis-prenyl unit, are different during the cis-prenyl chain elongation reaction.

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We measured the concentrations of acetyl-CoA and malonyl-CoA in shoots and roots of corn (Zea mays, L., cv. “Peter Corn”). Acetyl-CoA and malonyl-CoA concentrations were found to be relatively constant in shoots and in roots under a light-dark cycle. Acetyl-CoA concentrations were lower in shoots than in roots, whereas malonyl-CoA concentrations were higher in shoots than in roots.

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Changes in gene expression levels of c-myc and CD38 were examined during the differentiation of HL-60 cells to granulocytes due to three nicotinic acid-related compounds. CD38 expression was increased by isonicotinic acid and all-trans-retinoic acid (ATRA). Nicotinamide and nicotinamide N-oxide drastically decreased c-myc expression, but isonicotinic acid had no effect, suggesting that these compounds differentiate HL-60 to granulocytes through different pathways. These results should provide useful information as to the mechanisms of cell differentiation.

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In many organisms, glycogen gives rise to 1,5-anhydro-D-fructose (AF), which is reduced to 1,5-anhydro-D-glucitol (AG). AF reductase, which catalyzes the latter reaction, was purified from pig liver, but mouse ortholog has not yet been reported. In the database, aldo-keto reductase family 1, member E1 (AKR1E1) showed highest homology to pig enzyme. We confirmed that cloned AKR1E1 is mouse ortholog based on enzymatic properties of purified recombinant protein.

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A conidia suspension of Magnaporthe grisea carried elicitor activity that induced the expression of defense-related genes and the production of H₂O₂ in suspension-cultured rice cells. The levels of H₂O₂ produced were dependent on fungal isolates and were correlated with the catalase activity in the supernatant fraction of each conidia suspension, not with gene-for-gene interactions.

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To determine the properties and structure of OsChia1b, a family 19 chitinase from Oryza sativa L. cv. Nipponbare (japonica ssp.), recombinant OsChia1b was produced in Esherichia coli cells and purified to homogeneity by chitin affinity column chromatography. OsChia1b was highly active against soluble chitinous substrate, but not against crystalline chitin, and clearly inhibited hyphal extension of Trichoderma reesei.

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Carrot Argonaute1 (C-Ago1) was isolated from a subtractive cDNA library to obtain somatic embryogenesis related genes. C-Ago1 has three conserved domains, which are found in all other Argonautes. C-Ago1 has specific expression during somatic embryogenesis, which indicates that microRNA gene expression controlling system is required for somatic embryogenesis.

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The substrate specificities of porcine and bovine enteropeptidases were investigated using the peptide Val-(Asp)₄-Lys-Ile-Val-Gly and its various analogs with mutations in the (Asp)₄-Lys sequence as substrates. The results indicated that in addition to P1 Lys, P2 Asp in the substrates is most important, that P3 Asp is additionally important, and that P5 Asp contributes somewhat to the susceptibility, and that P4 Asp is the least important. These results were essentially identical as between porcine and bovine enteropeptidases.

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The anti-allergic effect of a 70% ethanol extract from Dictamnus dasycarpus Turcz (DDT) was studied in mice. DDT at doses of 200 and 500 mg/kg inhibited the systemic anaphylactic shock induced by compound 48/80 in a dose-dependent manner. It also inhibited dose-dependently the scratching behavior induced by compound 48/80, histamine and serotonin. An increase in the vascular permeability induced by compound 48/80, histamine and serotonin was also inhibited by DDT. In an in vitro study, DDT inhibited the histamine released from rat peritoneal mast cells induced by compound 48/80. It seems likely from these findings that DDT was effective in antagonizing certain pharmacological effects induced by compound 48/80 that occurred via both histamine and serotonin released from mast cells. In conclusion, DDT may be effective in the relief of symptoms of allergic atopic dermatitis and other allergy-related diseases.

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The hypoglycemic effects of Eriobotrya japonica seeds were investigated in type 2 diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and KK-A^y mice. The rats and mice were fed on a diet containing 10% powdered Eriobotrya japonica seeds with the coat intact for 4 months. Although the blood glucose concentration in the OLETF rats fed on the control diet without Eriobotrya japonica seeds was increased with time, the concentration in the OLETF rats fed on the diet with Eriobotrya japonica seeds was consistently low throughout the experimental period and was comparable to the level in Long-Evans Tokushima Otsuka (LETO) rats which are normal non-diabetic rats. Serum insulin was significantly lower in the OLETF rats fed on the Eriobotrya japonica seed diet than in those fed on the control diet at the termination of the experimental period. Eriobotrya japonica seeds suppressed the increment of blood glucose for 4 months and also effectively improved the glucose tolerance in the KK-A^y mice, these actions being mainly exerted by the ethanol extract of the seeds. These results suggest that Eriobotrya japonica seeds had a hypoglycemic property and the effect is attributable to the components extracted by ethanol.

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We reexamined the hypothesis that increased mucin secretion by the ingestion of insoluble dietary fiber (IDF) could affect small intestinal nutrient absorption. Polystyrene foam (PSF) was used as IDF. Rats were fed a diet with or without 90 g of PSF/kg for 1, 2 and 4 wk. At the end of each period, a glucose and ovalbumin (OVA) solution was intubated after 12 h of food depletion, and the changes in serum concentrations of these components were monitored. Luminal mucin was measured as O-linked oligosaccharide chains and also determined by ELISA. In all periods, the luminal mucin content was greater in the PSF-fed group than in the fiber-free control. However, the changes in serum glucose and OVA concentrations were comparable between the groups at any time during any period. These results show that the enhancement of luminal mucin secretion lasted even after chronic ingestion of IDF, but that the increased luminal mucin content had no effect on the rate of luminal nutrient absorption.

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There is evidence for defects in the mechanisms that allow the activation of p53 in many of the cancers that retain wild-type p53. Reactivation of p53 has been suggested to be an effective strategy for cancer therapy in wild-type p53-retained tumor cells. In the present study, we attempted to reactivate p53 in HepG2 retaining wild-type p53 by quercetin, an ubiquitous bioactive plant flavonoid. Our results show that quercetin inhibited the proliferation of HepG2 cells through the induction of cell cycle arrest and apoptosis, as characterized by the cell cycle distribution and DNA fragmentation. Molecular data revealed that quercetin induced p53 phosphorylation and total p53 protein, but that it did not up-regulate p53 mRNA at the transcription level. Consequently, quercetin stimulated p21 expression and suppressed cyclin D1 expression in favor of cell cycle arrest. Quercetin also increased the ratio of Bax/Bcl-2 in favor of apoptosis with such treatment. Interestingly, quercetin inhibited p53 ubiquitination and extended the half-life (t_1⁄2) of p53 from 74 to 184 min. Quercetin also inhibited p53 mRNA degradation at the post-transcription stage. Silencing p53 with p53 small interfering RNA (siRNA) significantly abrogated the p53-dependent gene expression and apoptotic induction. Taken together, our data demonstrate that quercetin stabilized p53 at both the mRNA and protein levels to reactivate p53-dependent cell cycle arrest and apoptosis in HepG2 cells.

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Isorhamnetin 3-O-glucoside, which was contained together with isorhamnetin 3,7-di-O-glucoside in atsumi-kabu leaves, suppressed increases in the plasma ALT and AST activities of mice with liver injury induced by the injection of carbon tetrachloride, but no suppression by isorhamnetin 3,7-di-O-glucoside was apparent. This result indicates that the release of glucose at the 7-position in isorhamnetin 3,7-di-O-glucoside was very important to mitigating liver injury.

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The izumi shrimp (Plesionika izumiae Omori, 1971) is an unused resource which can be caught off the southern coast of Tokushima Prefecture. We have previously found that an izumi shrimp hydrolysate significantly inhibited the age-associated spontaneous increase in blood pressure in stroke-prone spontaneously hypertensive rats. In this present study, two angiotensin I-converting enzyme inhibitory peptides were isolated from an izumi shrimp hydrolysate by using high-performance liquid chromatography, and their amino acid sequences were determined to be Val-Trp-Tyr-His-Thr and Val-Trp. A single oral administration of synthetic Val-Trp-Tyr-His-Thr or Val-Trp significantly decreased the blood pressure in stroke-prone spontaneously hypertensive rats. The antigenicity and allergenicity of the izumi shrimp hydrolysate against BALB/c mice were very low. These results demonstrate that the angiotensin I-converting enzyme inhibitory peptides isolated from the izumi shrimp hydrolysate had an anti-hypertensive effect on rats.

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Rats were fed on a diet containing cholesterol (Chol) at a level corresponding to the standard Chol intake in humans, and the influence of heat-moisture-treated high-amylose cornstarch (HHA) on their serum Chol level was investigated. HHA decreased the serum level of Chol in rats fed on the diet containing 0.1% Chol, which corresponds to a Chol intake in humans of 800 mg/d, although the liver levels of Chol increased in these rats. HHA did not influence the fecal excretion of Chol/bile acids. It is possible that the decrease in serum Chol level in the rats fed on the high-Chol diet can be attributed to the promotion of Chol uptake in the liver.

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We evaluated the neurotrophic activity of dietary polyphenols by using primary cultures of fetal rat hippocampal neurons in a serum-free medium. Among the tested compounds, chlorogenic acid and its metabolite, m-coumaric acid, together with catechins and flavanone, were found to promote neuronal differentiation comparable to the phytochemical, honokiol, which has been reported to show potent neurotrophic activity. The present findings may contribute to the development of further neurotrophic studies on dietary polyphenols and their metabolites.

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A pectin was extracted from the peel of Citrus tankan with a yield of 2.75%. The uronic acid content was 80.0%, and the degree of methoxylation was 63.2%. The pectin was composed of D-GalA, D-Gal, L-Ara and L-Rha in the molar ratio of 100:11.3:3.6:2.6. The molecular weight was estimated to be approximately 9.2×10⁴. The pectin formed a gel by conventional procedures.

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In the present study, specific discrimination of chicken DNA contamination in processed foods using the polymerase chain reaction was investigated. The primer pair was designed to amplify a 102-bp fragment of the chicken mitochondrial 16S ribosomal RNA gene. While the DNA from chicken meat was amplified, the DNA from other poultry meat, mammalian meat, fish, shellfish, and cereals was not amplified. The primer amplified DNA fragments derived from model processed and nonprocessed food samples containing 0.001, 0.01, 0.1, 1, 10, and 100% chicken.

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Asaia lannaensis sp. nov. was described for two strains isolated from flowers of the spider lily collected in Chiang Mai, Thailand. The isolates produced acetic acid from ethanol on ethanol/calcium carbonate agar, differing from the type strains of Asaia bogorensis, Asaia siamensis, and Asaia krungthepensis, but did not grow in the presence of 0.35% acetic acid (v/v). The new species is the fourth of the genus Asaia, the family Acetobacteraceae.

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Tanticharoenia sakaeratensis gen. nov., sp. nov. is proposed for three strains isolated from soil collected in Thailand. The three strains, AC37^T, AC38, and AC39, were included within a lineage comprising the genera Asaia, Kozakia, Swaminathania, Neoasaia, Acetobacter, Gluconobacter, and Saccharibacter in a phylogenetic tree based on 16S rRNA gene sequences, but formed a quite different, independent cluster. Pair-wise sequence similarities of strain AC37^T were 96.5–92.1% to the type strains of Acetobacter aceti, Gluconobacter oxydans, Acidomonas methanolica, Gluconacetobacter liquefaciens, Asaia bogorensis, Kozakia baliensis, Swaminathania salitolerans, Saccharibacter floricola, Neoasaia chiangmaiensis, and Granulibacter bethesdensis. The three strains had DNA base compositions comprising respectively 65.6, 64.5, and 65.6 mol % G+C with a range of 1.1 mol %, and formed a single species. Phenotypically, the three strains did not oxidize acetate or lactate, but grew on 30% D-glucose (w/v). Chemotaxonomically, they had Q-10. The type strain is AC37^T (= BCC 15772^T = NBRC 103193^T).

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A small enzyme showing diaphorase activity was purified from culture supernatant of Clostridium kluyveri and its N-terminal amino acid sequence was determined. This sequence identified a gene (diaA) encoding a protein (DiaA) of 229 amino acids with a predicted molecular weight of 24,981 in the genomic DNA sequence database of C. kluyveri constructed by the Research Institute of Innovative Technology for the Earth. The predicted protein was composed of a flavin reductase-like domain and a rubredoxin-like domain from its N-terminus. The diaA gene was cloned into an expression vector and expressed in an Escherichia coli recombinant. Recombinant enzyme rDiaA showed NADH/NADPH diaphorase activity with 2,6-dichlorophenolindophenol and nitro blue tetrazolium. The enzyme was most active at pH 8.0 at 40 °C. The UV-visible absorption spectrum and thin layer chromatography (TLC) analyses indicated that one rDiaA molecule contained a tightly bound FMN molecule as a prosthetic group. An iron molecule was also detected in an enzyme molecule.

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Bifidobacteria and Bacteroides-like bacteria are strictly anaerobic nonpathogenic members of human intestinal microflora. Here we describe an analysis of the species and subspecies composition of these bacterial populations in healthy children using a combination of culture and molecular methods at two different time points. It was found that B. bifidum and B. longum are the most common dominant taxons in infants aged between 8 and 16 months. The majority of the infants carried several dominant Bifidobacterium strains belonging to different species. Examination of the dominant bifidoflora in some of these children after a 5-year period showed major shifts in both species and strain composition, but the dominant strains remained unchanged in two children. The majority of dominant Bacteroides-like isolates belonged to species B. vulgatus and B. uniformis, but members of genera Alistipes and Barnesiella were common too. In addition, a novel approach to species identification of Bacteroidales order bacteria using amplified ribosomal DNA restriction analysis (ARDRA) is described.

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The effects of pulverized petal of Rosa rugosa on the growth of 10 species of intestinal and pathogenic bacteria were investigated. Growth of bifidobacteria and lactobacilli was not affected by the addition of the petal in plate cultivation. However, the growth of Bacteroides vulgatus, Escherichia coli, Staphylococcus aureus, and Bacillus cereus was completely inhibited by the addition of 0.1, 0.5, 0.1, and 0.05% (w/v) of the petal respectively. In liquid cultivation, the addition of the petal (0.5%) stimulated the growth of Bifidobacterium breve and slightly inhibited the growth of Lactobacillus salivarius. But the growth of E. coli, S. aureus, B. cereus, and Salmonella sp. was inhibited by nearly 50%. Hydrolyzable tannins isolated from R. rugosa, rugosin D, and tellimagradin II showed antibacterial activities against E. coli, S. aureus, B. cereus, and Salmonella sp., but little or no effect against Bif. breve and L. salivarius. R. rugosa petal showed selective antibacterial activities against intestinal and pathogenic bacteria, and the selectivity resembled that of prebiotics such as oligosaccharides and dietary fiber. Hydrolyzable tannins in R. rugosa, such as rugosin D and tellimagradin II, must be active constituents.

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Two different membrane-bound enzymes oxidizing D-sorbitol are found in Gluconobacter frateurii THD32: pyroloquinoline quinone-dependent glycerol dehydrogenase (PQQ-GLDH) and FAD-dependent D-sorbitol dehydrogenase (FAD-SLDH). In this study, FAD-SLDH appeared to be induced by L-sorbose. A mutant defective in both enzymes grew as well as the wild-type strain did, indicating that both enzymes are dispensable for growth on D-sorbitol. The strain defective in PQQ-GLDH exhibited delayed L-sorbose production, and lower accumulation of it, corresponding to decreased oxidase activity for D-sorbitol in spite of high D-sorbitol dehydrogenase activity, was observed. In the mutant strain defective in PQQ-GLDH, oxidase activity with D-sorbitol was much more resistant to cyanide, and the H⁺/O ratio was lower than in either the wild-type strain or the mutant strain defective in FAD-SLDH. These results suggest that PQQ-GLDH connects efficiently to cytochrome bo₃ terminal oxidase and that it plays a major role in L-sorbose production. On the other hand, FAD-SLDH linked preferably to the cyanide-insensitive terminal oxidase, CIO.

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Two high-molecular-weight rubredoxin genes (hrb) from Clostridium thermocellum and Moorella thermoacetica were expressed in Escherichia coli and their translated products (Hrb) were characterized. M. thermoacetica Hrb showed strong diaphorase activity. In contrast, C. thermocellum Hrb, containing neither FMN nor FAD in the molecule, showed flavin reductase activity with moderate diaphorase activity. Both enzymes were optimally active at about 50 °C.

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