Synthetic studies on bioactive compounds are described, involving phytotoxins (tobacco wildfire disease toxin tabtoxinine-β-lactam and rice blast disease toxin pyricuol) a glutarimide antibiotic (actiketal) black vomit toxin (gizzerosine) and marine products (antifeedant pteroenone and serinol compound didemniserinolipid).

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Cells must balance energy-efficient growth with the ability to adapt rapidly to sudden changes in their environment. For example, in an environment rich in amino acids, cells do not expend energy for making amino acid biosynthetic enzymes. However, if the environment becomes depleted of amino acids (nutritional downshift), cells will be exposed to a lack of both the amino acid biosynthetic enzymes and the amino acids required to make these enzymes. To solve this dilemma, cells must use their own proteins as sources of amino acids in response to the nutritional downshift. Once amino acid biosynthetic enzymes start to accumulate, the cell is able to produce its own amino acids, and a new growth phase begins. In Escherichia coli, amino acid starvation leads to the accumulation of an unusual molecule, polyphosphate (polyP), a linear polymer of many hundreds of orthophosphate residues. Protein degradation in this bacterium appears to be triggered by the accumulation of polyP. PolyP forms a complex with the ATP-dependent Lon protease. The formation of a complex then enables Lon to degrade free ribosomal proteins. Certain very abundant ribosomal proteins can be the sacrificial substrates targeted for degradation at the onset of the downshift. Here I propose to call the polyP-Lon complex the “stringent protease,” and I discuss new insights of protein degradation control in bacteria.

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Several geometrical isomers of 3,13- and 2,13-octadecadien-1-ols and their acetates were synthesized starting from 1,8-octanediol or 1,9-nonanediol utilizing acetylene coupling reactions. In addition to commercially available compounds, all geometrical isomers of each dienyl compound were analyzed by NMR and GC-MS to accumulate chemical data for studies of sex pheromones secreted from clearwing moths classified into the family Sesiidae of Lepidoptera. Although acetoxy derivatives of the 3,13- and 2,13-dienes showed almost the same mass spectra, the alcohols were distinguished by comparing the relative intensities of [M−18]⁺ at m⁄z 248, indicating direct differentiation of the two positional isomers without derivatization. Furthermore, each geometrical isomer eluted from a high-polar GC column with a different retention time. Base on these data, a pheromone gland extract of a sesiid moth, Nokona pernix, was analyzed by GC-EAD and GC-MS, and two EAG-active components were identified, viz., the (3E,13Z)- and (3Z,13Z)-isomers of 3,13-octadecadien-1-ol in a ratio of 9:1. In the field, the synthetic compounds mixed in 9:1 ratio attracted N. pernix males well, while a single component scarcely attracted the males. The number of attracted males peaked in the middle of June, and a small second peak was observed in August.

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Two kaempferol glycosides were isolated from green tea seed extract (GTSE). After conducting a structure analysis, these two compounds were identified as kaempferol-3-O-[2-O-β-D-galactopyranosyl-6-O-α-L-rhamnopyranosyl]-β-D-glucopyranoside (compound 1) and kaempferol-3-O-[2-O-β-D-xylopyranosyl-6-O-α-L-rhanmopyranosyl]-β-D-glucopyranoside (compound 2). These two compounds were hydrolysed by o-glycolytic enzymes for the production of kaempferol. After performing several reactions, we found the optimum enzyme combination, a reaction with β-galactosidase and hesperidinase. Finally, we produced kaempferol of above 95% purity. The 5α-reductase inhibition activities of GTSE hydrolysate (GTSE-H) containing kaempferol were evaluated by the contact cell-based metabolic method using a stable HEK 293 cell line. GTSE-H showed a good inhibition effect on HEK 293 cell lines both type 1 and type 2 on 5α-reductase. Especially, GTSE-H inhibited type 2 with kaempferol content dependency. The results indicate that the inhibition activity of hydrolysate on 5α-reductase type 2 increases in accordance with kaempferol content.

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The antioxidative properties of five prenylated flavonoids, including new flavanone (2), from the root bark of Cudrania tricuspidata were examined against the ABTS, DPPH, and hydroxyl radicals. In most of the assays to determine their antioxidative properties, the ABTS activity was strongly correlated with DPPH because both methods are responsible for the same chemical property of hydrogen- or electron-donation to the antioxidant. On the other hand, the prenylated flavonoids (1–5) acted differently with both methods; namely, all the prenylated flavonoids strongly scavenged the ABTS radical (IC₅₀<10 μM), while they were inactive against the DPPH radical (IC₅₀>300 μM). Even though isolated 5,7,2′,4′,-tetrahydroxy-6,5′-diprenylflavanone (3) showed weak reducing power (746 mV) by cyclic voltammetry when compared to quercetin (394 mV), both had similar ABTS activity (IC₅₀<5 μM).

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Soluble and cell wall bound γ-glutamyltransferases (GGTs) were purified from radish (Raphanus sativus L.) cotyledons. Soluble GGTs (GGT I and II) had the same M_r of 63,000, and were composed of a heavy subunit (M_r, 42,000) and a light one (M_r, 21,000). The properties of GGT I and II were similar. Bound GGTs (GGT A and B) were purified to homogeneity from the pellet after the extraction of soluble GGTs. GGT A and B were monomeric proteins with an M_r of 61,000. The properties of GGT A and B were similar. Thus, bound GGTs were distinguished from soluble GGTs. The optimal pHs of soluble and bound GGTs were about 7.5. Both soluble and bound GGTs utilized glutathione, γ-L-glutamyl-p-nitroanilide, oxidized glutathione and the conjugate of glutathione with monobromobimane as substrates, and were inhibited by acivicin, but soluble GGTs were also distinguished from bound GGTs with regard to these properties.

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Phospholipase B (PLB) from the asporogenous yeast Candida utilis was purified to homogeneity from a culture broth. The apparent molecular mass was 90–110 kDa by SDS–PAGE. The enzyme had two pH optima, one acidic (pH 3.0) and the other alkaline (pH 7.5). At acidic pH the enzyme hydrolyzed all phospholipids tested without metal ions. On the other hand, the PLB showed substrate specificity and required metal ions for alkaline activity.
The cDNA sequence of the PLB was analyzed by a combination of several PCR procedures. The PLB encoded a protein consisting of 643 amino acids. The amino acid sequence contained a lipase consensus sequence (GxSxG) and catalytic arginine and aspartic acid motifs which were identified as the catalytic triad in the PLB from Kluyveromyces lactis, suggesting that the catalytic mechanism of the PLB is similar to that of cytosolic phospholipase A₂ (cPLA₂), found in mammalian tissues.

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The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is essential for the biosynthesis of complex carbohydrates such as hyaluronan in many cell types, and is required for detoxification of toxic compounds in the liver. We previously defined the 714 bp 5′-flanking region of the UGDH gene as the core promoter, with putative negative regulatory elements residing in the region upstream of it. In the present study, we delineated the region from nucleotide positions −1057 to −957 on the UGDH promoter to be responsible for the repression of promoter activity. A mutation at nucleotide −1003, which is contained within a motif predicted to be the response element for peroxisome proliferator receptor α (PPARα), abolished the suppression effect. DNA-protein interaction was observed at this motif by electrophoretic mobility shift assay. The proteins interacting with the PPRE-like repressor motif were purified by biotin-labeled DNA affinity chromatography. Subsequently, MALDI-TOF identified the purified proteins as a 62-kDa zinc finger and a 42-kDa β-actin protein. Hence in this study we report the presence of an inhibitory cis-element in the distal region of the UGDH promoter that interacts with putative transcriptional repressors for the negative regulation of the UGDH gene.

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1,2-α-Mannosidase catalyzes the specific cleavage of 1,2-α-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-α-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I α-mannosidase were conserved. Expression of the full length of 1,2-α-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-α-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 °C. With Man₉GlcNAc₂ as the substrate, Man₅GlcNAc₂ finally accumulated while hydrolysis of the 1,2-α-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-α-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.

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The effects of a hot water extract of the stem of Stevia rebaudiana on the smooth muscle of isolated guinea pig ileum were investigated. The butyl alcohol layer of the extract antagonized the contractions of the isolated guinea pig ileum induced by histamine (1×10⁻⁵ M) and acetylcholine (1×10⁻⁵ M) in a concentration-dependent manner. The butyl alcohol layer of the extract also showed inhibition of CaCl₂ (1×10⁻³–3.8×10⁻¹ M)-induced contractions. The antagonism of the extract was considered to be non-specific, but this action might be related to an influx of extracellular Ca²⁺.
With column chromatography preparation, the active component was assumed to be as stevioside. The antagonistic effects exerted by the stem extract of Stevia rebaudiana contributed to the gastroprotective activity of the extract in animals fed dietary histamine.

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Bacillus stearothermophilus SA0301 produces an extracellular oligo-1,6-glucosidase (bsO16G) that also hydrolyzes p-nitrophenyl α-D-glucoside (Tonozuka et al., J. Appl. Glycosci., 45, 397–400 (1998)). We cloned a gene for an enzyme hydrolyzing p-nitrophenyl α-D-glucoside, which was different from the one mentioned above, from B. stearothermophilus SA0301. The k₀⁄K_m values of bsO16G for isomaltotriose and isomaltose were 13.2 and 1.39 s⁻¹·mM⁻¹ respectively, while the newly cloned enzyme did not hydrolyze isomaltotriose, and the k₀⁄K_m value for isomaltose was 0.81 s⁻¹·mM⁻¹. The primary structure of the cloned enzyme more closely resembled those of trehalose-6-phosphate hydrolases than those of oligo-1,6-glucosidases, and the cloned enzyme hydrolyzed trehalose 6-phosphate. An open reading frame encoding a protein homologous to the trehalose-specific IIBC component of the phopshotransferase system was also found upstream of the gene for this enzyme.

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3,4-Dihydroxyacetophenone (3,4-DHAP) was evaluated for antimelanogenic activity. The tyrosinase inhibitory action by 3,4-DHAP using mushroom tyrosinase revealed a strong inhibitory effect. To further explore this matter, inhibition of tyrosinase and melanin content was measured in B16 melanoma cells (B16 cells). Further, tyrosinase and microphthalmia transcription factor (MITF) protein levels were determined by the Western blot method. Additionally, tyrosinase and MITF protein levels were reduced by 3,4-DHAP. Our data indicate that the antimelanogenic activity of 3,4-DHAP was probably due to its inhibition of tyrosinase activity and the suppression of tyrosinase and MITF protein levels.

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An extracellular phospholipase C was partially purified from Pseudomonas sp. strain KS3.2. The enzyme was composed of an approximately 18-kDa peptide. Maximal enzyme activity was found at pH 7.2 and 50 °C. The enzyme retained activity between pH 8 and 9, and 50% activity at about 52 °C for 30 min. The enzyme sample showed the highest activity on phosphatidylcholine and low activity toward other phospholipids.

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A putative endo-β-1,4-D-galactanase gene of Thermotoga maritima was cloned and overexpressed in Escherichia coli. The recombinant enzyme hydrolyzed pectic galactans and produced D-galactose, β-1,4-D-galactobiose, β-1,4-D-galactotriose, and β-1,4-D-galactotetraose. The enzyme displayed optimum activity at 90 °C and pH 7.0. It was slowly inactivated above pH 8.0 and below pH 5.0 and stable at temperatures up to 80 °C.

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The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.

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A water-soluble and neutral polysaccharide was extracted from the current pseudobulbs of Oncidium “Gower Ramsey” during the early inflorescence stage (flower stalk less than 4 cm) by hot water, precipitated with ethanol, and purified with an anion exchanger. From the data of monosaccharide composition and linkage and anomeric configuration analyses, the polysaccharide was identified as a linear β-1→4 linked mannan.

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A cDNA encoding a novel heptahelical receptor from the prothoracic glands of the silkworm, Bombyx mori was cloned and sequenced during screening of a prothoracicotropic hormone (PTTH) receptor. Orthologs of this receptor are found not only in insects, but also in the vertebrates. In B. mori, ubiquitous expression of the mRNA was observed in the larva. Also, a higher expression level in the prothoracic glands was observed before molting and metamorphosis and was impaired after pupal molting. But, further analysis is required to confirm whether this receptor cDNA encodes the PTTH receptor.

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Chitosanase (ChoA) from Mitsuaria chitosanitabida 3001 was successfully evolved with secretion efficiency and thermal stability. The inactive ChoA mutant (G151D) gene was used to mutate by an error-prone PCR technique and mutant genes that restored chitosanase activity were isolated. Two desirable mutants, designated M5S and M7T, were isolated. Two amino acids, Leu74 and Val75, in the signal peptide of ChoA were changed to Gln and Ile respectively in the M7T mutant, in addition to the G151D mutation. The L74Q/V75I double ChoA mutant was 1.5-fold higher in specific activity than wild-type ChoA due to efficient secretion of ChoA. One amino acid Asn222 was changed to Ser in the M5S mutant in addition to the G151D mutation. The N222S single ChoA mutant was 1.2-fold higher in specific activity and showed a 17% increase in thermal stability at 50 °C as compared with wild-type ChoA. This is the first study to achieve an evolutional increase in enzyme capability among chitosanses.

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Conjugated linoleic acid (CLA), a mixture of positional and geometric isomers of linoleic acid, has attracted considerable attention because of its potentially beneficial biologic effects both in vitro and in vivo. Our results clearly show the specific action of the 10trans,12cis-CLA isomer against hyperlipidemia and obesity in obese Otsuka Long-Evans Tokushima Fatty (OLETF) rats. After 2 weeks of feeding with 10t,12c-CLA, but not 9cis,11trans-CLA, abdominal adipose tissue weight and serum and hepatic lipid levels in OLETF rats were lower than those in linoleic acid-fed rats. These effects were attributable to suppressed fatty acid synthesis and enhanced fatty acid beta oxidation in the liver on a 10t,12c-CLA diet. Additionally, we showed that mRNA expression of fatty acid synthase, carnitine palmitoyltransferase, leptin, and sterol regulatory element binding protein-1 was also regulated by 10t,12c-CLA. We suppose that 10t,12c-CLA reveals hypolipidemic and anti-obese activity through the alteration of mRNA expressions in the liver and white adipose tissue.

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The safety and effectiveness were examined of the spirulina alga on bone metabolism in ovariectomized estrogen-deficient rats and hindlimb-unloaded mice. The dosage range was from an amount equal to that recommended in so-called health foods for humans (0.08 g/kg BW/day) to a 100-fold higher dose. The bone mineral density (BMD) of the whole femur and tibia of ovariectomized rats in the any spirulina-treated groups was not significantly different from that of the ovariectomized group, although BMD of the distal femur and proximal tibia was significantly lower in the spirulina-treated groups than in the ovariectomized group after a 6 week-experimental period. BMD of the femur and tibia was not affected by treatment with any dose of spirulina in hindlimb-unloaded mice. These results suggest that the intake of spirulina decreased BMD in the trabecular bone of rodents under estrogen-deficient conditions.

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The interaction of (+)-catechin with a lipid bilayer was examined by the spin probe method. The spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was dissolved in an aqueous dipalmitoylphosphatidylcholine (DPPC) dispersion containing (+)-catechin. The temperature dependence of the TEMPO parameter was measured. The increase of this parameter due to pretransition was eliminated by the addition of (+)-catechin, suggesting that it was adsorbed to the lipid membrane surface in the gel state, which hindered the change of the membrane from a flat to wavy structure. In the temperature region of the main transition, the TEMPO parameter increased rapidly, then gradually with increasing temperature, which could be explained by the eutectic phase diagram. The rotational correlation time of a spin probe 16-doxylstearic acid and the order parameter of 5-doxylstearic acid in the aqueous dispersion system of egg yolk phosphatidylcholine revealed that the motion of the alkyl chain in the liquid crystal state was hindered in the center of the membrane as well as near the surface by the adsorption of (+)-catechin.

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We investigated the effect of lactic acid bacteria (LAB) isolated from eight samples of commercially available Nozawana-zuke, a traditional Japanese pickle, on cytokine expression by mouse spleen cell cultures. The 12 isolated strains of LAB (Nz1–Nz12), which were identified as genus Lactobacillus or Leuconostoc by the API50CHL test, enhanced the expression of interferon (IFN)-γ with 6 h of culture. Ten of these 12 LAB, particularly Nz8, enhanced interleukin (IL)-12 p40 expression. The actinase E- and Benzonase-treated or untreated cell wall fraction of Nz8 enhanced both IFN-γ and IL-12 p40 expression, while the cell plasma fraction had little effect. In the presence of anti-toll like receptor 4 antibody, the effect of the cell wall fraction of Nz8 was significantly abrogated. These results suggest that some LAB from Nozawana-zuke have a T helper 1-type immunoenhancing effect.

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We investigated the hypolipidemic and antioxidative effects on male ICR mice of a glycoprotein isolated from Rhus verniciflua Stokes (RVS) fruit. The administration of the RVS glycoprotein (100 mg/kg) for two weeks resulted in a significant decrease in such plasma lipid levels as total cholesterol (TC), triglyceride (TG), and low-density lipoprotein (LDL). The levels of TC, TG and LDL in the hyperlipidemic model were significantly increased, whereas the high-density lipoprotein (HDL) level was considerably decreased. The 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity and the level of thiobarbituric acid-reactive substances (TBARS) were significantly elevated, whereas the production of nitric oxide (NO) was diminished. Moreover, the administration of the RVS glycoprotein prior to inducing hyperlipidemic mice suppressed the increase in the plasma lipid levels (TC, TG and LDL), and decrease in the HDL level in Triton WR-1339-induced hyperlipidemic mice. Furthermore, the RVS glycoprotein significantly inhibited the activity of HMG-CoA reductase and the levels of TBARS in the hyperlipidemic mice. In addition, the activities of detoxicant enzymes [catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx)] were gradually augmented after a supplement with the RVS glycoprotein. The results suggest that the RVS glycoprotein would be effective in preventing an increase in the plasma lipid levels and in improving the antioxidant levels. This protein might be useful as a therapeutic agent.

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The oxidation processes of linoleic acid in the presence of ferulic acid, and 1-pentyl, 1-hexyl and 1-heptyl ferulates were observed at various temperatures and different molar ratios of each additive to linoleic acid. The processes were analyzed based on a kinetic equation of the autocatalytic type to evaluate the oxidative rate constant, k, and the kinetic parameter, Y₀, by which the initiation period for the oxidation of linoleic acid was mainly governed. The k values for linoleic acid mixed with each of the alkyl ferulates were smaller than that for linoleic acid mixed with ferulic acid. The greater suppressive effect of the alkyl ferulates would be ascribable to their higher solubility in linoleic acid. Both the activation energy, E, and the frequency factor, k₀, for the oxidation of linoleic acid mixed with ferulic acid or pentyl ferulate decreased with increasing molar ratio of the additive to linoleic acid.

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Oyster extract was prepared by hydrolysis of oyster protein with proteases, Aloase (a protease from Bacillus subtilis), and Pancitase (a protease from Aspergillus oryzae). Rats were fed a diet containing 20% casein (the control diet) or 15% casein and 5% oyster extract (the oyster extract diet) as the protein source. The oyster extract diet exerted a significant reduction in serum cholesterol and liver triglyceride concentrations as compared with the control diet in Sprague-Dawley (SD) rats fed cholesterol-supplemented diets for 4 weeks. The activities of cytosolic fatty acid synthase and glucose-6-phosphate dehydrogenase were significantly lower in the oyster extract group than in the control group in the liver of SD rats. Hepatic cholesterol and triglyceride concentrations were significantly lower in spontaneously hypertensive (SH) rats and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, type 2 diabetic rats, fed the oyster extract diet, for 4 weeks and 4 months respectively, than in those fed the control diet in the cholesterol-free diet. Blood pressure was significantly lower in the oyster extract group than in the control group at the 2nd and 4th weeks after the beginning of feeding experimental diets in SH rats. These results suggest that oyster extract prepared by hydrolysis of oyster induces triglyceride-lowering activity in the liver through a decrease in hepatic lipogenesis in SD rats, and that it exerts the antihypertensive effect in SH rats.

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We examined the participation of the superoxide anion radical (O₂⁻) in the beneficial effects of L-ascorbic acid on heat-induced fish gel (Kamaboko). The generation of a thiyl radical (S·) in glutathione, ovalbumin, and actomyosin was examined by electron spin resonance spectroscopy coupled with spin trapping. O₂⁻ was provided by the photoactivation of riboflavin. The typical line shape for S· was observed with the glutathione and ovalbumin samples. A signal different from that for S· was detected with the actomyosin sample, and its intensity markedly decreased when the SH groups of actomyosin had been modified. The signal was eliminated when superoxide dismutase was added, but unaffected when catalase or an equivalent amount of heat-inactivated superoxide dismutase or catalase were added. These results suggest that S· in actomyosin was produced by the reaction with O₂⁻ and that the beneficial effects of L-ascorbic acid are due to a different mechanism in Kamaboko from that in bread.

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Peptides showing inhibitory activity against the angiotensin I-converting enzyme (ACE) were investigated from the fibroin fraction of discarded silk fabric. Fibroin, which was hydrolyzed with alcalase after partial hydrolysis with hot aqueous 40% CaCl₂, released two major active peptides showing ACE-inhibitory activity. The two peptides were identified as glycyl-valyl-glycyl-tyrosine (GVGY) and glycyl-valyl-glycyl-alanyl-glycyl-tyrosine (GVGAGY) by analyses with a protein sequencer and LC/MS/MS. GVGY, whose ACE-inhibitory activity has not previously been reported, showed a blood pressure-depressing effect on spontaneously hypertensive rat (SHR).

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We investigated whether lowering food intake by high phosphorus (P) diet influenced parathyroid hormone (PTH) actions, bone turnover markers, and kidney mineral concentration in rats. Rats in two of the three groups were respectively given free access to a control diet (C group) and a high P diet (HP group) for 21 days. Rats in another group (PF group) were pair-fed the control diet with the HP group. Compared to the C and PF groups, serum PTH concentration, urinary C-terminal telopeptide of type I collagen excretion, and kidney calcium and P concentrations were significantly higher in the HP group. Urinary excretion of cAMP was significantly lower in the HP group than in the C and PF groups. These results suggested that high P diet decreased PTH action in the kidney and increased bone resorption and kidney mineral concentrations independently of lowering food intake.

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We have isolated a difructose anhydride III (DFA III)-assimilating bacterium, Ruminococcus productus AHU1760, from human. After an acclimation period of 1 week, male Sprague-Dawley rats (5 weeks old) were divided into four groups (control diet, R. productus diet, DFA III diet, and R. productus + DFA III diet; n=8) and fed the assigned test diets for 2 weeks. The viable count of administered R. productus was 4.9×10⁷ CFU/d in R. productus-fed rats and 4.7×10⁷ CFU/d in R. productus + DFA III-fed rats. Survival in cecal content of this strain was confirmed by randomly amplified polymorphic DNA. The ratio of secondary bile acids in feces in R. productus + DFA III-fed rats decreased the same as that in rats fed only DFA III. The viable count of lactobacilli and bifidobacteria, known as beneficial bacteria, increased more in R. productus + DFA III-fed rats than in control or R. productus-fed rats. A combination of R. productus and DFA III might improve the balance of intestinal microbiota to a healthier condition.

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Bluegill-degrading bacteria were isolated from various environmental sources. Brevibacillus sp. BGM1 degraded bluegill efficiently at 50 °C, and its culture supernatant showed the highest peptide and amino acid concentrations as trichloroacetic acid (TCA) soluble fraction (ASF) (10.7 mg/ml) of all supernatants obtained with bluegill as a substrate. Strain BGM1 secreted a protease(s) into the medium, and the concentration of peptides and amino acids gradually increased. The fertile effect of the degraded bluegill products (DGP) on Brassica rapa was also investigated. The root hair density of B. rapa grown with DGP at a concentration of 30 μg peptides and amino acids/ml was about 1.7 times higher than when grown with the same concentration of undegraded bluegill. DGP was shown to increase root hair numbers and adventitious root formation. The results of this study suggest that a specific peptide(s) for promotion of root hair is produced from the order Perciformes with a protease(s) from BGM1.

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We analyzed sequences of the D1D2 domain of the 26S ribosomal RNA gene (26S rDNA sequence), and the region of internal transcribed spacer 1, 5.8S ribosomal RNA gene and internal transcribed spacer 2 (ITS sequence) of the miso and soy sauce fermentation yeasts, Candida etchellsii and Candida versatilis, in order to evaluate the usefulness of this sequence analysis for identification and typing of these two species. In the 26S rDNA sequence method, the numbers of base substitutions among C. etchellsii strains were up to 2 in 482 bp (99.6% similarity), and they were divided into three types (types A, B, and C). Those of C. versatilis strains were also up to 2 in 521 bp (99.6% similarity) and they were divided into three types (types 1, 2, and 3). In the ITS sequence method, those of C. etchellsii strains were zero in 433 bp (type a, 100% similarity). Those of C. versatilis were 5 in 409 bp (98.8% similarity), divided into 4 types (types I, II, III and IV). It was found that molecular methods based on the sequences of the 26S rDNA D1D2 domain and the ITS region were rapid and precise compared with the physiological method for the identification and typing of these two species.

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The gene encoding Leifsonia alcohol dehydrogenase (LSADH), a useful biocatalyst for producing (R)-chiral alcohols, was cloned from the genomic DNA of Leifsonia sp. S749. The gene contained an opening reading frame consisting of 756 nucleotides corresponding to 251 amino acid residues. The subunit molecular weight was calculated to be 24,999, which was consistent with that determined by polyacrylamide gel electrophoresis. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by three column chromatographies. The predicted amino acid sequence displayed 30–50% homology to known short chain alcohol dehydrogenase/reductases (SDRs); moreover, the NADH-binding site and the three catalytic residues in SDRs were conserved. The recombinant E. coli cells which overexpressed lsadh produced (R)-form chiral alcohols from ketones using 2-propanol as a hydrogen donor with the highest level of productivity ever reported and enantiomeric excess (e.e.).

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Bacillus subtilis strain FP-133, isolated from a fermented fish paste, synthesized two novel halotolerant extracellular proteases (expro-I and expro-II), showing activity and stability at concentrations of 0–20% (w/v) NaCl. Each protease was purified to homogeneity and characterized. The purified expro-I was a non-alkaline serine protease with an optimum pH of 7.5, although most serine proteases from Bacillus strains act at the alkaline side. The molecular mass of expro-I was 29 kDa. The purified expro-II was a metalloprotease with a molecular mass of 34 kDa. It was activated by Fe²⁺, which has never been reported as a bacterial protease activator. At a concentration of 7.5% (w/v) NaCl, both proteases preferred animal proteins to vegetable proteins as natural substrates. In addition, under saline conditions, expro-I and II showed high catalytic activity toward gelatin and casein respectively.

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Eikenella corrodens is known not only as one of the periodontopathogenic bacteria but also as a pathogen associated with many infectious diseases of humans. Dental plaque is a complex biofilm community comprised of many bacterial species. E. corrodens has a lectin on its cell surface that is thought to be involved in its pathogenicity. In this study, we found that E. corrodens forms a biofilm on a polystyrene surface. A biofilm was formed at the bottom of the wells in microtiter plates after 24 h. Microcolonies were observed as the amount of biofilm became larger. When anaerobic respiration was repressed due to nitrate limitation, the biofilm formed only at the air–water interface. Strain 1073 and HU, which have higher lectin activity, formed a biofilm more effectively than other strains. Biofilm formation was repressed by the addition of N-acetyl-D-galactosamine. These results suggest that the lectin on the surface of E. corrodens might be involved in biofilm formation.

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Glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 can form theanine from glutamic acid and ethylamine in a mixture where yeast fermentation of sugar is coupled for ATP regeneration (coupled fermentation with energy transfer). From a genomic DNA library of P. taetrolens Y-30, a clone containing 6 kbp insertional DNA fragment was selected by the PCR screening technique with specific oligonucleotide primers for the GS gene. The fragment had an open reading frame of the GS gene encoding a protein of 468 amino acids (molecular mass, 52 kDa). The deduced amino acid sequence showed a significant homology with that of P. syringae pv. tomato GS (97%), and all the amino acid residues were fully conserved, which concern with catalytic activity in other bacterial GS. A tyrosine residue for adenylylation of GS was also found, and in vivo adenylylation was confirmed in P. taetrolens Y-30. The isolated GS gene was ligated into an expression vector (pET21a), and expressed in Escherichia coli AD494 (DE3). The enzyme productivity in the expression system was 30-fold higher than that in P. taetrolens Y-30. Recombinant GS had the same properties as those of unnadenylylated intrinsic GS, and formed theanine in the mixture of coupled fermentation with energy transfer.

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We tested the effects of several combinations of bait and fish components of the yeast two-hybrid detection system for estrogenic activity. A combination of the full-length human estrogen receptor α with the nuclear receptor-binding domain of co-activator steroid receptor co-activator-1 (SRC-1) or transcriptional intermediate factor-2 (TIF-2) was most effective for estrogen-dependent induction of the chromosome-integrated UAS_GAL-CYC1^p-lacZ reporter construct among the two-hybrid systems so far tested.

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We investigated the degradation pathways and kinetics of 2,4-dichlorophenol (DCP) by an endemic soil fungus, Mortierella sp. (Zygomycetes). Mortierella sp. degraded 32% of added DCP (final concentration, 250 μM) within 1 h. We identified four aromatic metabolites and found two DCP degradation pathways (a hydroxylation pathway and a dechlorination pathway). This is the first report of a dechlorination pathway in Zygomycetes.

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Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production.

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