In the course of characteristic screening for antitumor substances, we isolated novel inhibitors against telomerase and the expression of molecular chaperone GRP78, designated telomestatin and versipelostatin, respectively. Telomestatin specifically acts on telomere sequence to stabilize the specific DNA structure called G-quadruplex and shows unique biological aspects that induce telomere dysfunction. Versipelostatin decreased the expression of GRP78 accompanied by a high level of cell death under glucose deprived conditions that mimicked the circumstances of a solid tumor.
Furthermore, a screening program for glutamate receptor antagonist to treat brain stroke resulted in the isolation of an AMPA/NMDA antagonist kaitocephalin. Kaitocephalin potently protected rat hippocampal neurons from kainate, AMPA, and NMDA excitotoxicity. It also inhibited the Ca²⁺ influx elicited by AMPA and NMDA, but not by kainate.
Detailed analysis for the mode of action mechanism of these compounds indicated novel and unique biological phenomena not revealed by molecular biological techniques.

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We synthesized new chiral fluorescence labeling reagents having a 2,3-anthracenedicarboximide group from D-glucosamine, and it was possible to introduce target alcohols at the anomeric positions of the reagents with β-selectivity by glycosidations. Especially, it was possible to use methyl glycoside reagent as a glycosyl donor with a Lewis acid and microwave irradiation, and it gave selectively β-glycoside while the reaction without microwave irradiation gave α- and β-mixed glycosides. Those reagents showed very high chiral discrimination ability, and they made it possible to separate the eight stereoisomers of 4,8,12,16-tetramethylheptadecanol by HPLC after derivatizations.

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The stereochemistry and efficiency of an allyl cyanate-to-isocyanate rearrangement for the construction of quaternary stereocenter with nitrogen substituent was investigated by the synthesis of (R)-α-methylphenylalanine. The rearrangement was found to be stereospecific, and the chirality of allyl carbamate was transferred to that of the quaternary carbon bearing isocyanate group. These results establish that an allyl cyanate-to-isocyanate rearrangement is a useful method for the synthesis of natural products, that contain the quaternary carbon with nitrogen substituent.

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Two distinct electroantennographycally active (EAG-active) components, A and B, and a weakly active component C were found in a solvent extract from virgin females of the Ishigaki strain of the tussock moth, Orgyia postica (Walker). Components A, B, and C were found in the extract of the females at 4.0, 0.5, and 4.0 ng/female respectively. Components A, B, and C were identified as (6Z,9Z,11S,12S)-11,12-epoxyhenicosa-6,9-diene [(11S,12S)-1: posticlure], (6Z)-henicos-6-en-11-one (2), and (6Z,9Z)-henicosa-6,9-diene (3), respectively. Component B was absent in the extract from the Okinawa strain, in which components A and C were present at 2.0 and 1.5 ng/female respectively. (11S,12S)-1 and the racemic mixture showed attractiveness for both the Okinawa and Ishigaki strains, whereas (11R,12R)-1 did not. The addition of 2 significantly reduced the trap catches with (11S,12S)-1 on the Okinawa strain which lacked 2, while there was no significant inhibitory effect on the Ishigaki strain. The addition of 3 to (11S,12S)-1 did not significantly affect trap catches at Ishigaki or Okinawa. This confirmed that the attractant pheromone of O. postica of the Ishigaki strain is also (11S,12S)-1.

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Dou-chi, a traditional soybean food fermented with Aspergillus sp., is usually used as a seasoning in Chinese food, and has also been used as a folk medicine in China and Taiwan. As 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavengers, four phenol compounds, one isoflavanone, eight isoflavones and one 4-pyrone have been isolated from dou-chi. Among these fourteen compounds, 3′-hydroxydaidzein, dihydrodaidzein and a 4-pyrone compound have not yet been isolated from soybean miso. The structure of the novel 4-pyrone compound, 3-((E)-2-carboxyethenyl)-5-(4-hydroxyphenyl)-4-pyrone-2-carboxylic acid was elucidated by using the same compound as that obtained from the biotransformation of daidzein. 3′-Hydroxydaidzein showed as high DPPH radical-scavenging activity as that of α-tocopherol, and 6-hydroxydaidzein had mushroom tyrosinase inhibitory activity with an IC₅₀ value of 10 μM. The order of estrogenic activity is as follows: genistein > daidzein >> 3′-hydroxydaidzein > 8-hydroxygenistein, using a green fluorescent protein expression system. Furthermore, the contents of isoflavones in the fermentation process of dou-chi were measured.

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(3Z,6Z,9S,10R)-9,10-Epoxy-3,6-henicosadiene (1) and (3Z,6Z,9S,10R)-9,10-epoxy-1,3,6-henicosatriene (5), pheromone components of the female fall webworm moth (Hyphantria cunea Drury), were synthesized by starting from (2S,3R)-2,3-epoxy-4-t-butyldimethylsilyloxy-1-butanol (8). Epoxide 8 was prepared by employing lipase-catalyzed asymmetric acetylation of (±)-8 as the key optical resolution step.

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Two novel antifungal compounds were isolated from a culture broth of Sterile Dark, an unidentified fungus isolated from the rhizosphere of wheat grown in a continuous cropping field. These compounds were elucidated to be phthalide-based compounds by spectroscopic analyses.

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An arginine residue in loop 4 connecting β strand 4 and α-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg²⁰⁴ in xylanase A from Bacillus halodurans C-125 (XynA) and Arg¹⁹⁶ in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k_cat⁄K_m and the pH-k_cat relationships of these mutant enzymes were measured. The pK_e2 and pK_es2 values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK_es2 value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK_a value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.

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A bacterium, identified as Microbacterium liquefaciens MIM-CG-9535-I, was isolated from a soil sample taken from the industrial site of a gelatin manufacturer. A new type of protease, which restrictively decomposes gelatin at one or two positions, was purified from the bacterial culture. The molecular mass of the purified enzyme was 21 kDa by SDS–polyacrylamide gel electrophoresis. The purified enzyme specifically degraded the α-chain of gelatin with a molecular weight of 100 kDa into two peptides of 60 kDa and 40 kDa. Native collagen was not a substrate for the enzyme.

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The sarcosine oxidase gene and nearby genes from Corynebacterium sp. U-96 were determined. The genes for serine hydroxymethyltransferase, the β, δ, α, and γ subunits of sarcosine oxidase, serine dehydratase, and 10-formyltetrahydrofolate hydrolase are arranged in this order. This suggests that the bacteria contain a cluster of genes for the catabolism of sarcosine to pyruvate. The possibility that the gene cluster is a merit for the cellular energy demands of the bacteria is discussed. Functional expression of sarcosine oxidase in Escherichia coli was accomplished, but the β subunit and the βδ complex were expressed at a low level as a soluble protein.

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Rice (Oryza sativa var. Nipponbare) was transformed with an artificial avidin gene. The features of this construct are as follows: (1) a signal peptide sequence derived from barley alpha amylase was added at the N-terminal region, (2) codon usage of the gene was optimized for rice, and (3) the gene was driven by rice glutelin GluB-1, an endosperm-specific promoter. Avidin was produced in the grain of the transgenic rice but not in the leaves. The concentration of avidin in the kernels was about 1,800 ppm. All larvae of the confused flour beetle (Tribolium confusum) and Angoumois grain moth (Sitotroga cerealella) died when fed transgenic avidin rice powder or kernels, respectively, whereas most of the test insects developed into adults when they were fed a nontransgenic rice control diet. Avidin extracted from the transgenic rice kernel lost most biotin-binding activity after 5 min heating at 95 °C.

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The ZKT gene from Arabidopsis encodes a polypeptide of 335 amino acid residues, with a calculated molecular mass of 37.4 kDa. ZKT is a member of a novel protein family present in the plant kingdom, which contains a PDZ, a K-box, and a TPR motif. A BLAST search indicated that the ZKT gene is a single gene in Arabidopsis and that ZKT homologs are present in soybean and rice but not in animals. The level of ZKT mRNA decreased after wounding. Antisera from rabbit immunized with recommbinant ZKT protein recognized a protein of 37 kDa in Arabidopsis. Western analysis with anti-ZKT antibody indicated that the level of ZKT protein does not change after wounding. The ZKT protein has consensus sequence motifs for phosphorylation. Immunoprecipitation with anti-ZKT antibody and western analysis with anti-phosphoamino acid antibody indicated that the ZKT protein is phosophorylated at the threonine and serine residues after wounding. These results suggest that the ZKT protein may act as a molecular adaptor regulated by phosphorylation in wound responses.

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The heat shock cognate 70-4 protein gene promoter (HSC70-4p) from Bombyx mori (BmHSC70-4p) is an ideal candidate for the transgenic silkworm due to its high transcriptional activity, and the homologous region 3 from Bombyx mori nucleopolyhedrovirus (BmNPVhr3) functions as an enhancer for several promoters. Using luciferase as a reporter gene and transient expression system in vivo and in vitro, we found that BmNPVhr3 can significantly increase the transcriptional activity of BmHSC70-4p and HSC70-4p from Bombyx mandarina (BmandHSC70-4p). Moreover, the transcriptional activity of the combination of BmHSC70-4p and BmNPVhr3 changed with developmental stages and hormone titers. These results suggest that the combination of BmHSC70-4 promoter and BmNPVhr3 enhancer is more effective candidate for the transgene or stable cell expression system in Bombyx mori.

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Three Δ6 desaturase-defective mutants, designated YB214, HR95, and ST66, were newly isolated from Mortierella alpina 1S-4, after treating wild-type spores with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). These three mutants and Mut49, isolated previously, are capable of accumulating 5,11,14-cis-eicosatrienoic acid (20:3Δ5). Two functional Δ6 desaturases (Δ6I and Δ6II) were found to exist in M. alpina 1S-4. The mutation sites on the Δ6I gene in the Δ6 desaturase-defective mutants were identified. The mutations each resulted in an amino acid replacement (W314Stop, T375K, and G390D) in Δ6I from ST66, HR95, and YB214 respectively, and uncorrected transcription of the Δ6I gene in Mut49 was caused by disappearance of the GT-terminal of the second intron, resulting in low Δ6 desaturation activity in these mutants. On the other hand, there was no mutation site on the Δ6II genes of the mutants.

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It is generally believed that only L-amino acids are acceptable in protein synthesis, though some D-amino acids, including D-tyrosine, D-aspartate, and D-tryptophan are known to be bound enzymatically to tRNAs. In this report, we newly show that D-histidine and D-lysine are also able to be the substrates of respective Escherichia coli aminoacyl-tRNA synthetases.

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The jasmonic acid (JA)-responsive gene RERJ1 isolated from suspension-cultured rice cells encodes a transcription factor with a basic helix-loop-helix motif. In this study, we found that RERJ1 is also expressed in rice plants in response to JA, and that its expression in rice leaves is up-regulated by exposure to wounding and drought stress. It is also suggested that JA but not abscisic acid is involved in the up-regulation of RERJ1 expression caused by wounding and drought stress.

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For study of the self-association of the product of φ29 gene 1, one variant which has a substitution at the 71^st amino acid was used. By glycerol gradient sedimentation, the product of wild-type gene 1 existed both as large aggregate and oligomer, whereas the variant was detected as a single peak of monomer size. According to experiments using His-tagged proteins and Ni-NTA magnetic beads, the variant made only a little self-associated complex. From these results, a site essential for self-association was suggested to exist close to the carboxyl terminus of the product of φ29 gene 1.

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XIP-I and TAXI-I are wheat (Triticum aestivum L) grain proteins that inhibit microbial xylanases used in food processing. Although their biochemical properties and structural features were established recently, very little is known about their expression and their family members in wheat plants. To clarify the role of these xylanase inhibitor proteins in plant defense, we examined the expression of the XIP-type genes in response to a variety of biotic and abiotic signals. Although Xip-I was not expressed in flowering spikelets inoculated with Fusarium graminearum, transcription of Xip-I was greatly enhanced in Erysiphe graminis-infected leaves. Thus, unlike Taxi-I, Xip-I is pathogen-inducible, and unlike Taxi-III and Taxi-IV, its expression depends on the type of the pathogen and/or infected tissue. Xip-I was expressed when the leaves were wounded, and its expression was significantly elevated by treatment with methyl jasmonate (MeJA). The different expression profiles of XIP- and TAXI-type genes suggest distinct roles in plant defense.

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A hot-water extract of adzuki was obtained by boiling beans of adzuki (Vigna angularis). This hot-water extract was fractionated using HP-20 column chromatography. Its distilled water fraction (WEx) was found to stimulate tyrosinase activity in cultured mouse B16 melanoma cells and hair color pigmentation in C3H mice. At concentrations of 1–3 mg/ml, WEx stimulated melanogenesis without inhibiting cell growth. During this effect, WEx activated tyrosinase-inducing activity in the cells, but did not activate tyrosinase, which exists at an intracellular level. In this study, WEx increased cyclic adenosine-3′,5′-monophospate (cAMP) content in the cells and protein kinase A (PKA) activity, and stimulated translocation of cytosolic protein kinase C (PKC) to the membrane-bound PKC. These results suggest that the addition of WEx activates the adenylcyclase and protein kinase pathways and, as a result, stimulates melanogenesis. WEx was found to have pigmentation activity on hair color in C3H mice. It might be useful in anti-graying, protecting human skin from irradiation.

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Dioxins cause various adverse effects through transformation of aryl hydrocarbon receptor (AhR). In this study, we investigated whether black tea extract and its components, theaflavins, suppress AhR transformation in vitro. First, we confirmed that black tea extract strongly suppressed AhR transformation compared to green and oolong tea, although the catechin contents did not change significantly among the extracts. Then we isolated four theaflavins as active compounds from black tea leaves. They suppressed 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation in a dose-dependent manner. The IC₅₀ values of theaflavin, theaflavin-3-gallate, theaflavin-3′-gallate, and theaflavin-3,3′-digallate (Tfdg) were 4.5, 2.3, 2.2, and 0.7 μM, respectively. The suppressive effect of Tfdg was observed not only by pre-treatment but also by post-treatment. This suggests that theaflavins inhibit the binding of TCDD to the AhR and also the binding of the transformed AhR to the specific DNA-binding site as putative mechanisms.

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Total saponin of heat-processed ginseng (TSHG) stimulated the production of nitric oxide (NO) in interferon-γ (IFN-γ)-primed macrophages through the increased expression of inducible nitric oxide synthase (iNOS). However, TSHG by itself had a very weak effect on the NO synthesis without IFN-γ priming. The saponins of white ginseng inhibited the NO production in lipopolysaccharide (LPS)/IFN-γ activated macrophages rather than the stimulation of NO production found in IFN-γ primed macrophages. The NO production by TSHG-stimulated macrophages was inhibited by the NOS inhibitor (N^G-monomethyl-L-arginine (L-NMMA)) and nuclear factor-kappaB inhibitor (pyrrolidine dithiocarbamate (PDTC)). TSHG showed different serum-dependence from LPS on the activation of IFN-γ primed macrophages. This property of TSHG may explain the intensified anti-tumor properties of heat-processed ginseng through its immunostimulating activity.

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Dioxins induce adverse effects through transformation of the cytosolic aryl hydrocarbon receptor (AhR). Our previous study found that flavones and flavonols at dietary levels suppress AhR transformation. In the present study, we investigated whether 20 anthocyans dissolved in trifluoroacetic acid (TFA)–MeOH suppressed AhR transformation in a cell-free system and in Hepa-1c1c7 cells. Although four compounds at 50 μM suppressed 0.1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AhR transformation and their effects were dose-dependent in the cell-free system, they were ineffective at 0.5 μM, which is close to physiological concentration. Moreover, no anthocyan at 50 μM tested here suppressed 0.1 nM TCDD-induced AhR transformation in Hepa-1c1c7 cells. We also confirmed that protocatechuic acid and related compounds, which are possible metabolites of anthocyans, did not affect the transformation in the cell-free system. It is concluded that anthocyans are not suitable candidates for protection from dioxin toxicity.

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In order to elucidate the mechanism of the antihypertensive action of dried bonito (katsuobushi), we compared the effects of dried bonito extracts with those of captopril, an angiotensin I-converting enzyme (ACE) inhibitor, on aorta preparations isolated from rats. Dried bonito extracts (3×10⁻⁴ to 3×10⁻³ g/ml) more potently relaxed contractions induced by norepinephrine (10⁻⁷ M) than contractions induced by KCl (55.9 mM). Dried bonito extracts (3×10⁻³ g/ml) slightly inhibited 10⁻⁷ M angiotensin I-induced contractions. In contrast, captopril (10⁻⁸ to 10⁻⁷ M) did not affect 10⁻⁷ M norepinephrine- or 55.9 mM KCl-induced contractions, but a higher concentration of captopril (10⁻⁶ M) very slightly relaxed it. Captopril (10⁻⁸ to 10⁻⁶ M) markedly inhibited 10⁻⁷ M angiotensin I-induced contractions in a concentration-dependent manner. These results suggest that antihypertensive mechanism of action induced by dried bonito involves direct action on vascular smooth muscle in addition to ACE-inhibitory activity.

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Ovalbumin, a member of the serpin superfamily, is transformed into a thermostabilized form, S-ovalbumin, during storage of shell eggs or by an alkaline treatment of the isolated protein (ΔT_m=8 °C). As structural characteristics of S-ovalbumin, three serine residues (Ser164, Ser236 and Ser320) take the D-amino acid residue configuration, while the conformational change from non-thermostabilized native ovalbumin is very small (Yamasaki, M., Takahashi, N., and Hirose, M., J. Biol. Chem., 278, 35524–35530 (2003)). To assess the role of the structural characteristics on protein thermostabilization, ovalbumin and S-ovalbumin were denatured to eliminate the conformational modulation effects and then refolded. The denatured ovalbumin and S-ovalbumin were correctly refolded into the original non-denatured forms with the corresponding differential thermostability. There was essentially no difference in the disulfide structures of the native and refolded forms of ovalbumin and S-ovalbumin. These data are consistent with the view that the configuration inversion, which is the only chemical modification directly detected in S-ovalbumin so far, plays a central role in ovalbumin thermostabilization. The rate of refolding of S-ovalbumin was greater than that of ovalbumin, indicating the participation, at least in part, of an increased folding rate for thermodynamic stabilization.

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The Addition of a compound that lowers the intestinal uptake of fat and cholesterol might be an interesting strategy to reduce the risk of vascular disease. Partially hydrolyzed guar gum (PHGG) has been shown to have this effect in healthy volunteers after intake of a yogurt drink with 3 to 6% PHGG. In the present study a yogurt drink with 3% sunflower oil and 4% egg yolk was tested with 3% and 6% PHGG, and compared to a control without PHGG. Experiments were performed in a multi-compartmental model of the gastrointestinal tract, equipped to study the digestion and availability for absorption (bioaccessibility) of lipids. The results show that PHGG decreases the bioaccessibility of both fat and cholesterol in a dose-dependent manner. The bioaccessibility of fat was 79.4±1.7%, 70.8±2.5% and 60.1±1.1% for the control experiments and the experiments with 3% and 6% PHGG respectively. The bioaccessibility of cholesterol was 82.2±2.0%, 75.4±1.2% and 64.0±4.3% for the control and the experiments with 3% and 6% PHGG respectively. Additional experiments indicated that PHGG reduces bioaccessibility through the depletion flocculation mechanism. Depletion flocculation antagonizes the emulsification by bile salts and thus decreases lipolytic activity, resulting in a lower bioaccessibility of fat and cholesterol. Depletion flocculation with polymers might be an interesting mechanism, not described before, to reduce fat and cholesterol absorption.

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We evaluated the antioxidative activity of anthocyanins from an extract of the tuber of purple sweet potato (PSP) (Ipomoea batatas cultivar Ayamurasaki). Anthocyanins from PSP showed stronger 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity than anthocyanins from red cabbage, grape skin, elderberry, or purple corn, and eight major components of the anthocyanins from PSP showed higher levels of activity than ascorbic acid. In PSP anthocyanin-injected rats and PSP beverage-administered volunteers, DPPH radical-scavenging activity in the urine increased. The elevation of plasma transaminase activities induced by carbon tetrachloride was depressed in rats administered PSP anthocyanin solution. Two components, cyanidin 3-O-(2-O-(6-O-(E)-caffeoyl-β-D-glucopyranocyl)-β-D-glucopyranoide)-5-O-β-D-glucopyranoside and peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-β-D-glucopyranocyl)-β-D-glucopyranoide)-5-O-β-D-glucopyranoside, which were detected in the plasma, protected low density lipoprotein from oxidation at a physiological concentration. These results indicate that PSP anthocyanins have antioxidative activity in vivo as well as in vitro.

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Insulin receptor substrate-1 (IRS-1) plays a pivotal role in insulin signal transduction. It has been shown that the amino acids modulate insulin signaling at the level of IRS-1. Here we show that an amino acid unbalanced diet causes a reduction in serine phosphorylation as well as an elevation in insulin-induced tyrosine phosphorylation of IRS-1 in rat muscle. In fibroblasts and myotube cells, the effect of amino acid deprivation on IRS-1 phosphorylation was evident only when cells were pretreated with reagents causing hyperphosphorylation of serines of IRS-1. But, the target kinases of these reagents were not inactivated by amino acid deprivation, suggesting that amino acid deprivation activates serine/threonine phosphatase(s) of IRS-1. The phosphatases regulated by mammalian target of rapamycin do not appear to participate in the dephosphorylation either. These results suggest that amino acid deprivation dephosphorylates IRS-1 through unidentified serine/threonine phosphatases and thereby potentiates insulin signaling.

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We investigated the effects of dietary phosphorus (P) intake on the bone mineralization and calcium (Ca) absorption in adult female rats. Fifteen 16-wk-old female Wistar rats were divided into three groups, and respectively fed a low-P diet containing 0.15% P (LP), a control diet containing 0.5% P (C), and a high-P diet containing 1.5% P (HP) for 42 d. The apparent Ca absorption was significantly increased with decreasing dietary P level. The serum parathyroid hormone concentration was significantly lower in the LP group than in the C and HP groups. The serum osteocalcin concentration and urinary excretion of deoxypyridinoline were significantly higher in the HP groups than in the LP and C groups. The bone mineral density of the fifth lumbar vertebra was significantly increased with decreasing dietary P level. These results indicate that the low-P diet increased Ca absorption, this being effective for bone mineralization in adult female rats.

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Tea catechins, rich in (−)-epigallocatechin gallate and (−)-epicatechin gallate, or heat-treated tea catechins in which about 50% of the (−)-epigallocatechin gallate and (−)-epicatechin gallate in tea catechins was epimerized to (−)-gallocatechin gallate and (−)-catechin gallate, were fed to rats at 1% level for 23 d. Visceral fat deposition and the concentration of hepatic triacylglycerol were significantly lower in the tea catechin and heat-treated tea catechin groups than in the control group. The activities of fatty acid synthase and the malic enzyme in the liver cytosol were significantly lower in the two catechin groups than in the control group. In contrast, the activities of carnitine palmitoyltransferase and acyl-CoA oxidase in the liver homogenate were not significantly different among the three groups. These results suggest that the reduction in activities of enzymes related to hepatic fatty acid synthesis by the feeding of tea catechins or heat-treated tea catechins can cause reductions of hepatic triacylglycerol and possibly of visceral fat deposition.

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NADPH-dependent erythrose reductases (ERs) in erythritol-producing fungi, Trichosporonoides megachiliensis SNG-42, catalyze the reduction of D-erythrose. We previously characterized the biochemical properties of three isozymes of ERs (ER-I, ER-II, and ER-III). Using internal amino acid sequences of ER-III and ER-I with peptide mapping, we cloned three cDNAs (er1, 1121-bp (AB191474); er2, 1077-bp (AB191475); er3, 1119-bp (AB191476)). The er3 cDNA encoded a polypeptide 36,044 Da, and its deduced amino acid sequence was same as that of the native ER-III. The three recombinant enzymes expressed in Escherichia coli were purified to homogeneity. The recombinant enzymes of ER1, ER2, and ER3 showed similar electrophoretic properties to that of the native ER-I, ER-II, and ER-III on SDS– and Native– but not on IEF–PAGE. All three recombinant enzymes showed substrate specificity towards C-4 and C-3 aldehydes similar to that of the native ER-III. These results strongly suggest that cloned er1, er2, and er3 cDNAs encode erythrose reductases.

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We discovered FR207944 produced by Chaetomium sp. No. 217 in the course of screening for antifungal antibiotics from natural products. FR207944 is identical with fuscoatroside, described in the preceding paper as an anti-Aspergillus flavus agent. Determination of the relative stereochemistry of fuscoatroside was made formally by comparison with WF11605 (16-Oxo-FR207944). We confirmed the stereochemistry on the basis of single crystal X-ray analysis.

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Pseudomonas sp. strain AP-3 grows on benzoate, p-hydroxybenzoate, protocatechuate, and 2-aminophenol as sole carbon and energy source. This strain converted benzoate and p-hydroxybenzoate to catechol and protocatechuate respectively, which were metabolized via the ortho-cleavage pathway. The enzymes responsible for these reactions were shown to be inducible. In contrast, strain AP-3 constitutively expresses the enzymes involved in the metabolism of 2-aminophenol.

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The construction process of Bacillus subtilis strain with multiple mutations is accelerated by simultaneous use of a positive selection method. All the strains selected by neomycin acquired two simultaneous mutations at different genomic loci. The extremely low rate of false positives was accounted for by employing the improved method.

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