Oxygen electrodes and spectrophotometric analysis have been used to evaluate the contribution of H₂O₂, in addition to available chlorine, to the high redox potential of electrolyzed anode water (EAW) with potassium chloride as an electrolyte. H₂O₂ was added externally to EAW, and the reaction between H₂O₂ and the available chlorine in the water was examined. EAW has a low pH (2.5), a high concentration of dissolved oxygen, and extremely high redox potentials (19 mg/l and 1319 mV) when the available chlorine is at the concentration of about 580 μM. The addition of H₂O₂ to EAW led to H₂O₂ decomposition, and the amount of oxygen produced was equivalent to the amount of available chlorine. Oxygen production was reduced by ascorbic acid, and completely inhibited by 600 μM ascorbate. The rate of oxygen production was much affected by pH, and was slowest at or near pH 5.0. Rates were particularly high in alkaline solution. Absorbance at 235 nm (pH 3.0 and 5.0) and 292 nm (pH 10.0) decreased when H₂O₂ was added to the EAW at these pHs, and the extent of decrease was similar pH dependency to that of the oxygen production rate.
  Oxygen was not produced after H₂O₂ was added to EAW at pH 2.6 when available chlorine was absent, but oxygen was produced after potassium hypochlorite was added to such EAW. The oxygen production rates in EAW without available chlorine at pH 5.0 and 2.0, pH adjustment with KOH and HCl, respectively, were faster than the rate at pH 2.6, and fastest at pH 2.0.
  These results suggest that H₂O₂ or hydroxyl radicals derived from Fenton's reaction did not contribute to the high redox potential of EAW prepared with chlorine compounds as an electrolyte, so that the decomposition of H₂O₂ occurred rapidly with the reactions of chlorine and hypochlorite ions in EAW.

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  Tokyo Bay bottom sediments were analyzed for 2-ethyl-3-methylmaleimide, a degradation product of chlorophylls, which has been detected in ancient sediments. It was found in all sediments examined in concentrations of about 1 to 15 nmol/g- of dried sediment, and it was shown to be preserved for 100 years in the sediments. Its depth distribution agreed with that of the reported total organic carbon content of the sediments, reflecting a change in primary productivity. We concluded that this maleimide was produced under photic and oxygenic conditions in nature before the incorporation of photosynthesizing organisms into sediments.

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  Topsoil samples were collected from 36 different paddy fields in West Japan. Each soil sample was incubated with a basal salt-medium containing 0.2% OPPEO. Twelve samples possessed OPPEO-degrading activity, from which twelve cultures of OPPEO-degrading bacteria were isolated. The isolated bacteria grew on a medium containing 0.2% OPPEO as the sole carbon source, and OP2EO and OP3EO were accumulated in the medium under aerobic conditions. OP1EO and octylphenol, which have often been identified in surface water together with OP2EO, were not observed in this experiment. The bacterial isolates were gram negative and tentatively identified as Pseudomonas putida (10 isolates) and Burkholderia cepacia (one isolate) by BIOLOG and 16S rDNA RFLP analyses.

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  Tobacco YZI-1S cells exhibit a 150-fold greater resistance to the protoporphyrinogen oxidase (Protox)-inhibiting compound, S23142, from wild-type tobacco cells. To investigate the mechanism for this S23142 resistance, the protein level, enzymatic activity, and sensitivity to S23142 in two Protox isoenzymes (plastidal and mitochondrial forms) were examined. The level of mitochondrial Protox protein was greater, and its activity 5-times higher, in YZI-IS cells than in wild-type cells. Furthermore, the apparent IC₅₀ value of S23142 was about 20 nM, which is 20-fold higher than that observed in wild-type cells. In contrast, no differences were found in the plastidal Protox protein level, activity or its inhibition by S23142 between YZI-1S and wild-type cells. A southern blot analysis revealed that the mitochondrial Protox gene had been significantly amplified in the YZI-1S cells. These results suggest that the S23142 resistance of YZI-1S cells was due to the overproduction of mitochondrial Protox by gene amplification.

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  A core-class 2 tetrasaccharide-linked serine was synthesized in a convergent manner. The coupling reaction of disaccharide glycosyl donor 3 and acceptor 4 stereoselectively afforded tetrasaccharide 15, which was converted to glycosyl fluoride 20. Glycosylation of Fmoc serine allyl ester 5 with 20 produced α- and β-glycosides in 40% and 33% yields, respectively. α-Isomer 21 was converted into 1, a useful building block for the solid-phase synthesis of glycopeptides. On the other hand, 21 was N-deprotected and condensed with hydrophobic cholestanol through a succinyl spacer. The same compound was alternatively synthesized by coupling 20 and 28. Functional group manipulation and hydrogenation afforded core 2 tetrasaccharide-cholestanol conjugate 2.

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  Total syntheses of both enantiomers of dictyochromenol (1) and its (Z)-stereoisomer (2) were achieved with high enantiomeric purity. The results of this study reveal the relationship between the optical rotation of the resolved 1 enantiomers.

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  The chiral requirement on the α-methylbenzyl moiety of 2,4-diamino-6-chloro-s-triazine for sufficient inhibition of root growth was similar towards both rice and barnyard millet. With the monoalkylamino series, the most suitable configuration was markedly changed by the substituent on the other amino moiety. However, for the dialkylamino series, the (S)-enantiomer was an active inhibitor. Clear species selectivity between rice and barnyard millet was observed in the series for the (R)-enantiomers, providing high herbicidal activity toward Echinochloa plants and safety toward rice. The enantioselectivity against barnyard millet increased with increasing inhibitory activity of the active enantiomers, following Pfeiffer's rule. R-EtNH (3) controlled the growth of barnyardgrass with leaf-burning (LB) under paddy conditions, and S-EtNH (4) and S-Et2N (20) controlled the growth without LB. The RS-EtNH derivative is an interesting inhibitor controlling the growth of barn- yardgrass from the just-germinated stage (by the (R)-enantiomer) to early-middle growth stage (by the (S)-enantiomer).

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  Seven stilbenes, a new cis-ε-viniferin and the six known stilbenes, trans-resveratrol, trans-resveratrol-4′-O-β-D-glucopyranoside, trans-ε-viniferin, gnetin H, and suffruticosol A and B, were isolated and identified from seeds of Paeonia lactiflora. The antioxidative activity of these stilbene derivatives was evaluated against the 2-deoxyribose degradation and rat liver microsomal lipid peroxidation induced by the hydroxyl radical generated via a Fenton-type reaction. Among these stilbenes, trans-ε-viniferin and gnetin H significantly inhibited 2-deoxyribose degradation and lipid peroxidation in rat liver microsomes. In addition, cis-ε-viniferin, and suffruticosol A and B also exhibited moderate antioxidative activity. These results suggest that resveratrol dimers and trimers, together with resveratrol from seeds of Paeonia lactiflora may be useful as potential sources of natural antioxidants.

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  The absolute configuration of (+)-2,7(14),10-bis- abolatrien-1-ol-4-one, a peculiar sesquiterpenol in the Japanese cedar, Cryptomeria japonica, was determined as (1S,6R)-2,7(14),10-bisabolatrien-1-ol-4-one by comparing the specific rotation values of cryptomeriones respectively converted from (+)-2,7(14),10-bisabolatrien-1-ol-4-one and synthesized from (R)-(−)-carvone.

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  Glucosyltransferase and glucanotransferase involved in the production of cyclic tetrasaccharide (CTS; cyclo {→6}-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl- (1→)) from α-1,4-glucan were purified from Bacillus globisporus C11. The former was a 1,6-α-glucosyltransferase (6GT) catalyzing the α-1,6-transglucosylation of one glucosyl residue to the nonreducing end of maltooligosaccharides (MOS) to produce α-isomaltosyl-MOS from MOS. The latter was an isomaltosyl transferase (IMT) catalyzing α-1,3-, α-1,4-, and α,β-1,1-intermolecular transglycosylation of isomaltosyl residues. When IMT catalyzed α-1,3-transglycosylation, α-isomaltosyl-(1→3)-α-isomaltosyl-MOS was produced from α-isomaltosyl-MOS. In addition, IMT catalyzed cyclization, and produced CTS from α-isomaltosyl-(1→3)-α-isomaltosyl-MOS by intramolecular transglycosylation. Therefore, the mechanism of CTS synthesis from MOS by the two enzymes seemed to follow three steps:
  1) MOS→α-isomaltosyl-MOS (by 6GT),
  2) α-isomaltosyl-MOS→α-isomaltosyl-(1→3)-α- isomaltosyl-MOS (by IMT), and
  3) α-isomaltosyl-(1→3)-α-isomaltosyl-MOS→CTS +MOS (by IMT).
The molecular mass of 6GT was estimated to be 137 kDa by SDS-PAGE. The optimum pH and temperature for 6GT were pH 6.0 and 45°C, respectively. This enzyme was stable at from pH 5.5 to 10 and on being heated to 40°C for 60 min. 6GT was strongly activated and stabilized by various divalent cations. The molecular mass of IMT was estimated to be 102 kDa by SDS-PAGE. The optimum pH and temperature for IMT were pH 6.0 and 50°C, respectively. This enzyme was stable at from pH 4.5 to 9.0 and on being heated to 40°C for 60 min. Divalent cations had no effect on the stability or activity of this enzyme.

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  In higher plants, long-chain fatty acid hydroperoxides are intermediates in the synthesis of a diverse group of bioactive compounds. We used the reverse trascriptase-polymerase chain reaction to isolate a gene responsible for the oxidization of fatty acids from Petunia hybrida. A P450 cDNA not isolated earlier, CYP92B1, contained an open reading frame predicted to encode a polypeptide consisting of 510 amino acid residues. The transcript of the cyp92B1 gene was expressed at a high level in the early stage of flower development. CYP92B1 cDNA was expressed in a yeast, Saccharomyces cerevisiae, and recombinant yeast microsomes containing CYP92B1, a hemoprotein, metabolized lauric acid, linoleic acid, and linolenic acid.

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  A bacterial trehalose phosphorylase (TPase; EC 2.4.1.64) was purified from the culture supernatant of Bacillus stearothermophilus SK-1 to apparent homogeneity, and some properties were investigated. Furthermore, a gene from SK-1 responsible for the TPase was cloned by Southern hybridization with a degenerate oligonucleotide probe synthesized on the basis of the N-terminal sequence of the purified enzyme. The M_r of the enzyme was estimated to be 150,000 by gel filtration and 83,000 by SDS-PAGE, so the enzyme is likely to be a homodimer. The enzyme had optimum activity at pH 7.0-8.0 or nearby and the optimum temperature was about 75°C. The deduced amino acid sequence of the SK-1 TPase encodes a theoretical protein with a M_r of 87,950. Alignment of amino acid sequences with a maltose phosphorylase from Lactobacillus brevis the crystal structure and active site of which had been analyzed suggested that these two phosphorylases evolved from a common ancestor. The Escherichia coli cells harboring the plasmid containing the cloned TPase gene had about 100 times the activity of SK-1.

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  We have been studying a lysozyme derivative, called mPEG-lysozyme, in which Lys 33 is bound with a monomethoxypolyethylene glycol derivative. Here, we examined the surface hydrophobicity of the derivative and also its interactions with lipopolysaccharides and lipid bilayers. These properties may affect the antimicrobial activity of mPEG-lysozyme toward Gram-negative microorganisms. The lysozyme derivative had more than 150% of the antimicrobial activity for such microorganisms with that of native lysozyme taken to be 100%. Spectroscopic analyses indicated that mPEG-lysozyme bound to lipopolysaccharides with higher affinity than lysozyme, because of the high surface hydrophobicity of the derivative. In an experiment on carboxyfluorescein-leakage, mPEG-lysozyme strongly interacted with liposomes constructed from phosphatidylcholine, releasing carboxyfluorescein from the liposomes more effectively than lysozyme did. mPEG-lysozyme may perturb the outer membrane of Gram-negative microorganisms, gaining itself access to the peptidoglycan layers of the bacterium.

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  The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-β-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.

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  An anticoagulant was purified from a Chinese herb, Taraxacum platycarpum. Its activity was heat-labile, and was decreased by incubation with subtilisin Carlburg or proteinase K, indicating that the active component was a protein. The protein had a molecular mass of 31 kDa by gel filtration and 33 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, so it probably was a monomer. When present at the concentration of 70, 255, and 873 nM, respectively, the protein doubled the thrombin time, prothrombin time, and activated partial thromboplastin time. It inhibited thrombin and kallikrein, but did not hydrolyze fibrinogen. The protein bound the anion-binding exosite of thrombin, competing with the fibrinogen binding site. In addition, the protein caused the murine macrophage cell line Raw 264.7 to produce cyclooxygenase-2, nitric oxide synthase, nitric oxide, and tumor necrosis factor-α.

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  Hydrogen peroxide is a well-known mediator of apoptosis. As a mechanism for H₂O₂-induced apoptosis, both a mitochondrial Cyt.c-dependent pathway and a lysosome-mediated pathway have been suggested. However, the relative roles of and the relation between these two pathways in H₂O₂-induced apoptosis remain to be discovered. In this study, to find the relative roles of the lysosomal and mitochondrial pathways, the effects of E-64-d, a cell-permeable inhibitor of lysosomal cysteine proteases, on apoptosis caused by H₂O₂ in HL-60 cells were investigated. It was found that the concentration of H₂O₂ strongly affected the inhibitory effect of E-64-d on the apoptosis in HL-60 cells: dose-dependent inhibition (up to 40%) of both DNA fragmentation and caspase-3 activation was observed when a high concentration of H₂O₂ (50 μM) was used to induce apoptosis, but no inhibitory effect was detected when a low concentration (10 μM) was used. Consistent with these observations, apparent lysosomal destabilization was observed only with 50 μM H₂O₂. The release of mitochondrial Cyt.c, in contrast, was observed at both 10 μM and 50 μM. These results indicated that the mitochondrial Cyt.c-mediated pathway predominates in the H₂O₂-induced apoptosis in HL-60 cells and the lysosomal mediated pathway is partially involved when high concentrations of H₂O₂ are used to induce apoptosis.

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  An exoinulinase, P-I, was purified from the culture filtrate of Penicillium sp. strain TN-88 grown on inulin. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis with an apparent M_r of 81 kDa. The purified enzyme had extremely high specific activity, 743 U/mg, toward inulin. Inulinase activity was optimal at pH 4.0 and 55°C. A genomic DNA and cDNAs encoding this protein were cloned and sequenced. The exoinulinase gene (inuD) was present as a single copy in the genome. An open reading frame of 2,106 bp was interrupted by a single intron of 56 bp, and encoded a 25-amino acid signal peptide and a 677-amino acid mature protein. The mature protein contained two Cys residues and eight potential N-linked glycosylation sites. The 5′-noncoding region had a putative CAAT box at position −239. Four distinct transcription start points were observed at positions −98 (A), −91 (A), −80 (A), and −76 (A) from the start codon. The exoinulinase gene inuD was located 860-bp upstream of the previously reported endoinulinase gene inuC in the opposite direction of transcription.

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  Nine proteinase inhibitors, I-VIIa, VIIb, and VIII, were isolated from wild soja seeds by ammonium sulfate fractionation and successive chromatographies on SP-Toyopearl 650M, Sephacryl S-200SF, and DEAE-Toyopearl 650S columns. Reverse-phase HPLC finally gave pure inhibitors. All of the inhibitors inhibited trypsin with dissociation constants of 3.2-6.2×10^-9 M. Some of the inhibitors inhibited chymotrypsin and elastase as well. Two inhibitors (VIIb and VIII) with a molecular weight of 20,000 were classified as a soybean Kunitz inhibitor family. Others (I-VIIa) had a molecular weight of about 8,000, and were stable to heat and extreme pH, suggesting that these belonged to the Bowman-Birk inhibitor family. Partial amino acid sequences of four inhibitors were also analyzed. The complete sequence of inhibitor IV was ascertained from the nucleotide sequences of cDNA clones encoding isoinhibitors homologous to soybean C-II.

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  The effects of several respiration inhibitors on photosystem II (PS II) were investigated. Among the agents tested, piericidin A and thenoyltrifluoroacetone (TTFA) inhibited the photosynthetic electron transport of spinach as measured from chlorophyll (Chl) fluorescence parameters (Fm′−F)/Fm′ and Fv/Fm. Using specific donors and acceptors of electrons, we identified the sites of inhibition in and around the PS II complex; the site of inhibition by TTFA was between Q_A, primary quinone acceptor in PS II, and Q_B, secondary quinone acceptor, in the acceptor side of P680, the reaction center Chl of PS II, while inhibition by piericidin A of the acceptor side was downstream of Q_B, out of the PS II complex. Both agents also inhibited the donor side of P680, probably between tyrosine-161 of the reaction center protein of PS II and P680.

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  The transcriptional activator protein Gcn4p increases the transcription of many genes that code for amino acid synthesis genes during amino acid starvation in Saccharomyces cerevisiae. Here we showed that after the disruption of C-GCN4, a homolog in Candida maltosa of GCN4 in S. cerevisiae, formed pseudohyphae in minimal medium. This is the first report that a GCN4 homolog is involved in the control of morphological transition.

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  Two basic proteins, HLP-1 and HLP-2, were isolated from brewer's barley grain (Hordeum vulgare L.) and characterized as glycoproteins with molecular masses of 16 and 13 kDa and pI values of 7.4 and 8.8, respectively. They could bind sugars, metal ions, and both hydrophobic and hydrophylic molecules of low molecular mass. These characteristics may be related to their potential plant-protecting role.

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  Results of in situ RNA-RNA hybridization showed the presence of transcripts of the Lentinula edodes hydrophobin 1 gene, Le.hyd1, everywhere in the mycelial tissues of developing fruiting bodies except for the top parts of the pileus (cap) and for the prehymenophore. A high level of the transcript was detected in the parts surrounding the prehymenophore.

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  Human tyrosine tRNA and fly alanine, histidine, and initiator methionine tRNAs are generally cleavable internally by bacterial ribonuclease P ribozyme. The unusual internal cleavage reaction of tRNA, called hyperprocessing, occurs when the cloverleaf structure of the tRNA molecule is denatured to form a double-hairpin-like structure. The hyperprocessing reaction of these tRNAs requires magnesium ions. We analyzed details of this reaction using human tyrosine tRNA and Escherichia coli RNase P ribozyme. The usual processing reaction occurred efficiently with magnesium at 5 mM, but for the hyperprpocessing reaction, higher concentrations were needed. With such high concentrations, hyperprocessing cleaved both mature tRNA and tRNA precursor as substrates. When mature tRNA was the substrate, the apparent K_M was almost the same as in the usual reaction, but k_cat was smaller. These results indicated that the occurrence of hyperprocessing depends on the magnesium ion concentration, and suggested that magnesium ions contribute to the recognition of the shape of the substrate by bacterial RNase P enzymes.

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  A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.

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  Elsewhere, we characterized the structure of twelve N-glycans purified from royal jelly glycoproteins (Kimura, Y. et al., Biosci. Biotechnol. Biochem., 64, 2109-2120 (2000)). Structural analysis showed that the typical high-mannose type structure (Man9-4GlcNAc2) accounts for about 72% of total N-glycans, a biantennary-type structure (GlcNAc2Man3GlcNAc2) about 8%, and a hybrid-type structure (GlcNAc1Man4GlcNAc2) about 3%. During structural analysis of minor N-glycans of royal jelly glycoproteins, we found that one had an N-acetyl-galactosaminyl residue at the non reducing end; most of such residues have been found in N-glycans of mammalian glycoproteins. By exoglycosidase digestion, methylation analysis, ion-spray (IS)-MS analysis, and ¹H NMR spectroscopy, we identified the structure of the N-glycan containing GalNAc as; GlcNAcβ1-2Manα1-6(GalNAcβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1- 4GlcNAc. This result suggested that a β1-4 GalNAc transferase is present in hypopharyngeal and mandibular glands of honeybees.

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  Linoleic acid was encapsulated with a soluble soybean polysaccharide, gum arabic, or a mixture of both together with maltodextrin, and the oxidation process of the encapsulated acid was measured at 37°C and at a relative humidity of 12%. The soybean polysaccharide was more effective for encapsulating the acid and suppressing the oxidation of the encapsulated acid than gum arabic. A mixture of the soybean polysaccharide and maltodextrin was also effective for this purpose when the weight fraction of the polysaccharide was equal to or greater than 0.75.

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  Hypoallergenic wheat flour produced by modification with cellulase and actinase showed inhibitory activity against ovalbumin permeation in an in vitro model by using the Caco-2 cell monolayer. The activity was found in the cellulase preparation used for producing the flour. An active compound was isolated by HPLC and identified as Trp-Ser-Asn-Ser-Gly-Asn-Phe-Val-Gly-Gly-Lys by ¹H-NMR data and Edman degradation. The undecapeptide, some oligopeptides with the N-terminal sequences and Trp ethyl ester showed activity at 10^-7 M, acetyl Trp being active at 10^-2 M. These data suggest that the Trp residue without a free carboxyl group would be required for the inhibitory activity of ovalbumin absorption through the intestinal tract.

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  We investigated the effects of oolong tea on the basic metabolism of plasma lipids in mice under restraint stress. When a lipid emulsion (Intralipid 20%; a lipid emulsion containing 20% soybean oil) was injected intravenously into mice, the restraint stress prolonged the half-life (T_1/2) of elimination for plasma triglyceride (TG) from 28.7 to 55.5 min. The elimination rate per minute was 48.2% in stressed mice with the rate in starved control mice as 100%. Therefore, TG metabolism was disrupted by the stress, and the use of TG as an energy source decreased. We found that the metabolism of lipids significantly response to the restrained stress in the present study. Plasma TG was 515.9±29.9 mg/dl 35 min after Intralipid administration in control stressed mice, 478.7±26.7 mg/dl in the stressed group given caffeine 100 mg/kg of body weight, and 418.3±18.4 mg/dl in the stressed group given 1000 mg/kg oolong tea, an improvement by 7.2% and 18.9%, respectively, with the value for the untreated control group. The intake of oolong tea alleviated the stress-induced decrease in the rate of blood lipid metabolism; this effect may have arisen from some nonspecific stress-relieving property of the tea or from acceleration of lipid metabolism by properties of polyphenols, etc. in tea. Oolong tea had anti-stress effects on plasma TG metabolism, and the effects did not depend on caffeine.

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  Various kinds of high-molecular-mass polyphenols such as condensed tannins, hydrolyzable tannins, and polymerized anthocyanins, were readily characterized by a new size-exclusion HPLC method. This rapid analytical method may also be useful for the profiling of molecular mass distribution of polyphenolic constituents in many kinds of food materials.

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  The gene, designated hep, coding for a heparinase that degrades both heparin and heparan sulfate, was cloned from Bacillus circulans HpT298. Nucleotide sequence analysis showed that the open reading frame of the hep gene consists of 3,150 bp, encoding a precursor protein of 1,050 amino acids with a molecular mass of 116.5 kDa. A homology search found that the deduced amino acid sequence has partial similarity with enzymes belonging to the family of acidic polysaccharide lyases that degrade chondroitin sulfate and hyaluronic acid. Recombinant mature heparinase (111.2 kDa) was produced by the addition of IPTG from Escherichia coli harboring pETHEP with an open reading frame of the mature hep gene and was purified to homogeneity by SDS-polyacrylamide gel electrophoresis. Analyses of substrate specificity and degraded disaccharides indicated that the recombinant enzyme acts on both heparin and HS, as does heparinase purified from the wild-type strain.

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  We designed a polymerase chain reaction that allows us to clone two types of putative reverse transcriptase genes from 11 species of ectomycorrhizal basidiomycetes that belong to the genus Tricholoma. One corresponds to the putative gene of marY1, a long terminal repeat (LTR) retroelement from Tricholoma matsutake, and the other, marY2N, a LINE-like non-LTR (L1-like) retroelement from this fungus. Putative protein products predicted from nucleotide sequencing of cloned fragments were phylogenetically analyzed. marY1-like elements had a parallel phylogenetic relationship with no apparent correlation to a current taxonomic profile, while marY2N-like elements showed a vertical one in relation to host-plant species. Data suggest that marY1-like elements and marY2N-like elements have evolved independently, and the evolution of marY1-like elements could have occurred later than the evolution of marY2N-like elements in the species of Tricholoma.

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  A bacterial isolate, Pseudomonas putida CN-T19, could grow in a two-phase medium with toluene up to 50% (v/v). Changes in fatty acid composition and membrane fluidity of the isolate were investigated to understand how this microorganism responds toluene. The changes in the ratios of unsaturated to saturated fatty acids were insignificant between cells grown with and without toluene. The changes in the ratio of cis- to trans-fatty acids of C_16:1 and C_18:1 was, however, significantly lower in cells grown with toluene than cells grown without toluene, giving approximately 1.3 and 9.7, respectively. Toluene had a fluidizing effect on the membrane of cells grown without toluene, resulting in decrease in membrane polarization ratio. Less fluidizing effect of toluene on the membrane of cells grown with toluene was observed, giving 11% of polarization percentage, which was significantly lower than 53% in cells grown without toluene. These results suggest that cis/trans isomeration of C_16:1 and C_18:1 makes cell membranes more rigid to respond toluene, and is an adaptive strategy allowing P. putida CN-T19 to grow in the presence of organic solvent.

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  Eleven strains of lactobacilli were tested for their ability to induce the murine macrophage-like cell line J774.1 to secrete cytokines. Some of the bacteria tested induce the production of interleukin(IL) 6, IL-12, and tumor necrosis factor α (TNF-α) by J774.1 cells. Seven strains also induced the production of IL-10. However, no IL-1β was produced. Lactobacillus acidophilus TMC 0356 significantly induced the production of more IL-6, IL-10, IL-12, and TNF-α than the other bacteria tested (p<0.0001; ANOVA). These results suggest that lactobacilli can activate macrophages to secrete both inflammatory and anti-inflammatory cytokines. Selected strains might be used to bring about pro or antiinflammatory immune reactions.

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  Biotransformation of L-lysine (L-Lys) to L-pipecolic acid (L-PA) using lat-expressing Escherichia coli has been reported (Fujii et al., Biosci. Biotechnol. Biochem., 66, 622-627 (2002)). The rate-limiting step of this biotransformation seemes to be the transport of L-Lys into cells. To improve the L-PA production rate, we attempted to increase the rate of L-Lys uptake. E. coli BL21 carrying a plasmid with lat and lysP (pRH125) caused a 5-fold increase in the rate of L-PA production above the level of cells carrying a plasmid with lat (pRH124). Moreover, E. coli BL21 carrying a plasmid with lat, lysP, and yeiE (pRH127) caused a 6.4-fold increase in the rate of L-PA production above the level of cells carrying pRH124. Our results from RT-PCR experiments and the sequence similarity of YeiE to LysR transcriptional regulators suggest the possibility that yeiE expression induces lysP expression. The amplification of lysP, or rather both lysP and yeiE, increases the rate of L-PA production using lat-expressing E. coli.

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  Among 45 Bacillus subtilis strains isolated from non-salted types of fermented soybeans produced in several Southeast Asian countries, 20 had the insertion sequence IS4Bsu1 in the chromosome. In contrast, none of 49 B. subtilis strains of non-food origin contained IS4Bsu1. Frequent occurrence of this mobile DNA element in the soybean-fermenting B. subtilis would reflect the fact that few strains flourish on soybeans and thereby contribute to soybean fermentation.

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