Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.9 2002)

Behavior of Hydrogen Peroxide in Electrolyzed Anode Water
Kazuo HARADA p.1783

Isolation of Bacterial Strains that Produce the Endocrine Disruptor, Octylphenol Diethoxylates, in Paddy Fields
Eriko NISHIO,1 Yayoi ICHIKI,1 Hiroto TAMURA,2 Shiro MORITA,1 Katuji WATANABE,3 and Hiromichi YOSHIKAWA1, p.1792

Resistance to Protoporphyrinogen Oxidase-inhibiting Compound S23142 from Overproduction of Mitochondrial Protoporphyrinogen Oxidase by Gene Amplification in Photomixotrophic Tobacco Cells
Naohide WATANABE,1 Seiji TAKAYAMA,1 Shigeo YOSHIDA,2 Akira ISOGAI,1 and Fang-Sik CHE1, p.1799

Purification and Characterization of Glucosyltransferase and Glucanotransferase Involved in the Production of Cyclic Tetrasaccharide in Bacillus globisporus C11
Tomoyuki NISHIMOTO, Hajime AGA, Kazuhisa MUKAI, Takaharu HASHIMOTO, Hikaru WATANABE, Michio KUBOTA, Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA p.1806

CYP92B1, A Cytochrome P450, Expressed in Petunia Flower Buds, That Catalyzes Monooxidation of Long-Chain Fatty Acids
Mariana PETKOVA-ANDONOVA, Hiromasa IMAISHI, and Hideo OHKAWA p.1819

Oxidation of Linoleic Acid Encapsulated with Soluble Soybean Polysaccharide by Spray-drying
Yasumasa MINEMOTO,1 Xu FANG,2 Koichi HAKAMATA,2 Yoshiyuki WATANABE,2 Shuji ADACHI,2, Tadashi KOMETANI,1 and Ryuichi MATSUNO2 p.1829

Characterization of Trehalose Phosphorylase from Bacillus stearothermophilus SK-1 and Nucleotide Sequence of the Corresponding Gene
Yasushi INOUE, Keiko ISHII, Tetsuji TOMITA, Tsuneya YATAKE, and Fumio FUKUI p.1835

2-Ethyl-3-methylmaleimide in Tokyo Bay Sediments Providing the First Evidence for its Formation from Chlorophylls in the Present Photic and Oxygenic Zone
Masaki KOZONO,1 Shinya NOMOTO,1, Hajime MITA,1 Ryoshi ISHIWATARI,2,†† and Akira SHIMOYAMA1, p.1844

Interactions of a Lysozyme-Monomethoxypolyethylene Glycol Conjugate with Lipopolysaccharides and Lipid Bilayers and Effects of Conjugate on Gram-negative Bacteria
Yuichi NODAKE, Kenta IWASAKI, and Nobuyuki YAMASAKI p.1848

Accumulation of Maize Response Regulator Proteins in Mesophyll Cells after Cytokinin Treatment
Atsushi DEJI,1 Hitoshi SAKAKIBARA,2, Shinya OKUMURA,1 Tsukasa MATSUDA,1 Yuji ISHIDA,3 Shigehiro YAMADA,3 Toshihiko KOMARI,3 Tomoaki KUBO,3 Tomoyuki YAMAYA,2 and Tatsuo SUGIYAMA1,2 p.1853

Anticoagulant from Taraxacum platycarpum
Soo-In YUN, Hong-Rae CHO,1 and Hye-Seon CHOI p.1859

Involvement of Lysosomal Cysteine Proteases in Hydrogen Peroxide-induced Apoptosis in HL-60 Cells
Rumi ISHISAKA, Kozo UTSUMI,＊＊ and Toshihiko UTSUMI, p.1865

Cloning, Sequencing, and Expression of the Gene from Bacillus circulans That Codes for a Heparinase That Degrades Both Heparin and Heparan Sulfate
Eiichi YOSHIDA,1 Shinji ARAKAWA,1 Taizo MATSUNAGA,1 Shigeki TORIUMI,1 Shinji TOKUYAMA,1 Kiyoshi MORIKAWA,2 and Yasutaka TAHARA1, p.1873

Genetic Evidence That Two Types of Retroelements Evolved through Different Pathways in Ectomycorrhizal Homobasidiomycetes Tricholoma spp.
Hitoshi MURATA,1, Katsuhiko BABASAKI,1 Yasumasa MIYAZAKI,1 and Akiyoshi YAMADA2
p.1880

Purification and Properties of an Extracellular Exoinulinase from Penicillium sp. Strain TN-88 and Sequence Analysis of the Encoding Gene
Satoshi MORIYAMA, Hidetoshi AKIMOTO, Norio SUETSUGU, Soushi KAWASAKI, Toyohiko NAKAMURA, and Kazuyoshi OHTA p.1887

Purification and Characterization of Proteinase Inhibitors from Wild Soja (Glycine soja) Seeds
Masanobu DESHIMARU, Ryuji HANAMOTO, Chiho KUSANO, Shingo YOSHIMI, and Shigeyuki TERADA p.1897

Synthesis of Core-class 2 O-Linked Glycopeptides: a Benzyl-protected Tetrasaccharyl Serine and its Derivative Carrying a Hydrophobic Cholestanyl Group
Jun WATABE,1 Latika SINGH,2 Yuko NAKAHARA,2 Yukishige ITO,2 Hironobu HOJO,1 and Yoshiaki NAKAHARA1,2, p.1904

Total Synthesis of Both Enantiomers of Dictyochromenol and their (Z)-Isomers
Kota AOKI,1 Mihoko TAKAHASHI,1 Masaru HASHIMOTO,1, Toshikatsu OKUNO,1 Kazuya KURATA,2 and Minoru SUZUKI3 p.1915

Inhibition of Photosystem II of Spinach by the Respiration Inhibitors Piericidin A and Thenoyltrifluoroacetone
Nobuhiro IKEZAWA, Kentaro IFUKU, Tsuyoshi ENDO, and Fumihiko SATO p.1925

An Active Compound against Allergen Absorption in Hypoallergenic Wheat Flour Produced by Enzymatic Modification
Shoko TESAKI,1 Jun WATANABE,2 Soichi TANABE,3 Kei SONOYAMA,2 Eri FUKUSHI,2 Jun KAWABATA,2 and Michiko WATANABE1, p.1930

Note
Pseudohyphal Growth in a Dimorphic Yeast, Candida maltosa, after Disruption of the C-GCN4 Gene, a Homolog of Saccharomyces cerevisiae GCN4
Hiroaki TAKAKU, Hiroyuki HORIUCHI, Masamichi TAKAGI, and Akinori OHTA p.1936

Note
Isolation and Characterization of Highly Liganded Protein from Brewer′s Barley Grain
Stanislava GORJANOVI<Oh><0193><Wa>C,1, Ana CVETKOVI<Oh><0193><Wa>C,1 Desanka SU<Oh><0189><Wa>ZNJEVI<Oh><0193><Wa>C,1 Milo<Oh><0190><Wa>s BELJANSKI,1 Miroslav VRVI<Oh><0193><Wa>C,2 and Jovan HRANISAVLJEVI<Oh><0193><Wa>C1 p.1940

Note
Changes in Membrane Fluidity and Fatty Acid Composition of Pseudomonas putida CN-T19 in Response to Toluene
In Seon KIM,1, Jae Han SHIM,2 and Yong Tack SUH2 p.1945

Note
Distribution of Hydrophobin 1 Gene Transcript in Developing Fruiting Bodies of Lentinula edodes
Hiroo NISHIZAWA,a Yasumasa MIYAZAKI,b Shinya KANEKO,c and Kazuo SHISHIDO p.1951

Note
Effects of Oolong Tea on Metabolism of Plasma Fat in Mice under Restraint Stress
Hiroshi KURIHARA, Harukazu FUKAMI, Hirofumi KODA, Nobuo TSURUOKA, Namino SUGIURA, Hiroshi SHIBATA, and Takaharu TANAKA p.1955

Note
Enantioselective Effects of Optically Active α-Methylbenzyl-s-triazine on the Root Growth of Rice and Echinochloa Plants and Their Herbicidal Activity
Hiroyoshi OMOKAWA and Akiko TABEI p.1959

Note
Cytokine Production by the Murine Macrophage Cell Line J774.1 after Exposure to Lactobacilli
Hirotsugu MORITA,1 Fang HE,1, Tetsuo FUSE,1 Arthur C. OUWEHAND,2 Hideo HASHIMOTO,1 Masataka HOSODA,1 Koko MIZUMACHI,3 and Jun-Ichi KURISAKI4 p.1963

Note
Kinetics of Hyperprocessing Reaction of Human Tyrosine tRNA by Ribonuclease P Ribozyme from Escherichia coli
Tomoaki ANDO, Terumichi TANAKA, Yoshiaki HORI, and Yo KIKUCHI p.1967

Note
Characterization of Polymerized Polyphenols by Size-exclusion HPLC
Akio YANAGIDA,1, Toshihiko SHOJI,2 and Tomomasa KANDA3 p.1972

Note
Gene Encoding a Trehalose Phosphorylase from Thermoanaerobacter brockii ATCC 35047
Kazuhiko MARUTA, Kazuhisa MUKAI, Hiroshi YAMASHITA, Michio KUBOTA, Hiroto CHAEN, Shigeharu FUKUDA, and Masashi KURIMOTO p.1976

Note
Increase in the Rate of L-Pipecolic Acid Production Using lat-Expressing Escherichia coli by lysP and yeiE Amplification
Tadashi FUJII, Yasuhide ARITOKU, Hitosi AGEMATU, and Hiroshi TSUNEKAWA p.1981

Note
Occurrence of GalNAcβ1-4GlcNAc Unit in N-Glycan of Royal Jelly Glycoprotein
Mariko KIMURA,1 Yoichiro HAMA,2 Kazunori TSUMURA,1 Kiyoshi OKIHARA,3 Hiroyuki SUGIMOTO,3 Hideo YAMADA,3 and Yoshinobu KIMURA1, p.1985

Note
Antioxidative Activity of Resveratrol and Its Derivatives Isolated from Seeds of Paeonia lactiflora
Hyo Jin KIM,1 Eun Ju CHANG,1 Sung Hee CHO,1 Shin Kyo CHUNG,2 Heui Dong PARK,2 and Sang Won CHOI1, p.1990

Note
Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis Strains Isolated from Fermented Soybean Foods in Southeast Asia
Keitarou KIMURA, Yasuhiro INATSU, and Yoshifumi ITOH p.1994

Note
Determination of the Absolute Configuration of (＋)-2,7(14),10-Bisabolatrien- 1-ol-4-one from Japanese Cedar, Cryptomeria japonica
Chul-Sa KIM, Jun MORISAWA, Nobuhiro NISHIYAMA, Takehiro KASHIWAGI, Shin-ich TEBAYASHI, and Michio HORIIKE p.1997

-1-
Behavior of Hydrogen Peroxide in Electrolyzed Anode Water

Kazuo HARADA

Department of Research and Development, Hokkaido Electric Power Co., Inc. 2-1 Tsuishikari, Ebetsu, Hokkaido 067-0033, Japan

Received June 25, 2001; Accepted May 7, 2002
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.
Key words: electrolyzed anode water (EAW); hydrogen peroxide; oxygen production

-2-
Isolation of Bacterial Strains that Produce the Endocrine Disruptor, Octylphenol Diethoxylates, in Paddy Fields

Eriko NISHIO,1 Yayoi ICHIKI,1 Hiroto TAMURA,2 Shiro MORITA,1 Katuji WATANABE,3 and Hiromichi YOSHIKAWA1

1Department of Environmental Chemistry, Kyushu Kyoritsu University, Kitakyushu 807-8585, Japan 2Department of Applied Biochemistry, Meijyo University, Nagoya 468-8502, Japan 3Department of Agro-Environment Resource Research, Laboratory of Soil Microbiology, National Agricultural Research Center for Kyushu Okinawa Region, Kikuchi-gun, Kumamoto 861-1192, Japan

Received November 5, 2001; Accepted May 9, 2002
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.
Key words: octylphenol polyethoxylate; octylphenol diethoxylate; biodegradation; endocrine disruptor; paddy field

-3-
Resistance to Protoporphyrinogen Oxidase-inhibiting Compound S23142 from Overproduction of Mitochondrial Protoporphyrinogen Oxidase by Gene Amplification in Photomixotrophic Tobacco Cells

Naohide WATANABE,1 Seiji TAKAYAMA,1 Shigeo YOSHIDA,2 Akira ISOGAI,1 and Fang-Sik CHE1

1Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), Takayama 8916-5, Ikoma, Nara 630-0101, Japan 2Plant Function Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama, 351-0198, Japan

Received November 13, 2001; Accepted May 22, 2002
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.
Key words: gene amplification; herbicide resistance; mitochondria; protoporphyrin IX; protoporphyrinogen oxidase

-4-
Purification and Characterization of Glucosyltransferase and Glucanotransferase Involved in the Production of Cyclic Tetrasaccharide in Bacillus globisporus C11

Tomoyuki NISHIMOTO, Hajime AGA, Kazuhisa MUKAI, Takaharu HASHIMOTO, Hikaru WATANABE, Michio KUBOTA, Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA

Hayashibara Biochemical Laboratories, Inc. 7-7 Amase-minami machi, Okayama 700-0834, Japan

Received December 3, 2001; Accepted May 8, 2002
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.
Key words: cyclic tetrasaccharide; Bacillus globisporus; 1,6-α-glucosyltransferase; isomaltosyl transferase; cyclization

-5-
CYP92B1, A Cytochrome P450, Expressed in Petunia Flower Buds, That Catalyzes Monooxidation of Long-Chain Fatty Acids

Mariana PETKOVA-ANDONOVA, Hiromasa IMAISHI, and Hideo OHKAWA

Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada-ku, Kobe 657-8501, Japan

Received December 13, 2001; Accepted April 30, 2002
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.
Key words: cytochrome P450; Petunia hybrida; lauric acid; linoleic acid; linolenic acid

-6-
Oxidation of Linoleic Acid Encapsulated with Soluble Soybean Polysaccharide by Spray-drying

Yasumasa MINEMOTO,1 Xu FANG,2 Koichi HAKAMATA,2 Yoshiyuki WATANABE,2 Shuji ADACHI,2, Tadashi KOMETANI,1 and Ryuichi MATSUNO2

1Department of Chemical and Biochemical Engineering, Toyama National College of Technology, 13 Hongo, Toyama 939-8630, Japan 2Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received February 4, 2002; Accepted May 21, 2002
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.
Key words: microencapsulation; soybean polysaccharide; oxidation; linoleic acid; spray-drying

-7-
Characterization of Trehalose Phosphorylase from Bacillus stearothermophilus SK-1 and Nucleotide Sequence of the Corresponding Gene

Yasushi INOUE, Keiko ISHII, Tetsuji TOMITA, Tsuneya YATAKE, and Fumio FUKUI

Showa Sangyo Co., Ltd., 16, Sakura 1-chome, Tsukubashi, Ibaraki 305-0003, Japan

Received February 18, 2002; Accepted May 13, 2002
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 Mr 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 Mr 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.
Key words: trehalose phosphorylase; trehalose phosphorylase gene; Bacillus stearothermophilus

-8-
2-Ethyl-3-methylmaleimide in Tokyo Bay Sediments Providing the First Evidence for its Formation from Chlorophylls in the Present Photic and Oxygenic Zone

Masaki KOZONO,1 Shinya NOMOTO,1, Hajime MITA,1 Ryoshi ISHIWATARI,2,†† and Akira SHIMOYAMA1

1Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan 2Department of Chemistry, Tokyo Metropolitan University, 1-1 Minami-ohsawa, Hachioji, Tokyo 192-0397, Japan

Received February 19, 2002; Accepted May 10, 2002
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.
Key words: maleimide; porphyrin; chlorophyll; sediment

-9-
Interactions of a Lysozyme-Monomethoxypolyethylene Glycol Conjugate with Lipopolysaccharides and Lipid Bilayers and Effects of Conjugate on Gram-negative Bacteria

Yuichi NODAKE, Kenta IWASAKI, and Nobuyuki YAMASAKI

Laboratory of Biochemistry, Division of Bioresource and Bioenvironmental Sciences, Graduate School of Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Recieved February 22, 2002; Accepted April 15, 2002
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.
Key words: lysozyme; monomethoxypolyethylene glycol; antimicrobial activity; lipopolysaccharide; liposome

-10-
Accumulation of Maize Response Regulator Proteins in Mesophyll Cells after Cytokinin Treatment

Atsushi DEJI,1 Hitoshi SAKAKIBARA,2, Shinya OKUMURA,1 Tsukasa MATSUDA,1 Yuji ISHIDA,3 Shigehiro YAMADA,3 Toshihiko KOMARI,3 Tomoaki KUBO,3 Tomoyuki YAMAYA,2 and Tatsuo SUGIYAMA1,2

1Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan 2RIKEN (The Institute of Physical and Chemical Research), Plant Science Center, 1-7-22, Suehiro, Tsurumi, Yokohama 230-0045, Japan 3ORYNOVA K.K., 700 Higashibara, Iwata, Shizuoka 428-0802, Japan

Received February 25, 2002; Accepted April 5, 2002
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.
Key words: cytokinin; His-Asp phosphorelay; mesophyll cell; response regulator; Zea mays

-11-
Anticoagulant from Taraxacum platycarpum

Soo-In YUN, Hong-Rae CHO,1 and Hye-Seon CHOI

Department of Biological Sciences and Immunomodulation Research Center, University of Ulsan, Ulsan 680-749, Korea 1Department of General Surgery, Ulsan University Hospital, Ulsan, Korea

Received February 25, 2002; Accepted April 18, 2002
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.
Key words: anticoagulatory; antithrombotic; cyclooxygenase-2; nitric oxide synthase; Taraxacum platycarpum

-12-
Involvement of Lysosomal Cysteine Proteases in Hydrogen Peroxide-induced Apoptosis in HL-60 Cells

Rumi ISHISAKA, Kozo UTSUMI,＊＊ and Toshihiko UTSUMI

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida, Yamaguchi 753-8515, Japan ＊＊Institute of Medical Science, Kurashiki Medical Center, 250 Bakuro-cho, Kurashiki 710-8522, Japan

Received February 27, 2002; Accepted April 27, 2002
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.
Key words: apoptosis; lysosomes; mitochondria; HL-60 cells; hydrogen peroxide

-13-
Cloning, Sequencing, and Expression of the Gene from Bacillus circulans That Codes for a Heparinase That Degrades Both Heparin and Heparan Sulfate

Eiichi YOSHIDA,1 Shinji ARAKAWA,1 Taizo MATSUNAGA,1 Shigeki TORIUMI,1 Shinji TOKUYAMA,1 Kiyoshi MORIKAWA,2 and Yasutaka TAHARA1

1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan 2Central Research Laboratories, Seikagaku Corp., Higashiyamato, Tokyo 207-0021, Japan

Received February 28, 2002; Accepted May 8, 2002
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.
Key words: heparin; heparan sulfate; heparinase gene; Bacillus circulans

-14-
Genetic Evidence That Two Types of Retroelements Evolved through Different Pathways in Ectomycorrhizal Homobasidiomycetes Tricholoma spp.

Hitoshi MURATA,1, Katsuhiko BABASAKI,1 Yasumasa MIYAZAKI,1 and Akiyoshi YAMADA2

1Department of Applied Microbiology and Mushroom Sciences, Forestry ＆ Forest Products Research Institute, Kukizaki, Ibaraki 305-8687, Japan 2School of Agricultural Sciences, Shinshu University, Minami Minowa 8304, Nagano 399-4598, Japan

Received March 1, 2002; Accepted April 24, 2002
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.
Key words: basidiomycetes; ectomycorrhizal fungi; molecular evolution; reverse transcriptase; Tricholoma matsutake

-15-
Purification and Properties of an Extracellular Exoinulinase from Penicillium sp. Strain TN-88 and Sequence Analysis of the Encoding Gene

Satoshi MORIYAMA, Hidetoshi AKIMOTO, Norio SUETSUGU, Soushi KAWASAKI, Toyohiko NAKAMURA, and Kazuyoshi OHTA

Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Miyazaki University, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan

Received March 4, 2002; Accepted April 23, 2002
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 Mr 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.
Key words: exoinulinase; extracellular enzyme; gene cluster; inulin; Penicillium sp.

-16-
Purification and Characterization of Proteinase Inhibitors from Wild Soja (Glycine soja) Seeds

Masanobu DESHIMARU, Ryuji HANAMOTO, Chiho KUSANO, Shingo YOSHIMI, and Shigeyuki TERADA

Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan

Received March 11, 2002; Accepted May 2, 2002
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.
Key words: cDNA cloning; Glycine max; isoinhibitors; purification; wild soja seeds

-17-
Synthesis of Core-class 2 O-Linked Glycopeptides: a Benzyl-protected Tetrasaccharyl Serine and its Derivative Carrying a Hydrophobic Cholestanyl Group

Jun WATABE,1 Latika SINGH,2 Yuko NAKAHARA,2 Yukishige ITO,2 Hironobu HOJO,1 and Yoshiaki NAKAHARA1,2

1Institute of Glycotechnology, Department of Applied Biochemistry, Tokai University, Kitakaname 1117, Hiratsuka-shi, Kanagawa 259--1292, Japan 2The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan CREST, Japan Science and Technology Corporation (JST)

Received March 20, 2002; Accepted April 25, 2002
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.
Key words: O-glycopeptide; core-class 2; cholestanol; glycosylation; serine

-18-
Total Synthesis of Both Enantiomers of Dictyochromenol and their (Z)-Isomers

Kota AOKI,1 Mihoko TAKAHASHI,1 Masaru HASHIMOTO,1, Toshikatsu OKUNO,1 Kazuya KURATA,2 and Minoru SUZUKI3

1Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo- Cho 3, Hirosaki 036-8561, Japan 2Department of Material and Environmental Engineering, Hakodate National College of Technology, Hakodate 042-8501, Japan 3Laboratory of Biofunctional Chemistry, Graduate School of Enviromental Earth Science, Hokkaido University, Sapporo 060-0810, Japan

Received March 22, 2002; Accepted May 16, 2002
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.
Key words: dictyochromenol; total synthesis; optical rotation; feedingdeterrent

-19-
Inhibition of Photosystem II of Spinach by the Respiration Inhibitors Piericidin A and Thenoyltrifluoroacetone

Nobuhiro IKEZAWA, Kentaro IFUKU, Tsuyoshi ENDO, and Fumihiko SATO

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8502, Japan

Received April 1, 2002; Accepted April 18, 2002
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 QA, primary quinone acceptor in PS II, and QB, 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 QB, 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.
Key words: chlorophyll fluorescence; photosystem II inhibitors; piericidin A; spinach; thenoyltrifluoroacetone

-20-
An Active Compound against Allergen Absorption in Hypoallergenic Wheat Flour Produced by Enzymatic Modification

Shoko TESAKI,1 Jun WATANABE,2 Soichi TANABE,3 Kei SONOYAMA,2 Eri FUKUSHI,2 Jun KAWABATA,2 and Michiko WATANABE1

1Department of Nutrition and Life-science, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan 2Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan 3Faculty of Applied Biological Science, Hiroshima University, Hiroshima 739-8528, Japan

Received April 15, 2002; Accepted May 20, 2002
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.
Key words: Caco-2; hypoallergenic wheat flour; endo-1,4-β-xylanase; allergen absorption

-21-
Note
Pseudohyphal Growth in a Dimorphic Yeast, Candida maltosa, after Disruption of the C-GCN4 Gene, a Homolog of Saccharomyces cerevisiae GCN4

Hiroaki TAKAKU, Hiroyuki HORIUCHI, Masamichi TAKAGI, and Akinori OHTA

Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received December 10, 2001; Accepted April 12, 2002
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.
Key words: GCN4; Candida maltosa; pseudohyphal growth; morphogenesis

-22-
Note
Isolation and Characterization of Highly Liganded Protein from Brewer′s Barley Grain

Stanislava GORJANOVI<Oh><0193><Wa>C,1, Ana CVETKOVI<Oh><0193><Wa>C,1 Desanka SU<Oh><0189><Wa>ZNJEVI<Oh><0193><Wa>C,1 Milo<Oh><0190><Wa>s BELJANSKI,1 Miroslav VRVI<Oh><0193><Wa>C,2 and Jovan HRANISAVLJEVI<Oh><0193><Wa>C1

1Institute of General and Physical Chemistry, P.O. Box 551, Studentski trg 12, 11001 Belgrade, Yugoslavia 2Faculty of Chemistry, University of Belgrade, P.O. Box 158, Studentski trg, 11001 Belgrade, Yugoslavia

Received January 7, 2002; Accepted April 5, 2002
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.
Key words: barley grain; highly liganded proteins; metal-binding proteins; plant-protecting role

-23-
Note
Changes in Membrane Fluidity and Fatty Acid Composition of Pseudomonas putida CN-T19 in Response to Toluene

In Seon KIM,1, Jae Han SHIM,2 and Yong Tack SUH2

1Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta T6G 2G6, Canada 2Division of Applied Bioscience and Biotechnology, Institute of Agricultural Science and Technology, College of Agriculture and Life Science, Chonnam National University, Kwang-Ju 500-757, South Korea

Received Junuary 28, 2002; Accepted April 1, 2002
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 C16:1 and C18:1 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 C16:1 and C18:1 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.
Key words: Pseudomonas; solvent tolerance; fatty acid; cell membrane

-24-
Note
Distribution of Hydrophobin 1 Gene Transcript in Developing Fruiting Bodies of Lentinula edodes

Hiroo NISHIZAWA,a Yasumasa MIYAZAKI,b Shinya KANEKO,c and Kazuo SHISHIDO

Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

Received February 4, 2002; Accepted April 30, 2002
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.
Key words: hydrophobin gene expression; fruiting body; hymenophore; in situ RNA-RNA hybridization; Lentinula edodes

-25-
Note
Effects of Oolong Tea on Metabolism of Plasma Fat in Mice under Restraint Stress

Hiroshi KURIHARA, Harukazu FUKAMI, Hirofumi KODA, Nobuo TSURUOKA, Namino SUGIURA, Hiroshi SHIBATA, and Takaharu TANAKA

Institute for Health Care Science, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan

Received February 12, 2002; Accepted April 20, 2002
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 (T1/2) 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.
Key words: oolong tea; energy metabolism; restraint stress; Intralipid; triglyceride

-26-
Note
Enantioselective Effects of Optically Active α-Methylbenzyl-s-triazine on the Root Growth of Rice and Echinochloa Plants and Their Herbicidal Activity

Hiroyoshi OMOKAWA and Akiko TABEI

Center for Research on Wild Plants, Utsunomiya University, 350 Mine, Utsunomiya 321-8505, Japan

Received February 12, 2002; Accepted May 10, 2002
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).
Key words: enantioselectivity; optically active s-triazine; Pfeiffer′s rule; herbicidal activity

-27-
Note
Cytokine Production by the Murine Macrophage Cell Line J774.1 after Exposure to Lactobacilli

Hirotsugu MORITA,1 Fang HE,1, Tetsuo FUSE,1 Arthur C. OUWEHAND,2 Hideo HASHIMOTO,1 Masataka HOSODA,1 Koko MIZUMACHI,3 and Jun-Ichi KURISAKI4

1Technical Research Laboratory, Takanashi Milk Products Co., Ltd., Yokohama, Kanagawa 241-0023, Japan 2Departments of Biochemistry and Food Chemistry, University of Turku, Fin-20014 Turku, Finland 3National Institutes of Livestock and Grassland Science, Ikenodai, Kukizaki, Inashiki, Ibaraki 305-0901, Japan 4National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan

Received February 18, 2002; Accepted May 2, 2002
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.
Key words: cytokine; macrophage; Lactobacillus; inflammation; probiotics

-28-
Note
Kinetics of Hyperprocessing Reaction of Human Tyrosine tRNA by Ribonuclease P Ribozyme from Escherichia coli

Tomoaki ANDO, Terumichi TANAKA, Yoshiaki HORI, and Yo KIKUCHI

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan

Received February 20, 2002; Accepted April 26, 2002
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 KM was almost the same as in the usual reaction, but kcat 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.
Key words: Escherichia coli; tRNA; RNase P; hyperprocessing; tyrosine

-29-
Note
Characterization of Polymerized Polyphenols by Size-exclusion HPLC

Akio YANAGIDA,1, Toshihiko SHOJI,2 and Tomomasa KANDA3

1Department of Analytical Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan 2Fundamental Research Laboratory, Asahi Breweries, Ltd., 1-1-21 Midori, Moriya, Kitasoma-gun, Ibaraki 302-0106, Japan 3Hirosaki Plant, The Nikka Whisky Distilling Co., Ltd., 2-1-1 Sakae-machi, Hirosaki, Aomori 036-8336, Japan

Received February 26, 2002; Accepted April 30, 2002
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.
Key words: size-exclusion chromatography; polyphenols; procyanidins; tannins; anthocyanins

-30-
Note
Gene Encoding a Trehalose Phosphorylase from Thermoanaerobacter brockii ATCC 35047

Kazuhiko MARUTA, Kazuhisa MUKAI, Hiroshi YAMASHITA, Michio KUBOTA, Hiroto CHAEN, Shigeharu FUKUDA, and Masashi KURIMOTO

Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amase-minami machi, Okayama 700-0834, Japan

Received March 8, 2002; Accepted May 1, 2002
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.
Key words: trehalose; phosphorylase; nucleotide sequence; Thermoanaerobacter brockii

-31-
Note
Increase in the Rate of L-Pipecolic Acid Production Using lat-Expressing Escherichia coli by lysP and yeiE Amplification

Tadashi FUJII, Yasuhide ARITOKU, Hitosi AGEMATU, and Hiroshi TSUNEKAWA

Bioresource Laboratories, Mercian Corp., 4-9-1 Johnan, Fujisawa 251-0057, Japan

Received March 8, 2002; Accepted April 17, 2002
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.
Key words: lat; lysP; yeiE; L-pipecolic acid; proC

-32-
Note
Occurrence of GalNAcβ1-4GlcNAc Unit in N-Glycan of Royal Jelly Glycoprotein

Mariko KIMURA,1 Yoichiro HAMA,2 Kazunori TSUMURA,1 Kiyoshi OKIHARA,3 Hiroyuki SUGIMOTO,3 Hideo YAMADA,3 and Yoshinobu KIMURA1

1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1, Okayama 700-8530, Japan 2Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Honjyo 1, Saga, 840-8502, Japan 3Yamada Apiculture Center, Inc. Ichiba 194, Kagamino-Cho, Tomada-Gun, Okayama 708-0393, Japan

Received March 11, 2002; Accepted April 6, 2002
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.
Key words: insect N-glycan; N-glycan containing GalNAc; royal jelly glycoproteins; Apis mellifera

-33-
Note
Antioxidative Activity of Resveratrol and Its Derivatives Isolated from Seeds of Paeonia lactiflora

Hyo Jin KIM,1 Eun Ju CHANG,1 Sung Hee CHO,1 Shin Kyo CHUNG,2 Heui Dong PARK,2 and Sang Won CHOI1

1Department of Food Science and Nutrition, Catholic University of Daegu, Hayang, Gyeongbuk 712-702, Korea 2Department of Food Science and Technology, Kyungpook National University, Daegu 702-701, Korea

Received March 13, 2002; Accepted May 7, 2002
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.
Key words: Paeonia lactiflora; stilbene derivative; antioxidative activity; 2-deoxyribose degradation; rat liver microsome

-34-
Note
Frequency of the Insertion Sequence IS4Bsu1 among Bacillus subtilis Strains Isolated from Fermented Soybean Foods in Southeast Asia

Keitarou KIMURA, Yasuhiro INATSU, and Yoshifumi ITOH

Division of Applied Microbiology, National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan

Received April 1, 2002; Accepted April 26, 2002
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.
Key words: Bacillus subtilis; insertion sequence; soybean fermentation; Southern blotting

-35-
Note
Determination of the Absolute Configuration of (＋)-2,7(14),10-Bisabolatrien- 1-ol-4-one from Japanese Cedar, Cryptomeria japonica

Chul-Sa KIM, Jun MORISAWA, Nobuhiro NISHIYAMA, Takehiro KASHIWAGI, Shin-ich TEBAYASHI, and Michio HORIIKE

Department of Bioresources Science, Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku 783-8502, Japan

Received April 8, 2002; Accepted May 13, 2002
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.
Key words: (1S,6R) - 2,7(14),10 - bisabolatrien - 1 - ol-4-one; (＋)-2,7(14),10-bisabolatrien-1-ol-4-one; cryptomerione; Cryptomeria japonica