Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.2 2001)

Kazuaki HIRASAWA, Yoshitaka ISHII, Morio KOBAYASHI, Kenichi KOIZUMI,
and Kenji MARUHASHI・・・

Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center,
1900 Sodeshi-cho, Shimizu-shi, Shizuoka 424-0037, Japan

Received May 18, 2000; Accepted October 5, 2000
Rhodococcus erythropolis KA2-5-1 can desulfurize dibenzothiophene (DBT) into 2-hydroxybiphenyl. A cryptic plasmid, pRC4, which was derived from R. rhodochrous IFO3338, was combined with an Escherichia coli vector to construct an E. coli-Rhodococcus shuttle vector. The complete nucleotide sequence of 2582-bp pRC4 was analyzed. Based on the characteristics of its putative replication genes, pRC4 was assigned to the family of pAL5000-related replicons. The desulfurization gene cluster, dszABC, and the related reductase gene, dszD, cloned from KA2-5-1, were reintroduced into KA2-5-1 and efficiently expressed. The DBT desulfurization ability of the transformant carrying two dszABC clusters and one dszD on the vector was about 4-fold higher than that of the parent strain, and the transformant also showed improved desulfurization activity for light gas oil (LGO). Sulfur components in LGO before and after the reaction were analyzed with gas chromatography-atomic emission detection.
Key words: desulfurization; dibenzothiophene; Rhodococcus; plasmid; light gas oil

-2-
Isolation and Characterization of Enterocin SE-K4 Produced by Thermophilic
Enterococci, Enterococcus faecalis K-4

Tomoko EGUCHI,2 Kazuyo KAMINAKA,1 Jun SHIMA,1,・・・ Shinichi KAWAMOTO,1 Katsumi MORI,1
Seong-Hyun CHOI,2 Katsumi DOI,2 Sadahiro OHMOMO,3 and Seiya OGATA2

1National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki,
Fukuoka 812-8581, Japan
3National Institute of Animal Industry, 2 Ikenodai, Kukisaki, Ibaraki 305-0901, Japan

Received May 24, 2000; Accepted September 28, 2000
Enterococcus sp. K-4, with a bacteriocin-like activity against E. faecium, was isolated from grass silage in Thailand. Morphological, physiological, and phylogenetic studies clearly identified strain K-4 as a strain of E. faecalis. Strain K-4 produced a maximal amount of bacteriocin at 43--45°C. We purified, for the first time, the bacteriocin produced at high temperature by E. faecalis to homogeneity, using adsorption on cells of the producer strain and reversed-phase liquid chromatography. The bacteriocin, designated enterocin SE-K4, is a peptide of about 5 kDa as measured by SDS-PAGE, and Mass spectrometry analysis found the molecular mass of 5356.2, which is in good agreement. The amino acid sequencing of the N-terminal end of enterocin SE-K4 showed apparent sequence similarity to class IIa bacteriocins. Enterocin SE-K4 was active against E. faecium, E. faecalis, Bacillus subtilis, Clostridium beijerinckii, and Listeria monocytogenes. Enterocin SE-K4 is very heat stable.
Key words: bacteriocin; enterocin; thermophilic enterococci; natural food preservatives; starter of silage production

-3-
New Classification System for Oxygenase Components Involved
in Ring-Hydroxylating Oxygenations

Jeong-Won NAM, Hideaki NOJIRI, Takako YOSHIDA, Hiroshi HABE,
Hisakazu YAMANE, and Toshio OMORI・・・

Biotechnology Research Center, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan

Received June 6, 2000; Accepted October 17, 2000
Batie et al. [Chemistry and Biochemistry of Flavoenzymes, 3, 543--556 (1991)] proposed a classification system for ring-hydroxylating oxygenases in which the oxygenases are grouped into three classes in terms of the number of constituent components and the nature of the redox centers. But in recent years, many ring-hydroxylating oxygenases have been newly identified and characterized, and found difficult to classify into these three classes. Typical examples are carbazole 1,9a-dioxygenase and 2-oxo-1,2-dihydroquinoline 8-monooxygenase, which have been classified into class III and class IB, respectively, from biochemical characteristics. However, a phylogenetic study showed that the terminal oxygenases of both are closely related to class IA. Because this discrepancy derived from counting all the components together, here we proposed a new scheme based on the homology of the amino acid sequences of the α subunits of the terminal oxygenase components. This new scheme strongly reflects the actual phylogenetic affiliation of the terminal oxygenase component. By comparing their sequences pairwise using the CLUSTAL W program, 54 oxygenase components were classified into 4 groups (groups I, II, III, and IV). While group I contains broad-range oxygenases sharing low homology, groups II, III, and IV contain some typical oxygenases: benzoate/toluate dioxygenases for group II, naphthalene/polycyclic aromatic hydrocarbon dioxygenases for group III, and benzene/toluene/biphenyl dioxygenases for group IV. Our new scheme is simple and powerful, since an oxygenase component can be nearly automatically grouped when the DNA sequence is available, and it fits very well with the phylogenetic affiliation.
Key words: classification; ring-hydroxylating oxygenases; terminal oxygenase component; α-subunit

-4-
Effect of Dietary Short-Chain Fructooligosaccharides on the Cecal Microflora
in Gastrectomized Rats

Kensuke SAKAI,・・・ Kazuyoshi ARAMAKI, Misao TAKASAKI, Hiromi INABA,
Takahisa TOKUNAGA, and Atsutane OHTA

Bioscience Laboratories, Meiji Seika Kaisha Ltd., 5-3-1 Chiyoda, Sakado-shi, Saitama 350-0289, Japan

Received June 6, 2000; Accepted September 14, 2000
Total gastric resection is known to lead to changes in the microflora in the whole gastrointestinal tract. Dietary short-chain fructooligosaccharides (Sc-FOS) have been shown to also induce a change in the microflora in the large bowel by promoting an increase in the numbers of Bifidobacterium and Lactobacillus which have beneficial effects on the host. In the present study, 4-week-old male Sprague-Dawley rats received total gastrectomy or laparotomy, and each of these surgically treated groups was randomly divided into two experimental diet groups and given a 7.5％ Sc-FOS diet or control diet. Enumeration and identification of the cecal bacteria was performed by using selective and non-selective media. In the gastrectomized rats, the total bacterial count, and the counts of Bacteroidaceae and Enterobacteriaceae were higher than those in the sham-operated rats. Sc-FOS promoted an increase in the numbers of Bifidobacterium and Lactobacillus, In the rats fed on the Sc-FOS diet, the predominant type of bacteria was Lactobacillus and in the rats fed on the control diet, it was Bacteroidaceae irrespective of gastrectomy. We confirmed that both gastrectomy and dietary Sc-FOS changed the composition of cecal microflora in the rats. Dietary Sc-FOS in the gastrectomized rats increased the proportions of Lactobacillus relative to other types of bacteria to levels similar to those seen in healthy normal rats, and decreased the proportion of Bacteroidaceae.
Key words: gastrectomy; short-chain fructooligosaccharides; cecal microflora

-5-
Synthesis, Absolute Configuration and Biological Activity of Both Enantiomers
of 2-(5,6-Dichloro-3-indolyl)propionic Acid: New Dichloroindole Auxins

Masato KATAYAMA,1,・・・ Yasuhito KATO,2 and Shingo MARUMO3

1Department of Chemistry, National Industrial Research Institute of Nagoya (NIRIN), 1-1 Hirate-cho,
Kita-ku, Nagoya 462-8510, Japan
2Ageo ＆ Fine Chemicals Research Laboratory, Nippon Kayaku Co. Ltd., 225-1 Koshikiya, Ageo,
Saitama 362-0064, Japan
3Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8601, Japan

Received July 4, 2000; Accepted October 12, 2000
Racemic 2-(5,6-dichloro-3-indolyl)propionic acid (5,6-Cl2-2-IPA) was synthesized from 5,6-dichloroindole-3-acetic acid (5,6-Cl2-IAA) by successive esterification, methoxycarbonylation, methylation, and double hydrolysis. The racemate was converted to the diastereomeric esters of (S)-(--)-1-phenylethyl alcohol. These were separated by HPLC into two optically active diastereomers and then hydrolyzed with p-TsOH to the optically active enantiomers of 5,6-Cl2-2-IPA. The absolute configurations of both the 5,6-Cl2-2-IPA enantiomers were determined by comparing the 1H-NMR spectra of their diastereomeric (S)-(--)-1-phenylethyl esters with those of the diastereomeric (S)-(--)-1-phenylethyl esters of 2-(3-indolyl)propionic acid (2-IPA) whose absolute configurations are already known.
There was no essential difference between (S)-(+)- and (R)-(--)-5,6-Cl2-2-IPA in hypocotyl growth-inhibiting activity toward Chinese cabbage, but their inhibitory activities were stronger than that of the potent mother auxin, 5,6-Cl2-IAA. No essential difference in the coleoptile elongating activity of Avena sativa was apparent for the enantiomers, this activity being about one-third that of 5,6-Cl2-IAA.
Key words: (R) - (--) - 2 - (5,6 - dichloro - 3 - indolyl)propionic acid; (S)-(+)-2-(5,6-dichloro-3-
indolyl)propionic acid; dichloroindole auxin; Avena coleoptile elongation; hypocotyl growth inhibition

-6-
Storage-dependent Degradation of 57-kDa Protein in Royal Jelly:
a Possible Marker for Freshness

Masaki KAMAKURA,1,・・・ Toshiyuki FUKUDA,1 Makoto FUKUSHIMA,1 and Masami YONEKURA2

1POLA R＆D Laboratories, POLA Corporation, 560 Kashio-cho, Totsuka-ku, Yokohama 244-0812, Japan
2School of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan

Received July 10, 2000; Accepted October 18, 2000
In order to find a marker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40°C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4°C to 50°C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.
Key words: royal jelly; 57-kDa protein; storage-dependent degradation; marker for freshness; quality control

-7-
Identification of Collagen as a New Fish Allergen

Yuki HAMADA, Yuji NAGASHIMA, and Kazuo SHIOMI・・・

Department of Food Science and Technology, Tokyo University of Fisheries, Konan-4, Minato-ku,
Tokyo 108-8477, Japan

Received July 12, 2000; Accepted September 28, 2000
This study was intended to identify a high molecular weight allergen that had been detected in fish. Analyses by ELISA of five protein fractions prepared from bigeye tuna muscle showed that the high molecular weight allergen was contained in the myostromal protein fraction. Based on the results of SDS-PAGE, immunoblotting and amino acid analysis of the myostromal protein fraction, the high molecular weight allergen was judged to be collagen. Five of the eight patient sera used were found to react to the bigeye tuna collagen. In competitive ELISA inhibition experiments, the bigeye tuna collagen almost completely inhibited the IgE reactivity to the heated extracts from five species of fish, suggesting that collagen is commonly allergic regardless of fish species. However, no antigenic cross-reactivity was observed between collagens from fish and other animals.
Key words: allergen; bigeye tuna; collagen; fish; gelatin

-8-
Conformational Change in a Single Molecular Species, β3, of β-Conglycinin
in Acidic Ethanol Solution

Kazunobu TSUMURA, Makoto ENATSU, Koichi KURAMORI, Shimpei MORITA, Wataru KUGIMIYA,
Masahiro KUWADA,＊ Yuki SHIMURA,＊＊ and Hideyo HASUMI＊＊

Novelty Materials Research Institute, Tsukuba R＆D Center, Fuji Oil Co., Ltd. 4-3 Kinunodai, Yawara,
Tsukuba-gun, Ibaraki 300-2497, Japan
＊Department of Biochemistry, Kitasato University School of Medicine. 1-15-1, Kitasato, Sagamihara,
Kanagawa 228-8555, Japan
＊＊Department of Biophysical Chemistry, Kitasato University School of Medicine. 1-15-1, Kitasato,
Sagamihara, Kanagawa 228-8555, Japan

Received July 17, 2000; Accepted October 5, 2000
Several physicochemical experiments were done to obtain further information on the conformational changes occurring in β-conglycinin in acidic-ethanol solution, using a single molecular species of this protein, β3. By far-UV circular dichroism (CD), a transition from β-sheet to α-helical structure was observed upon addition of acidic-ethanol, and the α-helix content was found to reach 76％ in 70％ ethanol (pH 2). From analyses of near-UV CD and difference absorption spectra, it was found that the tertiary structure of the β3 species was significantly altered at ethanol concentrations between 10 and 20％. The profiles of binding of 1-anilinonaphthalene-8-sulfonic acid to the β3 species during acidic-ethanol denaturation were indicative of the existence of intermediate conformers in the molten globule-like denaturation state. By measuring Fourier transform infrared spectra and estimating the Stokes radius by dynamic light scattering, the β3 molecules were found to aggregate with an increase in ethanol concentration.
Key words: soybean protein; conformational change; β-conglycinin; ethanol

-9-
Kinetic Analysis of Microbial Desulfurization of Model and Light Gas Oils
Containing Multiple Alkyl Dibenzothiophenes

Morio KOBAYASHI, Keizo HORIUCHI,・・・ Osamu YOSHIKAWA, Kazuaki HIRASAWA,
Yoshitaka ISHII, Kazuhito FUJINO, Hiroshi SUGIYAMA, and Kenji MARUHASHI

Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center,
1900 Sodeshi-Cho, Shimizu-Shi, Shizuoka 424-0037, Japan

Received July 17, 2000; Accepted October 16, 2000
The reaction mechanism of biodesulfurization was investigated using whole cells of Rhodococcus erythropolis KA2-5-1, which have the ability to convert dibenzothiophene(DBT) into 2-hydroxybiphenyl. The desulfurization patterns of alkyl DBTs were represented by the Michaeis-Menten equation. The values of rate constants, the limiting maximal velocity (Vmax) and Michaelis constant (Km), for desulfurization of alkyl DBTs were calculated.
The relative desulfurization activities of various alkyl DBTs were reduced in proportion to the total carbon numbers of alkyl substituent groups. Alkyl DBTs that had a total of six carbons of alkyl substituent groups were not desulfurized. The type or position of alkyl substituent groups had little effect on desulfurization activity.
The desulfurization activity of each alkyl DBT, when mixed together, was reduced. This phenomenon was caused by apparent competitive inhibition of substrates. Using the apparent competitive inhibition model, the desulfurization pattern of a multiple components system containing alkyl DBTs was elucidated. This model was also applicable for biodesulfurization of light gas oil.
Key words: desulfurization; alkyl dibenzothiophene; light gas oil; Rhodococcus

-10-
Synthesis of the Enantiomers of Some Methyl-branched Cuticular
Hydrocarbons of the Ant, Diacamma sp.・・・

Kaoru MARUKAWA, Hirosato TAKIKAWA, and Kenji MORI・・・・・・

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan

Received July 17, 2000; Accepted October 11, 2000
The enantiomers of 3-methylpentacosane, 3-methylheptacosane, 3-methylnonacosane, 13-methylheptacosane, and 5-methylheptacosane were synthesized by starting from the enantiomers of 2-methylbutyl bromide or citronellol. These methyl-branched alkanes are the characteristic components of the cuticular hydrocarbons of queen of the ant, Diacamma sp.
Key words: methyl-branched alkane; optically active alkane; queen of ant; Diacamma sp.; cuticular hydrocarbon

-11-
Interactions of Dietary Fats and Proteins on Fatty Acid Composition
of Immune Cells and LTB4 Production by Peritoneal Exudate Cells of Rats

Shihoko KAKU, Shin-ichi YUNOKI,＊ Ken-ichi OHKURA,＊ Michihiro SUGANO,
Michiko NONAKA,＊ Hirofumi TACHIBANA,＊ and Koji YAMADA＊

Study of Food and Health Environment, Department of Environmental and Symbiotic Sciences,
Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan
＊Department of Bioscience and Biotechnology, Division of Bioenvironmental Sciences, Graduate School
of Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received July 21, 2000; Accepted September 28, 2000
The interaction of dietary fats and proteins on lipid parameters of rats was studied using safflower oil (linoleic acid-rich), borage oil (γ-linolenic acid-rich) or perilla oil (α-linolenic acid-rich) in combination with casein or soybean protein. The experiment was focused on the fatty acid composition of immune cells and the leukotriene B4 production by peritoneal exudate cells. Serum total cholesterol, triglyceride, and phospholipid levels were low in perilla oil-fed or soybean protein-fed rats. Fatty acid compositions of serum and liver phospholipids reflected those of dietary fats. However, feeding borage oil resulted in a marked increase in the proportion of dihomo-γ-linolenic acid in phospholipids of peritoneal exudate cells, spleen lymphocytes, and mesenteric lymph node lymphocytes in relation to those of liver and serum. It is suggested that activities of metabolic n-6 polyunsaturated fatty acids are different between immune and other tissues. In addition, the magnitude of the reduction of the proportion of linoleic acid of perilla oil in immune cells was considerably more moderate than serum and liver, indicating a different degree of interference of α-linolenic acid with linoleic acid metabolism. Leukotriene B4 release from peritoneal exudate cells was in the order of safflower oil>borage oil>perilla oil groups as reflecting the proportion of arachidonic acid, and tended to be lower in soybean protein-fed groups. These suggest an anti-
inflammatory property of γ-linolenic acid as well as α-linolenic acid tended to be strengthened when they were combined with soybean protein than with casein.
Key words: lymphocyte; soybean protein; α-linolenic acid; γ-linolenic acid; lipid metabolism

-12-
Molecular Cloning, DNA Sequence, and Expression of the Gene Encoding
for Thermostable Pectate Lyase of Thermophilic Bacillus sp. TS 47

Makoto TAKAO, Tetsuko NAKANIWA, Kentaro YOSHIKAWA,
Takao TERASHITA, and Takuo SAKAI・・・

Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, 3327-204,
Nakamachi, Nara 631-8505, Japan

Received July 26, 2000; Accepted October 11, 2000
The gene that encodes a thermostable pectate lyase (called PL 47), from Bacillus sp. TS 47, was cloned, sequenced, and expressed in mesophilic B. subtilis. The gene contained an open reading frame consisting of 1326 bp, which encoded 441 amino acids. The deduced amino acid sequence of the mature enzyme (416 amino acids with a calculated molecular mass of 47,262 Da), showed 52％ similarity with PL (BsPel) from mesophilic B. subtilis SO113. The structure-based alignment of the deduced amino acid sequence of PL 47 with that of BsPel suggested that PL 47 might have a parallel β-helix structure with three long loops. The amino acids making up PL 47 are richer in hydrophobic amino acids and glutamic acid than BsPel. The hydropathy profile of PL 47 indicated that the amino acid sequences around putative calcium binding sites are more hydrophobic than the same region of BsPel. The gene product expressed in B. subtilis as the host was stable up to 70°C and the reaction was optimal around 70°C, as well as native PL 47.
Key words: gene cloning; thermophile; Bacillus; pectate lyase; thermostable enzyme

-13-
Growth of Nisin-Producing Lactococci in Cooked Rice Supplemented
with Soybean Extract and its Application to Inhibition
of Bacillus subtilis in Rice Miso

Takeo KATO,・・・ Lisa INUZUKA,＊ Masami KONDO,＊ and Tsukasa MATSUDA＊＊

・・・Food Research Institute, Aichi Prefectural Government, 2-1-1 Shinpukuji-cho, Nishi-ku,
Nagoya 451-0083, Japan＊Faculty of Life Study, Sugiyamajogakuen University, Hoshigaokamotomachi, chikusa-ku,
Nagoya 464-8662, Japan
＊＊Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8601, Japan

Received July 27, 2000; Accepted September 27, 2000
Lactic acid fermentation of cooked rice and rice koji by supplementation with soybean extract (SBE) and its application to rice miso fermentation were investigated. By supplementing the cooked rice with SBE, lactic acid bacteria (LAB) grew well without any unfavorable effects on the rice such as off-flavor or coloration. Lactococcus lactis subsp. lactis IFO12007 (Lc. lactis, a producer of the bacteriocin nisin) proliferated at 108~9 cells/g after 24 h of incubation and produced high activity of nisin. The fermented rice with Lc. lactis strongly inhibited not only Bacillus subtilis ATCC19659 but also the other Bacillus strains. While some strains of LAB markedly inhibited the growth of Asp. oryzae, resulting in failure of koji fermentation, Lc. lactis did not affect the growth of these molds. When Lc. lactis was used for rice miso fermentation as a lactic acid starter culture, Lc. lactis rapidly proliferated and produced high nisin activity of 6,400 IU/g, in the steamed rice, resulting in complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation. The rice miso after 12 weeks of aging had a suitable pH, and favorable taste and color. Furthermore, hyposalting of rice miso could be done without difficulty by lactic acid fermentation of both rice and soybeans.
Key words: nisin; Lactococcus lactis; lactic acid fermentation; rice miso; Bacillus inhibition

-14-
LysR-type Transcriptional Regulator ChiR Is Essential for Production
of All Chitinases and a Chitin-Binding Protein, CBP21,
in Serratia marcescens 2170

Kazushi SUZUKI,1,§ Taku UCHIYAMA,2 Megumi SUZUKI,2 Naoki NIKAIDOU,1,2
Miguel REGUE,3 and Takeshi WATANABE1,2,・・・

1Department of Applied Biological Chemistry, Faculty of Agriculture, and 2Department of Biosystem Science,
Graduate School of Science and Technology, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan
3Department of Microbiology and Parasitology, Health Sciences Division, Faculty of Pharmacy,
University of Barcelona, Barcelona 08028, Spain

Received July 28, 2000; Accepted September 20, 2000
To identify the genes required for chitinase production by Serratia marcescens 2170, various Tn5 mutants somehow defective in chitinase production were isolated in a previous study. In order to identify the mutated gene in one of the chitinase-deficient mutants, N1, DNA regions flanking the Tn5 insertion were cloned and sequenced. Sequence comparison showed that the mutation occurred in the ORF located between chiB and cbp, which encode chitinase B and chitin-binding protein CBP21, respectively. The ORF encodes a 313-amino acid polypeptide which has significant similarity with various LysR-type transcriptional regulators, and thus the gene was designated chiR. Targeted mutagenesis confirmed that disruption of the chiR gene results in the phenotype of N1. Gel mobility shift assays using partially purified ChiR protein demonstrated that this protein specifically binds to the intergenic region between chiR and cbp. These results strongly suggest that ChiR is a LysR-type transcriptional regulator which is essential for production of all chitinases and CBP21.
Key words: Serratia marcescens; LysR-type transcriptional regulator; chitinase; chitin binding protein

-15-
Changes in Hematological Parameters of Athletes after Receiving Daily Dose
of a Mixture of 12 Amino Acids for One Month during the Middle-
and Long-distance Running Training

Masaru OHTANI,1,・・・ Kimiaki MARUYAMA,2 Shihoko SUZUKI,3 Masaaki SUGITA,4
and Kando KOBAYASHI1

1Department of Life Sciences (Sports Sciences), Graduate School of Arts and Sciences,
The University of Tokyo, Tokyo 153-8902, Japan
2Department of Life Science, Meiji University, Kawasaki 214-8571, Japan
3National Institute of Fitness and Sports in Kanoya, Kagoshima 891-2393, Japan
4Department of Health ＆ Physical Education, Mie University, Tsu City 514-8507, Japan

Received July 28, 2000; Accepted October 23, 2000
Previous studies have shown that a mixture of amino acids, consisting of 9 essential amino acids and 3 non-
essential amino acids was effective in facilitating muscle recovery from athletic activities. In this study, the objective was to determine whether this amino acid mixture improved the physical condition and associated blood parameters of athletes in training when administered for a prolonged period. Thirteen college middle- and long-distance runners were placed in a 6-month experiment and received the amino acid mixture at the dose of 2.2 g/day for one month, 4.4 g/day for one month, and 6.6 g/day for one month with washout periods between test periods. The physical condition was scored and blood samples were collected before and after each test period. When the subjects received 2.2 g of the amino acid mixture three times a day, the physical condition was significantly improved along with increases in red blood cell count, hemoglobin, hematocrit, serum albumin, and fasting glucose, and a decrease in creatine phophokinase (p<0.05), suggesting increased hematopoiesis and glycogenesis, and rapid alleviation of muscle inflammation by the amino acid mixture.
Key words: amino acids; exercise; sports medicine; hematology; blood biochemistry

-16-
Characterization of a Metalloenzyme from a Wild Mushroom,
Tricholoma saponaceum

Jun-Ho KIM and Yang Sun KIM＊

Department of Chemistry, Sangji University, Wonju 220-702, Korea
＊Division of Molecular Life Science and Center for Cell Signaling Research, Ewha Womans University,
Seoul 120-750, Korea

Received July 31, 2000; Accepted September 19, 2000
Two kinds of metalloendopeptidases from the fruiting bodies of Tricholoma saponaceum (TSMEP1 and TSMEP2) have been purified, and TSMEP1 has been characterized based on their fibrinolytic activity. The enzymes have the same N-terminal amino acid sequence, Ala-Leu-Tyr-Val-Gly-X-Ser-Pro-X-Gln-Gln-Ser-Leu-Leu-Val, but slightly different molecular weights of 18,147 and 17,947, as measured by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry. The N-terminal sequence do not match with any known protein or open reading frame. TSMEP1 hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, human IgG, hemoglobin, or urokinase. The enzyme hydrolyzes both Aα and Bβ subunits of human fibrinogen with equal efficiency but didn′t show any reactivity for the γ form of human fibrinogen. The enzymatic activity is strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzymes are metalloproteases. No inhibition was found with phenylmethylsulfonyl fluoride (PMSF), L-trans-epoxysuccinyl leucylamido-(4-guanidino)-butane (E-64), pepstatin and 2-mercaptoethanol. The activity of the purified enzyme was increased by Mg2+, Fe2+, Zn2+, and Co2+, and slightly decreased by Ca2+, but the enzyme activity was dramatically decreased by Cu2+, and totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and keep the high activity from pH 7.5 to 9, suggesting that the purified enzyme was a basic protease. The enzyme was stable up to 30°C and the maximum fibrinolytic activity was at 55°C.
Key words: metalloprotease; mushroom; Tricholoma saponaceum; fibrinolytic activity; MALDI

-17-
Antitumor Effect of Photodynamic Therapy with Zincphyrin,
Zinc-coproporphyrin III, in Mice

Minoru TORIYA,1,・・・ Megumi YAMAMOTO,2,・・・・・・ Kenji SAEKI,1 Yoshie FUJII,1
and Kazuhiko MATSUMOTO1,・・・・・・・・・

1Institute for Life Science Research and 2Department of Diagnostics Research and Development,
Asahi Chemical Industry Co., Ltd., 632-1 Mifuku, Ohito-cho, Tagata-gun, Shizuoka 410-2321, Japan

Received August 7, 2000; Accepted October 11, 2000
We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptmyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78--100 μg/ml with visible light of 27.2 mW・min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5--50 mg/kg with light irradiation of 75.48 mW・min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 μg/ml within 20 min, and the concentration remained at 4.2 μg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW・min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.
Key words: zinc-coproporphyrin III; photosensitizer; photodynamic therapy; cancer; Streptomyces

-18-
Feeding Unsaponifiable Compounds from Rice Bran Oil Does Not Alter
Hepatic mRNA Abundance for Cholesterol Metabolism-related Proteins
in Hypercholesterolemic Rats

Koji NAGAO,＊ Masao SATO,・・・ Miyuki TAKENAKA, Miyuki ANDO, Masako IWAMOTO,
and Katsumi IMAIZUMI

Laboratory of Nutrition Chemistry, Division of Bioscience and Biotechnology, Faculty of Agriculture,
Graduate School Kyushu University, Fukuoka 812-8581, Japan

＊Research Fellow of the Japan Society for the Promotion of Science
Received August 9, 2000; Accepted October 4, 2000
The hypocholesterolemic effect of rice bran oil (RBO) is defined in human and animal experiments which indicate the presence of active component(s) in the unsaponifiable fraction, but the detailed mechanism is not known yet. Exogenously hypercholesterolemic (ExHC) rats were fed for 2 weeks on a 0.5％ cholesterol diet supplemented with 10％ each of RBO, RBO-simulated oil (RBOSO) in its fatty acid composition, or RBOSO plus 0.25％ unsaponifiable compounds (UC) from RBO. Rats fed RBO or the UC resulted in lowing serum and liver cholesterol concentration and preventing reduction of high density lipoproteinic-cholesterol. Dietary RBO or the UC led to an elevation of fecal neutral sterol excretion, but no significant change in fecal bile acid excretion or in hepatic abundance of mRNAs for 3-hydroxy-3-methylglutaryl-CoA reductase, cholesterol-7α-hydroxylase, and low density lipoprotein receptor. Besides, serum and liver α-tocopherol concentrations were lowered in RBO or the UC-fed rats. These results show that the UC in RBO leads to a decreased serum cholesterol concentration by interrupting the absorption of intestinal hydrophobic compounds rather than by modifying cholesterol metabolism in the liver.
Key words: rice bran oil; unsaponifiable compounds; hypocholesterolemic effect; plant sterols; exogenously hypercholesterolemic

-19-
Occurrence of 11-Hydroxyjasmonic Acid Glucoside in Leaflets
of Potato Plants (Solanum tuberosum L.)

Hideyuki MATSUURA, Yasuhiro OHKUBO, and Teruhiko YOSHIHARA・・・

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan

Received August 15, 2000; Accepted October 16, 2000
In order to examine the occurrence of 11-hydroxyjasmonic acid glucoside in potato plants, a synthesis of 11-hydroxyjasmonic acid was accomplished, and the synthetic compound was employed as a standard for an LC-SIM analysis. The existence of 11-hydroxyjasmonic acid glucoside was proved by the LC-SIM analysis.
Key words: Solanum tuberosum L.; potato micro-tuber inducing stimulus; jasmonoid; liquid chromatography-selected ion monitoring (LC-SIM)

-20-
A Transgenic Apple Callus Showing Reduced Polyphenol Oxidase Activity
and Lower Browning Potential

Masatsune MURATA,a,・・・ Makiyo NISHIMURA,a Nanae MURAI,a Miyoshi HARUTA,a
Seiichi HOMMA,a and Yuji ITOHb

aDepartment of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112-8610, Japan
bNational Institute of Agrobiological Resources, 2425 Kamimurata, Omiya-cho, Ibaraki 319-22, Japan

Received August 21, 2000; Accepted October 17, 2000
Polyphenol oxidase (PPO) is responsible for enzymatic browning of apples. Apples lacking PPO activity might be useful not only for the food industry but also for studies of the metabolism of polyphenols and the function of PPO. Transgenic apple calli were prepared by using Agrobacterium tumefaciens carrying the kanamycin (KM) resistant gene and antisense PPO gene. Four KM-resistant callus lines were obtained from 356 leaf explants. Among these transgenic calli, three calli grew on the medium containing KM at the same rate as non-transgenic callus on the medium without KM. One callus line had an antisense PPO gene, in which the amount and activity of PPO were reduced to half the amount and activity in non-transgenic callus. The browning potential of this line, which was estimated by adding chlorogenic acid, was also half the browning potential of non-transgenic callus.
Key words: Malus×domestica (apple); enzymatic browning; polyphenol oxidase; antisense; transgenic plant

-21-
Construction of a Chimeric Shuttle Plasmid via a Heterodimer System:
Secretion of an scFv Protein from Bacillus brevis Cells Capable
of Inhibiting Hemagglutination

Teruaki SHIROZA,1,・・・ Yasuko SHIBATA,1 Mitsuo HAYAKAWA,1 Noriko SHINOZAKI,2
Kazuo FUKUSHIMA,2 Shigezo UDAKA,3 and Yoshimitsu ABIKO1

1Department of Biochemistry and 2Department of Microbiology, Nihon University School of Dentistry
at Matsudo, Matsudo, Chiba 271-8587, Japan
3Department of Fermentation Science, Tokyo University of Agriculture, Setagaya, Tokyo, 156-8502, Japan

Received August 24, 2000; Accepted October 2, 2000
Passive immunization is an attractive therapy for preventing oral diseases including dental caries and periodontal disease. For this purpose, we attempted to produce a single chain variable fragment, scFv, which inhibited hemagglutination using the Bacillus brevis protein-producing system. To accomplish this, a novel strategy, a heterodimer system, was used for the construction of a chimeric shuttle plasmid. Initially, a set of new plasmids, kanamycin-resistant donor and erythromycin-resistant general cloning plasmids, were constructed. p15A ori was a common replication origin in these plasmids, while the pUB110 rep and minus origin (MO) were cloned into the donor plasmid. Next, the secretion domain of the B. subtilis α-amylase gene and the G2-4 gene, coding for the scFv protein, were cloned into the general cloning plasmid and fused by PCR. Both the donor plasmid and the general cloning plasmid containing the fused gene were digested with NotI and them ligated, a dimeric plasmid being constructed. The key restriction sites, AscI, are arranged such that the pUB110 rep-MO moiety was switched from the donor to the general cloning plasmid following AscI digestion. The chimeric shuttle plasmid was readily constructed by simple re-circularization and a B. brevis transformant producing the scFv protein in the culture fluid was isolated.
Key words: scFv secretion; Bacillus brevis; pUB110; inhibition of hemagglutination; passive immunization

-22-
Effects of Buckwheat in a Renal Ischemia-Reperfusion Model

Takako YOKOZAWA,1,・・・ Hajime FUJII,2 Kenichi KOSUNA,2 and Gen-ichiro NONAKA2

1Institute of Natural Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani,
Toyama 930-0194, Japan
2Amino Up Chemical Co., Ltd., 363-32 Shin-Ei, Sapporo 004-0839, Japan

Received August 29, 2000; Accepted October 16, 2000
Experiments were done to find whether buckwheat extract ameliorates the renal injury induced by ischemia-reperfusion. In ischemic-reperfused control rats, the activities of antioxidative enzymes in renal tissue and blood and renal parameters deviated from the normal range, indicating dysfunction of the kidneys. In contrast, when buckwheat extract was given orally for 20 consecutive days before ischemia and reperfusion, the activities of the antioxidation enzymes superoxide dismutase, catalase, and glutathione peroxidase were higher, while thiobarbituric acid-reactive substance levels in serum and renal tissue were lower in the treated rats than in the controls. Decreased levels of urea nitrogen and creatinine in serum demonstrated a protective effect against the renal dysfunction caused by ischemia and recirculation. On the other hand, it was demonstrated that buckwheat extract had a protective effect on cultured proximal tubule cells subjected to hypoxia-reoxygenation, probably by preventing oxygen free radicals from attacking the cell membranes.
Key words: buckwheat; ischemia-reperfusion; renal injury; LLC-PK1

-23-
The Deletion of Amino-Terminal Domain in Thermoactinomyces vulgaris
R-47 α-Amylases: Effects of domain N on Activity, Specificity,
Stability and Dimerization

Takehiro YOKOTA,1 Takashi TONOZUKA,1 Shigehiro KAMITORI,2 and Yoshiyuki SAKANO1,・・・

1Department of Applied Biological Science, Tokyo University of Agriculture and Technology,
3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan
2Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology,
2-24-16 Naka-machi, Koganei-shi, Tokyo 184-8588, Japan

Received September 1, 2000; Accepted October 5, 2000
Thermoactinomyces vulgaris R-47 α-amylases, TVA I and TVA II, have a domain N, which is an extra structure in the family 13 enzymes. To investigate the roles of domain N in TVAs, we constructed TVAs-ΔN mutants which are deleted in domain N, and Y14,16,68A and Y41,82,95A mutants of TVA II. TVAs-ΔN were unstable under alkaline conditions, and their thermal stabilities were 10°C lower than that of wild-types. The specific activities of TVAs-ΔN for pullulan, starch, cyclodextrins, and oligosaccharides were drastically decreased, being about 1,500- to 10,000-fold smaller than those of wild-types. The kcat values of Y14,16,68A and Y41,82,95A for all tested substrates were markedly decreased, and the Km value of Y14,16,68A for α-CD and maltotriose were 25- and 3-fold larger, and that of Y41,82,92A for starch was 10-fold larger than that of the wild-type. TVA I and TVAs-ΔN in solution are a monomer, while TVA II is a homo-dimer, calculated by their molecular masses. These results suggest domain N in TVAs is an important structure for stabilization of enzymes, recognition and hydrolysis of substartes, and dimerization of TVA II.
Key words: α-Amylase; TVA; domain N; family 13

-24-
Sweetness of Sweet Protein Thaumatin Is More Thermoresistant
under Acid Conditions Than under Neutral or Alkaline Conditions

Ryosuke KANEKO and Naofumi KITABATAKE

Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan

Received September 7, 2000; Accepted October 6, 2000
Thermostability of thaumatin and mechanisms of thermoinactivation were examined at 80°C in the pH range from 2 to 10. The sweetness of thaumatin disappeared on heating at pH above 7 for 15 min, but the sweetness remained even after heating at 80°C for 4 h at pH 2. This indicated that the sweet protein thaumatin is more thermoresistant under acid conditions than under neutral or alkaline conditions. Prolonged heating of thaumatin under acid conditions slowly reduced sweetness, and produced a heterogeneous population of molecules, all of which was soluble and monomeric. The resultant molecules were clearly distinct from those generated by heating at pH above 7. Hydrolysis of peptide bonds and other irreversible chemical reactions slowly took place in the molecule heated under acid conditions, and it would be, in part, a cause of thermoinactivation of thaumatin under acid conditions. The thermostability of thaumatin and the mechanism of thermoinactivation were largely dependent on pH.
Key words: thaumatin; thermoinactivation; thermostability; pH dependence; hydrolysis of peptide bond

-25-
Inhibition of Vero Cell Cytotoxic Activity in Escherichia coli O157:H7
Lysates by Globotriaosylceramide, Gb3, from Bovine Milk

Shinobu WATARAI,1,・・・ Tana,1 Kaoru INOUE,2 Yasunori KUSHI,3 Emiko ISOGAI,4
Kenji YOKOTA,2 Katsumi NAKA,5 Keiji OGUMA,2 and Hiroshi KODAMA1

1Laboratory of Veterinary Immunology, Division of Veterinary Science, Graduate School of Agriculture
and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
2Department of Bacteriology, Okayama University Medical School, Shikata-cho, Okayama 700-8558, Japan
3Department of Biochemistry, Faculty of Medicine, Tokyo Medical and Dental University, Yushima,
Bunkyo-ku, Tokyo 113, Japan
4Department of Preventive Dentistry, Health Sciences University of Hokkaido, Hokkaido 061-0293, Japan
5Department of Cell Chemistry, Institute of Cellular and Molecular Biology, Okayama University Medical
School, Shikata-cho, Okayama 700-8558, Japan

Received September 11, 2000; Accepted October 7, 2000
In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-
induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2.
Key words: bovine milk; Gb3; glycolipids; O-157:H7; verotoxin

-26-
Note
Characterization of the Pro-aminopeptidase from Aeromonas caviae T-64

Zhen-Zhong ZHANG,a Satoru NIRASAWA,a,・・・ Yoshiaki NAKAJIMA,a
Michiteru YOSHIDA,b Isao KUSAKABE,c and Kiyoshi HAYASHIa

aApplied Enzymology Laboratory, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan
bDepartment of Biological Science and Technology, Science University of Tokyo, Noda, Chiba 278-8510, Japan
cInstitute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

Received June 5, 2000; Accepted October 12, 2000
The pro-aminopeptidase from Aeromonas caviae T-64 (pro-apAC) had maximal activity at 60°C and was more stable than mature apAC at temperature up to 65°C for 1 hour. The pH stability of pro-apAC ranged from 4.0 to 8.0, which is broader than the range for the mature apAC. The kcat/Km of pro-apAC was 1.4％ to 24％ of that of mature apAC.
Key words: Aeromonas caviae; aminopeptidase; enzyme stability; pro-enzyme; substrate specificity

-27-
Note
Purification and Characterization of an Aminopeptidase
from the Edible Basidiomycete Grifola frondosa

Toshikazu NISHIWAKI・・・ and Kiyoshi HAYASHI

National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, 2-1-2,
Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received June 7, 2000; Accepted October 4, 2000
An aminopeptidase was purified 178-fold from an extract of Grifola frondosa by ammonium sulfate precipitation and a series of column chromatographies on phenyl-Toyopearl, Sephadex G-25, and Mono-Q. The molecular mass of the enzyme was estimated to be 27 kDa and 30 kDa by gel filtration and SDS-PAGE, respectively. The enzyme had an optimum pH of 8.5 and was stable between pH 6.0 and pH 10.5, and it also had a high level of heat stability. The enzyme was inactivated by EDTA and o-phenanthroline, and it was also strongly inhibited by bestatin, but no inhibitory effect of DFP was observed. The enzyme preferentially hydrolyzed peptides containing hydrophobic residues in the N-terminal position.
Key words: Grifola frondosa; aminopeptidase; purification; enzyme characteristics

-28-
Note
Induction of Nitric Oxide Synthase and Subsequent Production
of Nitric Oxide Not Involved in Interferon-γ-induced Hyperpermeability
of Caco-2 Intestinal Epithelial Monolayers

Makoto SATAKE, Hirohito WATANABE, Yusei MIYAMOTO,＊ and Makoto SHIMIZU・・・

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Tokyo 113-8657, Japan

Received June 30, 2000; Accepted October 12, 2000
Caco-2 cell monolayers exposed to 1000 U/ml interferon-γ (IFN-γ) for 6 days elicited inducible nitric oxide synthase (iNOS) expression and increased translayer permeability. This iNOS increase was blocked by pyrrolidinedithiocarbamate (an inhibitor of iNOS induction) but it did not suppress the hyperpermeability response. Furthermore, 2,2?-(hydroxynitrosohydrazino)
bis-ethanamine (a NO donor) did not increase monolayer permeability. Therefore, IFN-γ-induced hyperpermeability is not due to its induction of iNOS activity and resulting increases in NO levels.
Key words: Caco-2 cells; tight junction; IFN-γ; iNOS; NO

-29-
Note
Lack of Effect of the Abnormal Fatty Acid Metabolism in NC/Nga
Mice on Their Atopic Dermatitis

Seiji KAWAMOTO,・・・ Maiko KITA, Mizuho HAMADA, Tsunehiro AKI,
Seiko SHIGETA, Osamu SUZUKI, and Kazuhisa ONO

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, 1-4-1 Kagamiyama, Higashi-Hiroshima 739-8527, Japan

Received July 3, 2000; Accepted September 12, 2000
Although clinical evidence has suggested that dysregulated fatty acid metabolism is associated with atopic disorders, the molecular basis for such a correlation remains to be demonstrated. In the present study, we analyzed the fatty acid composition in peripheral blood cells of NC/Nga mice, a model for atopic dermatitis (AD). We found that arachidonic acid significantly accumulated in mice with the AD manifestation. In addition, the leucotriene B4-releasing ability upon calcium ionophore A23187 stimulation was potentiated in blood cells. An arachidonic acid accumulation was not apparent in the non-atopic BALB/c strain, but was still observed in healthy NC/Nga mice fed under specific pathogen-free conditions. These results indicate that a disturbed fatty acid metabolism in NC/Nga mice was not a trigger factor for their dermatitis development.
Key words: arachidonic acid; atopic dermatitis; leucotriene B4・; NC/Nga mice; polyunsaturated fatty acid

-30-
Note
Occurrence of a Novel Lyase Catalyzing β-Elimination Reaction toward
threo-3-Chloro-L-aspartate in Pseudomonas putida TPU 7151

Yasuo KATO and Yasuhisa ASANO・・・

Biotechnology Research Center, Faculty of Engineering, Toyama Prefectural University,
Kosugi, Toyama 939-0398, Japan

Received July12, 2000; Accepted October 7, 2000
A bacterium, Pseudomonas putida TPU 7151, which degrades threo-3-chloro-L-aspartate, was isolated from soil and the enzyme responsible for the degradation of the amino acid was partially purified from the cell-free extract of the strain. The enzyme, which required PLP for its reaction, catalyzed a stoichiometric β-elimination reaction of threo-3-chloro-L-aspartate to form oxaloacetate, Cl−, and NH+4. The enzyme was active toward only threo-3-chloro-L-aspartate and L-cysteine, but did not catalyze a β-replacement reaction. The enzyme can be classified in a new group of PLP-dependent amino acid-lyases [EC 4.2.1.-].
Key words: lyase; screening; enzyme; β-elimination reaction; Pseudomonas

-31-
Note
Production of Galactooligosaccharides from Lactose Using a β-Glucosidase
from Thermus sp. Z-1

Kumiko AKIYAMA, Mitsunori TAKASE,＊ Koki HORIKOSHI,＊＊ and Shigeo OKONOGI

Graduate School of Practical Sciences, Showa Women′s University, 1-7 Taishido, Setagaya-ku,
Tokyo 154-8533, Japan
＊Nutritional Science Laboratory, Morinaga Milk Industry Co., Ltd., 1-83, 5-chome, Higashihara, Zama-city,
Kanagawa, 228-8583, Japan
＊＊Faculty of Life Science, Toyo University, 1-1-1 Izumino, Itakura-machi, Oura-gun, Gunma 374-0113, Japan

Received July 18, 2000; Accepted October 3, 2000
A thermostable β-glucosidase from Thermus sp. Z-1 that not only hydrolyzes β-glucosides but also β-galactosides was shown to efficiently produce oligosaccharides during hydrolysis of lactose. The yield of oligosaccharides was more than 40％ for 0.88 M lactose solution at 70°C at pH 7.0. The major product was a trisaccharide, 3?-galactosyllactose, formed by a galactosyltransfer reaction.
Key words: Thermus sp. Z-1; galactooligosaccharides; β-glucosidase

-32-
Note
Isolation and Identification of 2-Phenylethyl Disaccharide Glycosides and Mono
Glycosides from Rose Flowers, and Their Potential Role in Scent Formation

Shuzo WATANABE,1 Ikue HASHIMOTO,1 Kentaro HAYASHI,1 Kensuke YAGI,1 Tatsuo ASAI,2
Holger KNAPP,3 Markus STRAUBINGER,4 Peter WINTERHALTER,3 and Naoharu WATANABE1,・・・

1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan
2University Farm, Faculty of Agriculture, Shizuoka University, 63 Kariyado, Fujieda,
Shizuoka 426-0001, Japan
3Institute of Food Chemistry, Technical University, Braunschweig, Schleinitzsstrasse 20,
D-38106 Braunschwig, Germany
4Institute of Pharmacy and Food Chemistry, University of Erlangen-N"urnberg, Schuhstrasse 19,
D-91052 Erlangen, Germany

Received July 28, 2000; Accepted September 14, 2000
2-Phenylethyl 6-O-α-L-arabinofuranosyl-β-D-glucopyranoside (1), and its 6-O-β-D-xylopyraranosyl-β-D-glucopyranoside (2) were identified in the flowers of Rosa damascena Mill. harvested at the full bloom stage. 2-Phenylethyl β-D-glucopyranoside (3) and its β-D-galactopyranoside (4) together with 1 and 2 were also found in the flower buds harvested 44 hrs before the opening stage. Their potential role in scent formation is discussed.
Key words: Rosa damascena Mill.; 2-phenylethyl 6-O-α - L - arabinofuranosyl - β - D-glucopyranoside; 2-phenylethyl 6-O-β-D-xylopyranosyl-β-D-glucopyranoside; 2-phenylethyl β-D-glucopyranoside; aroma formation

-33-
Note
A Simple and Rapid Method for Intra- and Interspecific Transformation
of Bacillus subtilis on Solid Media by DNA in Protoplast Lysates

Takashi AKAMATSU・・・ and Hisataka TAGUCHI

Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan

Received August 7, 2000; Accepted October 11, 2000
A simple method for intra- and interspecific transformation of Bacillus subtilis on solid media has been devised with DNA in protoplast lysates, 0.8％ agar, glutamate, and yeast extract. The transformation frequency is 2.3×103 transformants per μg DNA, 10--20 times higher than that for conventional transformation on solid media.1) The method can be applicable to transformation in microtiter plates.
Key words: Bacillus subtilis; DNA; transformation; solid media; microtiter plates

-34-
Note
An in Vitro Approach to the Evaluation of the Cross Talk between
Intestinal Epithelium and Macrophages

Hiroki KANZATO, Mariko MANABE,＊ and Makoto SHIMIZU・・・

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
＊Faculty of Human Life and Science, Doshisha Women′s Collage of Liberal Arts,
Kamigyo-ku, Kyoto 602-0893, Japan

Received August 8, 2000; Accepted October 22, 2000
The intestinal epithelium acts as a mucosal barrier by varying their signals to immune cells within the intestine. To observe the cross talk between intestinal epithelium and macrophages, we establish a Caco-2---THP-1 co-culture system. Using this co-culture system, we suggested that paracrine factors of intestinal epithelium increased the phagocytic capacity of intestinal monocytes/macrophages to be ready for immune and inflammatory responses.
Key words: phagocytosis; co-culture; intestinal barrier; Caco-2; THP-1

-35-
Note
Inhibitory Activity of Berberine on DNA Strand Cleavage Induced
by Hydrogen Peroxide and Cytochrome c

Dong-Seong CHOI,a Sang-Jin KIM,a and Mun Yhung JUNGb,・・・

aDivision of Bioscience and Biotechnology,
bDivision of Food Science and Technology, Woosuk University, Samrae-Up, Wanju-Kun,
Jeonbuk Province 565-701, Korea

Received August 9, 2000; Accepted September 29, 2000
The inhibitory activity of berberine on the DNA single-strand cleavage induced by hydrogen peroxide and cytochrome c was measured. Berberine effectively inhibited single-strand cleavage of DNA and its effectiveness was concentration-dependent. As the berberine concentration increased, the inhibitory activity against the DNA single-strand cleavage increased. The treatments with 1, 5, 10, 50, and 100 μM berberine showed 7.7, 10.8, 32.2, 39.5, and 51.6％ inhibition of DNA cleavage. This inhibitory activity of berberine against the DNA single-strand cleavage has never been reported previously. The inhibitory activity of berberine against DNA cleavage was stronger than caffeic acid and ascorbic acid. Berberine did not show strong hydroxyl radical scavenging activity, but showed strong superoxide anion radical quenching ability.
Key words: berberine; DNA strand cleavage; hydrogen peroxide; cytochrome c; superoxide anion

-36-
Note
Isolation and Characterization of the Enterobacter cloacae cheR
Mutant Defective in Phosphate Taxis

Junichi KATO, Chinatsu NAGATA, Li YANG, Akio KURODA, Tsukasa IKEDA,
Noboru TAKIGUCHI, and Hisao OHTAKE

Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima,
Hiroshima 739-8527, Japan

Received August 10, 2000; Accepted September 25, 2000
A chemotaxis-defective mutant of Enterobacter cloacae IFO3320, designated EC1, was isolated after N-methyl-N?-nitro-N-nitrosoguanidine (NTG) mutagenesis. Computer-assisted capillary assays showed that EC1 failed to show chemotactic responses to peptone and inorganic phosphate (Pi). Cloning and sequence analysis showed that EC1 is a cheR mutant, suggesting that Pi taxis by E. cloacae is dependent on a methyl-accepting chemotaxis protein(s) (MCP). EC1 was further mutagenized with NTG to construct cheR pstS and cheR pstA double mutants. A recombinant plasmid pECT01.2, which contained the E. cloacae cheR gene, restored the ability of these double mutants to show chemotaxis toward peptone but not Pi. These results suggest that the phosphate-specific transport (Pst) system, together with a MCP(s), is required for detecting Pi in E. cloacae.
Key words: chemotaxis; inorganic phosphate; methyl-accepting chemotaxis protein; cheR; Enterobacter cloacae

-37-
Note
Apoptosis-inducing Activity of High Molecular Weight Fractions
of Tea Extracts

Sumio HAYAKAWA, Takashi KIMURA, Kouichi SAEKI, Yu KOYAMA, Yutaka AOYAGI,＊
Tadataka NORO,＊＊ Yoshiyuki NAKAMURA,＊＊＊ and Mamoru ISEMURA・・・

School of Food and Nutritional Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
＊Third Division, Internal Medicine, Niigata University School of Medicine, Niigata 951-8122, Japan
＊＊Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Yada,
Shizuoka 422-8526, Japan
＊＊＊School of Pharmaceutical Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan

Received August 15, 2000; Accepted October 5, 2000
High molecular weight fractions of green tea, black tea, oolong tea, and pu-erh tea were found to induce apoptosis in human monoblastic leukemia U937 cells by examination of their ability to inhibit cell proliferation and to induce apoptotic body formation and DNA ladder formation. These tea fractions were also shown to induce apoptosis in stomach cancer MKN-45 cells. In addition to known antitumor-promoting activity of tea high molecular weight fractions, their apoptosis-inducing activity may contribute to cancer chemopreventive effects of tea.
Key words: epigallocatechin gallate; stomach cancer; tea; chemoprevention

-38-
Note
Synthesis and Absolute Configuration of MQ-A3 [1-(14?-Methylhexadecanoyl)
pyrrolidine], a Novel Aliphatic Pyrrolidine Amide
from the Tropical Convolvulaceous Species

Arata YAJIMA and Goro YABUTA＊

Department of Fermentation Science, Faculty of Applied Biological Science, Tokyo University of Agriculture,
Sakuragaoka 1-1-1, Setagaya-ku, Tokyo 156-8502, Japan

Received August 22, 2000; Accepted October 13, 2000
A novel pyrrolidine amide (MQ-A3) isolated from the tropical convolvulaceous species was synthesized in 5 steps by starting from commercially available 12-bromododecanol and (S)-2-methylbutylbromide. The absolute configuration of the natural product was confirmed by a comparison of the specific rotation values.
Key words: pyrrolidine amide; Ipomoea aquatica; Merremia quinquefolia; synthesis; absolute configuration

-39-
Note
Dietary Curdlan Increases Proliferation of Bifidobacteria in The Cecum of Rats

Jun SHIMIZU,・・・ Noboru TSUCHIHASHI,＊ Keita KUDOH, Masahiro WADA,
Toshichika TAKITA, and Satoshi INNAMI

Department of Nutritional Science, Faculty of Applied Bio-Science, Tokyo University of Agriculture,
Sakuragaoka, Setagaya-Ku, Tokyo 156-8502, Japan
＊Department of Nutrition, Chiba College of Health Science, Wakaba, Mihama-Ku, Chiba 261-0014, Japan

Received August 22, 2000; Accepted October 20, 2000
Significant increases in the amounts of short-chain fatty acids and lactate, and in numbers of bifidobacteria were observed in the cecum of curdlan (CD) -fed rats as compared with those of cellulose-fed ones. The in vitro proliferation of 5 species of bifidobacteria was markedly increased in the cultures containing the supernatant obtained from the cecal contents of CD-fed rats. These findings suggest that bifidus factors have been produced in the cecum of CD-fed rats.
Key words: curdlan; dietary fiber; cecal microflora; bifidobacteria; rat

-40-
Note
Production of Monoclonal Antibody against Scrippsiella trochoidea Cysts
and Its Application to Analysis during Cyst Formation and Enzyme-linked
Immunosorbent Assay

Katsuichiro OKAZAKI,・・・ Takeshi IWAOKA, Noriko MURAKAMI,
Kazuhiko ICHIMI, and Shigeru MONTANI

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan

Received August 23, 2000; Accepted September 21, 2000
A monoclonal antibody was produced by the fusion of mouse myeloma cells with spleen cells from a mouse immunized with the cysts of Scrippsiella trochoidea, marine phytoplankton. Immunofluorescence microscopic observation showed that the antibody reacted with the spines on the cyst but not with the vegetative cells and cyst walls. An enzyme-linked immunosorbent assay could be used to measure the cysts in muddy bottom sediments using the purified antibody conjugated with horseradish peroxidase.
Key words: monoclonal antibody; Scrippsiella; cysts; phytoplankton; red tide

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Aplysiallene, a New Bromoallene as an Na, K-ATPase Inhibitor
from the Sea Hare, Aplysia kurodai

Yoshihiro OKAMOTO,・・・ Naoko NITANDA, Makoto OJIKA,・・・・・・ and Youji SAKAGAMI

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received August 29, 2000; Accepted September 20, 2000
A new bromoallene metabolite, named aplysiallene, was isolated from the Japanese sea hare, Aplysia kurodai, as an Na, K-ATPase inhibitor. Its structure was elucidated by spectroscopic methods. The known metabolites, laurinterol and debromolaurinterol, isolated from this animal were also evaluated for their Na, K-ATPase inhibitory activity.
Key words: aplysiallene; Aplysia kurodai; Na, K-ATPase; enzyme inhibitors; marine metabolites

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A Simultaneous Assay Method for L-Glutamate and L-Pyroglutamate Contents
in Soy Sauce Using a 5-Oxoprolinase (without ATP Hydrolyzing Activity)

Atsuhisa NISHIMURA,＊a,b Homare ITOH,a Hiroshi OYAMA,b Sawao MURAO, and Kohei ODAb

aResearch Laboratory, Ichibiki Co., Ltd., Toyohashi, Aichi 441-8019, Japan
bDepartment of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology,
Sakyo-ku, Kyoto, 606-8585, Japan

Received August 30, 2000; Accepted September 25, 2000
L-Glutamine and L-glutamate, which are important flavor components in soy sauce, are converted to L-pyroglutamate during brewing. Therefore, it is necessary that the L-glutamate and L-pyroglutamate contents can be measured accurately. We developed a simultaneous assay method for L-glutamate and L-pyroglutamate by using 5-oxoprolinase (without ATP hydrolyzing activity) and glutamate oxidase. By this method, the L-pyroglutamate could be measured accurately in a range of 0.05 to 1.0 mM in the presence of 1.0 mM L-glutamate. This system is effective for process and quality controls.
Key words: L-pyroglutamate; L-glutamate; assay; 5-oxoprolinase; soy sauce

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Actinidic Acid, a New Triterpene Phytoalexin from Unripe Kiwi Fruit

El Hassane LAHLOU,1 Nobuhiro HIRAI,1,2,＊ Tsunashi KAMO,1,2
Mitsuya TSUDA,3 and Hajime OHIGASHI2

1CREST, Japan Science and Technology Corporation (JST), Hon-cho 4-1-8, Kawaguchi 332-0012, Japan
2Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
3Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan

Received August 30, 2000; Accepted October 24, 2000
Seven phytoalexins (1-7), including a new compound, were isolated from the peel of unripe kiwi fruit (Actinidia deliciosa cv. Golden King) that had been wounded and inoculated with Colletotrichum musae. The new phytoalexin (1) was identified as 2α,3β,23-trihydroxy-12,20(30)-ursadien-28-oic acid, and named actinidic acid. Phytoalexins 2-6 are known triterpenes but have not previously been described as phytoalexins. Phytoalexin 7 is the same triterpene as the phytoalexin of nectarine fruit.
Key words: Actinidia deliciosa; actinidic acid; kiwi fruit; phytoalexin; 2α,3β,23-trihydroxy-12,20(30)-ursadien-28-oic acid

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(-)-Semivioxanthin, A New Abscisic Active Compound against Hinoki
Cypress Leaves Isolated from Cryptosporiopsis abietina

Hiroshi YADA,1 Hiroji SATO,2・・・ Hiroaki TOSHIMA,3 Megumi DEURA,3 and Akitami ICHIHARA3

1National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba,
Ibaraki 305-8642, Japan
2Laboratory of Science of Plant Resources, Experimental Farms, Faculty of Agriculture, Hokkaido University,
Sapporo 060-0811, Japan
3Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan

Received September 4, 2000; Accepted September 20, 2000
The new naphthopyrone, (-)-semivioxanthin (1) was isolated from Cryptosporiopsis abietina. The structure of 1 was determined as the reversed optical isomer of semivioxanthin by comparing its spectroscopic data with those of semivioxanthin. Compound 1 exhibited abscisic activity against Hinoki cypress leaves and antifungal activity against Cladosporium herbarum.
Key words: semivioxanthin; Cryptosporiopsis abietina; abscisic activity against Hinoki cypress leaves; 3,4-dihydro-9,10-dihydroxy - 7 - methoxy - 3 - methyl - 1 - oxo-1H-naphtho[2,3-c]pyran