Contents and Abstracts of Latest Issue of BBB

(Vol.66 No.4 2002)

Elucidation of the Toxic Mechanism of the Plasticizers, Phthalic Acid Esters,
Putative Endocrine Disrupters: Effects of Dietary Di(2-ethylhexyl)phthalate
on the Metabolism of Tryptophan to Niacin in Rats
Tsutomu FUKUWATARI,† Yuko SUZUKI, Etsuro SUGIMOTO, and Katsumi SHIBATA p.705

Effects of Dietary Powdered Green Tea and Theanine on Tumor Growth
and Endogenous Hyperlipidemia in Hepatoma-bearing Rats
Guoying ZHANG, Yutaka MIURA, and Kazumi YAGASAKI† p.711

NADH Oxidation by Manganese Peroxidase with or without α-Hydroxy Acid
Tetsuya DEGUCHI, Masaaki MATSUBARA, and Tomoaki NISHIDA＊ p.717

Coupling Reaction and Properties of Poly(ethylene glycol)-linked
Phospholipases A2
Ismael D. BIANCO,1,† Jos<Oh><0225><Wa>e J. DANIELE,2 Cristina DELGADO,3,†† Derek FISHER,3,††
Gillian E. FRANCIS,3,†† and Gerardo D. FIDELIO2 p.722

Characteristics of Lysophosphatidylcholine in Its Inhibition
of Taurine Uptake by Human Intestinal Caco-2 Cells
Koji ISHIZUKA, Yusei MIYAMOTO,＊ Hideo SATSU, Ryuichiro SATO, and Makoto SHIMIZU† p.730

Heterogeneity of Dehydrogenases of Stenotrophomonas maltophilia
Showing Dye-linked Activity with Polypropylene Glycols
Shinjiro TACHIBANA,1 Fusako KAWAI,2 and Masaaki YASUDA1,† p.737

Synthesis of the Glycosidic Precursor of Isomeric Marmelo Lactones,
Volatile Components of the Quince Fruit, Cydonia oblonga
Hiroki SHIMIZU and Takeshi KITAHARA† 743

Autoxidation Kinetic Analysis of Docosahexaenoic Acid Ethyl Ester
and Docosahexaenoic Triglyceride with Oxygen Sensor
Hidefumi YOSHII,† Takeshi FURUTA, Hirokazu SIGA, Shigeru MORIYAMA,＊ Takashi BABA,＊
Kazuaki MARUYAMA,＊ Yoshihisa MISAWA＊＊, Noriaki HATA,＊＊ and Pekka LINKO＊
p.749

Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile,
Shewanella sp., and Expression of the Recombinant Enzyme†
Takeshi MURAKAWA, Hiroshi YAMAGATA, Hiroki TSURUTA,†† and Yasuo AIZONO†††
p.754

Arbuscular Mycorrhizal Fungus-promoted Accumulation of Two
New Triterpenoids in Cucumber Roots
Kohki AKIYAMA† and Hideo HAYASHI p.762

Kinetic Analysis of the Effect of (−)-Epigallocatechin Gallate
on the DNA Scission Induced by Fe(II)
Yasunori OHASHI,1 Koichi YOSHINAGA,2 Hisashi YOSHIOKA,3 and Hiroe YOSHIOKA1,†
p.770

Purification and Characterization of a Novel Polysaccharide Involved
in the Pellicle Produced by a Thermotolerant Acetobacter Strain†
Somporn MOONMANGMEE, Hirohide TOYAMA, Osao ADACHI, Gunjana THEERAGOOL,1
Napha LOTONG,1 and Kazunobu MATSUSHITA†† p.777

Apoptosis Induction by Lectin Isolated from the Mushroom
Boletopsis leucomelas in U937 Cells
Yu KOYAMA, Yuko KATSUNO, Noriyuki MIYOSHI, Sumio HAYAKAWA, Takashi MITA,
Haruhiko MUTO,1 Satoko ISEMURA,2 Yutaka AOYAGI,3 and Mamoru ISEMURA† p.784

Gene Expression during Pi Deficiency in Pholiota nameko:
Accumulation of mRNAs for Two Transporters
Yuji TASAKI,1 Yuki KAMIYA,2 A. AZWAN,1 Takashi HARA,2 and Toshio JOH2† p.790

Isolation and Characterization of A β-Primeverosidase-like endo-manner
β-Glycosidase from Aspergillus fumigatus AP-20
Shigeru YAMAMOTO,1 Masamichi OKADA,1 Taichi USUI,2 and Kanzo SAKATA3,† p.801

Growth-phase Dependent Expression of the Mevalonate Pathway
in a Terpenoid Antibiotic-producing Streptomyces Strain
Yoshimitsu HAMANO,1 Tohru DAIRI,1,† Masahiro YAMAMOTO,1 Tomohisa KUZUYAMA,2
Nobuya ITOH,1 and Haruo SETO2,† p.808

Purification and Characterization of 31-kDa Palm Pollen Glycoprotein
(Ela g Bd 31 K), Which is Recognized by IgE from Palm Pollinosis Patients＊
Yoshinobu KIMURA,1,† Megumi MAEDA,1 Mariko KIMURA,1 Oi Ming LAI,2
Siang Hee TAN,3 Sook Mei HON,4 and Fook Tim CHEW4 p.820

Lymphatic Transport of Dietary Cholesterol Oxidation Products,
Cholesterol and Triacylglycerols in Rats
Hiroko TOMOYORI, Octavio CARVAJAL, Masahiro NAKAYAMA, Taiji KISHI,
Masao SATO, Ikuo IKEDA, and Katsumi IMAIZUMI† p.828

Comparison of the Apoptosis-inducing Abilities of Various
Protein Synthesis Inhibitors in U937 Cells
Ai KAGEYAMA, Izumi KUSANO, Tadashi TAMURA, Tatsuya ODA,†
and Tsuyoshi MURAMATSU p.835

The Biological and Structural Similarity between Lunularic Acid
and Abscisic Acid
Hiromichi YOSHIKAWA,1,† Yayoi ICHIKI,1 Keiko (DOI) SAKAKIBARA,1
Hiroto TAMURA,2 and Masahito SUIKO3 p.840

Detection of 2-Deoxy-D-ribose Radicals Generated by the Reaction
with the Hydroxyl Radical using a Rapid Flow-ESR Method
Yasunori OHASHI,1 Hisashi YOSHIOKA,2 and Hiroe YOSHIOKA1,† p.847

Note
Oral Administration of Swine Gastric Mucin Alters Levels of Serum
and Urine Nitric Oxide, and Lipid Metabolism in Mice
Abdelkrim KHEDARA and Misao MIWA† p.853

Note
Prevention of Browning in Potato with a Heat-treated Onion Extract
Min-Kyung LEE, Young-Mai KIM, Na-Young KIM, Gi-Nahm KIM,
Seok-Hwan KIM, Keuk-Seung BANG, and Inshik PARK† p.856

Note
Acid and Heat Stability of the Anticoagulative Activity
of an Onion Extract
Gi-Nahm KIM, Min-Kyung LEE, and Inshik PARK†p.859

Note
Isolation and Identification of Dihydrochrysanolide and Its 1-Epimer
from Chrysanthemum coronarium L.
Kyung Dong LEE,1 Min Suk YANG,1 Tae Jung HA,1 Ki Min PARK,2 and Ki Hun PARK1,†
p.862

Note
Response of the Ice-nucleating Bacterium Pantoea ananas
KUIN-3 during Cold Acclimation
Noriko KODA, Yukari INADA, Satoshi NAKAYAMA, Hidehisa KAWAHARA,
and Hitoshi OBATA p.866

Note
Increase in Oxidative Stress in Kidneys of Diabetic Akita Mice
Yuki UENO,1 Fumihiko HORIO,1 Koji UCHIDA,1 Michitaka NAITO,2 Hideki NOMURA,2
Yoji KATO,3 Takanori TSUDA,4 Shinya TOYOKUNI,5 and Toshihiko OSAWA1,† p.869

Note
Temperature Dependence of Kinetic Parameters for Hyperthermophilic
Glutamate Dehydrogenase from Aeropyrum pernix K1
Mohammad W. BHUIYA, Haruhiko SAKURABA, and Toshihisa OHSHIMA† p.873

Note
5-Halogenated Thymidine Analogues Induce a Senescence-like
Phenomenon in HeLa Cells
Eriko MICHISHITA, Naoto MATSUMURA, Toshihiro KURAHASHI, Toshikazu SUZUKI,
Hideki OGINO, Michihiko FUJII, and Dai AYUSAWA p.877

Note
Salts and Glycine Increase Reversibility and Decrease Aggregation
during Thermal Unfolding of Ribonuclease-A
Yoshiko KITA and Tsutomu ARAKAWA p.880

Note
Formation of New Lipoaminopeptides, Acremostatins A, B, and C,
by Co-cultivation of Acremonium sp. Tbp-5 and Mycogone rosea
DSM 12973
Thomas DEGENKOLB,1,†† Stephan HEINZE,1 Brigitte SCHLEGEL,1 Gary STROBEL,2
and Udo GR<Oh><0196><Wa>AFE1,† p.883

Note
Stereoselective Synthesis of (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic
Acid via C--H Insertion of Alkylidenecarbene
Susumu OHIRA,† Megumi AKIYAMA, Kumiko KAMIHARA, Yuichi ISODA, and
Atsuhito KUBOKI p.887

Note
Med, a Cell-surface Localized Protein Regulating a Competence
Transcription Factor Gene, comK, in Bacillus subtilis
Mitsuo OGURA,† Hitoshi HASHIMOTO, and Teruo TANAKA p.892

Note
Organization and Transcriptional Characterization of Catechol
Degradation Genes Involved in Carbazole Degradation
by Pseudomonas resinovorans Strain CA10
Hideaki NOJIRI, Kana MAEDA, Hiroyo SEKIGUCHI, Masaaki URATA, Masaki SHINTANI,
Takako YOSHIDA, Hiroshi HABE, and Toshio OMORI p.897

Note
New Medium Composition for High Betacyanin Production
by a Cell Suspension Culture of Table Beet (Beta vulgaris L.)
Toru AKITA,† Yasuhiko HINA, and Toyoyuki NISHI p.902

Note
5-Bromo-2′-deoxyuridine Efficiently Suppresses Division Potential
of the Yeast Saccharomyces cerevisiae
Michihiko FUJII, Hisasi ITO, Tsutomu HASEGAWA, Toshikazu SUZUKI,
Noritaka ADACHI,＊ and Dai AYUSAWA p.906

Note
Expression of Nicotiana glutinosa Ribonucleases in Escherichia coli
Madoka HINO, Shin KAWANO, and Makoto KIMURA† p.910

Note
Prevention by Polyhydroxysteroids and Saponins of Asterina pectinifera
of the Desquamation of Stratum Corneum Cells
Kazuyo UKAI,1 Atsufumi IWANAGA,2 Hiroshi NAGAI,1 and Michio NAMIKOSHI1,†
p.913

Note
Conjugated Linoleic Acid Reduces Body Fats and Cytokine Levels of Mice
Asuka AKAHOSHI,1 Yuki GOTO,1 Kayoko MURAO,1 Tomoko MIYAZAKI,1
Masao YAMASAKI,2 Michiko NONAKA,2 Koji YAMADA,2 and Michihiro SUGANO1 p.916

Note
Antimicrobial Activity of Perilla Seed Polyphenols
against Oral Pathogenic Bacteria
Hiroyo YAMAMOTO and Tomohiko OGAWA† p.921

Note
Synthesis of (R)-1,3-Butanediol by Enantioselective Oxidation Using
Whole Recombinant Escherichia coli Cells Expressing (S)-Specific
Secondary Alcohol Dehydrogenase
Hiroaki YAMAMOTO,† Akinobu MATSUYAMA, and Yoshinori KOBAYASHI p.925

Preliminary Communication
α-Glucosidase Mutant Catalyzes <Oh><0192><0192>α<Wa>-Glycosynthase′′-type Reaction
Masayuki OKUYAMA, Haruhide MORI, Kotomi WATANABE,
Atsuo KIMURA,† and Seiya CHIBA p.928

Preliminary Communication
Biosynthesis of Vitamin B6 in Rhizobium: In Vitro Synthesis of Pyridoxine
from 1-Deoxy-D-xylulose and 4-Hydroxy-L-threonine
Masaaki TAZOE,† Keiko ICHIKAWA, and Tatsuo HOSHINO p.934

-1-
Elucidation of the Toxic Mechanism of the Plasticizers, Phthalic Acid Esters,
Putative Endocrine Disrupters: Effects of Dietary Di(2-ethylhexyl)phthalate
on the Metabolism of Tryptophan to Niacin in Rats

Tsutomu FUKUWATARI,† Yuko SUZUKI, Etsuro SUGIMOTO, and Katsumi SHIBATA

Laboratory of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures,
The University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan

Received July 23, 2001; Accepted December 4, 2001
We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. In the present experiment, the effect of di(2-ethylhexyl)phthalate (DEHP) on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP were investigated to elucidate the toxic mechanism of phthalic acid esters (PhE). Rats were fed with a diet containing 0％, 0.01％, 0.05％, 0.1％, 0.5％, 1.0％, or 3.0％ DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05％; the conversion ratio was about 2％ in the control group, whereas it was 28％ in the 3.0％ DEHP group. It is suggested that the inhibition of α-amino-
β-carboxymuconate-ε-semialdehyde decarboxylase
(ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the tryptophan-
niacin metabolism.
Key words: phthalic acid ester; di(2-ethylhexyl)phthalate; conversion ratio of tryptophan to niacin; endocrine disrupter; niacin toxicity

-2-
Effects of Dietary Powdered Green Tea and Theanine on Tumor Growth
and Endogenous Hyperlipidemia in Hepatoma-bearing Rats

Guoying ZHANG, Yutaka MIURA, and Kazumi YAGASAKI†

Department of Applied Biological Science, Tokyo Noko University, Saiwaicho 3-5-8, Fuchu,
Tokyo 183-8509, Japan

Received July 24, 2001; Accepted December 9, 2001
The effects of dietary powdered green tea (PGT) and theanine on in vivo hepatoma growth and cancerous hyperlipidemia were investigated in rats that had been implanted with a rat ascites hepatoma cell line of AH109A cells. The hepatoma-bearing rats were fed with a 20％ casein diet (20C), 20C containing 2％ PGT, or 20C containing 0.1％ theanine for 14 days. Dietary PGT significantly and time-dependently reduced the solid tumor volume and weight as did dietary theanine. The hepatoma-induced endogenous hyperlipidemia, which was characterized by rises in the serum cholesterol (hypercholesterolemia) and triglyceride (hypertriglyceridemia) levels, was significantly suppressed by PGT and theanine supplementation. Bile acid excretion into the feces was significantly higher in the PGT- and theanine-fed rats than in the control rats. This inhibition of hypercholesterolemia may have resulted from tumor growth suppression as well as increased excretion of steroids from the body. These results suggest that PGT had both anti-proliferative activity toward hepatoma cells and hypolipidemic activity in the hepatoma bearing rats. They also suggest that theanine was, at least in part, responsible for the PGT actions.
Key words: green tea; hepatoma; hyperlipidemia; theanine; tumor growth

-3-
NADH Oxidation by Manganese Peroxidase with or without α-Hydroxy Acid

Tetsuya DEGUCHI, Masaaki MATSUBARA, and Tomoaki NISHIDA＊

Chemical and Environmental Technology Laboratory, KOBE STEEL, LTD., Kobe 651-2271, Japan
＊Department of Forest Resources Science, Faculty of Agriculture, Shizuoka University,
Shizuoka 422-8529, Japan

Received August 3, 2001; Accepted November 28, 2001
NADH oxidation by manganese peroxidase (MnP) was done in a reaction mixture including either α-hydroxy acid or acetate. The oxidation in the former reaction mixture was inhibited by a catalase and was accelerated by exogenous H2O2, while the oxidation in the latter reaction mixture was inhibited by a superoxide dismutase and was not accelerated by the exogenous H2O2. These results indicated that there are significant differences between the two reaction systems, particularly, in the active oxygen species involved in the reactions. Additionally, the experiment of MnP reduction with Mn(II) suggests that MnP has a separate catalytic activity other than an oxidation of Mn(II) to Mn(III) in the reaction mixture including acetate.
Key words: manganese peroxidase; NADH; nylon degradation

-4-
Coupling Reaction and Properties of Poly(ethylene glycol)-linked
Phospholipases A2

Ismael D. BIANCO,1,† Jos<Oh><0225><Wa>e J. DANIELE,2 Cristina DELGADO,3,†† Derek FISHER,3,††
Gillian E. FRANCIS,3,†† and Gerardo D. FIDELIO2

1CEPROCOR-Agencia C<Oh><0225><Wa>ordoba Ciencia S.E., CP 5164 Santa Mar<Oh><0225><0131><Wa>a de Punilla, C<Oh><0225><Wa>ordoba, Argentina
2Departamento de Qu<Oh><0225><0131><Wa>mica Biol<Oh><0225><Wa>ogica-CIQUIBIC-Facultad de Ciencias Qu<Oh><0225><0131><Wa>micas, Universidad Nacional
de C<Oh><0225><Wa>ordoba, Agencia postal 4, C.C. 61, 5000 C<Oh><0225><Wa>ordoba, Argentina
3Molecular Cell Pathology Laboratory, Royal Free Hospital School of Medicine, University of London,
London, U.K.

Received August 20, 2001; Accepted November 19, 2001
Secretory phospholipases A2 (PLA2) from Naja naja naja (cobra snake) venom, from Bothrops neuwiedii (crotalid snake) venom (two isoforms) and from bee venom were modified with tresylated monomethoxy poly(ethylene glycol) (TMPEG). The kinetic and inflammatory properties of the adducts (PEG-PLA2) were measured. As found by gel permeation chromatography, 95--100％ of P-1 PLA2 from B. neuwiedii and PLA2 from N. naja naja venom change their chromatographic mobility after TMPEG treatment. By contrast, only 50--60％ of both P-3-PLA2 from B. neuwiedii and PLA2 from bee venom modify their elution profile from Superdex 75.
All the modified proteins preserved the enzymatic activity toward phospholipid monolayers, but with a reduced specific activity and greater lag times than the unmodified controls. These results suggest that the PEG-PLA2 complexes would have an altered interaction with lipid membranes. The PEG-linked proteins preserve their edema-inducing activity evaluated by the rat hind-paw edema test except for N. naja naja PEG-PLA2 in which inflammatory activity was significatively decreased. Altogether, the results show a partial dissociation of catalytic and inflammatory activities of Group II and III secretory PLA2s after their modification with PEG.
Key words: poly(ethylene glycol); phospholipase A2; enzyme kinetics; lipid-monolayers; edema

-5-
Characteristics of Lysophosphatidylcholine in Its Inhibition
of Taurine Uptake by Human Intestinal Caco-2 Cells

Koji ISHIZUKA, Yusei MIYAMOTO,＊ Hideo SATSU, Ryuichiro SATO, and Makoto SHIMIZU†

Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
＊Department of Integrated Life Sciences, The University of Tokyo, Kashiwa-shi, Chiba 277-8562, Japan

Received August 30, 2001; Accepted December 9, 2001
The characteristics of lysophosphatidylcholine (LPC) in its inhibition of the taurine uptake by human intestinal Caco-2 cells were investigated. By treating the cells with 200 μM of LPC, the taurine uptake was rapidly decreased by approximately 60％. This decrease was accompanied by an increase in the Km value for the uptake. A rapid uptake of LPC itself by the cells was also observed. The inhibitory activity of LPC was specific to the uptake of taurine and certain amino acids, while the uptake of glucose, glutamic acid and peptide (glycylglutamine) was not affected by LPC. The activity was dependent on the structure of a polar head and the bound fatty acid. The phosphorylcholine residue was likely to have played an important role, and surface active LPC with fatty acids of C14 or longer was highly inhibitory. These results suggest that the interaction of LPC with the taurine transporter in the intestinal cell membrane was the cause of the reduced taurine uptake.
Key words: taurine; Caco-2; small intestine; transporter; lysophosphatidylcholine

-6-
Heterogeneity of Dehydrogenases of Stenotrophomonas maltophilia
Showing Dye-linked Activity with Polypropylene Glycols

Shinjiro TACHIBANA,1 Fusako KAWAI,2 and Masaaki YASUDA1,†

1Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus,
1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan
2Research Institute for Bioresources, Okayama University, 2-20-1 Chuo, Kurashiki,
Okayama 710-0046, Japan

Received August 31, 2001; Accepted December 17, 2001
Distinct enzyme activities were found in extracts from Stenotrophomonas maltophilia showing dye-linked oxidation of polypropylene glycols. The activities were induced when polypropylene glycols served as sole carbon and energy sources for the bacterium. In the logarithmic phase, most of the enzyme activities (88％) were found in the cytoplasm. In the stationary phase, more than half of the activities (54％) were found on the membrane, but significant activities were also distributed in the periplasm (34％) and the cytoplasm (12％). The enzyme activities differed from each other in their localization, time of induction in the growth cycle, specificity toward electron acceptor, and electrophoresis mobility.
Key words: polypropylene glycol; alcohol dehydrogenase; dye-linked dehydrogenase; Stenotrophomonas maltophilia

-7-
Synthesis of the Glycosidic Precursor of Isomeric Marmelo Lactones,
Volatile Components of the Quince Fruit, Cydonia oblonga

Hiroki SHIMIZU and Takeshi KITAHARA†

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received September 10, 2001; Accepted November 12, 2001
The glucosidic precursor of marmelo lactones was synthesized by employing a common intermediate which had been used for the synthesis of the glucosidic precursor of marmelo oxides. The synthesis was performed by modifying the former procedure. Monochloroacetyl was adopted to protect both the glucose and aglycon hydroxyl groups for selective transesterification in the presence of the glycosyl ester. Glycosylation of the aglycon carboxyl group with 1-α-bromopermonochloroacetylglucose and final selective alcoholysis yielded the target glucoside.
Key words: quince fruit; Cydonia oblonga Mill.; glycosidic precursor; volatile component; selective alcoholysis

-8-
Autoxidation Kinetic Analysis of Docosahexaenoic Acid Ethyl Ester
and Docosahexaenoic Triglyceride with Oxygen Sensor

Hidefumi YOSHII,† Takeshi FURUTA, Hirokazu SIGA, Shigeru MORIYAMA,＊ Takashi BABA,＊
Kazuaki MARUYAMA,＊ Yoshihisa MISAWA＊＊, Noriaki HATA,＊＊ and Pekka LINKO＊

Department of Biotechnology, Tottori University, Tottori 680-0945, Japan
＊R＆D Div., Maruha. Corporation, Tsukuba 300-4200, Japan
＊＊Harima Chemicals Inc., Tsukuba 300-2635, Japan
＊＊＊Department of Chemical Technology, Helsinki University of Technology, Espoo,
FIN-02015 HUT, Finland

Received September 11, 2001; Accepted November 19, 2001
The application of ω-3 polyunsaturated fatty acids (PUFAs) as food additives is restricted by their chemically quite reactive properties. However, quantitative analyses of the oxidative kinetics of PUFAs are very few compared to other studies on food chemistry.
In this study, the autoxidation kinetics of ethyl docosahexaenoate (DHAEE), docosahexaenoic triglyceride (DHA oil), and emulsified DHA oil were investigated with an oxygen sensor. The autocatalytic reaction rate constants for DHAEE, DHA oil, and the emulsified DHA oil with 20％(w/v) GA, 20％ SSPS, or 20％ SSPS containing 5％ soy protein were obtained at 35, 50, and 70°C. A plot of the natural logarithm of the frequency factor, ln ka0, vs. the activation energy, Ea, demonstrated that ln ka0 against Ea fitted well with a single straight line both for the data from this study and for other reported results. This implies that the chemical compensation relationship holds between ka0 and Ea for PUFA and emulsified DHA oil.
Key words: autoxidation; ethyl docosahexaenoate; kinetic energy; DHA oil

-9-
Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile,
Shewanella sp., and Expression of the Recombinant Enzyme†

Takeshi MURAKAWA, Hiroshi YAMAGATA, Hiroki TSURUTA,†† and Yasuo AIZONO†††

Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture,
Kobe University, Nada-ku, Kobe, Hyogo 657-8501, Japan

Received September 14, 2001; Accepted December 5, 2001
A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246--2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single α-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.
Key words: alkaline phosphatase; cold-active enzyme; flexible structure; psychrophile

-10-
Arbuscular Mycorrhizal Fungus-promoted Accumulation of Two
New Triterpenoids in Cucumber Roots

Kohki AKIYAMA† and Hideo HAYASHI

Natural Products Chemistry, Division of Applied Biological Chemistry, Graduate School of Agriculture
and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan

Received September 21, 2001; Accepted November 12, 2001
Cucumber (Cucumis sativus L.) roots were analyzed by HPLC and TLC for their levels of secondary metabolites upon inoculation with the arbuscular mycorrhizal fungus, Glomus caledonium. Three compounds in EtOAc extracts from the mycorrhizal roots showed significant increases six weeks after inoculation. These compounds were isolated by column chromatography and determined to be two novel triterpenes, 2β-hydroxybryonolic acid (2β,3β-dihydroxy-D:C-friedoolean-8-en-29-oic acid) and 3β-bryoferulic acid [3β-
O-trans-ferulyl-D:C-friedooleana-7,9(11)-diene-29-oic
acid], and the known triterpene, bryonolic acid, by spectroscopic methods. Time-course experiments showed that the levels of the three terpenoids in cucumber roots were significantly increased by the application of a 53-μm sieving from a soil inoculum of the arbuscular mycorrhizal fungus containing soil microbes but no mycorrhizal fungi, and that mycorrhizal colonization further promoted the terpenoid accumulation. Inoculation with Glomus mosseae also enhanced the accumulation of the triterpenes, whereas no accumulation was observed by inoculating with the fungal pathogen, Fusarium oxysporum f. sp. cucumerinum. 2β-Hydroxybryonolic acid was also isolated from the roots of melon and watermelon.
Key words: arbuscular mycorrhizal fungus; soil microbe; triterpenoid; cucumber; Cucurbitaceae

-11-
Kinetic Analysis of the Effect of (−)-Epigallocatechin Gallate
on the DNA Scission Induced by Fe(II)

Yasunori OHASHI,1 Koichi YOSHINAGA,2 Hisashi YOSHIOKA,3 and Hiroe YOSHIOKA1,†

1Radiochemistry Research Laboratory, 2Faculty of Science, Shizuoka University, 836 Ohya,
Shizuoka-shi 422-8529, Japan
3Institute for Environmental Science, University of Shizuoka, 52-1 Yada, Shizuoka-shi 422-8526, Japan

Received September 21, 2001; Accepted November 19, 2001
The DNA strand scission induced by Fe(II) in a citrate buffer solution and the effect of (−)-epigallocatechin gallate (EGCg) were kinetically analyzed. The rate of consumption of dissolved oxygen by Fe(II) in each of these solutions was measured and paralleled that DNA scission. Coordinated EGCg accelerated these reactions. Curves of the time-course characteristics of DNA scission were simulated by using the rate constant of oxygen consumption and by assuming that scission with the hydroxyl radical (OH), which was formed from the dissolved oxygen, proceeded competitively with the scavenging of OH by citrate, Cl− ions and EGCg added. Free EGCg acted as a DNA scission inhibitor to scavenge OH, in contrast to the case of the coordinated one. This analysis is useful for estimating the rate constant of the reaction between an antioxidant and OH, and might provide a new method for measuring the OH-scavenging activity.
Key words: tea catechin; iron complex; hydroxyl radical; DNA strand scission; kinetics

-12-
Purification and Characterization of a Novel Polysaccharide Involved
in the Pellicle Produced by a Thermotolerant Acetobacter Strain†

Somporn MOONMANGMEE, Hirohide TOYAMA, Osao ADACHI, Gunjana THEERAGOOL,1
Napha LOTONG,1 and Kazunobu MATSUSHITA††

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan
1Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

Received September 28, 2001; Accepted December 8, 2001
Acetobacter strains able to produce a thick pellicle at 37°C were screened among many thermotolerant strains isolated from fruits in Thailand. As a result, Acetobacter sp. SKU 1100 was selected as the producer of a relatively thick pellicle even when cultured at higher temperatures such as 37°C or 40°C. This strain could produce a pellicle polysaccharide in a shaking submerged culture as well as under static culture conditions. The polysaccharide was found to be attached to the bacterial cells. Although the polysaccharide production was higher at 30°C than at 37°C in shaking submerged culture, the productivity in static culture was not decreased even at higher temperatures. The membrane-attached polysaccharide was purified from the SKU 1100 strain by cell disruptions using either ultrasonic treatment or lysozyme treatment, followed by ultracentrifugation, enzyme treatments, dialysis against SDS, DEAE-cellulose column chromatography, alcohol precipitation, and gel filtration chromatography. The polysaccharide purified by the sonic treatment and also by the mild conditions using lysozyme treatment had the same average molecular mass of 120 kDa. The purified polysaccharide was composed of three different monosaccharides; glucose, galactose, and rhamnose, in an approximately equimolar ratio of 1:1:1.
Key words: thermotolerant acetic acid bacteria; Acetobacter species; pellicle polysaccharide formation; novel polysaccharide

-13-
Apoptosis Induction by Lectin Isolated from the Mushroom
Boletopsis leucomelas in U937 Cells

Yu KOYAMA, Yuko KATSUNO, Noriyuki MIYOSHI, Sumio HAYAKAWA, Takashi MITA,
Haruhiko MUTO,1 Satoko ISEMURA,2 Yutaka AOYAGI,3 and Mamoru ISEMURA†

School of Food and Nutritional Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
1Shizuoka Prefecture Forestry and Forest Products Research Institute, Negata, Hamakita,
Shizuoka 434-0016, Japan
2Nippon Dental University Junior Collage at Niigata, Niigata 951-8151, Japan
3Department of Third Internal Medicine, Niigata University School of Medicine, Niigata 951-8122, Japan

Received September 28, 2001; Accepted November 16, 2001
A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N′-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.
Key words: lectin; mushroom; apoptosis; tumor cells; affinity chromatography

-14-
Gene Expression during Pi Deficiency in Pholiota nameko:
Accumulation of mRNAs for Two Transporters

Yuji TASAKI,1 Yuki KAMIYA,2 A. AZWAN,1 Takashi HARA,2 and Toshio JOH2†

1Graduate School of Science and Technology, and 2Faculty of Agriculture, Niigata University, 2-8050,
Ikarashi, Niigata 950-2181, Japan

Received October 1, 2001; Accepted November 29, 2001
The effects of Pi deficiency on gene expression in Pholiota nameko were examined. A cDNA library was constructed from poly(A)＋ RNA isolated from mycelia cultured in Pi-depleted (P−) media, and 150 clones corresponding to Pi deficiency-inducible (pdi) genes were selected by differential hybridization with probes prepared from poly(A)＋ RNAs from the mycelia cultured in the Pi-supplied (P＋) and P− media. These clones were considered to derive from 31 genes by cross-hybridization. Northern blot analysis showed that these pdi genes were expressed in various patterns during Pi deficiency. Among the clones, the DNA sequences of pdi85 and pdi343 were analyzed. The deduced amino acid sequences indicated that they have structural similarities to Pi and metabolite transporters.
Key words: gene expression; metabolite transporter; Pholiota nameko; Pi deficiency; Pi transporter

-15-
Isolation and Characterization of A β-Primeverosidase-like endo-manner
β-Glycosidase from Aspergillus fumigatus AP-20

Shigeru YAMAMOTO,1 Masamichi OKADA,1 Taichi USUI,2 and Kanzo SAKATA3,†

1Amano Enzyme Inc. Gifu R＆D Center, Kakamigahara, Gifu 509-0108, Japan
2Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Shizuoka 422-8529, Japan
3Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan

Received October 9, 2001; Accepted November 29, 2001
A novel β-glycosidase-producing microorganism was isolated from soil and identified as Aspergillus fumigatus AP-20 based on its taxonomical characteristics. The enzyme was found to be an extracellular protein in the culture of the isolated fungus and was purified 88-fold by fractionation with ammonium sulfate followed by successive column chromatographies on phenyl-Sepharose HP and Mono P HR. The molecular mass was estimated to be 47 kDa by SDS-PAGE and the isoelectric point to be pH 6.0 by isoelectric focusing. The purified enzyme was highly specific for a substrate, p-nitrophenyl β-primeveroside (6-O-β-D-xylopyranosyl-β-D-glucopyranoside), which was cleaved in an endo-manner into primeverose and p-nitrophenol.
Key words: β-glycosidase; Aspergillus fumigatus; purification; characterization; β-primeverosidase

-16-
Growth-phase Dependent Expression of the Mevalonate Pathway
in a Terpenoid Antibiotic-producing Streptomyces Strain

Yoshimitsu HAMANO,1 Tohru DAIRI,1,† Masahiro YAMAMOTO,1 Tomohisa KUZUYAMA,2
Nobuya ITOH,1 and Haruo SETO2,†

1Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan
2Institute of Molecular and Celluar Biosciences, The University of Tokyo, Bunkyo-ku, 113-0032, Japan

Received October 12, 2001; Accepted November 19, 2001
Streptomyces griseolosporeus MF730-N6, a terpenoid antibiotic-terpentecin (Tp) producer, has both the nonmevalonate and mevalonate pathways for the formation of IPP. The Tp biosynthetic gene (ter) and the mevalonate pathway gene cluster (mev) including an HMG-CoA reductase gene (hmgr) had previously been cloned from strain MF730-N6. In this study, two distinct dxs genes (dxs 1 and dxs 2) and a dxr gene, which encode DXP synthases and DXP reductoisomerase, and participate in the initial and the second step of the nonmevalonate pathway, respectively, were cloned. These gene products were expressed in E. coli and confirmed to have the expected activities. The dxs 1, dxs 2, dxr, mev, and ter genes were used for Northern blot and primer extension analyses to examine temporal expression of these genes together with a gap gene coding for GAP dehydrogenase, which was also cloned in this study and used as an internal control. Transcripts of the dxs 1, dxs 2, dxr, and gap genes were detected throughout the cultivation. On the other hand, messages of the mev and ter genes were not detected at early growth phase but appeared when Tp production started. These results suggested that the nonmevalonate pathway and the mevalonate pathway were mainly used for the primary metabolism and the secondary metabolism, respectively, and that both of the two dxs genes were actually transcribed in this strain.
Key words: Streptomyces; mevalonate pathway; terpenoid; Northern blotting; terpentecin

-17-
Purification and Characterization of 31-kDa Palm Pollen Glycoprotein
(Ela g Bd 31 K), Which is Recognized by IgE from Palm Pollinosis Patients＊

Yoshinobu KIMURA,1,† Megumi MAEDA,1 Mariko KIMURA,1 Oi Ming LAI,2
Siang Hee TAN,3 Sook Mei HON,4 and Fook Tim CHEW4

1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University,
Okayama 700-8530, Japan
2Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia,
43400 Serdang Selangor, Malaysia
3Genome Centre, Institute Bioscience, Universiti Putra Malaysia, 43400 Serdang Selangor, Malaysia
4Department of Biological Sciences, National University of Singapore, Lower Kent Ridge Road,
Singapore 119074, Singapore

Received October 12, 2001; Accepted November 22, 2001
A basic glycoprotein, which was recognized by IgE from oil palm pollinosis patients, has been purified from oil palm pollen (Elaeis guineensis Jacq.), which is a strong allergen and causes severe pollinosis in Malaysia and Singapore. Soluble proteins were extracted from defatted palm pollen with both Tris-HCl buffer (pH 7.8) and Na-acetate buffer (pH 4.0). The allergenic glycoprotein was purified from the total extract to homogeneity with 0.4％ yield by a combination of DEAE- and CM-cellulose, SP-HPLC, and gel filtration. The purified oil palm pollen glycoprotein with molecular mass of 31 kDa was recognized by the β1-2 xylose specific antibody, suggesting this basic glycoprotein bears plant complex type N-glycan(s). The palm pollen basic glycoprotein, designated Ela g Bd 31 K, was recognized by IgE of palm pollinosis patients, suggesting Ela g Bd 31 K should be one of the palm pollen allergens. The preliminary structural analysis of N-glycans linked to glycoproteins of palm pollens showed that the antigenic N-glycans having α1-3 fucose and β1-2 xylose residues (GlcNAc2<10-48>0Man3Xyl1Fuc1<10-48>0GlcNAc2) actually occur on the palm pollen glycoproteins, in addition to the high-mannose type structures (Man9<10-48>5GlcNAc2).
Key words: pollen allergy; oil palm pollen allergen; antigenic N-glycan; plant glycoprotein; Elaeis guineensis Jacq.

-18-
Lymphatic Transport of Dietary Cholesterol Oxidation Products,
Cholesterol and Triacylglycerols in Rats

Hiroko TOMOYORI, Octavio CARVAJAL, Masahiro NAKAYAMA, Taiji KISHI,
Masao SATO, Ikuo IKEDA, and Katsumi IMAIZUMI†

Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan

Received October 15, 2001; Accepted November 19, 2001
Rats were fed on a diet containing 0.5％ cholesterol oxidation products (oxysterols) or 0.5％ cholesterol for 30 min, and their lymph was collected for 7 h. The amount of each of the individual oxysterols absorbed in the lymph depended on the ingested amounts, but the recovery was the highest for 5α,6α-epoxycholesterol (10.5％), this being followed by 7-ketocholesterol (5.8％), cholestanetriol (5.2％), 7β-hydroxycholesterol (4.8％), 7α-hydroxycholesterol (3.4％), 5β,6β-epoxycholesterol (2.2％), and 25-hydroxycholesterol (1.8％). A diet enriched with oxysterol, but not cholesterol, resulted in increased transport of triacylglycerols in the lymph. These results suggest that the absorption rate of oxysterols depends on the type, and indicate that the effect of dietary oxysterols on the lymphatic transport of triacylglycerols differs from that of dietary cholesterol. It therefore remains to be determined which oxysterol was responsible for the triacyglycerol transport.
Key words: cholesterol; triacylglycerols; lymph; oxysterols

-19-
Comparison of the Apoptosis-inducing Abilities of Various
Protein Synthesis Inhibitors in U937 Cells

Ai KAGEYAMA, Izumi KUSANO, Tadashi TAMURA, Tatsuya ODA,†
and Tsuyoshi MURAMATSU

Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi,
Nagasaki 852-8521, Japan

Received October 18, 2001; Accepted December 11, 2001
We compared the abilities of ricin, diphtheria toxin, cycloheximide, and anisomycin to induce apoptosis, using human myeloid leukemia U937 cells at the concentration of each toxin at which almost complete protein synthesis inhibition was attained within 3 h. Among these toxins, anisomycin was found to be the most potent apoptosis inducer. After a 6-h exposure to anisomycin (1 μg/ml), nearly 95％ of the cells had apoptotic nuclear morphological changes, while 53％, 30％, and 10％ of the cells showed apoptotic changes after exposure to ricin (0.1 μg/ml), diphtheria toxin (10 μg/ml), and cycloheximide (10 μg/ml), respectively. Furthermore, a rapid increase in caspase-3-like activity was observed in anisomycin-treated cells. A similar increase in caspase-3-like activity was also observed in ricin-treated cells on a slower time schedule. However, only a slight increase in the protease activity was induced by diphtheria toxin or cycloheximide even after 6 h of incubation. Since both ricin and anisomycin are known to act on 28S ribosomal RNA, our results suggest that this action mechanism may be responsible for their potent apoptosis induction, and protein synthesis inhibition alone is not sufficient to induce apoptosis.
Key words: protein synthesis inhibitors; apoptosis; 28S ribosomal RNA; ribotoxic stress

-20-
The Biological and Structural Similarity between Lunularic Acid
and Abscisic Acid

Hiromichi YOSHIKAWA,1,† Yayoi ICHIKI,1 Keiko (DOI) SAKAKIBARA,1
Hiroto TAMURA,2 and Masahito SUIKO3

1Department of Environmental Chemistry, Kyushu Kyoritsu University, Yahatanishi-ku,
Kitakyushu 807-8585, Japan
2Department of Applied Biological Chemistry, Meijo University, Tempaku-ku, Nagoya 468-8502, Japan
3Department of Biological Sciences, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan

Received November 5, 2001; Accepted December 13, 2001
Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA3)-induced α-amylase induction in embryoless barley seeds at 120 μM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 μM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C1-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.
Key words: lunularic acid; abscisic acid; α-amylase induction; immunoassay; computer chemistry

-21-
Detection of 2-Deoxy-D-ribose Radicals Generated by the Reaction
with the Hydroxyl Radical using a Rapid Flow-ESR Method

Yasunori OHASHI,1 Hisashi YOSHIOKA,2 and Hiroe YOSHIOKA1,†

1Radiochemistry Research Laboratory, Faculty of Science, Shizuoka University, 836 Ohya,
Shizuoka-shi 422-8529, Japan
2Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-shi 422-8526, Japan

Received November 7, 2001; Accepted December 17, 2001
ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO<09-25>) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO<09-25> at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.
Key words: deoxyribose radical; flow ESR method; hydroxyl radical; mixing resonator

-22-
Note
Oral Administration of Swine Gastric Mucin Alters Levels of Serum
and Urine Nitric Oxide, and Lipid Metabolism in Mice

Abdelkrim KHEDARA and Misao MIWA†

National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan

Received June 6, 2001; Accepted December 8, 2001
Mice were fed with swine gastric mucin in a basal diet for 5 weeks. In 5 week-old mice, a 2％ mucin diet significantly decreased nitric oxide levels of serum and liver. Reduction of serum total cholesterol and triglyceride and increase of HDL-cholesterol level were also significant with the mucin diet. In 14 month-old mice, the mucin diet was less effective.
Key words: nitric oxide (NO); cholesterol; triglyceride; mucin; mice

-23-
Note
Prevention of Browning in Potato with a Heat-treated Onion Extract

Min-Kyung LEE, Young-Mai KIM, Na-Young KIM, Gi-Nahm KIM,
Seok-Hwan KIM, Keuk-Seung BANG, and Inshik PARK†

Faculty of Food Science, Dong-A University, Pusan 604-714, Korea

Received July 16, 2001; Accepted November 1, 2001
The inhibitory effect of an onion extract on browning of potato was investigated. The addition of the heated onion extract to potato exhibited a marked inhibitory effect on potato polyphenol oxidase and the formation of a brown color. The inhibitory effect of the onion extract was dependent upon its heating temperature. The addition of both glycine and glucose increased the inhibitory effect of the onion extract toward potato polyphenol oxidase.
Key words: onion extract; browning; potato; polyphenol oxidase

-24-
Note
Acid and Heat Stability of the Anticoagulative Activity
of an Onion Extract

Gi-Nahm KIM, Min-Kyung LEE, and Inshik PARK†

Faculty of Food Science, Dong-A University, Pusan 604-714, Korea

Received August 3, 2001; Accepted December 4, 2001
An onion extract possessed anticoagulative activity apparently by inhibiting thrombin, and it was dependent upon the concentration of onion extract added. Boiling the onion extract at 100°C for 30 min made no difference to its anticoagulative activity compared to that of fresh onion. The anticoagulative activity of the onion extract was also retained after an acid treatment by incubating at pH 2.0 for 4 hrs. However, dialyis of the extract substantially eliminated the anticoagulative activity. Therefore, the anticoagulant in the onion extract seems to have been a heat- and acid-stable substance of low molecular weight.
Key words: thrombin inhibitor; onion; heat stability; acid stability

-25-
Note
Isolation and Identification of Dihydrochrysanolide and Its 1-Epimer
from Chrysanthemum coronarium L.

Kyung Dong LEE,1 Min Suk YANG,1 Tae Jung HA,1 Ki Min PARK,2 and Ki Hun PARK1,†

1Department of Agricultural Chemistry, Division of Applied Life Science, Gyeongsang National University,
Chinju 660-701, Korea
2The Research Institute of Natural Science, Gyeongsang National University, Chinju 660-701, Korea

Received August 10, 2001; Accepted November 9, 2001
A new sesquiterpene lactone (1) was isolated with known dihydrochrysanolide derivatives (2 and 3) from the flowers of Chrysanthemum coronarium L., and their structures were identified by spectroscopic data. The stereochemistry of the epimers (1 and 2) was determined from NOESY data and an X-ray crystallographic analysis. The isolated compounds (1--3) were examined for their cytotoxic activity against such human cell lines as A549, PC-3 and HCT-15.
Key words: Chrysanthemum coronarium L.; sesquiterpene lactone; dihydrochrysanolide; 1-epi-dihydrochrysanolide; cytotoxic activity

-26-
Note
Response of the Ice-nucleating Bacterium Pantoea ananas
KUIN-3 during Cold Acclimation

Noriko KODA, Yukari INADA, Satoshi NAKAYAMA, Hidehisa KAWAHARA,
and Hitoshi OBATA

Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35, Yamate-cho,
Suita, Osaka 564-8680, Japan

Received August 27, 2001; Accepted December 12, 2001
The ice-nucleating bacterium Pantoea ananas KUIN-3 accumulated glucose in cells following a shift in temperature (10°C) from the optimum growth temperature (30°C). This accumulation might be caused by the activation of glucose-6-phosphatase. Although this strain after culturing at 30°C was harmed by freezing, the cryotolerance of this strain was reached about 80％ after cold acclimation at 10°C.
Key words: ice-nucleating bacterium; cold acclimation; Pantoea ananas KUIN-3; accumulation of glucose

-27-
Note
Increase in Oxidative Stress in Kidneys of Diabetic Akita Mice

Yuki UENO,1 Fumihiko HORIO,1 Koji UCHIDA,1 Michitaka NAITO,2 Hideki NOMURA,2
Yoji KATO,3 Takanori TSUDA,4 Shinya TOYOKUNI,5 and Toshihiko OSAWA1,†

1Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan
2Nagoya University, Graduate School of Medicine, Nagoya 466-8550, Japan
3School of Humanities for Environmental Policy and Technology, Himeji Institute of Technology,
Himeji 670-0092, Japan
4Junior College Division, Tokaigakuen University, Nagoya 468-8514, Japan
5Department of Pathology and Biology of Disease, Graduate School of Medicine, Kyoto University,
Kyoto 606-8501, Japan

Received September 3, 2001; Accepted November 22, 2001
New biomarkers for oxidative damage, were used to identify whether hyperglycemia caused oxidative stress in diabetic Akita mice. At 13 weeks of age, the tissues of these mice were obtained, and the levels of Nε-(hexanonyl)lysine (HEL) and dityrosine (DT) were measured, these being related to lipid peroxide-derived protein covalent modification and protein cross-linking. The levels of HEL and DT in the kidneys of Akita mice were significantly increased compared with the control mice without any accumulation of thiobarbituric acid reactive substances and 4-hydroxy-2-nonenal-modified protein. Immunopositive staining was clearly observed in the kidneys of the Akita mice when using the anti-HEL antibody or anti-DT antibody. These results suggest that hyperglycemia in Akita mice induced oxidative stress and increased these markers in the kidneys.
Key words: diabetes; oxidative stress; Nε-(hexanonyl)lysine; dityrosine; lipid peroxidation

-28-
Note
Temperature Dependence of Kinetic Parameters for Hyperthermophilic
Glutamate Dehydrogenase from Aeropyrum pernix K1

Mohammad W. BHUIYA, Haruhiko SAKURABA, and Toshihisa OHSHIMA†

Department of Biological Science and Technology, Faculty of Engineering, The University of Tokushima,
Tokushima 770-8506, Japan

Received September 10, 2001; Accepted November 29, 2001
The temperature dependence of the steady-state kinetic parameters for a glutamate dehydrogenase from Aeropyrum pernix K1 was investigated. The enzyme showed a biphasic kinetic characteristic for L-glutamate and a monophasic one for NADP at 50--90°C. At low concentrations of L-glutamate the Km decreased from 2.02 to 0.56 mM and the catalytic efficiency (Vmax/Km) markedly increased (4--150 μmol・mg−1・mM−1) along with the increase of temperature from 50 to 90°C. At high concentrations of the substrate the Km was fairly high and approximately constant (around 225 mM), and the catalytic efficiency was low and its temperature-dependent change was small. The Km (0.039 mM) for NADP did not change with the increase of temperature. In the reductive amination, the Kms for 2-oxoglutarate (1.81 and 9.37 mM at low and high levels of ammonia, respectively) were independent on temperature, but the Kms for ammonia and NADPH rose from 86 to 185 mM and 0.050 to 0.175 mM, respectively.
Key words: Aeropyrum pernix K1; steady state kinetics; glutamate dehydrogenase; hyperthermophile; Km

-29-
Note
5-Halogenated Thymidine Analogues Induce a Senescence-like
Phenomenon in HeLa Cells

Eriko MICHISHITA, Naoto MATSUMURA, Toshihiro KURAHASHI, Toshikazu SUZUKI,
Hideki OGINO, Michihiko FUJII, and Dai AYUSAWA

Division of Biochemistry, Kihara Institute for Biological Research and Graduate School of Integrated Science,
Yokohama City University, Maioka-cho 641-12, Totsuka-ku, Yokohama 244-0813, Japan

Received September 12, 2001; Accepted December 13, 2001
We tested various thymidine analogues for induction of a senescence-like phenomenon in HeLa cells. CldU, BrdU, and IdU similarly induced the morphology of senescent cells and typical senescence markers. Thymidine analogues other than 5-halogenated forms caused only cell death. BrdU efficiently killed the cells in cooperation with irradiation with light and a brief treatment with Hoechst 33258, but CldU did not at all. 5-Halogenated thymidine analogues were thus shown to be specific inducers of cellular senescence in mammalian cells.
Key words: 5-halogenated deoxyuridine; senescence; DNA damage; HeLa cell

-30-
Note
Salts and Glycine Increase Reversibility and Decrease Aggregation
during Thermal Unfolding of Ribonuclease-A

Yoshiko KITA and Tsutomu ARAKAWA

Alliance Protein Laboratories, 3957 Corte Cancion, Thousand Oaks, CA 91360, USA

Received September 17, 2001; Accepted November 2, 2001
Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63％. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75°C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3°C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.
Key words: thermal unfolding; native gel; aggregation; ribonuclease-A

-31-
Note
Formation of New Lipoaminopeptides, Acremostatins A, B, and C,
by Co-cultivation of Acremonium sp. Tbp-5 and Mycogone rosea
DSM 12973

Thomas DEGENKOLB,1,†† Stephan HEINZE,1 Brigitte SCHLEGEL,1 Gary STROBEL,2
and Udo GR<Oh><0196><Wa>AFE1,†

1Hans-Kn<Oh><0228><Wa>oll-Institute for Natural Products Research, Beutenbergstra<Oh><0223><Wa>e 11a, D-07745 Jena, Germany
2Department of Plant Pathology, Montana State University, Bozeman, Montana 59717, USA

Received September 25, 2001; Accepted November 20, 2001
Formation of new lipoaminopeptides, acremostatins A, B, and C, was observed during co-cultivation of Acremonium sp. Tbp-5 and Mycogone rosea DSM 12973. Thus, co-cultivation of microorganisms producing related products could be suggested as a suitable way towards diversification of microbial structures.
Key words: co-cultivation; lipoaminopeptides; acremostatins; Acremonium; Mycogone rosea

-32-
Note
Stereoselective Synthesis of (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic
Acid via C--H Insertion of Alkylidenecarbene

Susumu OHIRA,† Megumi AKIYAMA, Kumiko KAMIHARA, Yuichi ISODA, and
Atsuhito KUBOKI

Department of Biological Chemistry, Faculty of Science, Okayama University of Science, 1-1 Ridai-cho,
Okayama 700-0005, Japan

Received October 1, 2001; Accepted November 16, 2001
(1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD), a potent agonist of metabotropic glutamate receptors, was synthesized from L-serine. The chiral quaternary center was constructed by C--H insertion of the alkylidenecarbene, this being generated by the reaction between lithiotrimethylsilyldiazomethane and the corresponding ketone.
Key words: metabotropic glutamate agonist; amino acid; chiral quaternary center; C--H insertion; alkylidenecarbene

-33-
Note
Med, a Cell-surface Localized Protein Regulating a Competence
Transcription Factor Gene, comK, in Bacillus subtilis

Mitsuo OGURA,† Hitoshi HASHIMOTO, and Teruo TANAKA

Department of Marine Science and Technology, Tokai University, 3-20-1 Orido, Shimizu,
Shizuoka 424-8610, Japan

Received October 1, 2001; Accepted November 16, 2001
Med was found as a positive regulator for comK, a master regulator for late competence genes. It was found by Western analysis that the ComK level was decreased in a med mutant. Experiments using an alkaline phosphatase fusion with Med and Western analysis of Med were done because a putative lipo-modification signal is found at the N-terminus of Med. The results obtained are consistent with the localization of Med at the cell surface. An implication of the cell-surface localization of Med is discussed in terms of comK regulation.
Key words: competence development; protein localization; Med

-34-
Note
Organization and Transcriptional Characterization of Catechol
Degradation Genes Involved in Carbazole Degradation
by Pseudomonas resinovorans Strain CA10

Hideaki NOJIRI, Kana MAEDA, Hiroyo SEKIGUCHI, Masaaki URATA, Masaki SHINTANI,
Takako YOSHIDA, Hiroshi HABE, and Toshio OMORI

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

Received October 11, 2001; Accepted November 16, 2001
Pseudomonas resinovorans strain CA10 assimilates catechol, which is an intermediate of carbazole degradation, by ortho cleavage pathway enzymes encoded by the catR, catBCA operon. Cat proteins of strain CA10 were very similar to those of P. putida, although the relatedness in non-coding regions was not high. It was found that catBCA genes were induced in carbazole-grown cells as a single transcriptional unit.
Key words: carbazole; catechol; cat operon; Pseudomonas resinovorans strain CA10

-35-
Note
New Medium Composition for High Betacyanin Production
by a Cell Suspension Culture of Table Beet (Beta vulgaris L.)

Toru AKITA,† Yasuhiko HINA, and Toyoyuki NISHI

The Nippon Shinyaku Institute for Botanical Research, 39 Sakanotsuji-cho, Ohyake,
Yamashina-ku, Kyoto 607-8182, Japan

Received October 15, 2001; Accepted December 5, 2001
The effect of a revised Linsmaier-Skoog (LS) medium on betacyanin production was investigated in suspension cultures of table beet (Beta vulgaris L.). The effects of a high iron concentration and low concentration of zinc on betacyanin production were not cumulative. The composition of the new revised medium for high betacyanin production was established by reducing the concentration of inorganic nitrogen (30 mM), modifying the ratio of ammonium to nitrate (1:14), reducing the concentration of zinc (0.0003 mM), and removing copper and cobalt. The revised LS medium enabled the maximum betacyanin yield of 550 mg/l to be obtained from a 14-day culture. This medium promoted the betacyanin production in three types of cell line differing in the betacyanin productivity. The betacyanin productivity (40 mg/l・day) was higher than that quoted in any other previous reports.
Key words: Beta vulgaris L.; betacyanin; cell suspension culture; culture medium

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Note
5-Bromo-2′-deoxyuridine Efficiently Suppresses Division Potential
of the Yeast Saccharomyces cerevisiae

Michihiko FUJII, Hisasi ITO, Tsutomu HASEGAWA, Toshikazu SUZUKI,
Noritaka ADACHI,＊ and Dai AYUSAWA

Division of Biochemistry and ＊Division of Molecular Cell Genetics, Kihara Institute for Biological Research
and Graduate School of Integrated Science, Yokohama City University, Maioka-cho 641-12, Totsuka-ku,
Yokohama 244-0813, Japan

Received October 17, 2001; Accepted November 28, 2001
We established a thymidine-auxotrophic strain of the yeast Saccharomyces cerevisiae to examine biological effects of thymidine analogues. 5-Bromo-2′-deoxyuridine efficiently suppressed the division potential of yeast showing morphology similar to senescent cells.
Key words: yeast; thymidine auxotroph; 5-bromo-2′-deoxyuridine; growth; senescence

-37-
Note
Expression of Nicotiana glutinosa Ribonucleases in Escherichia coli

Madoka HINO, Shin KAWANO, and Makoto KIMURA†

Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan

Received October 18, 2001; Accepted November 27, 2001
We previously isolated from Nicotiana glutinosa leaves three distinct cDNA clones, NGR1, NGR2, and NGR3, encoding a wound-inducible RNase NW, and putative RNases NGR2 and NGR3, respectively. In this study, we produced RNases NW and NGR3 in Escherichia coli and purified them to homogeneity. RNase NGR3 had non-absolute specificity toward polynucleotides, although RNase NW preferentially cleaved polyinosinic acid (Poly I). Both RNases NW and NGR3 were more active toward diribonucleoside monophosphates ApG, CpU, and GpU. Furthermore, kinetic parameters for RNase NW (Km, 0.778 mM and kcat, 1938 min−1) and RNase NGR3 (Km, 0.548 mM and kcat, 408 min−1) were calculated using GpU as a substrate.
Key words: expression; Nicotiana glutinosa; S-like RNase; TMV infection; wounding

-38-
Note
Prevention by Polyhydroxysteroids and Saponins of Asterina pectinifera
of the Desquamation of Stratum Corneum Cells

Kazuyo UKAI,1 Atsufumi IWANAGA,2 Hiroshi NAGAI,1 and Michio NAMIKOSHI1,†

1Department of Ocean Sciences, Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku,
Tokyo 108-8477, Japan
2Research ＆ Development Division, Sansho Seiyaku Co., Ltd., 2-26-7 Ohike, Ohnojo-shi,
Fukuoka 816-8550, Japan

Received October 25, 2001; Accepted November 30, 2001
Two polyhydroxysteroids and three steroidal saponins (pectiniosides A, B and C) were isolated as bioactive substances from Asterina pectinifera. These compounds inhibited the release of guinea-pig skin stratum corneum cells by 48--67％ (control, 0％) at 2 mg/ml. It is suggested that the bioactivity of these compounds is connected with the remedial and preventive effects of an aqueous extract of A. pectinifera on rough human skin.
Key words: Asterina pectinifera; polyhydroxysteroid; steroidal saponin; desquamation; stratum corneum

-39-
Note
Conjugated Linoleic Acid Reduces Body Fats and Cytokine Levels of Mice

Asuka AKAHOSHI,1 Yuki GOTO,1 Kayoko MURAO,1 Tomoko MIYAZAKI,1
Masao YAMASAKI,2 Michiko NONAKA,2 Koji YAMADA,2 and Michihiro SUGANO1

1Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide,
Kumamoto 862-8502, Japan
2Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University, Higashi-ku,
Fukuoka 812-8581, Japan

Received October 26, 2001; Accepted December 17, 2001
In order to discover the effect of CLA on the body fat size and serum cytokine levels, four groups of male mice were fed diets containing either 1％ linoleic acid (LA) or conjugated linoleic acid (CLA) with or without 0.2％ sesamin for 8 weeks. The weight gain and feed efficiency were significantly lower in the CLA groups. CLA significantly reduced relative weights (g/100 g body weight) of epididymal and perirenal adipose tissues, in particular the former. Concentrations of serum TNF-α and leptin were significantly reduced by dietary CLA. Sesamin did not show additional effects in all of these parameters. There was a positive correlation between cytokine production and body-fat reducing potential of CLA. These results indicated that mice appeared to be a hyperresponder to dietary CLA insofar as the reduction of body fat size is concerned.
Key words: conjugated linoleic acid; sesamin; adipose tissue; leptin; TNF-α

-40-
Note
Antimicrobial Activity of Perilla Seed Polyphenols
against Oral Pathogenic Bacteria

Hiroyo YAMAMOTO and Tomohiko OGAWA†

Department of Oral Microbiology, Asahi University School of Dentistry, 1851-1 Hozumi,
Hozumi-Cho, Motosu-Gun, Gifu 501-0296, Japan

Received October 26, 2001; Accepted December 10, 2001
A perilla seed (Perilla frutescens Britton var. japonica Hara) extract was examined for its antimicrobial activity against oral cariogenic streptococci and periodontopathic Porphyromonas gingivalis. Luteolin, one of the components of perilla seed, showed the strongest antimicrobial effect among the phenolic compounds. According to our results, perilla seed may be the source of an antimicrobial agent that could prevent dental caries and periodontal diseases.
Key words: perilla seed; luteolin; polyphenols; antimicrobial activity

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Note
Synthesis of (R)-1,3-Butanediol by Enantioselective Oxidation Using
Whole Recombinant Escherichia coli Cells Expressing (S)-Specific
Secondary Alcohol Dehydrogenase

Hiroaki YAMAMOTO,† Akinobu MATSUYAMA, and Yoshinori KOBAYASHI

Tsukuba Research Center, Daicel Chemical Industries, Ltd., 27 Miyukigaoka, Tsukuba,
Ibaraki 305-0841, Japan

Received November 6, 2001; Accepted December 5, 2001
The synthesis of (R)-1,3-butanediol (BDO) from its racemate was studied using whole cells of recombinant Escherichia coli expressing an (S)-specific secondary alcohol dehydrogenase (CpSADH) from Candida parapsilosis by enantioselective oxidation. Under the optimized conditions, the yield of (R)-1,3-BDO reached 72.6 g/l, with a molar recovery yield of 48.4％ from a racemate of 15％ and an optical purity of 95％ ee.
Key words: secondary alcohol dehydrogenase; (R)-1,3-butanediol; NAD＾{＋}-regeneration; enantioselective oxidation

-42-
Preliminary Communication
α-Glucosidase Mutant Catalyzes <Oh><0192><0192>α<Wa>-Glycosynthase′′-type Reaction

Masayuki OKUYAMA, Haruhide MORI, Kotomi WATANABE,
Atsuo KIMURA,† and Seiya CHIBA

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

Received November 26, 2001; Accepted December 20, 2001
Replacement of the catalytic nucleophile Asp481 by glycine in Schizosaccharomyces pombe α-glucosidase eliminated the hydrolytic activity. The mutant enzyme (D481G) was found to catalyze the formation of an α-glucosidic linkage from β-glucosyl fluoride and 4-nitrophenyl (PNP) α-glucoside to produce two kinds of PNP α-diglucosides, α-isomaltoside and α-maltoside. The two products were not hydrolyzed by D481G, giving 41 and 29％ yields of PNP α-isomaltoside and α-maltoside, respectively. PNP monoglycosides, such as α-xyloside, α-mannoside, or β-glucoside, acted as the substrate, but PNP α-galactoside and maltose could not. No detectable product was observed in the combination of α-glucosyl fluoride and PNP α-glucoside. This study is the first report on an <Oh><0192><0192>α<Wa>-glycosynthase′′-type reaction to form an α-glycosidic linkage.
Key words: α-glucosidase; catalytic nucleophile mutant; glycosynthase; oligosaccharide production

-43-
Preliminary Communication
Biosynthesis of Vitamin B6 in Rhizobium: In Vitro Synthesis of Pyridoxine
from 1-Deoxy-D-xylulose and 4-Hydroxy-L-threonine

Masaaki TAZOE,† Keiko ICHIKAWA, and Tatsuo HOSHINO

Department of Applied Microbiology, Nippon Roche Research Center, Kamakura,
Kanagawa 247-8530, Japan

Received December 24, 2001; Accepted December 25, 2001
Pyridoxine (vitamin B6) in Rhizobium is synthesized from 1-deoxy-D-xylulose and 4-hydroxy-L-threonine. To define the pathway enzymatically, we established an enzyme reaction system with a crude enzyme solution of R. meliloti IFO14782. The enzyme reaction system required NAD＋, NADP＋, and ATP as coenzymes, and differed from the E. coli enzyme reaction system comprising PdxA and PdxJ proteins, which requires only NAD＋ for formation of pyridoxine 5′-phosphate from 1-deoxy-D-xylulose 5-phosphate and 4-(phosphohydroxy)-L-threonine.
Key words: vitamin B6 in vitro synthesis; Rhizobium; 1-deoxy-D-xylulose; 4-hydroxy-L-threonine; pyridoxine<QL>